RU2775063C2 - Polypeptides recruiting t-cells, capable of binding cd123 and alpha/beta tcr - Google Patents

Abstract

FIELD: biochemistry.

SUBSTANCE: invention relates to the field of biochemistry, in particular to polypeptide, which redirects T-cells for the destruction of CD123-expressing cells. A structure for redirecting T-cells, containing the specified polypeptide; nucleic acid encoding the specified polypeptide or the specified structure; an expression vector and a host cell, containing the specified nucleic acid; and a composition containing the specified polypeptide are also disclosed. A method for the production of the specified polypeptide is disclosed.

EFFECT: invention has a capability of effective treatment of diseases associated with CD123-expressing cells.

20 cl, 50 ex, 62 dwg, 44 tbl

Description

The technical field to which the invention belongs

The present invention provides multispecific T cell recruiting polypeptides that comprise an immunoglobulin single variable domain specifically binding to a T cell receptor (TCR) constant domain on a T cell and one or more immunoglobulin single variable domains binding CD123 expressed on a cell- targets. The present invention also relates to monovalent CD123 binding polypeptides for use in these multispecific polypeptides. The invention also relates to nucleic acids encoding these polypeptides, as well as vectors, hosts and methods for producing polypeptides according to the invention. The invention also relates to methods of treatment using the polypeptides of the invention and kits in which they are provided.

Prior Art

CD123 (interleukin 3 receptor α-subunit, IL-3Rα) is a 75 kDa glycoprotein that is converted to 43 kDa upon cleavage with N-glycosidase (Sato et al. 1993, Blood 82: 752-761). CD123 consists of three HEK cellular domains, a transmembrane domain, and a short intracellular region. The N-terminal HEK cellular domain contributes significantly to the interaction of CD123 with IL-3, while the intracellular region is required for signaling (Barry et al. 1997, Blood 89: 842-852). CD123 specifically binds IL-3 with low affinity. Heterodimerization of CD123 with a common β subunit (βc), which does not itself bind to IL-3, results in the formation of IL-3R, a high affinity receptor for IL-3. The βc subunit plays an important role in signal transduction and as such elicits a number of biological functions (Hara et al., 1996 Stem cells 14: 605-618).

While the βc subunit is expressed on the surface of various cells, CD123 expression is more limited to IL-3 responsive cells such as hematopoietic stem/progenitor cells, monocytes, megakaryocytes, B lymphocytes, and plasmacytoid dendritic cells. Binding of IL-3 stimulates the proliferation and differentiation of hematopoietic cells. During the maturation of these cells, CD123 expression gradually decreases and cannot be detected in mature lymphocytes and granulocytes.

CD123 is reported to be highly expressed in leukemia stem cells (LSC) and is associated with the initiation and development of many diseases such as acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), and hairy cell leukemia (HCL). Details of CD123 and its associated clinical applications in leukemia are mentioned in a review by Liu et al. (2015 Life Sciences 122: 59-64). Given the difference in CD123 expression on normal hematopoietic stem cells and LSCs, CD123 is an interesting therapeutic target in hematological cancers.

AML is a clonal malignant disorder derived from a small population of LSC cells overexpressing CD123. AML is characterized by the proliferation of myeloid progenitor cells in the bone marrow and peripheral blood and leads to the destruction of normal hematopoiesis. Although therapeutic regimens and supportive care for AML patients have improved over the years, no major changes have occurred in standard treatment options over the past three decades. For an overview of new approaches and treatment options for AML, see Medinger et al. (2016 Leukemia Research Reports 6: 39-49). Currently, only 35-40% of patients under 60 years of age are cured of this disease. For older patients (>60 years), the overall prognosis remains poor. Allogeneic hematopoietic stem cell transplantation currently offers the best chance of a cure. Therefore, there remains a need for new therapeutic agents for the treatment of AML.

A possible strategy for the prevention of AML and the treatment of recurrences is the use of immunotherapy, which is a rapidly growing area of cancer research. Immunotherapy directs the body's immune system, and in particular T cells, to malignant tumor cells.

Cytotoxic T cells (CTL) are T lymphocytes that kill malignant tumor cells, cells that are infected (especially by viruses), or cells that are damaged in other ways. T lymphocytes (or T cells) express the T cell receptor or TCR molecule and the CD3 receptor on the cell surface. The αβ TCR-CD3 complex (or "TCR complex") consists of six different type I single-stranded transmembrane proteins: the TCRα and TCRβ chains, which form a TCR heterodimer responsible for ligand recognition, and the HEK valently linked CD3γ, CD3δ CD3ε, and ζ chains, which carry cytoplasmic sequence motifs that are phosphorylated upon receptor activation and recruit a large number of signaling components (Call et al., 2004, Molecular Immunology 40: 1295-1305).

Both the α and β chains of the T cell receptor consist of a constant domain and a variable domain. Physiologically, the αβ chains of the T-cell receptor recognize the peptide-loaded MHC complex and bind upon interaction with CD3 chains. These CD3 chains subsequently transmit the binding signal to the intracellular environment.

Taking into account the potential of natural cytotoxic T-lymphocytes (CTL) to promote cell lysis, various strategies have been studied to recruit immune cells to mediate the destruction of tumor cells. However, detection of specific T cell responses depends on the expression of MHC molecules by malignant tumor cells and on the presence, production, transport, and presentation of specific peptide antigens. More recent developments have attempted an alternative approach combining the benefits of immunotherapy with antibody therapy by recruiting all of the patient's T cells in a polyclonal manner using recombinant antibody technologies: "bispecificity".

Bispecific antibodies have been developed that have a tumor recognition portion on one arm (target-binding arm) while the other arm of the molecule has specificity for T-cell antigen (effector-binding arm), mainly CD3. By simultaneously binding the two arms to their respective antigens, T-lymphocytes are targeted and activated in the tumor cell, where they can perform their cytolytic function.

The concept of using bispecific antibodies to activate T cells against tumor cells was described over 20 years ago, but manufacturing problems and clinical failures have led to stagnation. Further progress was made when smaller format bispecific preparations were developed, resulting from the reduction of antibodies to their variable fragments.

Although the first T-cell recruitment format, Blinatumomab (a BiTE molecule that recognizes CD19 and CD3), was approved by the FDA in December 2014 for second-line therapy, many hurdles had to be overcome. The first clinical trials of Blinatumomab were prematurely terminated due to neurological adverse events, cytokine release syndrome and infections on the one hand, and the absence of objective clinical reactions or clear signs of biological activity on the other hand.

As a treatment option for AML, MacroGenics has recently developed MGD006, CD3 x CD123 bispecific DARTs (double affinity retargeting molecules). As described in Hussaini et al. (2016 Blood 127: 122-131), MGD006 are able to recognize CD123-positive leukemic cells and induce T-cell activation, resulting in the killing of CD123-overexpressing tumor cells in vitro and in vivo. However, DART also activates CD25, a marker of T cell activation, on T cells when incubated with the CD123-negative K562 ^GFP cell line (Figure 1D, Hussaini et al., 2016). In addition, target-independent killing was observed with two CD123-negative cell lines (Figure 2B, Hussaini et al., 2016). Therefore, there may be safety concerns with these DARTs due to this target-independent T-cell activation.

To minimize the risk of adverse effects and systemic side effects such as cytokine storms, maximum care must be taken in selecting both the tumor antigen arm and the T-cell antigen arm. The latter must bind to the constant domain of the TCR complex in a monovalent manner and may not trigger T cell signaling in the absence of target malignant tumor cells. Only specific binding of both arms to their targets (tumor and T-cell antigen) can induce the formation of cytolytic synapses and subsequent destruction of tumor cells. The specificity of the tumor recognition arm for its antigen is a necessary condition to avoid off-target binding, which will inevitably lead to target-independent T-cell activation.

In addition to being effective, MGD006, like blinatumomab, is very small in size and lacks an Fc domain. Therefore, MGD006 would require continuous intravenous infusion, which would not contribute to patient compliance. MacroGenics is now attempting to address this issue by merging the Fc domain with their next generation DARTs (WO2015026892), which not only makes the molecule larger, but may also introduce manufacturing issues and other Fc functions. The larger format with Fc is expected to have a better PK but reintroduces the risk of off-target activity.

Therefore, there remains a need for alternative bispecific CD123 x T-cell antigen-binding polypeptides with minimal target-independent T-cell activation, in which the half-life can be adapted.

The essence of the invention

The invention solves this problem by providing multispecific polypeptides containing an immunoglobulin single variable domain (ISV) that specifically binds to the T cell receptor (TCR) constant domain and one or more ISVs that specifically bind CD123. In a specific aspect, the polypeptide redirects T cells to cells expressing CD123 and induces T cell-mediated killing.

The combination of T-cell receptor-binding ISV and CD123-binding ISV was specifically chosen to result in efficient T-cell activation at CD123-expressing cells, while target-independent T-cell activation appears to be minimal. .

Thus, in a first aspect, the present invention provides a polypeptide that redirects T cells to kill CD123 expressing cells, comprising one immunoglobulin single variable domain (ISV) that specifically binds the T cell receptor (TCR) and one or more ISVs. , which specifically binds CD123, where the ISV that specifically binds TCR (essentially) consists of 4 framework regions (FR1-FR4, respectively) and 3 complementarity-determining regions (CDR1-CDR3, respectively), in which:

i) CDR1 is selected from the group consisting of:

a) SEQ ID NO: 181-191; or

b) amino acid sequences that have 4, 3, 2 or 1 amino acid differences with the amino acid sequence of one of SEQ ID NOs: 181-191; provided that an ISV comprising a CDR1 with a 4, 3, 2, or 1 amino acid difference binds the TCR with the same, about the same, or higher affinity as compared to binding to an ISV comprising a CDR1 without a 4, 3, 2 difference or 1 amino acid, where the specified affinity, measured using surface plasmon resonance;

and/or

ii) CDR2 is selected from the group consisting of:

c) SEQ ID NOS: 192-217; or

d) amino acid sequences that have a 4, 3, 2 or 1 amino acid difference from the amino acid sequence of one of SEQ ID NOs: 192-217; provided that an ISV comprising a CDR2 with a 4, 3, 2, or 1 amino acid difference binds the TCR with the same, about the same, or higher affinity as compared to binding of an ISV comprising a CDR2 with no difference in 4, 3, 2, or 1 amino acid, where the specified affinity is measured using surface plasmon resonance;

and/or

iii) CDR3 is selected from the group consisting of:

e) SEQ ID NOS: 218-225; or

f) amino acid sequences with a difference of 4, 3, 2 or 1 amino acid with the amino acid sequence of one of SEQ ID NOs: 218-225; provided that an ISV comprising a CDR3 with a 4, 3, 2, or 1 amino acid difference binds the TCR with the same, about the same, or higher affinity than an ISV comprising a CDR3 with no 4, 3, 2 or 2 amino acid difference 1 amino acid, where the specified affinity is measured using surface plasmon resonance;

and where one or more ISVs that specifically bind CD123 (substantially) consist of 4 framework regions (FR1-FR4, respectively) and 3 complementarity-determining regions (CDR1-CDR3, respectively), in which:

i) CDR1 is selected from the group consisting of:

a) SEQ ID NO: 11-16; or

b) amino acid sequences with a difference of 4, 3, 2 or 1 amino acid with the amino acid sequence of one of SEQ ID NOs: 11-16; provided that an ISV comprising a CDR1 with a 4, 3, 2, or 1 amino acid difference binds CD123 with the same, about the same, or higher affinity as compared to binding of an ISV comprising a CDR1 with no difference in 4, 3, 2, or 1 amino acid, where the specified affinity is measured using surface plasmon resonance;

and/or

ii) CDR2 is selected from the group consisting of:

c) SEQ ID NO: 17-20; or

d) amino acid sequences with a difference of 4, 3, 2 or 1 amino acid with the amino acid sequence of one of SEQ ID NOs: 17-20; provided that an ISV comprising a CDR2 with a 4, 3, 2, or 1 amino acid difference binds CD123 with the same, about the same, or higher affinity than an ISV comprising a CDR2 with no 4, 3, 2, or 2 amino acid difference 1 amino acid, where the specified affinity is measured using surface plasmon resonance;

and/or

iii) CDR3 is selected from the group consisting of:

e) SEQ ID NOS: 21-25; or

f) amino acid sequences with a difference of 4, 3, 2 or 1 amino acid with the amino acid sequence of one of SEQ ID NOs: 21-25; provided that an ISV comprising a CDR3 with a 4, 3, 2, or 1 amino acid difference binds CD123 with the same, about the same, or higher binding affinity than an ISV comprising a CDR3 with no difference in 4, 3, 2, or 1 amino acid, where the specified affinity is measured using surface plasmon resonance.

In a further aspect, the present invention relates to a polypeptide described herein, wherein an ISV that specifically binds a TCR (substantially) consists of 4 framework regions (FR1-FR4, respectively) and 3 complementarity-determining regions (CDR1-CDR3, respectively) , wherein:

i) CDR1 is selected from the group consisting of:

a) SEQ ID NO: 181-191; or

b) amino acid sequences with a difference of 4, 3, 2 or 1 amino acid with the amino acid sequence of one of SEQ ID NOs: 181-191; provided that an ISV comprising a CDR1 with a 4, 3, 2, or 1 amino acid difference binds the TCR with the same, about the same, or higher affinity as compared to binding to an ISV comprising a CDR1 without a 4, 3, 2 difference or 1 amino acid, where the specified affinity is measured using surface plasmon resonance;

and

ii) CDR2 is selected from the group consisting of:

c) SEQ ID NOS: 192-217; or

d) amino acid sequences with a difference of 4, 3, 2 or 1 amino acid with the amino acid sequence of one of SEQ ID NOs: 192-217; provided that an ISV comprising a CDR2 with a 4, 3, 2, or 1 amino acid difference binds the TCR with the same, about the same, or higher affinity as compared to binding of an ISV comprising a CDR2 with no difference in 4, 3, 2, or 1 amino acid, where the specified affinity is measured using surface plasmon resonance;

and

iii) CDR3 is selected from the group consisting of:

e) SEQ ID NOS: 218-225; or

f) amino acid sequences with a difference of 4, 3, 2 or 1 amino acid with the amino acid sequence of one of SEQ ID NOs: 218-225; provided that an ISV comprising a CDR3 with a 4, 3, 2, or 1 amino acid difference binds the TCR with the same, about the same, or higher affinity than an ISV comprising a CDR3 with no 4, 3, 2 or 2 amino acid difference 1 amino acid, where the specified affinity is measured using surface plasmon resonance;

and where one or more ISVs that specifically bind CD123 (substantially) consist of 4 framework regions (FR1-FR4, respectively) and 3 complementarity-determining regions (CDR1-CDR3, respectively), in which:

i) CDR1 is selected from the group consisting of:

a) SEQ ID NO: 11-16; or

b) amino acid sequences with a difference of 4, 3, 2 or 1 amino acid with the amino acid sequence of one of SEQ ID NOs: 11-16; provided that an ISV comprising a CDR1 with a 4, 3, 2, or 1 amino acid difference binds CD123 with the same, about the same, or higher affinity as compared to binding of an ISV comprising a CDR1 with no difference in 4, 3, 2, or 1 amino acid, where the specified affinity is measured using surface plasmon resonance;

and

ii) CDR2 is selected from the group consisting of:

c) SEQ ID NO: 17-20; or

d) amino acid sequences with a difference of 4, 3, 2 or 1 amino acid with the amino acid sequence of one of SEQ ID NOs: 17-20; provided that an ISV comprising a CDR2 with a 4, 3, 2, or 1 amino acid difference binds CD123 with the same, about the same, or higher affinity than an ISV comprising a CDR2 with no 4, 3, 2, or 2 amino acid difference 1 amino acid, where the specified affinity is measured using surface plasmon resonance;

and

iii) CDR3 is selected from the group consisting of:

e) SEQ ID NOS: 21-25; or

f) amino acid sequences with a difference of 4, 3, 2 or 1 amino acid with the amino acid sequence of one of SEQ ID NOs: 21-25; provided that an ISV comprising a CDR3 with a 4, 3, 2, or 1 amino acid difference binds CD123 with the same, about the same, or higher binding affinity than an ISV comprising a CDR3 with no difference in 4, 3, 2, or 1 amino acid, where the specified affinity is measured using surface plasmon resonance.

In a further aspect, the present invention relates to a polypeptide described herein, wherein an ISV that specifically binds a TCR (substantially) consists of 4 framework regions (FR1-FR4, respectively) and 3 complementarity-determining regions (CDR1-CDR3, respectively) , wherein:

i) CDR1 is selected from the group consisting of:

a) SEQ ID NO: 181-191; or

b) amino acid sequences with a difference of 4, 3, 2 or 1 amino acid with the amino acid sequence of one of SEQ ID NOs: 181-191, where the difference of 4, 3, 2 or 1 amino acid is present at positions 2, 4, 5, 6, 8 and/or 10 CDR1 (positions 27, 29, 30, 31, 33 and/or 35 according to Kabat numbering); provided that an ISV comprising a CDR1 with a 4, 3, 2, or 1 amino acid difference binds the TCR with the same, about the same, or higher affinity as compared to binding to an ISV comprising a CDR1 without a 4, 3, 2 difference or 1 amino acid, where the specified affinity is measured using surface plasmon resonance;

and

ii) CDR2 is selected from the group consisting of:

c) SEQ ID NOS: 192-217; or

d) amino acid sequences with a difference of 4, 3, 2 or 1 amino acid with the amino acid sequence of one of SEQ ID NOs: 192-217, where the difference of 4, 3, 2 or 1 amino acid is present at positions 1, 3, 5, 7, 8 and/or 9 CDR2 (positions 50, 52, 54, 56, 57 and/or 58 according to Kabat numbering); provided that an ISV comprising a CDR2 with a 4, 3, 2, or 1 amino acid difference binds the TCR with the same, about the same, or higher affinity as compared to binding of an ISV comprising a CDR2 with no difference in 4, 3, 2, or 1 amino acid, where the specified affinity is measured using surface plasmon resonance;

and

iii) CDR3 is selected from the group consisting of:

e) SEQ ID NOS: 218-225; or

f) amino acid sequences with a difference of 4, 3, 2 or 1 amino acid with an amino acid sequence of one of SEQ ID NOs: 218-225, where the difference of 4, 3, 2 or 1 amino acid is present at positions 1, 4, 5 and/or 8 CDR3 (positions 95, 98, 99 and/or 101 according to Kabat numbering); provided that an ISV comprising a CDR3 with a 4, 3, 2, or 1 amino acid difference binds the TCR with the same, about the same, or higher affinity than an ISV comprising a CDR3 with no 4, 3, 2 or 2 amino acid difference 1 amino acid, where the specified affinity is measured using surface plasmon resonance;

and where ISV that specifically binds CD123 is as described below.

In one aspect, the CDR1 included in the ISV that specifically binds the TCR may be selected from the group consisting of:

a) SEQ ID NO: 181; or

b) amino acid sequences with a difference of 4, 3, 2 or 1 amino acid from the amino acid sequence of SEQ ID NO: 181, where

- in position 2 D has been changed to A, S, E or G;

- in position 4 H has been changed to Y;

- in position 5 K has been changed to L;

- in position 6 I was changed to L;

- in position 8, F has been changed to I or V; and/or

- in position 10 G has been changed to S;

provided that an ISV comprising a CDR1 with a 4, 3, 2, or 1 amino acid difference binds the TCR with the same, about the same, or higher affinity as compared to binding of an ISV comprising a CDR1 with no difference in 4, 3, 2, or 1 amino acid, where the specified affinity is measured using surface plasmon resonance.

In addition, or in addition, the CDR2 included in the ISV that specifically binds the TCR may be selected from the group consisting of:

a) SEQ ID NO: 192; or

b) amino acid sequences with a difference of 4, 3, 2 or 1 amino acid from the amino acid sequence of SEQ ID NO: 192, where

- in position 1 H has been changed to T or R;

- in position 3 S has been changed to T or A;

- in position 5 G has been changed to S or A;

- in position 7 Q has been changed to D, E, T, A or V;

- in position 8, T was changed to A or V; and/or

- in position 9, D has been changed to A, Q, N, V or S;

provided that an ISV comprising a CDR2 with a 4, 3, 2, or 1 amino acid difference binds the TCR with the same, about the same, or higher affinity as compared to binding of an ISV comprising a CDR2 with no difference in 4, 3, 2, or 1 amino acid, where the specified affinity is measured using surface plasmon resonance.

In addition, or in addition, the CDR3 included in the ISV that specifically binds the TCR may be selected from the group consisting of:

a) SEQ ID NO: 218; or

b) amino acid sequences with a difference of 4, 3, 2 or 1 amino acid from the amino acid sequence of SEQ ID NO: 218, where

- in position 1, F has been changed to Y, L or G;

- in position 4 I was changed to L;

- in position 5 Y has been changed to W; and/or

- in position 8 D has been changed to N or S;

provided that an ISV comprising a CDR3 with a 4, 3, 2, or 1 amino acid difference binds the TCR with the same, about the same, or higher affinity than an ISV comprising a CDR3 with no 4, 3, 2 or 2 amino acid difference 1 amino acid, where the specified affinity is measured using surface plasmon resonance.

Accordingly, the present invention provides a polypeptide as described herein, wherein an ISV that specifically binds a TCR (substantially) consists of 4 framework regions (FR1-FR4, respectively) and 3 complementarity-determining regions (CDR1-CDR3, respectively), in which:

i) CDR1 is selected from the group consisting of:

a) SEQ ID NO: 181; or

b) amino acid sequences with a difference of 4, 3, 2 or 1 amino acid from the amino acid sequence of SEQ ID NO: 181, where

- in position 2 D has been changed to A, S, E or G;

- in position 4 H has been changed to Y;

- in position 5 K has been changed to L;

- in position 6 I was changed to L;

- in position 8, F has been changed to I or V; and/or

- in position 10 G has been changed to S;

provided that a polypeptide comprising a CDR1 with a 4, 3, 2, or 1 amino acid difference binds the TCR with the same, about the same, or higher affinity as compared to binding by a polypeptide containing a CDR1 with no difference in 4, 3, 2, or 1 amino acid, where the specified affinity is measured using surface plasmon resonance;

and

ii) CDR2 is selected from the group consisting of:

c) SEQ ID NO: 192; or

d) amino acid sequences with a difference of 4, 3, 2 or 1 amino acid from the amino acid sequence of SEQ ID NO: 192, where

- in position 1 H has been changed to T or R;

- in position 3 S has been changed to T or A;

- in position 5 G has been changed to S or A;

- in position 7 Q has been changed to D, E, T, A or V;

- in position 8, T was changed to A or V; and/or

- in position 9, D has been changed to A, Q, N, V or S;

provided that a polypeptide containing a CDR2 with a 4, 3, 2, or 1 amino acid difference binds the TCR with the same, about the same, or higher affinity as compared to binding by a polypeptide containing a CDR2 with no difference in 4, 3, 2, or 1 amino acid, where the specified affinity is measured using surface plasmon resonance;

and

iii) CDR3 is selected from the group consisting of:

e) SEQ ID NO: 218; or

f) amino acid sequences with a difference of 4, 3, 2 or 1 amino acid from the amino acid sequence of SEQ ID NO: 218, where

- in position 1, F has been changed to Y, L or G;

- in position 4 I was changed to L;

- in position 5 Y has been changed to W; and/or

- in position 8 D has been changed to N or S;

provided that a polypeptide containing a CDR3 with a difference of 4, 3, 2, or 1 amino acid binds the TCR with the same, approximately the same, or higher affinity as compared to binding by a polypeptide containing a CDR3 with no difference of 4, 3, 2, or 1 an amino acid wherein said affinity is measured by surface plasmon resonance;

and where ISV that specifically binds CD123 is as described below.

In a preferred aspect, an ISV that specifically binds TCRs (substantially) consists of 4 framework regions (FR1-FR4, respectively) and 3 complementarity-determining regions (CDR1-CDR3, respectively), in which CDR1 is selected from the group consisting of SEQ ID NOs: 181-191, CDR2 is selected from the group consisting of SEQ ID NOs: 192-217, and CDR3 is selected from the group consisting of SEQ ID NOs: 218-225.

Accordingly, the present invention relates to a polypeptide comprising an ISV that specifically binds a TCR (essentially) consisting of 4 framework regions (FR1-FR4, respectively) and 3 complementarity-determining regions (CDR1-CDR3, respectively), in which CDR1 is selected from from the group consisting of SEQ ID NO: 181-191, CDR2 is selected from the group consisting of SEQ ID NO: 192-217, and CDR3 is selected from the group consisting of SEQ ID NO: 218-225 and containing an ISV that specifically binds to CD123 , as described below.

In a further aspect, the present invention relates to a polypeptide described herein, wherein an ISV that specifically binds a TCR (substantially) consists of 4 framework regions (FR1-FR4, respectively) and 3 complementarity-determining regions (CDR1-CDR3, respectively) wherein CDR1 is SEQ ID NO: 181, CDR2 is SEQ ID NO: 192, and CDR3 is SEQ ID NO: 218, and wherein the ISV that specifically binds CD123 is as further described herein.

Preferred ISVs for use in a polypeptide of the invention may be selected from the group consisting of SEQ ID NOs: 42 and 78-180, or from ISVs that have greater than 80%, greater than 85%, greater than 90%, greater than 95% sequence identity, or even more than 99% with one of SEQ ID NOs: 42 and 78-180. Accordingly, the present invention provides a polypeptide as described herein, wherein the ISV that specifically binds a TCR is selected from the group consisting of SEQ ID NOs: 42 and 78-180, or from ISVs that have greater than 80% sequence identity, greater than 85%, greater than 90%, greater than 95%, or even greater than 99% with one of SEQ ID NOs: 42 and 78-180, and wherein the ISV that specifically binds CD123 is as described hereinafter.

An ISV that specifically binds a TCR may be present at any position in a polypeptide of the invention. Preferably, an ISV that specifically binds the TCR is present at the N-terminus of the polypeptide of the invention. Accordingly, in a further aspect, the present invention provides a polypeptide as described herein, wherein an ISV that specifically binds a TCR is located at the N-terminus of the polypeptide.

The polypeptide of the invention further encompasses one or more ISVs. The ISVs for use in the polypeptide of the invention were specifically selected for their high specificity for CD123 present in target cells expressing CD123.

Therefore, in a further aspect, the present invention relates to a polypeptide described herein, wherein the ISV that specifically binds TCR is as described herein, and where one or more ISVs that specifically bind CD123 (substantially) consist of 4 framework regions (FR1) FR4, respectively) and 3 complementarity-determining regions (CDR1-CDR3, respectively), in which:

i) CDR1 is selected from the group consisting of:

a) SEQ ID NO: 11-16; or

b) amino acid sequences with a difference of 4, 3, 2 or 1 amino acid with the amino acid sequence of one of SEQ ID NOs: 11-16, where the difference of 4, 3, 2 or 1 amino acid is present at positions 3, 6, 7 and/or 8 CDR1 (positions 28, 31, 32 and/or 33 according to Kabat numbering); provided that an ISV comprising a CDR1 with a 4, 3, 2, or 1 amino acid difference binds CD123 with the same, about the same, or higher binding affinity than an ISV comprising a CDR1 with no difference in 4, 3, 2, or 1 amino acid, where the specified affinity is measured using surface plasmon resonance;

and

ii) CDR2 is selected from the group consisting of:

c) SEQ ID NO: 17-20; or

d) amino acid sequences with a difference of 3, 2 or 1 amino acid with the amino acid sequence of one of SEQ ID NOs: 17-20, where the difference of 3, 2 or 1 amino acid is present at positions 3 to 6 and/or 10 of CDR2 (positions 52, 54 and/or 58 according to Kabat numbering); provided that an ISV comprising a CDR2 with a 3, 2, or 1 amino acid difference binds CD123 with the same, about the same, or higher affinity as compared to binding of an ISV comprising a CDR2 with no 3, 2, or 1 amino acid difference, wherein the specified affinity is measured using surface plasmon resonance;

and

iii) CDR3 is selected from the group consisting of:

e) SEQ ID NOS: 21-25; or

f) amino acid sequences with a difference of 3, 2 or 1 amino acid with the amino acid sequence of one of SEQ ID NOs: 21-25, where the difference of 3, 2 or 1 amino acid is present at positions 3, 4 and/or 5 in CDR3 (position 97 , 98 and/or 99 according to Kabat numbering); provided that an ISV comprising a CDR3 with a 3, 2, or 1 amino acid difference binds CD123 with the same, about the same, or higher affinity than an ISV comprising a CDR3 with no 3, 2, or 1 amino acid difference, wherein: this affinity was measured using surface plasmon resonance.

The present invention has identified ISVs that specifically bind CD123 to selected antigen-binding sites or paratopes. In one aspect, an ISV that specifically binds CD123 binds to an epitope that is associated with 56A10 ISV (ie, an ISV that belongs to the same family as 56A10 or an ISV that belongs to 56A10).

In one aspect, the CDR1 included in the ISV that specifically binds CD123 may be selected from the group consisting of:

a) SEQ ID NO: 11; or

b) amino acid sequences with a difference of 4, 3, 2 or 1 amino acid from the amino acid sequence of SEQ ID NO: 11, where

- in position 3, T has been changed to S or P;

- in position 6 I was changed to S;

- in position 7 N has been changed to D; and/or

- in position 8 D has been changed to V or A;

provided that an ISV comprising a CDR1 with a 4, 3, 2, or 1 amino acid difference binds CD123 with the same, about the same, or higher binding affinity than an ISV comprising a CDR1 with no difference in 4, 3, 2, or 1 amino acid, where the specified affinity is measured using surface plasmon resonance.

In addition, or in addition, the CDR2 included in the ISV that specifically binds the TCR may be SEQ ID NO: 17.

In addition, or in addition, the CDR3 included in the ISV that specifically binds the TCR may be selected from the group consisting of:

a) SEQ ID NO: 21; or

b) amino acid sequences with a difference of 1 amino acid from the amino acid sequence of SEQ ID NO: 21, where

- in position 3 P has been changed to A;

provided that an ISV comprising a CDR3 with a 1 amino acid difference binds CD123 with the same, approximately the same, or higher affinity as compared to binding to an ISV comprising a CDR3 with no 1 amino acid difference, said affinity being measured by surface plasmon resonance,

Accordingly, the present invention relates to a polypeptide as described herein, wherein the ISV that specifically binds a TCR is as described herein, and wherein one or more ISVs that specifically bind CD123 (substantially) consist of 4 framework regions (FR1-FR4, respectively) and 3 complementarity-determining regions (CDR1-CDR3, respectively), in which:

i) CDR1 is selected from the group consisting of:

a) SEQ ID NO: 11; or

b) amino acid sequences with a difference of 4, 3, 2 or 1 amino acid from the amino acid sequence of SEQ ID NO: 11, where

- in position 3, T has been changed to S or P;

- in position 6 I was changed to S;

- in position 7 N has been changed to D; and/or

- in position 8 D has been changed to V or A;

provided that a polypeptide comprising a CDR1 with a 4, 3, 2, or 1 amino acid difference binds CD123 with the same, about the same, or higher affinity as compared to binding by a polypeptide containing a CDR1 with no difference in 4, 3, 2, or 1 amino acid, where the specified affinity is measured using surface plasmon resonance;

and

ii) CDR2 is SEQ ID NO: 17;

and

iii) CDR3 is selected from the group consisting of:

c) SEQ ID NO: 21; or

d) amino acid sequences with a difference of 1 amino acid from the amino acid sequence of SEQ ID NO: 21, where

- in position 3 P has been changed to A;

provided that a polypeptide containing a CDR3 with a 1 amino acid difference binds CD123 with the same, about the same, or higher affinity as compared to binding to a polypeptide containing a CDR3 with no 1 amino acid difference, where said affinity is measured by surface plasmon resonance,

In a preferred aspect, an ISV that specifically binds CD123 (substantially) consists of 4 framework regions (FR1-FR4, respectively) and 3 complementarity-determining regions (CDR1-CDR3, respectively), in which the CDR1 is selected from the group consisting of SEQ ID NO: 11-15, CDR2 is SEQ ID NO: 17, and CDR3 is selected from the group consisting of SEQ ID NO: 21-22.

Accordingly, the present invention provides a polypeptide comprising an ISV that specifically binds a TCR as described herein, and comprising one or more ISVs that specifically bind CD123 (essentially) consisting of 4 framework regions (FR1-FR4, respectively) and 3 complementarity determining regions (CDR1-CDR3, respectively), where CDR1 is selected from the group consisting of SEQ ID NO: 11-15, CDR2 is SEQ ID NO: 17, and CDR3 is selected from the group consisting of SEQ ID NO: 21 -22.

In an additional aspect, the present invention relates to a polypeptide described herein, where the ISV that specifically binds TCR is as described herein, and where one or more ISVs that specifically bind CD123 (essentially) consists of 4 framework regions (FR1-FR4, respectively) and 3 complementarity-determining regions (CDR1-CDR3, respectively), wherein CDR1 is SEQ ID NO: 11, CDR2 is SEQ ID NO: 17, and CDR3 is SEQ ID NO: 21 .

Preferred ISVs for use in a polypeptide of the invention may be selected from the group consisting of SEQ ID NOs: 1-6, or from ISVs that have greater than 80%, greater than 85%, greater than 90%, greater than 95%, or even greater than 99% sequence identity. % with one of SEQ ID NOs: 1-6. Accordingly, the present invention also relates to a polypeptide described herein, wherein the ISV that specifically binds TCR is as described herein, and where one or more ISVs that specifically bind CD123 are selected from the group consisting of SEQ ID NOs: 1-6, or from ISVs that have greater than 80%, greater than 85%, greater than 90%, greater than 95%, or even greater than 99% sequence identity with one of SEQ ID NOs: 1-6.

In another aspect, an ISV that specifically binds CD123 binds to an epitope that binds to the 55F03 nanobody (ie, an ISV that belongs to the same family as 55F03 or an ISV that belongs to 55F03).

In one aspect, the CDR1 included in the ISV that specifically binds CD123 is SEQ ID NO: 16.

In addition, or in addition, the CDR2 included in the ISV that specifically binds CD123 may be selected from the group consisting of:

a) SEQ ID NO: 18; or

b) amino acid sequences that have a 3, 2 or 1 amino acid difference from the amino acid sequence of SEQ ID NO: 18, where

- in position 3 Y has been changed to W;

- in position 6 N has been changed to S; and/or

- in position 10 Q has been changed to E;

provided that an ISV comprising a CDR2 with a 3, 2, or 1 amino acid difference binds CD123 with the same, about the same, or higher affinity as compared to binding of an ISV comprising a CDR2 with no 3, 2, or 1 amino acid difference, wherein this affinity was measured using surface plasmon resonance.

In addition, or in addition, the CDR3 included in the ISV that specifically binds CD123 may be selected from the group consisting of:

a) SEQ ID NO: 23; or

b) amino acid sequences with a difference of 2 or 1 amino acid from the amino acid sequence of SEQ ID NO: 23, where

- in position 4, E was changed to R; and/or

- in position 5, T was changed to D or Y;

provided that an ISV comprising a CDR3 with a 2 or 1 amino acid difference binds CD123 with the same, about the same, or higher affinity as compared to binding of an ISV comprising a CDR3 with no 2 or 1 amino acid difference, wherein said affinity is measured with using surface plasmon resonance.

Accordingly, the present invention relates to a polypeptide as described herein, wherein the ISV that specifically binds a TCR is as described herein, and wherein one or more ISVs that specifically bind CD123 (substantially) consist of 4 framework regions (FR1-FR4, respectively) and 3 complementarity-determining regions (CDR1-CDR3, respectively), in which:

i) CDR1 is SEQ ID NO: 16;

and

ii) CDR2 is selected from the group consisting of:

a) SEQ ID NO: 18; or

b) amino acid sequences with a difference of 3, 2 or 1 amino acid from the amino acid sequence of SEQ ID NO: 18, where

- in position 3 Y has been changed to W;

- in position 6 N has been changed to S; and/or

- in position 10 Q has been changed to E;

provided that a polypeptide containing a CDR2 with a 3, 2, or 1 amino acid difference binds CD123 with the same, about the same, or higher affinity as compared to binding by a polypeptide containing a CDR2 with no 3, 2, or 1 amino acid difference, where the specified affinity is measured using surface plasmon resonance;

and

iii) CDR3 is selected from the group consisting of:

c) SEQ ID NO: 23; or

d) amino acid sequences with a difference of 2 or 1 amino acid from the amino acid sequence of SEQ ID NO: 23, where

- in position 4, E was changed to R; and/or

- in position 5, T was changed to D or Y;

provided that a polypeptide containing a CDR3 with a difference of 2 or 1 amino acid binds CD123 with the same, about the same or higher affinity compared to binding by a polypeptide containing a CDR3 with no difference of 2 or 1 amino acid, where the specified affinity is measured using surface plasmon resonance.

In a preferred aspect, an ISV that specifically binds CD123 (substantially) consists of 4 framework regions (FR1-FR4, respectively) and 3 complementarity-determining regions (CDR1-CDR3, respectively), wherein CDR1 is SEQ ID NO: 16, CDR2 is selected from the group consisting of SEQ ID NOs: 18-20 and CDR3 is selected from the group consisting of SEQ ID NOs: 23-25.

Accordingly, the present invention provides a polypeptide comprising an ISV that specifically binds a TCR as described herein, and comprising one or more ISVs that specifically bind CD123 (substantially) consisting of 4 framework regions (FR1-FR4, respectively) and 3 complementarity determining regions (CDR1-CDR3, respectively), where CDR1 is SEQ ID NO: 16, CDR2 is selected from the group consisting of SEQ ID NO: 18-20, and CDR3 is selected from the group consisting of SEQ ID NO: 23 -25.

In an additional aspect, the present invention relates to a polypeptide described herein, where the ISV that specifically binds TCR is as described herein, and where one or more ISVs that specifically bind CD123 (essentially) consists of 4 framework regions (FR1-FR4, respectively) and 3 complementarity-determining regions (CDR1-CDR3, respectively), wherein CDR1 is SEQ ID NO: 16, CDR2 is SEQ ID NO: 18, and CDR3 is SEQ ID NO: 23 .

Preferred ISVs for use in a polypeptide of the invention may be selected from the group consisting of SEQ ID NOs: 7-10, or from ISVs that have greater than 80%, greater than 85%, greater than 90%, greater than 95% or even greater sequence identity. 99% with one of SEQ ID NOs: 7-10. Accordingly, in a further aspect, the present invention relates to a polypeptide described herein, wherein an ISV that specifically binds a TCR is as described herein, and wherein one or more ISVs that specifically bind CD123 are selected from the group consisting of from SEQ ID NO: 7-10 or from ISVs that have more than 80%, more than 85%, more than 90%, more than 95%, or even more than 99% sequence identity with one of SEQ ID NO: 7-10.

The polypeptide of the invention may include one ISV that specifically binds CD123, or more than one ISV that specifically binds CD123, such as two, three, or even more. In a further aspect, the present invention relates to a polypeptide described herein, comprising an ISV that specifically binds a TCR as described herein, and comprising two or more ISVs that specifically bind CD123, preferably two.

The two or more, preferably two, ISVs included in a polypeptide of the invention may be any ISV that specifically binds to CD123 as described herein. The two or more, preferably two, ISVs included in a polypeptide of the invention may be the same ISV (ie, the same amino acid sequence) or they may be different ISVs (ie, a different amino acid sequence). In one aspect, the present invention provides a polypeptide as described wherein two or more ISVs that specifically bind CD123 are biparatopic, comprising a first ISV and a second ISV, wherein the first ISV binds to an epitope on CD123 that is different from the epitope on CD123 that binds second ISV.

Preferably two or more, preferably two ISVs that specifically bind CD123 are the 56A10 ISV and the 55F03 ISV. Accordingly, in one aspect, the present invention provides a polypeptide as described herein, wherein the first ISV is selected from ISVs related to 56A10 and the second ISV is selected from ISVs related to 55F03.

Two or more, preferably two, ISVs that specifically bind CD123 may be present at any position in the polypeptide of the invention. In one aspect, the present invention relates to a polypeptide described herein, where the second ISV is located at the N-terminus of the first ISV. In another aspect, the present invention relates to a polypeptide described herein, where the second ISV is located at the C-terminus of the first ISV.

The ISVs present in a polypeptide of the invention may be any of the ISVs known in the art and described hereinafter. In one aspect, the ISVs present in a polypeptide of the invention are selected from a single domain antibody, dAb, nanobody, V _HH , humanized V _HH , overblown VH , or V _HH that has been generated by affinity maturation. Accordingly, in a further aspect, the present invention provides a polypeptide as described herein, wherein an ISV that specifically binds a TCR and one or more ISVs that specifically bind CD123 (essentially) are comprised of a single domain antibody, dAb, nanobody, V _HH , humanized V _HH , camel VH or V _HH that was obtained by affinity maturation.

Preferred polypeptides of the invention are selected from the group consisting of SEQ ID NOs: 47, 49, 52, 53, 55, 56 and 58-61, or from polypeptides that have sequence identity greater than 80%, greater than 85%, greater than 90%, more than 95% or even more than 99% with one of SEQ ID NOs: 47, 49, 52, 53, 55, 56 and 58-61.

More preferably, the polypeptide is selected from the group consisting of SEQ ID NOs: 47, 49, 52, 53, 55, 56 and 58-61.

As discussed above, the polypeptide of the invention redirects T cells to kill cells expressing CD123. In one aspect, the present invention relates to a polypeptide described in this document, where the specified polypeptide induces the activation of T cells.

In a further aspect, the present invention relates to a polypeptide described herein, wherein said T cell activation is independent of MHC recognition.

In an additional aspect, the present invention relates to a polypeptide described herein, where the specified activation of T cells depends on the presentation to the T cell of the specified polypeptide associated with CD123 on the target cell.

In an additional aspect, the present invention relates to a polypeptide described herein, where the specified activation of T cells causes one or more cellular responses in the specified T cells, and the specified cellular response is selected from the group consisting of proliferation, differentiation, secretion of cytokines, release cytotoxic effector molecules, cytotoxic activity, expression of activation markers and redirected lysis of target cells.

In a specific aspect, T cell activation induced by a polypeptide of the invention causes death of CD123-expressing cells with an average EC50 of 1 nM to 1 pM, e.g., an average EC50 of 500 pM or less, such as less than 400, 300, 200, or 100 pM or even less, such as less than 90, 80, 70, 60, 50, 40, or 30 pM or even less, wherein said EC50 value is preferably determined in a TOPRO3 flow cytometric assay using MOLM-13 cells as target cells and human T cells as effector cells at a ratio of effector cells to target cells of 10:1.

In another specific aspect, T cell activation induced by a polypeptide of the invention causes lysis of cells expressing CD123 with an average percentage of lysis greater than about 10%, such as 15%, 16%, 17%, 18%, 19%. or 20% or even more, e.g. more than 25% or even more than 30%, said percent lysis is preferably determined in a TOPRO3 flow cytometry analysis using MOLM-13 cells as target cells and human T cells in as effector cells at a ratio of effector target cells of 10:1.

In another specific aspect, the present invention provides a polypeptide as described herein, wherein said T cell activation induced by the polypeptide of the invention causes IFN-γ secretion with an average EC50 of 100 nM to 10 pM, e.g., an average EC50 of 50 nM or less, such as less than 40, 30, 20, 10 or 9 nM or even less, such as less than 8, 7, 6, 5, 4, 3, 2 or 1 nM or even less, such as less than 500 pM or even less, eg less than 400, 300, 200 or 100 pM or even less, said EC ₅₀ value is preferably determined in an ELISA-based assay.

In an additional aspect, the present invention relates to a polypeptide described herein, where the specified activation of T cells causes the proliferation of these T cells.

As discussed above, the polypeptides of the present invention are chosen such that target independent activation of T cells should be minimal. Therefore, in an additional aspect, the present invention relates to a polypeptide described herein, where the activation of T cells in the absence of CD123 positive cells is minimal.

More specifically, the lysis of CD123 negative cells induced by T cell activation by the polypeptides of the present invention is no more than about 10%, such as 9% or less, such as 8%, 7%, or 6% or even less, wherein said lysis preferably defined as the mean percent lysis in a TOPRO3 flow cytometry assay using U-937 cells as target cells and human T cells as effector cells at a target effector cell ratio of 10:1.

The present invention also relates to structural elements, ie. ISVs that make up the polypeptides of the invention. Accordingly, the present invention also relates to a polypeptide that is an ISV that specifically binds CD123 and that includes or (essentially) consists of 4 framework regions (FR1-FR4, respectively) and 3 complementarity-determining regions (CDR1-CDR3, respectively) , wherein:

i) CDR1 is selected from the group consisting of:

a) SEQ ID NO: 11-16; or

b) amino acid sequences with a difference of 4, 3, 2 or 1 amino acid with the amino acid sequence of one of SEQ ID NOs: 11-16; provided that a polypeptide comprising a CDR1 with a 4, 3, 2, or 1 amino acid difference binds CD123 with the same, about the same, or higher affinity as compared to binding by a polypeptide containing a CDR1 with no difference in 4, 3, 2, or 1 amino acid, where the specified affinity is measured using surface plasmon resonance;

and/or

ii) CDR2 is selected from the group consisting of:

c) SEQ ID NO: 17-20; or

d) amino acid sequences with a difference of 4, 3, 2 or 1 amino acid with the amino acid sequence of one of SEQ ID NOs: 17-20; provided that a polypeptide containing a CDR2 with a 4, 3, 2, or 1 amino acid difference binds CD123 with the same, about the same, or higher affinity as compared to binding by a polypeptide containing a CDR2 with no difference in 4, 3, 2, or 1 amino acid, where the specified affinity is measured using surface plasmon resonance;

and/or

iii) CDR3 is selected from the group consisting of:

e) SEQ ID NOS: 21-25; or

f) amino acid sequences with a difference of 4, 3, 2 or 1 amino acid with the amino acid sequence of one of SEQ ID NOs: 21-25; provided that a polypeptide containing a CDR3 with a difference of 4, 3, 2, or 1 amino acid binds CD123 with the same, about the same, or higher affinity as compared to binding by a polypeptide containing a CDR3 with no difference in 4, 3, 2, or 1 amino acid, where the specified affinity is measured using surface plasmon resonance.

More preferably, a polypeptide that is an ISV that specifically binds CD123 contains or (essentially) consists of 4 framework regions (FR1-FR4, respectively) and 3 complementarity-determining regions (CDR1-CDR3, respectively) in which:

i) CDR1 is selected from the group consisting of:

a) SEQ ID NO: 11-16; or

b) amino acid sequences with a difference of 4, 3, 2 or 1 amino acid with the amino acid sequence of one of SEQ ID NOs: 11-16; provided that a polypeptide comprising a CDR1 with a 4, 3, 2, or 1 amino acid difference binds CD123 with the same, about the same, or higher affinity as compared to binding to a polypeptide containing a CDR1 with no difference in 4, 3, 2 or 1 amino acid, where the specified affinity is measured using surface plasmon resonance;

and/or

ii) CDR2 is selected from the group consisting of:

c) SEQ ID NO: 17-20; or

d) amino acid sequences with a difference of 4, 3, 2 or 1 amino acid with the amino acid sequence of one of SEQ ID NOs: 17-20; provided that a polypeptide containing a CDR2 with a 4, 3, 2, or 1 amino acid difference binds CD123 with the same, about the same, or higher affinity as compared to binding by a polypeptide containing a CDR2 with no difference in 4, 3, 2, or 1 amino acid, where the specified affinity is measured using surface plasmon resonance;

and/or

iii) CDR3 is selected from the group consisting of:

e) SEQ ID NOS: 21-25; or

f) amino acid sequences with a difference of 4, 3, 2 or 1 amino acid with the amino acid sequence of one of SEQ ID NOs: 21-25; provided that a polypeptide containing a CDR3 with a difference of 4, 3, 2, or 1 amino acid binds CD123 with the same, about the same, or higher affinity as compared to binding by a polypeptide containing a CDR3 with no difference in 4, 3, 2, or 1 amino acid, where the specified affinity is measured using surface plasmon resonance.

In a further aspect, the present invention also relates to a polypeptide as described above, which includes or (essentially) consists of 4 framework regions (FR1-FR4, respectively) and 3 complementarity-determining regions (CDR1-CDR3, respectively), in which:

i) CDR1 is selected from the group consisting of:

a) SEQ ID NO: 11-16; or

b) amino acid sequences with a difference of 4, 3, 2 or 1 amino acid with the amino acid sequence of one of SEQ ID NOs: 11-16, where the difference of 4, 3, 2 or 1 amino acid is present at positions 3, 6, 7 and/or 8 CDR1 (positions 28, 31, 32 and/or 33 according to Kabat numbering); provided that a polypeptide comprising a CDR1 with a 4, 3, 2, or 1 amino acid difference binds CD123 with the same, about the same, or higher affinity as compared to binding by a polypeptide containing a CDR1 with no difference in 4, 3, 2, or 1 amino acid, where the specified affinity is measured using surface plasmon resonance;

and/or

ii) CDR2 is selected from the group consisting of:

c) SEQ ID NO: 17-20; or

d) amino acid sequences with a difference of 3, 2 or 1 amino acid with the amino acid sequence of one of SEQ ID NOs: 17-20, where the difference of 3, 2 or 1 amino acid is present at positions 3 to 6 and/or 10 of CDR2 (positions 52, 54 and/or 58 according to Kabat numbering); provided that a polypeptide containing a CDR2 with a 3, 2, or 1 amino acid difference binds CD123 with the same, about the same, or higher affinity as compared to binding by a polypeptide containing a CDR2 with no 3, 2, or 1 amino acid difference, where the specified affinity is measured using surface plasmon resonance;

and/or

iii) CDR3 is selected from the group consisting of:

e) SEQ ID NOS: 21-25; or

f) amino acid sequences with a difference of 3, 2 or 1 amino acid with the amino acid sequence of one of SEQ ID NOs: 21-25, where the difference of 3, 2 or 1 amino acid is present at positions 3, 4 and/or 5 of CDR3 (positions 97, 98 and/or 99 according to Kabat numbering); provided that a polypeptide containing a CDR3 with a 3, 2, or 1 amino acid difference binds CD123 with the same, about the same, or higher affinity as compared to binding by a polypeptide containing a CDR3 with no 3, 2, or 1 amino acid difference, where this affinity was measured using surface plasmon resonance.

The present invention has identified ISVs that specifically bind CD123 through selected antigen-binding sites or paratopes. In one aspect, an ISV that specifically binds CD123 binds to an epitope that binds to 56A10 ISV (ie, an ISV that belongs to the same family as 56A10 or an ISV that belongs to 56A10).

Accordingly, in one aspect, the CDR1 included in the ISV that specifically binds CD123 may be selected from the group consisting of:

a) SEQ ID NO: 11; or

b) amino acid sequences with a difference of 4, 3, 2 or 1 amino acid from the amino acid sequence of SEQ ID NO: 11, where

- in position 3, T has been changed to S or P;

- in position 6 I was changed to S;

- in position 7 N has been changed to D; and/or

- in position 8 D has been changed to V or A;

provided that a polypeptide comprising a CDR1 with a 4, 3, 2, or 1 amino acid difference binds CD123 with the same, about the same, or higher affinity as compared to binding by a polypeptide containing a CDR1 with no difference in 4, 3, 2, or 1 amino acid, where the specified affinity is measured using surface plasmon resonance.

In addition, or in addition, a CDR2 included in ISV that specifically binds CD123 is SEQ ID NO: 17.

In addition, or in addition, the CDR3 included in the ISV that specifically binds CD123 may be selected from the group consisting of:

a) SEQ ID NO: 21; or

b) amino acid sequences with a difference of 1 amino acid from the amino acid sequence of SEQ ID NO: 21, where

- in position 3 P has been changed to A;

provided that a polypeptide containing a CDR3 with a 1 amino acid difference binds CD123 with the same, about the same, or higher affinity as compared to binding by a polypeptide containing a CDR3 with no 1 amino acid difference, where said affinity is measured by surface plasmon resonance ,

Accordingly, the present invention also relates to a polypeptide as described above, which includes or (essentially) consists of 4 framework regions (FR1-FR4, respectively) and 3 complementarity-determining regions (CDR1-CDR3, respectively), in which:

i) CDR1 is selected from the group consisting of:

a) SEQ ID NO: 11; or

b) amino acid sequences with a difference of 4, 3, 2 or 1 amino acid from the amino acid sequence of SEQ ID NO: 11, where

- in position 3, T has been changed to S or P;

- in position 6 I was changed to S;

- in position 7 N has been changed to D; and/or

- in position 8 D has been changed to V or A;

provided that a polypeptide comprising a CDR1 with a 4, 3, 2, or 1 amino acid difference binds CD123 with the same, about the same, or higher affinity as compared to binding by a polypeptide containing a CDR1 with no difference in 4, 3, 2, or 1 amino acid, where the specified affinity is measured using surface plasmon resonance;

and

ii) CDR2 is SEQ ID NO: 17;

and

iii) CDR3 is selected from the group consisting of:

c) SEQ ID NO: 21; or

d) amino acid sequences with a difference of 1 amino acid from the amino acid sequence of SEQ ID NO: 21, where

- in position 3 P has been changed to A;

provided that a polypeptide containing a CDR3 with a 1 amino acid difference binds CD123 with the same, about the same, or higher affinity as compared to binding by a polypeptide containing a CDR3 with no 1 amino acid difference, where said affinity is measured by surface plasmon resonance ,

In an additional aspect, the present invention relates to a polypeptide described herein, in which CDR1 is selected from the group consisting of SEQ ID NO: 11-15, CDR2 is SEQ ID NO: 17, and CDR3 is selected from the group consisting of SEQ ID NO: 17. NO: 21-22. Preferably, CDR1 is SEQ ID NO: 11, CDR2 is SEQ ID NO: 17, and CDR3 is SEQ ID NO: 21.

Preferred ISVs of the invention relating to 56A10 may be selected from the group consisting of SEQ ID NOs: 1-6, or from polypeptides that have sequence identity greater than 80%, greater than 85%, greater than 90%, greater than 95%, or even greater. 99% with one of SEQ ID NOs: 1-6. Accordingly, in an additional aspect, the present invention relates to a polypeptide described herein, where the polypeptide is selected from the group consisting of SEQ ID NO: 1-6 or from polypeptides that have a sequence identity of more than 80%, more than 85%, more than 90 %, more than 95% or even more than 99% with one of SEQ ID NOs: 1-6. Preferably the polypeptide is selected from the group consisting of SEQ ID NOs: 1-6.

In one aspect, a polypeptide of the invention binds to human CD123 expressed on MOLM-13 cells with an average EC50 between 10 nM and 100 pM, e.g., an average EC50 of 5 nM or less, e.g., less than 4, 3, 2, or 1 nM or even less, preferably by flow cytometry.

In another aspect, the polypeptide of the invention binds to human CD123 with an average KD value of 10 nM to 100 pM, e.g., an average KD value of 5 nM or less, for example, less than 4, 3, or 2 nM or even less, said KD value is preferably determined by surface plasmon resonance.

In yet another aspect, an ISV that specifically binds CD123 binds to an epitope that is associated with 55F03 ISV (ie, an ISV that belongs to the same family as 55F03 or an ISV that belongs to 55F03).

Accordingly, in one aspect, the CDR1 included in the ISV that specifically binds CD123 is SEQ ID NO: 16.

In addition, or in addition, the CDR2 included in the ISV that specifically binds CD123 may be selected from the group consisting of:

a) SEQ ID NO: 18; or

b) amino acid sequences with a difference of 3, 2 or 1 amino acid from the amino acid sequence of SEQ ID NO: 18, where

- in position 3 Y has been changed to W;

- in position 6 N has been changed to S; and/or

- in position 10 Q has been changed to E;

provided that a polypeptide containing a CDR2 with a 3, 2, or 1 amino acid difference binds CD123 with the same, about the same, or higher affinity as compared to binding by a polypeptide containing a CDR2 with no 3, 2, or 1 amino acid difference, where this affinity was measured using surface plasmon resonance.

In addition, or in addition, the CDR3 included in the ISV that specifically binds CD123 may be selected from the group consisting of:

a) SEQ ID NO: 23; or

b) amino acid sequences with a difference of 2 or 1 amino acid from the amino acid sequence of SEQ ID NO: 23, where

- in position 4, E was changed to R; and/or

- in position 5, T was changed to D or Y;

provided that a polypeptide containing a CDR3 with a difference of 2 or 1 amino acid binds CD123 with the same, about the same or higher affinity compared to binding by a polypeptide containing a CDR3 with no difference of 2 or 1 amino acid, where the specified affinity is measured using surface plasmon resonance.

Accordingly, the present invention also relates to a polypeptide as described above, which includes or (essentially) consists of 4 framework regions (FR1-FR4, respectively) and 3 complementarity-determining regions (CDR1-CDR3, respectively), in which:

i) CDR1 is SEQ ID NO: 16;

and

ii) CDR2 is selected from the group consisting of:

a) SEQ ID NO: 18; or

b) amino acid sequences with a difference of 3, 2 or 1 amino acid from the amino acid sequence of SEQ ID NO: 18, where

- in position 3 Y has been changed to W;

- in position 6 N has been changed to S; and/or

- in position 10 Q has been changed to E;

provided that a polypeptide containing a CDR2 with a 3, 2, or 1 amino acid difference binds CD123 with the same, about the same, or higher affinity as compared to binding by a polypeptide containing a CDR2 with no 3, 2, or 1 amino acid difference, where the specified affinity is measured using surface plasmon resonance;

and

iii) CDR3 is selected from the group consisting of:

c) SEQ ID NO: 23; or

d) amino acid sequences with a difference of 2 or 1 amino acid from the amino acid sequence of SEQ ID NO: 23, where

- in position 4, E was changed to R; and/or

- in position 5, T was changed to D or Y;

provided that a polypeptide containing a CDR3 with a difference of 2 or 1 amino acid binds CD123 with the same, about the same or higher affinity compared to binding by a polypeptide containing a CDR3 with no difference of 2 or 1 amino acid, where the specified affinity is measured using surface plasmon resonance.

In an additional aspect, the present invention relates to a polypeptide described herein, in which CDR1 is SEQ ID NO: 16, CDR2 is selected from the group consisting of SEQ ID NO: 18-20, and CDR3 is selected from the group consisting of SEQ ID NO: 18-20 NO: 23-25. Preferably, CDR1 is SEQ ID NO: 16, CDR2 is SEQ ID NO: 18, and CDR3 is SEQ ID NO: 23.

Preferred ISVs of the invention relating to 56A10 may be selected from the group consisting of SEQ ID NOs: 7-10 or from polypeptides that have sequence identity greater than 80%, greater than 85%, greater than 90%, greater than 95%, or even greater. 99% with one of SEQ ID NOs: 7-10. Accordingly, in an additional aspect, the present invention relates to a polypeptide described herein, where the polypeptide is selected from the group consisting of SEQ ID NO: 7-10 or from polypeptides that have a sequence identity of more than 80%, more than 85%, more than 90 %, more than 95% or even more than 99% with one of SEQ ID NO: 7-10. Preferably, the polypeptide is selected from the group consisting of SEQ ID NO: 7-10.

In one aspect, a polypeptide of the invention binds to human CD123 expressed on MOLM-13 cells with an average EC50 of 10 μM to 100 nM, e.g., an average EC50 of 5 μM or less, e.g., less than 4, 3, 2, or 1 µM or even less, preferably by flow cytometry.

In another aspect, a polypeptide of the invention binds to human CD123 with a mean KD of 1 μM to 10 nM, e.g., a mean KD of 500 nM or less, such as less than 400, 300, or 200 nM or less, where said KD is preferred. determined by surface plasmon resonance.

In a further aspect, the present invention relates to a polypeptide that cross-blocks CD123 binding of at least one of the polypeptides described herein, or that cross-blocks CD123 binding of one of the polypeptides of SEQ ID NOS: 1-10.

In a further aspect, the present invention relates to a polypeptide that is cross-blocked from binding to CD123 by at least one of the polypeptides described herein, or that is cross-blocked from binding to CD123 by one of the polypeptides of SEQ ID NOS: 1-10.

A polypeptide that specifically binds CD123 as described herein preferably (essentially) consists of a single domain antibody, dAb, nanobody, V _HH , humanized V _HH , overblown VH or V _HH that has been generated by affinity maturation.

A polypeptide of the invention that specifically binds CD123 may contain one or more ISVs that specifically bind CD123. Accordingly, in a further aspect, the present invention provides a polypeptide comprising two or more ISVs, preferably two, that specifically bind CD123. In a preferred aspect, two or more ISVs, preferably two ISVs, that specifically bind CD123 are selected from the 56A10 ISV group or from the 55F03 ISV group.

In a further aspect, the present invention relates to a polypeptide that specifically binds CD123, comprising two ISVs that specifically bind CD123, where the ISVs are selected from the group of ISVs related to 56A10 or from the group of ISVs related to 55F03.

A polypeptide of the invention comprising two or more ISVs, preferably two ISVs that specifically bind CD123, is preferably biparatopic, comprising a first ISV and a second ISV, wherein the first ISV binds to an epitope on CD123 that is different from the epitope on CD123 bound by the second ISV. In a preferred aspect, the first ISV is selected from the group of ISVs related to 56A10 and the second ISV is selected from the group of ISVs related to 55F03.

ISVs may be present at any position in the biparatopic polypeptide of the invention that binds CD123. In one aspect, the second ISV is located at the N-terminus of the first ISV. In another aspect, the second ISV is located at the C-terminus of the first ISV.

The ISVs present in a polypeptide of the invention may be directly linked to each other, or they may be linked via one or more linkers, preferably peptidic linkers. Accordingly, in a further aspect, the present invention provides a polypeptide as described herein, wherein the ISVs are directly linked to each other or linked to each other via a linker. Preferred linkers for use in the polypeptides of the invention are shown in Table B-3 (SEQ ID NOs: 325 to 336). As such, in a further aspect, the present invention relates to a polypeptide described herein, wherein the linker is selected from the group consisting of SEQ ID NOs: 325 to 336.

The present invention further encompasses constructs (also referred to herein as "construct(s) of the invention") that comprise the polypeptide described herein and further comprise one or more other groups, residues, fragments, or linking units, optionally linked through one or more peptide linkers.

In a further aspect, said one or more other groups, residues, fragments, or binding units may provide a construct with an increased half-life compared to the corresponding polypeptide without one or more other groups, residues, fragments, or binding units. Mentioned one or more other groups, residues, fragments or binding units that provide a polypeptide with an extended half-life, can be any molecule that provides retention of the polypeptide in the serum. In one aspect, one or more other groups, residues, fragments, or binding units that provide the polypeptide with an increased half-life are selected from the group consisting of a polyethylene glycol molecule, serum proteins or fragments thereof, binding units that can bind to serum proteins, the Fc part and small proteins or peptides that can bind to serum proteins.

Accordingly, in one aspect, the present invention provides a construct as described herein, wherein said one or more other groups, residues, fragments, or linking units that provide a construct with an extended half-life is selected from the group consisting of serum albumin ( such as human serum albumin) or serum immunoglobulin (such as IgG).

In another aspect, the present invention provides a construct as described herein, wherein said one or more other binding units that provide an extended half-life construct are selected from the group consisting of binding units that can bind to serum albumin (such as human serum albumin) or serum immunoglobulin (such as IgG). Preferably, said one or more other binding units that provide the polypeptide with an extended half-life is an ISV that binds serum albumin. In a further aspect, said ISV that binds serum albumin may (essentially) consist of a single domain antibody, dAb, nanobody, V _HH , humanized V _HH , or overblown VH.

The preferred ISV for use in the constructs described herein is an ISV that binds serum albumin and is (essentially) composed of 4 framework regions (FR1-FR4, respectively) and 3 complementarity-determining regions (CDR1-CDR3, respectively), wherein CDR1 is GFTFSSFGMS (SEQ ID NO: 363) or GFTFRSFGMS (SEQ ID NO: 364), CDR2 is SISGSGSDTL (SEQ ID NO: 365) and CDR3 is GGSLSR (SEQ ID NO: 366). Preferred ISVs that bind serum albumin are selected from the group consisting of SEQ ID NOs: 43 and 351-362.

With regard to the polypeptides of the invention, other groups, residues, fragments or linking units, such as ISV, can be directly linked to each other or connected to each other through a linker. In a further aspect, the present invention provides the construct described herein wherein the linker is selected from the group consisting of SEQ ID NOs: 325-336.

Preferred constructs of the invention may be selected from the group consisting of SEQ ID NOs: 63-67 or constructs that have greater than 80%, greater than 85%, greater than 90%, greater than 95%, or even greater than 99% sequence identity with one of the SEQs. ID NO: 63-67, preferably SEQ ID NO: 63-67.

The constructs of the invention can be sequence-optimized, for example, to make the construct more human-like, improve the expression of the constructs, increase the storage stability of the constructs, and/or make the constructs less likely to be bound by serum-preexisting antibodies. In one aspect, the present invention provides the construct described herein, further comprising a C-terminal extension (X)n, wherein n is 1 to 5, such as 1, 2, 3, 4, or 5, and wherein X is natural amino acid, preferably not cysteine. Preferred constructs are selected from the group consisting of SEQ ID NOs: 338-342.

The present invention also relates to nucleic acids encoding polypeptides and constructs (which are such that they can be obtained by expression of the nucleic acid encoding them), as defined in this document. In one aspect, the nucleic acid as described herein is in the form of a genetic construct.

The present invention also relates to an expression vector containing a nucleic acid as defined herein.

The present invention also relates to a host or host cell containing a nucleic acid as defined herein or an expression vector as defined herein.

In a further aspect, the present invention provides a method for producing a polypeptide or construct (i.e., those that can be obtained by expression of a nucleic acid encoding them) as defined herein, said method at least comprising the steps of:

a) expression in a suitable host cell or host organism or other suitable nucleic acid expression system as defined herein; optionally followed by:

b) isolating and/or purifying the polypeptide or construct as defined herein.

In an additional aspect, the present invention relates to a composition containing at least one polypeptide or construct as defined herein, or a nucleic acid as defined herein. In one aspect, the composition is a pharmaceutical composition. In one aspect, the composition further comprises at least one pharmaceutically acceptable carrier, diluent or excipient and/or adjuvant and optionally contains one or more additional pharmaceutically active polypeptides and/or compounds.

The present invention also relates to a polypeptide described herein, a construct described herein, or a composition described herein for use as a drug. In a further aspect, the present invention relates to the use of a polypeptide described herein or a composition described herein for the manufacture of a medicament. In a further aspect, the present invention relates to a polypeptide described herein, a construct described herein, or a composition described herein for use in the prevention, treatment and/or alleviation of a disease or condition associated with CD123. The present invention also relates to a method for preventing, treating, and/or improving a CD123-associated disease or condition, comprising the step of administering to a subject in need thereof a pharmaceutically active amount of a polypeptide described herein, a construct described herein, or a composition, described in this document. The present invention also relates to the use of a polypeptide described herein, a construct described herein, or a composition described herein for the manufacture of a medicament for the prevention, treatment and/or amelioration of a disease or condition associated with CD123. Without limitation, a CD123-associated disease or condition may be a proliferative disease or an inflammatory condition. Accordingly, in a further aspect, the present invention relates to a polypeptide described herein, a construct described herein, or a composition described herein for use in the prevention, treatment and/or amelioration of a proliferative disease or inflammatory condition. The present invention also relates to a method for preventing, treating and/or alleviating a proliferative disease or inflammatory condition, comprising the step of administering to a subject in need thereof a pharmaceutically active amount of a polypeptide described herein, a construct as described herein, or a composition described in this document. The present invention also relates to the use of a polypeptide described herein, a construct described herein, or a composition described herein for the manufacture of a medicament for the prevention, treatment and/or amelioration of a proliferative disease or inflammatory condition.

Without limitation, the proliferative disease may be a cancer. Accordingly, in a further aspect, the present invention provides a polypeptide as described herein, a construct as described herein, or a composition as described herein for use in the prevention, treatment and/or amelioration of cancer. The present invention also relates to a method for preventing, treating and/or alleviating a cancer comprising the step of administering to a subject in need thereof a pharmaceutically active amount of a polypeptide described herein, a construct described herein, or a composition described herein. The present invention also relates to the use of a polypeptide described herein, a construct described herein, or a composition described herein, for the manufacture of a medicament for the prevention, treatment and/or amelioration of an oncological disease.

The cancer to be treated by the method of the invention may be any cancer known to be treated by killing cells by targeting through CD123. Cancers that express CD123 on aberrantly proliferating cells include, but are not limited to, lymphomas (including Burkitt's lymphoma, Hodgkin's lymphoma, and non-Hodgkin's lymphoma), leukemias (including acute myeloid leukemia, chronic myeloid leukemia, B-cell acute lymphoblastic leukemia, chronic lymphocytic leukemia and hairy cell leukemia), myelodysplastic syndrome, neoplasm of blast plasmacytoid dendritic cells, systemic mastocytosis and multiple myeloma. Accordingly, in a further aspect, the present invention relates to a polypeptide described herein, a construct described herein, a composition described herein for use in the prevention, treatment and/or amelioration of a cancer selected from lymphomas (including Burkitt, Hodgkin's lymphoma, and non-Hodgkin's lymphoma), leukemias (including acute myeloid leukemia, chronic myeloid leukemia, B-cell acute lymphoblastic leukemia, chronic lymphocytic leukemia, and hairy cell leukemia), myelodysplastic syndrome, blast plasmacytoid dendritic cell neoplasms, systemic mastocytosis, and multiple myeloma. The present invention also relates to a method for preventing, treating and/or alleviating a cancer selected from lymphomas (including Burkitt's lymphoma, Hodgkin's lymphoma and non-Hodgkin's lymphoma), leukemias (including acute myeloid leukemia, chronic myeloid leukemia, B-cell acute lymphoblastic leukemia , chronic lymphocytic leukemia and hairy cell leukemia), myelodysplastic syndrome, neoplasms of blast plasmacytoid dendritic cells, systemic mastocytosis, and multiple myeloma, comprising the step of administering to a subject in need thereof a pharmaceutically active amount of a polypeptide described herein, a construct described herein, or compositions described in this document. The present invention also relates to the use of a polypeptide described herein, a construct described herein, or a composition described herein, for the manufacture of a medicament for the prevention, treatment and/or alleviation of a condition in an oncological disease selected from the group consisting of of lymphomas (including Burkitt's lymphoma, Hodgkin's lymphoma and non-Hodgkin's lymphoma), leukemias (including acute myeloid leukemia, chronic myeloid leukemia, B-cell acute lymphoblastic leukemia, chronic lymphocytic leukemia and hairy cell leukemia), myelodysplastic syndrome, neoplasms of blast plasmacytoid dendritic cells , systemic mastocytosis and multiple myeloma.

The inflammatory condition to be treated by the method of the invention may be any inflammatory condition known to be treated by killing cells via CD123 targeting. Inflammatory conditions known to involve CD123 expression on cells include, but are not limited to, autoimmune lupus (SLE), allergies, asthma, and rheumatoid arthritis. Accordingly, in a further aspect, the present invention relates to a polypeptide described herein, a construct described herein, a composition described herein for use in the prevention, treatment and/or alleviation of an inflammatory condition selected from the group consisting of autoimmune lupus (SLE), allergies, asthma and rheumatoid arthritis. The present invention also relates to a method for the prevention, treatment and/or alleviation of an inflammatory condition selected from the group consisting of autoimmune lupus (SLE), allergies, asthma and rheumatoid arthritis, comprising the step of administering to an object in need thereof a pharmaceutically active amount of a polypeptide described in herein, the construct described herein, or the composition described herein. The present invention also relates to the use of a polypeptide described herein, a construct described herein, or a composition described herein, for the manufacture of a medicament for the prevention, treatment and/or amelioration of an inflammatory condition selected from the group consisting of autoimmune lupus (SLE), allergies, asthma and rheumatoid arthritis.

The polypeptides, constructs and compositions of the present invention may also be used in combination with another therapeutic drug. Accordingly, in a further aspect, the present invention provides a polypeptide as described herein, a construct as described herein, a composition as described herein, for use in combination therapy.

The present invention also relates to the method described herein, where the treatment is a combination treatment.

In a further aspect, the present invention relates to the use of a polypeptide described herein, a construct described herein, or a composition described herein, for the manufacture of a medicament for the prevention, treatment and/or amelioration of the condition described herein, wherein treatment is a combination treatment.

In a further aspect, the present invention provides a kit comprising a polypeptide as described herein, a construct as described herein, a nucleic acid as described herein, an expression vector as described herein, or a host or host cell as described herein. document.

Brief description of the drawings

Figure 1. Evaluation of human TCR/CD3 and human CD3 expression on transfected CHO, HEK293 and Llana cell lines using 100 nM anti-human TCR α/β antibody (clone BW242/412) (black) and 100 nM anti-human CD3 antibody (clone OKT-3) (gray). The MCF value (mean channel fluorescence) was plotted for each cell line. The x-axis shows cell type and transfected genes; CD3 indicates transfection with the CD3 complex (epsilon, delta, gamma and zeta chains), huTCR indicates transfection with TCR α/β chains, where the variable domain used is in brackets.

Figure 2. Evaluation of the quality of soluble recombinant TCR α/β cynomolgus monkey proteins using antibody clone R73, against TCR of non-human/rat primates; anti-human TCR α/β antibodies (solid circles) and an irrelevant anti-egg lysozyme nanobody (cAblys) (open circles). The OD value was plotted against the nanobody concentration.

Figure 3. Dose-dependent binding of the monovalent anti-TCR nanobody T0170055A02 to human TCR/CD3 expressed on CHO-K1 cells (Figure 3A) and to primary human T cells (Figure 3B). Dose-dependent binding of the monovalent anti-TCR nanobody T0170056G05 to human TCR/CD3 expressed on CHO-K1 cells (Figure 3C) and to primary human T cells (Figure 3D). The MCF value (mean channel fluorescence) was plotted against nanobody concentration.

Figure 4. Dose-dependent binding of anti-TCR monovalent nanobodies T0170055A02 (Figure 4A) and T0170056G05 (Figure 4B) to HEK293H to human TCR (2IAN)/CD3 (solid circle), human HEK293H CD3 (cross) and to the HEK293H control cell line (open circles) ). The MCF value (mean channel fluorescence) was plotted against nanobody concentration.

Figure 5. Dose-dependent binding of monovalent anti-TCR nanobodies T0170055A02 (Figure 4A, solid circles) and T0170056G05 (Figure 4B, solid circles) and irrelevant nanobody (Figure 4A and Figure 4B, open circles) to soluble recombinant human TCR α/β(2XN9) -protein with "lightning". OD at 450 nm was plotted against nanobody concentration.

Figure 6. Kinetic analysis of T01700055A02 (Figure 6A) and T01700056G05 (Figure 6B) on the interaction of soluble recombinant human TCR α/β protein (2XN9)-protein with a zipper using BioLayer interferometry on an Octet RED384 instrument. The analyte concentrations used were: 1000, 333, 111, 37, 12.3, 4.1 and 1.4 nM. The Langmuir equation fit to the kinetic data is indicated by black lines, while the sensograms are represented by gray lines.

Figure 7. Dose-dependent binding of monovalent anti-TCR nanobodies T0170055A02 (Figure 7A, solid circles) and T0170056G05 (Figure 7B, solid circles) and an irrelevant nanobody (Figure 7A and Figure 7B, open circles) to soluble recombinant cynomolgus monkey TCR α/β protein with "lightning". OD at 450 nm was plotted against nanobody concentration.

Figure 8. Kinetic analysis of T0170055A02 (Figure 8A) and T0170056G05 (Figure 8B) on the interaction of soluble recombinant TCRα/β cynomolgus monkey protein with zipper using BioLayer interferometry on an Octet RED384 instrument. The analyte concentrations used were: 1000, 333, 111, 37, 12.3, 4.1 and 1.4 nM. The Langmuir equation fit to the kinetic data is indicated by black lines, while the sensograms are represented by gray lines.

Figure 9. Activation data for bead-bound monovalent anti-TCR nanobodies (Figure 9A). Activation data with monovalent anti-TCR nanobodies presented in solution (Figure 9B). Activation was measured by monitoring CD69 activation on primary human T cells. The MCF value (mean channel fluorescence) was plotted for each nanobody.

Figure 10. Evaluation of human CD123 expression on HEK293 Flp-In, HEK293 Flp-In CD123 cynomolgus monkey, CHO Flp-In, and CHO Flp-In human CD123 using an anti-CD123 antibody (BD Biosciences cat. no. 554527) (black) and isotype control (eBioscience, cat. no. 16-4724-85), followed by labeling with PE-labeled goat anti-mouse antibody (Jackson Immunoresearch lab. Inc., cat. no. 115-116-071) (grey), using a flow cytometry. The MFI value (mean channel fluorescence intensity) was plotted for each cell line.

Figure 11. Evaluation of human CD123 expression in U-937, MOLM-13, KG1a and NCI-H929 cells using an anti-CD123 antibody labeled with APC (BD Biosciences, Cat. No. 560087) (black) and an isotype control labeled with APC ( Biolegend cat # 400220) (gray) using flow cytometry. The MFI value (mean channel fluorescence intensity) was plotted for each cell line.

Figure 12. Dose-dependent binding of the monovalent anti-CD123 nanobody A0110056A10 to MOLM-13 cells (Figure 12A) and KG1a cells (Figure 12C). Dose-dependent binding of the monovalent anti-CD123 nanobody A0110055F03 to MOLM-13 cells (Figure 12B) and KG1a cells (Figure 12D). The MFI value (mean channel fluorescence intensity) was plotted against concentration.

Figure 13. Dose-dependent binding of Alexa647-labeled A0110056A10 to Flp-In parental CHO cells (open symbol) and CD123-transfected human CHO cells (solid symbol) (Figure 13A). Dose-dependent binding of Alexa647-labeled A0110056A10 to parental HEK Flp-In cells (open symbol) and HEK cells transfected with cynomolgus monkey CD123 (solid symbol) (Figure 13B). The MFI value (mean channel fluorescence intensity) was plotted against concentration.

Figure 14. Dose-dependent binding of A0110055F03 to parent Flp-In CHO cells (open symbol) and CHO cells transfected with human CD123 (solid symbol) (Figure 14A). Dose-dependent binding of A0110055F03 to parental HEK Flp-In cells (open symbol) and HEK cells transfected with cynomolgus monkey CD123 (solid symbol) (Figure 14B). The MFI value (mean channel fluorescence intensity) was plotted against concentration.

Figure 15. Dose-dependent binding of the monovalent anti-CD123 nanobody A0110056A10-Alexa 647 to MOLM-13 cells (Figure 15A) and to Flp-In CHO cells transfected with human CD123 (Figure 15B). The MFI value (mean channel fluorescence intensity) was plotted against concentration.

Figure 16. Dose-dependent competition of monovalent nanobodies A1010056A10 (squares) and A0110055F03 (circles) with Alexa 647 labeled A0110056A10 for binding to human CD123 on MOLM-13 cells (Figure 16A) and on CHO Flp-In cells transfected with human CD123 (Figure 16B) . The MFI value (mean channel fluorescence intensity) was plotted against concentration.

Figure 17. Dose-dependent binding of APC-labeled mouse anti-human CD123 antibody (clone 7G3) to human CD123 on MOLM-13 cells (Figure 17A) and on Flp-In CHO cells transfected with human CD123 (Figure 17B). The MFI value (mean channel fluorescence intensity) was plotted against concentration.

Figure 18. Dose-dependent competition of monovalent nanobodies A0110056A10 (squares) and A0110055F03 (circles) with APC-labeled mouse anti-human CD123 antibody (clone 7G3) for binding to CD123 expressed on MOLM-13 cells (Figure 18A) or CHO Flp-In cells, transfected with huCD123 (Figure 18B). The MFI value (mean channel fluorescence intensity) was plotted against concentration.

Figure 19. Dose-dependent binding of mouse anti-human CD123 antibody (clone 7G3) to self-biotinylated recombinant CD123 protein (R&D Systems cat. no. 301-R3/CF). OD at 450 nm is measured as a function of concentration.

Figure 20. Dose-dependent competition of monovalent anti-CD123 nanobodies A0110056A10 (squares) and A0110055F03 (solid circles) with mouse monoclonal anti-CD123 antibody (clone 7G3) (BD Biosciences, Cat no. 554527) for binding to the CD123 protein, in ELISA ( figure 20A). The irrelevant anti-egg lysozyme nanobody cAbLys (open circles) and mouse anti-CD123 monoclonal antibody (clone 7G3) in solution (stars) were taken together as negative and positive controls, respectively (Figure 20B). OD at 450 nm is measured as a function of concentration.

Figure 21. Dose-dependent binding of monovalent anti-CD123 nanobody A0110056A10-Alexa 647 to MOLM-13 cells (Figure 21A), to CHO Flp-In cells transfected with human CD123 (Figure 21B) and to HEK Flp-In cells transfected with cynomolgus CD123 (figure 21C). The MFI value (mean channel fluorescence intensity) was plotted against concentration.

Figure 22. Dose dependent competition of multivalent CD123/TCR binding polypeptides with Alexa647-A0110056A10 for binding to CD123 expressed on MOLM-13 cells (Figure 22A) and to huCD123 transfected into Flp-In CHO cells (Figure 22C) or cyCD123, transfected into HEK cells Flp-In (figure 22B). The irrelevant multivalent T017000129 polypeptide was taken in parallel as a negative control. The MFI value (mean channel fluorescence intensity) was plotted against concentration.

Figure 23. Dose dependent competition of multivalent CD123/TCR binding polypeptides with biotinylated T0170056G05 for binding to human TCR/CD3 expressed on CHO-K1 cells. The MFI value (mean channel fluorescence intensity) was plotted against concentration.

Figure 24. Dose-dependent competition of multivalent CD123/TCR binding polypeptides with T017000099 for binding to CD3/TCR expressed on HSC-F. Monovalent His-tagged T017000125 was taken as a positive control. The MFI value (mean channel fluorescence intensity) was plotted against concentration.

Figure 25. Dose-dependent retargeting killing of CD123-expressing MOLM-13 cells by human effector T cells, in a flow cytometric assay with multivalent CD123/TCR-binding polypeptides at an effector to target ratio of 10:1. A0110056A10, T017000132 and T017000129 were taken together as a negative control. The percentage of cell death (% TOPRO-positive cells) was plotted against the concentration of the construct.

Figure 26. Dose-dependent retargeted killing of CD123-expressing KG1a cells by human effector T cells in a flow cytometric assay with multivalent CD123/TCR-binding polypeptides at an effector to target ratio of 10:1. A0110056A10, T017000129 and T017000132 were taken in parallel as negative controls. The percentage of cell death (% TOPRO-positive cells) was plotted against the concentration of the construct.

Figure 27. Dose-dependent retargeted killing of CD123-expressing MOLM-13 cells by cynomolgus monkey effector T cells in a flow cytometric assay with multivalent CD123/TCR-binding polypeptides at an effector to target ratio of 10:1. A0110056A10 was taken in parallel as a negative control. The percentage of cell death (% TOPRO-positive cells) was plotted against the concentration of the construct.

Figure 28. Dose-dependent retargeted killing of CD123-expressing KG1a cells by cynomolgus monkey effector T cells in a flow cytometric assay with multivalent CD123/TCR-binding polypeptides at an effector to target ratio of 8. Several irrelevant constructs were taken in parallel as negative controls. The percentage of cell death (% TOPRO-positive cells) was plotted against the concentration of the construct.

Figure 29. Dose-dependent T cell activation (CD25 upregulation) by multivalent CD123/TCR binding polypeptides on CD4/CD8+ selected T cells during redirection of cynomolgus monkey effector T cells to kill human CD123 positive MOLM-13 cells, after incubation period 72 hours. The MFI (mean fluorescence intensity) of CD4/CD8+ selected T cells was plotted against the concentration of the constructs.

Figure 30. Dose-dependent retargeted killing by human effector T cells of transfected human CD123 CHO Flp-In cells in an xCELLigence-based assay using T017000139 (solid diamonds) at an effector to target ratio of 15:1. Monovalent nanobodies A0110056A10, T0170056G05 and irrelevant construct T017000129 were taken in parallel as a negative control. The cell index (CI) after an incubation period of 50 h was plotted against the concentration of the multispecific polypeptide.

Figure 31. Monovalent building blocks and multispecific polypeptides in a human effector T cell redirected killing assay using the CD123-negative Flp-In CHO control line in an xCELLigence based xCELLigence assay at a 15:1 effector to target ratio. The CI after an incubation period of 50 h was plotted against the concentration of the multispecific polypeptide.

Figure 32. Effect of monovalent building blocks and multispecific CD123/TCR binding polypeptides on the growth of a CD123 transfected cell line (Figure 32A) and a control cell line (Figure 32B) in the absence of T cells. The CI after an incubation period of 50 h was plotted against the concentration of the multispecific polypeptide.

Figure 33. Dose-dependent retargeted killing by cynomolgus effector T cells of HEK Flp-In cells transfected with cynomolgus CD123 in an xCELLigence-based assay using T017000139 (filled diamonds) at an effector to target ratio of 15:1. The monovalent nanobody, T0170056G05 and the irrelevant construct T017000129 were taken in parallel as a negative control. The CI after an incubation period of 80 hours was plotted against the concentration of the multispecific polypeptide.

Figure 34. Monovalent building block and multispecific polypeptides in a cynomolgus monkey redirected killing assay using the CD123-negative control HEK Flp-In cell line. The CI after an incubation period of 80 hours was plotted against the concentration of the multispecific polypeptide.

Figure 35. Effect of monovalent building blocks and multispecific CD123/TCR binding polypeptides on the growth of a CD123 transfected cell line (Figure 35A) and a control cell line (Figure 35B) in the absence of T cells. The CI after an incubation period of 80 hours was plotted against the concentration of the multispecific polypeptide.

Figure 36. Dose-dependent cytokine production by human effector T cells (Figure 36A) or cynomolgus monkey effector T cells (Figure 36B) during redirected killing by human T cells dependent on the multispecific CD123/TCR binding polypeptide of CHP Flp target cells -In expressing human CD123 at an effector to target ratio of 10:1. INF-γ production was measured after 72 hours. The OD value was plotted against concentration.

Figure 37. Dose dependent cytokine production by effector T cells during redirected T cell killing dependent on the multispecific CD123/TCR binding polypeptide of CHO Flp-In target cells expressing human CD123 at an effector to target ratio of 10:1. IL-6 production was measured after 72 hours. The pg/ml value was plotted against the concentration.

Figure 38. Redirected autologous T cell mediated depletion of CD123+ pDCs and basophils by multivalent CD123/TCR binding polypeptides in healthy human (Figure 38A) and cynomolgus monkey (Figure 38B) PBMC samples after an incubation period of 5 hours. The percentage of Lin-/CD123+ cells (pDC and basophils) was plotted against the concentration of the constructs.

Figure 39. Redirected autologous T cell depletion of monocytes by multivalent CD123/TCR binding polypeptides in healthy human PBMC samples after an incubation period of 5 hours (Figure 39A) and 24 hours (Figure 39B). The percentage of monocytes (CD14+ cells) was plotted against the concentration of constructs.

Figure 40. Dose-dependent upregulation of CD69, human T cell activation by multivalent CD123/TCR binding polypeptides on CD3+ selected T cells during redirected T cell killing of autologous CD123 positive cells after a 24 hour incubation period. The MFI (mean fluorescence intensity) of CD3+ selected T cells was plotted against the concentration of the constructs.

Figure 41. Dose-dependent characterization of monovalent nanobodies and the irrelevant multivalent polypeptide T017000129 in redirected killing by human effector T cells (Figure 41A) or cynomolgus monkey (Figure 41B) of human CD123 KG1a cells in a flow cytometric assay at an effector to target ratio of 10:1 . T017000139 (filled diamonds) was taken in parallel as a positive control. The percentage of cell death (% TOPRO-positive cells) was plotted against the concentration of the construct.

Figure 42. Dose-dependent characterization of monovalent nanobodies and the irrelevant multivalent polypeptide T017000129 for retargeted killing by human effector T cells (Figure 42A) or cynomolgus monkey (Figure 42B and Figure 42C) of human CD123 MOLM-13 cells in a flow cytometric assay at effector ratio to the target 10:1. T017000139 (filled diamonds) was taken in parallel as a positive control. The percentage of cell death (% TOPRO-positive cells) was plotted against the concentration of the construct.

Figure 43. Dose-dependent production of cytokines by human effector T cells during multispecific CD123/TCR-binding polypeptide-dependent retargeted killing by T cells of target cells MOLM-13 (Figure 43A and Figure 43C) and KG1a (Figure 43B) at an effector to targets 10:1. The production of human IL-6 (Figure 43C) and human IFN-β (Figure 43A and Figure 43B) was measured after 72 hours. The cytokine concentration was plotted against the concentration.

Figures 44A and 44B. Dose-dependent characterization of target-independent retargeted killing by human effector T cells by multispecific CD123/TCR-binding polypeptides in a flow cytometric assay using the CD123-negative NCI-H929 cell line. The percentage of cell death (% TOPRO-positive cells) was plotted against the concentration of the construct.

Figure 45. Dose-dependent characterization of target-independent retargeted killing by human (Figure 45A) or cynomolgus monkey (Figure 45B) effector T cells by multispecific CD123/TCR-binding polypeptides in a flow cytometric assay using a CD123-negative cell line U937. The percentage of cell death (% TOPRO-positive cells) was plotted against the concentration of the construct.

Figure 46. Dose-dependent reading of T cell activation by multivalent CD123/TCR binding polypeptides on CD4/CD8+ selected T cells during killing by effector T cells of CD123 negative U-937 cells (Figure 46A) and during killing by effector T cells. human cells of CD123 negative NCI-H929 cells (Figure 46B) after an incubation period of 72 hours. The MFI (mean fluorescence intensity) of CD4/CD8+ selected T cells was plotted against the concentration of the constructs.

Figure 47. Effect of multispecific CD123/TCR binding polypeptides on cytokine production using human effector T cells and NCI-H929 target cells at an effector to target ratio of 10:1. The production of human IL-6 (Figure 47B) and human IFN-β (Figure 47A) was measured. The OD value of the amount of cytokine was plotted against concentration.

Figures 48A and 48B. Dose-dependent T cell proliferation of effector human T cells due to multispecific polypeptides under conditions of redirected death of MOLM-13 target cells at an effector-target ratio of 10:1. CPM (pulses per minute) was plotted against concentration.

Figure 49. Dose-dependent T cell proliferation of human effector T cells due to multispecific polypeptides in the absence of target cells. CPM (pulses per minute) was plotted against concentration.

Figure 50. Lytic potential of non-activated and pre-activated T cells in the presence of T017000114 and MOLM-13 cells at various E:T ratios. The percentage of cell death was plotted against the concentration of the construct.

Figure 51. Lytic potential of non-activated and pre-activated T cells in the presence of T017000139 and KG1a cells at various E:T ratios. The percentage of cell death was plotted against the concentration of the construct.

Figure 52. Dose-dependent retargeted killing by human T cells (Figures 52A and 52B) and cynomolgus monkeys (Figures 52C and 52D) of MOLM-13 cells in the absence or presence of serum albumin in a flow cytometric assay due to multivalent CD123/TCR- binding polypeptides at an effector to target ratio of 10:1. The irrelevant multivalent polypeptide T017000129 and the monovalent structural elements A0110056A10 and T0170056G05 were taken in parallel as a negative control. Percent cell death (% TOPRO positive cells) was plotted against polypeptide concentration.

Figure 53. Dose-dependent retargeted killing by human T cells (Figures 53A, 53B and 53C) and cynomolgus monkey (Figure 53D, 53E and 53F) of KG1a cells in the absence or presence of serum albumin in a multivalent CD123/TCR flow cytometry assay -binding polypeptides at an effector to target ratio of 10:1. The irrelevant multivalent polypeptides A022600009 (in the presence or absence of SA) and T017000129, as well as the monovalent building blocks A0110056A10 and T0170056G05 were taken in parallel as negative controls. The percentage of cell death (% TOPRO-positive cells) was plotted against the concentration of the construct.

Figure 54. Dose-dependent cytokine production by human T cells during redirected killing by T cells of MOLM-13 due to multispecific CD123/TCR binding polypeptides at an effector to target ratio of 10:1. The production of human IL-6 (FIG. 54B) and human IFN-β (FIG. 54A) was measured. The amount of cytokines is plotted against concentration.

Figure 55. Dose-dependent T cell proliferation of human effector T cells due to multispecific HLE polypeptides under conditions of redirected killing of MOLM-13 target cells at an effector to target ratio of 10:1. CPM (pulses per minute) was plotted against concentration.

Figure 56. Redirected depletion of CD123+ pDCs and basophils by autologous T cells due to multivalent CD123/TCR binding HLE polypeptides in healthy human PBMC samples after an incubation period of 5 hours. The percentage of Lin-/CD123+ cells (pDC and basophils) was plotted against the concentration of the constructs.

Figure 57. Redirected depletion of CD123+ pDCs and basophils by autologous T cells due to multivalent CD123/TCR binding HLE polypeptides in healthy cynomolgus monkey PBMC samples in vitro after an incubation period of 5 hours. The percentage of Lin-/CD123+ cells (pDC and basophils) was plotted against the concentration of the constructs.

Figure 58 Redirected monocyte depletion by autologous T cells due to multivalent CD123/TCR binding polypeptides in healthy human PBMC samples after a 24 hour incubation period. The percentage of monocytes (CD14 + cells) was plotted against the concentration of constructs.

Figure 59. Peripheral blood T cell count of treated cynomolgus monkeys over time. The absolute counts of CD4+ CD3+ T cells (Figure 59A) and CD8+ CD3+ T cells (Figure 59B) per µl of blood are expressed as mean ± SEM over time for different treatment groups: positive control (open circles, n = 2) irrelevant polypeptide/ TCR polypeptide (cross, n = 4), CD123/TCR polypeptide (black triangle, n = 4). Gray bars represent periods of continuous infusion.

Figure 60. The number of CD123+CD14 - cells in the peripheral blood of the treated cynomolgus monkeys depending on time. The absolute number of CD123+ CD14- cells per µl of blood is expressed as the mean ± SEM for different treatment groups: positive control (open circles, n = 2), irrelevant TCR polypeptide/polypeptide (crossover, n = 4), CD123/TCR polypeptide (black triangle , n = 4). Gray bars represent periods of continuous infusion.

Figure 61. Expression of PD-1 on CD4+ CD3+ and CD8+ CD3+ T cells as a function of time. The frequency of CD4+CD3+ T cells (Figure 61A) and CD8+CD3+ T cells (Figure 61B) in blood is expressed as mean ± SEM for different treatment groups: positive control (open circles, n = 2), irrelevant polypeptide/TCR polypeptide (cross, n = 4), CD123/TCR polypeptide (black triangle, n = 4). Gray bars represent periods of continuous infusion.

Figure 62. Serum interleukin-6 in treated cynomolgus monkeys as a function of time. Serum IL-6 concentration is expressed as mean ± SEM (pg/mL) for different treatment groups: positive control (open circles, n=2), irrelevant/TCR polypeptide (crossover, n=4), CD123/polypeptide/ TCR (black triangle, n = 4). Gray bars represent periods of continuous infusion.

Detailed description

Definitions

Unless otherwise indicated or defined, all terms used have their usual meaning in the art, which will be understood by a person skilled in the art. Reference is made, for example, to standard manuals such as Sambrook et al. (1989, Molecular Cloning: A Laboratory Manual (2nd ^Ed .) Vols. 1-3, Cold Spring Harbor Laboratory Press), F. Ausubel et al. (1987, Current protocols in molecular biology, Green Publishing and Wiley Interscience, New York), Lewin (1985, Genes II, John Wiley & Sons, New York, NY), Old et al. (1981, Principles of Gene Manipulation: An Introduction to Genetic Engineering (2nd ^Ed .) University of California Press, Berkeley, CA), Roitt et al. (2001, Immunology (6th Ed.) Mosby/Elsevier, Edinburgh), ^Roitt et al. (2001, Roitt's Essential Immunology (10th Ed.) Blackwell Publishing, UK), and Janeway et al. (2005, ^{Immunobiology} (6th Ed.) Garland Science Publishing/Churchill Livingstone, New York, 2005), as well as the general prior art cited herein.

Unless otherwise indicated, all methods, steps, methods and manipulations that are not specifically described in detail can be performed and have been performed in a manner known per se, as will be understood by a person skilled in the art. Hereby, for example, we again refer to the standard manual and the general prior art referred to in this document and in other sources cited therein; and, for example, the following reviews Presta (2006, Adv. Drug Deliv. Rev. 58(5-6): 640-56), Levin and Weiss (2006, Mol. Biosyst. 2(1): 49-57), Irving et al. (2001, J. Immunol. Methods 248(1-2): 31-45), Schmitz et al. (2000, Placenta 21 Suppl. A: S106-12), Gonzales et al. (2005, Tumor Biol. 26(1): 31-43), which describe protein engineering techniques such as affinity maturation and other techniques to improve the specificity and other desired properties of proteins such as immunoglobulins.

As used herein, the term "sequence" (for example, in terms of "immunoglobulin sequence", "antibody sequence", "variable domain sequence", "V _HH sequence", or "protein sequence") should generally be understood to include the corresponding amino acid sequence, as well as nucleic acids or nucleotide sequences encoding them, unless the context requires a more limited interpretation.

Amino acid sequences are interpreted as meaning a single amino acid or a straight chain of two or more amino acids, depending on the context. Nucleotide sequences are interpreted as an unbranched sequence of 3 or more nucleotides.

Amino acids are those L-amino acids commonly found in naturally occurring proteins and are listed in Table B-1 below. Those amino acid sequences that contain D-amino acids are not to be covered by this definition. Any amino acid sequence that contains post-translationally modified amino acids can be described as an amino acid sequence that is originally translated using the symbols shown in the table below, with the positions changed; for example, hydroxylation or glycosylation, but these modifications need not be explicitly shown in the amino acid sequence. Any peptide or protein that can be expressed as links to modified sequences, crosslinks and end cap structures, non-peptidyl links, etc. is covered by this definition.

The terms "protein", "peptide", "protein/peptide" and "polypeptide" are used interchangeably throughout the description, and each has the same meaning for the purposes of this disclosure. Each term refers to an organic compound consisting of a linear chain of two or more amino acids. A compound may have ten or more amino acids; twenty-five or more amino acids; fifty or more amino acids; one hundred or more amino acids, two hundred or more amino acids, and even three hundred or more amino acids. One of skill in the art will appreciate that polypeptides generally contain fewer amino acids than proteins, although there is no art-recognized cut-off point for the number of amino acids that distinguish polypeptides and proteins; that polypeptides can be obtained by chemical synthesis or recombinant methods; and that proteins are usually produced in vitro or in vivo by recombinant methods known in the art.

Amino acid residues will be specified according to the standard three-letter or one-letter amino acid code. We hereby refer to Table A-2 on page 48 in WO 08/020079.

Table B-1. Common amino acids 1-letter 3 letter The code The code Name A Ala alanine B asx Aspartic acid or asparagine C Cys Cysteine D asp Aspartic acid E Glu Glutamic acid F Phe Phenylalanine G gly Glycine H His Histidine I ile isoleucine J Xle Isoleucine or leucine K Lys Lysine L Leu leucine M Met methionine N Asn asparagine O pyl pyrrolysine P Pro Proline Q Gln glutamine R Arg Arginine S Ser serine T Thr Threonine U Scy selenocysteine V Val valine W trp Tryptophan X xxx Unusual or unspecified Y Tyr Tyrosine Z glx Glutamic acid or glutamine

A nucleic acid or amino acid is considered to be "(substantially) isolated (form)", for example, in comparison with the reaction medium or culture medium from which it was obtained - when it has been separated from at least one other component, with which it is normally associated in said source or medium, such as another nucleic acid, another protein/polypeptide, another biological component or macromolecule, or at least one contaminant, impurity or minor component. In particular, a nucleic acid or amino acid is considered "(substantially) isolated" when it is at least 2-fold purified, in particular at least 10-fold, more particularly at least 100-fold, and up to 1000 or more times. A nucleic acid or amino acid that is "(substantially) in isolated form" is preferably substantially homogeneous as determined by a suitable method such as a suitable chromatographic method such as polyacrylamide gel electrophoresis.

Unless the context clearly requires otherwise, throughout the specification and claims, the words "comprises", "comprising" and the like are to be construed in an all-encompassing sense, and not in an exclusive or exhaustive sense; those. in the sense of "including, but not limited to".

For example, when a nucleotide sequence, amino acid sequence, or polypeptide is said to "comprise" another nucleotide sequence, amino acid sequence, or polypeptide, or "essentially consist of" another nucleotide sequence, amino acid sequence, or polypeptide, this may mean that the latter nucleotide sequence, amino acid the sequence or polypeptide has been included in the first mentioned nucleotide sequence, amino acid sequence or polypeptide, respectively, but more often this usually means that the first mentioned nucleotide sequence, amino acid sequence or polypeptide includes within its sequence a section of nucleotides or amino acid residues, respectively, which has such the same nucleotide sequence or amino acid sequence, respectively, as the last sequence, regardless of how the first said sequence was actually created or obtained (which, for example, may be any suitable method described herein). By way of non-limiting example, when a polypeptide of the invention is considered to contain an immunoglobulin single variable domain, this may mean that said immunoglobulin single variable domain sequence has been included in the sequence of the polypeptide of the invention, but more commonly, it usually means that the polypeptide of the invention contains in its sequence sequence of immunoglobulin single variable domains, regardless of how was created or received the specified polypeptide according to the invention. Furthermore, when a nucleic acid or nucleotide sequence is considered to comprise another nucleotide sequence, the first mentioned nucleic acid or nucleotide sequence is preferably such that when it is expressed in an expression product (e.g., a polypeptide), the amino acid sequence encoded by the latter nucleotide sequence is part said expression product (in other words, the last nucleotide sequence is in the same reading frame as the first mentioned larger nucleic acid or nucleotide sequence).

By "essentially consists of" is meant that the immunoglobulin single variable domain used in the method of the invention is either exactly the same as the polypeptide of the invention or corresponds to a polypeptide of the invention that has a limited number of amino acid residues, e.g., 1-20 amino acids. residues, e.g. 1-10 amino acid residues and preferably 1-6 amino acid residues such as 1, 2, 3, 4, 5 or 6 amino acid residues added at the amino-terminus, at the carboxy-terminus or both at the amino-terminus and at the carboxy-terminus of the immunoglobulin single variable domain.

By "consist of" is meant that the immunoglobulin single variable domain used in the method of the invention is exactly the same as the polypeptide of the invention.

For the purposes of comparing two or more nucleotide sequences, the percentage of "sequence identity" between the first nucleotide sequence and the second nucleotide sequence can be calculated by dividing [the number of nucleotides in the first nucleotide sequence that are identical to the nucleotides at the corresponding positions in the second nucleotide sequence] by [total number of nucleotides in the first nucleotide sequence] and multiplying by [100%], where each deletion, insertion, substitution or addition of a nucleotide in the second nucleotide sequence compared to the first nucleotide sequence is considered as a difference in one nucleotide (position). Otherwise, the degree of sequence identity between two or more nucleotide sequences can be calculated using a known computer algorithm for sequence alignment, such as NCBI Blast v2.0, using standard settings. Some other methods, computer algorithms and settings for determining the degree of sequence identity, for example, are described in WO 04/037999, EP 0967284, EP 1085089, WO 00/55318, WO 00/78972, WO 98/49185 and GB 2357768. percent "sequence identity" between two nucleotide sequences in accordance with the calculation method described above, the nucleotide sequence with the most nucleotides will be taken as the "first" nucleotide sequence, and the other nucleotide sequence will be taken as the "second" nucleotide sequence.

For the purposes of comparing two or more amino acid sequences, the percentage of "sequence identity" between the first amino acid sequence and the second amino acid sequence (also referred to herein as "amino acid identity") can be calculated by dividing the [number of amino acid residues in the first amino acid sequence that are identical to the amino acid residues at the respective positions in the second amino acid sequence] by [total number of amino acid residues in the first amino acid sequence] and multiplying by [100%] in which each deletion, insertion, substitution or addition of an amino acid residue in the second amino acid sequence compared to the first amino acid sequence is considered as a difference in one amino acid residue (position), i.e. as "amino acid difference" as defined herein. Otherwise, the degree of sequence identity between two amino acid sequences can be calculated using a known computer algorithm such as those mentioned above for determining the degree of sequence identity for nucleotide sequences, in this case using standard settings. Generally, to determine the percentage of "sequence identity" between two amino acid sequences according to the calculation method described above, the amino acid sequence with the most amino acid residues will be taken as the "first" amino acid sequence, and the other amino acid sequence will be taken as the "second" amino acid sequence.

In addition, when determining the degree of sequence identity between two amino acid sequences, one of skill in the art may take into account so-called "conservative" amino acid substitutions, which can generally be described as amino acid substitutions in which an amino acid residue is replaced by another amino acid residue with a similar chemical structure. and which has little or no effect on the function, activity, or other biological properties of the polypeptide. Such conservative amino acid substitutions are well known in the art, for example from WO 04/037999, GB 335768, WO 98/49185, WO 00/46383 and WO 01/09300; and (preferred) types and/or combinations of such substitutions may be selected on the basis of the respective disclosures in WO 04/037999 as well as WO 98/49185 and additional references cited therein.

Such conservative substitutions are preferably substitutions in which one amino acid in the following groups (a) to (e) is replaced by another amino acid residue within the same group: (a) small, non-polar or slightly polar residues: Ala, Ser, Thr , Pro and Gly; (b) polar, negatively charged residues and their (uncharged) amides: Asp, Asn, Glu and Gln; (c) polar positively charged residues: His, Arg and Lys; (d) large aliphatic, non-polar residues: Met, Leu, Ile, Val and Cys; and (e) aromatic residues: Phe, Tyr and Trp. Particularly preferred conservative substitutions are: Ala to Gly or Ser; Arg to Lys; Asn to Gln or to His; Asp to Glu; Cys to Ser; Gln to Asn; Glu to Asp; Gly on Ala or Pro; His to Asn or to Gln; Ile on Leu or on Val; Leu to Ile or to Val; Lys to Arg, to Gln or to Glu; Met on Leu, on Tyr or on Ile; Phe to Met, to Leu or to Tyr; Ser to Thr; Thr to Ser; Trp to Tyr Tyr to Trp; and/or Phe to Val, to Ile or to Leu.

Any amino acid substitutions applied to the polypeptides described herein can also be based on the analysis of amino acid variation frequencies between homologous proteins of different species developed by Schulz et al. (“Principles of Protein Structure”, Springer-Verlag, 1978), and on the analysis of structuring potentials developed by Chou and Fasman (Biochemistry 13: 211, 1974; Adv. Enzymol., 47: 45-149, 1978), and on the analysis hydrophobicity patterns in proteins developed by Eisenberg et al. (1984, Proc. Natl. Acad Sci. USA 81: 140-144), Kyte and Doolittle (1981, J. Molec. Biol. 157: 105-132), and Goldman et al. (1986, Ann. Rev. Biophys. Chem. 15: 321-353, 1986), which are all incorporated herein by reference in their entirety. Information on the primary, secondary and tertiary structure of nanobodies is given in the description in this document and in the general prior art mentioned above. In addition, the crystal structure of the V _HH domain from llama is given for this purpose, for example, in Desmyter et al. (1996, Nature Structural Biology, 3: 803), Spinelli et al. (1996, Natural Structural Biology, 3: 752-757) and Decanniere et al. (1999, Structure, 7(4): 361). Further information on the HEK amino acid residues that in conventional V _H domains form the V _H /V _L interface and potential overblown substitutions at these positions can be found in the prior art mentioned above.

Amino acid sequences and nucleic acid sequences are considered "exactly the same" if they have 100% sequence identity (as defined herein) throughout their length.

When comparing two amino acid sequences, the term "amino acid difference" refers to the insertion, deletion or substitution of one amino acid residue at the position of the first sequence compared to the second sequence; it is understood that two amino acid sequences may contain one, two or more such amino acid differences. More specifically, in the amino acid sequences and/or polypeptides of the present invention, the term "amino acid difference" refers to the insertion, deletion or substitution of one amino acid residue at the position of the CDR sequence indicated in b), d) or f) compared to the CDR sequence, respectively , a), c) or e); it should be understood that the CDR sequence b), d) and f) may contain one, two or a maximum of three such amino acid differences compared to the CDR sequence, respectively, a), c) or e).

An "amino acid difference" can be any one, two, three, or up to four substitutions, deletions, or insertions, or any combination thereof, that either improves the properties of the polypeptide of the invention, or at least does not detract too much from the desired properties or out of balance. or combinations of desired properties of a polypeptide of the invention. In this respect, the resulting polypeptide of the invention should at least bind the CD123 or T cell receptor with the same, about the same, or higher affinity as compared to a polypeptide containing one or more CDR sequences without one, two, three, or at most four substitutions, deletions or insertions, where the specified affinity is measured using surface plasmon resonance.

In this regard, the amino acid sequence according to b), d) and/or f) may be an amino acid sequence that is derived from the amino acid sequence according to a), c) and/or e), respectively, by affinity maturation using one or more affinity maturation techniques known per se.

For example, and depending on the host organism used to express the polypeptide of the invention, such deletions and/or substitutions can be engineered to remove one or more sites for post-translational modification (such as one or more glycosylation sites), which is within the competence of a person skilled in the art.

"Affinity" means the strength or stability of a molecular interaction. Affinity is usually represented as K _D , or dissociation constant, which has units of mol/liter (or M). Affinity can also be expressed as an association constant, K _A , which is equal to 1/K _D and has units of (mol/liter) ^-1 (or M ^-1 ). In the present description, the stability of the interaction between two molecules will be mainly expressed in terms of the K _D value of their interaction; it is clear to the person skilled in the art that given the ratio K _A = 1/K _D , the determination of the strength of the molecular interaction from its K _D value can also be used to calculate the corresponding value of K _A. The K _D value characterizes the strength of the molecular interaction also in a thermodynamic sense, since it is related to the change in the free energy (DG) of binding according to the well-known relation DG = RT.ln (K _D ) (which is equivalent to DG = -RT.ln (K _A )), where R is equal to the gas constant, T is equal to the absolute temperature, ln is the natural logarithm.

K _D for biological interactions that are considered significant (eg, specific) typically range from 10 ^-10 M (0.1 nM) to 10 ^-5 M (10,000 nM). The stronger the interaction, the smaller its K _D.

K _D can also be expressed as the ratio of the dissociation rate constant of the complex, denoted k _off , to its association rate constant, denoted k _on (so K _D = k _off /k _on and K _A = k _on /K _off ). The dissociation rate k _off has units of s ^-1 (where s ^is the SI designation for a second). The association rate k _on has units of M ^-1 s ^-1 . The association rate can vary from 10 ² M ^-1 s ^-1 to about 10 ⁷ M ^-1 s ^-1 , approaching the diffusion-limited association rate constant for bimolecular interactions. The dissociation rate is related to the half-life of a given molecular interaction by the ratio t _1/2 = ln(2)/k _off . The dissociation rate can vary from 10 ^-6 s ^-1 (about an irreversible complex with _1/2 from several days) to 1 s ^-1 (t _1/2 = 0.69 s).

Specific binding of an antigen-binding protein, such as ISVD, to an antigen or antigenic determinant can be determined by any suitable method known per se, including, for example, the Scatchard assay and/or competitive binding assays such as radioimmunoassays (RIA ), enzyme immunoassays (EIA) and competition sandwich assays and their various variants known per se in the art; and other methods mentioned in this document.

The affinity of a molecular interaction between two molecules can be measured using various methods known per se, such as the well-known surface plasmon resonance (SPR) biosensor method (see, for example, Ober et al. 2001, Intern. Immunology 13: 1551-1559 ). The term "Surface Plasmon Resonance" as used herein refers to an optical phenomenon that allows real-time analysis of biospecific interactions by detecting changes in protein concentrations in a biosensor array where one molecule is immobilized on a biosensor chip and another molecule passes over the immobilized molecule in flow conditions, giving k _on , k _off measurements and hence K _D (or K _A ) values. This can be done, for example, using the well known BIAcore® system (BIAcore International AB, GE Healthcare company, Uppsala, Sweden and Piscataway, NJ). For further descriptions, see Jonsson et al. (1993, Ann. Biol. Clin. 51: 19-26), Jonsson et al. (1991 Biotechniques 11: 620-627), Johnsson et al. (1995, J. Mol. Recognit. 8: 125-131) and Johnnson et al. (1991, Anal. Biochem. 198: 268-277).

Another well-known biosensor method for determining the affinity of biomolecular interactions is biolayer interferometry (BLI) (see, for example, Abdiche et al. 2008, Anal. Biochem. 377: 209-217). The term "biolayer interferometry" or "BLI", as used herein, refers to a label-free optical technique that analyzes the interference pattern of light reflected from two surfaces: an internal reference layer (reference beam) and an immobilized protein layer at the biosensor tip ( signal beam). A change in the number of molecules bound to the biosensor tip causes a shift in the interference pattern, reported as a wavelength shift (nm), the magnitude of which is a direct measure of the number of molecules bound to the surface of the biosensor tip. Because interactions can be measured in real time, rates and associations and dissociations and affinities can be determined. BLI can, for example, be performed using well-known Octet® systems (ForteBio, a division of Pall Life Sciences, Menlo Park, USA).

Alternatively, affinity can be measured in a kinetic exclusion (KinExA) assay (see e.g. Drake et al. 2004, Anal. Biochem., 328:35-43) using the KinExA® platform (Sapidyne Instruments Inc, Boise, USA). As used herein, the term "KinExA" refers to a method for measuring true equilibrium binding affinity and kinetics of unmodified molecules in solution. Equilibrated solutions of the antibody/antigen complex are passed through a column of beads pre-coated with the antigen (or antibody), which allows the free antibody (or antigen) to bind to the coating molecule. The antibody (or antigen) thus detected is captured by the fluorescently labeled protein that binds the antibody (or antigen).

The GYROLAB® Immunoassay System provides a platform for automated bioassay and rapid sample analysis (Fraley et al. 2013, Bioanalysis 5: 1765-74).

One of skill in the art will also appreciate that the measured K _D may correspond to the apparent K _D if the measurement process somehow affects the true binding affinity of the putative molecules, such as artifacts associated with applying a single molecule to the biosensor. In addition, apparent K _D can be measured if one molecule contains more than one recognition site for another molecule. In such a situation, the measured affinity can be affected by the avidity of the interaction between the two molecules. As will be appreciated by one of skill in the art, and as described on pages 53-56 of WO 08/020079, the dissociation constant may be an actual or apparent dissociation constant. Methods for determining the dissociation constant are known to those skilled in the art, which, for example, include the methods mentioned on pages 53-56 of WO 08/020079.

The terms "epitope" and "antigenic determinant", which may be used interchangeably, refer to the part of a macromolecule, such as a polypeptide or protein, that is recognized by antigen-binding molecules such as immunoglobulins, conventional antibodies, immunoglobulin single variable domains and/or polypeptides of the invention and, in particular, the antigen-binding site of said molecules. Epitopes define the minimal binding site of an immunoglobulin and thus represent the target of immunoglobulin specificity.

The portion of an antigen-binding molecule (such as an immunoglobulin, a conventional antibody, an immunoglobulin single variable domain, and/or a polypeptide of the invention) that recognizes an epitope is referred to as a "paratope".

A polypeptide (such as an immunoglobulin, an antibody, an immunoglobulin single variable domain, a polypeptide of the invention, or usually an antigen-binding molecule or fragment thereof) that can "bind (to)" or "specifically bind (to)" that which "has affinity" and/ or "has specificity" for a particular epitope, antigen, or protein (or at least one part, fragment, or epitope thereof), is said to be "against" or "directed against" the specified epitope, antigen, or protein, or is a "binding" molecule with respect to such an epitope, antigen or protein, or is said to be an "anti" epitope, "anti" antigen, or "anti" protein (eg, "anti"-CD123 or "anti"-TCR).

The term "specificity" has the meaning given to it in paragraph n) on pages 53-56 in WO 08/020079; and as mentioned herein refers to the number of different types of antigens or antigenic determinants to which a particular antigen-binding molecule or antigen-binding protein (such as an immunoglobulin single variable domain and/or polypeptide of the invention) can bind. The specificity of an antigen-binding protein can be determined based on affinity and/or avidity, as described on pages 53-56 in WO 08/020079 (incorporated herein by reference), which also describes some preferred methods for measuring binding between an antigen-binding molecule ( such as an immunoglobulin single variable domain and/or a polypeptide of the invention) and a corresponding antigen. Typically, antigen-binding proteins (such as immunoglobulin single variable domains and/or polypeptides of the invention) will bind to their antigen with a dissociation constant (K _D ) of 10 ^-5 to 10 ^-12 mol/l or less and preferably 10 ^-7 up to 10 ^-12 mol/liter or less, and more preferably 10 ^-8 to 10 ^-12 mol/liter (i.e., with an association constant (K _A ) of 10 ⁵ to 10 ¹² liters/mol or more, and preferably 10 ⁷ up to 10 ¹² l/mol or more, and more preferably from 10 ⁸ to 10 ¹² l/mol). Generally, any K _D value greater than 10 ⁴ mol/liter (or any K _A value below 10 ⁴ M ^-1 ) is considered to indicate non-specific binding. Preferably, the monospecific polypeptide of the invention will bind to the antigen of interest with an affinity of less than 500 nM, preferably less than 200 nM, more preferably less than 10 nM, for example, for example, from 10 to 5 nM, for example, less than 10 nM. less than 5 nM, less than 3 nM, less than 2 nM, for example, 10 nM - 1 nM, 5 nM - 1 nM or even less. The specific binding of an antigen-binding protein to an antigen or antigenic determinant may be determined by any suitable method known per se, including, for example, Scatchard and/or competitive binding assays such as radioimmunoassays (RIA), enzyme-linked immunosorbent assays (ELISA), and sandwich assays of competition, as well as various variations thereof known per se in the art; and other methods mentioned in this document. As will be appreciated by one of skill in the art, and as described on pages 53-56 of WO 08/020079, the dissociation constant may be an actual or apparent dissociation constant. Methods for determining the dissociation constant are known to those skilled in the art and, for example, include the methods mentioned on pages 53-56 of WO 08/020079.

One approach that can be used to assess affinity is the two-step ELISA (enzyme-linked immunosorbent assay) procedure from Friguet et al. (1985, J. Immunol. Methods 77: 305-19). This method establishes a solution phase binding equilibrium measurement and avoids possible artifacts associated with the adsorption of one of the molecules on a carrier such as a plastic.

However, accurate measurement of K _D can be quite laborious, and as a result, often apparent K _D values are determined to estimate the binding strength of two molecules. It should be noted that, since all measurements are performed in a consistent manner (e.g., keeping the analysis conditions unchanged), apparent K _D measurements can be used as an approximation to the true K _D and, therefore, in this document, K _D and apparent K _D should be considered with equal importance. or relevance.

Finally, it should be noted that in many situations an experienced scientist can judge the convenience of determining the binding affinity relative to some reference molecule. For example, to assess the strength of the binding between molecules A and B, one can, for example, use a control molecule C which is known to bind to B and which is appropriately labeled with a fluorophore or chromophore group or other chemical moiety such as biotin for easy detection in ELISA or FACS (fluorescence activated cell sorting) or other format (fluorophore for fluorescence detection, chromophore for light absorption detection, biotin for streptavidin-mediated ELISA detection). Typically, the reference molecule C is kept at a fixed concentration and the concentration of A is changed for a given concentration or amount of B. The result is an IC ₅₀ value corresponding to the concentration of A, at which the signal measured for C in the absence of A is halved. Provided that K _{D ref} , K _D of the reference molecule, is known as well as the total concentration c _ref of the reference molecule, the apparent K _D for the AB interaction can be obtained from the following formula: K _D = IC ₅₀ /(1 + c _ref /K _Dref ). Note that if c _ref << K _{D ref} , K _D ≈ IC ₅₀ . Provided that the IC ₅₀ measurement is performed in a consistent manner (e.g., keeping c _ref fixed) for the compared binders, the IC ₅₀ can evaluate the strength or stability of the molecular interaction, and this measurement is evaluated as equivalent to K _D or apparent K _D throughout the text.

The half-maximal inhibitory concentration (IC ₅₀ ) is a measure of the effectiveness of a compound in inhibiting a biological or biochemical function, such as a pharmacological effect. This quantitative measure indicates how much ISV (e.g., nanobodies) (inhibitor) is needed to inhibit a given biological process (or process component, i.e. enzyme, cell, cell receptor, chemotaxis, anaplasia, metastasis, invasiveness, etc.) half. In other words, it is half the maximum (50%) inhibitory concentration (IC) of the substance (50% IC or IC ₅₀ ). The IC ₅₀ of a drug can be determined by plotting a dose-response curve and examining the effect of different concentrations of an antagonist, such as an ISVD (eg, nanobody) of the invention, on the change in agonist activity. IC ₅₀ values can be calculated for a given antagonist, such as an ISVD (eg, nanobodies) of the invention, by determining the concentration required to inhibit half of the agonist's maximum biological response.

The term half maximum effective concentration (EC ₅₀ ) refers to the concentration of a compound that elicits a response halfway between baseline and maximum after a specified exposure time. In the present context, it is used as a measure of the effectiveness of a polypeptide, ISV (eg, nanobodies). The EC _{50 of} the graduated dose-response curve represents the concentration of a compound at which 50% of its maximum effect is observed. The concentration is preferably expressed in molar units.

In biological systems, small changes in ligand concentration usually result in rapid changes in response, following a sigmoidal function. The inflection point of the curve, at which the increase in reaction with increasing ligand concentration begins to slow down, is the EC ₅₀ . This can be determined mathematically by deriving the best fitting line. Relying on a graph for evaluation is convenient in most cases. In the case where EC ₅₀ is given in the examples section, the experiments were designed to reflect K _D as accurately as possible. In other words, the EC ₅₀ values can then be considered as K _D values.

This also applies to IC ₅₀ , which is a measure of the inhibition of a compound (50% inhibition). For competitive binding assays and functional antagonist assays, IC ₅₀ is the most common summary measure of the dose-response curve. For agonist/stimulant assays, the most common summary measure is the EC ₅₀ .

The inhibitory constant, Ki, is a measure of how strong an inhibitor is; this is the concentration required to obtain half of the maximum inhibition. The absolute inhibition constant K _i can be calculated using the Cheng-Prusoff equation:

,

,

in which [L] is a fixed ligand concentration.

An immunoglobulin single variable domain and/or polypeptide is said to be "specific" for a first target or antigen compared to a second target or antigen when it binds to the first antigen with an affinity (described above, and respectively expressed as a K _D value, K _A , speed k _off and/or speed k _on ) which is at least 10 times, for example at least 100 times, and preferably at least 1000 times, and up to 10000 times or even better than the affinity with which the immunoglobulin single variable domain and/or polypeptide binds to the second target or antigen. For example, an immunoglobulin single variable domain and/or polypeptide can bind to a first target or antigen with a K _D value that is at least 10 times smaller, such as at least 100 times smaller, and preferably at least , 1000 times less, for example, 10000 times less or even less than the K _D with which said immunoglobulin single variable domain and/or polypeptide binds to the second target or antigen. Preferably, if the immunoglobulin single variable domain and/or polypeptide is "specific for" the first target or antigen as compared to the second target or antigen, then it is directed against (as defined herein) said first target or antigen, but is not directed against said first target or antigen. second target or antigen.

An amino acid sequence, such as, for example, an immunoglobulin single variable domain or a polypeptide of the invention, is considered to be "cross-reactive" to two different antigens or antigenic determinants (such as, for example, serum albumin from two different mammalian species), such as , for example, human serum albumin and cynomolgus monkey serum albumin, such as, for example, CD123 from different mammalian species, such as, for example, human CD123, and cynomolgus monkey CD123, such as, for example, TCR from different mammalian species, such as, for example, human TCR and cynomolgus TCR) if it is specific (as defined herein) for these different antigens or antigenic determinants.

The terms "(cross) blocking", "(cross) blocking", "(cross) blocking", "competitive linking", "(cross)-compete", "(cross)-compete" and "(cross) competition" are used interchangeably herein, means the ability of an immunoglobulin, antibody, immunoglobulin single variable domain, polypeptide, or other binding agent to interfere with the binding of other immunoglobulins, antibodies, immunoglobulin single variable domains, polypeptides, or binding agents to a given target. The extent to which an immunoglobulin, antibody, immunoglobulin single variable domain, polypeptide, or other binding agent is able to interfere with the binding of another to a target, and therefore whether it can be said to be cross-blocked in accordance with the invention, can be determined using competitive assays. One particularly suitable quantitative cross-blocking assay is described in the examples and includes, for example, a fluorescent activated cell sorting (FACS) binding assay with CD123 expressed on cells. The degree of (cross) blocking can be measured by the (reduced) channel fluorescence. Another suitable cross-blocking quantification uses the Biacore instrument, which can measure the extent of interactions using surface plasmon resonance technology. Another suitable quantitative cross-blocking assay uses an ELISA-based approach to measure competition between immunoglobulins, antibodies, immunoglobulin single variable domains, polypeptides, or other binding agents for their binding to a target.

The following generally describes a suitable FACS assay to determine if it cross-blocks an immunoglobulin, antibody, immunoglobulin single variable domain, polypeptide, or other binding agent or is capable of cross-blocking in accordance with the invention. It is understood that the assay can be used with any of the immunoglobulins, antibodies, immunoglobulin single variable domains, polypeptides, or other binding agents described herein. The FACS instrument (eg FACSArray; Becton Dickinson) operates in accordance with the manufacturer's recommendations.

To evaluate "(cross) blocking" or "(cross) competition" between two binding agents (such as, for example, two immunoglobulin single variable domains and/or nanobodies) for binding to CD123, a competitive FACS experiment can be performed using cells (such as such as endogenously CD123-expressing MOLM-13 cell lines or Flp-In™-CHO cells overexpressing human CD123). Various reagents can be used for detection, including, for example, anti-ANTI-FLAG® M2 monoclonal antibody (Sigma-Aldrich, cat. no. F1804), anti-C-myc monoclonal antibody (Sigma-Aldrich, cat. no. WH0004609M2), ANTI monoclonal antibody -HIS TAG (Sigma -Aldrich, cat. no. SAB1305538), each of which is labeled differently. A wide range of fluorophores can be used as labels in flow cytometry (e.g., PE (R-phycoerythrin), 7-aminoactinomycin D (7-AAD), acridine orange, various forms of Alexa Fluor (such as, for example, Alexa647), allophycocyanin (APC ), AmCyan, Aminocoumarin, APC Cy5, APC Cy7, APC-H7, APC/Alexa Fluor 750, AsRed2, Azami-Green, Azurite, B ODIPY FL C5-ceramide, BCECF-AM, DiBAC2(3) bis-oxonol, BODIPY -FL, Calcein, Calcein AM, Caroxy-H2DCFDA, Cascade Blue, Cascade Yellow, Cell Tracker Green, Cerulean, CFSE, Chromomycin A3, CM-H2DCFDA, Cy2, Cy3, Cy3.5, Cy3B, Cy5, Cy5.5, Cy7 , CyPet, DAF-FM, DAF-FM diacetate, DAPI, DCFH (2'7'-dichorodihydrofluorescein), DHR, dihydrocalcein AM, dihydrorhoadamin, dihydrotidium, DiLC1 (5), DiOC6 (3), DiOC7 (3), dOCe- Red, DRAQ5, Dronpa-Green, various forms of DsRed dTomato, various forms of DyLight, E.coli BioParticles AF488, E2-Crimson, E2-Orange, EBFP2, ECFP, various forms of eFluor, EGFP, EGFP*, Emerald, eqFP650, eqFP670, ER-Tracker Blue-White DPX, br ethidium omide, Express2, EYFP, Fc OxyBurst Green, Fc OxyBurst Green 123, FITC, Fluo-3, Fluo-4, Fluorescein, Fura-2, Fura-Red, GFPuv, H2DCFDA, HcRed1, Hoechst Blue (33258), Hoechst Red (33342), Hydroxycoumarin, HyPer, Indo-1, Indo-1 Blue (low Ca2+), Indo-1 Violet (high Ca2+), iRFP, J-Red, JC-1, JC-9, Katushka (TurboFP635) , Katushka2 Kusabira-Orange, LDS 751, Lyssamine Rhodamine B, Live/Dead various forms, Lucifer yellow, Lucifer Yellow CH, Lyso Tracker Blue, Lyso Tracker Green, Lyso Tracker Red, mAmertrine, Marina Blue, mBanana, mCFP, mCherry, mCitrine , methoxycoumarin, mHoneyDew, Midoriishi-Cyan, mithramycin, Mito Tracker Deep Red, Mito Tracker Green, Mito Tracker Orange, Mito Tracker Red, MitoFluor Green, mKate (TagFP635), mKate2, mKeima, mKeima-Red, mKO, mKOk, mNeptune, monochlorobiman, mOrange2, mRaspberry, mPlum, mRFP1, mStrawberry, mTangerine, mTarquoise, mTFP1, mTFP1 (Teal), NBD, OxyBurst Green H2DCFDA, OxyBurst Green H2HFF BSA, Pacific Blue, PE (R-phycoerythrin), Cy5, PE 5, Cy5 NP Cy7, PE Texas Red, PE-Cy5 conjugates, PE-Cy7 conjugates, PerCP (peridinine-chlorophyll protein), PerCP Cy5.5, PhiYFP, PhiYFP-m, propidium iodide (PI), various forms of Qdot, Red 613, Tomato RFP, Rhod-2, S65A, S65C, S65L, S65T, Singlet Oxygen Sensor Green, Sirius, SNARF, Superfolder GFP, SYTOX Blue, SYTOX Green, SYTOX Orange, T-Sapphire, TagBFP, TagCFP, TagGFP, TagRFP, TagRFP657, TagYFP, tdTomato, Texas Red, Thiazole Orange, TMRE, TMRM, Topaz, TOTO-1, TO-PRO-1, TRITC, TRITC TruRed, TurboFP602, TurboFP635, TurboGFP, TurboRFP, TurboYFP, Venus, Vybrant CycleDye Violet, GFP wild-type, X-Rhodamine, Y66F, Y66H, Y66W, YOYO-1, YPet, ZsGreen1, ZsYellow1, Zymosan A BioParticles AF488 (see more at: http://www.thefcn.org/flow-fluorochromes). Fluorophores or simply "fluorescent agents" are typically attached to an antibody (eg, immunoglobulin single variable domains such as nanobodies) that recognizes CD123 or to an antibody that is used as a detection reagent. Various conjugated antibodies are available, such as, but not limited to, for example, Alexa Fluor®, DyLight®, rhodamine, PE, FITC, and Cy3 conjugated antibodies. Each fluorophore has a characteristic excitation peak and emission wavelength. The combination of labels that can be used will depend on the wavelength of the lamp(s) or laser(s) used to excite the fluorophore and the available detectors.

To assess competition between the two test binding agents (named A and B*) for binding to CD123, a dilution series of cold (without any label) binding agent A is added to cells (e.g. 100,000) along with labeled binding agent B* . The concentration of B* in the test mixture should be high enough to easily saturate the binding sites on CD123 expressed on the cells. The concentration of B* binding agent that saturates binding sites for that binding agent on CD123 expressed on cells can be determined by a series of B* titrations on cells expressing CD123 and determining the EC ₅₀ value for binding. For saturating concentration operation, Coupling Agent B* at 100 times the EC ₅₀ concentration can be used.

After incubating the cells with a mixture of A and B* and washing the cells, a FACS reading can be performed. First, selection frames are set on intact cells, determined by the scattering profile, and the total amount of channel fluorescence is recorded.

A separate solution of binding agent B* is also prepared. The binding agent in these solutions must be in the same buffer and at the same concentration as in the test mixture (with binding agents A and B*). This separate solution is also added to the cells. After incubation and cell washing, readings can be performed on FACS. First, selection frames are set on intact cells, determined by the scattering profile, and the total amount of channel fluorescence is recorded.

Decreased fluorescence for cells incubated with a mixture of A and B* compared to fluorescence for cells incubated with B* solution alone indicates that binding agent A (cross) blocks binding to binding agent B* when binding to CD123, expressed on cells.

The cross-blocking immunoglobulin, antibody, immunoglobulin single variable domain, polypeptide, or other binding agent of the invention is one that will bind to CD123 in the aforementioned FACS cross-blocking assay, so that during the assay and in the presence of a second immunoglobulin, antibody, immunoglobulin single variable domain, polypeptide, or other binding agent, the detectable fluorescence is 80% to 0.1% (e.g., 80% to 4%) of the maximum fluorescence (measured for a single labeled immunoglobulin, antibody, immunoglobulin single variable domain, polypeptide, or other binding agent), in particular from 75% to 0.1% (for example, from 75% to 4%) of the maximum fluorescence, and more specifically from 70% to 0.1% (for example, from 70% to 4% ) from maximum fluorescence (as just defined above).

Competition between two test binding agents (referred to as A* and B*) for binding to CD123 can also be assessed by adding both binding agents, each with a different fluorophore, to cells expressing CD123. After incubation and cell washing, FACS readings can be performed. Detection frames are set for each fluorophore and the total amount of fluorescence in the channel is recorded. Reduction and/or absence of fluorescence of one of the fluorophores indicates (cross) blocking by binding agents to bind to CD123 expressed on cells.

The following generally describes a suitable Biacore assay for determining whether an immunoglobulin, antibody, immunoglobulin single variable domain, polypeptide, or other binding agent is cross-linked or cross-blockable in accordance with the invention. It is understood that the assay can be used with any of the immunoglobulins, antibodies, immunoglobulin single variable domains, polypeptides, or other binding agents described herein. The Biacore instrument (eg Biacore 3000) is operated according to the manufacturer's recommendations. Thus, in one cross-blocking assay, a target protein (eg, CD123) is coupled to the Biacore CM5 chip using standard amine group coupling to produce a surface that is coated with the target. Typically, 200-800 target resonant units will be bound to the chip (an amount that gives easily measurable levels of binding, but which is easily saturable by the concentrations of the test reagent used). The two binding agents to be tested (named A* and B*), to be evaluated for their ability to cross-block each other, are mixed at a one to one molar ratio of binding sites in an appropriate buffer to create a test mixture. When calculating concentrations based on binding sites, it is assumed that the molecular weight of the binding agent is equal to the total molecular weight of the binding agent divided by the number of target binding sites on that binding agent. The concentration of each binding agent in the test mixture should be high enough to readily saturate the binding sites of that binding agent to the target molecules captured on the Biacore chip. the binding agents in the mixture have the same molar concentration (based on binding), and this concentration is usually from 1.00 to 1.5 micromolar (based on the binding site). Separate solutions containing only A* and B* are also prepared. A* and B* in these solutions must be in the same buffer and at the same concentration as in the test mixture. The test mixture is passed through the target-coated Biacore chip and the total number of bindings is recorded. The chip is then processed in such a way as to remove the associated binding agents without damaging the target associated with the chip. This is usually done by treating the chip with 30 mM HCl for 60 seconds. Solution A* is then passed through the target-coated surface and the number of bindings recorded. The chip is processed again to remove all associated binding agents without damaging the target associated with the chip. Solution B* is then passed through the target-coated surface and the amount of binder is recorded. Next, the maximum theoretical binding of a mixture of A* and B* is calculated, which is the sum of the binding of each binding agent when passing only on the target surface. If the actual recorded binding of the mixture is less than this theoretical maximum, then the two binding agents are said to cross-block each other. Thus, in general, the cross-blocking immunoglobulin, antibody, immunoglobulin single variable domain, polypeptide, or other binding agent of the invention is one that will bind to the target in the above Biacore cross-blocking assay, so that during the assay and in the presence of a second immunoglobulin, antibody, immunoglobulin single variable domain, polypeptide, or other binding agent, reported binding is 80% to 0.1% (e.g., 80% to 4%) of the maximum theoretical binding, in particular, 75% to 0.1 %. (e.g., 75% to 4%) of the maximum theoretical binding, and more specifically, 70% to 0.1% (e.g., 70% to 4%) of the maximum theoretical binding (as just defined above) of the two immunoglobulins , antibodies, immunoglobulin single variable domains, polypeptides, or binding agents in combination. The Biacore assay described above is a primary assay used to determine whether immunoglobulins, antibodies, immunoglobulin single variable domains, a polypeptide, or other binding agents cross-block each other in accordance with the invention. In rare cases, certain immunoglobulins, antibodies, immunoglobulin single variable domains, polypeptides, or other binding agents may not bind to a target chemically bonded with an amine to the CM5 Biacore chip (this usually occurs when the appropriate binding site on the target is masked or the bond to the chip is destroyed ). In such cases, cross-blocking can be determined using a labeled version of the target, for example, a version labeled with a His tag at the N-terminus. In this particular format, the anti-His antibody will bind to the Biacore chip and then the His-tagged target will pass over the surface of the chip and be captured by the anti-His antibody. The cross-blocking assay should be carried out essentially as described above, except that after each chip refresh cycle, a new His-tagged target will be loaded back onto the anti-His coated surface. In addition to the example given using an N-terminally His-tagged target, a C-terminally His-tagged target may be used as an alternative. In addition, various other labels and combinations of label-binding proteins that are known in the art can be used for such a cross-blocking assay (e.g., HA label with anti-HA antibodies; FLAG label with anti-FLAG antibodies; biotin label with streptavidin).

The following generally describes an ELISA assay to determine whether an immunoglobulin, antibody, immunoglobulin single variable domain, polypeptide, or other anti-target binding agent (e.g., CD123) is cross-linked or cross-blockable as defined herein. . It is understood that the assay can be used with any of the immunoglobulins, antibodies, immunoglobulin single variable domains, polypeptides, or other binding agents described herein. The general principle of the assay is to have an immunoglobulin, antibody, immunoglobulin single variable domain, polypeptide, or binding agent that is directed against a target coated on the wells of an ELISA plate. An excess amount of a second, potentially cross-blocking immunoglobulin, antibody, immunoglobulin single variable domain, polypeptide, or other anti-target binding agent is added to the solution (ie, not bound to the ELISA plate). A limited amount of target is then added to the wells. The immunoglobulin, antibody, immunoglobulin single variable domain, polypeptide or other binding agent of the coating and the immunoglobulin, antibody, immunoglobulin single variable domain, polypeptide or other binding agent in solution compete to bind a limited number of target molecules. The plate is washed to remove excess target that has not been bound by an immunoglobulin, antibody, immunoglobulin single variable domain, polypeptide, or other binding agent, and to remove a second dissolved immunoglobulin, antibody, immunoglobulin single variable domain, polypeptide, or other binding agent, and any a complex formed between a second dissolved immunoglobulin, antibody, immunoglobulin single variable domain, polypeptide, or other binding agent, and a target. The amount of bound target is then measured using a reagent that is suitable for detecting the target. An immunoglobulin, antibody, immunoglobulin single variable domain, polypeptide, or other binding agent in solution that is capable of cross-blocking the coated immunoglobulin, antibody, immunoglobulin single variable domain, polypeptide, or other binding agent can cause a reduction in the number of target molecules that the coated immunoglobulin, antibody, immunoglobulin single variable domain, polypeptide, or other binding agent can bind relative to the number of target molecules that the immunoglobulin, antibody, immunoglobulin single variable domain, polypeptide, or other coating binding agent can bind in the absence of a second dissolved immunoglobulin, antibody, immunoglobulin single variable domain , polypeptide or other binding agent. When the first immunoglobulin, antibody, immunoglobulin single variable domain, polypeptide, or other binding agent, such as Ab-X, is selected as the immobilized immunoglobulin, antibody, immunoglobulin single variable domain, polypeptide, or other binding agent, it is applied to the wells plate for ELISA, after which the plates are blocked with an appropriate blocking solution to minimize non-specific binding of reagents that are added subsequently. An excess of a second immunoglobulin, antibody, immunoglobulin single variable domain, polypeptide, or other binding agent, i. Ab-Y is then added to the ELISA plate such that the number of moles of Ab-Y target binding sites per well is at least 10 times greater than the number of moles of Ab-X target binding sites that were used per well when ELISA plate coating. The target is then added such that the number of moles of target added per well is at least 25 times lower than the number of moles of Ab-X target binding sites that were used to coat each well. After a suitable incubation period, the ELISA plate is washed and a target detection reagent is added to measure the amount of target specifically bound to the coated immunoglobulin, antibody, immunoglobulin single variable domain, polypeptide or other anti-target binding agent (in this case Ab-X). Assay background signal is defined as the signal produced in wells with an immunoglobulin, antibody, immunoglobulin single variable domain, polypeptide or other binding agent coating (in this case Ab-X), a second immunoglobulin single variable domain, polypeptide or other binding agent in the liquid phase (in this case Ab-Y), target buffer only (i.e., no target), and target detection reagents. A positive control signal for assay is defined as the signal generated in wells with an immunoglobulin, antibody, immunoglobulin single variable domain, polypeptide, or other coating binding agent (in this case, Ab-X), buffer only for a second immunoglobulin, antibody, immunoglobulin single variable domain, a polypeptide or other binding agent in the liquid phase (ie, without a second immunoglobulin, antibody, immunoglobulin single variable domain, polypeptide or other binding agent), a target, and reagents for determining the target. The ELISA assay can be run such that the positive control signal is at least 6 times the background signal. To avoid any artifacts (eg, significantly different target affinities between Ab-X and Ab-Y) resulting from the choice of which immunoglobulin, antibody, immunoglobulin single variable domain, polypeptide, or other binding agent is used as the coating immunoglobulin , antibody, immunoglobulin single variable domain, polypeptide or other binding agent, which immunoglobulin, antibody, immunoglobulin single variable domain, polypeptide or other binding agent is used as a second (competitive), cross-blocking assay can be performed in two formats: 1) format 1 , wherein Ab-X is an immunoglobulin, antibody, immunoglobulin single variable domain, polypeptide, or other binding agent that is applied to the ELISA plate, and wherein Ab-Y is a competitive immunoglobulin, antibody, immunoglobulin single variable domain , polypeptide, or other binding agent that is in solution, and 2) format 2, where Ab-Y is an immunoglobulin, antibody, immunoglobulin single variable domain, polypeptide, or other binding agent that is applied to the ELISA plate, and Ab-X is a competitive immunoglobulin, antibody, immunoglobulin single variable domain, polypeptide, or other binding agent that is in solution. Ab-X and Ab-Y are defined as cross-blocking if, in format 1 or format 2, an anti-target immunoglobulin, antibody, immunoglobulin single variable domain, polypeptide, or other binding agent in the liquid phase is capable of causing a reduction from 60% to 100 %, specifically from 70% to 100%, and more specifically from 80% to 100% of the target detection signal (i.e., the amount of target associated with a coating immunoglobulin, antibody, immunoglobulin single variable domain, polypeptide, or other binding agent) compared to a target detection signal obtained in the absence of an immunoglobulin, antibody, immunoglobulin single variable domain, polypeptide, or other binding agent directed against the target in the liquid phase (ie, in the positive control wells).

Other methods for determining whether an immunoglobulin, antibody, immunoglobulin single variable domain, polypeptide, or other binding agent directed against a target is (cross)-blocking, (cross) blocking capable, competitively binding, or is (cross)-competing, as defined in this document are described, for example, in Xiao-Chi Jia et al. (2004, Journal of Immunological Methods 288: 91–98), Miller et al. (2011, Journal of Immunological Methods 365: 118-125) and/or in the methods described herein (see, for example, example 7).

As used herein, the term "CD123" refers to the α-subunit of the interleukin 3 receptor (IL-3R).

As used herein, the term "TCR" refers to the T-cell receptor, which consists of TCRα and TCRβ chains. Both α and β TCR chains consist of a constant domain and a variable domain. The polypeptides and immunoglobulin single variable domains of the present invention bind to the TCR constant domain.

The "half-life" of a polypeptide of the invention can generally be defined as described in point o) on page 57 of WO 08/020079 and, as mentioned therein, refers to the time taken for the serum concentration of the polypeptide to decrease in vivo by 50%, for example, due to degradation of the polypeptide and/or clearance or sequestration of the polypeptide by natural mechanisms. The in vivo half-life of a polypeptide of the invention can be determined by any known method, such as pharmacokinetic analysis. Suitable methods will be understood by a person skilled in the art and may, for example, generally be as described in point o) on page 57 in WO 08/020079. As also mentioned in o) on page 57 of WO 08/020079, the half-life can be expressed using parameters such as t1/2-alpha, t1/2-beta and area under the curve (AUC). We hereby refer, for example, to standard reference books such as Kenneth et al. (1986, Chemical Stability of Pharmaceuticals: A Handbook for Pharmacists, John Wiley & Sons Inc) and M Gibaldi and D Perron (1982, Pharmacokinetics, Marcel Dekker, 2nd Rev. ^Ed ., 1982). The terms "increase in half-life" or "increased half-life" are also defined in paragraph o) on page 57 of WO 08/020079 and specifically refer to an increase in t1/2-β, either with or without an increase in t1/2- α and/or AUC or both.

Unless otherwise indicated, the terms "immunoglobulin" and "immunoglobulin sequence" as used herein to refer to a heavy chain antibody or conventional 4-chain antibody are used as a generic term to include both the full length antibody and their individual chains, and also all parts, domains, or fragments thereof (including, but not limited to, antigen-binding domains or fragments such as V _HH domains or V _H /V _L domains, respectively).

As used herein, the term "domain" (of a polypeptide or protein) refers to a complex protein structure that has the ability to maintain its tertiary structure independently of the rest of the protein. Typically, domains are responsible for discrete functional properties of proteins and in many cases can be added, removed, or transferred to other proteins without losing the function of the remainder of the protein and/or domain.

As used herein, the term "immunoglobulin domain" refers to the globular region of an antibody chain (such as, for example, a conventional 4-chain or heavy chain antibody chain) or a polypeptide that essentially consists of such a globular region. Immunoglobulin domains are characterized in that they retain the immunoglobulin folding characteristic of antibody molecules, which consists of a bilayer sandwich of about seven antiparallel beta strands arranged in two beta sheets, optionally stabilized by a conserved disulfide bond.

As used herein, the term "immunoglobulin variable domain" means an immunoglobulin domain essentially consisting of four "framework regions", which are referred to in this field and hereinafter as "framework region 1" or "FR1"; as "wireframe 2" or "FR2"; as "wireframe 3" or "FR3"; and as "frame region 4" or "FR4", respectively; wherein the framework regions are interrupted by three "complementarity determining regions" or "CDRs", which are referred to in this area and below as "complementarity determination region 1" or "CDR1"; as "complementarity detection region 2" or "CDR2"; and as "complementarity detection region 3" or "CDR3", respectively. Thus, the general structure or sequence of an immunoglobulin variable domain can be specified as follows: FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4. It is the variable domain (or domains) of an immunoglobulin that confers specificity of an antibody to an antigen because it contains an antigen-binding site.

The term "immunoglobulin single variable domain", used interchangeably with "single variable domain", defines molecules in which an antigen binding site is present and constituted by a single immunoglobulin domain. This sets immunoglobulin single variable domains apart from "regular" immunoglobulins or fragments thereof, in which two immunoglobulin domains, in particular two variable domains, interact to form an antigen-binding site. Typically, in conventional immunoglobulins, the heavy chain variable domain (VH) and the light chain variable domain (VL) interact to form an antigen binding site. In this case, complementarity determining regions (CDRs) of both VH and VL will contribute to the creation of an antigen binding site, ie. a total of 6 CDRs will be involved in creating the antigen binding site.

In view of the above definition, the antigen-binding domain of a conventional 4-chain antibody (such as an IgG, IgM, IgA, IgD, or IgE molecule; known in the art) or a Fab fragment, an F(ab')2 fragment, an Fv fragment, such as a disulfide-linked Fv either an scFv fragment or a diabody (all known in the art) derived from such a conventional 4 chain antibody are generally not considered an immunoglobulin single variable domain because, in these cases, binding to the corresponding antigen epitope is usually not a single (single) an immunoglobulin domain, but a pair of (associated) immunoglobulin domains such as light and heavy chain variable domains, i. using a VH-VL pair of immunoglobulin domains that co-bind to an epitope of the corresponding antigen.

In contrast, immunoglobulin single variable domains are able to specifically bind to an antigen epitope without pairing with an additional immunoglobulin variable domain. The immunoglobulin single variable domain binding site is formed by a single VH/V _HH or VL domain. Therefore, the antigen binding site of an immunoglobulin single variable domain is formed by no more than three CDRs.

Thus, a single variable domain may be a light chain variable region sequence (eg, a VL sequence) or a suitable fragment thereof; or a heavy chain variable region sequence (eg, VH sequence or VHH sequence) or a suitable fragment thereof; since such a domain is capable of forming a single antigen-binding unit (i.e., a functional antigen-binding unit that essentially consists of a single variable domain such that one antigen-binding domain does not need to interact with another variable domain to form a functional antigen-binding unit).

In one aspect of the invention, the immunoglobulin single variable domains are heavy chain variable domain sequences (eg, VH sequence); more specifically, the immunoglobulin single variable domains can be heavy chain variable domain sequences that are derived from a conventional four-chain antibody, or heavy chain variable domain sequences that are derived from a heavy chain-only antibody.

For example, an immunoglobulin single variable domain can be a (single) domain antibody (or an amino acid suitable for use as a (single) domain antibody), a "dAb" or a dAb (or an amino acid suitable for use as a dAb), or a nanobody. (as defined herein and including, without limitation, V _HH ); other single variable domains, or any suitable fragment of any of them.

In particular, the immunoglobulin single variable domain may be Nanotel® (as defined herein) or a suitable fragment thereof. [Note: Nanobody®, Nanobody® and Nanoclone® are registered trademarks of Ablynx N.V.]. For a general description of Nanotel, we hereby refer to the following description below, as well as to the prior art referred to in this document, for example, described in WO 08/020079 (p. 16).

The "V _HH domains", also known as V _HH , V _H H domains, V _HH antibody fragments, and V _HH antibodies, were originally described as the antigen-binding immunoglobulin (variable) domain of "heavy chain-only antibodies" (t ie "antibodies lacking light chains", Hamers-Casterman et al Nature 363: 446-448, 1993). The term "V _HH domain" was chosen to distinguish these variable domains from the heavy chain variable domains that are present in conventional 4 chain antibodies (referred to herein as "V _H domains" or "VH domains") and from light chain variable domains that are present in conventional 4 chain antibodies (referred to herein as "V _L domains" or "VL domains"). For a further description of V _HH and Nanotel, see the review article Muyldermans (Reviews in Molecular Biotechnology 74: 277-302, 2001), as well as the following patent applications, which are cited as prior art: WO 94/04678, WO 95/04079 and WO 96/34103 from Vrije Universiteit Brussel; WO 94/25591, WO 99/37681, WO 00/40968, WO 00/43507, WO 00/65057, WO 01/40310, WO 01/44301, EP 1134231 and WO 02/48193 from Unilever; WO 97/49805, WO 01/21817, WO 03/035694, WO 03/054016 and WO 03/055527 from Vlaams Instituut voor Biotechnologie (VIB); WO 03/050531 from Algonomics NV and Ablynx NV; WO 01/90190 from the National Research Council of Canada; WO 03/025020 (= EP 1433793) from the Antibody Institute; and WO 04/041867, WO 04/041862, WO 04/041865, WO 04/041863, WO 04/062551, WO 05/044858, WO 06/40153, WO 06/079372, WO 06/122786, WO 086/1227 and WO 06/122825 from Ablynx NV and further published patent applications from Ablynx NV. We also hereby refer to the additional prior art mentioned in these applications and, in particular, to the list of references mentioned on pages 41-43 of International Application WO 06/040153, the list and sources of which are incorporated herein by reference. As described in these references, Nanobodies (in particular, V _HH sequences and partially humanized Nanobodies) in particular can be characterized by the presence of one or more "characteristic residues" in one or more framework sequences. Further description of nanobodies, including humanization and/or overblending of nanobodies, as well as other modifications, parts or fragments, derivatives or "nanobody fusion molecules", multivalent constructs (including HEK which are non-limiting examples of linker sequences), and various modifications to increase the half-life of nanobodies and their compositions with them, can be found, for example, in WO 08/101985 and WO 08/142164. For a further general description of Nanobodies, we hereby refer to the prior art cited herein, such as, for example, described in WO 08/020079 (p. 16).

"Domain Antibodies", also known as "Dab", "Domain Antibodies" and "dAb" (the terms "Domain Antibodies" and "dAb", used as trademarks by the GlaxoSmithKline group) have been described, for example, in EP 0368684, Ward et al. (1989, Nature 341: 544-546), Holt et al. (Tends in Biotechnology 21: 484-490, 2003) and WO 03/002609, as well as, for example, WO 04/068820, WO 06/030220, WO 06/003388 and other published patent applications of Domantis Ltd. Domain antibodies substantially correspond to non-camelid mammalian VH or VL domains, in particular from human 4 chain antibodies. To bind an epitope as a single antigen-binding domain, ie. without conjugation to a VL or VH domain, respectively, a specific selection of such antigen-binding properties is required, for example, using libraries of sequences of single human VH or VL domains. Domain antibodies have, like V _HH , a molecular weight of about 13 to about 16 kDa and, when derived from fully human sequences, do not require humanization, eg, for therapeutic use in humans.

It should also be noted that individual variable domains can be derived from HEKs of certain shark species (e.g. so-called "IgNAR domains", see e.g. WO 05/18629), although this is less preferred in the context of the present invention as they are non-mammalian. .

Thus, within the meaning of the present invention, the term "immunoglobulin single variable domain" or "single variable domain" includes polypeptides that are derived from a non-human source, preferably a camel, preferably a heavy chain-only camel antibody. They can be humanized as described above. In addition, the term includes polypeptides derived from non-camelid sources, such as mice or humans, that have been "overtowed" as described, for example, in Davies and Riechmann (FEBS 339: 285-290, 1994; Biotechnol. 13 : 475-479; 1996, Prot. Eng. 9: 531-537) and Riechmann and Muyldermans (1999, J. Immunol. Methods 231: 25-38).

Domain V amino acid residues_HH numbered according to the common numbering for V domains_h, given in Kabat et al. ("Sequence of proteins of immunological interest", US Public Health Services, NIH Bethesda, MD, Publication No. 91), applied to V domains_HH camelids as shown, for example, in figure 2 in Riechmann and Muyldermans (1999, J. Immunol. Methods 231: 25-38). Alternative ways of numbering amino acid residues of V domains_h, which can also be applied similarly to domains V_hh, known in the field. However, in the present description, claims and figures, Kabat numbering applicable to V domains will be followed._HHas described above, unless otherwise noted.

It should be noted that - as is well known in the art for V _H domains and for V _HH domains - the total number of amino acid residues in each of the CDRs may vary and may not correspond to the total number of amino acid residues indicated by Kabat numbering (i.e., one or several positions according to the Kabat numbering may not be occupied in the actual sequence, or the actual sequence may contain more amino acid residues than the number allowed by the Kabat numbering). This means that, in general, the Kabat numbering may or may not correspond to the actual numbering of the amino acid residues in the actual sequence. The total number of amino acid residues in the V _H domain and the V _HH domain will typically be in the range of 110 to 120, often 112 to 115. It should be noted, however, that shorter and longer sequences may also be suitable for the purposes described in this document.

The determination of the CDR regions may also be performed in accordance with various methods. In the definition of CDR according to Kabat, FR1 from V _HH includes amino acid residues at positions 1-30, CDR1 from V _HH includes amino acid residues at positions 31-35, FR2 from V _HH includes amino acids at positions 36-49, CDR2 from V _HH includes amino acid residues at positions 50-65, FR3 from V _HH includes amino acid residues at positions 66-94, CDR3 from V _HH includes amino acid residues at positions 95-102, and FR4 from V _HH includes amino acid residues at positions 103-113.

(Eds., Antibody Engineering, vol 2, Springer Verlag Heidelberg Berlin, Martin, Chapter 3, pp. 33-51, 2010). Согласно этому способу FR1 включает аминокислотные остатки в положениях 1-25, CDR1 включает аминокислотные остатки в положениях 26-35, FR2 включает аминокислоты в положениях 36-49, CDR2 включает аминокислотные остатки в положениях 50- 58, FR3 включает аминокислотные остатки в положениях 59-94, CDR3 включает аминокислотные остатки в положениях 95-102, а FR4 включает аминокислотные остатки в положениях 103-113 (по нумерации Kabat).However, in the present application, the CDR sequences have been determined according to Kontermann and

(Eds., Antibody Engineering, vol 2, Springer Verlag Heidelberg Berlin, Martin, Chapter 3, pp. 33-51, 2010). According to this method, FR1 includes amino acid residues at positions 1-25, CDR1 includes amino acid residues at positions 26-35, FR2 includes amino acids at positions 36-49, CDR2 includes amino acid residues at positions 50-58, FR3 includes amino acid residues at positions 59- 94, CDR3 includes amino acid residues at positions 95-102 and FR4 includes amino acid residues at positions 103-113 (Kabat numbering).

Immunoglobulin single variable domains such as domain antibodies and nanobodies (including _HH V domains) can be humanized. In particular, humanized immunoglobulin single variable domains of a humanized immunoglobulin, such as Nanobodies (including V _HH domains), may be immunoglobulin single variable domains as generally defined in the preceding paragraphs, but in which at least one amino acid is present. a residue (and in particular at least one of the framework residues) that is and/or corresponds to a humanizing substitution (as defined herein). Potentially useful humanizing substitutions can be identified by comparing the framework sequence of a naturally occurring V _HH sequence with the corresponding framework sequence of one or more closely related human V _H sequences, followed by one or more of the potentially useful humanizing substitutions (or combinations thereof) determined in this way. can be introduced into said V _HH sequence (by any method known per se, as further described herein) and the resulting humanized V _HH sequences can be tested for target affinity, stability, ease and level of expression and/or to other desired properties. Thus, with a limited degree of trial and error, other suitable humanizing substitutions (or suitable combinations thereof) may be determined by one of ordinary skill in the art based on the description disclosed herein. In addition, based on the above (framework regions), an immunoglobulin single variable domain such as Nanobody (including V _HH domains) can be partially humanized or fully humanized.

Immunoglobulin single variable domains such as domain antibodies and Nanobodies (including V _HH domains and humanized V _HH domains) can also be subjected to affinity maturation by introducing one or more changes in the amino acid sequence of one or more CDRs, which changes result in improved affinity. the resulting immunoglobulin single variable domain to its corresponding antigen compared to the corresponding parent molecule. The affinity matured immunoglobulin single variable domain molecules of the invention can be prepared by methods known in the art, for example, as described by Marks et al. (1992, Biotechnology 10: 779-783), Barbas, et al. (1994, Proc. Nat. Acad. Sci, USA 91: 3809-3813), Shier et al. (1995, Gene 169: 147-155), Yelton et al. (Immunol. 155: 1994-2004), Jackson et al. (J. Immunol. 154: 3310-9, 1995), Hawkins et al. (1995, J. MoI. Biol. 226: 889-896), Johnson and Hawkins (1996, Affinity maturation of antibodies using phage display, Oxford University Press).

The process of constructing/selecting and/or producing a polypeptide starting from an immunoglobulin single variable domain, such as a domain antibody or nanobody, is also referred to herein as "formatting" said immunoglobulin single variable domain; and the immunoglobulin single variable domain that is part of the polypeptide is said to be "formatted" or "in the format" of said polypeptide. Examples of methods in which an immunoglobulin single variable domain can be formatted and examples of such formats will be understood by one of skill in the art based on the disclosure herein; and such formatted immunoglobulin single variable domain form a further aspect of the invention.

For example, and without limitation, one or more immunoglobulin single variable domains can be used as a "binding unit", "binding domain", or "building block" (these terms are used interchangeably) to produce a polypeptide that may optionally contain one or more additional immunoglobulin single variable domains that can serve as a binding unit (ie, against the same or a different epitope on CD123 and/or against one or more antigens, proteins, or targets other than CD123, such as, for example, TCR).

Monovalent polypeptides contain or essentially consist of only one binding unit (such as, for example, immunoglobulin single variable domains). Polypeptides that include two or more binding units (such as, for example, immunoglobulin single variable domains) will also be referred to herein as "multivalent" polypeptides, and binding units/immunoglobulin single variable domains present in such polypeptides will also be referred to. herein as being in "multivalent format". For example, a "bivalent" polypeptide may contain two immunoglobulin single variable domains, optionally linked through a linker sequence, while a "trivalent" polypeptide may contain three immunoglobulin single variable domains, optionally linked through two linker sequences; while a "tetravalent" polypeptide may contain four immunoglobulin single variable domains, optionally linked via three linker sequences, and so on.

In a multivalent polypeptide, two or more immunoglobulin single variable domains may be the same or different and may be directed against the same antigen or antigenic determinant (e.g., against the same part(s) or epitope(s), or against different parts or epitopes) or may alternatively be directed against different antigens or antigenic determinants; or any suitable combination thereof. Polypeptides that contain at least two binding units (such as, for example, immunoglobulin single variable domains), in which at least one binding unit is directed against the first antigen (i.e. CD123) and at least , one binding unit directed against a second antigen (i.e., other than CD123) will also be referred to as "multispecific" polypeptides, and binding units (such as, for example, immunoglobulin single variable domains) present in such polypeptides will also be referred to in this document as being in a "multi-specific format". Thus, for example, a "bispecific" polypeptide of the invention is a polypeptide that contains at least an immunoglobulin single variable domain directed against a first antigen (i.e., CD123) and one additional immunoglobulin single variable domain directed against a second antigen (i.e., other than CD123, such as, for example, TCR), while a "trispecific" polypeptide of the invention is a polypeptide that contains at least one immunoglobulin single variable domain directed against the first antigen (i.e., TCR). e. CD123), another immunoglobulin single variable domain directed against a second antigen (i.e., other than CD123, such as, for example, TCR) and at least one additional immunoglobulin single variable domain directed against a third antigen ( i.e. different from both CD123 and the second antigen); etc.

Polypeptides that are directed against a single antigen will also be referred to as "monospecific" polypeptides. Such "monospecific" polypeptides may be monovalent polypeptides containing only one binding unit (such as, for example, an immunoglobulin single variable domain) directed against a single antigen (eg, TCR or CD123). Such "monospecific" polypeptides may also be multivalent polypeptides containing two or more immunoglobulin single variable domains directed against the same antigen. Such "monospecific" multivalent polypeptides may be directed against the same portion(s) or epitope(s) of the same antigen, or against different portions or epitopes of the same antigen) (eg, CD123).

Polypeptides that contain two or more binding units directed against different parts or epitopes on the same antigen are also referred to as "multiparatope" polypeptides. As such, "multiparatopic" polypeptides, such as, for example, "biparatopic" polypeptides or "triparatopic" polypeptides, contain or essentially consist of two or more binding units, each of which has a different paratope (as will be further described in this document; see chapter on monospecific polypeptides of the invention).

Polypeptides of the invention

The present invention relates to polypeptides that redirect T cells to kill CD123 expressing cells. The ability of these polypeptides to perform this function is due to their multispecific format. The multispecific polypeptides of the present invention (referred to as "the multispecific polypeptide(s) of the present invention") comprise an immunoglobulin single variable domain (ISV) that specifically binds a T cell receptor (TCR) and one or more ISVs. that specifically bind CD123.

The invention also relates to monovalent polypeptides that can be used as a binding unit or structural element in such a multispecific polypeptide according to the invention. Accordingly, in one aspect, the invention provides ISVs that specifically bind TCRs. In another aspect, the invention provides ISVs that specifically bind CD123. These monovalent polypeptides bind to only one antigen and will therefore be referred to as "monospecific(s) polypeptide(s) of the invention".

ISVs that specifically bind CD123 can be further formatted to form multivalent polypeptides, which are also included in the invention. Such multivalent polypeptides contain two or more ISVs that specifically bind CD123. These multivalent polypeptides bind only one antigen (ie CD123) and will therefore also be referred to as "monospecific(s) polypeptide(s) of the invention".

The monospecific(s) polypeptide(s) of the invention and the multispecific(s) polypeptide(s) of the invention are further described herein and are commonly referred to as "polypeptide(s) of the invention".

1. Monospecific polypeptides of the invention

1.1 Monospecific polypeptides that bind TCR

The present invention relates to a monospecific polypeptide that specifically binds a TCR. Preferably, such a monospecific polypeptide of the invention is monovalent. In a preferred aspect, the monospecific polypeptide is an immunoglobulin single variable domain, which will be referred to herein as "immunoglobulin single variable domain(s) of the invention" or "ISV of the invention".

The T cell receptor (also referred to herein as TCR) is a heterodimer that consists of a chain of TCRα and TCRβ. Both α and β TCR chains consist of a constant domain and a variable domain. The polypeptides of the invention bind specifically to the constant domain of the TCR.

The T cell receptor is part of the TCR complex. As used herein, the terms "TCR complex" or "TCRαβ-CD3 complex" refer to the T cell receptor complex present on the surface of T cells (see Kuhns et al. 2006, Immunity 24: 133-139). The TCR complex consists of six different type I transmembrane proteins: the TCRα and TCRβ chains, which form a TCR heterodimer responsible for ligand recognition, and the HEK valently linked CD3γ, CD3δ, CD3ε, and ζ chains, which carry cytoplasmic sequence motifs that are phosphorylated upon receptor activation. and recruit a large number of signaling components. The sequences for human CD3 and human TCR α/β constant domains are shown in Table A-8 (SEQ ID NOs: 70-75; see also UniProt Identifiers: CD3 delta: P04234, CD3 gamma: P09693, CD3 epsilon: P07766, CD3 zeta : P20963, TCR alpha: P01848 and TCR beta: refers to P01850).

In one aspect, the present invention relates to a polypeptide described herein, which binds to the constant domain of the T cell receptor (TCRα) (SEQ ID NO: 74) and/or the constant domain of the T cell receptor (TCRβ) (SEQ ID NO: 75) or their polymorphic variants or isoforms.

Isoforms are alternative protein sequences that can be generated from the same gene by one or a combination of biological events such as use of an alternative promoter, alternative splicing, alternative initiation, and ribosomal frameshifting, all of which are known in the art.

Only after rigorous methods of immunization, screening and selection, the authors of the present invention were able to identify ISVs that bind to the TCR constant domains. A sequence cluster was identified comprising 104 clones with similarities and differences in CDR1, CDR2 and CDR3 (see Table A-5). The corresponding sequence alignment is shown (Table A-1).

Accordingly, the present invention provides polypeptides that are ISVs selected from the group consisting of SEQ ID NOs: 42 and 78-180 (see also Table A-5). In a further aspect, the polypeptide is selected from the group consisting of SEQ ID NOs: 42 and 78-180, or from polypeptides that have greater than 80%, greater than 85%, greater than 90%, greater than 95%, or even greater than 99% sequence identity with one from SEQ ID NOs: 42 and 78-180.

Accordingly, the present invention relates to a polypeptide that binds TCR and includes or (essentially) consists of 4 framework regions (FR1-FR4, respectively) and 3 complementarity-determining regions (CDR1-CDR3, respectively), in which CDR1 has the amino acid sequence GX ₁ VX ₂ X ₃ X ₄ NX ₅ LX ₆ wherein X ₁ is D, A, S, E or G, X ₂ is H or Y, X ₃ is K or L, X ₄ is I or L, X ₅ means F, I or V, and X ₆ means G or S.

In a further aspect, the present invention relates to a polypeptide that binds a TCR and which comprises or (essentially) consists of 4 framework regions (FR1-FR4, respectively) and 3 complementarity-determining regions (CDR1-CDR3, respectively), in which CDR2 has the amino acid sequence X ₁ IX ₂ IX ₃ DX ₄ X ₅ X ₆ wherein X ₁ is H, T or R, X ₂ is S, T or A, X ₃ is G, S or A, X ₄ is Q, D, E, T, A or V, X ₅ is T, A or V and X ₆ is D, A, Q, N, V or S.

In a further aspect, the present invention relates to a polypeptide that binds TCR and which includes or (essentially) consists of 4 framework regions (FR1-FR4, respectively) and 3 complementarity-determining regions (CDR1-CDR3, respectively), in which CDR3 has the amino acid the sequence X ₁ SR X ₂ X ₃ PYX ₄ Y, wherein X ₁ is F, Y, G, L or K, X ₂ is I or L, X ₃ is Y or W and X ₄ is D, N or S.

Preferred CDR sequences for use in the polypeptides of the invention, as well as preferred combinations of CDR sequences, are shown in Table A-5.

Accordingly, the present invention relates to a polypeptide, preferably an ISV, which specifically binds a TCR and which comprises or essentially consists of 4 framework regions (FR1-FR4, respectively) and 3 complementarity-determining regions (CDR1-CDR3, respectively), in which:

i) CDR1 is selected from the group consisting of:

a) SEQ ID NOS: 181-191; or

b) amino acid sequences with a difference of 4, 3, 2 or 1 amino acid with the amino acid sequence of one of SEQ ID NOs: 181-191; provided that an ISV comprising a CDR1 with a 4, 3, 2, or 1 amino acid difference binds the TCR with approximately the same or higher affinity as compared to binding to an ISV comprising a CDR1 with no 4, 3, 2, or 1 amino acid difference, where the specified affinity is measured using surface plasmon resonance;

and/or

ii) CDR2 is selected from the group consisting of:

c) SEQ ID NOS: 192-217; or

d) amino acid sequences with a difference of 4, 3, 2 or 1 amino acid with the amino acid sequence of one of SEQ ID NOs: 192-217; provided that an ISV comprising a CDR2 with a 4, 3, 2, or 1 amino acid difference binds the TCR with approximately the same or higher affinity as compared to binding of an ISV comprising a CDR2 with no 4, 3, 2, or 1 amino acid difference, wherein said affinity measured by surface plasmon resonance;

and/or

iii) CDR3 is selected from the group consisting of:

e) SEQ ID NOS: 218-225; or

f) amino acid sequences with a difference of 4, 3, 2 or 1 amino acid with the amino acid sequence of one of SEQ ID NOs: 218-225; provided that an ISV comprising a CDR3 with a 4, 3, 2, or 1 amino acid difference binds the TCR with approximately the same or higher affinity as compared to binding of an ISV comprising a CDR3 with no 4, 3, 2, or 1 amino acid difference, wherein this affinity was measured using surface plasmon resonance.

In a further aspect, the present invention provides a polypeptide, preferably an ISV, wherein:

i) CDR1 is selected from the group consisting of:

a) SEQ ID NOS: 181-191; or

b) amino acid sequences with a difference of 4, 3, 2 or 1 amino acid with the amino acid sequence of one of SEQ ID NOs: 181-191; provided that a polypeptide comprising a CDR1 with a 4, 3, 2, or 1 amino acid difference binds the TCR with approximately the same or higher affinity as compared to binding by a polypeptide containing a CDR1 with no 4, 3, 2, or 1 amino acid difference, wherein the specified affinity is measured using surface plasmon resonance;

and

ii) CDR2 is selected from the group consisting of:

c) SEQ ID NOS: 192-217; or

d) amino acid sequences with a difference of 4, 3, 2 or 1 amino acid with the amino acid sequence of one of SEQ ID NOs: 192-217; provided that a polypeptide containing a CDR2 with a difference of 4, 3, 2, or 1 amino acid binds the TCR with approximately the same or higher affinity compared to binding by a polypeptide containing a CDR2 with no difference of 4, 3, 2, or 1 amino acid, where said affinity measured by surface plasmon resonance;

and

iii) CDR3 is selected from the group consisting of:

e) SEQ ID NOS: 218-225; or

f) amino acid sequences with a difference of 4, 3, 2 or 1 amino acid with the amino acid sequence of one of SEQ ID NOs: 218-225; provided that a polypeptide containing a CDR3 with a difference of 4, 3, 2, or 1 amino acid binds the TCR with approximately the same or higher affinity compared to binding by a polypeptide containing a CDR3 with no difference of 4, 3, 2, or 1 amino acid, where this affinity was measured using surface plasmon resonance.

In particular, the present invention relates to a polypeptide, preferably an ISV, in which:

i) CDR1 is selected from the group consisting of:

a) SEQ ID NOS: 181-191; or

b) amino acid sequences with a difference of 4, 3, 2 or 1 amino acid with the amino acid sequence of one of SEQ ID NOs: 181-191, where the difference of 4, 3, 2 or 1 amino acid is present at positions 2, 4, 5, 6, 8 and/or 10 CDR1 (positions 27, 29, 30, 31, 33 and/or 35 according to Kabat numbering); provided that a polypeptide comprising a CDR1 with a 4, 3, 2, or 1 amino acid difference binds the TCR with approximately the same or higher affinity as compared to binding by a polypeptide containing a CDR1 with no 4, 3, 2, or 1 amino acid difference, wherein the specified affinity is measured using surface plasmon resonance;

and

ii) CDR2 is selected from the group consisting of:

c) SEQ ID NOS: 192-217; or

d) amino acid sequences with a difference of 4, 3, 2 or 1 amino acid with the amino acid sequence of one of SEQ ID NOs: 192-217, where the difference of 4, 3, 2 or 1 amino acid is present at positions 1, 3, 5, 7, 8 and/or 9 CDR2 (positions 50, 52, 54, 56, 57 and/or 58 according to Kabat numbering); provided that a polypeptide containing a CDR2 with a difference of 4, 3, 2, or 1 amino acid binds the TCR with approximately the same or higher affinity compared to binding by a polypeptide containing a CDR2 with no difference of 4, 3, 2, or 1 amino acid, where the specified affinity is measured using surface plasmon resonance;

and

iii) CDR3 is selected from the group consisting of:

e) SEQ ID NOS: 218-225; or

f) amino acid sequences with a difference of 4, 3, 2 or 1 amino acid with an amino acid sequence of one of SEQ ID NOs: 218-225, where the difference of 4, 3, 2 or 1 amino acid is present at positions 1, 4, 5 and/or 8 CDR3 (positions 95, 98, 99 and/or 101 according to Kabat numbering); provided that a polypeptide containing a CDR3 with a difference of 4, 3, 2, or 1 amino acid binds the TCR with approximately the same or higher affinity compared to binding by a polypeptide containing a CDR3 with no difference of 4, 3, 2, or 1 amino acid, where this affinity was measured using surface plasmon resonance.

In another aspect, the present invention provides a polypeptide, preferably an ISV, wherein the CDR1 is selected from the group consisting of:

a) SEQ ID NO: 181; or

b) amino acid sequences with a difference of 4, 3, 2 or 1 amino acid from the amino acid sequence of SEQ ID NO: 181, where

- in position 2 D has been changed to A, S, E or G;

- in position 4 H has been changed to Y;

- in position 5 K has been changed to L;

- in position 6 I was changed to L;

- in position 8, F has been changed to I or V; and/or

- in position 10 G has been changed to S;

provided that a polypeptide comprising a CDR1 with a 4, 3, 2, or 1 amino acid difference binds the TCR with approximately the same or higher affinity as compared to binding by a polypeptide containing a CDR1 with no 4, 3, 2, or 1 amino acid difference, wherein this affinity was measured using surface plasmon resonance.

In another aspect, the present invention provides a polypeptide, preferably an ISV, wherein the CDR2 is selected from the group consisting of:

a) SEQ ID NO: 192; or

b) amino acid sequences with a difference of 4, 3, 2 or 1 amino acid from the amino acid sequence of SEQ ID NO: 192, where

- in position 1 H has been changed to T or R;

- in position 3 S has been changed to T or A;

- in position 5 G has been changed to S or A;

- in position 7 Q has been changed to D, E, T, A or V;

- in position 8, T was changed to A or V; and/or

- in position 9, D has been changed to A, Q, N, V or S;

provided that a polypeptide containing a CDR2 with a difference of 4, 3, 2, or 1 amino acid binds the TCR with approximately the same or higher affinity compared to binding by a polypeptide containing a CDR2 with no difference of 4, 3, 2, or 1 amino acid, where this affinity was measured using surface plasmon resonance.

In another aspect, the present invention provides a polypeptide, preferably an ISV, wherein the CDR3 is selected from the group consisting of:

a) SEQ ID NO: 218; or

b) amino acid sequences with a difference of 4, 3, 2 or 1 amino acid from the amino acid sequence of SEQ ID NO: 218, where

- in position 1, F has been changed to Y, L or G;

- in position 4 I was changed to L;

- in position 5 Y has been changed to W; and/or

- in position 8 D has been changed to N or S;

provided that a polypeptide containing a CDR3 with a difference of 4, 3, 2, or 1 amino acid binds the TCR with approximately the same or higher affinity compared to binding by a polypeptide containing a CDR3 with no difference of 4, 3, 2, or 1 amino acid, where this affinity was measured using surface plasmon resonance.

Accordingly, the present invention provides a polypeptide, preferably an ISV, wherein:

i) CDR1 is selected from the group consisting of:

a) SEQ ID NO: 181; or

b) amino acid sequences with a difference of 4, 3, 2 or 1 amino acid from the amino acid sequence of SEQ ID NO: 181, where

- in position 2 D has been changed to A, S, E or G;

- in position 4 H has been changed to Y;

- in position 5 K has been changed to L;

- in position 6 I was changed to L;

- in position 8, F has been changed to I or V; and/or

- in position 10 G has been changed to S;

provided that a polypeptide comprising a CDR1 with a 4, 3, 2, or 1 amino acid difference binds the TCR with approximately the same or higher affinity as compared to binding by a polypeptide containing a CDR1 with no 4, 3, 2, or 1 amino acid difference, wherein said affinity measured by surface plasmon resonance;

and

ii) CDR2 is selected from the group consisting of:

c) SEQ ID NO: 192; or

d) amino acid sequences with a difference of 4, 3, 2 or 1 amino acid from the amino acid sequence of SEQ ID NO: 192, where

- in position 1 H has been changed to T or R;

- in position 3 S has been changed to T or A;

- in position 5 G has been changed to S or A;

- in position 7 Q has been changed to D, E, T, A or V;

- in position 8, T was changed to A or V; and/or

- in position 9, D has been changed to A, Q, N, V or S;

provided that a polypeptide containing a CDR2 with a difference of 4, 3, 2, or 1 amino acid binds the TCR with approximately the same or higher affinity compared to binding by a polypeptide containing a CDR2 with no difference of 4, 3, 2, or 1 amino acid, where the specified affinity is measured using surface plasmon resonance;

and

iii) CDR3 is selected from the group consisting of:

e) SEQ ID NO: 218; or

f) amino acid sequences with a difference of 4, 3, 2 or 1 amino acid from the amino acid sequence of SEQ ID NO: 218, where

- in position 1, F has been changed to Y, L or G;

- in position 4 I was changed to L;

- in position 5 Y has been changed to W; and/or

- in position 8 D has been changed to N or S;

provided that a polypeptide containing a CDR3 with a difference of 4, 3, 2, or 1 amino acid binds the TCR with approximately the same or higher affinity compared to binding by a polypeptide containing a CDR3 with no difference of 4, 3, 2, or 1 amino acid, where this affinity was measured using surface plasmon resonance.

In another aspect, the present invention relates to a polypeptide, preferably an ISV, wherein CDR1 is selected from the group consisting of SEQ ID NOs: 181-191, CDR2 is selected from the group consisting of SEQ ID NOs: 192-217, and CDR3 is selected from the group, consisting of SEQ ID NO: 218-225.

Accordingly, in a preferred aspect, the present invention relates to a polypeptide, preferably an ISV, wherein CDR1 is SEQ ID NO: 181, CDR2 is SEQ ID NO: 192, and CDR3 is SEQ ID NO: 218.

In general, the combinations of CDRs listed in Table A-5 (ie, those listed on the same row in Table A-5) are preferred. Thus, it is generally preferred that when the CDR in the ISV is the CDR sequence mentioned in Table A-5, or is suitably selected from the group consisting of CDR sequences that have a 4, 3, 2, or only 1 amino acid difference with sequence of the CDRs listed in Table A-5, that at least one, and preferably both, other CDRs are suitably selected from the CDR sequences that belong to the same combination in Table A-5 (i.e., mentioned on the same line in table A-5) or suitably selected from the group consisting of CDR sequences that have a difference of 4, 3, 2, or only 1 amino acid with a CDR sequence belonging to the same combination.

The present invention also relates to a polypeptide, preferably an ISV, which cross-blocks the binding to the TCR of at least one of the polypeptides described herein and/or which cross-blocks the binding to the TCR of at least one of the polypeptides described in this document.

The polypeptides of the present invention specifically bind TCRs on the surface of effector cells such as T cells. In a "monovalent" format, the monovalent polypeptides of the invention that bind TCR cause minimal or no T cell activation.

As used herein, the term "effector cell" means a cell containing a TCR complex, preferably an immune cell such as a T cell, preferably a CD4 ⁺ T helper cell (also known as a CD4 cell, T helper cell, or T4 cell) , more preferably a cytotoxic T cell (also known as a T _C cell, CTL or CD8 ⁺ T cell) or a natural killer T cell (NKT cell). In HEK aspects, the cell is present in vivo. In HEK, in some aspects, the cell is present in vitro. The effector cell according to the invention relates in particular to mammalian cells, preferably primate cells and even more preferably human cells.

"T cell activation" as used herein refers to one or more cellular responses of T cells, e.g. cytotoxic T cells, e.g. selected from: proliferation, differentiation, secretion of cytokines, release of a cytotoxic effector molecule, cytotoxic activity, marker expression activation and redirected lysis of target cells.

The monospecific polypeptide of the invention binds to a T cell receptor (TCR) constant domain with a mean K _D of 100 nM to 10 pM, e.g. a mean K _D of 90 nM or less, even more preferably a mean K _D of 80 nM or less, such as less than 70, 60, 50, 40, 30, 20, 10, 5 nM or even less, such as less than 4, 3, 2 or 1 nM, such as less than 500, 400, 300, 200, 100, 90, 80, 70, 60, 50, 40, 30, 20 pM or even less, such as less than 10 pM. Preferably, K _D is determined using Kinexa, BLI or SPR, for example, as determined using Proteon. For example, the specified K _D is determined as indicated in the "Examples" section.

A monospecific polypeptide of the invention binds to a TCR with an EC50 value between 100 nM and 1 pM, e.g., an average EC50 value of 100 nM or less, even more preferably an average EC50 value of 90 nM or less, e.g., less than 80, 70, 60, 50 , 40, 30, 20, 10, 5 nM or even less, such as less than 4, 3, 2 or 1 nM or even less, such as less than 500, 400, 300 200, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 5 pM or even less, such as less than 4 pM. Said average EC50 is preferably determined by FACS, Biacore or ELISA, for example said EC50 is determined as indicated in the Examples section.

The examples show that K _D correlates well with EC50.

In a further aspect, a monospecific polypeptide, as described herein, has an association rate constant (k _on ) for (or binding to) a TCR selected from the group consisting of at least about 10 ² M ^-1 s ^-1 , at least about 10 ³ M ^-1 s ^-1 , at least about 10 ⁴ M ^-1 s ^-1 , at least about 10 ⁵ M ^-1 s ^-1 , at least about 10 ⁶ M ^{- 1} s ^-1 , 10 ⁷ M ^-1 s ^-1 , at least about 10 ⁸ M ^-1 s ^-1 , at least about 10 ⁹ M ^-1 s ^-1 and at least about 10 ¹⁰ M ^-1 s ^-1 , preferably as measured by surface plasmon resonance or as performed in the examples section.

In a further aspect, a monospecific polypeptide as described herein has a dissociation rate constant (k _off ) for (or binding to) a TCR selected from the group consisting of at most about 10 ^-3 s ^-1 , at most about 10 ^-4 s ^-1 , at most about 10 ^-5 s ^-1 , at most about 10 ^-6 s ^-1 , at most about 10 ^-7 s ^-1 , at most about 10 ^-8 s ^-1 at most about 10 ^-9 s ^-1 and at most about 10 ^-10 s ^-1 , preferably as measured by surface plasmon resonance or as performed in the examples section.

The monospecific polypeptides and/or immunoglobulin single variable domains of the invention that bind TCRs may have framework sequences, which are preferably (suitable combinations of) immunoglobulin framework sequences or framework sequences that have been derived from immunoglobulin framework sequences (e.g., by sequence optimization, such as humanization or overblown). For example, framework sequences can be framework sequences derived from an immunoglobulin single variable domain, such as a light chain variable domain (eg, V _L sequence) and/or from a heavy chain variable domain (eg, V _H sequence). In one particularly preferred aspect, the framework sequences are either framework sequences that have been derived from a V _HH sequence (in which said framework sequences may optionally be partially or fully humanized) or are conventional V _H sequences that have been overblown.

Framework sequences may preferably be such that the monospecific polypeptide and/or immunoglobulin single variable domain is a domain antibody (or an amino acid sequence that is suitable for use as a domain antibody); a single domain antibody (or an amino acid that is suitable for use as a single domain antibody); "dAb" (or an amino acid that is suitable for use as a dAb); nanobody; V _HH ; humanized V _HH ; overblown V _H ; or V _HH , which was obtained by affinity maturation. Again, suitable framework sequences will be understood by those skilled in the art, for example, based on standard reference books and the further disclosure and prior art referred to herein.

In particular, the framework sequences present in the monospecific polypeptides of the invention may contain one or more characteristic residues (as defined in WO 08/020079 (Tables A-3-A-8)), such that the monospecific polypeptide of the invention is a nanobody. Some preferred, but non-limiting examples (suitable combinations) of such framework sequences will become apparent from further disclosure herein (see, for example, Table A-5). Typically, nanobodies (particularly V _HH , partially or fully humanized V _HH and overblown V _H ) may in particular be characterized by the presence of one or more "characteristic residues" in one or more framework sequences (for example, as further described in WO 08 /020079, page 61, line 24 - page 98, line 3).

More specifically, the invention relates to polypeptides containing or (essentially) consisting of at least an immunoglobulin single variable domain, which is an amino acid sequence with the (general) structure

FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4

wherein FR1-FR4 refer to framework regions 1-4, respectively, and wherein CDR1-CDR3 refer to complementarity-determining regions 1-3, respectively, and which:

i) has at least 80%, more preferably 90%, even more preferably 95% amino acid identity with at least one of the amino acid sequences of SEQ ID NOS: 42 and 78-180 (see Table A-5) where, for purposes of determining the degree of amino acid identity, amino acid residues that form CDR sequences are not considered. In this regard, we also hereby refer to Table A-5, which lists framework sequences 1 (SEQ ID NO: 226-250), framework sequences 2 (SEQ ID NO: 251-276), framework sequences 3 (SEQ ID NO: 277 -319) and framework sequences 4 (SEQ ID NOs: 320-324) of immunoglobulin single variable domains SEQ ID NOs: 42 and 78-180 (see Table A-5); or

and in which:

ii) preferably one or more amino acid residues at positions 11, 37, 44, 45, 47, 83, 84, 103, 104 and 108 according to Kabat numbering are selected from representative residues mentioned in Tables A-3 to A-8 WO 08/020079.

The present invention also relates to a number of sequence-optimized polypeptides and/or immunoglobulin single variable domains.

In particular, the sequence-optimized polypeptides and/or immunoglobulin single variable domains of the invention may be the amino acid sequences that are generally defined for immunoglobulin single variable domains in the preceding paragraphs, but in which at least one amino acid residue is present ( and in particular in at least one of the framework residues) which is and/or which corresponds to a humanizing substitution (as defined herein). Some preferred, but non-limiting, humanizing substitutions (and suitable combinations thereof) will become apparent to those skilled in the art based on the disclosure herein. In addition, or otherwise, other potentially useful humanizing substitutions can be identified by comparing the framework sequence of a naturally occurring _HH V sequence with the corresponding framework sequence of one or more closely related human VH sequences, followed by one or more of the potentially useful humanizing substitutions ( or combinations thereof) so defined can be introduced into said V _HH sequence (by any method known per se, as further described herein) and the resulting humanized V _HH sequences can be tested for target affinity, stability, the ease and level of expression and/or other desired properties. Thus, with a limited degree of trial and error, other suitable humanizing substitutions (or suitable combinations thereof) may be determined by one of ordinary skill in the art based on the description disclosed herein. In addition, based on the above (framework region) immunoglobulin individual variable domains can be partially humanized or fully humanized.

The present invention also relates to sequence-optimized polypeptides and/or immunoglobulin single variable domains that can show improved expression and/or increased storage stability during stability studies. The sequence-optimized polypeptides and/or ISVs of the present invention may exhibit reduced post-translational N-terminal modification by pyroglutamate and therefore may have increased product stability. In addition, sequence-optimized polypeptides and/or ISVs of the present invention may exhibit other improved properties such as, for example, less immunogenicity, improved binding characteristics (suitably measured and/or expressed as a K _D value (actual or apparent), KA values (actual or apparent), _{k on} rate and/or k _off rate or alternatively EC ₅₀ values as further described herein) for TCR, improved affinity and/or improved avidity for TCR.

Some particularly preferred sequence-optimized immunoglobulin single variable domains of the invention are sequence-optimized variants of the immunoglobulin single variable domains of SEQ ID NOs: 42 and 78-180.

Thus, some other preferred immunoglobulin single variable domains of the invention are nanobodies that can bind (as defined herein) to TCRs and that:

i) is a sequence-optimized variant of one of the immunoglobulin single variable domains with the sequences of SEQ ID NOs: 42 and 78-180; and/or

ii) have at least 80% amino acid identity with at least one of the immunoglobulin single variable domains of SEQ ID NOs: 42 and 78-180 (see Table A-5), in which, for purposes of determining the amino acid degree identity, amino acid residues that form CDR sequences are not considered; In this regard, we also hereby refer to Table A-5, which lists framework sequences 1 (SEQ ID NO: 226-250), framework sequences 2 (SEQ ID NO: 251-276), framework sequences 3 (SEQ ID NO: 277 -319) and framework sequences 4 (SEQ ID NOs: 320-324) of immunoglobulin single variable domains SEQ ID NOs: 42 and 78-180 (see Table A-5);

and in which:

iii) preferably one or more amino acid residues at positions 11, 37, 44, 45, 47, 83, 84, 103, 104 and 108 according to Kabat numbering are selected from representative residues mentioned in Table A-3 - Table A-8 WO 08/020079.

The sequence-optimized polypeptides and/or immunoglobulin single variable domains of the invention may also contain the specific mutations/amino acid residues described in the following co-pending US Provisional Applications, all entitled "Improved Immunoglobulin Variable Domains": US 61/994552, filed in 16 May 16 2014; US 61/014015, filed June 18, 2014; U.S. Patent 62/040167, filed August 21, 2014; and US 62/047,560, filed September 8, 2014 (all assigned to Ablynx NV), as well as WO 2015/173325, which was based on these provisional applications and was published on November 19, 2015.

In particular, sequence-optimized polypeptides and/or immunoglobulin single variable domains of the invention may suitably contain (i) K or Q at position 112; or (ii) K or Q at position 110 in combination with V at position 11; or (iii) T at position 89; or (iv) L at position 89 with K or Q at position 110; or (v) V at position 11 and L at position 89; or any suitable combination of (i)-(v).

As also described in said co-pending US Provisional Applications, when the polypeptides and/or immunoglobulin single variable domains of the invention comprise mutations according to one of (i) to (v) above (or a suitable combination thereof):

the amino acid residue at position 11 is preferably selected from L, V or K (and most preferably V); and

the amino acid residue at position 14 is preferably selected from A or P; and

the amino acid residue at position 41 is preferably selected from A or P; and

the amino acid residue at position 89 is preferably selected from T, V or L; and

the amino acid residue at position 108 is preferably selected from Q or L; and

the amino acid residue at position 110 is preferably selected from T, K or Q; and

the amino acid residue at position 112 is preferably selected from S, K or Q.

As mentioned in these co-pending US Provisional Applications, these mutations are effective in preventing or reducing the binding of so-called "pre-existing antibodies" to polypeptides, immunoglobulin single variable domains and/or constructs of the invention. For this purpose, the polypeptides and/or immunoglobulin single variable domains of the invention may also contain (optionally in combination with the indicated mutations) a C-terminal extension (X)n (in which n is from 1 to 10, preferably from 1 to 5), such as 1, 2, 3, 4 or 5 (and preferably 1 or 2, e.g. 1); and each X is a (preferably naturally occurring) amino acid residue which is independently selected and preferably independently selected from the group consisting of alanine (A ), glycine (G), valine (V), leucine (L) or isoleucine (I)), for which we hereby again refer to the said US Provisional Applications as well as to WO 12/175741. In particular, the polypeptide and/or immunoglobulin single variable domain of the invention may include such a C-terminal extension when it forms the C-terminal end of a protein, polypeptide or other construct containing the same (again, as further described in the provisional applications mentioned above USA and also WO 12/175741).

Accordingly, the present invention relates to a polypeptide described herein, further comprising a C-terminal extension (X)n, in which n is from 1 to 5, for example, 1, 2, 3, 4 or 5, and in which X represents is a naturally occurring amino acid, preferably not cysteine.

These polypeptides of the invention, and in particular the immunoglobulin single variable domains containing the CDR sequences of the invention, are particularly suitable for use as a building block or binding unit for the production of multispecific polypeptides, such as the multispecific polypeptides of the invention.

Accordingly, the monospecific polypeptides of the invention that bind the TCR may be substantially in isolated form (as defined herein), or they may form part of a protein or polypeptide, which may include or consist essentially of a single polypeptide or ISV that binds TCR, and which may optionally further include one or more additional amino acid sequences (all optionally linked via one or more suitable linkers).

Accordingly, the present invention also relates to a protein or polypeptide that comprises or essentially consists of a single monospecific polypeptide of the invention (or suitable fragments thereof). In a further aspect, the monospecific polypeptides of the invention that bind a TCR may form part of a multispecific polypeptide that may include, or essentially consist of, a single ISV that binds a TCR, and which may optionally further comprise one or more additional ISVs that specifically bind another target, such as CD123, and which may optionally further include one or more additional amino acid sequences (all optionally linked via one or more suitable linkers).

The monospecific polypeptides of the invention are thus used as a binding unit or structural element in such a protein or polypeptide to provide a multispecific polypeptide of the invention as described herein (for multispecific polypeptides containing one or more V _HH domains and their preparation also hereby refer to Conrath et al (2001, J. Biol. Chem. 276: 7346-7350) and also, for example, WO 96/34103, WO 99/23221 and WO 2010/115998). Thus, the present invention also relates to a polypeptide that is a monovalent construct comprising or essentially consisting of a single monovalent polypeptide that binds the TCR.

1.2 CD123 binding polypeptides

The present invention relates to a monospecific polypeptide that specifically binds CD123, preferably human and/or cynomolgus monkey CD123. In a preferred aspect, the monospecific polypeptide is an immunoglobulin single variable domain, also referred to herein as an "immunoglobulin single variable domain of the invention" or "ISV of the invention".

CD123 is also known as the α subunit of the interleukin 3 receptor (IL-3Rα). The sequences of human CD123 and cynomolgus monkey CD123 are shown in Table A-8 (SEQ ID NOS: 68-69; see also human CD123: NCBI RefSeq NP_002174 and cynomolgus monkey CD123: NCBI genbank no. EHH61867.1).

In one aspect, the present invention provides a monospecific polypeptide described herein that binds to human CD123 (SEQ ID NO: 68).

Monospecific polypeptides that bind CD123 have been carefully selected for their specificity for CD123. The polypeptides of the invention exhibit highly specific binding to CD123 when formatted in the multispecific format of the invention (ie, a format including one ISV that binds TCR and one or more ISVs that bind CD123). As such, off-target binding is avoided and target independent T cell activation is minimal, as further shown herein.

The authors identified 2 nanobody clusters (Example 12) that exhibited highly specific binding to CD123. When cluster representatives were formatted into a multispecific polypeptide of the invention (as described below), only minimal target-independent T cell activation was observed, indicating high specificity of cluster representatives. The corresponding alignments are shown (see Table A-2 for nanobodies associated with (family members of) 56A10 nanobodies (i.e., nanobodies belonging to the same family as 56A10 nanobody), and Table A-3 for nanobodies associated with (members of the family) 55F03 nanobodies (i.e., nanobodies belonging to the same family as 55F03 nanobody).

"Nanobody family", "V _HH family" or "family" as used herein refers to a group of nanobody and/or V _HH sequences that are the same length (i.e. they have the same number of amino acids within their sequence) , and of which the amino acid sequence between position 8 and position 106 (according to Kabat numbering) has an amino acid sequence identity of 89% or more.

Accordingly, the present invention provides polypeptides, preferably ISVs, selected from the group consisting of SEQ ID NOs: 1-10 (see also Table A-4). In a further aspect, the polypeptide is selected from the group consisting of SEQ ID NOs: 1-10, or from polypeptides that have greater than 80%, greater than 85%, greater than 90%, greater than 95%, or even greater than 99% sequence identity with one of the SEQs. ID NO: 1-10.

Accordingly, the present invention relates to a polypeptide, preferably an ISV, which specifically binds CD123 and which comprises or essentially consists of 4 framework regions (FR1-FR4, respectively) and 3 complementarity-determining regions (CDR1-CDR3, respectively), in which:

i) CDR1 is selected from the group consisting of:

a) SEQ ID NO: 11-16; or

b) amino acid sequences with a difference of 4, 3, 2 or 1 amino acid with the amino acid sequence of one of SEQ ID NOs: 11-16; provided that a polypeptide comprising a CDR1 with a 4, 3, 2, or 1 amino acid difference binds CD123 with approximately the same or higher affinity as compared to binding by a polypeptide containing a CDR1 with no 4, 3, 2, or 1 amino acid difference, wherein said affinity measured by surface plasmon resonance;

and/or

ii) CDR2 is selected from the group consisting of:

c) SEQ ID NO: 17-20; or

d) amino acid sequences with a difference of 4, 3, 2 or 1 amino acid with the amino acid sequence of one of SEQ ID NOs: 17-20; provided that a polypeptide containing a CDR2 with a difference of 4, 3, 2, or 1 amino acid binds CD123 with approximately the same or higher affinity compared to binding by a polypeptide containing a CDR2 with no difference of 4, 3, 2, or 1 amino acid, where the specified affinity is measured using surface plasmon resonance;

and/or

iii) CDR3 is selected from the group consisting of:

e) SEQ ID NOS: 21-25; or

f) amino acid sequences with a difference of 4, 3, 2 or 1 amino acid with the amino acid sequence of one of SEQ ID NOs: 21-25; provided that a polypeptide containing a CDR3 with a difference of 4, 3, 2, or 1 amino acid binds CD123 with approximately the same or higher affinity compared to binding by a polypeptide containing a CDR3 with no difference of 4, 3, 2, or 1 amino acid, where this affinity was measured using surface plasmon resonance.

In a further aspect, the present invention relates to a polypeptide, preferably an ISV, that specifically binds CD123 and that comprises or essentially consists of 4 framework regions (FR1-FR4, respectively) and 3 complementarity-determining regions (CDR1-CDR3, respectively), wherein:

i) CDR1 is selected from the group consisting of:

a) SEQ ID NO: 11-16; or

b) amino acid sequences with a difference of 4, 3, 2 or 1 amino acid with the amino acid sequence of one of SEQ ID NOs: 11-16; provided that a polypeptide comprising a CDR1 with a 4, 3, 2, or 1 amino acid difference binds CD123 with approximately the same or higher affinity as compared to binding by a polypeptide containing a CDR1 with no 4, 3, 2, or 1 amino acid difference, wherein the specified affinity is measured using surface plasmon resonance;

and

ii) CDR2 is selected from the group consisting of:

c) SEQ ID NO: 17-20; or

d) amino acid sequences with a difference of 4, 3, 2 or 1 amino acid with the amino acid sequence of one of SEQ ID NOs: 17-20; provided that a polypeptide containing a CDR2 with a difference of 4, 3, 2, or 1 amino acid binds CD123 with approximately the same or higher affinity compared to binding by a polypeptide containing a CDR2 with no difference of 4, 3, 2, or 1 amino acid, where the specified affinity is measured using surface plasmon resonance;

and

iii) CDR3 is selected from the group consisting of:

e) SEQ ID NOS: 21-25; or

f) amino acid sequences with a difference of 4, 3, 2 or 1 amino acid with the amino acid sequence of one of SEQ ID NOs: 21-25; provided that a polypeptide containing a CDR3 with a difference of 4, 3, 2, or 1 amino acid binds CD123 with approximately the same or higher affinity compared to binding by a polypeptide containing a CDR3 with no difference of 4, 3, 2, or 1 amino acid, where this affinity was measured using surface plasmon resonance.

In a further aspect, a polypeptide of the invention, preferably an ISV, contains or essentially consists of 4 framework regions (FR1-FR4, respectively) and 3 complementarity-determining regions (CDR1-CDR3, respectively), in which

i) CDR1 is selected from the group consisting of:

a) SEQ ID NO: 11-16; or

b) amino acid sequences with a difference of 4, 3, 2 or 1 amino acid with the amino acid sequence of one of SEQ ID NOs: 11-16, where the difference of 4, 3, 2 or 1 amino acid is present at positions 3, 6, 7 and/or 8 CDR1 (positions 28, 31, 32 and/or 33 according to Kabat numbering); provided that a polypeptide comprising a CDR1 with a 4, 3, 2, or 1 amino acid difference binds CD123 with approximately the same or higher affinity as compared to binding by a polypeptide containing a CDR1 with no 4, 3, 2, or 1 amino acid difference, wherein the specified affinity is measured using surface plasmon resonance;

and/or

ii) CDR2 is selected from the group consisting of:

c) SEQ ID NO: 17-20; or

d) amino acid sequences with a difference of 3, 2 or 1 amino acid with the amino acid sequence of one of SEQ ID NOs: 17-20, where the difference of 3, 2 or 1 amino acid is present at positions 3 to 6 and/or 10 of CDR2 (positions 52, 54 and/or 58 according to Kabat numbering); provided that a polypeptide containing a CDR2 with a 3, 2, or 1 amino acid difference binds CD123 with approximately the same or higher affinity compared to binding by a polypeptide containing a CDR2 with no 3, 2, or 1 amino acid difference, where said affinity is measured with using surface plasmon resonance;

and/or

iii) CDR3 is selected from the group consisting of:

e) SEQ ID NOS: 21-25; or

f) amino acid sequences with a difference of 3, 2 or 1 amino acid with the amino acid sequence of one of SEQ ID NOs: 21-25, where the difference of 3, 2 or 1 amino acid is present at positions 3, 4 and/or 5 of CDR3 (positions 97, 98 and/or 99 according to Kabat numbering); provided that a polypeptide containing a CDR3 with a difference of 3, 2, or 1 amino acid binds CD123 with approximately the same or higher affinity compared to binding by a polypeptide containing a CDR3 with no difference of 3, 2, or 1 amino acid, where the specified affinity is measured using surface plasmon resonance.

In a further aspect, a polypeptide of the invention, preferably an ISV, contains or essentially consists of 4 framework regions (FR1-FR4, respectively) and 3 complementarity-determining regions (CDR1-CDR3, respectively), in which

i) CDR1 is selected from the group consisting of:

a) SEQ ID NO: 11-16; or

b) amino acid sequences with a difference of 4, 3, 2 or 1 amino acid with the amino acid sequence of one of SEQ ID NOs: 11-16, where the difference of 4, 3, 2 or 1 amino acid is present at positions 3, 6, 7 and/or 8 CDR1 (positions 28, 31, 32 and/or 33 according to Kabat numbering); provided that a polypeptide comprising a CDR1 with a 4, 3, 2, or 1 amino acid difference binds CD123 with approximately the same or higher affinity as compared to binding by a polypeptide containing a CDR1 with no 4, 3, 2, or 1 amino acid difference, wherein the specified affinity is measured using surface plasmon resonance;

and

ii) CDR2 is selected from the group consisting of:

c) SEQ ID NO: 17-20; or

d) amino acid sequences with a difference of 3, 2 or 1 amino acid with the amino acid sequence of one of SEQ ID NOs: 17-20, where the difference of 3, 2 or 1 amino acid is present at positions 3 to 6 and/or 10 of CDR2 (positions 52, 54 and/or 58 according to Kabat numbering); provided that a polypeptide containing a CDR2 with a 3, 2, or 1 amino acid difference binds CD123 with approximately the same or higher affinity compared to binding by a polypeptide containing a CDR2 with no 3, 2, or 1 amino acid difference, where said affinity is measured with using surface plasmon resonance;

and

iii) CDR3 is selected from the group consisting of:

e) SEQ ID NOS: 21-25; or

f) amino acid sequences with a difference of 3, 2 or 1 amino acid with the amino acid sequence of one of SEQ ID NOs: 21-25, where the difference of 3, 2 or 1 amino acid is present at positions 3, 4 and/or 5 of CDR3 (positions 97, 98 and/or 99 according to Kabat numbering); provided that a polypeptide containing a CDR3 with a difference of 3, 2, or 1 amino acid binds CD123 with approximately the same or higher affinity compared to binding by a polypeptide containing a CDR3 with no difference of 3, 2, or 1 amino acid, where the specified affinity is measured using surface plasmon resonance.

In one aspect, polypeptides, preferably ISVs, of the invention may have greater than 80%, greater than 85%, greater than 90%, greater than 95%, or even greater than 99% sequence identity with one of SEQ ID NOs: 1-6 (see also Table A -four). These polypeptides are referred to herein as "polypeptide(s) related to 56A10" or "ISV related to 56A10".

Accordingly, the present invention relates to a polypeptide, preferably an ISV, wherein the CDR1 is selected from the group consisting of:

a) SEQ ID NO: 11; or

b) amino acid sequences with a difference of 4, 3, 2 or 1 amino acid from the amino acid sequence of SEQ ID NO: 11, where

- in position 3, T has been changed to S or P;

- in position 6 I was changed to S;

- in position 7 N has been changed to D; and/or

- in position 8 D has been changed to V or A;

provided that a polypeptide comprising a CDR1 with a 4, 3, 2, or 1 amino acid difference binds CD123 with approximately the same or higher affinity as compared to binding by a polypeptide containing a CDR1 with no 4, 3, 2, or 1 amino acid difference, wherein this affinity was measured using surface plasmon resonance.

In a further aspect, the present invention relates to a polypeptide, preferably an ISV, wherein the CDR2 is SEQ ID NO: 17.

In a further aspect, the present invention relates to a polypeptide, preferably an ISV, wherein the CDR3 is selected from the group consisting of:

a) SEQ ID NO: 21; or

b) amino acid sequences with a difference of 1 amino acid from the amino acid sequence of SEQ ID NO: 21, where

- in position 3 P has been changed to A;

provided that a polypeptide containing a CDR3 with a 1 amino acid difference binds CD123 with approximately the same or higher affinity as compared to binding by a polypeptide containing a CDR3 with no 1 amino acid difference, where said affinity is measured by surface plasmon resonance.

Accordingly, the present invention provides a polypeptide, preferably an ISV, wherein:

i) CDR1 is selected from the group consisting of:

a) SEQ ID NO: 11; or

b) amino acid sequences with a difference of 4, 3, 2 or 1 amino acid from the amino acid sequence of SEQ ID NO: 11, where

- in position 3, T has been changed to S or P;

- in position 6 I was changed to S;

- in position 7 N has been changed to D; and/or

- in position 8 D has been changed to V or A;

provided that a polypeptide comprising a CDR1 with a 4, 3, 2, or 1 amino acid difference binds CD123 with approximately the same or higher affinity as compared to binding by a polypeptide containing a CDR1 with no 4, 3, 2, or 1 amino acid difference, wherein the specified affinity is measured using surface plasmon resonance;

and

ii) CDR2 is SEQ ID NO: 17;

and

iii) CDR3 is selected from the group consisting of:

c) SEQ ID NO: 21; or

d) amino acid sequences with a difference of 1 amino acid from the amino acid sequence of SEQ ID NO: 21, where

- in position 3 P has been changed to A;

provided that a polypeptide comprising a CDR3 with a 1 amino acid difference binds CD123 with approximately the same or higher affinity as compared to binding by a polypeptide comprising a CDR3 with no 1 amino acid difference, where said affinity is measured by surface plasmon resonance.

In another aspect, the present invention relates to a polypeptide, preferably an ISV, as described herein, in which CDR1 is selected from the group consisting of SEQ ID NO: 11-15, CDR2 is SEQ ID NO: 17, and CDR3 is selected from the group, consisting of SEQ ID NO: 21-22.

Accordingly, in a preferred aspect, the present invention relates to a polypeptide, preferably an ISV, as described herein, wherein CDR1 is SEQ ID NO: 11, CDR2 is SEQ ID NO: 17, and CDR3 is SEQ ID NO: 21.

Accordingly, the present invention provides polypeptides that are ISVs selected from the group consisting of SEQ ID NOs: 1-6.

Polypeptides or ISVs related to 56A10 were selected for their exceptional specificity for CD123. Binding of the polypeptides of the invention can be measured in suitable binding assays, including, but not limited to, flow cytometry analysis. In such a flow cytometry assay, cells that endogenously express CD123 (such as, for example, MOLM-13 or KG1a cells) can be used. Alternatively, cells that are transfected to overexpress CD123 (such as, for example, CHO-K1 huCD123 or HEK293 cyno CD123) can be used. Suitable cell lines will become apparent from the examples provided herein.

A polypeptide, preferably an ISV, of the invention can bind CD123 expressed on cells or cells expressing CD123 with an average EC ₅₀ between 10 nM and 100 pM.

More specifically, a polypeptide, preferably an ISV, of the invention binds to human CD123 expressed on MOLM-13 cells with an average EC50 between 10 nM and 100 pM, e.g. an average EC50 of 5 nM or less, e.g. less than 4, 3, 2 or 1 nM or even less, preferably by flow cytometry.

A polypeptide, preferably an ISV, of the invention binds to human CD123 expressed on CHO-K1 cells with an average EC ₅₀ of 10 nM to 100 pM, e.g. an average EC ₅₀ of 5 nM or less, e.g. less than 4.3, 2 or 1 nM or even less, preferably by flow cytometry.

A polypeptide, preferably an ISV, of the invention binds to cynomolgus monkey CD123 expressed on HEK293 cells with an average EC ₅₀ between 10 nM and 100 pM, for example an average EC ₅₀ of 5 nM or less, for example less than 4 or 3 nM or even less preferred by flow cytometry.

The binding of polypeptides, preferably ISV, according to the invention can also be measured using SPR.

As such, a polypeptide, preferably an ISV, of the invention can bind to human CD123 with an average K _D value between 10 nM and 100 pM, for example, with an average K _D value of 5 nM or less, for example, less than 4, 3, or 2 nM, or even however, said K _D value is preferably determined by surface plasmon resonance.

Accordingly, the present invention provides a polypeptide or ISV as described herein, wherein said mean K _D or EC ₅₀ is determined by flow cytometry or SPR, e.g., said K _D or EC ₅₀ is determined as set forth in the examples section.

The examples show that K _D measured in SPR correlates well with EC ₅₀ measured in flow cytometry.

In another aspect, the polypeptides of the invention may have greater than 80%, greater than 85%, greater than 90%, greater than 95%, or even greater than 99% sequence identity with one of SEQ ID NOs: 7-10 (see also Table A-4). These polypeptides are referred to herein as "polypeptide(s) related to 55F03" or "ISV related to 55F03".

Accordingly, the present invention relates to a polypeptide, preferably an ISV, wherein the CDR1 is SEQ ID NO: 16.

In a further aspect, the present invention relates to a polypeptide, preferably an ISV, wherein the CDR2 is selected from the group consisting of:

a) SEQ ID NO: 18; or

b) amino acid sequences with a difference of 3, 2 or 1 amino acid from the amino acid sequence of SEQ ID NO: 18, where

- in position 3 Y has been changed to W;

- in position 6 N has been changed to S; and/or

- in position 10 Q has been changed to E;

provided that a polypeptide containing a CDR2 with a 3, 2, or 1 amino acid difference binds CD123 with approximately the same or higher affinity compared to binding by a polypeptide containing a CDR2 with no 3, 2, or 1 amino acid difference, where said affinity is measured with using surface plasmon resonance.

In a further aspect, the present invention relates to a polypeptide, preferably an ISV, wherein the CDR3 is selected from the group consisting of:

a) SEQ ID NO: 23; or

b) amino acid sequences with a difference of 2 or 1 amino acid from the amino acid sequence of SEQ ID NO: 23, where

- in position 4, E was changed to R; and/or

- in position 5, T was changed to D or Y;

provided that a polypeptide containing CDR3 with a difference of 2 or 1 amino acid binds CD123 with approximately the same or higher affinity compared to binding by a polypeptide containing CDR3 with no difference of 2 or 1 amino acid, where the specified affinity is measured using surface plasmon resonance .

Accordingly, the present invention provides a polypeptide, preferably an ISV, wherein:

i) CDR1 is SEQ ID NO: 16;

and

ii) CDR2 is selected from the group consisting of:

a) SEQ ID NO: 18; or

b) amino acid sequences with a difference of 3, 2 or 1 amino acid from the amino acid sequence of SEQ ID NO: 18, where

- in position 3 Y has been changed to W;

- in position 6 N has been changed to S; and/or

- in position 10 Q has been changed to E;

provided that a polypeptide containing a CDR2 with a 3, 2, or 1 amino acid difference binds CD123 with approximately the same or higher affinity compared to binding by a polypeptide containing a CDR2 with no 3, 2, or 1 amino acid difference, where said affinity is measured with using surface plasmon resonance;

and

iii) CDR3 is selected from the group consisting of:

c) SEQ ID NO: 23; or

d) amino acid sequences with a difference of 2 or 1 amino acid from the amino acid sequence of SEQ ID NO: 23, where

- in position 4, E was changed to R; and/or

- in position 5, T was changed to D or Y;

provided that a polypeptide containing CDR3 with a difference of 2 or 1 amino acid binds CD123 with approximately the same or higher affinity compared to binding by a polypeptide containing CDR3 with no difference of 2 or 1 amino acid, where the specified affinity is measured using surface plasmon resonance .

In another aspect, the present invention relates to a polypeptide, preferably an ISV, as described herein, wherein CDR1 is SEQ ID NO: 16, CDR2 is selected from the group consisting of SEQ ID NO: 18-20, and CDR3 is selected from the group, consisting of SEQ ID NO: 23-25.

Accordingly, in a preferred aspect, the present invention relates to a polypeptide, preferably an ISV, as described herein, wherein CDR1 is SEQ ID NO: 16, CDR2 is SEQ ID NO: 18, and CDR3 is SEQ ID NO: 23.

Preferred polypeptides and/or ISVs are selected from the group consisting of SEQ ID NOs: 7-10.

Polypeptides or ISVs related to 55F03 were selected for their exceptional specificity for CD123. Binding of the polypeptides of the invention can be measured in suitable binding assays, including, but not limited to, flow cytometry and SPR assays, as described herein.

A polypeptide, preferably an ISV, of the invention can bind CD123 expressed on cells or cells expressing CD123 with an average EC50 between 10 μM and 100 nM.

More specifically, a polypeptide, preferably an ISV, of the invention binds to human CD123 expressed on MOLM-13 cells with an average EC50 of 10 μM to 100 nM, e.g. an average EC50 of 5 μM or less, e.g. less than 4, 3, 2 or 1 μM or even less, preferably by flow cytometry.

A polypeptide, preferably an ISV, of the invention binds to human CD123 expressed on CHO-K1 cells with an average EC50 between 100 nM and 1 nM, e.g. an average EC50 of 50 nM or less, such as less than 40, 30, 20 or 10 nM or even less, such as less than 9, 8 or 7 nM or even less, preferably as measured by flow cytometry.

A polypeptide, preferably an ISV, of the invention binds to cynomolgus monkey CD123 expressed on HEK293 cells with an average EC50 between 10 nM and 100 pM, for example an average EC50 of 5 nM or less, for example less than 4 or 3 nM or even less, preferably by flow cytometry.

In a further aspect, the present invention relates to a polypeptide or ISV that binds to human CD123 with a mean K _D of 1 μM to 10 nM, such as a mean K _D of 500 nM or less, such as less than 400, 300, or 200 nM or even less, said K _D value is preferably determined by surface plasmon resonance.

Accordingly, the present invention provides a polypeptide or ISV as described herein, wherein said mean K _D or EC ₅₀ is determined by flow cytometry or SPR, e.g., said K _D or EC ₅₀ is determined as set forth in the examples section.

In the examples, it was shown that K _D measured in SPR correlated well with EC50 as determined in a flow cytometric analysis using MOLM-13 cells.

In general, the combinations of CDRs listed in Table A-4 (ie, those listed on the same row in Table A-4) are preferred. Thus, it is generally preferred that when the CDR in the ISV is the CDR sequence mentioned in Table A-4, or is suitably selected from the group consisting of CDR sequences that have a 4, 3, 2, or only 1 amino acid difference with sequence of the CDRs listed in Table A-4, then at least one, and preferably both, other CDRs are suitably selected from the CDR sequences that belong to the same combination in Table A-4 (i.e., mentioned on the same line in table A-4) or suitably selected from the group consisting of CDR sequences that have a difference of 4, 3, 2, or only 1 amino acid with the CDR sequence(s) belonging to the same combination. Exemplary polypeptides of the present invention having the CDRs described above are shown in Table A-4.

The present invention also relates to a polypeptide, preferably an ISV, which specifically binds CD123, which cross-blocks the binding to CD123 of at least one of the polypeptides described herein and/or selected from SEQ ID NOs: 1-10, and/or that is cross-blocked from binding to CD123 by at least one of the polypeptides described herein and/or selected from SEQ ID NOs: 1-10.

The invention also relates to a monospecific polypeptide that includes or (essentially) consists of two or more ISVs that bind CD123. In such a multivalent (monospecific) polypeptide, also referred to herein as "multivalent polypeptide(s) of the invention", two or more ISVs that bind CD123 may optionally be connected via one or more peptide linkers, as further described herein.

Accordingly, the present invention relates to a polypeptide comprising two or more ISVs that specifically bind CD123, where the ISVs are selected from the group of ISVs related to 56A10 or from the group of ISVs related to 55F03.

In a more specific aspect, the present invention relates to polypeptides containing two ISVs that specifically bind CD123, where the ISVs are selected from the group of ISVs related to 56A10 or from the group of ISVs related to 55F03.

In such a multivalent monospecific polypeptide of the invention, two or more immunoglobulin single variable domains may be the same or different and may be directed against the same CD123 antigenic determinant (e.g. against the same portion(s) or epitope(s) of CD123) or may alternatively be directed against different antigenic determinants of CD123 or against different parts or epitopes of CD123; or any suitable combination thereof. For example, a divalent polypeptide of the invention may include (a) two immunoglobulin single variable domains; (b) a first immunoglobulin single variable domain directed against the first CD123 antigenic determinant and a second immunoglobulin single variable domain directed against the same CD123 antigenic determinant that is different from the first immunoglobulin variable domain; or (c) a first immunoglobulin variable domain directed against a first CD123 antigenic determinant and a second immunoglobulin variable domain directed against another CD123 antigenic determinant.

The trivalent polypeptide of the invention may be any of the above, further comprising (a) an identical immunoglobulin single variable domain; (b) another immunoglobulin single variable domain directed against the same CD123 antigenic determinant; or (c) another immunoglobulin single variable domain directed against another CD123 antigenic determinant.

As such, in one aspect, the monospecific polypeptide of the invention may be a multiparatopic polypeptide, such as, for example, a biparatopic polypeptide. As used herein, the term "biparatopic" (antigen-) binding molecule or "biparatopic" polypeptide means a polypeptide containing at least two (i.e., two or more) immunoglobulin single variable domains, wherein the "first" immunoglobulin single variable domain the domain is directed against CD123 and the "second" immunoglobulin single variable domain is directed against CD123, and where these "first" and "second" immunoglobulin single variable domains have a different paratope. Accordingly, a biparatopic polypeptide comprises or consists of two or more immunoglobulin single variable domains that are directed against CD123, wherein at least one "first" immunoglobulin single variable domain is directed against a first epitope on CD123 and at least one "second The immunoglobulin single variable domain is directed against a second epitope on CD123 that is different from the first epitope on CD123.

Accordingly, the present invention provides polypeptides wherein two or more ISVs that specifically bind CD123 are biparatopic, comprising a first ISV and a second ISV, wherein the first ISV binds to an epitope on CD123 that is different from the epitope on CD123 bound via the second ISV. Such(s) polypeptide(s) is also referred to herein as "biparatopic(s) polypeptide(s) according to the invention".

In a further aspect, the present invention relates to a (biparatopic) polypeptide described herein, wherein the first ISV is selected from the group of ISVs related to 56A10 and the second ISV is selected from the group of ISVs related to 55F03.

In an additional aspect, the present invention relates to a polypeptide described herein, where the second ISV is located at the N-terminus of the first ISV. Such a polypeptide includes an ISV related to 55F03 at the N-terminus of an ISV related to 56A10.

In an additional aspect, the present invention relates to a polypeptide described herein, where the second ISV is located at the C-terminus of the first ISV. Such a polypeptide includes an ISV related to 55F03 at the C-terminus of an ISV related to 56A10.

The biparatopic polypeptides of the invention may have improved binding affinity for CD123 compared to the corresponding monovalent polypeptide due to active binding, also referred to as "avidity".

Avidity is the affinity of a polypeptide, i. the ligand is able to bind through two (or more) pharmacophores (ISVs) in which multiple interactions are synergized to enhance "apparent" affinity. Avidity is a measure of the strength of binding between a polypeptide of the invention and the corresponding antigens or antigenic determinants. A polypeptide of the invention is capable of binding through its two (or more) building blocks, such as ISVs, to at least two targets or antigenic determinants in which multiple interactions, for example, of the first building block or ISV binding to the first target or first antigenic determinant, and a second structural element or ISV that binds to the second target or second antigenic determinant are synergized to enhance the "apparent" affinity. Avidity is related both to the affinity between an antigenic determinant and its antigen-binding site on the antigen-binding molecule, and to the number of suitable binding sites present on the antigen-binding molecules. For example, and without limitation, polypeptides that contain two or more structural elements, such as ISVs, directed against different targets in the cell or different antigenic determinants can (and usually will) bind with higher avidity than either of the individual monomers or individual building blocks such as, for example, the monovalent ISVs contained in the polypeptides of the invention.

The monospecific polypeptides of the invention comprise or (essentially) consist of one or more immunoglobulin single variable domains. The framework sequences of these ISVs are preferably a (suitable combination) of immunoglobulin framework sequences or framework sequences that have been derived from immunoglobulin framework sequences (eg, by sequence optimization such as humanization or overblown). For example, framework sequences can be framework sequences derived from an immunoglobulin single variable domain, such as a light chain variable domain (eg, V _L sequence), and/or from a heavy chain variable domain (eg, V _H sequence). In one particularly preferred aspect, the framework sequences are either framework sequences that have been derived from a V _HH sequence (in which said framework sequences may optionally be partially or fully humanized) or are conventional V _H sequences that have been overblown.

Framework sequences may preferably be such that the ISV included in the monospecific polypeptide of the invention is a domain antibody (or an amino acid sequence that is suitable for use as a domain antibody); a single domain antibody (or an amino acid that is suitable for use as a single domain antibody); "dAb" (or an amino acid that is suitable for use as a dAb); nanobody; V _HH ; humanized V _HH ; overblown V _H ; or V _HH , which was obtained by affinity maturation. Again, suitable framework sequences will be understood by those skilled in the art, for example, based on standard reference books and the further disclosure and prior art referred to herein.

In particular, the framework sequences present in the monospecific polypeptides of the invention may include one or more characteristic residues (as defined in WO 08/020079 (Tables A-3-A-8)), so that the monospecific polypeptide of the invention is a nanobody. Some preferred, but non-limiting examples (suitable combinations) of such framework sequences will become apparent from the further disclosure herein (see, for example, Table A-4). Typically, nanobodies (particularly V _HH , partially or fully humanized V _HH and overblown V _H ) may in particular be characterized by the presence of one or more "characteristic residues" in one or more framework sequences (such as those further described in the application WO 08/020079, page 61, line 24 - page 98, line 3).

More specifically, the invention relates to polypeptides containing at least one immunoglobulin single variable domain, which is an amino acid sequence with the (general) structure

FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4

wherein FR1-FR4 refer to framework regions 1-4, respectively, and wherein CDR1-CDR3 refer to complementarity determination regions 1-3, respectively, and which:

i) have at least 80%, more preferably 90%, even more preferably 95% amino acid identity with at least one of the amino acid sequences of SEQ ID NOs: 1-10 (see Table A-4), which, for the purposes of determining the degree of amino acid identity, amino acid residues that form CDR sequences are not taken into account. In this regard, we also hereby refer to Table A-4, which lists Structure 1 sequences (SEQ ID NO: 26-29), Structure 2 sequences (SEQ ID NO: 30-33), Frame 3 sequences (SEQ ID NO: 34 -39) and framework sequences 4 (SEQ ID NOs: 40-41) of immunoglobulin single variable domains SEQ ID NOs: 1-10 (see Table A-4); or

and in which:

ii) preferably one or more amino acid residues at positions 11, 37, 44, 45, 47, 83, 84, 103, 104 and 108 according to Kabat numbering are selected from representative residues mentioned in Tables A-3 to A-8 from WO 08/020079.

The present invention also relates to a number of sequence-optimized polypeptides and/or immunoglobulin single variable domains.

In particular, the sequence-optimized polypeptides and/or immunoglobulin single variable domains of the invention may be the amino acid sequences that are generally defined for immunoglobulin single variable domains in the preceding paragraphs, but in which at least one amino acid residue is present ( and in particular, at least one of the framework residues which is and/or which corresponds to a humanizing substitution (as defined herein) Some preferred, but non-limiting, humanizing substitutions (and suitable combinations thereof) will become apparent to those skilled in the art In addition, or alternatively, other potentially useful humanizing substitutions can be established by comparing the framework sequence of a naturally occurring V _HH sequence with the corresponding framework sequence of one or more closely related human VH sequences, after which one or more of the potentially useful humanizing substitutions (or combinations thereof) so determined can be introduced into said _HH V sequence (by any means known per se, as further described herein), and the resulting humanized _HH V sequences can be tested for target affinity, stability, ease and level of expression, and/or other desired properties. Thus, with a limited degree of trial and error, other suitable humanizing substitutions (or suitable combinations thereof) may be determined by one of ordinary skill in the art based on the description disclosed herein. In addition, based on the above (framework region) immunoglobulin individual variable domains can be partially humanized or fully humanized.

The present invention also relates to sequence-optimized polypeptides and/or immunoglobulin single variable domains that can show improved expression and/or increased storage stability during stability studies. The sequence-optimized polypeptides and/or ISVs of the present invention may exhibit reduced post-translational N-terminal modification by pyroglutamate and therefore have increased product stability. In addition, sequence-optimized polypeptides and/or ISVs of the present invention may exhibit other improved properties such as, for example, less immunogenicity, improved binding characteristics (suitably measured and/or expressed as a K _D value (actual or apparent), KA values (actual or apparent), k _on rate and/or k _off rate, or alternatively as an EC ₅₀ value, as further described herein) with _CD123 , improved affinity and/or improved avidity for CD123.

HEK particularly preferred sequence optimized immunoglobulin single variable domains of the invention are sequence optimized immunoglobulin single variable domain variants of SEQ ID NOs: 1-10.

Thus, HEK which other preferred immunoglobulin single variable domains of the invention are nanobodies that can bind (as defined herein) to CD123 and that:

i) is a sequence-optimized variant of one of the immunoglobulin single variable domains with the sequence of SEQ ID NOs: 1-10; and/or

ii) have at least 80% amino acid identity with at least one of the immunoglobulin single variable domains of SEQ ID NOs: 1-10 (see Table A-4), in which, for purposes of determining the degree of amino acid identity, the amino acids residues that form CDR sequences are not counted; in this regard, we also hereby refer to Table A-4, which lists framework sequences 1 (SEQ ID NO: 26-29), framework sequences 2 (SEQ ID NO: 30-33), framework sequences 3 (SEQ ID NO: 34 -39) and framework sequences 4 (SEQ ID NOs: 40-41) of immunoglobulin single variable domains SEQ ID NOs: 1-10 (see Table A-4);

and in which:

iii) preferably one or more amino acid residues at positions 11, 37, 44, 45, 47, 83, 84, 103, 104 and 108 according to Kabat numbering are selected from representative residues mentioned in Table A-3 - Table A-8 from WO 08/020079.

The polypeptides and/or immunoglobulin single variable domains of the invention may also contain the specific mutations/amino acid residues described in the following concurrent US Provisional Applications, all of which are entitled "Improved Immunoglobulin Variable Domains": US 61/994552 filed May 16, 2014 ; US 61/014015, filed June 18, 2014; U.S. Patent 62/040167, filed August 21, 2014; and US 62/047,560, filed September 8, 2014 (all assigned to Ablynx NV), as well as WO 2015/173325, which was based on these provisional applications and was published on November 19, 2015.

In particular, the polypeptides and/or immunoglobulin single variable domains of the invention may suitably contain (i) K or Q at position 112; or (ii) K or Q at position 110 in combination with V at position 11; or (iii) T at position 89; or (iv) L at position 89 with K or Q at position 110; or (v) V at position 11 and L at position 89; or any suitable combination of (i)-(v).

As also described in said co-pending US Provisional Applications, when the polypeptide and/or immunoglobulin single variable domains of the invention contain mutations according to one of (i) to (v) above (or a suitable combination thereof):

the amino acid residue at position 11 is preferably selected from L, V or K (and most preferably V); and

the amino acid residue at position 14 is preferably selected from A or P; and

the amino acid residue at position 41 is preferably selected from A or P; and

the amino acid residue at position 89 is preferably selected from T, V or L; and

the amino acid residue at position 108 is preferably selected from Q or L; and

the amino acid residue at position 110 is preferably selected from T, K or Q; and

the amino acid residue at position 112 is preferably selected from S, K or Q.

As mentioned in these co-pending US Provisional Applications, these mutations are effective in preventing or reducing the binding of so-called "pre-existing antibodies" to immunoglobulin polypeptides and/or single variable domains and/or constructs of the invention. For this purpose, the polypeptides and/or immunoglobulin single variable domains of the invention may also contain (optionally in combination with the indicated mutations) a C-terminal extension (X)n (in which n is from 1 to 10, preferably from 1 to 5), such as 1, 2, 3, 4 or 5 (and preferably 1 or 2, e.g. 1); and each X is a (preferably naturally occurring) amino acid residue which is independently selected and preferably independently selected from the group consisting of alanine (A ), glycine (G), valine (V), leucine (L) or isoleucine (I)), for which we hereby again refer to the said US provisional applications as well as to WO 12/175741. In particular, one variable domain of a polypeptide and/or immunoglobulin of the invention may contain such a C-terminal extension when it forms the C-terminal end of a protein, polypeptide, or other construct containing it (again, as further described in the U.S. Provisional Applications cited above). , as well as WO 12/175741).

Accordingly, the present invention relates to a polypeptide described herein, further comprising a C-terminal extension (X)n, in which n is from 1 to 5, for example, 1, 2, 3, 4 or 5, and in which X represents is a naturally occurring amino acid, preferably not cysteine.

These polypeptides of the invention, and in particular the immunoglobulin single variable domains containing the CDR sequences of the invention, are particularly suitable for use as a building block or linker for the production of multivalent or multispecific polypeptides.

Accordingly, the monospecific polypeptides of the invention that bind CD123 may be substantially in isolated form (as defined herein), or they may form part of a protein or polypeptide, which may include or essentially consist of one or more ISVs, that bind CD123, and which may optionally further comprise one or more additional amino acid sequences (all optionally linked via one or more suitable linkers).

Accordingly, the present invention also relates to a protein or polypeptide that comprises or essentially consists of one or more monospecific polypeptides of the invention (or suitable fragments thereof). In a further aspect, the monospecific polypeptides of the invention that bind CD123 may form part of a multispecific polypeptide that may include or essentially consist of one or more ISVs that bind CD123 and which may further comprise one ISV that specifically binds to another target, such as, for example, a TCR, and which may optionally further comprise one or more additional amino acid sequences (all optionally linked via one or more suitable linkers).

The monospecific polypeptides of the invention are thus used as a binding unit or structural element in such a protein or polypeptide to provide a multispecific polypeptide of the invention as described herein (for multispecific polypeptides containing one or more V _HH domains and their preparation , we hereby also refer to Conrath et al (2001, J. Biol. Chem. 276: 7346-7350) and also, for example, WO 96/34103, WO 99/23221 and WO 2010/115998).

2. Multispecific polypeptides

The invention also relates to multispecific polypeptides containing or (essentially) consisting of two or more structural elements (such as at least two monospecific polypeptides or ISV according to the invention), in which at least one structural element is directed against the first antigen (ie CD123) and at least one structural element directed against the second antigen (ie TCR). These multispecific polypeptides are also referred to herein as "the multispecific(s) polypeptide(s) of the invention". Preferred immunoglobulin single variable domains for use in these multispecific polypeptides of the invention are the monospecific polypeptides of the invention (as previously described).

As described hereinafter, additional binding units such as immunoglobulin single variable domains having different antigenic specificity (ie other than CD123 and TCR) can be linked to the multispecific polypeptides of the invention. By combining immunoglobulin single variable domains with three or more specificities, trispecific, tetraspecific, etc. can be obtained. designs. These multispecific polypeptides are also referred to herein as "(multispecific) polypeptide(s) of the invention" or "construct(s) of the invention".

Thus, for example, a "bispecific polypeptide of the invention" is a polypeptide that contains or (essentially) consists of at least an immunoglobulin single variable domain against a first antigen (i.e., CD123) and at least one additional immunoglobulin variable domain against the second antigen (i.e., TCR), while a "trispecific polypeptide of the invention" is a polypeptide that contains or (essentially) consists of at least an immunoglobulin single variable domain against the first antigen ( i.e., CD123), at least one additional immunoglobulin single variable domain against a second antigen (i.e., TCR) and at least one additional immunoglobulin single variable domain against a third antigen (i.e., different from CD123 and TCR), etc. The immunoglobulin single variable domains may optionally be linked via one or more peptide linkers, as further described herein.

Accordingly, the present invention relates to polypeptides containing or (essentially) consisting of an immunoglobulin single variable domain that specifically binds a TCR and one or more ISVs that specifically bind CD123. In a further aspect, the present invention also relates to polypeptides comprising or (essentially) consisting of an immunoglobulin single variable domain that specifically binds a TCR and two or more ISVs that specifically bind CD123. Some non-limiting examples of such multispecific polypeptides or constructs thereof will become apparent from the further description provided herein.

It is understood (as also demonstrated in the Examples section) that an ISV that specifically binds TCR and one or more ISVs that specifically bind CD123 can be placed in any order in a polypeptide of the invention. More specifically, in one aspect, the TCR binding ISV is located at the N-terminus and the CD123-binding ISV is located at the C-terminus. In another aspect, one or more CD123 binding ISVs are located at the N-terminus and TCR-binding ISVs are located at the C-terminus. In another aspect, one or more ISVs that bind CD123 are located at the N-terminus, an ISV that binds TCRs are located centrally, and one or more additional ISVs that bind CD123 are located at the C-terminus. In a preferred aspect, the invention provides a polypeptide wherein an ISV that specifically binds a TCR is located at the N-terminus of the polypeptide.

In some aspects, the multispecific polypeptides of the invention comprise two or more ISVs that specifically bind CD123. In one aspect, two or more ISVs that specifically bind CD123 bind to the same epitope on CD123. In one aspect, such multispecific polypeptides of the invention may contain two or more ISVs related to 56A10. In another aspect, such polypeptides of the invention comprise two or more ISVs related to 55F03.

In a more preferred aspect, two or more ISVs that specifically bind CD123 bind to a different epitope. Accordingly, the present invention provides a multispecific polypeptide wherein two or more ISVs that specifically bind CD123 are biparatopic, comprising a first ISV and a second ISV, wherein the first ISV binds to an epitope on CD123 that is different from the epitope on CD123 bound by the second ISV. .

More specifically, the present invention relates to a multispecific polypeptide of the invention, wherein the first ISV that binds CD123 is selected from ISVs related to 56A10 and the second ISV that binds CD123 is selected from ISVs related to 55F03. As previously discussed, these biparatopic polypeptides of the invention have improved binding affinity for CD123 compared to the monospecific polypeptides of the invention due to active binding, also referred to as avidity.

It is understood (as also demonstrated in the Examples section) that ISVs that bind CD123 can be placed in any order in the multispecific polypeptide of the invention. More specifically, in one aspect, the second ISV (ie ISV related to 55F03) is located at the N-terminus of the first ISV (ie ISV related to 56A10). In another aspect, the second ISV (ie, ISV related to 55F03) is located at the C-terminus of the first ISV (ie, ISV related to 56A10). Some non-limiting examples of such multispecific constructs will become apparent from the further description provided herein.

Typically, the multispecific polypeptides of the invention combine high affinity and high specificity for antigen recognition on a target cell with T cell activation resulting in activation that is independent of the native specificity of T cells.

A "target cell", as used herein, is a cell that presents a particular antigen (ie, CD123) on its surface. In one aspect, a "target cell" is a cell that overexpresses CD123. In a preferred aspect, such a target cell is associated with a disease associated with CD123. In an even more preferred aspect, the target cell is a cancer cell that (over)expresses CD123. The term "cancer" refers to a pathological condition in mammals, which is usually characterized by impaired cell proliferation or survival.

"T cell activation" as used herein refers to one or more cellular responses of T cells, e.g., cytotoxic T cells, such as those selected from: proliferation, differentiation, secretion of cytokines, release of a cytotoxic effector molecule, cytotoxic activity, marker expression activation and redirected lysis of target cells. Used in this document, the term "cellular(s) response(s)" refers to the response of the cells as a result of intracellular signaling during the assembly of the TCR complex.

The mode of action of polypeptides that bind to both a cell surface molecule (such as, for example, CD123) on a target cell and the TCR of a T cell is well known. Bringing the T cell into close proximity to the target cell (such as, for example, a cell expressing CD123), ie. involvement of said T cell and clustering of the TCR complex results in T cell activation and subsequent killing of the target cell by the T cell. In the present invention, this process is used to combat a disease associated with CD123, such as a proliferative disease or an inflammatory condition. Typically, T cells are equipped with granules containing a lethal combination of pore-forming proteins called perforins and cell-death proteases called granzymes. Preferably, these proteins are delivered to target cells (such as, for example, cells expressing CD123) via the cytolytic synapse that is formed when T cells are in close proximity to the target cell that is to be killed. Typically, close proximity between a T cell and a target cell is achieved by binding the T cell to the MHC/peptide complex using the appropriate T cell receptor. The polypeptides of the present invention bring the T cell into close proximity to the target cell in the absence of a T cell receptor/MHC interaction.

Accordingly, the present invention provides a multispecific polypeptide as described herein, wherein said polypeptide directs a T cell to a target cell. Accordingly, the polypeptide(s) of the present invention "redirect(s) T cells to kill cells expressing CD123", which means that the polypeptide(s) of the present invention bring(s) the T cell into such close proximity to the cell, expressing CD123 that it is destroyed.

With one arm (ISV that binds TCR), the multispecific polypeptide of the invention binds to the constant domain of the TCR subunit, the protein component of the T cell receptor signal transduction complex on T cells. On the other arm (one or more ISVs that bind CD123), the multispecific polypeptide binds to CD123 on target cells. Preferably, T cell activation is only observed when the multispecific polypeptides are presented to T cells at the (site) CD123-expressing cells. Dependence of the antigen on target cells (ie, cells expressing CD123) for activation results in a favorable safety profile. The multispecific polypeptides of the invention exhibit highly specific binding to CD123. As such, off-target binding is avoided and target-independent T-cell activation is minimal, as illustrated herein. In one aspect, the multispecific polypeptides transiently bind T cells and target cells. Preferably, the multispecific polypeptide can induce the activation of quiescent polyclonal T cells, such as CD4 ⁺ and/or CD8 ⁺ T cells, for highly redirected lysis of target cells (ie cells expressing CD123). Preferably, the T cell is directed to the next target cell after the first target cell is lysed.

In one aspect, the present invention provides a multispecific polypeptide as described herein, wherein said multispecific polypeptide induces T cell activation.

In a further aspect, the present invention provides a multispecific polypeptide wherein said T cell activation is independent of MHC recognition.

"T cell activation independent of MHC recognition" as used herein refers to T cell activation that is independent of the binding of the MHC/peptide complex on the target cell to its corresponding T cell receptor on the T cell. If the T cell is in close proximity to the target cell, the target cell will be killed. Typically, close proximity between a T cell and a target cell is achieved by binding the T cell to the MHC/peptide complex using the appropriate T cell receptor. The multispecific polypeptides of the present invention bring the T cell into close proximity to the target cell in the absence of a T cell receptor/MHC interaction. The multispecific polypeptides bind to CD123 on the target cell and, as such, are presented to and associated with T cells, resulting in T cell activation and killing of the target cell.

Accordingly, in a further aspect, the present invention relates to a multispecific polypeptide, wherein said T cell activation is dependent on the presentation of said CD123-associated polypeptide on the target cell, the T cell.

In a further aspect, the present invention relates to a multispecific polypeptide, wherein said T cell activation induces one or more cellular responses by said T cells, said cellular response being selected from the group consisting of proliferation, differentiation, cytokine secretion, release of a cytotoxic effector molecule, cytotoxic activity, expression of activation markers, and redirected lysis of target cells.

Suitable assays for measuring T cell activation are known in the art, for example as described in WO 99/54440 or Schlereth et al. (2005, Cancer Immunol. Immunother. 20:1-12) or as shown in the examples or below.

Without limitation, T cell activation by the polypeptides of the invention can be measured by monitoring the upregulation of CD69, CD25 and various cell adhesion molecules, de novo expression and/or cytokine release (e.g., IFN-γ, TNF-α, IL-6, IL-2, IL-4 and IL-10), increased granzyme and perforin expression and/or cell proliferation, membrane blebbing, procaspase 3 and/or 7 activation, nuclear DNA fragmentation and/or caspase substrate cleavage, poly(ADP-ribose ) polymerase. Preferably, redirected lysis of target cells by multispecific polypeptides is independent of T cell receptor specificity, the presence of MHC class I and/or β2 microglobulin, and/or any costimulatory stimuli.

The polypeptides of the invention exhibit redirected lysis in vitro with previously unstimulated (ie, not activated) peripheral polyclonal CD8 ⁺ and CD4 ⁺ positive T cells, as further explained herein. Redirected lysis of target cells by recruiting T cells with the polypeptides of the invention includes the formation of a cytolytic synapse and the delivery of perforin and granzymes. Cell lysis by T cells has been described, for example, by Atkinson and Bleackley (1995, Crit. Rev. Immunol 15(3-4): 359-384). Preferably, the polypeptide of the present invention provides for the killing of target cells, for example, malignant tumor cells, by stimulating T cells to form pores and delivering pro-apoptotic components of cytotoxic T cell granules. Preferably, the T cells involved are capable of successive lysis of target cells. In vitro with the polypeptides of the invention, redirected lysis occurs at low picomolar concentrations, suggesting that very low amounts of the polypeptides of the invention must be bound to target cells to trigger T cells. As shown in the examples, a low ratio of effector target cells may indicate sequential lysis of target cells and demonstrates the high activity of the polypeptides of the invention.

As used herein, the term "activity" is a measure of the biological activity of an agent, such as a monospecific or multispecific polypeptide, ISV, or nanobody. Specific activity is a function of the amount of a polypeptide of the invention required for its specific effect. It is measured simply as the reciprocal of the IC ₅₀ for that polypeptide. With respect to the multispecific polypeptides of the invention, this refers to the ability of said polypeptide of the invention to induce T cell activation. The effectiveness of an agent can be determined by any suitable method known in the art, such as, for example, as described in the experimental section. Cell culture-based activity assays are often the preferred format for determining biological activity because they measure the physiological response elicited by an agent and can provide results within a relatively short period of time. Various types of cellular assays may be used based on the mechanism of action of the product, including, but not limited to, proliferation assays, cytotoxicity assays, cell killing assays, reporter gene assays, cell surface receptor binding assays, and assays to measure induction/inhibition of functionally important proteins or other signaling molecules (such as phosphorylated proteins, enzymes, cytokines, cAMP, and the like), T cell-mediated tumor cell killing assays (eg, as set forth in the Examples section), are all well known in the art. The results of a cell-based efficacy analysis can be expressed as "relative activity" as determined by comparing the multispecific polypeptide of the invention with the response obtained for the corresponding reference monovalent ISV, for example, a polypeptide containing only one ISV or one nanobody, optionally additionally containing an irrelevant nanobody (see experimental section).

The "potency" of a polypeptide according to the invention measures the maximum strength of the effect itself, at saturating concentrations of the polypeptide. Efficacy indicates the maximum response achievable from a polypeptide of the invention. It refers to the ability of a polypeptide to produce the desired (therapeutic) effect.

In one aspect, the multispecific polypeptide of the invention activates T cells resulting in the killing of CD123 expressing cells (such as MOLM-13 or KG1a cells) with an average EC ₅₀ between 10 nM and 1 pM as determined in a flow cytometric assay. (see also example 25).

More specifically, the polypeptide of the present invention induces T cell activation, wherein said T cell activation causes death of CD123 expressing cells (such as _MOLM -13 cells) with an average EC50 of 1 nM to 1 pM, e.g., an average EC50 of 500 pM or less, such as less than 400, 300, 200 or 100 pM or even less, such as less than 90, 80, 70, 60, 50, 40 or 30 pM or even less, the indicated EC ₅₀ value for the example is determined in the analysis based on flow cytometry as measured by TOPRO3 using MOLM-13 cells as target cells and human T cells as effector cells at a ratio of effector cells to target cells of 10:1.

More specifically, the polypeptide of the invention induces T cell activation, wherein said T cell activation causes lysis of CD123 expressing cells (such as MOLM-13 cells) with an average percentage of lysis greater than about 10%, such as 15%, 16% , 17%, 18%, 19%, or 20% or even more, for example, more than 25% or even more than 30%, the indicated percentage of lysis, for example, is determined in the analysis based on flow cytometry from TOPRO3 data using MOLM-13 cells as target cells and human T cells as effector cells at a ratio of effector cells to target cells of 10:1.

More specifically, the polypeptide of the present invention induces T cell activation, wherein said T cell activation causes death of CD123 expressing cells (such as _KG1a cells) with an average EC50 of 10 nM to 10 pM, for example an average EC50. 5 nM or less, such as less than 4, 3, 2, or 1 nM, or even less, such as less than 90, 80, 70, or 60 pM or even less, the specified EC50 value, for example, is determined in the analysis based on flow cytometry according to with TOPRO3 using KG1a cells as target cells and human T cells as effector cells at a ratio of effector cells to target cells of 10:1.

More specifically, the polypeptide of the invention induces T cell activation, wherein said T cell activation causes lysis of CD123 expressing cells (such as KG1a cells) with an average percentage of lysis greater than about 10%, such as 15%, 16%, 17 % or 18% or even more, for example, more than 24%, the specified percentage of lysis, for example, is determined in the analysis based on flow cytometry according to TOPRO3 using KG1a cells as target cells and human T cells as effector cells when the ratio of effector cells and target cells is 10:1.

In another aspect, the multispecific polypeptides of the invention activate T cells and may, as such, induce cytokine secretion. Accordingly, the polypeptides induce the secretion of IFN-γ or IL-6 with an average EC ₅₀ of 100 nM to 10 pM. (see also example 30).

More specifically, the polypeptide of the present invention induces T cell activation, wherein said T cell activation causes secretion of IFN-γ with an average EC ₅₀ value of 100 nM to 10 pM, for example, with an average EC ₅₀ value of 50 nM or less, for example, less than 40, 30, 20, 10 or 9 nM or even less such as less than 8, 7, 6, 5, 4, 3, 2 or 1 nM or even less such as less than 500 pM or even less such as less than 400 , 300, 200 or 100 pM or even less, the indicated value of EC50, for example, is determined in the analysis based on ELISA, as, for example, further explained in example 30.

More specifically, the polypeptide of the present invention induces T cell activation, wherein said T cell activation causes secretion of IL-6 with an average EC ₅₀ value of 100 nM to 10 pM, for example, with an average EC ₅₀ value of 50 nM or less, for example, less than 40, 30, 20 or 10 nM or even less, such as less than 9, 8, 7, 6, 5, 4, 3, 2 or 1 nM or even less, such as less than 500 pM or even less, such as less than 400, 300, 200 or 100 pM or even less, the indicated EC ₅₀ value, for example, is determined in an ELISA-based assay, as, for example, further explained in example 30.

In another aspect, the multispecific polypeptides of the invention induce depletion of plasmacytoid cells (pDCs) and basophils (see also Example 31).

Accordingly, the present invention relates to a polypeptide, where the specified activation of T cells causes the depletion of plasmacytoid cells (pDC) and basophils.

In another aspect, the multispecific polypeptides of the invention can further induce T cell proliferation (see also Example 39).

Accordingly, the present invention relates to a polypeptide, where the specified activation of T cells causes the proliferation of these T cells.

The multispecific polypeptides of the invention comprise one or more ISVs that specifically bind CD123, which have been carefully selected for their specificity. As such, the multispecific polypeptides of the invention exhibit highly specific binding to CD123, allowing them to kill CD123 expressing target cells. In contrast, in the absence of cells expressing CD123, only minimal killing was observed, highlighting the safety of the polypeptides of the invention.

Accordingly, in another aspect, the present invention provides a polypeptide in which T cell activation in the absence of CD123 positive cells is minimal (see also Examples 36 to 38).

More specifically, the present invention relates to a polypeptide wherein T cell activation-induced lysis of CD123-negative cells is no more than about 10%, such as 9% or less, such as 8%, 7% or 6% or even less, said lysis, for example, defined as the mean percent lysis in a TOPRO3 flow cytometric assay using CD123-negative cells such as U-937 or NCI-H929 cells as target cells and human T cells as effector cells when the ratio of effector cells and target cells is 10:1.

More specifically, the present invention relates to a polypeptide that does not induce the secretion of IFN-γ and IL-6 in the presence of CD123 negative cells, said secretion being determined, for example, in an ELISA-based assay.

The inventors found that certain multispecific polypeptides of the invention, comprising a TCR binding ISV of the invention and one or more CD123 binding ISVs of the invention, were particularly useful in targeting T cells to kill CD123 expressing cells. With these multispecific polypeptides of the invention, T cell activation was minimal in the absence of cells expressing CD123.

Accordingly, the present invention relates to a multispecific polypeptide that redirects T cells to kill CD123-expressing cells, comprising an immunoglobulin single variable domain (ISV) that specifically binds a T cell receptor (TCR) and one or more ISVs that specifically bind CD123, where the ISV that specifically binds the TCR essentially consists of 4 framework regions (FR1-FR4, respectively) and 3 complementarity-determining regions (CDR1-CDR3, respectively), in which:

i) CDR1 is selected from the group consisting of:

a) SEQ ID NO: 181-191; or

b) amino acid sequences with a difference of 4, 3, 2 or 1 amino acid with the amino acid sequence of one of SEQ ID NOs: 181-191; provided that an ISV comprising a CDR1 with a 4, 3, 2, or 1 amino acid difference binds the TCR with approximately the same or higher affinity as compared to binding to an ISV comprising a CDR1 with no 4, 3, 2, or 1 amino acid difference, where the specified affinity is measured using surface plasmon resonance;

and/or

ii) CDR2 is selected from the group consisting of:

c) SEQ ID NOS: 192-217; or

d) amino acid sequences with a difference of 4, 3, 2 or 1 amino acid with the amino acid sequence of one of SEQ ID NOs: 192-217; provided that an ISV comprising a CDR2 with a 4, 3, 2, or 1 amino acid difference binds the TCR with approximately the same or higher affinity as compared to binding of an ISV comprising a CDR2 with no 4, 3, 2, or 1 amino acid difference, wherein said affinity measured by surface plasmon resonance;

and/or

iii) CDR3 is selected from the group consisting of:

e) SEQ ID NOS: 218-225; or

f) amino acid sequences with a difference of 4, 3, 2 or 1 amino acid with the amino acid sequence of one of SEQ ID NOs: 218-225; provided that an ISV comprising a CDR3 with a 4, 3, 2, or 1 amino acid difference binds the TCR with approximately the same or higher affinity as compared to binding of an ISV comprising a CDR3 with no 4, 3, 2, or 1 amino acid difference, wherein said affinity measured by surface plasmon resonance;

and where one or more ISVs that specifically bind CD123 essentially consist of 4 framework regions (FR1-FR4, respectively) and 3 complementarity-determining regions (CDR1-CDR3, respectively), in which:

i) CDR1 is selected from the group consisting of:

a) SEQ ID NO: 11-16; or

b) amino acid sequences with a difference of 4, 3, 2 or 1 amino acid with the amino acid sequence of one of SEQ ID NOs: 11-16; provided that an ISV comprising a CDR1 with a 4, 3, 2, or 1 amino acid difference binds CD123 with approximately the same or higher affinity as compared to binding to an ISV comprising a CDR1 with no 4, 3, 2, or 1 amino acid difference, where the specified affinity is measured using surface plasmon resonance;

and/or

ii) CDR2 is selected from the group consisting of:

c) SEQ ID NO: 17-20; or

d) amino acid sequences with a difference of 4, 3, 2 or 1 amino acid with the amino acid sequence of one of SEQ ID NOs: 17-20; provided that an ISV comprising a CDR2 with a 4, 3, 2, or 1 amino acid difference binds CD123 with approximately the same or higher affinity as compared to binding of an ISV comprising a CDR2 with no 4, 3, 2, or 1 amino acid difference, wherein said affinity measured by surface plasmon resonance;

and/or

iii) CDR3 is selected from the group consisting of:

e) SEQ ID NOS: 21-25; or

f) amino acid sequences with a difference of 4, 3, 2 or 1 amino acid with the amino acid sequence of one of SEQ ID NOs: 21-25; provided that an ISV comprising a CDR3 with a 4, 3, 2, or 1 amino acid difference binds CD123 with approximately the same or higher affinity as compared to binding to an ISV comprising a CDR3 with no 4, 3, 2, or 1 amino acid difference, where the specified affinity is measured using surface plasmon resonance.

In a further aspect, the present invention relates to a multispecific polypeptide, wherein an ISV that specifically binds a TCR essentially consists of 4 framework regions (FR1-FR4, respectively) and 3 complementarity-determining regions (CDR1-CDR3, respectively), in which:

i) CDR1 is selected from the group consisting of:

a) SEQ ID NO: 181-191; or

b) amino acid sequences with a difference of 4, 3, 2 or 1 amino acid with the amino acid sequence of one of SEQ ID NOs: 181-191; provided that an ISV comprising a CDR1 with a 4, 3, 2, or 1 amino acid difference binds the TCR with approximately the same or higher affinity as compared to binding to an ISV comprising a CDR1 with no 4, 3, 2, or 1 amino acid difference, where the specified affinity is measured using surface plasmon resonance;

and

ii) CDR2 is selected from the group consisting of:

c) SEQ ID NOS: 192-217; or

d) amino acid sequences with a difference of 4, 3, 2 or 1 amino acid with the amino acid sequence of one of SEQ ID NOs: 192-217; provided that an ISV comprising a CDR2 with a 4, 3, 2, or 1 amino acid difference binds the TCR with approximately the same or higher affinity as compared to binding of an ISV comprising a CDR2 with no 4, 3, 2, or 1 amino acid difference, wherein said affinity measured by surface plasmon resonance;

and

iii) CDR3 is selected from the group consisting of:

e) SEQ ID NOS: 218-225; or

f) amino acid sequences with a difference of 4, 3, 2 or 1 amino acid with the amino acid sequence of one of SEQ ID NOs: 218-225; provided that an ISV comprising a CDR3 with a 4, 3, 2, or 1 amino acid difference binds the TCR with approximately the same or higher affinity as compared to binding of an ISV comprising a CDR3 with no 4, 3, 2, or 1 amino acid difference, wherein said affinity measured by surface plasmon resonance;

and where one or more ISVs that specifically bind CD123 essentially consist of 4 framework regions (FR1-FR4, respectively) and 3 complementarity-determining regions (CDR1-CDR3, respectively) in which:

i) CDR1 is selected from the group consisting of:

a) SEQ ID NO: 11-16; or

b) amino acid sequences with a difference of 4, 3, 2 or 1 amino acid with the amino acid sequence of one of SEQ ID NOs: 11-16; provided that an ISV comprising a CDR1 with a 4, 3, 2, or 1 amino acid difference binds CD123 with approximately the same or higher affinity as compared to binding to an ISV comprising a CDR1 with no 4, 3, 2, or 1 amino acid difference, where the specified affinity is measured using surface plasmon resonance;

and

ii) CDR2 is selected from the group consisting of:

c) SEQ ID NO: 17-20; or

d) amino acid sequences with a difference of 4, 3, 2 or 1 amino acid with the amino acid sequence of one of SEQ ID NOs: 17-20; provided that an ISV comprising a CDR2 with a 4, 3, 2, or 1 amino acid difference binds CD123 with approximately the same or higher affinity as compared to binding of an ISV comprising a CDR2 with no 4, 3, 2, or 1 amino acid difference, wherein said affinity measured by surface plasmon resonance;

and

iii) CDR3 is selected from the group consisting of:

e) SEQ ID NOS: 21-25; or

f) amino acid sequences with a difference of 4, 3, 2 or 1 amino acid with the amino acid sequence of one of SEQ ID NOs: 21-25; provided that an ISV comprising a CDR3 with a 4, 3, 2, or 1 amino acid difference binds CD123 with approximately the same or higher affinity as compared to binding to an ISV comprising a CDR3 with no 4, 3, 2, or 1 amino acid difference, where the specified affinity is measured using surface plasmon resonance.

In a specific aspect, the present invention relates to a multispecific polypeptide, wherein the ISV that specifically binds the TCR is essentially composed of 4 framework regions (FR1-FR4, respectively) and 3 complementarity-determining regions (CDR1-CDR3, respectively), in which:

i) CDR1 is selected from the group consisting of:

a) SEQ ID NO: 181-191; or

b) amino acid sequences with a difference of 4, 3, 2 or 1 amino acid with the amino acid sequence of one of SEQ ID NOs: 181-191, where the difference of 4, 3, 2 or 1 amino acid is present at positions 2, 4, 5, 6, 8 and/or 10 CDR1 (positions 27, 29, 30, 31, 33 and/or 35 according to Kabat numbering); provided that an ISV comprising a CDR1 with a 4, 3, 2, or 1 amino acid difference binds the TCR with approximately the same or higher affinity as compared to binding to an ISV comprising a CDR1 with no 4, 3, 2, or 1 amino acid difference, where the specified affinity is measured using surface plasmon resonance;

and

ii) CDR2 is selected from the group consisting of:

c) SEQ ID NOS: 192-217; or

d) amino acid sequences with a difference of 4, 3, 2 or 1 amino acid with the amino acid sequence of one of SEQ ID NOs: 192-217, where the difference of 4, 3, 2 or 1 amino acid is present at positions 1, 3, 5, 7, 8 and/or 9 CDR2 (positions 50, 52, 54, 56, 57 and/or 58 according to Kabat numbering); provided that an ISV comprising a CDR2 with a 4, 3, 2, or 1 amino acid difference binds the TCR with approximately the same or higher affinity as compared to binding of an ISV comprising a CDR2 with no 4, 3, 2, or 1 amino acid difference, wherein said affinity measured by surface plasmon resonance;

and

iii) CDR3 is selected from the group consisting of:

e) SEQ ID NOS: 218-225; or

f) amino acid sequences with a difference of 4, 3, 2 or 1 amino acid with an amino acid sequence of one of SEQ ID NOs: 218-225, where the difference of 4, 3, 2 or 1 amino acid is present at positions 1, 4, 5 and/or 8 CDR3 (positions 95, 98, 99 and/or 101 according to Kabat numbering); provided that an ISV comprising a CDR3 with a 4, 3, 2, or 1 amino acid difference binds the TCR with approximately the same or higher affinity as compared to binding of an ISV comprising a CDR3 with no 4, 3, 2, or 1 amino acid difference, wherein said affinity measured by surface plasmon resonance;

and where ISV that specifically binds CD123 is as described later in this document.

In another aspect, the present invention relates to a multispecific polypeptide as described above, wherein the ISV that specifically binds a TCR is essentially composed of 4 framework regions (FR1-FR4, respectively) and 3 complementarity-determining regions (CDR1-CDR3, respectively), in which CDR1 is selected from the group consisting of:

a) SEQ ID NO: 181; or

b) amino acid sequences with a difference of 4, 3, 2 or 1 amino acid from the amino acid sequence of SEQ ID NO: 181, where

- in position 2 D has been changed to A, S, E or G;

- in position 4 H has been changed to Y;

- in position 5 K has been changed to L;

- in position 6 I was changed to L;

- in position 8, F has been changed to I or V; and/or

- in position 10 G has been changed to S;

provided that an ISV comprising a CDR1 with a 4, 3, 2, or 1 amino acid difference binds the TCR with approximately the same or higher affinity as compared to binding to an ISV comprising a CDR1 with no 4, 3, 2, or 1 amino acid difference, where the specified affinity is measured using surface plasmon resonance.

In another aspect, the present invention relates to a multispecific polypeptide as described above, wherein the ISV that specifically binds a TCR is essentially composed of 4 framework regions (FR1-FR4, respectively) and 3 complementarity-determining regions (CDR1-CDR3, respectively), in in which CDR2 is selected from the group consisting of:

a) SEQ ID NO: 192; or

b) amino acid sequences with a difference of 4, 3, 2 or 1 amino acid from the amino acid sequence of SEQ ID NO: 192, where

- in position 1 H has been changed to T or R;

- in position 3 S has been changed to T or A;

- in position 5 G has been changed to S or A;

- in position 7 Q has been changed to D, E, T, A or V;

- in position 8, T was changed to A or V; and/or

- in position 9, D has been changed to A, Q, N, V or S;

provided that an ISV comprising a CDR2 with a 4, 3, 2, or 1 amino acid difference binds the TCR with approximately the same or higher affinity as compared to binding of an ISV comprising a CDR2 with no 4, 3, 2, or 1 amino acid difference, wherein this affinity was measured using surface plasmon resonance.

In another aspect, the present invention relates to a multispecific polypeptide as described above, wherein the ISV that specifically binds a TCR is essentially composed of 4 framework regions (FR1-FR4, respectively) and 3 complementarity-determining regions (CDR1-CDR3, respectively), in in which CDR3 is selected from the group consisting of:

a) SEQ ID NO: 218; or

b) amino acid sequences with a difference of 4, 3, 2 or 1 amino acid from the amino acid sequence of SEQ ID NO: 218, where

- in position 1, F has been changed to Y, L or G;

- in position 4 I was changed to L;

- in position 5 Y has been changed to W; and/or

- in position 8 D has been changed to N or S;

provided that an ISV comprising a CDR3 with a 4, 3, 2, or 1 amino acid difference binds the TCR with approximately the same or higher affinity as compared to binding of an ISV comprising a CDR3 with no 4, 3, 2, or 1 amino acid difference, wherein this affinity was measured using surface plasmon resonance.

Accordingly, the present invention relates to a multispecific polypeptide as described above, wherein an ISV that specifically binds a TCR essentially consists of 4 framework regions (FR1-FR4, respectively) and 3 complementarity-determining regions (CDR1-CDR3, respectively), in which :

i) CDR1 is selected from the group consisting of:

a) SEQ ID NO: 181; or

b) amino acid sequences with a difference of 4, 3, 2 or 1 amino acid from the amino acid sequence of SEQ ID NO: 181, where

- in position 2 D has been changed to A, S, E or G;

- in position 4 H has been changed to Y;

- in position 5 K has been changed to L;

- in position 6 I was changed to L;

- in position 8, F has been changed to I or V; and/or

- in position 10 G has been changed to S;

provided that an ISV comprising a CDR1 with a 4, 3, 2, or 1 amino acid difference binds the TCR with approximately the same or higher affinity as compared to binding to an ISV comprising a CDR1 with no 4, 3, 2, or 1 amino acid difference, where the specified affinity is measured using surface plasmon resonance;

and

ii) CDR2 is selected from the group consisting of:

c) SEQ ID NO: 192; or

d) amino acid sequences with a difference of 4, 3, 2 or 1 amino acid from the amino acid sequence of SEQ ID NO: 192, where

- in position 1 H has been changed to T or R;

- in position 3 S has been changed to T or A;

- in position 5 G has been changed to S or A;

- in position 7 Q has been changed to D, E, T, A or V;

- in position 8, T was changed to A or V; and/or

- in position 9, D has been changed to A, Q, N, V or S;

provided that an ISV comprising a CDR2 with a 4, 3, 2, or 1 amino acid difference binds the TCR with approximately the same or higher affinity as compared to binding of an ISV comprising a CDR2 with no 4, 3, 2, or 1 amino acid difference, wherein said affinity measured by surface plasmon resonance;

and

iii) CDR3 is selected from the group consisting of:

e) SEQ ID NO: 218; or

f) amino acid sequences with a difference of 4, 3, 2 or 1 amino acid from the amino acid sequence of SEQ ID NO: 218, where

- in position 1, F has been changed to Y, L or G;

- in position 4 I was changed to L;

- in position 5 Y has been changed to W; and/or

- in position 8 D has been changed to N or S;

provided that an ISV comprising a CDR3 with a 4, 3, 2, or 1 amino acid difference binds the TCR with approximately the same or higher affinity as compared to binding of an ISV comprising a CDR3 with no 4, 3, 2, or 1 amino acid difference, wherein said affinity measured by surface plasmon resonance;

and where ISV that specifically binds CD123 is as described later in this document.

In another aspect, the present invention relates to a multispecific polypeptide, where the ISV that specifically binds the TCR is essentially composed of 4 framework regions (FR1-FR4, respectively) and 3 complementarity-determining regions (CDR1-CDR3, respectively), in which CDR1 is selected from the group consisting of SEQ ID NOs: 181-191, CDR2 selected from the group consisting of SEQ ID NOs: 192-217, and CDR3 selected from the group consisting of SEQ ID NOs: 218-225 and in which the ISV which specifically binds CD123 as described later in this document.

Accordingly, the present invention relates to a multispecific polypeptide, wherein the ISV that specifically binds the TCR is essentially composed of 4 framework regions (FR1-FR4, respectively) and 3 complementarity-determining regions (CDR1-CDR3, respectively), in which CDR1 is SEQ ID NO: 181, CDR2 is SEQ ID NO: 192 and CDR3 is SEQ ID NO: 218, and wherein the ISV that specifically binds CD123 is as described hereinafter.

In a preferred aspect, the present invention provides a multispecific polypeptide wherein the ISV that specifically binds the TCR is selected from the group consisting of SEQ ID NOs: 42 and 78-180, or from ISVs that have greater than 80% sequence identity. greater than 85%, greater than 90%, greater than 95%, or even greater than 99% with one of SEQ ID NOs: 42 and 78-180 and wherein the ISV that specifically binds CD123 is as described hereinafter.

In addition to the TCR binding ISV as described above, in the multispecific polypeptides of the invention, one or more ISVs that specifically bind CD123 are 56A10 and/or 55F03.

Accordingly, the present invention relates to a multispecific polypeptide, wherein the ISV that specifically binds TCR is as described herein, and where one or more ISVs that specifically bind CD123 are essentially composed of 4 framework regions (FR1-FR4, respectively) and 3 complementarity-determining regions (CDR1-CDR3, respectively), in which:

i) CDR1 is selected from the group consisting of:

a) SEQ ID NO: 11-16; or

b) amino acid sequences with a difference of 4, 3, 2 or 1 amino acid with the amino acid sequence of one of SEQ ID NOs: 11-16, where the difference of 4, 3, 2 or 1 amino acid is present at positions 3, 6, 7 and/or 8 CDR1 (positions 28, 31, 32 and/or 33 according to Kabat numbering); provided that an ISV comprising a CDR1 with a 4, 3, 2, or 1 amino acid difference binds CD123 with approximately the same or higher affinity as compared to binding to an ISV comprising a CDR1 with no 4, 3, 2, or 1 amino acid difference, where the specified affinity is measured using surface plasmon resonance;

and

ii) CDR2 is selected from the group consisting of:

c) SEQ ID NO: 17-20; or

d) amino acid sequences with a difference of 3, 2 or 1 amino acid with the amino acid sequence of one of SEQ ID NOs: 17-20, where the difference of 3, 2 or 1 amino acid is present at positions 3 to 6 and/or 10 of CDR2 (positions 52, 54 and/or 58 according to Kabat numbering); provided that an ISV comprising a CDR2 with a 3, 2, or 1 amino acid difference binds CD123 with approximately the same or higher affinity as compared to binding of an ISV comprising a CDR2 with no 3, 2, or 1 amino acid difference, wherein said affinity is measured with using surface plasmon resonance;

and

iii) CDR3 is selected from the group consisting of:

e) SEQ ID NOS: 21-25; or

f) amino acid sequences with a difference of 3, 2 or 1 amino acid with the amino acid sequence of one of SEQ ID NOs: 21-25, where the difference of 3, 2 or 1 amino acid is present at positions 3, 4 and/or 5 of CDR3 (positions 97, 98 and/or 99 according to Kabat numbering); provided that an ISV comprising a CDR3 with a 3, 2, or 1 amino acid difference binds CD123 with approximately the same or higher affinity as compared to binding to an ISV comprising a CDR3 with no 3, 2, or 1 amino acid difference, where said affinity is measured using surface plasmon resonance.

In one aspect, one or more ISVs that specifically binds CD123 may be an ISV related to 56A10.

Accordingly, the present invention relates to a multispecific polypeptide as described above, wherein one or more ISVs that specifically bind CD123 are essentially composed of 4 framework regions (FR1-FR4, respectively) and 3 complementarity-determining regions (CDR1-CDR3, respectively) where CDR1 is selected from the group consisting of:

a) SEQ ID NO: 11; or

b) amino acid sequences with a difference of 4, 3, 2 or 1 amino acid from the amino acid sequence of SEQ ID NO: 11, where

- in position 3, T has been changed to S or P;

- in position 6 I was changed to S;

- in position 7 N has been changed to D; and/or

- in position 8 D has been changed to V or A;

provided that an ISV comprising a CDR1 with a 4, 3, 2, or 1 amino acid difference binds CD123 with approximately the same or higher affinity as compared to binding to an ISV comprising a CDR1 with no 4, 3, 2, or 1 amino acid difference, where the specified affinity is measured using surface plasmon resonance.

In another aspect, the present invention relates to a multispecific polypeptide as described above, wherein one or more ISVs that specifically bind CD123 are essentially composed of 4 framework regions (FR1-FR4, respectively) and 3 complementarity-determining regions (CDR1-CDR3, respectively). ), where CDR2 is SEQ ID NO: 17.

In another aspect, the present invention relates to a multispecific polypeptide as described above, wherein one or more ISVs that specifically bind CD123 are essentially composed of 4 framework regions (FR1-FR4, respectively) and 3 complementarity-determining regions (CDR1-CDR3, respectively). ), where CDR3 is selected from the group consisting of:

a) SEQ ID NO: 21; or

b) amino acid sequences with a difference of 1 amino acid from the amino acid sequence of SEQ ID NO: 21, where

- in position 3 P has been changed to A;

provided that an ISV comprising a CDR3 with a 1 amino acid difference binds CD123 with approximately the same or higher affinity as compared to binding to an ISV comprising a CDR3 without a 1 amino acid difference, said affinity being measured by surface plasmon resonance.

Accordingly, the present invention relates to a multispecific polypeptide, wherein the ISV that specifically binds TCR is as described herein, and where one or more ISVs that specifically bind CD123 essentially consists of 4 framework regions (FR1-FR4, respectively) and 3 complementarity-determining regions (CDR1-CDR3, respectively), in which:

i) CDR1 is selected from the group consisting of:

a) SEQ ID NO: 11; or

b) amino acid sequences with a difference of 4, 3, 2 or 1 amino acid from the amino acid sequence of SEQ ID NO: 11, where

- in position 3, T has been changed to S or P;

- in position 6 I was changed to S;

- in position 7 N has been changed to D; and/or

- in position 8 D has been changed to V or A;

provided that a polypeptide comprising a CDR1 with a 4, 3, 2, or 1 amino acid difference binds CD123 with approximately the same or higher affinity as compared to binding by a polypeptide containing a CDR1 with no 4, 3, 2, or 1 amino acid difference, wherein said affinity measured by surface plasmon resonance;

and

ii) CDR2 is SEQ ID NO: 17;

and

iii) CDR3 is selected from the group consisting of:

c) SEQ ID NO: 21; or

d) amino acid sequences with a difference of 1 amino acid from the amino acid sequence of SEQ ID NO: 21, where

- in position 3 P has been changed to A;

provided that a polypeptide containing a CDR3 with a 1 amino acid difference binds CD123 with approximately the same or higher affinity as compared to binding by a polypeptide containing a CDR3 with no 1 amino acid difference, where said affinity is measured by surface plasmon resonance.

In another aspect, the present invention relates to a multispecific polypeptide, where the ISV that specifically binds TCR is as described herein, and where one or more ISVs that specifically bind CD123, essentially consists of 4 framework regions (FR1-FR4 , respectively) and 3 complementaries. defining regions (CDR1-CDR3, respectively), in which CDR1 is selected from the group consisting of SEQ ID NO: 11-15, CDR2 is SEQ ID NO: 17, and CDR3 is selected from the group consisting of SEQ ID NO: 21 - 22.

Accordingly, the present invention relates to a multispecific polypeptide, wherein the ISV that specifically binds TCR is as described herein, and where one or more ISVs that specifically bind CD123 essentially consists of 4 framework regions (FR1-FR4, respectively) and 3 complementarity determining regions (CDR1-CDR3, respectively), where CDR1 is SEQ ID NO: 11, CDR2 is SEQ ID NO: 17, and CDR3 is SEQ ID NO: 21.

In a preferred aspect, the present invention relates to a multispecific polypeptide, wherein the ISV that specifically binds the TCR is as described herein, and where one or more ISVs that specifically bind CD123 are selected from the group consisting of SEQ ID NO: 1 -6 or from ISVs that have greater than 80%, greater than 85%, greater than 90%, greater than 95%, or even greater than 99% sequence identity with one of SEQ ID NOs: 1-6.

In addition to or in addition to the above, an ISV that specifically binds CD123 may be an ISV related to 55F03.

Accordingly, the present invention also relates to a multispecific polypeptide as described above, wherein one or more ISVs that specifically bind CD123 are essentially composed of 4 framework regions (FR1-FR4, respectively) and 3 complementarity-determining regions (CDR1-CDR3, respectively). ), where CDR1 is SEQ ID NO: 16.

In another aspect, the present invention relates to a multispecific polypeptide as described above, wherein one or more ISVs that specifically bind CD123 are essentially composed of 4 framework regions (FR1-FR4, respectively) and 3 complementarity-determining regions (CDR1-CDR3, respectively). ), where CDR2 is selected from the group consisting of:

a) SEQ ID NO: 18; or

b) amino acid sequences with a difference of 3, 2 or 1 amino acid from the amino acid sequence of SEQ ID NO: 18, where

- in position 3 Y has been changed to W;

- in position 6 N has been changed to S; and/or

- in position 10 Q has been changed to E;

provided that an ISV comprising a CDR2 with a 3, 2, or 1 amino acid difference binds CD123 with approximately the same or higher affinity as compared to binding of an ISV comprising a CDR2 with no 3, 2, or 1 amino acid difference, wherein said affinity is measured with using surface plasmon resonance.

In another aspect, the present invention relates to a multispecific polypeptide as described above, wherein one or more ISVs that specifically bind CD123 are essentially composed of 4 framework regions (FR1-FR4, respectively) and 3 complementarity-determining regions (CDR1-CDR3, respectively). ), where CDR3 is selected from the group consisting of:

a) SEQ ID NO: 23; or

b) amino acid sequences with a difference of 2 or 1 amino acid from the amino acid sequence of SEQ ID NO: 23, where

- in position 4, E was changed to R; and/or

- in position 5, T was changed to D or Y;

provided that an ISV comprising a CDR3 with a 2 or 1 amino acid difference binds CD123 with approximately the same or higher affinity as compared to binding to an ISV comprising a CDR3 with no 2 or 1 amino acid difference, where said affinity is measured by surface plasmon resonance.

Accordingly, the present invention relates to a multispecific polypeptide, wherein the ISV that specifically binds TCR is as described herein, and where one or more ISVs that specifically bind CD123 essentially consists of 4 framework regions (FR1-FR4, respectively) and 3 complementarity-determining regions (CDR1-CDR3, respectively), in which:

i) CDR1 is SEQ ID NO: 16;

and

ii) CDR2 is selected from the group consisting of:

a) SEQ ID NO: 18; or

b) amino acid sequences with a difference of 3, 2 or 1 amino acid from the amino acid sequence of SEQ ID NO: 18, where

- in position 3 Y has been changed to W;

- in position 6 N has been changed to S; and/or

- in position 10 Q has been changed to E;

provided that a polypeptide containing a CDR2 with a difference of 3, 2, or 1 amino acid binds CD123 with approximately the same or higher affinity compared to binding by a polypeptide containing a CDR2 with no difference of 3, 2, or 1 amino acid, where the specified affinity is measured with using surface plasmon resonance;

and

iii) CDR3 is selected from the group consisting of:

c) SEQ ID NO: 23; or

d) amino acid sequences with a difference of 2 or 1 amino acid from the amino acid sequence of SEQ ID NO: 23, where

- in position 4, E was changed to R; and/or

- in position 5, T was changed to D or Y;

provided that a polypeptide containing CDR3 with a difference of 2 or 1 amino acid binds CD123 with approximately the same or higher affinity compared to binding by a polypeptide containing CDR3 with no difference of 2 or 1 amino acid, where the specified affinity is measured using surface plasmon resonance .

In another aspect, the present invention relates to a multispecific polypeptide, where the ISV that specifically binds TCR is as described herein, and where one or more ISVs that specifically bind CD123, essentially consists of 4 framework regions (FR1-FR4 , respectively) and 3 complementarity defining regions (CDR1-CDR3, respectively), in which CDR1 is SEQ ID NO: 16, CDR2 is selected from the group consisting of SEQ ID NO: 18-20, and CDR3 is selected from the group consisting of SEQ ID NO: 23-25.

Accordingly, the present invention relates to a multispecific polypeptide, wherein the ISV that specifically binds TCR is as described herein, and where one or more ISVs that specifically bind CD123 essentially consists of 4 framework regions (FR1-FR4, respectively) and 3 complementarity determining regions (CDR1-CDR3, respectively), where CDR1 is SEQ ID NO: 16, CDR2 is SEQ ID NO: 18, and CDR3 is SEQ ID NO: 23.

In a preferred aspect, the present invention relates to a multispecific polypeptide, where the ISV that specifically binds the TCR is as described herein, and where one or more ISVs that specifically bind CD123 are selected from the group consisting of SEQ ID NO: 7 -10 or from ISVs that have greater than 80%, greater than 85%, greater than 90%, greater than 95%, or even greater than 99% sequence identity with one of SEQ ID NOs: 7-10.

As detailed for monospecific polypeptides, the immunoglobulin single variable domains present in a multispecific polypeptide of the invention may consist of a light chain variable domain sequence (eg, a V _L sequence) or a heavy chain variable domain sequence (eg, a V _H sequence); they may consist of a heavy chain variable domain sequence that is derived from a conventional four-chain antibody, or a heavy chain variable domain sequence that is derived from a heavy chain-only antibody. In a preferred aspect, they consist of a domain antibody (or an amino acid that is suitable for use as a domain antibody), a single domain antibody (or an amino acid that is suitable for use as a single domain antibody), a "dAb" (or an amino acid that is suitable for use in as a dAb), a nanobody (including but not limited to V _HH ), a humanized V _HH , an overblown V _H , or an affinity matured V _HH sequence. The immunoglobulin single variable domains may consist of a partially or fully humanized nanobody or a partially or fully humanized V _HH . The immunoglobulin single variable domains may also contain mutations (as described herein) that are effective in preventing or reducing the binding of so-called "pre-existing antibodies" to the immunoglobulin single variable domains and constructs of the invention. In a preferred aspect of the invention, the immunoglobulin single variable domains included in the multispecific polypeptide of the invention are one or more monospecific polypeptides of the invention, as defined herein.

Preferred polypeptides of the invention may be selected from the group consisting of SEQ ID NOS: 47, 49, 52, 53, 55, 56 and 58-61 (see also Table A-7). In a further aspect, the polypeptide is selected from the group consisting of SEQ ID NOs: 47, 49, 52, 53, 55, 56, and 58-61, or from polypeptides that have sequence identity greater than 80%, greater than 85%, greater than 90%, more than 95% or even more than 99% with one of SEQ ID NOs: 47, 49, 52, 53, 55, 56 and 58-61.

The multispecific polypeptides of the invention may contain one or more other groups, residues, fragments, or linking units to form the "polypeptide(s) of the invention" or "construct(s) of the invention", as further described herein. For example, such a binding unit may be an amino acid sequence that increases the half-life (also referred to herein as "extended half-life" and "extended half-life construct") of the polypeptide. According to a specific but non-limiting aspect of the invention, the polypeptides of the invention may thus comprise, in addition to one or more anti-CD123 immunoglobulin single variable domains and one anti-TCR immunoglobulin single variable domain, at least one anti-TCR immunoglobulin single variable domain. serum albumin (such as human serum albumin). Accordingly, the present invention relates to the construct described herein, wherein said binding unit that provides the polypeptide with an extended half-life is an immunoglobulin single variable domain that binds serum albumin. In a further aspect, the present invention relates to the construct described herein, wherein said ISV that binds serum albumin essentially consists of a single domain antibody, dAb, nanobody, V _HH , humanized V _HH , or overblown VH.

In a preferred aspect, the ISV that binds serum albumin is selected from the group consisting of SEQ ID NO 43 or 351-362.

In a preferred aspect, the ISVs are directly linked to each other or linked to each other via a linker.

Preferred constructs of the invention may be selected from the group of constructs consisting of SEQ ID NOs: 63-67, or constructs that have greater than 80%, greater than 85%, greater than 90%, greater than 95%, or even greater than 99% sequence identity with one of SEQ ID NOs: 63-67.

In a preferred aspect, the construct is selected from the group consisting of SEQ ID NOs: 63-67.

After their administration, the extended half-life constructs of the invention are not immediately removed by renal clearance. Thus, prolonging the half-life will contribute to a favorable PK profile. Accordingly, there will be no need for continuous intravenous infusion, and therefore patient compliance with the regimen and treatment regimen will be improved. In a specific aspect, the constructs of the present invention do not require continuous infusion.

Also, as detailed for monospecific polypeptides, multispecific polypeptides of the invention or constructs of the invention may further contain mutations that are effective in preventing or reducing the binding of so-called "pre-existing antibodies" to the polypeptides and constructs of the invention. For this purpose, the polypeptides and constructs of the invention may contain a C-terminal extension (X)n (in which n is 1 to 10, preferably 1 to 5, such as 1, 2, 3, 4 or 5 (and preferably 1 or 2, e.g. 1), and each X is a (preferably naturally occurring) amino acid residue which is independently selected and preferably independently selected from the group consisting of alanine (A), glycine (G), valine (V) leucine (L ) or isoleucine (I)), as described herein.

Accordingly, the present invention provides a polypeptide or construct of the invention further comprising a C-terminal extension (X)n wherein n is 1 to 5, such as 1, 2, 3, 4 or 5, and wherein X is naturally occurring amino acid, preferably without cysteine.

More specifically, the present invention relates to a polypeptide or construct, where the specified polypeptide or construct is selected from the group consisting of SEQ ID NO: 338-342.

The method for producing multivalent or multispecific polypeptides of the invention may include at least the steps of linking two or more immunoglobulin single variable domains, monovalent polypeptides and/or monospecific polypeptides of the invention and, for example, one or more linkers together in a suitable manner. The immunoglobulin single variable domains, monovalent polypeptides and/or monospecific polypeptides of the invention (and linkers) can be linked by any method known in the art and as further described herein. Preferred methods include linking nucleic acid sequences that encode immunoglobulin single variable domains, monovalent polypeptides and/or monospecific polypeptides of the invention (and linkers) to obtain a genetic construct that expresses a multivalent or multispecific polypeptide. Methods for linking amino acids or nucleic acids will be understood by one of skill in the art, and again hereby reference is made to standard guidelines such as Sambrook et al. and Ausubel et al. mentioned above as well as examples below.

Accordingly, the present invention also relates to the use of an immunoglobulin single variable domain, a monovalent polypeptide and/or a monospecific polypeptide of the invention in the preparation of a multivalent polypeptide or a multispecific polypeptide of the invention. A method for producing a multivalent or multispecific polypeptide will include linking one or more immunoglobulin single variable domains and/or polypeptides of the invention to at least one additional immunoglobulin single variable domain, monovalent polypeptide and/or monospecific polypeptide of the invention, optionally via one or more linkers . An immunoglobulin single variable domain, a monovalent polypeptide and/or a monospecific polypeptide of the invention is then used as a binding domain or link in the provision and/or production of a multivalent or multispecific polypeptide comprising two (e.g., in a divalent polypeptide), three (e.g., in a trivalent polypeptide) or more (eg, in a multivalent polypeptide) binding units. In this regard, an immunoglobulin single variable domain, a monovalent polypeptide and/or a monospecific polypeptide of the invention can be used as a binding domain or binding unit in providing and/or obtaining a multivalent or multispecific, such as a bispecific or trispecific polypeptide, containing two, three or more binding units.

Accordingly, the present invention also relates to the use of an immunoglobulin single variable domain and/or, in particular, a monovalent or monospecific polypeptide of the invention (as described herein) in the preparation of a multivalent or multispecific polypeptide. A method for producing a multivalent or multispecific polypeptide will include linking an immunoglobulin single variable domain, a monovalent polypeptide and/or a monospecific polypeptide of the invention to at least one additional immunoglobulin single variable domain, a monovalent polypeptide and/or a monospecific polypeptide of the invention, optionally through one or multiple linkers (as further described herein).

Invention designs

The monospecific polypeptide of the invention and the multispecific polypeptide of the invention may or may not additionally contain one or more other groups, residues, fragments or linking units (these monovalent polypeptides as well as multivalent polypeptides (with or without additional groups, residues, fragments or linking units) all are referred to as "the structure(s) of the invention"). If present, such additional groups, residues, fragments, or linking units may or may not provide additional functionality for the immunoglobulin single variable domain (and/or the polypeptide in which it is present) and may or may not change properties of the immunoglobulin single variable domain.

For example, such additional groups, residues, fragments, or linking units may be one or more additional amino acid sequences such that the polypeptide is a (fusion) protein or (fusion) polypeptide. In a preferred, but non-limiting aspect, said one or more other groups, residues, fragments or binding units are immunoglobulins. Even more preferably, said one or more other groups, residues, fragments or binding units are immunoglobulin single variable domains selected from the group consisting of domain antibodies, amino acids suitable for use as domain antibodies, single domain antibodies, amino acids suitable for use as a single domain antibody, a "dAb", amino acids suitable for use as a dAb, or a nanobody (such as, for example, V _HH , humanized V _HH or overblown V _H ).

As described above, additional binding units such as immunoglobulin single variable domains having different antigenic specificity can be linked to form multispecific constructs. By combining immunoglobulin single variable domains with two or more specificities, bispecific, trispecific, and so on can be obtained. designs. For example, a polypeptide of the invention may comprise a monospecific or multispecific polypeptide of the invention and one or more immunoglobulin single variable domains against a different target (ie, other than CD123 or TCR). Such designs and their modifications, which a person skilled in the art can easily foresee, are covered by the term "design of the invention" as used in this document.

In the constructs described above, one, two or more immunoglobulin single variable domains and one or more groups, residues, fragments or binding units can be linked directly to each other and/or through one or more suitable linkers or spacers. For example, when one or more groups, residues, fragments, or linking units are amino acid sequences, linkers can also be amino acid sequences such that the resulting construct is a fusion (protein) or fusion (polypeptide).

One or more additional groups, residues, fragments or linking units may be any suitable and/or desired amino acid sequence. Additional amino acid sequences may or may not change, transform or otherwise affect the (biological) properties of the polypeptide of the invention and may or may not add additional functionality to the polypeptide of the invention. Preferably, the additional amino acid sequence is such that it confers one or more desired properties or functionality to the polypeptide of the invention.

An example of such amino acid sequences will be understood by those skilled in the art and can generally include all amino acid sequences that are used in fusion peptides based on conventional antibodies and fragments thereof (including, but not limited to, ScFv antibodies and single domain antibodies). We hereby refer, for example, to the review by Holliger and Hudson (2005, Nature Biotechnology 23: 1126-1136).

For example, such an amino acid sequence may be an amino acid sequence that increases half-life, solubility or absorption, reduces immunogenicity or toxicity, eliminates or reduces unwanted side effects and/or confers other beneficial properties and/or reduces undesirable properties of the construct of the invention compared to the polypeptide of the invention as such. Some non-limiting examples of such amino acid sequences are serum proteins such as human serum albumin (see e.g. WO 00/27435) or hapten molecules (e.g. haptens that are recognized by circulating antibodies, see e.g. WO 98/22141).

In a specific but non-limiting aspect of the invention, which will be further described herein, a construct of the invention may have an increased serum half-life (as further described herein) compared to the immunoglobulin single variable domain or polypeptide from which it was derived. . For example, a single variable domain or polypeptide of an immunoglobulin of the invention may be linked (chemically or otherwise) to one or more groups or moieties that increase half-life (such as a polyethylene glycol (PEG) molecule) to provide a polypeptide derivative of the invention with an increased half-life. half-life.

In one particular aspect of the invention, a construct is provided that has an increased half-life compared to the corresponding polypeptide of the invention. Examples of constructs of the invention that contain such half-life-prolonging portions, for example, include, but are not limited to, constructs in which the variable domains of a single immunoglobulin are suitably linked to one or more serum proteins or fragments thereof (such as (human) serum albumin or suitable fragments thereof) or with one or more binding units (such as, for example, domain antibodies, amino acids suitable for use as a domain antibody, single domain antibodies, amino acids suitable for use as a single domain antibody, "DAb", amino acids , which are suitable for use as dAbs, nanobodies, V _HH , humanized V _HH or overblown V _H ) that can bind to serum proteins (such as serum albumin (such as human serum albumin)), serum immunoglobulins (such as IgG) , transferrin or one and c other whey proteins listed in WO 04/003019; polypeptides in which an immunoglobulin single variable domain is linked to an Fc portion (such as a human Fc) or to a suitable portion or fragment thereof; or constructs in which one or more immunoglobulin single variable domains are suitably linked to one or more small proteins or peptides that can bind to serum proteins (such as, but not limited to, the proteins and peptides described in WO 91/01743, WO 01/45746 or WO 02/076489). We also hereby refer to the dAbs described in WO 03/002609 and WO 04/003019 and Harmsen et al. (2005, Vaccine 23: 4926-4942); to EP 0368684 as well as WO 08/028977, WO 08/043821, WO 08/043822, WO 08/068280, WO 09/127691 and WO 11/095545 from Ablynx NV.

According to a specific but non-limiting aspect of the invention, the constructs of the invention may comprise, in addition to one or more anti-CD123 immunoglobulin single variable domains and/or an anti-TCR immunoglobulin single variable domain, at least one immunoglobulin single variable domain that binds serum albumin (such as human serum albumin). Accordingly, the present invention relates to the construct described herein, wherein said binding unit that provides the construct with an extended half-life is an immunoglobulin single variable domain that binds serum albumin. In a further aspect, the present invention relates to the construct described herein, wherein said ISV that binds serum albumin essentially consists of a single domain antibody, dAb, nanobody, V _HH , humanized V _HH , or overblown VH.

The ISV that binds serum albumin can be any ISV as described in the art.

In one aspect, the immunoglobulin single variable domain that binds human serum albumin may be as commonly described in the Ablynx NV applications cited above (see, for example, WO 04/062551). Some preferred nanobodies that provide an extended half-life and that can be used in the constructs of the invention include the ALB-1-ALB-10 nanobodies disclosed in WO 06/122787 (see Tables II and III) as well as the nanobodies disclosed in WO 2012/175400 or WO 2015/173325 (e.g. SEQ ID NO: 1-11 in WO 2012/175400, SEQ ID NO: 19 in WO 2015/173325) and nanobodies from Provisional Applications US 62/256 841, US 62/ 335,746, US 62/349,294 and the corresponding international application WO 2017/085172 from Assignee entitled "Improved Serum Albumin Binding Agents", which refers to the priority of these three US Provisional Applications."

In one aspect, the present invention relates to a polypeptide described herein, where the specified ISV, which binds serum albumin, essentially consists of 4 framework regions (FR1-FR4, respectively) and 3 complementarity-determining regions (CDR1-CDR3, respectively), wherein CDR1 is GFTFSSFGMS (SEQ ID NO: 363) or GFTFRSFGMS (SEQ ID NO: 364), CDR2 is SISGSGSDTL (SEQ ID NO: 365) and CDR3 is GGSLSR (SEQ ID NO: 366).

Some particularly preferred nanobodies that provide an extended half-life and that can be used in the constructs of the invention include immunoglobulin single variable domains, also referred to as Alb8, Alb23, Alb129, Alb132, Alb11, Alb11(S112K)-A, Alb82, Alb82-A, Alb82-AA, Alb82-AAA, Alb82-G, Alb82-GG, Alb82-GGG (Table B-2).

Accordingly, the present invention relates to the construct described herein, wherein said ISV that binds serum albumin is selected from the group consisting of SEQ ID NO 43 or 351-362.

Table B-2. Immunoglobulin single variable domains that bind HSA for use in constructs of the invention ISV SEQID NO Subsequence Alb8 43 EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS Alb23 351 EVQLLESGGGLVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS Alb129 352 EVQLVESGGGVVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTATYYCTIGGSLSRSSQGTLVTVSSA Alb132 353 EVQLVESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTATYYCTIGGSLSRSSQGTLVTVSSA Alb11 354 EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS Alb11(S112K)-A 355 EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVKVSSA Alb82 356 EVQLVESGGGVVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSS Alb82-A 357 EVQLVESGGGVVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSSA Alb82-AA 358 EVQLVESGGGVVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSSAA Alb82-AAA 359 EVQLVESGGGVVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSSAAA Alb82-G 360 EVQLVESGGGVVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTALYYCTIGGSLSRSSSQGTLVTVSSG Alb82-GG 361 EVQLVESGGGVVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSSGG Alb82-GGG 362 EVQLVESGGGVVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSSGGG

Generally, extended half-life constructs of the invention preferably have a half-life that is at least 1.5 times, preferably at least 2 times, such as at least 5 times, such as at least at least 10 times or more than 20 times greater than the half-life of the corresponding immunoglobulin single variable domain or polypeptide of the invention per se.

Generally, extended half-life constructs of the invention preferably have a half-life that is increased by more than 1 hour, preferably more than 2 hours, more preferably more than 6 hours, such as more than 12 hours or even more than 24 hours. 48 or 72 hours compared to the half-life of the respective variable domain or polypeptide of the respective immunoglobulin of the invention per se.

In another preferred, but non-limiting aspect, such constructs of the invention exhibit a human serum half-life of at least about 12 hours, preferably at least 24 hours, more preferably at least 48 hours, even more preferably at least 72 hours or more. For example, compounds or polypeptides of the invention may have a half-life of at least 5 days (e.g., 5 to 10 days), preferably at least 9 days (e.g., about 9 to 14 days), more preferably at least about 10 days (e.g., 10 to 15 days) or at least about 11 days (e.g., 11 to 16 days), more preferably at least about 12 days (e.g., 12 up to 18 days or more) or more than 14 days (for example, from 14 to 19 days).

In the present invention, it has been demonstrated that the inclusion of an albumin-targeted binding unit in the construct, as such, has no significant effect on the potency or efficacy obtained. Although there was a slight loss of potency/activity in the presence of HSA, the half-life of the TCR-binding multispecific polypeptides was still effective in killing cells expressing CD123. Albumin-based drug delivery has been shown to be useful in achieving improved cancer therapy, mainly due to its passive targeting of tumors due to enhanced permeability and retention effect and increased demand for albumin by tumor cells as an energy and amino acid source.

In one specific aspect, one or more polypeptides of the invention may be linked (optionally via a suitable linker or hinge region) to one or more constant domains (e.g., 2 or 3 constant domains that can be used as an Fc moiety/to form Fc moieties), an Fc moiety, and/or one or more antibody moieties, fragments, or domains that confer one or more effector functions on a polypeptide of the invention and/or may confer the ability to bind to one or more Fc receptors. For example, for this purpose and without limitation, one or more additional amino acid sequences may comprise one or more C _H 2 and/or C _H 3 domains of an antibody, such as a heavy chain-only antibody (as described herein) and more preferably from a conventional human 4-chain antibody; and/or may form (part of) an Fc region, for example from IgG (eg from IgG1, IgG2, IgG3 or IgG4), from IgE or other human Ig such as IgA, IgD or IgM. For example, WO 94/04678 describes heavy chain-only antibodies comprising a camelid _HH V domain or a humanized derivative thereof (i.e., nanobody) in which the C _H 2 and/or C _H 3 domain of Camelidae C is replaced by a human C domain. _H 2 and C _H 3 to provide an immunoglobulin that consists of 2 heavy chains each containing the nanobody and human C _H 2 and C _H 3 domains (but without the C _H 1 domain), the immunoglobulin having an effector function provided by the domains C _H 2 and C _H 3 and whose immunoglobulin can function in the absence of any light chains. Other amino acid sequences that may be appropriately linked to the polypeptides of the invention to provide effector function will be understood by those skilled in the art and may be selected based on the desired effector function(s). We hereby refer, for example, to WO 04/058820, WO 99/42077, WO 02/056910 and WO 05/017148, as well as to the review by Holliger and Hudson, supra; and WO 09/068628. Linking a polypeptide of the invention to an Fc portion may also result in an increased half-life compared to the corresponding polypeptide of the invention. For some applications, it may also be appropriate or even preferable to use the Fc portion and/or constant domains (ie, _CH 2 and/or _CH 3 domains) that provide an extended half-life without any biologically significant effector function. Other suitable constructs containing one or more polypeptides of the invention and one or more constant domains with extended in vivo half-life will be understood by one skilled in the art and may, for example, include polypeptides linked to a C _H 3 domain, optionally via a linker sequence. In general, any fusion protein or derivatives with extended half-life will preferably have a molecular weight greater than 50 kDa, the threshold for renal absorption.

In another specific, but non-limiting, aspect, the polypeptides of the invention may be linked (optionally via a suitable linker or hinge region) to naturally occurring synthetic or semi-synthetic constant domains (or analogs, variants, mutants, portions, or fragments thereof) that have a reduced propensity to (or have little or no tendency) to self-associate into dimers (ie, compared to the constant domains that naturally occur in conventional 4-chain antibodies). Such monomeric (ie, non-self-associating) Fc chain variants, or fragments thereof, will be understood by those skilled in the art. For example, Helm et al. (J. Biol. Chem. 271: 7494, 1996) describe monomeric variants of Fc chains that can be used in the polypeptide chains of the invention.

In addition, such monomeric Fc chain variants are preferably such that they are still capable of binding to complement or the corresponding Fc receptors (depending on the Fc region from which they are derived) and/or such that they still have HEKs that or all effector functions of the Fc portion from which they are derived (or at a reduced level still suitable for the intended use). Alternatively, in such a polypeptide chain of the invention, the monomeric Fc chain may be used to impart an increased half-life to the polypeptide chain, in which case the monomeric Fc chain may also have no or essentially no effector functions.

Additional amino acid residues may or may not alter, transform or otherwise affect other (biological) properties of the polypeptide of the invention and may or may not add additional functionality to the polypeptide of the invention. For example, such amino acid residues:

a) may include an N-terminal Met residue, for example as a result of expression in a heterologous host cell or host organism.

b) may form a signal sequence or leader sequence that directs the secretion of the polypeptide from the host cell upon synthesis (e.g. to provide a pre-, pro-, or pre-pro form of the polypeptide of the invention, depending on the host cell used to express the polypeptide of the invention) . Suitable secretory leader peptides will be understood by those skilled in the art and may be as described below. Typically, such a leader sequence will be linked to the N-terminus of the polypeptide, although the invention is not limited to this in its broadest sense;

c) may form a "tag", eg an amino acid sequence or residue, which allows or facilitates purification of the polypeptide, eg using affinity methods directed against said sequence or residue. Thereafter, said sequence or residue may be removed (eg, by chemical or enzymatic cleavage) to produce a polypeptide (for this purpose, the label may optionally be linked to an amino acid sequence or polypeptide sequence via a cleavable linker sequence or contain a cleavable motif). Some preferred, but non-limiting examples of such residues are multiple histidine residues, glutathione residues, and a myc tag such as AAAEQKLISEEDLNGAA (SEQ ID NO: 367);

d) may be one or more amino acid residues that have been functionalized and/or that may serve as a site for the attachment of functional groups. Suitable amino acid residues and functional groups will be understood by those skilled in the art and include, without limitation, the amino acid residues and functional groups specified herein for derivatives of the polypeptides of the invention.

In the constructs of the invention, two or more building blocks, ISVs or nanobodies, and optionally one or more polypeptides, one or more other groups, drugs, agents, residues, fragments, or binding units can be directly linked to each other (as, for example, described in WO 99/23221) and/or may be linked to each other via one or more suitable spacers or linkers, or any combination thereof.

Suitable spacers or linkers for use in the constructs of the invention will be understood by those skilled in the art and can generally be any linker or spacer used in the art to link amino acid sequences and/or other groups, drugs, agents, residues, fragments, or linking units. Preferably, said linker or spacer is suitable for use in constructing polypeptides and/or constructs that are intended for pharmaceutical use.

Some particularly preferred spacers include spacers and linkers which are used in the art to link antibody fragments or antibody domains. These include the linkers mentioned in the general art cited above, as well as, for example, linkers that are used in the art to construct a diabody or ScFv fragments (however, it should be noted in this regard that while in diabodies and ScFv fragments the the linker sequence must have a length, degree of flexibility, and other properties that allow the respective V _H and V _L domains to combine to form a complete antigen-binding site, there is no particular limitation on the length or flexibility of the linker used in the polypeptide of the invention, since each immunoglobulin single variable the domain itself forms a complete antigen-binding site).

For example, the linker may be a suitable amino acid sequence, and in particular amino acid sequences of 1 to 50, preferably 1 to 30, eg 1 to 10 amino acid residues. Some preferred examples of such amino acid sequences include gly-ser linkers, e.g. of the (gly _x ser _y ) _z type, such as (e.g. (gly ₄ ser) ₃ or (gly ₃ ser ₂ ) ₃ , as described in WO 99/42077 and the GS30, GS15, GS9, and GS7 linkers described in the Ablynx applications referenced herein (see, for example, WO 06/040153 and WO 06/122825), as well as hinge regions, such as hinge regions from naturally occurring antibodies on heavy chain basis or similar sequences (such as those described in WO 94/04678).

Some other particularly preferred linkers are mentioned in Table B-3, of which 35GS (SEQ ID NO: 334) is particularly preferred.

Accordingly, the invention relates to polypeptides in which the ISVs are linked to each other via a linker selected from the group consisting of SEQ ID NOs: 325-336.

Table B-3. Linkers Linker SEQID NO Subsequence 5GS 325 GGGGS 7GS 326 SGGSGGS 9GS 327 GGGGSGGGS 10GS 328 GGGGSGGGGS 15GS 329 GGGGSGGGGSGGGGS 18GS 330 GGGGSGGGGSGGGGGGGS 20GS 331 GGGGSGGGGSGGGGSGGGGS 25GS 332 GGGGSGGGGSGGGGSGGGGSGGGGS 30GS 333 GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS 35GS 334 GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS 40GS 335 GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS Poly-A 336 AAA

Other suitable linkers generally include organic compounds or polymers, in particular those suitable for use in proteins for pharmaceutical use. For example, poly(ethylene glycol) groups have been used to link antibody domains, see eg WO 04/081026.

It is within the scope of the invention that the length, degree of flexibility and/or other properties of the linker(s) used (although not critical, as is usually the case for linkers used in ScFv fragments) can have an effect on the properties of the final polypeptide of the invention. including, but not limited to, affinity, specificity or avidity for CD123 and/or TCR or one or more other antigens. Based on the description disclosed herein, one skilled in the art will be able to determine the optimal linker(s) for use in a particular polypeptide of the invention, optionally after some limited routine experimentation.

For example, in multivalent or multispecific polypeptides of the invention that contain building blocks, ISVs or nanobodies directed against the first and second targets, the length and flexibility of the linker are preferably such that they allow each building block, ISV or nanobodies of the invention present in the polypeptide , bind to its cognate target, for example, an antigenic determinant on each of the targets. Again, based on the description disclosed herein, one skilled in the art will be able to determine the optimal linkers for use in a particular polypeptide of the invention, optionally after some limited routine experimentation.

It is also within the scope of the invention that the linker(s) used impart one or more other beneficial properties or functionality to the polypeptides or constructs of the invention and/or provide one or more sites for derivatization and/or attachment of functional groups (e.g., as described herein for the polypeptide derivatives of the invention). For example, linkers containing one or more charged amino acid residues can provide improved hydrophilic properties, while linkers that form or contain small epitopes or tags can be used for detection, identification and/or purification purposes. Again, based on the description disclosed herein, one skilled in the art will be able to determine the optimal linkers for use in a particular polypeptide of the invention, optionally after some limited routine experimentation.

Finally, when two or more linkers are used in the polypeptides or constructs of the invention, these linkers may be the same or different. Again, based on the description disclosed herein, one skilled in the art will be able to determine the optimal linkers for use in a particular polypeptide of the invention, optionally after some limited routine experimentation.

Typically, for ease of expression and production, a polypeptide or construct of the invention will be a linear polypeptide. However, the invention in its broadest sense is not limited to this. For example, when a polypeptide of the invention contains three or more amino acid sequences, ISVs or nanobodies, they can be linked using a linker with three or more "arms", each of which is "linked" to the amino acid sequence, ISV or nanobody, to provide a "star" construction. It is also possible, although usually less preferred, to use ring structures.

Accordingly, the present invention provides a polypeptide as described herein wherein said first ISV and said second ISV and optionally said third ISV and/or said ISV-binding serum albumin are directly linked to each other or linked through a linker.

Also included in the present invention are constructs that contain an immunoglobulin single variable domain or polypeptide of the invention and further contain labels or other functional moieties, eg, toxins, labels, radiochemicals, etc.

Otherwise, additional groups, residues, fragments or linking units may, for example, be chemical groups, residues, fragments, which themselves may or may not be biologically and/or pharmacologically active. For example, and without limitation, such groups can be linked to two or more immunoglobulin variable domains or monovalent polypeptides to provide a "derivative" of the polypeptide of the invention.

Accordingly, the invention in its broadest sense also includes derivatives of the polypeptides of the invention. Such derivatives can usually be obtained by modification, and in particular by chemical and/or biological (for example, enzymatic) modification of the polypeptides of the invention and/or one or more amino acid residues that form the polypeptide of the invention,

Examples of such modifications, as well as examples of amino acid residues in polypeptide sequences that can be modified in this way (i.e. either on the main chain of the protein, but preferably on the side chain), methods and methods that can be used to introduce such modifications, and the potential uses and benefits of such modifications will be apparent to those skilled in the art (see also Zangi et al. 2013, Nat. biotechnol. 31: 898-907).

For example, such modification may include the introduction (for example, by covalent linkage or any other suitable method) of one or more functional groups, residues or fragments in or on the polypeptide of the invention and, in particular, one or more functional groups, residues or fragments that confer one or more desired properties or functionality to a polypeptide of the invention. An example of such functional groups will be clear to a person skilled in the art.

For example, such modification may include the introduction (for example, by covalent linkage or any other suitable method) of one or more functional groups that increase the half-life, solubility and/or absorption of the polypeptide of the invention, which reduce the immunogenicity and/or toxicity of the polypeptide of the invention, which eliminate or reduce any undesirable side effects of the polypeptide of the invention, and/or which confer other beneficial properties and/or reduce undesirable properties of the polypeptide of the invention; or any combination of two or more of the above. Examples of such functional groups and methods of introducing them will be understood by one of skill in the art and may generally include all functional groups and methods mentioned in the general art cited above, as well as functional groups and methods known per se for modifying pharmaceutical proteins and, in in particular, for the modification of antibodies or antibody fragments (including ^ScFv and single domain antibodies), for example, hereby refer to Remington (1980, Pharmaceutical Sciences, 16th ed., Mack Publishing Co., Easton, PA, 1980). Such functional groups may, for example, be linked directly (eg, covalently) to the polypeptide of the invention, or optionally via a suitable linker or spacer, as again will be appreciated by one of skill in the art.

One specific example is a derivative polypeptide of the invention, where the polypeptide of the invention has been chemically modified to increase its half-life (eg, by PEGylation). This is one of the most widely used methods for increasing the half-life and/or reducing the immunogenicity of pharmaceutical proteins, which involves the attachment of a suitable pharmacologically acceptable polymer such as poly(ethylene glycol) (PEG) or a derivative thereof (such as methoxypoly(ethylene glycol) or mPEG). Generally, any suitable form of PEGylation can be used, such as the PEGylation used in the art for antibodies and antibody fragments (including but not limited to (single)domain antibodies and ScFv; we hereby refer to, for example, Chapman (2002, Nat. Biotechnol 54: 531-545), Veronese and Harris (2003, Adv. Drug Deliv. Rev. 54: 453-456), Harris and Chess (2003, Nat. Rev. Drug. Discov. 2: 214-221) and WO 04/060965 Various protein PEGylation reagents are also commercially available, for example from Nektar Therapeutics, USA.

Preferably, site-directed PEGylation is used, in particular via a cysteine residue (see, for example, Yang et al. (2003, Protein Engineering 16: 761-770)). For example, for this purpose, PEG can be attached to a cysteine residue that occurs naturally in the polypeptide of the invention, the polypeptide of the invention can be modified so that one or more cysteine residues are appropriately introduced for PEG attachment, or an amino acid sequence containing one or more cysteine residues for PEG attachment may be fused to the N- and/or C-terminus of a polypeptide of the invention, all using protein engineering techniques known per se to one of skill in the art.

Preferably, for the polypeptides of the invention, PEGs with a molecular weight greater than 5,000, such as greater than 10,000 and less than 200,000, such as less than 100,000, are used; for example, in the range from 20000 to 80000.

Another, generally less preferred modification involves N-linked or O-linked glycosylation, usually as part of a co-translational and/or post-translational modification, depending on the host cell used to express the polypeptide of the invention.

Another modification may include the introduction of one or more detectable labels or other signal-generating groups or fragments, depending on the intended use of the labeled polypeptide of the invention. Suitable labels and methods for attaching, using and detecting them will be understood by those skilled in the art and, for example, include, but are not limited to, fluorescent labels (such as fluorescein, isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine, and fluorescent metals such as ¹⁵² Eu or other metals from the lanthanide series), phosphorescent labels, chemiluminescent labels or bioluminescent labels (such as luminal, isoluminol, acridinium aromatic ester, imidazole, acridinium salts, oxalate ester, dioxetane or GFP and its analogues), radioisotopes (such as ³ H, ¹²⁵ I, ³² P, ³⁵ S, ¹⁴ C, ⁵¹ Cr, ³⁶ Cl, ⁵⁷ Co, ⁵⁸ Co, ⁵⁹ Fe and ⁷⁵ Se), metals, metal chelates or metal cations (for example, metal cations, such as ^99m Tc, ¹²³ I, ¹¹¹ In, ¹³¹ I, ⁹⁷ Ru, ⁶⁷ Cu, ⁶⁷ Ga and ⁶⁸ Ga or other metals or metal cations which are particularly suitable for use in in vivo, in vitro or in situ imaging diagnostics, such like ( ¹⁵⁷ Gd , ⁵⁵ Mn, ¹⁶² Dy, ⁵² Cr and ⁵⁶ Fe)), as well as chromophores and enzymes (such as malate dehydrogenase, staphylococcal nuclease, delta-V-steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate dehydrogenase, triose phosphate isomerase, biotinavidin peroxidase, horseradish ) peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, β-galactosidase, ribonuclease, urease, catalase, glucose-VI-phosphate dehydrogenase, glucoamylase and acetylcholinesterase). Other suitable labels will be understood by the person skilled in the art and, for example, include fragments that can be detected using NMR or EPR spectroscopy.

Such labeled polypeptides of the invention can, for example, be used for in vitro, in vivo or in situ assays (including immunoassays known per se such as ELISA, RIA, EIA and other "sandwich assays", etc.), and also for in vivo diagnostics. and for visualization purposes, depending on the choice of a specific label.

As will be appreciated by one of skill in the art, another modification may include the introduction of a chelate group, for example to chelate one of the metals or metal cations mentioned above. Suitable chelating groups, for example, include, without limitation, diethyl-enetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).

Another modification may include the introduction of a functional group that is one part of a specific binding pair, such as a biotin-(strept)avidin binding pair. Such a functional group can be used to link a polypeptide of the invention to another protein, polypeptide, or chemical entity that is linked to the other half of the binding pair, i.e. through the formation of a binding pair. For example, a polypeptide of the invention may be conjugated to biotin and linked to another protein, polypeptide, compound, or carrier conjugated to avidin or streptavidin. For example, such a conjugated polypeptide of the invention can be used as a reporter, for example, in a diagnostic system where the detectable signal producing agent is conjugated to avidin or streptavidin. Such binding pairs may, for example, also be used to link a polypeptide of the invention to a carrier, including carriers suitable for pharmaceutical purposes. One non-limiting example are the liposome formulations described by Cao and Suresh (2000, Journal of Drug Targeting 8: 257). Such binding pairs can also be used to bind a therapeutically active agent to a polypeptide of the invention.

Other potential chemical and enzymatic modifications will be apparent to those skilled in the art. Such modifications may also be introduced for research purposes (eg, to study function-activity relationships). We hereby refer, for example, to Lundblad and Bradshaw (1997, Biotechnol. Appl. Biochem. 26: 143-151).

Preferably, the derivatives are such that they bind to CD123 and/or TCR with affinity (suitably measured and/or expressed as K _D value (actual or apparent), K _A value (actual or apparent), k _on rate, and /or speed k _off , or, alternatively, as an IC ₅₀ value, as described later in this document), as defined herein (ie, as defined for the polypeptides of the invention).

Such polypeptides of the invention and derivatives thereof may also be in substantially isolated form (as defined herein).

The invention further relates to methods for producing polypeptides, nucleic acids, host cells and compositions described herein.

The polypeptides and constructs of the invention may be prepared in a manner known per se, as will be apparent to those skilled in the art from the following description. For example, the polypeptides and constructs of the invention can be prepared by any method known per se for the production of antibodies, and in particular for the production of antibody fragments (including, but not limited to (single)domain antibodies and ScFv fragments). Some preferred, but non-limiting, methods for producing polypeptides, constructs, and nucleic acids include those described herein.

The method for obtaining a polypeptide or construct (i.e. one that can be obtained by expression of a nucleic acid encoding it) according to the invention may include the following steps:

- expression in a suitable host cell or host organism (also referred to herein as a "host of the invention") or in another suitable nucleic acid expression system that encodes said polypeptide or construct of the invention (also referred to herein as a "nucleic acid according to the invention)

which is optionally accompanied by:

- isolation and/or purification of the polypeptide or construct according to the invention thus obtained.

In particular, such a method may include the steps of:

- culturing and/or maintaining a host of the invention under conditions which are such that said host of the invention expresses and/or produces at least one polypeptide or construct (i.e. one that can be obtained by expression of a nucleic acid encoding them) according to the invention;

which is optionally accompanied by:

- isolation and/or purification of the polypeptide or construct according to the invention thus obtained.

Accordingly, the present invention also relates to a nucleic acid or nucleotide sequence that encodes a polypeptide or construct (i.e., one that can be obtained by expression of a nucleic acid encoding it) of the invention (also referred to as "nucleic acid of the invention"). The nucleic acid of the invention may be in the form of single or double stranded DNA or RNA, and preferably in the form of double stranded DNA. For example, the nucleotide sequences of the invention may be genomic DNA, cDNA, or synthetic DNA (such as DNA using codons that has been specifically adapted for expression in the intended host cell or host organism).

According to one aspect of the invention, the nucleic acid of the invention is substantially isolated as defined herein. The nucleic acid of the invention may also be in the form, present and/or may be part of a vector such as, for example, a plasmid, cosmid or YAC, which may also be in substantially isolated form.

Nucleic acids of the invention can be prepared or obtained in a manner known per se, based on the information on the polypeptides or constructs (which are such that they can be obtained by expression of the nucleic acid encoding them) of the present invention given in this document and/ or may be isolated from a suitable natural source. Also, as will be understood by a person skilled in the art, to obtain a nucleic acid of the invention also several nucleotide sequences, such as at least one nucleotide sequence encoding an immunoglobulin single variable domain of the invention, and, for example, nucleic acids encoding one or multiple linkers may be linked to each other in a suitable manner.

Methods for obtaining nucleic acids according to the invention will be understood by a person skilled in the art and may, for example, include, but are not limited to, automatic DNA synthesis; site-directed mutagenesis; combining two or more naturally occurring and/or synthetic sequences (or two or more parts thereof), introducing mutations that result in the expression of a truncated expression product; introducing one or more restriction sites (for example, to create cassettes and/or regions that can be easily digested and/or ligated using suitable restriction enzymes), and/or introducing mutations via a PCR reaction using one or more "mismatched" primers. These and other methods will be apparent to those skilled in the art, and again reference is hereby made to standard manuals such as Sambrook et al. and Ausubel et al. mentioned above as well as examples below.

The nucleic acid of the invention may also be in the form of, be present in, and/or be part of a genetic construct, as will be understood by one of skill in the art. Such genetic constructs typically comprise at least one nucleic acid of the invention, which is optionally linked to one or more elements of the genetic constructs known per se, such as, for example, one or more suitable regulatory elements (such as suitable promoter(s) , enhancer(s), terminator(s), etc.) and other elements of genetic constructs mentioned in this document. Such genetic constructs containing at least one nucleic acid of the invention will also be referred to herein as "genetic constructs of the invention".

The genetic constructs of the invention may be DNA or RNA, and are preferably double-stranded DNA. The genetic constructs of the invention may also be in a form suitable for transformation of a putative host cell or host organism, in a form suitable for integration into the genomic DNA of a putative host cell, or in a form suitable for independent replication, maintenance and/or inheritance. in the intended host. For example, the genetic constructs of the invention may be in the form of a vector such as, for example, a plasmid, cosmid, YAC, viral vector, or transposon. In particular, the vector may be an expression vector, ie. a vector that can provide in vitro and/or in vivo expression (eg, in a suitable host cell, host organism, and/or expression system).

In a preferred but non-limiting aspect, the genetic construct of the invention comprises

a) at least one nucleic acid according to the invention; functionally related to

b) one or more regulatory elements such as a promoter and optionally a suitable terminator;

and possibly also

c) one or more additional elements of genetic constructs known per se;

in which the terms "regulatory element", "promoter", "terminator" and "operably linked" have their usual meaning in the art (as further described herein); and wherein said "additional elements" present in the genetic constructs may, for example, be 3' or 5' UTR sequences, leader sequences, selectable markers, expression markers/reporter genes, and/or elements that may facilitate or increase (efficiency) of transformation or integration. These and other suitable elements for such genetic constructs will be understood by one skilled in the art and may, for example, depend on the type of construct used; putative host cell or host organism; a method for expressing the nucleotide sequences of interest of the present invention (eg, via constitutive, transient, or inducible expression); and/or transformation method to be used. For example, regulatory sequences, promoters and terminators known per se for the expression and production of antibodies and antibody fragments (including but not limited to (single)domain antibodies and ScFv fragments) can be used in a substantially similar manner.

Preferably, in the genetic constructs of the invention, said at least one nucleic acid of the present invention and said regulatory elements, and optionally said one or more additional elements, are "operably linked" to each other, which generally means that they are in a functional relationship together. For example, a promoter is considered "operably linked" to a coding sequence if said promoter is capable of initiating or otherwise controlling/regulating transcription and/or expression of a coding sequence (in which said coding sequence is to be understood as being "under control" of said promoter). Typically, when two nucleotide sequences are operably linked, they will have the same orientation and will usually also be in the same reading frame. They will usually also be substantially contiguous, although this may not be required either.

Preferably, the regulatory and additional elements of the genetic constructs of the invention are such that they are capable of providing the intended biological function in the intended host cell or host organism.

For example, a promoter, enhancer, or terminator must be "functional" in the intended host cell or host organism, which implies that (for example) said promoter must be capable of initiating or otherwise controlling/regulating transcription and/or expression of a nucleotide sequence - e.g. , coding sequence - with which it is operatively linked (as defined herein).

Some particularly preferred promoters include, but are not limited to, promoters known per se to be expressed in the host cells mentioned herein; and in particular promoters for expression in bacterial or yeast cells such as those mentioned herein and/or used in the examples.

The selectable marker must be such that it allows - i.e. under appropriate selection conditions, host cells and/or host organisms that have been (successfully) transformed with a nucleotide sequence of the invention be distinguished from host cells/organisms that have not been (successfully) transformed. HEK Preferred, but non-limiting examples of such markers are genes that confer resistance to antibiotics (such as kanamycin or ampicillin), genes that confer temperature tolerance, or genes that allow maintenance of a host cell or host organism in the absence of certain factors, compounds and/or (food) components in the environment that are necessary for the survival of non-transformed cells or organisms.

The leader sequence should be such that - in the intended host cell or host organism - it allows the desired post-translational modifications and/or such that it directs the transcribed mRNA to the desired part or organelle of the cell. The leader sequence may also allow secretion of the expression product from said cell. As such, the leader sequence can be any pro-, pre-, or pre-pro sequence active in a host cell or host organism. Leader sequences may not be required for expression in a bacterial cell. For example, leader sequences known per se for the expression and production of antibodies and antibody fragments (including, but not limited to, single domain antibodies and ScFv fragments) can be used in a substantially similar manner.

The expression marker or reporter gene must be such that - in the host cell or host organism - it allows detection of the expression of the (gene or nucleotide sequence present in) the genetic construct. The expression marker may also optionally allow localization of the expressed product, for example, to a specific part or organelle of a cell and/or to (a) a specific cell(s), tissue(s), organ(s), or part(s) of a multicellular organism. Such reporter genes may also be expressed as a fusion protein with an ISV, polypeptide or construct of the invention. Some preferred, but non-limiting examples include fluorescent proteins such as GFP.

Some preferred, but non-limiting examples of suitable promoters, terminators and other elements include those that can be used for expression in the host cells mentioned herein; and in particular those suitable for expression in bacterial or yeast cells, such as those mentioned herein and/or those used in the examples below. For some (additional) non-limiting examples of promoters, selectable markers, leader sequences, expression markers and other elements that may be present/used in the genetic constructs of the invention, such as terminators, transcriptional and/or translational enhancers and/or integration factors - hereby reference to general guidelines such as Sambrook et al. and Ausubel et al. mentioned above, as well as the examples that are given in WO 95/07463, WO 96/23810, WO 95/07463, WO 95/21191, WO 97/11094, WO 97/42320, WO 98/06737, WO 98/21355, US 7207410, US 5693492 and EP 1085089. Other examples will be clear to the expert. We also hereby refer to the general prior art cited above and additional references cited herein.

The genetic constructs of the invention can generally be prepared by appropriately linking the nucleotide sequence(s) of the invention to one or more of the additional elements described above, for example, using the methods described in general guidelines such as Sambrook et al. and Ausubel et al., which are mentioned above.

Often the genetic constructs of the invention will be obtained by inserting the nucleotide sequence of the invention into a suitable (expression) vector known per se. Some preferred, but non-limiting examples of suitable expression vectors are those used in the examples below as well as those mentioned herein.

The nucleic acids of the invention and/or the genetic constructs of the invention can be used to transform a host cell or host organism, i.e. for the expression and/or production of a polypeptide or construct (ie so that it can be obtained by expression of a nucleic acid encoding them) according to the invention. The host is preferably non-human. Suitable hosts or host cells will be understood by those skilled in the art and may, for example, be any suitable fungal, prokaryotic or eukaryotic cell or cell line, or any suitable fungal, prokaryotic or (non-human) eukaryotic organism, for example:

- a bacterial strain, including, without limitation, gram-negative strains such as strains of Escherichia coli; Proteus, for example, Proteus Mirabilis; from Pseudomonas, eg Pseudomonas fluorescens; and Gram-positive strains such as Bacillus strains, for example Bacillus subtilis or Bacillus brevis; Streptomyces, e.g. Streptomyces lividans; Staphylococcus, for example, Staphylococcus carnosus; and Lactococcus, eg Lactococcus lactis;

- a fungal cell, including, without limitation, cells from species of Trichoderma, for example, Trichoderma reesei; from Neurospora, eg Neurospora crassa; from Sordaria, e.g. from Sordaria macrospora; from Aspergillus, for example from Aspergillus niger or from Aspergillus sojae; or from other filamentous fungi;

- a yeast cell, including, without limitation, cells from Saccharomyces species, for example, Saccharomyces cerevisiae; from Schizosaccharomyces, for example, Schizosaccharomyces pombe; from Pichia, for example, Pichia pastoris or Pichia methanolica; from Hansenula, e.g. Hansenula polymorpha; from Kluyveromyces, for example Kluyveromyces lactis; from Arxula, e.g. Arxula adeninivorans; from Yarrowia, eg Yarrowia lipolytica;

- an amphibian cell or cell line such as Xenopus oocytes;

insect-derived cells or cell lines, such as Lepidoptera-derived cells/cell lines, including, but not limited to, Spodoptera SF9 and Sf21 cells or Drosophila-derived cells/cell lines, such as Schneider and Kc cells;

a plant or plant cell, for example from tobacco plants; and/or

- a mammalian cell or cell line, for example, a cell or cell line derived from a human, a cell or cell line from mammals, including, without limitation, CHO cells, BHK cells (for example, BHK-21 cells) and human cells or cell lines , such as HeLa cells, COS (eg, COS-7) and PER.C6;

as well as all other host cells or hosts (non-human) known per se to express and produce antibodies and antibody fragments (including, but not limited to (single) domain antibodies and ScFv fragments) understood by one of skill in the art. Reference is also made to the general prior art cited above and also, for example, to WO 94/29457; W096/34103; WO 99/42077; Franken et al. (1998, Res Immunol. 149: 589-599); Riechmann and Muyldermans (1999), supra; van der Linden (2000, J. Biotechnol. 80: 261-270); Joosten et al. (2003, Microb. Cell Fact. 2:1); Joosten et al. 2005 (Appl. Microbiol. Biotechnol. 66: 384-392); and additional sources cited in this document.

The polypeptides or constructs of the invention can also be expressed as so-called "intracellular antibodies" as, for example, described in WO 94/02610, WO 95/22618 and US 7 004 940; WO 03/014960; in Cattaneo and Biocca (1997, "Intracellular Antibodies: Development and Application", Landes and Springer-Verlag); and in Kontermann (2004, Methods 34: 163-170).

The polypeptides or constructs of the invention can, for example, also be produced in the milk of transgenic mammals, for example, in the milk of rabbits, cows, goats or sheep (see, for example, US 6,741,957, US 6,304,489 and US 6,849,992 for general methods of introducing transgenes into mammals), in plants or plant parts, including, but not limited to, their leaves, flowers, fruits, seeds, roots or tubers (for example, in tobacco, corn, soybeans or alfalfa) or, for example, in silkworm pupae Bombix mori.

In addition, the polypeptides or constructs of the invention can also be expressed and/or produced in cell-free expression systems, and suitable examples of such systems will be apparent to those skilled in the art. Some preferred but non-limiting examples include expression in the wheat germ system; in rabbit reticulocyte lysates; or in the E. coli Zubay system.

Preferably, the invention uses an expression system (in vivo or in vitro), such as a bacterial expression system, which provides the polypeptides or constructs of the invention in a form suitable for pharmaceutical use, and such expression systems will again be understood by the skilled person. As will also be appreciated by one of skill in the art, polypeptides or constructs of the invention suitable for pharmaceutical use can be prepared using peptide synthesis techniques.

For industrial scale production, preferred heterologous hosts for (industrial) production of immunoglobulin single variable domain or immunoglobulin single variable domain containing polypeptide therapeutics include E. coli, Pichia pastoris, S. cerevisiae strains which are suitable for expression/production/fermentation in on a large scale, and in particular for pharmaceutical expression/production/fermentation on a large scale. Suitable examples of such strains will be apparent to those skilled in the art. Such strains and production/expression systems are also provided by companies such as Biovitrum (Uppsala, Sweden).

Alternatively, mammalian cell lines, in particular Chinese Hamster Ovary (CHO) cells, can be used for large scale expression/production/fermentation, and in particular for large scale pharmaceutical expression/production/fermentation. Again, such expression/production systems are also provided by some of the companies mentioned above.

The choice of a particular expression system will depend in part on the need for certain post-translational modifications, more specifically glycosylation. Production of a recombinant protein containing an immunoglobulin single variable domain for which glycosylation is desirable or necessary would require the use of mammalian expressing hosts that have the ability to glycosylate the expressed protein. In this regard, one of skill in the art will appreciate that the resulting glycosylation pattern (ie the type, number and position of residues attached) will depend on the cell or cell line that is used for expression. Preferably, either a human cell or cell line is used (i.e., resulting in a protein that essentially has a human glycosylation pattern), or another mammalian cell line is used that can provide a glycosylation pattern that is essentially and/or functionally the same. , as in human glycosylation or at least mimics human glycosylation. Typically, prokaryotic hosts such as E. coli do not have the ability to glycosylate proteins, and the use of lower eukaryotes such as yeast generally results in a glycosylation pattern that differs from human glycosylation. However, it should be understood that all of the aforementioned host cells and expression systems can be used in the invention depending on the desired polypeptide or construct to be obtained.

Thus, according to one non-limiting aspect of the invention, the polypeptide or construct of the invention is glycosylated. According to another non-limiting aspect of the invention, the polypeptide or construct of the invention is non-glycosylated.

According to one preferred, but non-limiting aspect of the invention, the polypeptide or construct of the invention is produced in a bacterial cell, in particular a bacterial cell suitable for large scale pharmaceutical production, such as cells of the strains mentioned above.

According to another preferred, but non-limiting, aspect of the invention, the polypeptide or construct of the invention is produced in a yeast cell, in particular a yeast cell suitable for large scale pharmaceutical production, such as the aforementioned cell species.

According to another preferred but non-limiting aspect of the invention, the polypeptide or construct of the invention is produced in a mammalian cell, in particular in a human cell or in a human cell line, and more specifically in a human cell or human cell line, which are suitable for large-scale pharmaceutical production. , such as the cell lines mentioned above.

When expression in a host cell is used to produce polypeptides or constructs of the invention, the polypeptides or constructs of the invention can be produced either intracellularly (eg, in the cytosol, periplasm, or inclusion bodies) and then isolated from the host cell and optionally further purified; or can be obtained extracellularly (eg, in the medium in which the host cells are cultured), and then isolated from the culture medium and optionally further purified. When eukaryotic host cells are used, extracellular production is generally preferred as this greatly facilitates further isolation and subsequent processing of the resulting polypeptides or constructs. Bacterial cells such as the E. coli strains mentioned above do not normally secrete proteins extracellularly except for a few classes of proteins such as toxins and hemolysin, and secretory production in E. coli refers to the translocation of proteins across the inner membrane into the periplasmic space. Periplasmic production provides several advantages over cytosolic production. For example, the N-terminal amino acid sequence of the secreted product may be identical to the natural gene product after cleavage of the secretion signal sequence by a specific signal peptidase. In addition, protease activity in the periplasm is significantly lower than in the cytoplasm. In addition, protein purification is easier due to fewer contaminating proteins in the periplasm. Another advantage is that the correct disulfide bonds can be formed since the periplasm provides a more oxidative environment than the cytoplasm. Proteins overexpressed in E. coli are often found in insoluble aggregates, so-called inclusion bodies. These inclusion bodies may be located in the cytosol or in the periplasm; extraction of biologically active proteins from these inclusion bodies requires a denaturation/refolding process. Many recombinant proteins, including therapeutic proteins, are isolated from inclusion bodies. Alternatively, as will be appreciated by one of skill in the art, recombinant strains of bacteria that have been genetically modified to secrete the protein of interest, and in particular the polypeptide or construct of the invention, may be used.

Thus, according to one non-limiting aspect of the invention, a polypeptide or construct of the invention is a polypeptide or construct that has been produced intracellularly and that has been isolated from a host cell, and in particular from a bacterial cell or from an inclusion body in a bacterial cell. According to another non-limiting aspect of the invention, a polypeptide or construct of the invention is a polypeptide or construct that has been produced extracellularly and that has been isolated from the medium in which the host cell is cultured.

Some preferred but non-limiting promoters for use with these host cells include:

- for expression in E. coli: the lac promoter (and its derivatives, such as the lacUV5 promoter); arabinose promoter; left (PL) and right (PR) lambda phage promoters; trp operon promoter; lac/trp hybrid promoters (tac and trc); the T7 promoter (more specifically, gene 10 of T7 phage) and other T-phage promoters; Tn10 tetracycline resistance gene promoter; engineered variants of the above promoters that include one or more copies of a foreign operator regulatory sequence;

- for expression in S. cerevisiae: constitutive: ADH1 (alcohol dehydrogenase 1), ENO (enolase), CYC1 (cytochrome c iso-1), GAPDH (glyceraldehyde-3-phosphate dehydrogenase), PGK1 (phosphoglycerate kinase), PYK1 (pyruvate kinase); regulated: GAL1,10,7 (enzymes of galactose metabolism), ADH2 (alcohol dehydrogenase 2), PHO5 (acid phosphatase), CUP1 (copper metallothionein); heterologous: CaMV (cauliflower mosaic virus 35S promoter);

- for expression in Pichia pastoris: AOX1 promoter (alcohol oxidase I);

- for expression in mammalian cells: immediate early enhancer/promoter of human cytomegalovirus (hCMV); a human cytomegalovirus (hCMV) early early variant that contains two tetracycline operator sequences so that the promoter can be regulated by the Tet repressor; herpes simplex virus thymidine kinase (TK) promoter; Rous sarcoma virus long terminal repeat (RSV LTR) enhancer/promoter; human, chimpanzee, mouse, or rat elongation factor 1α (hEF-1α) promoter; SV40 early promoter; long terminal repromoter of HIV-1; β actin promoter.

HEK preferred but non-limiting vectors for use with these host cells include:

- vectors for expression in mammalian cells: pMAMneo (Clontech), pcDNA3 (Invitrogen), pMC1neo (Stratagene), pSG5 (Stratagene), EBO-pSV2-neo (ATCC 37593), pBPV-1 (8-2) (ATCC 37110) , pdBPV-MMTneo (342-12) (ATCC 37224), pRSVgpt (ATCC37199), pRSVneo (ATCC37198), pSV2-dhfr (ATCC 37146), pUCTag (ATCC 37460) and 1ZD35 (ATCC 37565), as well as expression systems based on viruses, for example based on adenovirus;

- vectors for expression in bacterial cells: pET vectors (Novagen) and pQE vectors (Qiagen);

- vectors for expression in yeast or other fungal cells: expression vectors pYES2 (Invitrogen) and Pichia (Invitrogen);

- vectors for expression in insect cells: pBlueBacII (Invitrogen) and other baculovirus vectors;

vectors for expression in plants or plant cells: for example cauliflower or tobacco mosaic virus vectors, suitable Agrobacterium strains or Ti-plasmid vectors.

Some preferred, but non-limiting, secretory sequences for use with these host cells include:

- for use in bacterial cells such as E. coli: PelB, Bla, OmpA, OmpC, OmpF, OmpT, StII, PhoA, PhoE, MalE, Lpp, LamB, etc.; signal peptide TAT, secretion signal of the C-terminus of hemolysin;

- for use in yeast: mating factor preprosequence α, phosphatase (pho1), invertase (Suc), etc.;

- for use in mammalian cells: native signal if the target protein is of eukaryotic origin; mouse Ig κ chain signal peptide V-J2-C; etc.

Suitable methods for transforming a host cell or host cell of the invention will be apparent to those skilled in the art and may depend on the intended host cell/host organism and the genetic construct used. Again, we hereby refer to the manuals and patent applications mentioned above.

Following transformation, a step may be performed to identify and select those host cells or host organisms that have been successfully transformed with the nucleotide sequence/genetic construct of the invention. This may be, for example, a selection step based on a selectable marker present in the genetic construct of the invention, or a step involving the detection of a polypeptide or construct of the invention, for example, using specific antibodies.

A transformed host cell (which may be in the form of a stable cell line) or host organisms (which may be in the form of a stable mutant line or strain) form further aspects of the present invention.

Preferably, these host cells or host organisms are such that they express or (at least) are capable of expressing (e.g., under suitable conditions) a polypeptide or construct of the invention (and in the case of a host organism: in at least one cell, part, tissue or organ). The invention also includes the next generations, progeny and/or descendants of the host cell or host organism of the invention, which can be obtained, for example, by cell division or by sexual or asexual reproduction.

Accordingly, in another aspect, the invention relates to a host or host cell that expresses (or that, under appropriate circumstances, is capable of expressing) a polypeptide or construct of the invention; and/or which contains a nucleic acid encoding it. HEK preferred, but non-limiting examples of such hosts or host cells may be as generally described in WO 04/041867, WO 04/041865 or WO 09/068627. For example, the polypeptides or constructs of the invention may advantageously be expressed, produced or manufactured in a yeast strain such as a strain of Pichia pastoris. We also hereby refer to WO 04/25591, WO 10/125187, WO 11/003622 and WO 12/056000 which also describe the expression/production in Pichia and other hosts/host cells of immunoglobulin single variable domains and polypeptides containing them.

In order to obtain/achieve expression of polypeptides or constructs of the invention, the transformed host cell or transformed host organism can generally be stored, maintained and/or cultured under conditions under which the (desired) polypeptide or construct of the invention is expressed/produced. Suitable conditions will be understood by one skilled in the art and will generally depend on the host cell/host organism used, as well as on the regulatory elements that control the expression of the (relevant) nucleotide sequence of the invention. Again, we hereby refer to the guidelines and patent applications mentioned above in the paragraphs on the genetic constructs of the invention.

In general, suitable conditions may include the use of a suitable environment, the presence of a suitable food source and/or suitable nutrients, the use of a suitable temperature, and optionally the presence of a suitable inducing factor or compound (eg, when the nucleotide sequences of the invention are under the control of an inducible promoter); all of which may be selected by the skilled person. Again, under such conditions, the polypeptides or constructs of the invention may be expressed constitutively, transiently, or only upon appropriate induction.

One of ordinary skill in the art will also appreciate that a polypeptide or construct of the invention may (first) be provided in an immature form (as mentioned above), which may then be subjected to post-translational modification depending on the host cell/organism used. Also, a polypeptide or construct of the invention may be glycosylated, again depending on the host cell/host organism used.

The polypeptide or construct of the present invention can then be isolated from the host cell/host organism and/or from the medium in which said host cell or host organism has been cultured using methods known per se for protein isolation and/or purification, such as (preparative) chromatography and/or electrophoresis methods, differential precipitation methods, affinity methods (e.g. using a specific, cleavable amino acid sequence fused to a polypeptide or construct of the invention) and/or preparative immunological methods (i.e. using antibodies against the polypeptide or structures to be highlighted).

Compositions of the invention

The invention also relates to a product or composition comprising or comprising at least one polypeptide or construct of the invention and/or at least one nucleic acid of the invention, and optionally one or more other components of such compositions known to per se, i.e. to the intended use of the composition.

Generally, for pharmaceutical use, the polypeptides or constructs of the invention may be formulated into a pharmaceutical formulation or composition comprising at least one polypeptide or construct of the invention and at least one pharmaceutically acceptable carrier, diluent or excipient and/or an adjuvant; and optionally one or more other pharmaceutically active polypeptides and/or compounds. By way of non-limiting examples, such a composition may be in a form suitable for oral administration, for parenteral administration (e.g., intravenous, intraperitoneal, subcutaneous, intramuscular, intraluminal, intraarterial, or intrathecal administration), for topical administration, for administration by inhalation, skin patch, implant, a suppository, etc., with parenteral administration being preferred. Such suitable administration forms, which may be solid, semi-solid or liquid, depending on the mode of administration, as well as methods and carriers for use in their preparation, will be understood by a person skilled in the art and will be further described herein. Such a pharmaceutical preparation or composition will generally be referred to herein as a "pharmaceutical composition". A pharmaceutical preparation of a composition for use in an organism other than a human will generally be referred to herein as a "veterinary composition". Some preferred, but non-limiting examples of such compositions will become apparent from the further description provided herein.

Thus, in a further aspect, the invention relates to a pharmaceutical composition that comprises at least one polypeptide or construct of the invention and at least one suitable carrier, diluent, or excipient (i.e., suitable for pharmaceutical use), and , optionally, one or more additional active substances. In a specific aspect, the invention relates to a pharmaceutical composition that contains a polypeptide or construct according to the invention selected from any of SEQ ID NOs: 1-10, 47, 49, 52, 53, 55, 56, 58-61, 63-67, and 338-342 and at least one suitable carrier, diluent or excipient (i.e., suitable for pharmaceutical use) and optionally one or more other active substances.

The phrase "pharmaceutically acceptable" is used herein to refer to those compounds, materials, compositions and/or dosage forms which, under sound medical justification, are suitable for use in contact with human and animal tissues without undue toxicity, irritation, allergic reaction, or other problems. or complications commensurate with a reasonable benefit/risk ratio.

The phrase "pharmaceutically acceptable carrier", as used herein, means a pharmaceutically acceptable material, composition, or carrier, such as a liquid or solid excipient, diluent, excipient, or solvent, encapsulating material involved in the transfer or transport of a compound of the present invention from one organ or part of the body, to another organ or part of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not causing harm to the patient.

In general, the polypeptides and constructs of the invention may be formulated and administered by any suitable method known per se. We hereby refer, for example, to the general prior art mentioned above (and in particular to WO 04/041862, WO 04/041863, WO 04/041865, WO 04/041867 and WO 08/020079) as well as to standard guidelines such as Remington's Pharmaceutical Sciences, 18th ^Ed ., Mack Publishing Company, USA (1990), Remington, the Science and Practice of Pharmacy, ^21st Ed., Lippincott Williams and Wilkins (2005); or the Handbook of Therapeutic Antibodies (S. Dubel, Ed.), Wiley, Weinheim, 2007 (see, for example, pp. 252-255).

For example, the polypeptides or constructs of the invention may be formulated and administered by any method known per se for conventional antibodies and antibody fragments (including ScFvs and diabodies) and other pharmaceutically active proteins. Such formulations and methods for preparing them will be understood by those skilled in the art and, for example, include formulations suitable for parenteral administration (eg, intravenous, intraperitoneal, subcutaneous, intramuscular, intraluminal, intraarterial, or intrathecal administration).

Formulations for parenteral administration may, for example, be sterile solutions, suspensions, dispersions or emulsions which are suitable for infusion or injection. Suitable carriers or diluents for such formulations, for example, include, but are not limited to, those mentioned on page 143 of WO 08/020079. Generally, aqueous solutions or suspensions will be preferred.

The polypeptides or constructs of the invention may be administered intravenously or intraperitoneally by infusion or injection. Solutions of the polypeptides or constructs of the invention may be prepared in water, optionally mixed with a non-toxic surfactant. Dispersions can also be obtained in glycerol, liquid polyethylene glycols, triacetin and mixtures thereof, and in oils. Under normal conditions of storage and use, these preparations may contain a preservative to prevent the growth of microorganisms.

Pharmaceutical compositions suitable for parenteral administration include one or more immunoglobulin single variable domains, polypeptides or constructs in combination with one or more pharmaceutically acceptable sterile isotonic aqueous or non-aqueous solutions, dispersions, suspensions or emulsions or sterile powders, which can be reconstituted into sterile solutions or dispersions for injection immediately prior to use, which may contain sugars, alcohols, antioxidants, buffers, bacteriostats, solutes that render the composition isotonic with the blood of the intended recipient, or suspending or thickening agents.

Examples of suitable aqueous and non-aqueous carriers that can be used in pharmaceutical compositions include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like) and suitable mixtures thereof, vegetable oils such as olive oil, and injectable organic esters. such as ethyl oleate. Correct flow can be maintained, for example, by using coating materials such as lecithin, by maintaining the required particle size in the case of dispersions, and by using surfactants.

These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms on the compounds of the present invention can be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. It may also be desirable to include isotonic agents such as sugars, sodium chloride and the like in the compositions. In addition, prolonged absorption of an injectable pharmaceutical form may be caused by the inclusion of agents that delay absorption, such as aluminum monostearate and gelatin.

In some cases, in order to prolong the effect of the drug, it is desirable to slow down the absorption of the drug from subcutaneous or intramuscular injections. This can be achieved by using a liquid suspension of crystalline or amorphous material having poor water solubility. The rate of drug absorption depends on its rate of dissolution, which, in turn, may depend on crystal size and crystalline form. Alternatively, delayed absorption of a parenteral dosage form is achieved by dissolving or suspending the drug in an oil vehicle.

Injectable depot forms are obtained by forming microencapsular matrices of the compounds in question in biodegradable polymers such as polylactide-polyglycolide. Depending on the ratio of drug to polymer and the nature of the particular polymer used, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Injectable depot formulations are also prepared by administering the drug in liposomes or microemulsions that are compatible with body tissues.

Pharmaceutical dosage forms suitable for injection or infusion may include sterile aqueous solutions or dispersions or sterile powders containing the active ingredient which are adapted for the immediate preparation of sterile injectable or infusion solutions or dispersions, optionally encapsulated in liposomes. In all cases, the final dosage form must be sterile, fluid and stable under the conditions of manufacture and storage.

Sterile injectable solutions are prepared by incorporating the polypeptides or constructs of the invention in the required amount in an appropriate solvent with several other ingredients listed above as needed, followed by filter sterilization. In the case of sterile powders for the preparation of sterile injectable solutions, methods of preparation include vacuum and freeze drying processes which yield a powder of the active ingredient plus any additional ingredient of interest present in the pre-sterile filtered solutions.

The amount of polypeptides or constructs of the invention required for use in treatment will vary not only with the particular polypeptide or construct chosen, but also with the route of administration, the nature of the condition being treated, and the age and condition of the patient, and will ultimately be determined. at the discretion of the treating physician or physician. Also, the dosage of the polypeptides or constructs of the invention will vary.

The desired dose may conveniently be presented as a single dose or as divided doses administered at appropriate intervals, such as two, three, four or more sub-doses per day. The sub-dose itself may be further subdivided into, for example, several separate, freely distributed administrations.

The introduction may include long-term daily treatment. By "long term" is meant at least two weeks, and preferably several weeks, months or years. Necessary modifications within this dosage range can be determined by one of ordinary skill in the art using only routine experimentation provided herein. See Remington's Pharmaceutical Sciences (Martin, EW, ed. 4), Mack Publishing Co., Easton, PA. The dosage can also be adjusted by the individual physician in case of any complications.

In another aspect, kits are provided comprising a polypeptide or construct of the invention, a nucleic acid of the invention, an expression vector of the invention, or a host or host cell of the invention. The kit may also contain one or more vials containing the polypeptide or construct and instructions for use. The kit may also contain means for administering the polypeptide or construct of the invention, such as a syringe, infusor, or the like.

The use of polypeptides, constructs or compositions according to the invention

The invention further relates to the uses and uses of the polypeptides, constructs, nucleic acids, host cells, and compositions described herein, as well as methods for the prevention and/or treatment of diseases or conditions associated with CD123. Some preferred, but non-limiting applications and uses will become apparent from the further description provided herein.

The polypeptides, constructs and compositions of the present invention can generally be used to activate T cells at (site) CD123-expressing cells; for example, to lyse cells expressing CD123. Simultaneous binding of the polypeptides and constructs of the present invention to TCR on T cells and CD123 on tumor cells induces cell activation and subsequent lysis (destruction) of cells expressing CD123. When the polypeptides and constructs of the present invention are not bound to cells expressing CD123, they do not substantially activate T cells. As such, target-independent lysis (ie, cell lysis without CD123 expression) by the polypeptides and constructs of the present invention is minimal.

Accordingly, in one aspect, the polypeptides, constructs, and compositions of the present invention cause lysis of cells expressing CD123, with an average percentage of lysis of at least 10%, preferably at least 15%, such as at least 16% , 17%, 18%, 19%, or 20% or more, e.g., 30% or more compared to the number of cells expressing CD123 under the same conditions, but without the presence of a polypeptide or construct of the invention, measured by any suitable method known per se, for example, using one of the assays described herein (such as human T-cell mediated redirected killing assays based on flow cytometry, as described in the Examples section).

In addition, or at the same time, the lysis of CD123-negative cells induced by T cell activation, polypeptides, constructs and compositions of the present invention is no more than about 10%, for example, 9% or less, for example, 8, 7 or 6% or even less than the number of CD123-negative cells under the same conditions, but without the presence of a polypeptide or construct of the invention, measured by any suitable method known per se, for example, using one of the assays described herein (such as retargeted kill assays Human T cells based on flow cytometry as described in the Examples section).

Such killing of CD123 expressing cells may be advantageous in diseases or conditions in which the presence of such CD123 expressing cells is abundant and/or undesirable.

Accordingly, in one aspect, the present invention provides a polypeptide, construct or composition for use as a drug.

In a further aspect, the present invention provides a polypeptide or construct of the invention, or a composition containing them, for use in the prevention, treatment and/or amelioration of a CD123 associated disease or condition.

More specifically, the present invention provides a polypeptide or construct of the invention, or a composition comprising the same, for use in the prevention, treatment and/or amelioration of a CD123-associated disease or condition, wherein the CD123-associated disease or condition is a proliferative disease or inflammatory condition.

The invention also relates to a method for preventing, treating and/or ameliorating a disease or condition associated with CD123, said method comprising administering to a subject in need thereof a pharmaceutically active amount of a polypeptide or construct of the invention and/or a composition containing the same.

In particular, the present invention relates to a method as described above, wherein the disease or condition associated with CD123 is a proliferative disease or an inflammatory condition.

An inflammatory condition can be any inflammatory condition that can be prevented, treated and/or alleviated by killing cells expressing CD123.

In one aspect, the inflammatory condition is selected from the group consisting of autoimmune lupus (SLE), allergy, asthma, and rheumatoid arthritis.

Accordingly, the present invention provides a polypeptide, construct or composition for use in the prevention, treatment and/or reduction of an inflammatory condition, wherein said inflammatory condition is selected from the group consisting of autoimmune lupus (SLE), allergy, asthma, and rheumatoid arthritis.

Accordingly, the present invention also relates to methods for preventing, treating and/or alleviating an inflammatory condition, wherein said inflammatory condition is selected from the group consisting of autoimmune lupus (SLE), allergy, asthma, and rheumatoid arthritis, said method comprising administering to a subject in need of this, a pharmaceutically active amount of at least one polypeptide or construct according to the invention or composition according to the invention.

A proliferative disease can be any proliferative disease that can be prevented, treated and/or alleviated by killing CD123 expressing cells.

In one aspect, said proliferative disease is a cancer. Examples of cancers associated with CD123 overexpression will be understood by those skilled in the art based on the disclosure herein, and include, for example, without limitation, the following cancers: lymphomas (including Burkitt's lymphoma, Hodgkin's lymphoma, and non-Hodgkin's lymphoma), leukemias ( including acute myeloid leukemia, chronic myeloid leukemia, B-cell acute lymphoblastic leukemia, chronic lymphocytic leukemia and hairy cell leukemia), myelodysplastic syndrome, neoplasm of blast plasmacytoid dendritic cells, systemic mastocytosis and multiple myeloma.

Accordingly, the present invention relates to a polypeptide, construct or composition for use in the prevention, treatment and/or alleviation of an oncological disease, wherein said oncological disease is selected from the group consisting of lymphomas (including Burkitt's lymphoma, Hodgkin's lymphoma and non-Hodgkin's lymphoma), leukemias (including acute myeloid leukemia, chronic myeloid leukemia, B-cell acute lymphoblastic leukemia, chronic lymphocytic leukemia and hairy cell leukemia), myelodysplastic syndrome, neoplasms of blast plasmacytoid dendritic cells, systemic mastocytosis and multiple myeloma.

Accordingly, the present invention also relates to methods for preventing, treating and/or alleviating a cancer, wherein said cancer is selected from the group consisting of lymphomas (including Burkitt's lymphoma, Hodgkin's lymphoma, and non-Hodgkin's lymphoma), leukemias (including acute myeloid leukemia, chronic myeloid leukemia, B-cell acute lymphoblastic leukemia, chronic lymphocytic leukemia and hairy cell leukemia), myelodysplastic syndrome, blast plasmacytoid dendritic cell neoplasms, systemic mastocytosis and multiple myeloma, wherein said method comprises administering, to an object in need thereof, a pharmaceutically active amount, according to at least one polypeptide or construct of the invention or composition of the invention.

The invention also relates to the use of a polypeptide or a construct of the invention or a composition of the invention for the manufacture of a medicament.

In a further aspect, the present invention relates to the use of a polypeptide or construct of the invention, or a composition containing them, for the manufacture of a medicament for the prevention, treatment and/or amelioration of a CD123-associated disease or condition.

More specifically, the present invention relates to the use of a polypeptide or construct of the invention, or a composition containing them, for the manufacture of a medicament for the prevention, treatment and/or amelioration of a disease or condition associated with CD123, where the associated disease or condition is a proliferative disease or inflammatory condition.

In one aspect, the invention relates to the use of a polypeptide or construct of the invention, or a composition containing it, for the manufacture of a medicament for the prevention, treatment and/or amelioration of an inflammatory condition, wherein said inflammatory condition is selected from the group consisting of autoimmune lupus (SLE), allergies, asthma and rheumatoid arthritis.

In another aspect, the invention relates to the use of a polypeptide or construct of the invention, or a composition containing it, for the manufacture of a medicament for the prevention, treatment and/or amelioration of a condition of a proliferative disease, wherein said proliferative disease is an oncological disease. Examples of cancers associated with overexpression of CD123 will be understood by those skilled in the art based on the disclosure herein and, for example, include (but are not limited to) the following cancers: lymphomas (including Burkitt's lymphoma, Hodgkin's lymphoma, and non-Hodgkin's lymphoma), leukemias ( including acute myeloid leukemia, chronic myeloid leukemia, B-cell acute lymphoblastic leukemia, chronic lymphocytic leukemia and hairy cell leukemia), myelodysplastic syndrome, neoplasm of blast plasmacytoid dendritic cells, systemic mastocytosis and multiple myeloma.

Accordingly, the present invention also relates to the use of a polypeptide or a construct of the invention, or a composition containing them, for the manufacture of a medicament for the prevention, treatment and/or alleviation of a condition in an oncological disease, where said oncological disease is selected from the group consisting of lymphomas (including including Burkitt's lymphoma, Hodgkin's lymphoma and non-Hodgkin's lymphoma), leukemias (including acute myeloid leukemia, chronic myeloid leukemia, B-cell acute lymphoblastic leukemia, chronic lymphocytic leukemia and hairy cell leukemia), myelodysplastic syndrome, neoplasms of blast plasmacytoid dendritic cells, systemic mastocytosis and multiple myeloma.

In the context of the present invention, the term "prevention, treatment and/or alleviation" includes not only the prevention, treatment and/or alleviation of a disease, but also usually includes preventing the onset of a disease, slowing or changing the course of a disease, preventing or slowing the onset of one or more symptoms associated disease, reducing and/or ameliorating one or more symptoms associated with the disease, reducing the severity and/or duration of the disease and/or any symptoms associated with it, and/or preventing a further increase in the severity of the disease and/or any symptoms associated with it , prevention, reduction or elimination of any physiological damage caused by the disease, and, as a rule, any pharmacological action that is beneficial to the patient who is being treated.

Used interchangeably herein, the term "pharmaceutically effective amount" or "pharmaceutically active amount" refers to an amount sufficient to activate T cells in the presence of CD123 expressing cells. In the context of a CD123-associated disease, it refers to an amount of a polypeptide, construct, or pharmaceutical composition alone or in combination with another therapy that provides a therapeutic benefit in the prevention, treatment, and/or amelioration of a CD123-associated disease. Used in connection with the amount of a multispecific polypeptide or construct of the invention, the term may encompass an amount that improves overall therapy, reduces or avoids unwanted effects, or enhances therapeutic efficacy or synergy with another therapy.

As used herein, the term "therapy" refers to any protocol, method, and/or agent that can be used to treat, prevent, and/or treat a CD123-associated disease, such as an inflammatory condition or a proliferative disease. In some embodiments, the terms “therapies” and “therapy” refer to biological therapy, supportive therapy, and/or other therapies useful for the treatment, prevention, and/or management of a CD123-associated disease, such as an inflammatory condition or a proliferative disease, or one or several of its symptoms known to a person skilled in the art, for example, medical personnel.

In another aspect, the invention relates to a method of immunotherapy, and in particular to passive immunotherapy, which comprises administering to an object suffering from or at risk of a disease associated with CD123, a pharmaceutically active amount of a polypeptide or construct according to the invention and/or a pharmaceutical composition containing them.

The object to be treated may be any warm-blooded animal, but in particular it is a mammal and in particular a human. As will be appreciated by one of skill in the art, the subject to be treated will in particular be a human suffering from or at risk for the diseases and conditions mentioned herein.

Generally, the polypeptides or constructs of the invention and/or compositions containing them can be administered by any suitable route. For example (but not limited to), the polypeptides of the invention and compositions containing them can be administered orally, parenterally (for example, intravenously, intraperitoneally, subcutaneously, intramuscularly, intramuscularly, intraarterially or intrathecally, or any other route of administration that avoids the gastrointestinal tract) , intranasal, transdermal, topically, by suppository, by inhalation, again depending on the particular pharmaceutical composition or composition to be used. The physician will be able to select the appropriate route of administration and the appropriate pharmaceutical composition or composition for use in such administration, depending on the disease or disorder to be prevented or treated and other factors well known to the physician.

In a preferred aspect, the polypeptides or constructs of the invention, or compositions containing them, are administered intravenously (eg, but not limited to, by infusion or bolus) or subcutaneously.

The polypeptides or constructs of the invention and/or compositions containing them are administered in accordance with a treatment regimen that is suitable for preventing, treating and/or ameliorating a disease associated with CD123. The physician will generally be able to determine an appropriate treatment regimen, depending on factors such as the type of disease being treated, the stage of the disease, the severity of the disease and/or the severity of its symptoms, the particular polypeptide or construct of the invention to be used, the particular route of administration, and the pharmaceutical the composition or composition to be used, age, sex, weight, diet, general condition of the patient, and the like, well known to the physician.

Typically, the treatment regimen includes the introduction of one or more polypeptides or constructs according to the invention or one or more compositions containing them, in one or more pharmaceutically effective amounts or doses. The specific amount or doses to be administered may be determined by the physician, again based on the factors noted above.

Generally, to prevent, treat, and/or alleviate a CD123-associated disease, and depending on the type of CD123-associated disease (e.g., proliferative disorder (including cancer) or inflammatory condition) being treated, the stage of disease to be treated. be treated, the efficacy of the polypeptide or construct of the invention to be used, the particular route of administration, and the particular pharmaceutical formulation or composition used, the polypeptides of the invention will typically be administered in an amount of 1 gram to 1 microgram per kg of body weight per day. The physician will generally be able to determine the appropriate daily dose depending on the factors mentioned herein. It will also be clear that in certain cases the doctor may choose to deviate from these amounts, for example, based on the factors mentioned above and his expert judgment. As a general rule, some indication of administration amounts can be derived from the amounts typically administered for comparable conventional antibodies of antibody fragments against the same target in essentially the same way, however, taking into account differences in affinity/avidity, potency , biodistribution, half-life and the like are well known to the skilled person.

Generally, a single polypeptide or construct of the invention will be used in the above method. However, it is within the scope of the invention to use two or more polypeptides or constructs of the invention in combination.

The polypeptides or constructs of the invention, or compositions containing them, may also be used in combination with one or more other pharmaceutically active compounds or principles, i. as a combination treatment regimen, which may or may not result in a synergistic effect. Again, the physician will be able to select such additional compounds or principles, as well as an appropriate combination treatment regimen, based on the above factors and their expert judgment.

In particular, the polypeptides, constructs, and compositions of the invention may be used in combination with other pharmaceutically active compounds or principles that are or may be used in the prevention, treatment, and/or amelioration of a CD123-associated disease (e.g., a proliferative disorder (including cancers). ) or an inflammatory condition), which may or may not result in a synergistic effect. Examples of such compounds and principles, as well as routes, methods, and pharmaceutical compositions or compositions for their administration, will be understood by the physician.

Examples of such compounds and principles, as well as routes, methods and pharmaceutical compositions or compositions for their administration will be understood by the physician and include (but are not limited to): anthracyclines (daunurubicin, doxorubicin, idarubicin, mitoxantrone, rubidazon), cytarabine (AML), hematopoietic factors growth demethylating agents (such as decitabine or azacitidine), polytransretinoic acid, arsenic trioxide, DNA methyltransferase inhibitors, melphalan, prednisone, lenalidomide, cyclophosphamide, thalidomide, dexamethasone, bortezomib, fludarabine, corticosteroids, vincristine, rasburicase, L-asparaginase, PEGylated asparaginase, cladribine, pentostatin, adriamycin, bleomycin, vinblastine, dacarbazine; or any combination of them.

When two or more substances or active principles are to be used as part of a combination treatment regimen, they can be administered by the same route of administration or by different routes of administration at essentially the same time or at different time points (e.g., essentially simultaneously, sequentially or according to the alternating mode). When the substances or active principles are to be administered simultaneously via the same route of administration, they may be administered as different pharmaceutical compositions or compositions, or as part of a combined pharmaceutical composition or composition, as will be understood by the person skilled in the art.

In addition, when two or more active substances or active principles are to be used as part of a combined treatment regimen, each of the substances or active principles may be administered in the same amount and according to the same regimen as used when the compound or active principle is applied by itself. by itself, and such combined use may or may not result in a synergistic effect. However, when the combined use of two or more active substances or active principles results in a synergistic effect, it may also be possible to reduce the amount of one, several or all of the substances or active principles to be administered while still achieving the desired therapeutic effect. This may, for example, be useful in preventing, limiting or reducing any unwanted side effects that are associated with the use of one or more substances or active principles, when used in their usual amounts, while obtaining the desired pharmaceutical or therapeutic effect.

The effectiveness of the treatment regimen used in accordance with the invention can be determined and/or monitored by any method known per se for a given disease or disorder, as will be understood by the physician. The physician will also be able, if necessary and on a case-by-case basis, to change or modify a particular treatment regimen to achieve the desired therapeutic effect, to avoid, limit or reduce unwanted side effects, and / or to achieve an appropriate balance between achieving the desired therapeutic effect, on the one hand hand, and the prevention, limitation or reduction of unwanted side effects, on the other hand.

Typically, the treatment regimen will be followed until the desired therapeutic effect is achieved and/or until the desired therapeutic effect is to be maintained. Again, this can be determined by a doctor.

Further use of the polypeptides or constructs, nucleic acids, genetic constructs, and hosts and host cells of the invention will be apparent to those skilled in the art based on the disclosure herein.

The aspects illustrated and discussed in this specification are only intended to educate those skilled in the art in the best way known to the inventors for making and using the invention. Modifications and variations of the above aspects of the invention are possible without departing from the invention, as will be understood by those skilled in the art in light of the foregoing. Therefore, it should be understood that within the scope of the claims and their equivalents, the invention may be practiced otherwise than specifically described.

Further, the invention will be described using the following non-limiting preferred aspects, examples and figures.

The entire contents of all references (including references, issued patents, published patent applications, and pending patent applications) cited throughout this application are hereby incorporated by reference in their entirety, in particular for the description referenced above.

EXAMPLES

Example 1 TCR Related Materials and Methods

1.1. TCR αβ/CD3-transfected cell lines

Transient and stable cell lines CHO-K1 (ATCC: CCL-61), HEK293H (Life technologies, 11631-017), Llana (llama umbilical cord fibroblast cells) were obtained with recombinant overexpression of all 6 chains of the complete human T-cell receptor (TCR) complex. ). For this, the coding sequences of the TCR alpha (α) and TCR beta (β) chains were cloned into a pcDNA3.1-based vector, downstream of the CMV promoter, and a 2A-like viral peptide sequence was inserted between both chains in order to cause the ribosome to jump into the time of translation of the polyprotein. In the same vector, the coding sequences for the epsilon, delta, gamma, and zeta chains of the CD3 complex were cloned downstream from an additional CMV promoter, again using a 2A-like viral peptide sequence between the respective chains. In addition, a stable clone of HEK293H was obtained with recombinant overexpression of 4 chains of human CD3, as described above, using a single gene vector.

The sequences used for the constant domains of human CD3 and human TCR were obtained from UniProtKB (CD3 delta: P04234, CD3 gamma: P09693, CD3 epsilon: P07766, CD3 zeta: P20963, TCR α: P01848 and TCR β: P01850; SEQ ID NO: 70 -75, respectively). Sequences for human TCRα/β variable domains were derived from crystal structure sequences (PDB codes: 2IAN, 2XN9 and 3TOE) (human TCRα variable domains were derived from 2IAN, 2XN9 and 3TOE with SEQ ID NOS: 343, 76 and 345, respectively; human TCRβ variable domains were derived from 2IAN, 2XN9 and 3TOE with SEQ ID NOS: 344, 77 and 346, respectively).

Cell surface expression of the human T cell receptor complex was confirmed by flow cytometry using a functional mouse IgG2b anti-human TCRα/β antibody, clone BW242/412 (Miltenyi, 130-098-219) and a functional mouse IgG2a anti-CD3 antibody labeled with PE, clone OKT-3 (eBioscience, 12-0037) (figure 1).

1.2 Soluble recombinant TCR α/β proteins

Human and cynomolgus/rhesus monkey TCRα/β soluble proteins were produced in-house. The sequences of the extracellular portion of the human TCRα/β constant domain were obtained from UniProtKB (TCR α: P01848 and TCR β: P01850; SEQ ID NOs: 74 and 75, respectively). The human TCRα/β variable domains were derived from the crystal structure sequence (PDB code: 2XN9; SEQ ID NOs: 76 and 77, respectively, for the α and β chain).

The sequences for the extracellular portion of the cynomolgus/rhesus monkey TCRα/β constant domains were obtained from GenBank files EHH63463 and AEA41868, respectively (SEQ ID NOS: 347 and 348). The sequences for the cynomolgus/rhesus monkey TCRα/β variable domains were obtained from AEA41865 and AEA41866 (SEQ ID NOS: 349 and 350, respectively, for the α and β chain).

The extracellular domains of human TCRα/β(2XN9) or cynomolgus/rhesus TCRα/β were fused to the zipper protein coding sequence (O'Shea et al. 1993 Curr. Biol. 3 (10): 658-667), expressed in CHOK1SV (Lonza) cells using the GS Lonza™ gene expression system and then purified.

The quality of the TCR α/β zipper protein was assessed in an ELISA binding assay. Maxisorp 96-well ELISA plates (Nunc) were coated with 2 μg/ml soluble recombinant human TCRα/β(2XN9) zipper or soluble recombinant TCRα/β zipper. After overnight incubation, the plates were washed and blocked with PBS + 1% casein for 1 hour at room temperature. Plates were then incubated with serial dilutions of either FLAG labeled functional nanobody or functional mouse anti-TCRα/β non-human primate/rat IgG antibody, clone R73 (eBioscience, 16-5960) for 1 hour at room temperature, with shaking, again washed and incubated with anti-FLAG monoclonal antibody conjugated to M2-peroxidase (HRP) (Sigma, A8592), respectively, with peroxidase-conjugated rabbit antibody against mouse immunoglobulins (Dako, P0260). After 1 hour, a solution of TMB One (Promega, G7431) was added. The reaction was stopped with 2 M H ₂ SO ₄ and dose-dependent binding was determined by measuring the optical density at 450 nm using Tecan sunrise 4 (figure 2).

Example 2 Immunization of TCR/CD3 llamas, cloning of heavy chain-only antibody fragment repertoires and phage production

2.1. Immunization

It is believed that in camelids (eg llama and alpaca) antibodies containing only heavy chains against the α and/or β constant chains of the T cell receptor (TCR) are generated. Although the native T cell receptor complex consists of the CD3 chains (gamma, delta, epsilon and zeta) as well as the α- and β-chains of the TCR, it has been suggested that the absence of CD3 chains would facilitate access to the TCR constant domains. Moreover, the CD3 chains laterally surround and restrict access to the constant domains of the α- and β-chains of TCR. Contrary to our experience with other targets, generating an immune response against TCR α- or β-chains was not as easy as expected.

In the final approach, after the approval of the Ethics Committee (CRIA, LA1400575, Belgium-EC2012 #1), the authors attempted a complex immunization protocol with DNA encoding the T-cell complex. Briefly, 3 additional llamas were immunized with the human pVAX1-TCR(2IAN)/CD3 plasmid vector (described in Example 1.1) (Invitrogen, Carlsbad, CA, USA) and the human pVAX1-TCRα/β(2XN9)/CD3 plasmid vector (described in Example 1.1). example 1.1) (Invitrogen, Carlsbad, CA, USA) using standard protocols. Two llamas received an additional 1 subcutaneous injection of primary human T cells. Human T cells were harvested from buffy coats from healthy volunteers (Ghent Blood Bank) using RosetteSep (StemCell Technologies, 15061) followed by enrichment on Ficoll-Paque™ PLUS (GE Healthcare, 17-1440-03) according to manufacturer's instructions and stored in liquid nitrogen. After thawing, cells were washed and resuspended in D-PBS from Gibco and kept on ice until injection.

2.2. Cloning of antibody fragment repertoires containing only heavy chains and obtaining phages.

For each animal, blood samples were collected after administration of one type of immunization antigen. From these blood samples, PBMCs were prepared using Ficoll-Hypaque according to the manufacturer's instructions (Amersham Biosciences, Piscataway, NJ, USA). For each immunized llama, libraries were constructed by pooling all of the RNA isolated from samples originating from a specific subset of immunization regimens, ie. after one type of immunization antigen.

Briefly, the PCR amplified V _HH repertoire was cloned through specific restriction sites into a vector designed to facilitate phage display of the V _HH library. The vector was derived from pUC119. In frame with the V _HH coding sequence, the vector encodes a C-terminal 3xFLAG and a His6 tag. Phages were prepared according to standard protocols (see, for example, WO 04/041865, WO 04/041863, WO 04/062551, WO 05/044858 and other prior art sources and applications filed by Ablynx NV cited herein).

Example 3 Selection of V _HH TCR/CD3 Specific by Phage Display

The vast majority of the V _HH selected were directed against the variable regions of the TCR α or TCR β chains. Therefore, the authors had to develop different selection and counter-selection strategies.

Briefly, V _HH repertoires derived from all llamas and cloned as a phage library were used in various selection strategies using a variety of selection conditions. Screening using transfected human TCR/CD3 cell lines with the same variable domain used during immunization yielded only variable domain binding agents. For this reason, tools were used during selection involving transfected cells comprising various TCRα/β variable domains (described in Example 1.1), soluble protein (described in Example 1.2), or primary human T cells (isolated as described in Example 2.1) ), which have proven to be critical in the identification of constant domains. Additional variables during selection included the method of antigen presentation (in solution using cells or plated using proteins), antigen concentration, ortholog used (recombinant human or cynomolgus TCR α/β protein), and number of rounds of selection. All solid phase-coated selections were made in 96-well Maxisorp plates (Nunc, Wiesbaden, Germany).

The selection was carried out as follows. TCRα/β-CD3 antigen preparations for solid phase and liquid phase selection formats were presented as described above at several concentrations. After 2 hours of incubation with phage libraries followed by extensive washing, bound phages were eluted with trypsin (1 mg/ml) for 15 minutes. Trypsin protease activity was immediately neutralized by the application of 0.8 mM protease inhibitor ABSF. As a control, selections without antigen were carried out in parallel.

The resulting phages were used to infect E. coli to analyze individual V _HH clones. Periplasmic extracts were prepared according to standard protocols (see, e.g., WO 03/035694, WO 04/041865, WO 04/041863, WO 04/062551 and other prior art references and applications cited herein by Ablynx NV) .

Example 4 Screening, sequence analysis and purification

4.1. Screening for TCR/CD3-binding Nanobodies in Flow Cytometry Analysis

The periplasmic extracts were screened for binding to TCR/CD3 expressing cells using human TCR/CD3 transfected CHO-K1 or HEK293H cells and the appropriate CHO-K1 or HEK293H control lines in a mixed cell line scheme. For this, a large batch of control cell lines were labeled with 8 μM PKH26 and frozen. 5×10 ⁴ PKH-labeled control cells were mixed with 5×10 ⁴ target cells and incubated with periplasmic extracts for 30 minutes at 4° C. and washed 3 times. The cells were then incubated with 1 μg/ml of ANTI-FLAG® M2 monoclonal antibody (Sigma-Aldrich, F1804) for 30 minutes at 4°C, washed again and incubated for 30 minutes at 4°C with 5 μg/ml of goat antibodies , against mouse IgG labeled with allophycocyanin (APC) AffiniPure (Jackson Immunoresearch, 115-135-164). Samples were washed, resuspended in FACS buffer (D-PBS from Gibco, with 10% FBS from Sigma and 0.05% sodium azide from Merck) and then analyzed with a BD FACSArray. First, based on the distribution of FSC-SSC was selected population P1, which represented more than 80% of the entire cell population. Within these gates, 20,000 cells were counted at the time of data collection. Based on the PKH26-SSC distribution, a PKH labeled parent population and an unlabeled human TCR/CD3 target population were selected. For these two populations, the mean APC was calculated.

4.2 Screening for TCR/CD3-binding nanobodies in the human T cell activation assay

After several attempts, it turned out that the activation of purified human T cells with antibodies or nanobodies in accordance with standard protocols, i. coated on a 96-well plate was not sensitive enough (data not shown).

Another assay based on the activation of bead-associated T cells was developed to evaluate activity. Briefly, Dynabeads® with goat anti-mouse IgG (ThermoFisher Scientific, 11033) were coated with ANTI-FLAG® M2 monoclonal mouse antibody (Sigma-Aldrich, F1804) (15 μg/1E7 beads). After a 2 hour incubation period at 4° C., Dynabeads® were washed and incubated with 80 μl of periplasmic extract for 20 minutes at 4° C. with shaking. Unbound nanobodies were washed out before adding the bead complex together with soluble mouse anti-CD28 antibody (Pelicluster CD28 - Sanquin, M1650) to purified primary human T cells (isolated as described in Example 2.1). Unstimulated human T cells were used as controls. Briefly, Dynabeads® with goat anti-mouse IgG (ThermoFisher Scientific, 11033) coupled to ANTI-FLAG® M2 monoclonal mouse antibodies were incubated with 80 μl of a periplasmic extract containing irrelevant nanobodies. After removal of unbound nanobodies in a washing step, the irrelevant nanobody-bead complex was added to purified primary human T cells. After a 24-hour incubation at 37°C and 5% CO ₂ the activation status of human T cells was determined by measuring the level of expression of CD69 by flow cytometry using a mouse monoclonal antibody against human CD69PE (BD Biosciences, 557050).

4.3 Sequence analysis of obtained nanobodies

Nanobodies that received positive results in the flow cytometry screen and in the T cell activation assay were sequenced.

Sequence analysis led to the identification of nanobody T0170056G05 and various members of its family, representing a total of 104 different clones (SEQ ID NOS: 42 and 78-180). An appropriate alignment was performed (Table A-1).

The sequence variability of the CDR family members against T0170056G05 is shown in the tables below.

* In case position 5 is L, then position 6 is also L.

4.4 Purification of monovalent nanobodies

Two representative nanobodies of the identified family were selected and expressed in E. coli TG1 as triple Flag, His6-tagged proteins. Expression was induced by adding 1 mM IPTG and allowed to continue for 4 hours at 37°C. After centrifugation of cell cultures, periplasmic extracts were prepared by freezing-thawing of precipitates. These extracts were used as starting material and the nanobodies were purified by IMAC and size exclusion chromatography (SEC).

The nanobodies were purified to 95% purity as assessed by SDS-PAGE (data not shown).

Example 5 Binding of Anti-TCR Nanobodies to Human TCR/CD3 Expressed on CHO-K1 Cells and Purified Primary Human T Cells

The dose-dependent binding of purified monovalent anti-TCR nanobodies to human TCR α/β (2XN9)/CD3 expressed on CHO-K1 cells and to purified primary human T cells was assessed by flow cytometry. Briefly, cells were harvested and transferred to a 96-well V-bottom plate (1×10 ⁵ cells/well) and serial dilutions of nanobodies (with 1 μM) were allowed to bind for 30 minutes at 4° C. in FACS buffer. Cells were washed three times by centrifugation and labeled with monoclonal mouse antibodies ANTI-FLAG® M2 1 μg/ml (Sigma-Aldrich, F1804) for 30 minutes at 4°C, washed again and incubated for 30 min at 4°C with 5 μg/ml. ml R-phycoerythrin-labeled F(ab') fragment of goat anti-mouse IgG AffiniPure (Jackson Immunoresearch 115-116-071). After incubation, cells were washed 3 times with FACS buffer. Cells were then resuspended in FACS buffer supplemented with 5 nM TOPRO3 (Molecular Probes, T3605) to distinguish between live and dead cells that are removed during the gate selection procedure. Cells were analyzed using a FACS Array flow cytometer (BD Biosciences) and Flowing Software. First, based on the distribution of FSC-SSC was selected population P1, which represented more than 80% of the entire cell population. In these gates, 10,000 cells were counted during data collection. TOPRO+ cells (dead cells) were excluded from this population and the mean PE value was calculated.

The results are shown in Figure 3. The EC ₅₀ values obtained from the dose-response curve are shown in Table C-1.

Table C-1. EC ₅₀ (M) of anti-TCR monovalent nanobodies on binding human CHO-K1 TCRα/β(2XN9)-/CD3 cells and on binding of purified primary T cells determined by flow cytometry CHO-K1 TCRα/β(2XN9)/CD3 Primary human T cells Sample ID EC ₅₀ (M) 95%LCI 95% UCI _EC50 (M) 95%LCI 95% UCI T0170055A02 8.4E-09 7.2E-09 9.7E-09 9.1E-08 8.1E-08 1.0E-07 T0170056G05 8.9E-09 8.3E-09 9.4E-09 9.1E-08 8.3E-08 9.9E-08

The nanobodies are clearly associated with human TCR/CD3 expressed on CHO-K1 cells. The nanobodies also bind to purified primary human T cells, albeit with slightly less efficiency than human α/β(2XN9)/CD3 TCR CHO-K1 cells.

Example 6 Binding Epitope Determination

Binding of purified monovalent anti-TCR nanobodies to human TCRα/β(2IAN)/CD3 expressed on HEK293H cells was assessed and compared to binding to human CD3 transfected HEK293H cells by flow cytometry as described in Example 5. Cells were added dilution series T0170055A02 and T0170056G05 starting from 1 µM. The parental HEK293H cell line was included as a TCR/CD3 negative cell line.

The results are shown in Figure 4. The EC ₅₀ values obtained from the dose-response curve are shown in Table C-2.

Table C-2. EC ₅₀ (M) of monovalent nanobodies against human TCRα/β(2IAN)/CD3 or human CD3 expressed on HEK293H cells determined by flow cytometry HEK293H wt HEK293HCD3 HEK293H TCR/CD3 Sample ID _EC50 MCF at 1 µM _EC50 MCF at 1 µM _EC50 MCF at 1 µM T0170055A02 no match 246 no match +1194 5.5E-08 91229 T0170056G05 no match 299 no match 352 8.4E-08 86510

The nanobodies clearly bind to human TCR (2IAN)/CD3 expressed on HEK293H, but not to HEK293H cells transfected with human CD3 alone or to the parental HEK293H cell line. In conclusion, 2 clones were specific for binding to human TCRα/β. Binding to human CD3 was not observed.

Example 7 Binding of Anti-TCR Nanobodies to Soluble Recombinant Human TCRα/β Protein

7.1. Binding of anti-TCR nanobodies to human T-cell receptor protein in ELISA

Binding of purified monovalent TCR nanobodies to soluble recombinant human TCR α/β protein was assessed in ELISA (as described in Example 1.2) using 2 μg/ml of soluble recombinant recombinant human TCR α/β protein as a direct coating.

The results are shown in Figure 5. The EC ₅₀ values obtained from the dose-response curve are shown in Table C-3.

Table C-3. EC ₅₀ (M) of monovalent anti-TCR nanobodies upon binding of soluble recombinant TCR protein
human α/β (2XN9) as determined by ELISA Sample ID EC ₅₀ (M) 95%LCI 95% UCI T0170055A02 1.9E-09 1.7E-09 2.2E-09 T0170056G05 4.0E-09 3.5E-09 4.6E-09

Anti-TCR nanobodies bind to soluble recombinant human TCR α/β protein.

7.2. Binding of anti-TCR nanobodies to human T-cell receptor protein in BLI

Binding affinity was measured using biolayer interferometry (BLI) on an Octet RED384 instrument (Pall ForteBio Corp.). Recombinant human soluble TCRα/β(2XN9) zipper protein was covalently immobilized on amino-reactive sensors (ForteBio) via NHS/EDC coupling chemistry. For kinetic analysis, the sensors were first immersed in run buffer (10 mM Hepes, 150 mM NaCl, 0.05% p20, pH 7.4 from GE Healthcare Life Sciences) to determine baseline settings. The probes were then immersed in wells containing various concentrations of purified nanobodies (ranging from 1.4 nM to 1 mM) for the association step (180 s) and transferred to wells containing working buffer for the dissociation step (15 min). Affinity constants (KD) were calculated using a 1:1 interaction model using ForteBio data analysis software.

The results are shown in Figure 6. The binding characteristics are listed in Table C-4.

Table C-4. Kinetic analysis of monovalent anti-TCR nanobodies to bind soluble recombinant human TCR α/β (2XN9) protein as determined by the Octet RED384 instrument soluble TCRα/β(2XN9) human zipper protein Sample ID k _on (1/Ms) k _off (1/s) K _D (M) T0170055A02 4.9E+04 8.4E-04 1.7E-08 T0170056G05 5.0E+04 1.2E-03 2.4E-08

Binding affinity determined using BLI on human soluble TCRα/β (2XN9) zipper protein showed a correlation with affinity determined on human TCRα/β(2XN9)/CD3 CHO-K1 cells using flow cytometry (see See also example 5).

Example 8 Binding of Anti-TCR Nanobodies to Recombinant TCRα/β Soluble Protein

8.1. Binding of anti-TCR nanobodies to the cynomolgus monkey T-cell receptor protein in ELISA

Binding of purified monovalent anti-TCR nanobodies to recombinant soluble cynomolgus TCR α/β protein was assessed in ELISA (as described in Example 1.2) with 2 μg/ml, recombinant soluble cynomolgus TCR α/β zipper coated as a direct cover.

EC ₅₀ values derived from the dose-response curve are presented in Table C-5. An example result is shown in figure 7.

Table C-5. EC50 (M) of monovalent anti-TCR nanobodies for binding to recombinant cynomolgus monkey TCRα/β zipper protein determined by ELISA Sample ID EC ₅₀ (M) 95%LCI 95% UCI T0170055A02 1.6E-07 1.5E-07 1.7E-07 T0170056G05 7.7E-08 6.6E-08 9.1E-08

The results showed that anti-TCR nanobodies bind to recombinant cynomolgus monkey TCRα/β, a zipper protein.

8.2. Binding of anti-TCR nanobodies to the cynomolgus monkey T-cell receptor protein in BLI

The binding affinity of monovalent anti-TCR nanobodies was measured using biolayer interferometry (BLI) on an Octet RED384 instrument (Pall ForteBio Corp.) essentially as described in Example 7.2 using recombinant soluble cynomolgus monkey TCRα/β protein.

The results are shown in Figure 8. The binding characteristics of the anti-TCR nanobodies are shown in Table C-6.

Table C-6. Kinetic analysis of monovalent anti-TCR nanobodies upon binding to recombinant cynomolgus macaque TCRα/β, a zipper protein, performed using an Octet RED384 instrument Sample ID k _on (1/Ms) k _off (1/s) K _D (M) T0170055A02 1.1E+05 2.4E-02 2.1E-07 T0170056G05 1.1E+05 1.6E-02 1.5E-07

The nanobodies bind to the recombinant soluble TCRα/β protein of the cynomolgus monkey with an affinity 10 times lower than the recombinant soluble protein TCRα/β (2XN9) - with a "lightning".

Example 9 Determination of Activating Ability of Purified Primary Human T Cells

The functionality of the purified monovalent anti-TCR nanobodies was evaluated in a human T cell activation assay. Dynabeads® with goat anti-mouse IgG (ThermoFisher Scientific, 11033) were coated with ANTI-FLAG® M2 monoclonal mouse antibody (Sigma-Aldrich, F1804, 15 μg/1E7 beads). After a 2 hour incubation period at 4° C., Dynabeads® were washed and incubated with a fixed (1 μg) amount of purified Flag-labelled nanobody for 20 minutes at 4° C. with shaking. Unbound nanobodies were washed out before adding the bead complex along with soluble mouse anti-CD28 antibody (Pelicluster CD28 - Sanquin, M1650) to purified primary human T cells isolated (isolated as described in Example 2.1) from various healthy donors. In addition, the effect of monovalent TCR binding by nanobodies was evaluated by incubating the nanobody with purified primary human T cells without prior capture of Dynabeads® with anti-mouse IgG antibody in the presence of anti-CD28 antibody. The activation status of purified primary human T cells was monitored by measuring CD69 expression by flow cytometry using a mouse anti-human CD69PE monoclonal antibody (BD Biosciences, 557050) after incubation for 24 hours at 37° C. and 5% CO ₂ .

In conclusion, anti-TCR nanobodies showed clear CD69 activation upon capture on Dynabeads with anti-mouse IgG. The irrelevant nanobody showed no upregulation of CD69 (Figure 9A). In addition, none of the nanobodies presented in solution could activate purified primary human T cells, as measured by increased expression of CD69 (Figure 9B).

Example 10 Immunization of CD123 Lamas, Cloning of Heavy Chain Only Antibody Repertoires and Phage Generation

10.1. Immunization

Following standard protocols, three llamas were immunized with His-tagged recombinant human CD123 extracellular domain (R&D Systems, 301-R3/CF) via intramuscular injection in the neck, using Stimune as an adjuvant (Cedi Diagnostics, Lelystad, The Netherlands) .

Immune serum samples taken on day 35 were analyzed for antigen specific binding by ELISA with adsorbed hCD123. All llamas show an excellent IgG1-mediated serum response and a good to moderate heavy chain-mediated response against hCD123.

10.2 Cloning of heavy chain-only antibody fragment repertoires and phage production

For each animal, 100 ml blood samples were collected four and eight days after the last injection of immunizing antigen. PBMCs were prepared from these blood samples using Ficoll-Hypaque according to the manufacturer's instructions (Amersham Biosciences, Piscataway, NJ, USA). Libraries were created for each immunized llama by pooling total RNA isolated from different blood samples.

Briefly, the PCR amplified V _HH repertoire was cloned through specific restriction sites into a phagemid vector designed to facilitate phage display of the V _HH library. The vector was derived from pUC119. In frame with the V _HH encoding sequence, the vector encodes a 3xFLAG C-terminal tag and HIS6. Phages were prepared according to standard protocols (see, for example, WO 04/041865, WO 04/041863, WO 04/062551, WO 05/044858 and other prior art references and Ablynx NV applications cited herein).

Example 11 Selection of CD123-Specific V _HH Using Phage Display

V _HH repertoires derived from all llamas and cloned as a phagemid library were used in various selection strategies using a variety of selection conditions. Variables included: i) source of CD123 antigen (recombinant protein produced in human cells or full-length protein overexpressed on cells), ii) antigen presentation (in solution using a biotinylated recombinant ectodomain directly applied to plates using a non-biotinylated ectodomain fused to Fc) and iii) antigen concentration.

Briefly, HEK293T cells overexpressing CD123 (self-produced), biotinylated human CD123 (R&D Systems, 301-R3/CF, biotinylated in-house) and plated on human CD123-Fc plates (Sino Biologicals, 10518-H08H) were incubated for 1 h - 2 h with 2×E ¹¹ phage particles from different libraries, followed by intensive washing; bound phages were eluted with trypsin (1 mg/ml) for 15 minutes, and then the protease activity was immediately neutralized by the application of 0.8 mm ABSF protease inhibitor. As a control, selection was carried out in parallel with the parental cell line or without antigen.

The resulting phages were used to infect E. coli to analyze individual V _HH clones. Periplasmic extracts were prepared according to standard protocols (see, e.g., WO 03/035694, WO 04/041865, WO 04/041863, WO 04/062551 and other prior art references and applications cited herein by Ablynx NV) .

Example 12 Screening for CD123 binding nanobodies.

12.1. Screening in ELISA for binding

The periplasmic extracts were screened in an ELISA for binding to human CD123 (R&D Systems, 301-R3). For this microtiter plate was coated with human CD123 (1 μg/ml) and incubated overnight at 4°C. The plates were blocked for one hour at room temperature with 4% Marvel in PBS. The plates were washed with PBS-Tween. Periplasmic extracts (diluted 1/10 in PBS with 2% Marvel) were incubated for at least 1 hour at room temperature. The plates were washed with PBS-Tween, after which V _HH binding was detected with anti-FLAG M2 peroxidase (HRP) monoclonal antibodies (Sigma, A8592, 1/5000) in PBS with 1% Marvel. Staining was performed with esTMB substrate (Nalgene) and signals were measured after 15 minutes at 450 nm.

12.2. Screening by flow cytometry

The periplasmic extracts were screened in flow cytometry analysis on transiently transfected HEK293T-hCD123 cells in a 96-well format. In addition, binding was evaluated for endogenously IL-3R-expressing MOLM-13 cells to confirm binding to IL-3Rα in the presence of an IL-3Rβ partner in the heterodimeric receptor complex. For this, cells (1×10 ⁵ cells/well/0.1 ml) were incubated with periplasmic extracts (1:10 dilutions) for 30 minutes at 4°C in FACS buffer (D-PBS from Invitrogen, with 10% FBS from Sigma and 0.05% sodium azide from Merck). Cells were washed 3 times and incubated with 1 μg/ml monoclonal ANTI-FLAG® M2 antibody (Sigma-Aldrich, F1804) for 30 minutes at 4°C, washed again and incubated for 30 minutes at 4°C with goat antibody against mouse labeled antibodies labeled with RPE (Jackson) Immunoresearch, 115-116-071, 1:100). Samples were washed, incubated with TOPRO3 for dead cell staining in FACS buffer and fluorescence evaluated on a FACSArray (BD).

12.3. Nanobody sequencing

The nanobodies were sequenced and were positive in binding ELISA and in flow cytometry analysis. Sequence analysis led to the identification of nanobodies A0110056A10 and A0110055F03 and various members of their families. The corresponding alignments are shown in Table A-2 and Table A-3, respectively.

CDR sequence variability of family members against A0110056A10 is shown in the tables below.

* if position 3 is S, then position 7 is D;

** if position 6 is I, then position 8 is D.

The sequence variability of the CDR family members relative to A011005F03 is shown in the tables below.

* if position 6 is S, then position 10 ^is E.

* if position 4 is R, then position 5 is D or Y.

12.4 Purification of monovalent nanobodies

Representative nanobodies for each family were selected and expressed in E. coli TG1 as triple Flag, His6-tagged proteins. Expression was induced by adding 1 mm IPTG and carried out for 4 hours at 37°C. After centrifugation of cell cultures, periplasmic extracts were prepared by freezing-thawing of precipitates. These extracts were used as starting material and the nanobodies were purified by IMAC and size exclusion chromatography (SEC). The nanobodies were purified to 95% purity as assessed by SDS-PAGE (data not shown).

Example 13 Additional cell lines for characterization

13.1. Cell lines transfected with CD123

Stable cell lines HK293 Flp-In (Invitrogen, R750-07) and CHO Flp-In (Invitrogen, R758-07) with recombinant CD123 overexpression were generated using Flp-In™ site-directed recombination technology (Flp-In™ system for obtaining stable expression mammalian cell lines using Flp-recombinase-mediated integration (Invitrogen, K601001, K601002)). Thus, DNA integration occurs at a specific genomic location in the FRT (Flp recombination target) site by Flp recombinase (pOG44) derived from Saccharomyces cerevisiae. Both the Flp-In™ host cell line and the expression plasmid (pcDNA5) contain this FRT site, allowing for single homologous DNA recombination. The sequence for human CD123 was obtained from NCBI RefSeq NP_002174, the cynomolgus monkey CD123 sequence was obtained from NCBI genbank no. EHH61867.1 (SEQ ID NOS: 68 and 69, respectively). Cell surface expression of human and cynomolgus CD123 was confirmed by flow cytometry using a mouse anti-CD123 monoclonal antibody (BD Biosciences, 554527) and an isotype control mouse IgG2a (BD Bioscience, 16-4724-85). Briefly, cells (1×10 ⁵ cells/well) were harvested and transferred to a 96-well V-bottom plate (Greiner Bio-one, 651 180) and stained at 4°C with mouse monoclonal anti-CD123 antibody (0.25 µg/ml) and isotype control mouse IgG2a (0.25 µg/ml). After 30 min incubation, cells were pelleted by centrifugation and washed 3 times with FACS buffer (D-PBS from Gibco with 10% FBS (Sigma, F7524) and 0.05% sodium azide (Acros organic, 190380050)). Cells were then incubated with 5 μg/ml goat F(ab') fragment against mouse IgG labeled with AffiniPure R-phycoerythrin (Jackson Immunoresearch, 115-116-071) for 30 minutes at 4°C. After incubation, cells were washed 3 times with FACS buffer. Cells were then resuspended in FACS buffer supplemented with 5 nM TOPRO3 (Molecular Probes, T3605) to distinguish live from dead cells. Cells were analyzed using a FACS Array flow cytometer (BD Biosciences) and Flowing Software. First, based on the distribution of FSC-SSC was selected population P1, which represented more than 80% of the entire cell population. During the accumulation of data in these "gates" 10,000 cells were counted. TOPRO+ cells (dead cells) were excluded from this population and the mean PE value was calculated. The data is shown in figure 10.

13.2. Cell lines U937, MOLM-13, KG1a and NCI-H929

Human CD123 expression level on MOLM-13 (DSMZ, ACC-554), U-937 (ATCC®, CRL-1593.2™), KG1a (ATCC®, CCL246.1™) and NCI-H929 (DSMZ, ACC-163) was determined using an APC labeled anti-CD123 monoclonal antibody (BD Biosciences, 560087) and an APC labeled isotype control (Biolegend, 400220) in flow cytometry. Briefly, cells were harvested and suspended at a density of 1×10 ⁷ cells/ml in FACS buffer with 25 μg human Fc block (BD Biosciences, 564220) and incubated for 10 minutes at room temperature. The cells were then diluted to a cell concentration of 1×10 ⁶ cells/ml and transferred to a V-shaped 96-well plate (1×10 ⁵ cells/well). Cells were stained at 4° C. with APC-labeled anti-CD123 monoclonal mouse antibody (10-fold diluted) and APC-labeled isotype control (10-fold diluted). After 30 minutes incubation, cells were washed 3 times and resuspended in FACS buffer supplemented with 1 μg/ml propidium iodide (PI) (Sigma, P4170) for 30 minutes at 4°C and then analyzed using BD FACSCanto and Flowing software. First, based on the distribution of FSC-SSC was selected population P1, which represented more than 80% of the entire cell population. In P1, 10,000 cells were counted. PI+ cells (dead cells) were excluded from this population and the mean APC was calculated. The data is shown in figure 11.

In addition, the number of receptors per cell was determined using QIFIKIT (Dako, K0078) according to the manufacturer's instructions. Data are shown in Table C-7.

Table C-7. Number of CD123 molecules per cell MOLM-13 KG1a CD123 molecules/cell 6543 3353

Example 14 Binding of Monovalent Anti-CD123 Nanobodies to Cell Lines Endogenously Expressing CD123

Dose-dependent binding of purified monovalent anti-CD123 nanobodies to cell lines endogenously expressing CD123, MOLM-13 and KG1a was assessed by flow cytometry. Briefly, cells were harvested and transferred to a 96-well V-bottom plate (1×10 ⁵ cells/well) and serial dilutions of nanobodies (with 1 μM) were allowed to bind for 30 minutes at 4° C. in FACS buffer. Cells were washed three times by centrifugation and labeled with murine monoclonal antibody ANTI-FLAG® M2 (Sigma-Aldrich, F1804) for 30 minutes at 4°C, washed again and incubated for 30 minutes at 4°C with 5 μg/ml goat F( ab') fragment against mouse IgG labeled with R-phycoerythrin AffiniPure (Jackson Immunoresearch, 115-116-071). After incubation, cells were washed 3 times with FACS buffer. Cells were then resuspended in FACS buffer supplemented with 5 nM TOPRO3 (Molecular Probes, T3605) to distinguish between live and dead cells that are removed during gate selection. Cells were analyzed using a FACS Array flow cytometer (BD Biosciences) and Flowing software. First, based on the distribution of FSC-SSC was selected population P1, which represented more than 80% of the entire cell population. At this gate, 10,000 cells were counted at the time of data collection. TOPRO+ cells (dead cells) were excluded from this population and the mean PE value was calculated.

Binding of nanobodies to MOLM-13 and KG1a is shown in Figure 12. EC50 values derived from dose-response curves are shown in Table C-8.

Table C-8. EC ₅₀ (M) of monovalent anti-CD123 nanobodies for binding to MOLM-13 and KG1a determined by flow cytometry MOLM-13 KG1a Sample ID _EC50 (M) 95%LCI 95% UCI _EC50 (M) 95%LCI 95% UCI A0110056A10 6.3E-10 3.8E-10 8.8E-10 5.0E-10 2.4E-10 7.6E-10 A0110055F03 >1E-07 >1E-07

Both nanobodies were found to bind to cells endogenously expressing CD123 (MOLM-13, KG1a).

Example 15 Binding of Monovalent Anti-CD123 Nanobodies to CD123 on Transfected Cells

Dose-dependent binding of purified monovalent anti-CD123 nanobodies to CHO-K1 cells overexpressing human CD123 and HEK293 overexpressing cynomolgus CD123 was assessed by flow cytometry.

To detect A0110055F03 binding, cells were harvested and transferred to a 96-well V-bottom plate (1×10 ⁵ cells/well). Serial dilutions of A0110055F03 (starting at 100 nM) were allowed to bind for 30 minutes at 4° C. in FACS buffer. Cells were washed 3 times with FACS buffer by centrifugation and labeled with 1 μg/ml mouse monoclonal antibody ANTI-FLAG® M2 (Sigma-Aldrich, F1804) for 30 minutes at 4°C to detect bound nanobody. Detection was performed with 0.5 μg/ml goat anti-mouse F(ab') fragment IgG labeled with AffiniPure R-phycoerythrin (Jackson Immunoresearch, 115-116-071) for 30 minutes at 4°C. Cells were washed and incubated with TOPRO3 to stain for dead cells, which are then removed during a gate selection procedure. The cells were then analyzed using the BD FACSArray. First, based on the distribution of FSC-SSC was selected population P1, which represented more than 80% of the entire cell population. In these gates, 10,000 cells were counted during data collection. TOPRO+ cells (dead cells) were excluded from this population and the mean PE value was calculated.

To detect A0110056A10 binding, cells were harvested and transferred to a 96-well V-bottom plate (1×10 ⁵ cells/well). Serial dilutions of labeled Alexa647 A0110056A10 (starting at 100 nM) were allowed to bind for 30 minutes at 4° C. in FACS buffer. After 30 min incubation, cells were pelleted by centrifugation and washed 3 times with FACS buffer. Subsequently, cells were resuspended in FACS buffer supplemented with 1 μg/ml propidium iodide to distinguish between live and dead cells. Cells were analyzed using a FACS Array flow cytometer (BD Biosciences) and Flowing software. First, based on the distribution of FSC-SSC was selected population P1, which represented more than 80% of the entire cell population. In this gate, 10,000 cells were counted at the time of data collection. PI+ cells (dead cells) were excluded from this population and the mean APC value was calculated.

Binding of nanobodies to CD123 transfected cell lines and a control cell line is shown in Figure 13 and Figure 14 for nanobodies A0110056A10 and A0110055F03, respectively. EC ₅₀ values derived from dose-response curves are presented in Table C-9.

Table C-9. EC ₅₀ (M) of monovalent anti-CD123 nanobodies for binding to cells transfected with huCD123 and cyCD123 determined by flow cytometry HEK Flp-In cells transfected with cyCD123 CHO Flp-In cells transfected with huCD123 Sample ID _EC50 (M) 95%LCI 95% UCI _EC50 (M) 95%LCI 95% UCI A0110056A10 2.48E-09 1.90E-09 3.20E-09 9.74E-10 8.50E-10 1.11E-09 A0110055F03 2.53E-09 2.04E-09 3.14E-09 5.94E-09 1.00E-09 3.52E-08

Both nanobodies were found to bind to cell lines transfected with CD123. Both nanobodies are cross-reactive between human and cynomolgus CD123.

Example 16 Competition of nanobodies for binding to cell-expressed CD123 as measured by flow cytometry

To find out if CD123 nanobodies compete with each other, a competitive analysis of nanobodies was performed. To this end, a large batch of A0110056A10 has been tagged with Alexa647 and frozen. Cells were then harvested and transferred to the V-bottom of a 96-well plate (1×10 ⁵ cells/well) and mixed with serial dilutions of nanobodies and a fixed concentration of A0110056A10-Alexa647 (0.7 nM for MOLM-13, 0.5 nM for CHO Flp-In CD123 human). The concentrations of A0110056A10-Alexa647 used in the assay were below the EC ₅₀ value for A0110056A10-Alexa647 binding to the respective cells (binding curves are depicted in Figure 15). After a 90 minute incubation period at 4° C., A0110056A10-Alexa647 binding was determined by flow cytometry. To do this, the cells were washed 3 times and resuspended in FACS buffer with the addition of 1 μg/ml propidium iodide, incubated for 30 minutes at 4°C and then analyzed using BD FACSArray. First, based on the distribution of FSC-SSC was selected population P1, which represented more than 80% of the entire cell population. In P1, 10,000 cells were counted. PI+ cells (dead cells) were excluded from this population and the mean APC was calculated.

The results are shown in Figure 16. The IC ₅₀ values obtained from the dose-response curves are shown in Table C-10.

Table C-10. IC ₅₀ (M) of monovalent anti-CD123 nanobodies in competitive nanobodies A0110056A10 MOLM-13 CHO Flp-In CD123 human Sample ID IC ₅₀ (M) 95%LCI 95% UCI IC ₅₀ (M) 95%LCI 95% UCI A0110056A10 1.4E-09 1.1E-09 1.8E-09 6.7E-09 5.3E-09 8.5E-09 A0110055F03 >1E-07 / / /

As expected, unlabeled A0110056A10 competed with A0110056A10-Alexa647 for binding to CD123 on MOLM-13 cells and to human CD123 expressed on transfected CHO Flp-In cells. The A0110055F03 nanobody did not compete with A0110056A10-Alexa647 for CD123 binding on human CD123 transfected CHO Flp-In cells. In the MOLM-13 cell line, A0110055F03 only competed with A0110056A10-Alexa647 at the highest concentrations tested.

Example 17 Competition with mouse anti-CD123 monoclonal antibody (clone 7G3) for binding to cell-expressed CD123 as measured by flow cytometry

To test whether anti-CD123 nanobodies compete with mouse anti-CD123 monoclonal antibody (clone 7G3) for binding to human CD123 on cells, a mouse anti-CD123 monoclonal antibody (clone 7G3) competitive assay was performed using a technique based on flow cytometry as described in Example 16. To this end, serial dilutions of the nanobodies and the EC ₃₀ concentration of APC-labeled anti-CD123 monoclonal antibody (clone 7G3) (BD Biosciences, 560087) were incubated for 90 minutes with the cells, after which the binding of the antibody in the flow was determined. cytometry. Binding curves of APC-labelled anti-CD123 mouse monoclonal antibody (clone 7G3) to MOLM-13 and CD123-transfected human CHO Flp-In cells are shown in Figure 17.

The results of competitive experiments are shown in Figure 18. IC ₅₀ values derived from dose-response curves are shown in Table C-11.

Table C-11. IC ₅₀ (M) of monovalent anti-CD123 nanobodies in competition with mouse anti-CD123 monoclonal antibody (clone 7G3) MOLM-13 CHO Flp-In Human CD123 Sample ID IC ₅₀ (M) 95%LCI 95% UCI IC ₅₀ (M) 95%LCI 95% UCI A0110056A10 1.1E-09 1.1E-09 1.2E-09 6.5E-09 6.1E-09 6.9E-09 A0110055F03 4.8E-07 1.8E-07 1.2E-06

Nanobody A0110056A10 competed with mouse monoclonal anti-CD123 antibody (clone 7G3) for MOLM-13 and CHO Flp-In cells transfected with human CD123; therefore, the epitopes of the mouse anti-CD123 monoclonal antibody (clone 7G3) and the A0110056A10 nanobody at least partially overlap. A0110055F03 competed with a mouse monoclonal anti-CD123 antibody (clone 7G3) on MOLM-13 cells. The lack of competition with the mouse monoclonal anti-CD123 antibody (clone 7G3) on the CHO Flp-In cell line transfected with human CD123 may be the result of the lower affinity of the A0110055F03 nanobody for human CD123.

Example 18 Competition with mouse anti-CD123 monoclonal antibody (clone 7G3) for binding to recombinant human CD123 by ELISA

To investigate whether anti-CD123 nanobodies compete with mouse anti-CD123 monoclonal antibody (clone 7G3) for binding to recombinant human CD123 protein, a competitive assay was performed using an ELISA based technique. Briefly, a mouse monoclonal anti-CD123 antibody (clone 7G3) (BD Biosciences, 554527) was used as a coating at 1 μg/ml in PBS. After incubation overnight at 4°C, the plates were blocked with casein (1% in PBS) at room temperature. Then a serial dilution of monovalent anti-CD123 nanobodies and 4 nM recombinant CD123 protein, biotinylated in-house (R&D Systems, 301-R3/CF) was added and incubated for 1 hour at room temperature in PBS + 0.1% casein + 0 .05% Tween. The concentration of self-biotinylated recombinant CD123 protein was based on the EC30 value obtained by binding the self-biotinylated recombinant CD123 protein to a mouse monoclonal anti-CD123 antibody (clone 7G3) (binding curve depicted in Figure 19). An uncoated mouse monoclonal anti-CD123 antibody (clone 7G3) was taken in parallel as a positive control, and an irrelevant anti-egg lysozyme antibody, cAbLys, was taken in parallel as a negative control. The plates were washed with PBS + 0.05% Tween using a Tecan Hydrospeed washer and 7G3-bound biotinylated-CD123 was determined with 1 μg/ml extravidin peroxidase (Sigma, E2886) in PBS + 0.1% casein + 0.05% Tween followed by color development with esTMB substrate. The reaction was stopped with 1 M HCl and the absorbance at OD 450 nm was measured using Tecan Infinite M1000.

The results are shown in Figure 20. The IC ₅₀ value obtained from the dose-response curve is shown in Table C-12.

Table C-12. IC ₅₀ (M) of monovalent nanobody A010056A10 in competitive ELISA with 7G3 Sample ID IC ₅₀ (M) 95%LCI 95% UCI A0110056A10 3.1E-09 1.5E-09 6.5E-09

Competition was observed between A0110056A10 and a mouse monoclonal anti-CD123 antibody (clone 7G3) for binding to recombinant human CD123 protein. A0110055F03 does not compete with the mouse monoclonal anti-CD123 antibody (clone 7G3) for binding to recombinant human CD123 protein.

Example 19 Binding of Monovalent Anti-CD123 Nanobodies to Human CD123 Protein (SPR)

The binding affinity of the purified CD123-specific nanobodies for human CD123 was assessed using an SPR-based assay on a ProteOn XPR36 instrument. For this, recombinant CD123 (R&D Systems, 301-R3-025/CF) was immobilized on the CM5 chip by amine group coupling using EDC and NHS chemistry. Purified nanobodies were injected over 2 minutes at various concentrations (from 4.2 nM to 1000 nM) for kinetic analysis using the One-shot Kinetics™ approach. The flow rate was 45 μl/min and ProteOn working buffer (PBS, pH 7.4, 0.005% Tween 20) was used as running buffer. The dissociation time of the 1000 nM sample was 15 min. Association/dissociation data were assessed by fitting a 1:1 interaction model (Langmuir binding model).

Table C-13. Binding Characteristics of Monovalent Anti-CD123 Nanobodies Defined in ProteOn Using Human CD123 Protein Applied as Direct Coating Sample ID k _a [1/(M⋅s)] k _d (1/s) K _D (M) A0110056A10 5.3E+05 7.9E-04 1.5E-09 A0110055F03 2.5E+04 3.9E-03 1.6E-07

The K _D values of monovalent anti-CD123 nanobodies for binding to human CD123 correlated with cellular binding data. A0110056A10 has better affinity than A0110055F03.

Example 20 Construction of Multispecific CD123/TCR Polypeptides and Control Polypeptides

To obtain polypeptides capable of recruiting T cells, CD123 nanobodies were coupled to the anti-TCR (T cell receptor) nanobody T0170056G05 (SEQ ID NO: 42). The latter is a nanobody that specifically binds the TCRα/β constant domain. (See WO 2016/180969 entitled "T-cell recruitment polypeptides based on alpha/beta TCR reactivity", filed May 13, 2016 by Ablynx NV).

The therapeutic activity of polypeptides involved in T cells can be improved by simultaneously targeting multiple epitopes to a tumor associated antigen. Tumor cells can create an escape mechanism not only by down-regulating target antigens in therapy, but also by introducing (point) mutations. Simultaneous targeting of multiple epitopes on an antigen would likely reduce the chance of generating tumor escape variants from surveillance. In addition, targeting multiple epitopes in a single antigen can increase binding affinity (avidity effect). For this concept of multivalent tumor antigen targeting, two nanobodies responsive to the CD123 antigen were linked to the T0170056G05 nanobody against the TCR/CD3 complex.

The specific order of the respective nanobodies varied within the format. Effector and tumor nanobodies were genetically linked to the 35GS linker and subsequently expressed in yeast Pichia according to standard protocols (multispecific polypeptides). In parallel, irrelevant polypeptides were generated by replacing one or both of the tumor-reactive nanobodies with an irrelevant anti-egg white lysozyme nanobody cAbLys or anti-RSV nanobody RSV007B02 (Q108L).

The resulting polypeptides are listed in Table C-14.

Table C-14. Sample ID and description of multispecific polypeptides Sample ID SEQ
ID NO* Description T017000113 46 A0110055F03-35GS-cAbLys3(D1E,Q5V,A6E,Q108L)-35GS-T0170056G05-FLAG3-HIS6 T017000114 47 A0110055F03-35GS-A0110056A10-35GS-T0170056G05-FLAG3-HIS6 T017000115 48 A0110056A10-35GS-cAbLys3(D1E,Q5V,A6E,Q108L)-35GS-T0170056G05-FLAG3-HIS6 T017000116 49 A0110056A10-35GS-A0110055F03-35GS-T0170056G05-FLAG3-HIS6 T017000120 fifty cAbLys3(D1E,Q5V,A6E,Q108L)-35GS-A0110055F03-35GS-T0170056G05-FLAG3-HIS6 T017000121 51 cAbLys3(D1E,Q5V,A6E,Q108L)-35GS-A0110056A10-35GS-T0170056G05-FLAG3-HIS6 T017000125 42 T0170056G05-HIS6 T017000126 52 A0110055F03(E1D)-35GS-A0110056A10-35GS-T0170056G05-A T017000128 53 T0170056G05(E1D)-35GS-A0110056A10-A T017000129 54 T0170056G05(E1D)-35GS-RSV007B02(Q108L)-A T017000130 55 A0110056A10(E1D)-35GS-A0110055F03-35GS-T0170056G05-A T017000131 56 A0110056A10(E1D)-35GS-T0170056G05-A T017000132 57 RSV007B02(E1D,Q108L)-35GS-T0170056G05-A T017000134 58 A0110056A10(E1D)-35GS-T0170056G05-35GS-A0110055F03-A T017000135 59 A0110055F03(E1D)-35GS-T0170056G05-35GS-A0110056A10-A T017000138 60 T0170056G05(E1D)-35GS-A0110056A10-35GS-A0110055F03-A T017000139 61 T0170056G05(E1D)-35GS-A0110055F03-35GS-A0110056A10-A

*SEQ ID NOs correspond to multispecific polypeptide sequences without C-terminal tags or Ala extension.

Example 21 Competition between A0110056A10 and multispecific CD123/TCR polypeptides for binding to cell-expressed CD123 as measured by flow cytometry

Binding of multispecific CD123/TCR polypeptides to human or cynomolgus CD123 was assessed in the A0110056A10 competition assay as described in Example 16. After binding to huCD123 MOLM-13 and CHO Flp-In cells, binding to HEK Flp-In cells transfected with cynomolgus CD123 was assessed. .

To this end, the batch of A0110056A10 has been tagged with Alexa647 and frozen. The cells were then harvested and transferred to a 96-well V-bottom plate (1×10 ⁵ cells/well) and mixed with serial dilution of multispecific binding polypeptides (starting at 1 μM) and a fixed concentration of A0110056A10-Alexa647. The concentrations used in the assay (0.4 nM for MOLM-13, 0.9 nM for human CHO Flp-In CD123 and cynomolgus HEK Flp-In CD123) were below the EC ₅₀ value for A0110056A10-Alexa647 binding to the respective cells ( bonding curves are shown in figure 21). After an incubation period of 90 minutes at 4° C., A0110056A10-Alexa647 binding was determined by flow cytometry as described in Example 16. A0110056A10 and T017000129 were taken together as positive and negative controls, respectively.

The results are shown in Figure 22. The IC ₅₀ values obtained from the dose-response curves are shown in Table C-15 and Table C-16.

Table C-15. IC ₅₀ (M) of multispecific CD123/TCR polypeptides and controls in competitive assay with A0110056A10 nanobody on MOLM-13 cells MOLM-13 Sample ID IC50(M) 95%LCI 95% UCI T017000121 2.6E-08 2.2E-08 3.1E-08 T017000128 3.1E-09 2.6E-09 3.6E-09 T017000138 3.5E-09 3.0E-09 4.1E-09 T017000139 3.7E-09 3.1E-09 4.4E-09 T017000116 1.8E-09 1.5E-09 2.2E-09 A0110056A10 1.1E-09 8.7E-10 1.3E-09

Table C-16. IC ₅₀ (M) of CD123/TCR multispecific polypeptides and controls in a competitive assay with A0110056A10 nanobody on CD123-transfected cells HEK Flp-In cells transfected with cyCD123 CHO Flp-In cells transfected with huCD123 Sample ID IC ₅₀ (M) 95%LCI 95% UCI IC ₅₀ (M) 95%LCI 95% UCI T017000121 1.1E-07 9.6E-08 1.3E-07 2.3E-08 1.9E-08 2.7E-08 T017000128 3.1E-08 2.6E-08 3.6E-08 5.8E-09 5.4E-09 6.2E-09 T017000138 1.2E-08 1.0E-08 1.4E-08 3.6E-09 2.8E-09 4.6E-09 T017000139 1.1E-08 9.6E-09 1.3E-08 3.3E-09 2.7E-09 4.1E-09 T017000116 5.6E-09 4.8E-09 6.4E-09 1.6E-09 1.4E-09 1.9E-09 A0110056A10 7.0E-09 6.1E-09 8.1E-09 1.9E-09 1.7E-09 2.3E-09

All tested multispecific polypeptides showed binding to cells expressing human and cynomolgus CD123, confirming the cross-reactivity of human and cynomolgus CD123. A slight drop in affinity was observed for polypeptides that did not have A0110056A10 at the N-terminus.

Example 22 Competition between T0170056G05 and CD123/TCR multispecific polypeptides for binding to cell-expressed human TCR/CD3 as measured by flow cytometry

Binding of multispecific CD123/TCR polypeptides to human TCR/CD3 was evaluated in a competitive assay with T0170056G05 by flow cytometry. To this end, a large batch of T0170056G05 was labeled with biotin and frozen. CHO-K1 cells expressing human TCR/CD3 were then harvested and transferred to a 96-well V-bottom plate (1×10 ⁵ cells/well) and mixed with serial dilutions of multispecific binding polypeptides (starting at 1 μM) and fixed concentration of biotinylated T0170056G05 in FACS buffer. The concentration of biotinylated T0170056G05 used in the assay (30 nM) was below the EC50 value for cell binding (data not shown). After a 90 minute incubation period at 4° C., binding of the biotinylated T01700056G05 nanobody was determined by flow cytometry. For this, the cells were washed 3 times and resuspended in streptavidin-PE (Ebioscience, 12-4317-87, 1000-fold diluted) in FACS buffer and incubated for 30 min at 4°C. Cells were then washed 3 times and resuspended in FACS buffer + 1 μg/ml TOPRO (Molecular Probes, T3605) for 30 minutes at 4°C and then analyzed with BD FACSCanto. First, based on the distribution of FSC-SSC was selected population P1, which represented more than 80% of the entire cell population. In P1, 10,000 cells were counted. TOPRO+ cells (dead cells) were excluded from this population and the mean PE value was calculated. T0170056G05 was taken as a positive control.

The results are shown in Figure 23. The IC ₅₀ values obtained from the dose-response curves are shown in Table C-17.

Table C-17. IC ₅₀ (M) of multispecific CD123/TCR polypeptides and control nanobody in competitive assay with T0170056G05 CHO-K1 huTCR/CD3 Sample ID IC ₅₀ (M) 95%LCI 95% UCI T017000138 5.9E-08 5.5E-08 6.4E-08 T017000139 4.9E-08 4.5E-08 5.2E-08 T017000129 1.8E-07 1.7E-07 1.9E-07 T017000128 5.7E-08 5.3E-08 6.1E-08 T017000116 >1E-07 T0170056G05 5.5E-08 5.2E-08 5.9E-08

Binding of multispecific CD123/TCR polypeptides to human TCR/CD3 expressed on cells has been confirmed. A decrease in the CD123/TCR affinity of the T017000116 multispecific polypeptide compared to the monovalent TCR nanobody was observed due to the C-terminal position of the TCR nanobody.

Example 23 Competition Between T017000099 and Multispecific CD123/TCR Polypeptides for Binding to Cynomolgus TCR/CD3 Expressed on Cells by Flow Cytometry

Binding of multispecific CD123/TCR polypeptides to cynomolgus TCR/CD3 was assessed in a competitive assay with T01700099 (bivalent T0170056G01, SEQ ID NO: 337) by flow cytometry. To this end, HSC-F cells (JCRB, JCRB1164) expressing cynomolgus TCR/CD3 were harvested and transferred into a 96-well V-bottomed plate (1×10 ⁵ cells/well) and mixed with serial dilutions of multispecific CD123/ TCR (starting at 1 μM) and 500 nM T01700099 in FACS buffer. After a 90-minute incubation period at 4°C, cells were washed 3 times with FACS buffer by centrifugation and labeled with 1 µg/mL mouse monoclonal antibody ANTI-FLAG® M2 (Sigma-Aldrich, F1804) for 30 minutes at 4°C to detect bound T01700099. Detection was performed with 5 μg/ml goat anti-mouse IgG labeled with allophycocyanin (APC) AffiniPure (Jackson Immunoresearch, 115-135-164) for 30 minutes at 4°C. Cells were washed and incubated with propidium iodide to stain for dead cells, which were then removed during a gate selection procedure. The cells were then analyzed using the BD FACSArray. First, based on the distribution of FSC-SSC was selected population P1, which represented more than 80% of the entire cell population. At this gate, 10,000 cells were counted at the time of data collection. PI+ cells (dead cells) were excluded from this population and the mean APC was calculated. T017000125 were taken as a positive control.

The results are shown in Figure 24. The IC ₅₀ values obtained from the dose-response curves are shown in Table C-18.

Table C-18. IC ₅₀ (M) of multispecific CD123/TCR polypeptides and control nanobodies in competition with T017000099 HSC-F Sample ID IC ₅₀ (M) 95%LCI 95% UCI T017000125 3.7E-07 2.5E-07 5.3E-07 T017000128 6.3E-07 4.4E-07 8.8E-07 T017000129 5.2E-07 3.7E-07 7.2E-07 T017000138 4.4E-07 3.2E-07 6.2E-07 T017000139 6.4E-07 4.5E-07 9.2E-07

For all multispecific CD123/TCR polypeptides with a TCR-binding nanobody at the N-terminus, binding to cynomolgus TCR/CD3 was observed.

Example 24 Binding of monovalent nanobodies and multispecific polypeptides to the human CD123 protein (SPR)

Binding affinity for CD123/TCR multispecific polypeptides was assessed using SPR-based affinity determination on a ProteOn XPR36 instrument. For this, recombinant CD123 (R&D Systems, 301-R3-025/CF) was immobilized on the CM5 chip by amine group coupling using EDC and NHS chemistry. Purified nanobodies were injected over 2 minutes at various concentrations (from 4.2 nM to 1000 nM) using the One-shot Kinetics™ approach for kinetic analysis. The dissociation time of the 1000 nM sample was 15 min. The flow rate was 45 μl/min and ProteOn working buffer (PBS, pH 7.4, 0.005% Tween 20) was used as running buffer. Association/dissociation data were assessed by fitting a 1:1 interaction model (Langmuir binding model). Binding characteristics are listed in Table C-19.

Table C-19. Binding characteristics of multispecific polypeptides defined in ProteOn using human CD123 protein applied as direct coating Sample ID to _a [1/(M ⋅ s)] k _d (1/s) K _D (M) T017000120 1.5E+04 2.5E-03 1.7E-07 Values are indicative only due to incomplete regeneration of the previous polypeptide (T017000116) T017000121 4.0E+04 8.6E-04 2.2E-08 - T017000113 2.0E+04 3.2E-03 1.6E-07 T017000115 3.3E+05 5.1E-04 1.5E-09 Values are indicative only due to incomplete regeneration of the previous polypeptide (T017000114) T017000114 8.4E+04 1.9E-04 2.3E-09 T017000116 2.4E+05 2.1E-04 8.8E-10

The K _D of monovalent nanobodies and multispecific polypeptides for human IL-3Rα correlate with binding data on cells. The T017000116 polypeptide containing two structural elements against IL-3Rα had a better K _D .

Example 25 Redirected Human T Cell Mediated Killing of CD123 Target Cells by Multispecific CD123/TCR Polypeptides in a Flow Cytometry Assay

To evaluate whether multispecific CD123/TCR polypeptides are capable of killing tumor cells, cytotoxicity assays were performed with isolated human T cells as effector cells.

For this, human T cells were collected from buffy coats from healthy volunteers (Ghent Blood Bank) using RosetteSep (StemCell Technologies, 15061) followed by enrichment on Ficoll-Paque™ PLUS (GE Healthcare, 17-1440-03) according to manufacturer's instructions. The quality and purity of purified human T cells were tested with anti-CD3 (eBioscience, 12-0037-73), anti-CD8 (BDBiosciences, 555367), anti-CD4 (BD Biosciences, 345771), anti-CD45RO (BD Biosciences, 555493), anti-CD45RA (BDBiosciences, 550855), anti-CD19 (BDBiosciences, 555413), anti-CD25 (BDBiosciences, 557138), and anti-CD69 (BDBiosciences, 557050) fluorescently labeled antibodies in flow cytometry analysis. Cells were frozen in liquid nitrogen.

MOLM-13 and KG1a cells expressing human CD123 were labeled with 8 μM PKH-26 membrane dye using the PKH26 red fluorescent cell linker kit (Sigma, PKH26GL-1KT) according to the manufacturer's instructions and used as target cells. 2.5×10 ⁵ effector (i.e., primary human T cells) and 2.5×10 ⁴ target cells (i.e., PKH labeled MOLM-13 or KG1a cells) were co-incubated in 96-well V- figurative tablets (ratio of effectors and targets 10:1). To measure concentration-dependent cell lysis, serial dilutions of multispecific CD123/TCR polypeptides were added to cells and incubated for 18 h in an atmosphere with 5% CO ₂ at 37°C. The A0110056A10 nanobody and the T017000129 and T017000132 polypeptides were taken together as a negative control. After incubation, cells were pelleted by centrifugation and washed with FACS buffer. Cells were then resuspended in FACS buffer supplemented with 5 nM TOPRO3 (Molecular Probes, T3605) to distinguish live from dead cells. Cells were analyzed using a FACS Array flow cytometer (BD Biosciences). For each sample, a total sample volume of 80 μl was obtained. Gating was performed on PKH26 positive cells and TOPRO3 positive cells were identified in this population. T017000129, T017000132 and A0110056A10 were taken in parallel as negative controls.

Exemplary results are shown in Figure 25 and Figure 26 for MOLM-13 and KG1a cells, respectively. EC ₅₀ values are shown in Tables C-20 and C-21 for MOLM-13 and KG1a cells, respectively.

Table C-20. EC ₅₀ (M) and percent lysis of multispecific CD123/TCR polypeptides for targeted human T cell-mediated killing of MOLM-13 cells in a flow cytometric assay Sample ID _EC50 (M) 95%LCI 95% UCI % lysis T017000116 2.2E-10 1.1E-10 4.5E-10 17 T017000128 1.5E-10 9.4E-11 2.5E-10 twenty T017000135 1.1E-11 8.1E-12 1.5E-11 33 T017000138 2.3E-11 1.5E-11 3.7E-11 22 T017000139 2E-11 1.4E-11 2.9E-11 29 T017000134 3E-10 1E-10 9.2E-10 9

Table C-21. EC ₅₀ (M) and percent lysis of CD123/TCR multispecific polypeptides for redirected human T-cell-mediated killing in a flow cytometric assay Sample ID _EC50 (M) 95%LCI 95% UCI % lysis T017000114 2.4E-10 1.5E-10 3.6E-10 7 T017000116 4.2E-10 2.4E-10 7.3E-10 5 T017000128 8.7E-10 7.0E-10 1.1E-09 28 T017000135 5.1E-11 4.1E-11 6.5E-11 eighteen T017000138 2.8E-10 2.2E-10 3.4E-10 24 T017000139 1.1E-10 8.5E-11 1.3E-10 27

CD123/TCR ultraspecific polypeptides induced human T-cell mediated killing of CD123 positive cell lines. There was a clear preference for the position of the TCR nanobody in the multispecific polypeptide. In general, polypeptides with an anti-TCR nanobody in the N-terminal position showed better killing potential. T017000135, T017000138 and T013700139 polypeptides with two CD123 reactive nanobodies showed improved specific activity compared to T017000128 polypeptide with only one CD123 nanobody. These results demonstrated that multispecific CD123/TCR polypeptides can induce T-cell mediated killing of tumor target positive cells and that targeting multiple epitopes on a single antigen improves functionality (avidity effect).

In addition, a comparison of T017000138 and T017000139 polypeptides, which are both trivalent polypeptides with a reactive TCR nanobody at the N-terminal position, showed that there is an effect of CD123 nanobody orientation on activity and efficacy.

The monovalent CD123 building block and irrelevant polypeptides containing the TCR building block did not cause any killing of the target cell, suggesting the need to cross-link the T cell and the target cell with multispecific CD123/TCR polypeptides to induce killing.

The results were confirmed using purified T cells from different donors (data not shown).

Example 26 Redirected Killing of Target Cells of CD123 Mediated by Cynomolgus T Cells by Multispecific CD123/TCR Polypeptides in a Flow Cytometry Assay

To confirm the cross-reactivity of multispecific CD123/TCR polypeptides between human and cynomolgus TCR, the polypeptides were evaluated in the cynomolgus monkey T cell-mediated CD123-positive tumor cell killing assay. Briefly, multispecific polypeptides were incubated with 2.5×10 ⁴ PKH labeled target cells (i.e., MOLM-13 or KG1a cells) in the presence of 2.5×10 ⁵ effector cells (i.e., primary cynomolgus monkey T cells). ) corresponding to an effector to target cell ratio (Ratio E:T) of 10 to 1 as described in Example 25. T cells were isolated at LPT Laboratory of Pharmacology and Toxicology GmbH & Co. KG, using a T cell isolation kit (MACS, 130-091-993). The A0110056A10 nanobody and the T017000129 and T017000132 polypeptides were taken together as a negative control.

Exemplary results are shown in Figures 27 and Figures 28 for MOLM-13 and KG1a cells, respectively. EC ₅₀ values are shown in Table C-22 and Table C-23 for MOLM-13 and KG1a cells, respectively.

Table C-22. EC ₅₀ (M) and percent lysis of CD123/TCR multispecific polypeptides for targeted killing of MOLM-13 cells by cynomolgus monkey T cells in a flow cytometric analysis Sample ID _EC50 (M) 95%LCI 95% UCI % lysis T017000116 3.9E-10 2.4E-10 6.2E-10 fifteen T017000128 1.8E-10 1.5E-10 2.3E-10 fifty T017000138 1.7E-11 1.3E-11 2.2E-11 47 T017000139 1.1E-11 8.9E-12 1.4E-11 55

Table C-23. EC ₅₀ (M) and percent lysis of multispecific CD123/TCR polypeptides for cynomolgus monkey T-cell mediated KG1a cell killing in a flow cytometric analysis Sample ID _EC50 (M) 95%LCI 95% UCI % lysis T017000114 1.9E-10 1.3E-10 2.8E-10 7 T017000116 3.7E-10 2.9E-10 4.7E-10 fourteen T017000128 3.2E-10 2.7E-10 3.7E-10 38 T017000135 2.4E-11 2.0E-11 2.9E-11 22 T017000138 8.8E-11 7.6E-11 1.0E-10 38 T017000139 2.6E-11 2.3E-11 3.0E-11 42

All CD123/TCR multispecific polypeptides except T017000134 induced cynomolgus monkey T-cell mediated killing of CD123-positive MOLM-13 or KG1a cell lines. Polypeptides with a reactive TCR nanobody at the N-terminal position were the most potent and efficient. The T017000138 and T017000139 polypeptides, which are both trivalent polypeptides with two reactive CD123 nanobodies, showed improved activity compared to the T017000128 polypeptide, which contains only one reactive CD123 nanobody. Monovalent anti-CD123 nanobodies and irrelevant polypeptides containing the TCR building block did not induce killing of target cells, suggesting the need to cross-link T cells and target cells via multispecific CD123/TCR polypeptides to induce killing.

The results were confirmed using purified T cells from various cynomolgus monkeys (data not shown).

Example 27 Activation of Cynomolgus Macaque T Cells by Multispecific CD123/TCR Polypeptides During Redirected Killing of CD123 Target Cells Mediated by Cynomolgus Macaque T Cells

To monitor T cell activation after treatment of cynomolgus T cells and CD123-positive MOLM-13 cells with CD123/TCR multispecific polypeptides, the polypeptides were incubated for 72 h at 37°C with 2.5×10 ⁴ target cells in the presence of 2 .5×10 ⁵ primary T cells (E:T=10:1) as described in Example 26. Cynomolgus macaque T cell activation was measured by monitoring CD25 upregulation in the CD4/CD8 T cell population by flow cytometry .

To this end, after a 72-hour incubation, effector and target cells were collected by centrifugation and suspended in FACS buffer with 25 μg/ml Human BD Fc Block™ (BD Bioscience, 564220) and incubated for 10 minutes at room temperature (RT). Cells were then stained with monoclonal mouse anti-CD4-APC antibodies (Biolegend, 300505) (200-fold diluted), monoclonal mouse anti-CD8 APC antibodies (BDBiosciences, 555366) (50-fold diluted), and monoclonal anti-CD25 PE antibodies (BD Biosciences, 557138) (50-fold diluted) in FACS buffer for 30 minutes at 4°C. After incubation, cells were pelleted by centrifugation and washed with FACS buffer. Cells were then resuspended in FACS buffer and analyzed with a FACS Canto flow cytometer (BD Biosciences). For each sample, a total sample volume of 30 μl was obtained. T cells were sorted into selection frames based on the SSC-APC plot. The mean PE was calculated from this population.

The data is shown in Figure 29. The EC ₅₀ value obtained for T017000139 is shown in Table C-24.

Table C-24. EC ₅₀ (M) upregulation of CD25 on cynomolgus macaque T cell by T017000139 during redirected killing of MOLM-13 cells by cynomolgus macaque T cells in a flow cytometry-based assay Sample ID _EC50 (M) 95%LCI 95% UCI T017000139 6.0E-11 5.1E-11 7.1E-11

No upregulation of CD25 was observed when T cells were incubated with CD123-positive MOLM-13 target cells and monovalent TCR-binding building blocks or CD123, T0170056G05, A0110055F03 or A0110056A10 or the irrelevant multivalent T017000129 polypeptide. The data showed upregulation of CD25 on primary cynomolgus monkey T cells after incubation with CD123 positive target cells MOLM-13 and the T017000139 multispecific polypeptide.

MOLM-13 cells were killed in a dose dependent manner (Figure 25, Table C-22), indicating that the CD123/TCR multispecific binding polypeptide induced T cell activation during redirected killing.

Similarly, T cells were not activated when incubated with target cells and monovalent building blocks and an irrelevant multispecific polypeptide, indicating that the T cell and target cell need to be cross-linked with TCR/CD123 multispecific polypeptides to induce CD25 activation.

Example 28 Redirected Human T Cell Mediated Killing of CD123 Transfected Adhesive Target Cells by Multispecific CD123/TCR Binding Polypeptides in an xCELLigence Based Assay

TCR/CD123 binding polypeptides have been characterized for redirected human T cell mediated killing of transfected CD123 adherent target cells in an xCELLigence-based assay. This assay measures the fluctuations in impedance caused by the adherence of target cells to the electrode surface. T cells do not adhere and therefore do not interfere with impedance measurements. The xCELLigence instrument (Roche) quantifies electrical impedance changes by displaying them as a dimensionless parameter called cell index, which is directly proportional to the total area of a tissue culture well that is covered with cells. Briefly, the xCELLigence station was placed in an incubator at 37°C with 5% CO ₂ . 50 µl of assay medium was added to each well of an E-plate 96 (ACEA Biosciences; 05 232 368 001) and a blank read was performed on the xCELLigence system to measure background impedance in the absence of cells. Then 2×10 ⁴ CHO Flp-In cells transfected with human CD123 or control CHO Flp-In cells were seeded on E-plate 96 plates and 50 μl of serially diluted multispecific polypeptide was added. After 30 minutes at room temperature, 50 μl human T cells (3×10 ⁵ ) were added to each well to achieve a 15:1 effector to target ratio. The plate was placed on the xCELLigence station and the impedance was measured every 15 minutes for 3 days. The data was analyzed using the fixed time point specified in the results.

The IC ₅₀ values obtained from this assay are listed in Table C-25. The results are shown in figure 30, figure 31 and figure 32.

Table C-25. IC ₅₀ (M) T013700139 for redirected human T-cell mediated killing of adherent target cells transfected with human CD123 in an xCELLigence-based assay at a 15:1 effector to target ratio assayed 50 hours post seeding Sample ID IC50(M) 95%LCI 95% UCI T017000139 2.4E-10 2.0E-10 2.8E-10

The data obtained confirmed the results obtained in the kill assay based on flow cytometry, ie. multispecific CD123/TCR polypeptides can induce human T-cell mediated killing of CD123 positive cell lines (Figure 30), with no killing activity observed in the absence of T cells (Figure 32). In addition, only in the presence of the tumor target antigen CD123 was T-cell mediated killing observed (see Figure 31 for no killing in the case of the control cell line), indicating that the multispecific polypeptides are critically dependent on their target for induction of cytotoxicity.

The monovalent nanobodies A0110056A10 and T0170056G05 and the irrelevant polypeptide T017000129 did not induce killing of target cells, suggesting the need for cross-linking of T cells and target cells with a multispecific CD123/TCR binding polypeptide to induce killing.

The results were confirmed using purified T cells from different donors (data not shown).

Example 29 Cynomolgus T cell-mediated killing of CD123-transfected adherent target cells by multispecific CD123/TCR-binding polypeptides in an xCELLigence-based assay

To confirm the cross-reactivity of multispecific polypeptides, the polypeptides were evaluated in redirected killing by cynomolgus monkey T cells of adherent target cells transfected with cynomolgus CD123 in an xCELLigence-based assay as described in Example 28.

The IC ₅₀ values obtained from this assay are listed in Table C-26. The results are shown in figure 33, figure 34 and figure 35.

Table C-26. IC ₅₀ (M) T013700139 for redirected, T-cell mediated cynomolgus monkey killing of adherent target cells transfected with cynomolgus monkey CD123 in xCELLigence-based assay at 15:1 effector to target ratio analyzed 80 h post seeding Sample ID IC ₅₀ (M) 95%LCI 95% UCI T017000139 1.1E-11 2.7E-12 4.8E-11

The multispecific CD123/TCR T017000139 polypeptide could induce cynomolgus monkey T-cell mediated killing of CD123 positive cell lines (Figure 33), with no killing activity observed in the absence of T cells (Figure 35). In addition, in the presence of the tumor target antigen, cynomolgus monkey CD123, T-cell mediated killing was observed (Figure 34), indicating that the multispecific polypeptides are critically dependent on their target for induction of cytotoxicity.

The monovalent nanobodies A0110056A10 and T0170056G05 and the irrelevant T017000129 polypeptide do not induce killing of target cells, suggesting that T cells and target cells need to be cross-linked with a multispecific CD123/TCR binding polypeptide to induce killing.

Cross-reactivity between CD123 and the human cynomolgus monkey TCR of the T017000139 multispecific polypeptide was confirmed in xCELLigence-based kill assay.

The results were confirmed using purified T cells from different donors (data not shown).

Example 30 Effect of Multispecific CD123/TCR Binding Polypeptides on Cytokine Production During Redirected Killing

Cytokine release induction was monitored during human and cynomolgus T-cell mediated CD123 killing based on xCELLigence assay. Release of the cytokine IFN-γ and IL-6 was measured by ELISA. Briefly, CHO-K1 cells transfected with human CD123 (2×10 ⁴ cells/well) were seeded in an E-plate 96 in the presence of purified primary human or cynomolgus T cells (3×10 ⁵ cells/well) with serial dilution multispecific TCR/CD123 binding polypeptide (starting at 125 nM) or a fixed concentration (125 nM) of irrelevant polypeptides as described in Example 28. 72 h after adding primary human or cynomolgus T cells/polypeptides to the plates, IFN production was measured human -γ and human IL-6 by human primary T cells and cynomolgus monkey IFN-γ by primary cynomolgus monkey T cells in the supernatant.

The release of human IL-6 was measured in ELISA using the Quantikine human IL-6 ELISA kit (R&D systems, D6050) according to the manufacturer's instructions. IFN-γ release was determined as follows: Maxisorp 96-well ELISA plates (Nunc) were coated with anti-human IFN-γ antibodies (BDBiosciences, 551221), and respectively with cynomolgus monkey IFN-γ antibodies (BioLegend, 507502). After incubation overnight, the plates were washed and blocked with PBS + 2% BSA for 1 hour at room temperature. Plates were then incubated with 100 µl of supernatants (2-fold diluted) and 1 µg/ml of biotinylated anti-IFN-γ antibody (BD Biosciences, 554550) for 2 h 30 min with shaking, washed again and incubated with streptavidin-HRP (Dakocytomation , P0397). TMB One solution (Promega, G7431) was added after 30 minutes. The reaction was terminated with 2M H ₂ SO ₄ and polypeptide dose dependent IFN-γ production was determined by measuring absorbance at 405 nm using a Tecan sunrise 4.

The results for IFN-γ are shown in Figure 36. The results for IL-6 are shown in Figure 37. The EC ₅₀ values obtained from these assays are listed in Table C-27 and Table C-28.

Table C-27. EC ₅₀ (M) of TCR/CD123 binding polypeptides for IFN-γ secretion during redirected T-cell mediated killing of CD123-transfected adherent target cells in an xCELLigence-based assay Human T cells Cynomolgus macaque T cells Sample ID EC50(M) 95%LCI 95% UCI EC50(M) 95%LCI 95% UCI T017000128 8.6E-11 6.6E-11 1.1E-10 1.0E-10 3.7E-11 2.9E-10 T017000135 2.0E-09 1.3E-09 3.1E-09 / / / T017000138 4.0E-10 2.3E-10 7.0E-10 8.7E-10 2.1E-10 3.6E-09 T017000139 1.0E-09 5.9E-10 1.9E-09 2.3E-09 7.8E-10 6.6E-09 T017000116 8.1E-09 4.6E-09 1.4E-08 / / /

Table C-28. EC ₅₀ (M) of TCR/CD123 binding polypeptides for human IL-6 secretion during redirected T cell-mediated killing of CD123-transfected adherent target cells in an xCELLigence-based assay Human T cells Sample ID _EC50 (M) 95%LCI 95% UCI T017000128 6.9E-11 3.8E-11 1.3E-10 T017000135 5.9E-10 3.0E-10 1.2E-09 T017000138 3.8E-11 1.8E-11 8.1E-11 T017000139 4.8E-10 2.6E-10 9.0E-10 T017000116 9.6E-09 5.6E-09 1.6E-08

Cytokine production was observed when CD123 overexpressing CHO Flp-In cells and primary T cells were incubated with CD123/TCR binding polypeptides. The irrelevant polypeptides T017000129 and T017000132 do not induce cytokine production.

Example 31 Redirected Autologous T Cell Depletion of Plasmacytoid Dendritic Cells (pDCs) and Basophils by Multispecific CD123/TCR Polypeptides in Healthy PBMCs

Cryopreserved peripheral blood mononucleocytes (PBMCs) were thawed and washed with assay medium (RMPI 1640 + 10% FBS). 2×10 ⁵ PBMCs were incubated with serial dilutions of the multispecific polypeptides in 200 μl of assay medium in a 96-well V-shaped plate with a V-shaped bottom and incubated at 37° C. in a 5% CO2 incubator. At the indicated time points, cells were stained at 4°C with anti-CD14-APC (Biolegend, 301808), anti-CD16-APC (Biolegend, 302012), anti-CD19-APC (Biolegend, 302212), anti-CD20-APC (Biolegend, 302312), against CD56-APC (Biolegend, 318310), against CD123-PE (Biolegend, 306006) and against HLA-DR-FITC (Biolegend, 307603). Briefly, cells were harvested and washed once with FACS buffer (D-PBS from Gibco, with 10% FBS from Sigma and 0.05% sodium azide from Merck) and resuspended in 25 µl Human BD Fc Block™, 0.5 µg/ml (BD Biosciences, 564220, 1000-fold dilution) for 10 minutes at room temperature. Then 25 µl of antibody was added and incubated for 30 min at 4°C according to the manufacturer's instructions. A single well containing PBMC was resuspended in FACS buffer supplemented with 5 nM TOPRO3 (Molecular Probes, T3605) to distinguish between dead and live cells in the cell population. Samples were washed 3 times, resuspended in FACS buffer (D-PBS from Gibco, with 10% FBS from Sigma and 0.05% sodium azide from Merck) and then analyzed with a FACS Canto cytometer (BD) equipped with FACS Diva software. For each sample, a total sample volume of 75 μl was obtained. Data analysis was performed using FACS Diva and Flowing software.

The well containing the TOPRO dye was gated to exclude dead cells. Human and cynomolgus monkey PDCs and basophils were identified as negative for differentiation markers (CD16, CD20, CD19, CD56)/CD123 positive population.

The results are presented in figure 38.

Depletion of the CD123-positive human population and, accordingly, the cynomolgus monkey by multispecific CD123/TCR polypeptides was observed after an incubation period of 5 hours. When the targeted nanobody was replaced with an irrelevant nanobody, no depletion of the CD123 positive population was observed, indicating that the T cell and target cell need to be cross-linked with multispecific TCR/CD123 polypeptides to induce depletion.

The analysis was repeated using PBMC from 3 different human donors and PBMC from 2 different cynomolgus monkeys, confirming the functionality of the multispecific TCR/CD123 polypeptides.

Example 32 Redirected Depletion of Autologous Human T Cells in Monocytes by Multispecific CD123/TCR Binding Polypeptides in Healthy Human PBMC Samples

To evaluate monocyte depletion, the assay was performed as described above using cryopreserved human PBMCs that were thawed and washed with assay medium (RMPI 1640 + 10% FBS) and incubated with serial dilutions of the multispecific polypeptide for 5 or 24 hours. Cell staining was performed as described above. Monocytes were identified as a CD14+ population.

The results are shown in figure 39.

After 5 hours of incubation, no monocyte depletion was observed for the TCR/CD123 multispecific polypeptide and irrelevant nanobodies. After 24 hours, monocyte depletion was observed for multispecific TCR/CD123 polypeptides but not for irrelevant polypeptides. The assay was repeated using PBMCs from 3 different donors, confirming the functionality of the multispecific TCR/CD123 polypeptides.

Example 33 Activation of Human T Cells by Multispecific CD123/TCR Binding Polypeptides during Redirected T Cell Killing of Autologous CD123 Positive Cells in Healthy Human PBMC Samples

To characterize T cell activation during the depletion process mediated by multispecific TCR/CD123 polypeptides, analysis of autologous PBMCs was performed as described above, and human T cell activation status was monitored by measuring CD69 upregulation after 24 hours of incubation. Briefly, after a 24 hour incubation, cells were stained for 30 minutes at 4°C with monoclonal mouse anti-CD3-FITC antibody (BD Biosciences, 555332) for identification of human T cells and with monoclonal mouse human CD69-PE antibody (BD Biosciences, 557050) for evaluation of T-cell activation. Cells were washed 3 times, resuspended in FACS buffer (D-PBS from Gibco, with 10% FBS from Sigma and 0.05% sodium azide from Merck) and then analyzed with a FACS Canto Cytometer (BD) equipped with FACS Diva software. For each sample, a total sample volume of 75 μl was obtained. Data analysis was performed using FACS Diva and Flowing software.

Exemplary results are shown in figure 40.

The data showed dose dependent activation of CD69 on human CD3+ T cells when PBMCs were incubated with multispecific CD123/TCR binding polypeptides. Incubation with monovalent nanobodies or irrelevant polypeptides did not upregulate CD69.

Example 34 Characterization of Irrelevant Polypeptides for Targeted T Cell-Mediated Killing of CD123 Target Cells in a Flow Cytometry Assay

To evaluate the safety of the T0170056G05 TCR nanobody, an in-depth analysis of irrelevant polypeptides (monovalent nanobodies and T017000129 multivalent polypeptide) was performed in a T cell mediated retargeting assay at an E:T ratio of 10:1 as described in Examples 25 and 26. The T017000139 polypeptide was taken as a positive control.

Results using KG1a target cells are shown in Figure 41, results using MOLM-13 are shown in Figure 42. The EC ₅₀ values obtained are shown in Table C-29 and Table C-30.

Table C-29. EC ₅₀ (M) T017000139 for redirected T-cell mediated killing of CD123-positive KG1a cells in a flow cytometric assay Human T cells Cynomolgus macaque T cells Sample ID _EC50 (M) 95%LCI 95% UCI _EC50 (M) 95%LCI 95% UCI T017000139 8.1E-11 7.4E-11 8.9E-11 1.7E-11 9.7E-12 3.1E-11

Table C-30. EC ₅₀ (M) T017000139 for retargeted, T-cell mediated killing of CD123-positive MOLM-13 cells in a flow cytometric assay
Experiment 1 Cynomolgus macaque T cells Sample ID _EC50 (M) 95%LCI 95% UCI T017000139 6E-11 5.1E-11 7.1E-11

Experiment 2

Human T cells Cynomolgus macaque T cells Sample ID _EC50 (M) 95%LCI 95% UCI _EC50 (M) 95%LCI 95% UCI T017000139 3.0E-11 1.9E-11 4.7E-11 3.1E-11 1.9E-11 5.1E-11

The positive control T017000139 behaved as expected using human and cynomolgus T cells. Neither the monovalent building blocks nor the irrelevant T017000129 polypeptide (the anti-CD123 building blocks were replaced by irrelevant building blocks) caused the killing of CD123-positive cells, confirming the specificity of the multispecific TCR/CD123 polypeptides.

Example 35 Effect of Multispecific CD123/TCR Binding Polypeptides on Cytokine Production During Redirected Killing of Human Cells

Cytokine release induction was monitored during a human T cell-mediated CD123 cell killing assay based on FACS data. The release of human cytokines IFN-γ and IL-6 was measured by ELISA. Briefly, MOLM-13 or KG1a was seeded in a 96-well V-bottom plate (2×10 ⁴ cells/well) in the presence of purified primary human T cells (3×10 ⁵ cells/well) with serial dilution of multispecific TCR/ CD123 binding polypeptides, irrelevant polypeptides as described in Example 25. 72 hours after the addition of primary human T cells/polypeptides to the plates, the production of human IFN-γ and IL-6 by human primary T cells was measured in the supernatant as described in example 30.

The results are shown in figures 43A, 43B and 43C. The EC ₅₀ values obtained from this analysis are listed in Table C-31 and Table C-32.

Table C-31. EC ₅₀ (M) of TCR/CD123 binding polypeptides for human IFN-γ secretion during redirected T-cell mediated killing of CD123-positive MOLM-13 or KG1a cells in a flow cytometric assay MOLM-13 KG1a Sample ID _EC50 (M) 95%LCI 95% UCI _EC50 (M) 95%LCI 95% UCI T017000139 3.4E-10 2.5E-10 4.6E-10 3.2E-11 1.9E-11 5.4E-11

Table C-32. EC ₅₀ (M) of TCR/CD123 binding polypeptides for human IL-6 secretion during redirected T-cell mediated killing of CD123-positive MOLM-13 cells in a flow cytometric assay Sample ID _EC50 (M) 95%LCI 95% UCI T017000139 2.3E-11 1.5E-11 3.5E-11

Cytokine production was observed when MOLM-13 or KG1a cells and primary human T cells were incubated with CD123/TCR binding polypeptides. The irrelevant T017000129 polypeptide does not induce cytokine production.

Example 36 Characterization of target-independent retargeted killing by human or cynomolgus macaque effector T cells for multispecific CD123/TCR binding polypeptides in a flow cytometric assay using CD123-negative cell lines

To evaluate CD123-independent targeted killing by multispecific polypeptides, the CD123-negative U-937 and NCI-H929 cell lines were evaluated in a flow cytometry-based kill assay. U-937 and NCI-H929 cells were labeled with 8 μM PKH-26 membrane dye using the PKH26 red fluorescent cell linker kit (Sigma, PKH26GL-1KT) according to the manufacturer's instructions and used as target cells. The assay was performed as described in Examples 25 and 26 using primary human or cynomolgus T cells (E:T=10:1).

Exemplary results are shown in Figures 44 and 45 for NCI-H929 and U-937 cells, respectively.

The TCR/CD123 multispecific polypeptides and irrelevant polypeptides showed only minimal U-937 cell killing activity by redirected T cells (less than 6%), indicating that the multispecific polypeptides have good CD123 binding specificity.

Example 37 Characterization of T Cell Activation by Multispecific CD123/TCR Binding Polypeptides in an Effector T Cell Redirected Kill Assay Using CD123 Negative Cell Lines

To monitor T cell activation after treatment of T cells and CD123-negative cells with CD123/TCR multispecific polypeptides, the polypeptides were incubated for 24 hours at 37°C with 2.5×10 ⁴ U-937 target cells, and, respectively, NCI-H929 target cells in the presence of 2.5×10 ⁵ primary T cells (E:T = 10:1) as described in examples 25 and 26. T cell activation was assessed as described previously by monitoring CD25 activation after 72 hours of incubation on CD4/CD8 T cell populations was measured by flow cytometry as described in Example 27 using a monoclonal mouse anti-CD4-APC antibody (Biolegend, 300505), a monoclonal mouse anti-CD8 APC antibody ( BD Biosciences, 555366) and an anti-CD25 monoclonal antibody (BD Biosciences, 557138).

Exemplary results are shown in Figure 46.

Assessment of T cell activation after incubation with the multispecific polypeptides and CD123-negative U-937 or NCI-929 cell lines showed only minimal CD25 activation for any of the multispecific polypeptides. Thus, in the presence of CD123 negative target cells, only minimal T cell activation and T cell killing occurred.

Example 38 Characterization of Cytokine Production by Multispecific CD123/TCR Binding Polypeptides during Redirected Killing by Human Cells

Aspecific induction of cytokine release was monitored during human T-cell mediated killing assay based on FACS data. The release of the cytokine IFN-γ and IL-6 was measured using ELISA. Briefly, NCI-H929 was seeded in a 96-well V-bottom plate (2×10 ⁴ cells/well) in the presence of purified primary human T cells (3×10 ⁵ cells/well) with serial dilutions of multispecific TCR-binding polypeptides. /CD123, or irrelevant polypeptides, as described in Example 25. 72 hours after the addition of human primary T cells/polypeptides to the plates, the production of IFN-γ and IL-6 in human primary T cells in the supernatant was measured, as described in example 30. The results are shown in figure 47.

No cytokine production was observed when the CD123-negative NCI-H929 cell line and primary human T cells were incubated with CD123/TCR binding polypeptides.

Example 39 Effect of Multispecific CD123/TCR Binding Polypeptides on T Cell Proliferation During Redirected Killing

To investigate the effect of multispecific CD123/TCR binding polypeptides on human T cell proliferation, gamma irradiated (100 Gy) MOLM-13 cells were seeded in 96-well flat bottom microtiter plates (Greiner bio-one, 655 180, 2× 10 ⁴ cells/well) together with multispecific polypeptides and primary human T cells (2×10 ⁵ cells/well) and incubated for 72 hours at 37°C in a humidified atmosphere with 5×CO ₂ in air. The cells were then injected with 3H-thymidine (3H-Tdr, New England Nuclear, Boston, MA, specific activity 20 Ci/mM) over approximately 18 hours, collected on glass fiber filter strips, and then counted by liquid scintillation.

Exemplary results are shown in Figure 48.

Multispecific CD123/TCR polypeptides induced T cell proliferation in a dose dependent manner. For the irrelevant T017000129 polypeptide, T cell proliferation was not observed.

In parallel, the effect of multispecific polypeptides that bind CD123/TCR on the proliferation of human T cells in the absence of target cells was evaluated. To do this, multispecific polypeptides and primary human T cells (2×10 ⁵ cells/well) were incubated for 72 hours at 37° C. in a humidified atmosphere with 5×CO ₂ in air. Proliferation was measured as described above. The data is shown in figure 49.

Example 40 Lytic Efficacy of Pre-Activated T Cells Compared to Non-Activated T Cells

In order to test the lytic activity of T cells in response to a multi-day incubation period under stimulating conditions, primary human T cells (isolated as described in Example 25) were thawed and pre-activated using Dynabeads® Human T-Activator CD3/CD28 ( Gibco - Technologies, 11132D) at a ratio of T cells to beads of 2:1. After 3 days, the pellets were replaced with fresh pellets for another three days. The beads were then removed and pre-activated, and non-activated T cells were evaluated in the MOLM-13 target cell kill assay. Briefly, non-activated or pre-activated CD3/CD28 primary T cells from the same donor were mixed with PKH-labeled MOLM-13 cells at various E:T ratios (8:1, 2:1, 1:2, 1:4) and with serial dilutions of T017000114 or with PKH-labeled KG1A cells at various E:T ratios (2:1, 1:2, 1:4, 1:8) and with serial dilutions of T017000139. Cytotoxicity readings after 24 hours of incubation were performed as described in Example 25 above.

The results are shown in Figures 50 and 51 for MOLM-13 and KG1a cells, respectively. The EC ₅₀ values obtained in this assay are listed in Table C-33 and Table C-34 for MOLM-13 and KG1a cells, respectively.

Table C-33. EC ₅₀ (M) and percent lysis for T017000114 in redirected human T cell-mediated killing of MOLM-13 cells in a flow cytometric analysis using pre-activated and non-activated T cells Non-activated T cells Pre-activated T cells Sample ID _EC50 (M) 95%LCI 95% UCI % lysis _EC50 (M) 95%LCI 95% UCI % lysis T017000114
E:T ratio 8:1 1.4E-09 2.4E-10 7.8E-09 12 2.4E-10 1.9E-10 3.2E-10 43 T017000114
E:T ratio 2:1 1.9E-09 6.2E-12 6.0E-07 ten 6.8E-10 4.7E-10 9.9E-10 31 T017000114
Ratio E:T 1:2 1.0E-09 4.0E-10 2.6E-09 eighteen

Table C-34. EC ₅₀ (M) and percent lysis for T017000139 in redirected human T cell-mediated killing of KG1a cells in a flow cytometric analysis using pre-activated and non-activated T cells Non-activated T cells Pre-activated T cells Sample ID _EC50 (M) 95%LCI 95% UCI % lysis _EC50 (M) 95%LCI 95% UCI % lysis T017000139
E:T ratio 2:1 1.4E-10 8.1E-11 2.5E-10 19 4.5E-11 2.2E-11 9.1E-11 43 T017000139
Ratio E:T 1:2 7.7E-11 3.0E-12 2.0E-09 eight 4.9E-11 2.2E-11 1.1E-10 twenty T017000139
Ratio E:T 1:4 9.2E-11 2.8E-12 3.0E-09 four 5.8E-11 3.1E-11 1.1E-10 13 T017000139
E:T ratio 1:8 3.4E-11 1.9E-13 6.1E-09 3 5.6E-11 2.3E-11 1.4E-10 eight

Pre-activated T cells lysed target cells more strongly than non-activated T cells at all E:T ratios tested. Pre-stimulation of effector cells with anti-CD3/anti-CD28 resulted in higher lysis rates.

Example 41 Construction of Multispecific CD123/TCR Binding Polypeptides and Extended Half-Life (HLE) Control Polypeptides

ALB11 (SEQ ID NO: 43), a human serum albumin (HSA)-binding nanobody, has been fused to CD123/TCR binding polypeptides to increase the in vivo half-life of formatted molecules (WO 06/122787). A number of formats were generated with TCR α/β recruiting nanobodies at the N-terminus and nanobodies targeted at CD123 tumors or albumin-targeted nanobodies at the C-terminus using the 35GS linker and expressed as above. Irrelevant polypeptides were generated by replacing the tumor antigen-binding nanobodies with an irrelevant anti-RSV nanobody. An overview of the formats examined is shown in Table C-35.

Table C-35. Sample ID and description of HLE constructs Sample ID SEQID NO* Description A022600009 62 T0170056G05-35GS-RSV007B02(Q108L)-35GS-ALB11 T017000142 63 T0170056G05(E1D)-35GS-A0110056A10-35GS-ALB11-A T017000143 64 T0170056G05(E1D)-35GS-A0110056A10-35GS-A0110055F03-35GS-ALB11-A T017000144 65 T0170056G05(E1D)-35GS-A0110056A10-35GS-ALB11-35GS-A0110055F03-A T017000145 66 T0170056G05(E1D)-35GS-A0110055F03-35GS-A0110056A10-35GS-ALB11-A T017000146 67 T0170056G05(E1D)-35GS-A0110055F03-35GS-ALB11-35GS-A0110056A10-A

*SEQ ID NOs correspond to multispecific polypeptide sequences without C-terminal tags or Ala extension

Example 42 Albumin Binding Properties of ALB11 in Multispecific Recruiting Polypeptides

The binding affinity of the extended half-life multispecific polypeptides to human serum albumin (SA), and respectively cynomolgus monkey, was measured by SPR-based affinity determination on a Biacore T100 instrument. For this, human SA and, respectively, cynomolgus monkey SA (Sigma, A3782) (produced in-house) were immobilized on the CM5 chip by amine group coupling using EDC and NHS chemistry. TCR/CD123 binding polypeptides were administered over 2 minutes at various concentrations (6.2 to 500 nM) and allowed to dissociate for 15 minutes at a flow rate of 45 μl/min. Between injections of surface samples were regenerated with 10 mm glycine-HCl pH 1.5. HBS-EP+ was used as working buffer. Kinetic constants were calculated from sensograms using BIAEvaluation software with an algorithm using a 1:1 single cycle kinetics binding model. The affinity constant KD was calculated from the obtained association and dissociation rate constants ka and kd and is shown in Table C-36.

Table C-36. Albumin-binding properties of ALB11 in multispecific HLE polypeptides human SA SA cynomolgus monkey k _a (1/(M ⋅ s) k _d (1/s) K _D (M) k _a (1/(M ⋅ s) k _d (1/s) K _D (M) T017000142 1.1E+05 7.2E-03 6.9E-08 9.0E+04 7.3E-03 8.1E-08 T017000143 1.6E+05 9.5E-03 5.9E-08 1.2E+05 9.4E-03 7.6E-08 T017000144 9.1E+04 7.8E-03 8.5E-08 7.4E+04 8.1E-03 1.1E-07 T017000145 1.1E+05 8.5E-03 7.6E-08 1.0E+05 9.9E-03 9.8E-08 T017000146 8.4E+04 8.5E-03 1.0E-07 6.8E+04 8.8E-03 1.3E-07 A02260009 1.1E+05 8.0E-03 7.5E-08 8.5E+04 8.0E-03 9.4E-08 ALB11 5.3E+05 1.6E-03 3.0E-09 5.1E+05 1.6E-03 3.2E-09

Formatting the ALB11 building block into multispecific recruiting polypeptides resulted in an acceptable reduction in binding affinity for human and cynomolgus serum albumin.

Example 43 Redirected T Cell Mediated Killing of MOLM-13 Target Cells by CD123/TCR HLE Binding Polypeptides in a Flow Cytometry Assay

Since the addition of ALB11 nanobodies and binding to serum albumin (SA) can affect the activity of the polypeptides, CD123/TCR binding HLE polypeptides were evaluated for human and cynomolgus T cell-mediated retargeting of CD123-positive MOLM target cells. -13 based on flow cytometric analysis as described in Examples 25 and 26 in the absence or presence of 30 μM SA.

The EC ₅₀ values obtained from this analysis are listed in Table C-37. The results are presented in figure 52.

Table C-37. EC ₅₀ (M) and percent lysis for CD123/TCR-binding HLE polypeptides in redirected T cell-mediated killing of MOLM-13 cells in a flow cytometric analysis at an E:T ratio of 10:1 Human T cells Cynomolgus macaque T cells Sample ID %
lysis _EC50 (M) 95%LCI 95% UCI %
lysis _EC50 (M) 95%LCI 95% UCI T017000138 21 1.1E-10 8.8E-11 1.3E-10 54 1.5E-10 1.1E-10 2.1E-10 T017000144 23 2.7E-11 2.0E-11 3.6E-11 56 2.3E-11 1.7E-11 3.0E-11 T017000144+
SA 24 2.6E-10 1.9E-10 3.5E-10 52 1.1E-10 9.3E-11 1.2E-10 T017000139 twenty 2.0E-11 9.3E-12 4.4E-11 56 2.9E-11 2.3E-11 3.5E-11 T017000146 21 2.5E-11 57 2.9E-11 2.3E-11 3.7E-11 T017000146+ SA 25 2.3E-10 1.7E-10 2.9E-10 44 1.6E-10 1.3E-10 1.9E-10

All multispecific CD123/TCR-binding HLE polypeptides showed dose-dependent killing of MOLM-13 cells by both human and cynomolgus T cells. The inclusion of ALB11 in the polypeptide did not reduce activity. When adding HSA or CSA, a slight decrease in specific activity was observed, while this did not affect the effectiveness.

Example 44 Redirected T Cell Mediated Killing of KG1a Target Cells by CD123/TCR Binding HLE Polypeptides in a Flow Cytometry Assay

Extended half-life polypeptides binding TCR/CD123 were also evaluated for human and cynomolgus T cell-mediated retargeting of CD123 KG1a target cells based on flow cytometry analysis as described in Examples 24 and 25, in the absence or in the presence of 30 μM serum albumin.

The EC ₅₀ values obtained from this analysis are listed in Table C-38. The results are shown in figure 53.

Table C-38. EC ₅₀ (M) and percent lysis for CD123/TCR-binding HLE polypeptides in T cell-mediated redirected killing of KG1a cells in a flow cytometric analysis at an E:T ratio of 10:1 Human T cells Cynomolgus macaque T cells Sample ID %
lysis _EC50 (M) 95%LCI 95% UCI %
lysis _EC50 (M) 95%LCI 95% UCI T017000138 64 1.6E-10 1.3E-10 2.0E-10 21 2.8E-11 2.3E-11 3.4E-11 T017000143 62 1.9E-10 1.6E-10 2.3E-10 25 4.7E-11 2.9E-11 7.5E-11 T017000144 67 4.1E-11 3.4E-11 5.0E-11 28 6.3E-12 9.3E-13 4.3E-11 T017000143+SA 60 1.4E-09 1.0E-09 1.9E-09 25 8.6E-10 7.1E-10 1.0E-09 T017000144+SA 58 2.9E-10 2.4E-10 3.6E-10 29 6.6E-11 3.4E-11 1.3E-10 T017000139 67 8.1E-11 7.4E-11 8.9E-11 34 1.7E-11 9.7E-12 3.1E-11 T017000145 65 7.4E-11 6.4E-11 8.6E-11 35 1.6E-11 1.1E-11 2.4E-11 T017000146 64 6.6E-11 5.9E-11 7.4E-11 34 1.5E-11 7.2E-12 3.0E-11 T017000145+SA 51 5.9E-10 4.9E-10 7.2E-10 32 1.2E-10 7.4E-11 1.9E-10 T017000146+SA 60 6.8E-10 5.8E-10 8.0E-10 36 2.0E-10 1.1E-10 3.5E-10 T017000128 55 3.1E-10 3.0E-10 3.3E-10 32 2.8E-10 1.3E-10 6.1E-10 T017000142 59 6.8E-10 6.2E-10 7.5E-10 29 2.2E-10 1.4E-10 3.6E-10 T017000142+SA 48 3.1E-09 3.0E-09 3.3E-09 twenty 3.2E-10 2.9E-10 3.5E-10

All multispecific CD123/TCR-binding HLE polypeptides demonstrated dose-dependent killing of KG1a cells by both human and cynomolgus monkey T cells. The inclusion of ALB11 in the polypeptide did not reduce the specific activity. When adding HSA or CSA, a slight decrease in specific activity was observed, while this did not affect the effectiveness.

Example 45 Effect of T017000144 Multispecific CD123/TCR Binding HLE Polypeptide on Cytokine Production During Redirected Killing

Cytokine release induction was monitored during human T-cell mediated killing assay based on FACS data. The release of IFN-γ and IL-6 was measured using ELISA. Briefly, MOLM-13 (2×10 ⁴ cells/well) was plated in a 96-well V-bottomed plate in the presence of purified primary human T cells (3×10 ⁵ cells/well) with serial dilutions of multispecific polypeptides that bind to TCR/CD123, or irrelevant polypeptides as described in Example 35. 72 hours after the addition of primary human T cells/polypeptides to the plates, IFN-γ and IL-6 production was measured, with primary human T cells in the supernatant as described in example 30.

The EC ₅₀ values obtained in this analysis are listed in Table C-39 and Table C-40. The results are presented in figure 54.

Table C-39. EC ₅₀ (M) of TCR/CD123 binding polypeptides for human IFN-γ secretion during redirected, T-cell mediated killing of CD123-positive MOLM-13 cells in a flow cytometry-based assay Sample ID _EC50 (M) 95%LCI 95% UCI T017000139 3.4E-10 2.5E-10 4.6E-10 T017000144 1.9E-10 1.3E-10 2.9E-10

Table C-40. EC ₅₀ (M) of TCR/CD123 binding polypeptides for secretion of human IL-6 during redirected, T-cell mediated killing of CD123-positive MOLM-13 cells in a flow cytometry-based assay Sample ID _EC50 (M) 95%LCI 95% UCI T017000139 2.3E-11 1.5E-11 3.5E-11 T017000144 1.6E-11 9.9E-12 2.7E-11

Cytokine production was observed when MOLM-13 cells and primary human T cells were incubated with CD123/TCR-binding HLE polypeptide T017000144. The irrelevant T017000129 polypeptide did not induce cytokine production.

Example 46 Effect of Multispecific CD123/TCR Binding HLE Polypeptides on T Cell Proliferation During Redirected Killing

To investigate the effect of multispecific CD123/TCR-binding HLE polypeptides on human T cell proliferation, gamma-irradiated (100 Gy) MOLM-13 cells were seeded in 96-well flat bottom microtiter plates (2×10 ⁴ cells/well) together with multispecific HLE polypeptide T017000144 and primary human T cells (2×10 ⁵ cells/well) and incubated for 72 hours at 37°C in a humidified atmosphere with 5X CO ₂ in air in the absence of SA. T017000129 was taken as a negative control. Cells were then injected with 3H-thymidine (3H-Tdr, New England Nuclear, Boston, MA, specific activity 20 Ci/mM) over approximately 18 hours, collected on glass fiber filter strips, and then counted by liquid scintillation counting.

Exemplary results are shown in Figure 55.

The multispecific CD123/TCR binding HLE polypeptide T017000144 induced T cell proliferation in a dose dependent manner. For the irrelevant T017000129 polypeptide, T cell proliferation was not observed.

Example 47 Redirected Depletion of Plasmacytoid Dendritic Cells (pDCs) and Basophils by Autologous T Cells by Exposure to Multispecific CD123/TCR Binding HLE Polypeptides in Healthy Human PBMC and Healthy Cynomolgus PBMC Samples

HLE constructs were further evaluated in the analysis of autologous human and cynomolgus monkey PBMCs in the absence of SA as described in Example 31. differentiation, CD123-positive, HLA-DR-negative) multispecific polypeptides were assessed after an incubation period of 5 hours.

The results are shown in Figures 56 and 57 for human and cynomolgus PBMC, respectively.

Multispecific HLE polypeptides binding CD123/TCR were able to deplete CD123+ pDCs and basophils among human and cynomolgus monkey PBMCs with redirected T cells. T017000144 was the strongest polypeptide. The T017000142 polypeptide, consisting of one TCR structural element, ALB11 and only one CD123 structural element, showed no functionality in the PBMC assay after 5 hours. Cross-reactivity between human and cynomolgus monkeys of CD123/TCR-binding multispecific HLE polypeptides was confirmed under autologous conditions.

Example 48 Redirected Depletion of Autologous T Cells in Monocytes by Multispecific CD123/TCR Binding HLE Polypeptides in Healthy Human PBMC Samples

Depletion of monocytes (CD14+ cells) by multispecific HLE polypeptides in autologous human PBMC conditions was assessed after an incubation period of 24 h in the absence of SA. The analysis was carried out as described in example 32.

The results are presented in figure 58.

Multispecific HLE polypeptides are able to deplete CD123+ monocytes in human PBMCs with redirected T cells. T017000144 was the strongest polypeptide. The T017000142 polypeptide, consisting of one TCR structural element, ALB11, and only one structural element, CD123, demonstrated functionality in an autologous monocyte depletion assay after 24 hours.

Example 49 Efficacy and Safety in Vivo in a Non-Human Primate Model

The in vivo efficacy and safety of non-HLE and HLE multispecific CD123/TCR binding nanobodies was evaluated in a non-human primate model.

Animals treated with a control compound were included as positive controls. Treatment of non-HLE IRR/TCR polypeptides was used as a specificity control for the CD123 targeting fragment in the multispecific polypeptides. The control compound and non-specific multispecific CD123/TCR binding polypeptides were administered to cynomolgus monkeys by continuous intravenous infusion after a 7 day NaCl "pre-treatment" infusion. Non-HLE multispecific CD123/TCR binding polypeptides were administered for 4 weeks as a 4-day/3-day infusion at equimolar doses to the control compound in a weekly escalation schedule according to Table C-41. The HLE multispecific CD123/TCR binding polypeptide was administered to cynomolgus monkeys by intravenous bolus injections on days 1, 2, 3, 8, 15 and 22 on a weekly escalation schedule according to Table C-41.

Table C-41. Processing scheme Group Compound Dose levels (ng/kg/day) Path
introductions Test week 1 Test week 2 Test week 3 Test week 4 one Source D1-5: 100 D8-12: 300 D15-19: 600 D22-26: 1000 continuous 24-hour infusion 2 Irrelevant/TCR polypeptide D1-5: 49.5 D8-12: 148.4 D15-19: 296.8 D22-26: 494.6 continuous 24-hour infusion 3 Non-HLE CD123/TCR Polypeptide D1-5: 74.0 D8-12: 222.1 D15-19: 444.3 D22-26: 740.4 continuous 24-hour infusion four HLE CD123/TCR polypeptide D1: 0.6
D2: 0.4
D3: 0.34 D8: 2.21 D15: 4.42 D22: 7.13 intravenous bolus injection

T cell redistribution from blood was monitored by measuring T cell subsets as described in Table C-42 using flow cytometry and differential blood analysis on test days -7, d-4, d1 (pre-dose + 4 hours post-dose). ), d4, d8 (pre-dose + 4 hours post-dose), d11, d15 (pre-dose + 4 hours post-dose), d18, d22 (pre-dose + 4 hours post-dose), d25, d29, d32 and d36.

In vivo efficacy was determined by evaluating the percentage and number of CD123+ cells in PBMCs, as detailed in Table C-43, using flow cytometry and blood single cell counts on test days -7, d-4, d1 (pre-dose + 4 hours post-dose, d4, d8 (pre-dose + 4 hours post-dose), d11, d15 (pre-dose + 4 hours post-dose), d18, d22 (pre-dose + 4 hours post-dose) - dose), d25, d29, d32 and d36.

Safety was determined by assessing cytokines (IL-1β, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12 (p70), TNF-α, TNF-β, IFN -γ) in serum on test days -7, d-4, d1 (4 hours post-dose), d4, d8 (4 hours post-dose), d11, d15 (4 hours post-dose), d18, d22 (4 hours post-dose), d25, d29, d32 and d36.

Table C-42. Combinations of markers for phenotyping of blood T-cells Marker Cells Unit CD3+CD4+ T helper cells (Th) % PBMCs and cells/µl CD3+CD8+ Cytotoxic T cells (Tc) % PBMCs and cells/µl CD25+CD3+CD4+ activated Th % CD3+CD4+ and cells/µl CD25+CD3+CD8+ activated Tc % CD3+CD8+ and cells/µl PD1+CD3+CD4+ positive regulation of PD (protein
programmed cell death) in Th % CD3+CD4+ and cells/µl PD1+CD3+CD8+ PD activation at Tc % CD3+CD8+ and cells/µl CD25+PD1+CD3+CD4+ Upregulation of PD in activated Th % CD3 + CD4 + CD25 + cells/µl CD25+PD1+CD3+CD8+ Upregulation of PD in activated Tc % CD3+CD8+CD25+ and cells/µl

Table C-43. Combinations of markers to assess the depletion of CD123+ cells Marker Cells Unit CD14 - ^low CD123+ pDC (plamacytoid dendritic cells) based on marker CD123 and basophils and mDC (myeloid dendritic cells) % PBMCs and cells/µl CD14 - ^low CD303+ pDC based on CD303 marker % PBMCs and cells/µl CD14 - ^low CD123+ CD303+ overlap between CD123 and CD303 % PBMCs and cells/µl CD14+ monocytes % PBMCs and cells/µl CD14+CD123+ CD123+ monocytes % CD14+ cells/µl CD123+ Absolute target cell number based on CD123 % PBMCs and cells/µl CD303+ Absolute number of CD303 cells % PBMCs and cells/µl

Serum samples for PK analysis were collected on days -7, d-4, d1 (pre-dose + 4 hours post-dose), d4, d8 (pre-dose + 4 hours post-dose), d11, d15 (pre-dose + 4 hours post-dose). hours post-dose, d18, d22 (pre-dose + 4 hours post-dose), d25, d29, d32 and d36.

Serum samples for ADA analysis were collected on days -7, d1 (pre-dose), d8 (pre-dose), d15 (pre-dose), d22 (pre-dose), d29 and d36.

On days 4 and 25, blood T cells were isolated from all animals and tested in a depletion test as described in Example 25.

On the 29th day, the animals of the 2nd group were autopsied. On day 36, all remaining animals were autopsied.

Example 50 In Vivo Efficacy and Safety in a Non-Human Primate Model - Multispecific Nanobody Binding CD123/TCR Experimental Results

In vivo efficacy and safety of the T017000139 multispecific CD123/TCR binding polypeptide was evaluated in a non-human primate model.

Animals treated with a control compound (MGD006, Macrogenics) were included as positive controls. Treatment with the irrelevant/TCR binding polypeptide T017000129 was used as a specificity control for the CD123 targeting portion of the multispecific polypeptide. A control compound, an irrelevant protein/TCR binding polypeptide, and a multispecific CD123/TCR binding polypeptide were administered by continuous intravenous infusion after a 7 day NaCl infusion of cynomolgus monkeys for "pretreatment". The irrelevant protein/TCR binding polypeptide and the multispecific CD123/TCR binding polypeptide were administered for 4 weeks as a 4-day/3-day infusion at equal doses relative to the control compound in a weekly escalation schedule according to Table C-44.

Table C-44. Processing scheme Group Compound Dose levels (ng/kg/day) Path
introductions Test week 1 (D1-5) Test Week 2 (D8-12) Test week 3 (D15-19) Test week 4 (D22-26) one Control connection
(MGD006) 100 300 600 1000 continuous 24-hour infusion 2 Irrelevant Protein/TCR Binding Polypeptide
T=7 49.5 148.4 296.8 494.6 continuous 24-hour infusion 3 CD123/TCR polypeptide (T017000139) 74.0 222.1 444.3 740.4 continuous 24-hour infusion

T cell redistribution from blood was monitored by measuring T cell subsets using flow cytometry on test days -7, d-4, d1 (pre-dose + 4 hours post-dose), d4, d8 (pre-dose + 4 hours post-dose). administration), d11, d15 (pre-dose + 4 hours post-dose), d18, d22 (pre-dose + 4 hours post-dose), d25, d29, d32 and d36.

As shown in Figure 59, in the positive control group treated with the control compound, the number of circulating CD4 ⁺ CD3 ⁺ and CD8 ⁺ CD3 ⁺ T cells fluctuated during different dosing cycles, indicating directed migration and/or marginal standing of leukocytes rather than to exhaustion. In contrast, treatment with a CD123/TCR polypeptide or an irrelevant protein/TCR binding polypeptide did not result in a large fluctuation in the number of CD4 ⁺ CD3 ⁺ or CD8 ⁺ CD3 ⁺ T cells.

Circulating CD123 ⁺ CD14 ^- cell counts were studied as a pharmacodynamic endpoint for evaluating in vivo efficacy by measuring the number of CD123 ⁺ CD14 ^- cells in PBMCs using blood flow cytometry on test days -7, d-4, d1 (before administration + 4 hours post-dose, d4, d8 (pre-dose + 4 hours post-dose), d11, d15 (pre-dose + 4 hours post-dose), d18, d22 (pre-dose + 4 hours post-dose), d25, d29, d32 and d36.

The results are shown in Figure 60. CD123 ⁺ CD14 ^- cells were depleted in animals treated with control compound MGD006 (positive control), although loss of potency was observed by the 4th ^dosing cycle. Treatment with the CD123/TCR polypeptide caused a depletion of ^{CD123 +} CD14 ^- cells in the blood already from the first dosing cycle, which was maintained during the 4th and last dosing cycle. In animals treated with the irrelevant polypeptide/TCR, no significant depletion of CD123 ⁺ CD14 ^- cells was observed.

PD-1 expression was then examined as a surrogate marker to assess T cell depletion in vivo in circulating CD4+CD3+ T cells and CD8+CD3+ T cells. For this, PD-1 expression was measured in PBMC using blood flow cytometry on test days -7, d-4, d1 (pre-dose + 4 hours post-dose), d4, d8 (pre-dose + 4 hours post-dose) , d11, d15 (pre-dose + 4 hours post-dose), d18, d22 (pre-dose + 4 hours post-dose), d25, d29, d32 and d36.

The results are shown in Figure 61. PD-1 expression was strongly increased on most CD4+CD3+ T cells and CD8+CD3+ T cells in animals treated with control compound MGD006 (positive control) and was about half in CD4+CD3+ T- cells and CD8+CD3+ T cells after dosing has been discontinued. In contrast, PD-1 expression remained at baseline after treatment with CD123/TCR polypeptide or irrelevant polypeptide/TCR during dosing cycles.

Safety was assessed by assessing cytokines (IL-1β, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12 (p70), TNF-α, TNF-β, IFN-γ) in serum levels on test days -7, d-4, d1 (4 hours post-dose), d4, d8 (4 hours post-dose), d11, d15 (4 hours post-dose), d18, d22 (4 hours post-dose). dose administration) and d25.

The levels of the following cytokines remained below the detection limit of the assay: IL-1β, IL-2, IL-4, IL-5, IL-10, IL-12 (p70), TNF-α, TNF-β, and IFN-γ. Interleukin-6 was detected at low concentrations in the positive control group at the beginning of the first dosing cycle. This increase was temporary and only in one animal. In the CD123/TCR polypeptide group, one animal showed a detectable pre-dose IL-6 concentration and one animal showed a transient slight increase at the start of the second dosing cycle, indicating manipulation stress. In the irrelevant protein/TCR-treated polypeptide group, one animal showed a transient slight increase in IL-6 at the third dosing cycle, again indicating manipulation stress. The results of IL-6 measurements are shown in Figure 62.

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SEQUENCE LIST

<110> Ablynx NV

<120> T - Cell recruiting polypetides capable of binding CD123 and TCR

alpha/beta

<130> P16-014-PCT-1

<150> US 62/422,770

<151> 2016-11-16

<150> US62/557,208

<151> 2017-09-12

<160> 370

<170>PatentIn version 3.5

<210> 1

<211> 115

<212> PRT

<213> Artificial Sequence

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<223> Nanobody Sequence

<400> 1

Glu Val Gln Leu Val Lys Ser Gly Gly Gly Leu Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ile Thr Ser Lys Ile Asn

20 25 30

Asp Met Gly Trp Tyr Arg Gln Thr Pro Gly Asn Tyr Arg Glu Trp Val

35 40 45

Ala Ser Ile Thr Ala Thr Gly Thr Thr Asn Tyr Arg Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Ser Thr Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Thr Val Tyr Tyr Cys Asn

85 90 95

Thr Phe Pro Pro Ile Ser Asn Phe Trp Gly Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ser

115

<210> 2

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<212> PRT

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Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Asn Gly Ile Ser Ser Lys Ser Asp

20 25 30

Ala Met Gly Trp Tyr Arg Gln Thr Pro Gly Lys Tyr Arg Glu Trp Val

35 40 45

Ala Ser Ile Thr Ala Thr Gly Thr Thr Asn Tyr Arg Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Thr Val Tyr Tyr Cys Asn

85 90 95

Thr Phe Pro Ala Ile Ser Asn Phe Trp Gly Gln Gly Thr Gln Val Thr

100 105 110

Val Ser Ser

115

<210> 3

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<212> PRT

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Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Asn Gly Ile Thr Ser Lys Ser Asn

20 25 30

Ala Met Gly Trp Tyr Arg Gln Thr Pro Gly Lys Tyr Arg Glu Trp Val

35 40 45

Ala Ser Ile Thr Ala Thr Gly Thr Thr Asn Tyr Arg Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Thr Val Tyr Tyr Cys Asn

85 90 95

Thr Phe Pro Ala Ile Ser Asn Phe Trp Gly Gln Gly Thr Gln Val Thr

100 105 110

Val Ser Ser

115

<210> 4

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<212> PRT

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Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ile Pro Ser Lys Ile Asn

20 25 30

Asp Met Gly Trp Phe Arg Gln Thr Pro Gly Asn Tyr Arg Glu Trp Val

35 40 45

Ala Ser Ile Thr Ala Thr Gly Thr Thr Asn Tyr Arg Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Thr Val Tyr Tyr Cys Asn

85 90 95

Thr Phe Pro Pro Ile Ser Asn Phe Trp Gly Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ser

115

<210> 5

<211> 115

<212> PRT

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<400> 5

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ile Thr Ser Lys Ile Asn

20 25 30

Asp Met Gly Trp Tyr Arg Gln Thr Pro Gly Asn Tyr Arg Glu Trp Val

35 40 45

Ala Ser Ile Thr Ala Thr Gly Thr Thr Asn Tyr Arg Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Ser Thr Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Thr Val Tyr Tyr Cys Asn

85 90 95

Thr Phe Pro Pro Ile Ser Asn Phe Trp Gly Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ser

115

<210> 6

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<212> PRT

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Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ile Thr Ser Lys Ser Asn

20 25 30

Val Met Gly Trp Tyr Arg Gln Thr Pro Gly Lys Tyr Arg Glu Trp Val

35 40 45

Ala Ser Ile Thr Ala Thr Gly Thr Thr Asn Tyr Arg Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Thr Val Tyr Tyr Cys Asn

85 90 95

Thr Phe Pro Ala Ile Ser Asn Phe Trp Gly Gln Gly Thr Gln Val Thr

100 105 110

Val Ser Ser

115

<210> 7

<211> 125

<212> PRT

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Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly

1 5 10 15

Pro Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Ser Tyr

20 25 30

Val Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val

35 40 45

Ala Ala Ile Tyr Trp Ser Asn Gly Lys Thr Gln Tyr Thr Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Gly Asp Asn Ala Lys Asn Thr Val Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Asn Pro Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Val Ala Asp Lys Asp Glu Thr Gly Phe Arg Thr Leu Pro Ile Ala Tyr

100 105 110

Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120 125

<210> 8

<211> 125

<212> PRT

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<223> Nanobody Sequence

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Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Ser Tyr

20 25 30

Val Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val

35 40 45

Ala Ala Ile Tyr Trp Ser Ser Gly Lys Thr Glu Tyr Thr Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Leu Ser Gly Asp Asn Ala Lys Asn Thr Val Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Asn Pro Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Val Ala Asp Lys Asp Arg Asp Gly Phe Arg Thr Leu Pro Ile Ala Tyr

100 105 110

Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120 125

<210> 9

<211> 125

<212> PRT

<213> Artificial Sequence

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<223> Nanobody Sequence

<400> 9

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Ser Tyr

20 25 30

Val Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val

35 40 45

Ala Ala Ile Tyr Trp Ser Ser Gly Lys Thr Glu Tyr Thr Glu Ser Val

50 55 60

Lys Gly Arg Phe Thr Leu Ser Gly Asp Asn Ala Lys Asn Thr Val Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Asn Pro Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Val Ala Asp Lys Asp Arg Tyr Gly Phe Arg Thr Leu Pro Ile Ala Tyr

100 105 110

Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120 125

<210> 10

<211> 125

<212> PRT

<213> Artificial Sequence

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<223> Nanobody Sequence

<400> 10

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Ser Tyr

20 25 30

Val Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val

35 40 45

Ala Ala Ile Trp Trp Ser Ser Gly Lys Thr Glu Tyr Thr Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Leu Ser Gly Asp Asn Ala Lys Asn Thr Val Tyr

65 70 75 80

Leu Gln Met Asn Asn Leu Asn Pro Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Val Ala Asp Lys Asp Arg Asp Gly Phe Arg Thr Leu Pro Ile Ala Tyr

100 105 110

Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120 125

<210> 11

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR1

<400> 11

Gly Ile Thr Ser Lys Ile Asn Asp Met Gly

1 5 10

<210> 12

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<213> Artificial Sequence

<220>

<223> CDR1

<400> 12

Gly Ile Ser Ser Lys Ser Asp Ala Met Gly

1 5 10

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<213> Artificial Sequence

<220>

<223> CDR1

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Gly Ile Thr Ser Lys Ser Asn Ala Met Gly

1 5 10

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<220>

<223> CDR1

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Gly Ile Pro Ser Lys Ile Asn Asp Met Gly

1 5 10

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Gly Ile Thr Ser Lys Ser Asn Val Met Gly

1 5 10

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<220>

<223> CDR1

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Gly Arg Thr Phe Ser Ser Tyr Val Met Gly

1 5 10

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Ser Ile Thr Ala Thr Gly Thr Thr Asn

fifteen

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Ala Ile Tyr Trp Ser Asn Gly Lys Thr Gln

1 5 10

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Ala Ile Tyr Trp Ser Ser Gly Lys Thr Glu

1 5 10

<210> 20

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<220>

<223> CDR2

<400> 20

Ala Ile Trp Trp Ser Ser Gly Lys Thr Glu

1 5 10

<210> 21

<211> 7

<212> PRT

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<220>

<223> CDR3

<400> 21

Phe Pro Pro Ile Ser Asn Phe

fifteen

<210> 22

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR3

<400> 22

Phe Pro Ala Ile Ser Asn Phe

fifteen

<210> 23

<211> 16

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR3

<400> 23

Asp Lys Asp Glu Thr Gly Phe Arg Thr Leu Pro Ile Ala Tyr Asp Tyr

1 5 10 15

<210> 24

<211> 16

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR3

<400> 24

Asp Lys Asp Arg Asp Gly Phe Arg Thr Leu Pro Ile Ala Tyr Asp Tyr

1 5 10 15

<210> 25

<211> 16

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR3

<400> 25

Asp Lys Asp Arg Tyr Gly Phe Arg Thr Leu Pro Ile Ala Tyr Asp Tyr

1 5 10 15

<210> 26

<211> 25

<212> PRT

<213> Artificial Sequence

<220>

<223> FR1

<400> 26

Glu Val Gln Leu Val Lys Ser Gly Gly Gly Leu Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser

20 25

<210> 27

<211> 25

<212> PRT

<213> Artificial Sequence

<220>

<223> FR1

<400> 27

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Asn

20 25

<210> 28

<211> 25

<212> PRT

<213> Artificial Sequence

<220>

<223> FR1

<400> 28

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser

20 25

<210> 29

<211> 25

<212> PRT

<213> Artificial Sequence

<220>

<223> FR1

<400> 29

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly

1 5 10 15

Pro Leu Arg Leu Ser Cys Ala Ala Ser

20 25

<210> 30

<211> 14

<212> PRT

<213> Artificial Sequence

<220>

<223> FR2

<400> 30

Trp Tyr Arg Gln Thr Pro Gly Asn Tyr Arg Glu Trp Val Ala

1 5 10

<210> 31

<211> 14

<212> PRT

<213> Artificial Sequence

<220>

<223> FR2

<400> 31

Trp Tyr Arg Gln Thr Pro Gly Lys Tyr Arg Glu Trp Val Ala

1 5 10

<210> 32

<211> 14

<212> PRT

<213> Artificial Sequence

<220>

<223> FR2

<400> 32

Trp Phe Arg Gln Thr Pro Gly Asn Tyr Arg Glu Trp Val Ala

1 5 10

<210> 33

<211> 14

<212> PRT

<213> Artificial Sequence

<220>

<223> FR2

<400> 33

Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val Ala

1 5 10

<210> 34

<211> 39

<212> PRT

<213> Artificial Sequence

<220>

<223> FR3

<400> 34

Tyr Arg Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala

1 5 10 15

Lys Ser Thr Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr

20 25 30

Thr Val Tyr Tyr Cys Asn Thr

35

<210> 35

<211> 39

<212> PRT

<213> Artificial Sequence

<220>

<223> FR3

<400> 35

Tyr Arg Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala

1 5 10 15

Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr

20 25 30

Thr Val Tyr Tyr Cys Asn Thr

35

<210> 36

<211> 39

<212> PRT

<213> Artificial Sequence

<220>

<223> FR3

<400> 36

Tyr Thr Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Gly Asp Asn Ala

1 5 10 15

Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Asn Pro Glu Asp Thr

20 25 30

Ala Val Tyr Tyr Cys Val Ala

35

<210> 37

<211> 39

<212> PRT

<213> Artificial Sequence

<220>

<223> FR3

<400> 37

Tyr Thr Asp Ser Val Lys Gly Arg Phe Thr Leu Ser Gly Asp Asn Ala

1 5 10 15

Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Asn Pro Glu Asp Thr

20 25 30

Ala Val Tyr Tyr Cys Val Ala

35

<210> 38

<211> 39

<212> PRT

<213> Artificial Sequence

<220>

<223> FR3

<400> 38

Tyr Thr Glu Ser Val Lys Gly Arg Phe Thr Leu Ser Gly Asp Asn Ala

1 5 10 15

Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Asn Pro Glu Asp Thr

20 25 30

Ala Val Tyr Tyr Cys Val Ala

35

<210> 39

<211> 39

<212> PRT

<213> Artificial Sequence

<220>

<223> FR3

<400> 39

Tyr Thr Asp Ser Val Lys Gly Arg Phe Thr Leu Ser Gly Asp Asn Ala

1 5 10 15

Lys Asn Thr Val Tyr Leu Gln Met Asn Asn Leu Asn Pro Glu Asp Thr

20 25 30

Ala Val Tyr Tyr Cys Val Ala

35

<210> 40

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<223> FR4

<400> 40

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

1 5 10

<210> 41

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<223> FR4

<400> 41

Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

1 5 10

<210> 42

<211> 117

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody sequence

<400> 42

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Asp Val His Lys Ile Asn

20 25 30

Phe Leu Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Lys Val

35 40 45

Ala His Ile Ser Ile Gly Asp Gln Thr Asp Tyr Ala Asp Ser Ala Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Glu Ser Lys Asn Met Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Phe Cys Arg

85 90 95

Ala Phe Ser Arg Ile Tyr Pro Tyr Asp Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 43

<211> 115

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 43

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Asn

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe

20 25 30

Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Thr Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ser

115

<210> 44

<211> 127

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 44

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Asp

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Ser Tyr

20 25 30

Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val

35 40 45

Ala Ala Ile Ser Trp Ser Asp Gly Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ala Asp Leu Thr Ser Thr Asn Pro Gly Ser Tyr Ile Tyr Ile Trp

100 105 110

Ala Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120 125

<210> 45

<211> 133

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 45

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Ile Gly Pro Tyr

20 25 30

Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val

35 40 45

Ala Ala Ile Asn Met Gly Gly Gly Ile Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn Ala Lys Asn Thr Val Tyr

65 70 75 80

Leu Leu Met Asn Ser Leu Glu Pro Glu Asp Thr Ala Ile Tyr Tyr Cys

85 90 95

Ala Ala Asp Ser Thr Ile Tyr Ala Ser Tyr Tyr Glu Cys Gly His Gly

100 105 110

Leu Ser Thr Gly Gly Tyr Gly Tyr Asp Ser Trp Gly Gln Gly Thr Leu

115 120 125

Val Thr Val Ser Ser

130

<210> 46

<211> 445

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 46

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly

1 5 10 15

Pro Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Ser Tyr

20 25 30

Val Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val

35 40 45

Ala Ala Ile Tyr Trp Ser Asn Gly Lys Thr Gln Tyr Thr Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Gly Asp Asn Ala Lys Asn Thr Val Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Asn Pro Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Val Ala Asp Lys Asp Glu Thr Gly Phe Arg Thr Leu Pro Ile Ala Tyr

100 105 110

Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly

115 120 125

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

130 135 140

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

145 150 155 160

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly

165 170 175

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Ile Gly Pro Tyr

180 185 190

Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val

195 200 205

Ala Ala Ile Asn Met Gly Gly Gly Ile Thr Tyr Tyr Ala Asp Ser Val

210 215 220

Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn Ala Lys Asn Thr Val Tyr

225 230 235 240

Leu Leu Met Asn Ser Leu Glu Pro Glu Asp Thr Ala Ile Tyr Tyr Cys

245 250 255

Ala Ala Asp Ser Thr Ile Tyr Ala Ser Tyr Tyr Glu Cys Gly His Gly

260 265 270

Leu Ser Thr Gly Gly Tyr Gly Tyr Asp Ser Trp Gly Gln Gly Thr Leu

275 280 285

Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

290 295 300

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

305 310 315 320

Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly

325 330 335

Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Val Ala

340 345 350

Ser Gly Asp Val His Lys Ile Asn Phe Leu Gly Trp Tyr Arg Gln Ala

355 360 365

Pro Gly Lys Glu Arg Glu Lys Val Ala His Ile Ser Ile Gly Asp Gln

370 375 380

Thr Asp Tyr Ala Asp Ser Ala Lys Gly Arg Phe Thr Ile Ser Arg Asp

385 390 395 400

Glu Ser Lys Asn Met Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu

405 410 415

Asp Thr Ala Val Tyr Phe Cys Arg Ala Phe Ser Arg Ile Tyr Pro Tyr

420 425 430

Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

435 440 445

<210> 47

<211> 427

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 47

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly

1 5 10 15

Pro Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Ser Tyr

20 25 30

Val Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val

35 40 45

Ala Ala Ile Tyr Trp Ser Asn Gly Lys Thr Gln Tyr Thr Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Gly Asp Asn Ala Lys Asn Thr Val Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Asn Pro Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Val Ala Asp Lys Asp Glu Thr Gly Phe Arg Thr Leu Pro Ile Ala Tyr

100 105 110

Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly

115 120 125

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

130 135 140

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

145 150 155 160

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly

165 170 175

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ile Thr Ser Lys Ile Asn

180 185 190

Asp Met Gly Trp Tyr Arg Gln Thr Pro Gly Asn Tyr Arg Glu Trp Val

195 200 205

Ala Ser Ile Thr Ala Thr Gly Thr Thr Asn Tyr Arg Asp Ser Val Lys

210 215 220

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Ser Thr Val Tyr Leu

225 230 235 240

Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Thr Val Tyr Tyr Cys Asn

245 250 255

Thr Phe Pro Pro Ile Ser Asn Phe Trp Gly Gln Gly Thr Leu Val Thr

260 265 270

Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly

275 280 285

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

290 295 300

Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly

305 310 315 320

Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Val Ala Ser Gly

325 330 335

Asp Val His Lys Ile Asn Phe Leu Gly Trp Tyr Arg Gln Ala Pro Gly

340 345 350

Lys Glu Arg Glu Lys Val Ala His Ile Ser Ile Gly Asp Gln Thr Asp

355 360 365

Tyr Ala Asp Ser Ala Lys Gly Arg Phe Thr Ile Ser Arg Asp Glu Ser

370 375 380

Lys Asn Met Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr

385 390 395 400

Ala Val Tyr Phe Cys Arg Ala Phe Ser Arg Ile Tyr Pro Tyr Asp Tyr

405 410 415

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

420 425

<210> 48

<211> 435

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 48

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ile Thr Ser Lys Ile Asn

20 25 30

Asp Met Gly Trp Tyr Arg Gln Thr Pro Gly Asn Tyr Arg Glu Trp Val

35 40 45

Ala Ser Ile Thr Ala Thr Gly Thr Thr Asn Tyr Arg Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Ser Thr Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Thr Val Tyr Tyr Cys Asn

85 90 95

Thr Phe Pro Pro Ile Ser Asn Phe Trp Gly Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly

115 120 125

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

130 135 140

Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly

145 150 155 160

Ser Val Gln Ala Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly

165 170 175

Tyr Thr Ile Gly Pro Tyr Cys Met Gly Trp Phe Arg Gln Ala Pro Gly

180 185 190

Lys Glu Arg Glu Gly Val Ala Ala Ile Asn Met Gly Gly Gly Ile Thr

195 200 205

Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn

210 215 220

Ala Lys Asn Thr Val Tyr Leu Leu Met Asn Ser Leu Glu Pro Glu Asp

225 230 235 240

Thr Ala Ile Tyr Tyr Cys Ala Ala Asp Ser Thr Ile Tyr Ala Ser Tyr

245 250 255

Tyr Glu Cys Gly His Gly Leu Ser Thr Gly Gly Tyr Gly Tyr Asp Ser

260 265 270

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser

275 280 285

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

290 295 300

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val

305 310 315 320

Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu

325 330 335

Arg Leu Ser Cys Val Ala Ser Gly Asp Val His Lys Ile Asn Phe Leu

340 345 350

Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Lys Val Ala His

355 360 365

Ile Ser Ile Gly Asp Gln Thr Asp Tyr Ala Asp Ser Ala Lys Gly Arg

370 375 380

Phe Thr Ile Ser Arg Asp Glu Ser Lys Asn Met Val Tyr Leu Gln Met

385 390 395 400

Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Phe Cys Arg Ala Phe

405 410 415

Ser Arg Ile Tyr Pro Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr

420 425 430

Val Ser Ser

435

<210> 49

<211> 427

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 49

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ile Thr Ser Lys Ile Asn

20 25 30

Asp Met Gly Trp Tyr Arg Gln Thr Pro Gly Asn Tyr Arg Glu Trp Val

35 40 45

Ala Ser Ile Thr Ala Thr Gly Thr Thr Asn Tyr Arg Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Ser Thr Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Thr Val Tyr Tyr Cys Asn

85 90 95

Thr Phe Pro Pro Ile Ser Asn Phe Trp Gly Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly

115 120 125

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

130 135 140

Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly

145 150 155 160

Leu Val Gln Ala Gly Gly Pro Leu Arg Leu Ser Cys Ala Ala Ser Gly

165 170 175

Arg Thr Phe Ser Ser Tyr Val Met Gly Trp Phe Arg Gln Ala Pro Gly

180 185 190

Lys Glu Arg Glu Phe Val Ala Ala Ile Tyr Trp Ser Asn Gly Lys Thr

195 200 205

Gln Tyr Thr Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Gly Asp Asn

210 215 220

Ala Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Asn Pro Glu Asp

225 230 235 240

Thr Ala Val Tyr Tyr Cys Val Ala Asp Lys Asp Glu Thr Gly Phe Arg

245 250 255

Thr Leu Pro Ile Ala Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr

260 265 270

Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly

275 280 285

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

290 295 300

Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly

305 310 315 320

Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Val Ala Ser Gly

325 330 335

Asp Val His Lys Ile Asn Phe Leu Gly Trp Tyr Arg Gln Ala Pro Gly

340 345 350

Lys Glu Arg Glu Lys Val Ala His Ile Ser Ile Gly Asp Gln Thr Asp

355 360 365

Tyr Ala Asp Ser Ala Lys Gly Arg Phe Thr Ile Ser Arg Asp Glu Ser

370 375 380

Lys Asn Met Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr

385 390 395 400

Ala Val Tyr Phe Cys Arg Ala Phe Ser Arg Ile Tyr Pro Tyr Asp Tyr

405 410 415

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

420 425

<210> 50

<211> 445

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 50

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Ile Gly Pro Tyr

20 25 30

Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val

35 40 45

Ala Ala Ile Asn Met Gly Gly Gly Ile Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn Ala Lys Asn Thr Val Tyr

65 70 75 80

Leu Leu Met Asn Ser Leu Glu Pro Glu Asp Thr Ala Ile Tyr Tyr Cys

85 90 95

Ala Ala Asp Ser Thr Ile Tyr Ala Ser Tyr Tyr Glu Cys Gly His Gly

100 105 110

Leu Ser Thr Gly Gly Tyr Gly Tyr Asp Ser Trp Gly Gln Gly Thr Leu

115 120 125

Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

130 135 140

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

145 150 155 160

Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly

165 170 175

Gly Gly Leu Val Gln Ala Gly Gly Pro Leu Arg Leu Ser Cys Ala Ala

180 185 190

Ser Gly Arg Thr Phe Ser Ser Tyr Val Met Gly Trp Phe Arg Gln Ala

195 200 205

Pro Gly Lys Glu Arg Glu Phe Val Ala Ala Ile Tyr Trp Ser Asn Gly

210 215 220

Lys Thr Gln Tyr Thr Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Gly

225 230 235 240

Asp Asn Ala Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Asn Pro

245 250 255

Glu Asp Thr Ala Val Tyr Tyr Cys Val Ala Asp Lys Asp Glu Thr Gly

260 265 270

Phe Arg Thr Leu Pro Ile Ala Tyr Asp Tyr Trp Gly Gln Gly Thr Leu

275 280 285

Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

290 295 300

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

305 310 315 320

Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly

325 330 335

Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Val Ala

340 345 350

Ser Gly Asp Val His Lys Ile Asn Phe Leu Gly Trp Tyr Arg Gln Ala

355 360 365

Pro Gly Lys Glu Arg Glu Lys Val Ala His Ile Ser Ile Gly Asp Gln

370 375 380

Thr Asp Tyr Ala Asp Ser Ala Lys Gly Arg Phe Thr Ile Ser Arg Asp

385 390 395 400

Glu Ser Lys Asn Met Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu

405 410 415

Asp Thr Ala Val Tyr Phe Cys Arg Ala Phe Ser Arg Ile Tyr Pro Tyr

420 425 430

Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

435 440 445

<210> 51

<211> 435

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 51

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Ile Gly Pro Tyr

20 25 30

Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val

35 40 45

Ala Ala Ile Asn Met Gly Gly Gly Ile Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn Ala Lys Asn Thr Val Tyr

65 70 75 80

Leu Leu Met Asn Ser Leu Glu Pro Glu Asp Thr Ala Ile Tyr Tyr Cys

85 90 95

Ala Ala Asp Ser Thr Ile Tyr Ala Ser Tyr Tyr Glu Cys Gly His Gly

100 105 110

Leu Ser Thr Gly Gly Tyr Gly Tyr Asp Ser Trp Gly Gln Gly Thr Leu

115 120 125

Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

130 135 140

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

145 150 155 160

Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly

165 170 175

Gly Gly Leu Val Gln Ala Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala

180 185 190

Ser Gly Ile Thr Ser Lys Ile Asn Asp Met Gly Trp Tyr Arg Gln Thr

195 200 205

Pro Gly Asn Tyr Arg Glu Trp Val Ala Ser Ile Thr Ala Thr Gly Thr

210 215 220

Thr Asn Tyr Arg Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp

225 230 235 240

Asn Ala Lys Ser Thr Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu

245 250 255

Asp Thr Thr Val Tyr Tyr Cys Asn Thr Phe Pro Pro Ile Ser Asn Phe

260 265 270

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser

275 280 285

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

290 295 300

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val

305 310 315 320

Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu

325 330 335

Arg Leu Ser Cys Val Ala Ser Gly Asp Val His Lys Ile Asn Phe Leu

340 345 350

Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Lys Val Ala His

355 360 365

Ile Ser Ile Gly Asp Gln Thr Asp Tyr Ala Asp Ser Ala Lys Gly Arg

370 375 380

Phe Thr Ile Ser Arg Asp Glu Ser Lys Asn Met Val Tyr Leu Gln Met

385 390 395 400

Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Phe Cys Arg Ala Phe

405 410 415

Ser Arg Ile Tyr Pro Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr

420 425 430

Val Ser Ser

435

<210> 52

<211> 427

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 52

Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly

1 5 10 15

Pro Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Ser Tyr

20 25 30

Val Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val

35 40 45

Ala Ala Ile Tyr Trp Ser Asn Gly Lys Thr Gln Tyr Thr Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Gly Asp Asn Ala Lys Asn Thr Val Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Asn Pro Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Val Ala Asp Lys Asp Glu Thr Gly Phe Arg Thr Leu Pro Ile Ala Tyr

100 105 110

Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly

115 120 125

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

130 135 140

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

145 150 155 160

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly

165 170 175

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ile Thr Ser Lys Ile Asn

180 185 190

Asp Met Gly Trp Tyr Arg Gln Thr Pro Gly Asn Tyr Arg Glu Trp Val

195 200 205

Ala Ser Ile Thr Ala Thr Gly Thr Thr Asn Tyr Arg Asp Ser Val Lys

210 215 220

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Ser Thr Val Tyr Leu

225 230 235 240

Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Thr Val Tyr Tyr Cys Asn

245 250 255

Thr Phe Pro Pro Ile Ser Asn Phe Trp Gly Gln Gly Thr Leu Val Thr

260 265 270

Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly

275 280 285

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

290 295 300

Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly

305 310 315 320

Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Val Ala Ser Gly

325 330 335

Asp Val His Lys Ile Asn Phe Leu Gly Trp Tyr Arg Gln Ala Pro Gly

340 345 350

Lys Glu Arg Glu Lys Val Ala His Ile Ser Ile Gly Asp Gln Thr Asp

355 360 365

Tyr Ala Asp Ser Ala Lys Gly Arg Phe Thr Ile Ser Arg Asp Glu Ser

370 375 380

Lys Asn Met Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr

385 390 395 400

Ala Val Tyr Phe Cys Arg Ala Phe Ser Arg Ile Tyr Pro Tyr Asp Tyr

405 410 415

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

420 425

<210> 53

<211> 267

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 53

Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Asp Val His Lys Ile Asn

20 25 30

Phe Leu Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Lys Val

35 40 45

Ala His Ile Ser Ile Gly Asp Gln Thr Asp Tyr Ala Asp Ser Ala Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Glu Ser Lys Asn Met Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Phe Cys Arg

85 90 95

Ala Phe Ser Arg Ile Tyr Pro Tyr Asp Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

115 120 125

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

130 135 140

Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly

145 150 155 160

Gly Gly Leu Val Gln Ala Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala

165 170 175

Ser Gly Ile Thr Ser Lys Ile Asn Asp Met Gly Trp Tyr Arg Gln Thr

180 185 190

Pro Gly Asn Tyr Arg Glu Trp Val Ala Ser Ile Thr Ala Thr Gly Thr

195 200 205

Thr Asn Tyr Arg Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp

210 215 220

Asn Ala Lys Ser Thr Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu

225 230 235 240

Asp Thr Thr Val Tyr Tyr Cys Asn Thr Phe Pro Pro Ile Ser Asn Phe

245 250 255

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

260 265

<210> 54

<211> 279

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 54

Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Asp Val His Lys Ile Asn

20 25 30

Phe Leu Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Lys Val

35 40 45

Ala His Ile Ser Ile Gly Asp Gln Thr Asp Tyr Ala Asp Ser Ala Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Glu Ser Lys Asn Met Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Phe Cys Arg

85 90 95

Ala Phe Ser Arg Ile Tyr Pro Tyr Asp Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

115 120 125

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

130 135 140

Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly

145 150 155 160

Gly Gly Leu Val Gln Ala Gly Asp Ser Leu Arg Leu Ser Cys Ala Ala

165 170 175

Ser Gly Arg Thr Phe Ser Ser Tyr Ala Met Gly Trp Phe Arg Gln Ala

180 185 190

Pro Gly Lys Glu Arg Glu Phe Val Ala Ala Ile Ser Trp Ser Asp Gly

195 200 205

Ser Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg

210 215 220

Asp Asn Ala Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Lys Pro

225 230 235 240

Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ala Asp Leu Thr Ser Thr Asn

245 250 255

Pro Gly Ser Tyr Ile Tyr Ile Trp Ala Tyr Asp Tyr Trp Gly Gln Gly

260 265 270

Thr Leu Val Thr Val Ser Ser

275

<210> 55

<211> 427

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 55

Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ile Thr Ser Lys Ile Asn

20 25 30

Asp Met Gly Trp Tyr Arg Gln Thr Pro Gly Asn Tyr Arg Glu Trp Val

35 40 45

Ala Ser Ile Thr Ala Thr Gly Thr Thr Asn Tyr Arg Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Ser Thr Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Thr Val Tyr Tyr Cys Asn

85 90 95

Thr Phe Pro Pro Ile Ser Asn Phe Trp Gly Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly

115 120 125

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

130 135 140

Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly

145 150 155 160

Leu Val Gln Ala Gly Gly Pro Leu Arg Leu Ser Cys Ala Ala Ser Gly

165 170 175

Arg Thr Phe Ser Ser Tyr Val Met Gly Trp Phe Arg Gln Ala Pro Gly

180 185 190

Lys Glu Arg Glu Phe Val Ala Ala Ile Tyr Trp Ser Asn Gly Lys Thr

195 200 205

Gln Tyr Thr Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Gly Asp Asn

210 215 220

Ala Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Asn Pro Glu Asp

225 230 235 240

Thr Ala Val Tyr Tyr Cys Val Ala Asp Lys Asp Glu Thr Gly Phe Arg

245 250 255

Thr Leu Pro Ile Ala Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr

260 265 270

Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly

275 280 285

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

290 295 300

Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly

305 310 315 320

Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Val Ala Ser Gly

325 330 335

Asp Val His Lys Ile Asn Phe Leu Gly Trp Tyr Arg Gln Ala Pro Gly

340 345 350

Lys Glu Arg Glu Lys Val Ala His Ile Ser Ile Gly Asp Gln Thr Asp

355 360 365

Tyr Ala Asp Ser Ala Lys Gly Arg Phe Thr Ile Ser Arg Asp Glu Ser

370 375 380

Lys Asn Met Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr

385 390 395 400

Ala Val Tyr Phe Cys Arg Ala Phe Ser Arg Ile Tyr Pro Tyr Asp Tyr

405 410 415

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

420 425

<210> 56

<211> 267

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 56

Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ile Thr Ser Lys Ile Asn

20 25 30

Asp Met Gly Trp Tyr Arg Gln Thr Pro Gly Asn Tyr Arg Glu Trp Val

35 40 45

Ala Ser Ile Thr Ala Thr Gly Thr Thr Asn Tyr Arg Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Ser Thr Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Thr Val Tyr Tyr Cys Asn

85 90 95

Thr Phe Pro Pro Ile Ser Asn Phe Trp Gly Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly

115 120 125

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

130 135 140

Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly

145 150 155 160

Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Val Ala Ser Gly

165 170 175

Asp Val His Lys Ile Asn Phe Leu Gly Trp Tyr Arg Gln Ala Pro Gly

180 185 190

Lys Glu Arg Glu Lys Val Ala His Ile Ser Ile Gly Asp Gln Thr Asp

195 200 205

Tyr Ala Asp Ser Ala Lys Gly Arg Phe Thr Ile Ser Arg Asp Glu Ser

210 215 220

Lys Asn Met Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr

225 230 235 240

Ala Val Tyr Phe Cys Arg Ala Phe Ser Arg Ile Tyr Pro Tyr Asp Tyr

245 250 255

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

260 265

<210> 57

<211> 279

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 57

Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Asp

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Ser Tyr

20 25 30

Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val

35 40 45

Ala Ala Ile Ser Trp Ser Asp Gly Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ala Asp Leu Thr Ser Thr Asn Pro Gly Ser Tyr Ile Tyr Ile Trp

100 105 110

Ala Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly

115 120 125

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

130 135 140

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly

145 150 155 160

Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro

165 170 175

Gly Gly Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Asp Val His Lys

180 185 190

Ile Asn Phe Leu Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu

195 200 205

Lys Val Ala His Ile Ser Ile Gly Asp Gln Thr Asp Tyr Ala Asp Ser

210 215 220

Ala Lys Gly Arg Phe Thr Ile Ser Arg Asp Glu Ser Lys Asn Met Val

225 230 235 240

Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Phe

245 250 255

Cys Arg Ala Phe Ser Arg Ile Tyr Pro Tyr Asp Tyr Trp Gly Gln Gly

260 265 270

Thr Leu Val Thr Val Ser Ser

275

<210> 58

<211> 426

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 58

Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly Ser

1 5 10 15

Leu Arg Leu Ser Cys Ala Ala Ser Gly Ile Thr Ser Lys Ile Asn Asp

20 25 30

Met Gly Trp Tyr Arg Gln Thr Pro Gly Asn Tyr Arg Glu Trp Val Ala

35 40 45

Ser Ile Thr Ala Thr Gly Thr Thr Asn Tyr Arg Asp Ser Val Lys Gly

50 55 60

Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Ser Thr Val Tyr Leu Gln

65 70 75 80

Met Asn Ser Leu Lys Pro Glu Asp Thr Thr Val Tyr Tyr Cys Asn Thr

85 90 95

Phe Pro Pro Ile Ser Asn Phe Trp Gly Gln Gly Thr Leu Val Thr Val

100 105 110

Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

115 120 125

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

130 135 140

Gly Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu

145 150 155 160

Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Asp

165 170 175

Val His Lys Ile Asn Phe Leu Gly Trp Tyr Arg Gln Ala Pro Gly Lys

180 185 190

Glu Arg Glu Lys Val Ala His Ile Ser Ile Gly Asp Gln Thr Asp Tyr

195 200 205

Ala Asp Ser Ala Lys Gly Arg Phe Thr Ile Ser Arg Asp Glu Ser Lys

210 215 220

Asn Met Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala

225 230 235 240

Val Tyr Phe Cys Arg Ala Phe Ser Arg Ile Tyr Pro Tyr Asp Tyr Trp

245 250 255

Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly

260 265 270

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

275 280 285

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln

290 295 300

Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly Pro Leu Arg

305 310 315 320

Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Ser Tyr Val Met Gly

325 330 335

Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val Ala Ala Ile

340 345 350

Tyr Trp Ser Asn Gly Lys Thr Gln Tyr Thr Asp Ser Val Lys Gly Arg

355 360 365

Phe Thr Ile Ser Gly Asp Asn Ala Lys Asn Thr Val Tyr Leu Gln Met

370 375 380

Asn Ser Leu Asn Pro Glu Asp Thr Ala Val Tyr Tyr Cys Val Ala Asp

385 390 395 400

Lys Asp Glu Thr Gly Phe Arg Thr Leu Pro Ile Ala Tyr Asp Tyr Trp

405 410 415

Gly Gln Gly Thr Leu Val Thr Val Ser Ser

420 425

<210> 59

<211> 427

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 59

Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly

1 5 10 15

Pro Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Ser Tyr

20 25 30

Val Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val

35 40 45

Ala Ala Ile Tyr Trp Ser Asn Gly Lys Thr Gln Tyr Thr Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Gly Asp Asn Ala Lys Asn Thr Val Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Asn Pro Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Val Ala Asp Lys Asp Glu Thr Gly Phe Arg Thr Leu Pro Ile Ala Tyr

100 105 110

Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly

115 120 125

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

130 135 140

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

145 150 155 160

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

165 170 175

Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Asp Val His Lys Ile Asn

180 185 190

Phe Leu Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Lys Val

195 200 205

Ala His Ile Ser Ile Gly Asp Gln Thr Asp Tyr Ala Asp Ser Ala Lys

210 215 220

Gly Arg Phe Thr Ile Ser Arg Asp Glu Ser Lys Asn Met Val Tyr Leu

225 230 235 240

Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Phe Cys Arg

245 250 255

Ala Phe Ser Arg Ile Tyr Pro Tyr Asp Tyr Trp Gly Gln Gly Thr Leu

260 265 270

Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

275 280 285

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

290 295 300

Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly

305 310 315 320

Gly Gly Leu Val Gln Ala Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala

325 330 335

Ser Gly Ile Thr Ser Lys Ile Asn Asp Met Gly Trp Tyr Arg Gln Thr

340 345 350

Pro Gly Asn Tyr Arg Glu Trp Val Ala Ser Ile Thr Ala Thr Gly Thr

355 360 365

Thr Asn Tyr Arg Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp

370 375 380

Asn Ala Lys Ser Thr Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu

385 390 395 400

Asp Thr Thr Val Tyr Tyr Cys Asn Thr Phe Pro Pro Ile Ser Asn Phe

405 410 415

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

420 425

<210> 60

<211> 427

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 60

Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Asp Val His Lys Ile Asn

20 25 30

Phe Leu Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Lys Val

35 40 45

Ala His Ile Ser Ile Gly Asp Gln Thr Asp Tyr Ala Asp Ser Ala Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Glu Ser Lys Asn Met Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Phe Cys Arg

85 90 95

Ala Phe Ser Arg Ile Tyr Pro Tyr Asp Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

115 120 125

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

130 135 140

Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly

145 150 155 160

Gly Gly Leu Val Gln Ala Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala

165 170 175

Ser Gly Ile Thr Ser Lys Ile Asn Asp Met Gly Trp Tyr Arg Gln Thr

180 185 190

Pro Gly Asn Tyr Arg Glu Trp Val Ala Ser Ile Thr Ala Thr Gly Thr

195 200 205

Thr Asn Tyr Arg Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp

210 215 220

Asn Ala Lys Ser Thr Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu

225 230 235 240

Asp Thr Thr Val Tyr Tyr Cys Asn Thr Phe Pro Pro Ile Ser Asn Phe

245 250 255

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser

260 265 270

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

275 280 285

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val

290 295 300

Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly Pro Leu

305 310 315 320

Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Ser Tyr Val Met

325 330 335

Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val Ala Ala

340 345 350

Ile Tyr Trp Ser Asn Gly Lys Thr Gln Tyr Thr Asp Ser Val Lys Gly

355 360 365

Arg Phe Thr Ile Ser Gly Asp Asn Ala Lys Asn Thr Val Tyr Leu Gln

370 375 380

Met Asn Ser Leu Asn Pro Glu Asp Thr Ala Val Tyr Tyr Cys Val Ala

385 390 395 400

Asp Lys Asp Glu Thr Gly Phe Arg Thr Leu Pro Ile Ala Tyr Asp Tyr

405 410 415

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

420 425

<210> 61

<211> 427

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 61

Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Asp Val His Lys Ile Asn

20 25 30

Phe Leu Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Lys Val

35 40 45

Ala His Ile Ser Ile Gly Asp Gln Thr Asp Tyr Ala Asp Ser Ala Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Glu Ser Lys Asn Met Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Phe Cys Arg

85 90 95

Ala Phe Ser Arg Ile Tyr Pro Tyr Asp Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

115 120 125

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

130 135 140

Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly

145 150 155 160

Gly Gly Leu Val Gln Ala Gly Gly Pro Leu Arg Leu Ser Cys Ala Ala

165 170 175

Ser Gly Arg Thr Phe Ser Ser Tyr Val Met Gly Trp Phe Arg Gln Ala

180 185 190

Pro Gly Lys Glu Arg Glu Phe Val Ala Ala Ile Tyr Trp Ser Asn Gly

195 200 205

Lys Thr Gln Tyr Thr Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Gly

210 215 220

Asp Asn Ala Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Asn Pro

225 230 235 240

Glu Asp Thr Ala Val Tyr Tyr Cys Val Ala Asp Lys Asp Glu Thr Gly

245 250 255

Phe Arg Thr Leu Pro Ile Ala Tyr Asp Tyr Trp Gly Gln Gly Thr Leu

260 265 270

Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

275 280 285

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

290 295 300

Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly

305 310 315 320

Gly Gly Leu Val Gln Ala Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala

325 330 335

Ser Gly Ile Thr Ser Lys Ile Asn Asp Met Gly Trp Tyr Arg Gln Thr

340 345 350

Pro Gly Asn Tyr Arg Glu Trp Val Ala Ser Ile Thr Ala Thr Gly Thr

355 360 365

Thr Asn Tyr Arg Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp

370 375 380

Asn Ala Lys Ser Thr Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu

385 390 395 400

Asp Thr Thr Val Tyr Tyr Cys Asn Thr Phe Pro Pro Ile Ser Asn Phe

405 410 415

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

420 425

<210> 62

<211> 429

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 62

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Asp Val His Lys Ile Asn

20 25 30

Phe Leu Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Lys Val

35 40 45

Ala His Ile Ser Ile Gly Asp Gln Thr Asp Tyr Ala Asp Ser Ala Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Glu Ser Lys Asn Met Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Phe Cys Arg

85 90 95

Ala Phe Ser Arg Ile Tyr Pro Tyr Asp Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

115 120 125

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

130 135 140

Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly

145 150 155 160

Gly Gly Leu Val Gln Ala Gly Asp Ser Leu Arg Leu Ser Cys Ala Ala

165 170 175

Ser Gly Arg Thr Phe Ser Ser Tyr Ala Met Gly Trp Phe Arg Gln Ala

180 185 190

Pro Gly Lys Glu Arg Glu Phe Val Ala Ala Ile Ser Trp Ser Asp Gly

195 200 205

Ser Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg

210 215 220

Asp Asn Ala Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Lys Pro

225 230 235 240

Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ala Asp Leu Thr Ser Thr Asn

245 250 255

Pro Gly Ser Tyr Ile Tyr Ile Trp Ala Tyr Asp Tyr Trp Gly Gln Gly

260 265 270

Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

275 280 285

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

290 295 300

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val Glu

305 310 315 320

Ser Gly Gly Gly Leu Val Gln Pro Gly Asn Ser Leu Arg Leu Ser Cys

325 330 335

Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe Gly Met Ser Trp Val Arg

340 345 350

Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Ser Ile Ser Gly Ser

355 360 365

Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile

370 375 380

Ser Arg Asp Asn Ala Lys Thr Thr Leu Tyr Leu Gln Met Asn Ser Leu

385 390 395 400

Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys Thr Ile Gly Gly Ser Leu

405 410 415

Ser Arg Ser Ser Gln Gly Thr Leu Val Thr Val Ser Ser

420 425

<210> 63

<211> 417

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 63

Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Asp Val His Lys Ile Asn

20 25 30

Phe Leu Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Lys Val

35 40 45

Ala His Ile Ser Ile Gly Asp Gln Thr Asp Tyr Ala Asp Ser Ala Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Glu Ser Lys Asn Met Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Phe Cys Arg

85 90 95

Ala Phe Ser Arg Ile Tyr Pro Tyr Asp Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

115 120 125

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

130 135 140

Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly

145 150 155 160

Gly Gly Leu Val Gln Ala Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala

165 170 175

Ser Gly Ile Thr Ser Lys Ile Asn Asp Met Gly Trp Tyr Arg Gln Thr

180 185 190

Pro Gly Asn Tyr Arg Glu Trp Val Ala Ser Ile Thr Ala Thr Gly Thr

195 200 205

Thr Asn Tyr Arg Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp

210 215 220

Asn Ala Lys Ser Thr Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu

225 230 235 240

Asp Thr Thr Val Tyr Tyr Cys Asn Thr Phe Pro Pro Ile Ser Asn Phe

245 250 255

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser

260 265 270

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

275 280 285

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val

290 295 300

Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Asn Ser Leu

305 310 315 320

Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe Gly Met

325 330 335

Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Ser

340 345 350

Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val Lys Gly

355 360 365

Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Thr Thr Leu Tyr Leu Gln

370 375 380

Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys Thr Ile

385 390 395 400

Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Leu Val Thr Val Ser

405 410 415

Ser

<210> 64

<211> 577

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 64

Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Asp Val His Lys Ile Asn

20 25 30

Phe Leu Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Lys Val

35 40 45

Ala His Ile Ser Ile Gly Asp Gln Thr Asp Tyr Ala Asp Ser Ala Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Glu Ser Lys Asn Met Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Phe Cys Arg

85 90 95

Ala Phe Ser Arg Ile Tyr Pro Tyr Asp Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

115 120 125

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

130 135 140

Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly

145 150 155 160

Gly Gly Leu Val Gln Ala Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala

165 170 175

Ser Gly Ile Thr Ser Lys Ile Asn Asp Met Gly Trp Tyr Arg Gln Thr

180 185 190

Pro Gly Asn Tyr Arg Glu Trp Val Ala Ser Ile Thr Ala Thr Gly Thr

195 200 205

Thr Asn Tyr Arg Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp

210 215 220

Asn Ala Lys Ser Thr Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu

225 230 235 240

Asp Thr Thr Val Tyr Tyr Cys Asn Thr Phe Pro Pro Ile Ser Asn Phe

245 250 255

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser

260 265 270

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

275 280 285

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val

290 295 300

Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly Pro Leu

305 310 315 320

Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Ser Tyr Val Met

325 330 335

Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val Ala Ala

340 345 350

Ile Tyr Trp Ser Asn Gly Lys Thr Gln Tyr Thr Asp Ser Val Lys Gly

355 360 365

Arg Phe Thr Ile Ser Gly Asp Asn Ala Lys Asn Thr Val Tyr Leu Gln

370 375 380

Met Asn Ser Leu Asn Pro Glu Asp Thr Ala Val Tyr Tyr Cys Val Ala

385 390 395 400

Asp Lys Asp Glu Thr Gly Phe Arg Thr Leu Pro Ile Ala Tyr Asp Tyr

405 410 415

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser

420 425 430

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

435 440 445

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val

450 455 460

Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Asn Ser Leu

465 470 475 480

Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe Gly Met

485 490 495

Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Ser

500 505 510

Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val Lys Gly

515 520 525

Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Thr Thr Leu Tyr Leu Gln

530 535 540

Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys Thr Ile

545 550 555 560

Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Leu Val Thr Val Ser

565 570 575

Ser

<210> 65

<211> 577

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 65

Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Asp Val His Lys Ile Asn

20 25 30

Phe Leu Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Lys Val

35 40 45

Ala His Ile Ser Ile Gly Asp Gln Thr Asp Tyr Ala Asp Ser Ala Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Glu Ser Lys Asn Met Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Phe Cys Arg

85 90 95

Ala Phe Ser Arg Ile Tyr Pro Tyr Asp Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

115 120 125

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

130 135 140

Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly

145 150 155 160

Gly Gly Leu Val Gln Ala Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala

165 170 175

Ser Gly Ile Thr Ser Lys Ile Asn Asp Met Gly Trp Tyr Arg Gln Thr

180 185 190

Pro Gly Asn Tyr Arg Glu Trp Val Ala Ser Ile Thr Ala Thr Gly Thr

195 200 205

Thr Asn Tyr Arg Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp

210 215 220

Asn Ala Lys Ser Thr Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu

225 230 235 240

Asp Thr Thr Val Tyr Tyr Cys Asn Thr Phe Pro Pro Ile Ser Asn Phe

245 250 255

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser

260 265 270

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

275 280 285

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val

290 295 300

Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Asn Ser Leu

305 310 315 320

Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe Gly Met

325 330 335

Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Ser

340 345 350

Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val Lys Gly

355 360 365

Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Thr Thr Leu Tyr Leu Gln

370 375 380

Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys Thr Ile

385 390 395 400

Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Leu Val Thr Val Ser

405 410 415

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

420 425 430

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

435 440 445

Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val

450 455 460

Gln Ala Gly Gly Pro Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr

465 470 475 480

Phe Ser Ser Tyr Val Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu

485 490 495

Arg Glu Phe Val Ala Ala Ile Tyr Trp Ser Asn Gly Lys Thr Gln Tyr

500 505 510

Thr Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Gly Asp Asn Ala Lys

515 520 525

Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Asn Pro Glu Asp Thr Ala

530 535 540

Val Tyr Tyr Cys Val Ala Asp Lys Asp Glu Thr Gly Phe Arg Thr Leu

545 550 555 560

Pro Ile Ala Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser

565 570 575

Ser

<210> 66

<211> 577

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 66

Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Asp Val His Lys Ile Asn

20 25 30

Phe Leu Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Lys Val

35 40 45

Ala His Ile Ser Ile Gly Asp Gln Thr Asp Tyr Ala Asp Ser Ala Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Glu Ser Lys Asn Met Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Phe Cys Arg

85 90 95

Ala Phe Ser Arg Ile Tyr Pro Tyr Asp Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

115 120 125

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

130 135 140

Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly

145 150 155 160

Gly Gly Leu Val Gln Ala Gly Gly Pro Leu Arg Leu Ser Cys Ala Ala

165 170 175

Ser Gly Arg Thr Phe Ser Ser Tyr Val Met Gly Trp Phe Arg Gln Ala

180 185 190

Pro Gly Lys Glu Arg Glu Phe Val Ala Ala Ile Tyr Trp Ser Asn Gly

195 200 205

Lys Thr Gln Tyr Thr Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Gly

210 215 220

Asp Asn Ala Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Asn Pro

225 230 235 240

Glu Asp Thr Ala Val Tyr Tyr Cys Val Ala Asp Lys Asp Glu Thr Gly

245 250 255

Phe Arg Thr Leu Pro Ile Ala Tyr Asp Tyr Trp Gly Gln Gly Thr Leu

260 265 270

Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

275 280 285

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

290 295 300

Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly

305 310 315 320

Gly Gly Leu Val Gln Ala Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala

325 330 335

Ser Gly Ile Thr Ser Lys Ile Asn Asp Met Gly Trp Tyr Arg Gln Thr

340 345 350

Pro Gly Asn Tyr Arg Glu Trp Val Ala Ser Ile Thr Ala Thr Gly Thr

355 360 365

Thr Asn Tyr Arg Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp

370 375 380

Asn Ala Lys Ser Thr Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu

385 390 395 400

Asp Thr Thr Val Tyr Tyr Cys Asn Thr Phe Pro Pro Ile Ser Asn Phe

405 410 415

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser

420 425 430

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

435 440 445

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val

450 455 460

Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Asn Ser Leu

465 470 475 480

Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe Gly Met

485 490 495

Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Ser

500 505 510

Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val Lys Gly

515 520 525

Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Thr Thr Leu Tyr Leu Gln

530 535 540

Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys Thr Ile

545 550 555 560

Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Leu Val Thr Val Ser

565 570 575

Ser

<210> 67

<211> 577

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 67

Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Asp Val His Lys Ile Asn

20 25 30

Phe Leu Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Lys Val

35 40 45

Ala His Ile Ser Ile Gly Asp Gln Thr Asp Tyr Ala Asp Ser Ala Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Glu Ser Lys Asn Met Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Phe Cys Arg

85 90 95

Ala Phe Ser Arg Ile Tyr Pro Tyr Asp Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

115 120 125

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

130 135 140

Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly

145 150 155 160

Gly Gly Leu Val Gln Ala Gly Gly Pro Leu Arg Leu Ser Cys Ala Ala

165 170 175

Ser Gly Arg Thr Phe Ser Ser Tyr Val Met Gly Trp Phe Arg Gln Ala

180 185 190

Pro Gly Lys Glu Arg Glu Phe Val Ala Ala Ile Tyr Trp Ser Asn Gly

195 200 205

Lys Thr Gln Tyr Thr Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Gly

210 215 220

Asp Asn Ala Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Asn Pro

225 230 235 240

Glu Asp Thr Ala Val Tyr Tyr Cys Val Ala Asp Lys Asp Glu Thr Gly

245 250 255

Phe Arg Thr Leu Pro Ile Ala Tyr Asp Tyr Trp Gly Gln Gly Thr Leu

260 265 270

Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

275 280 285

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

290 295 300

Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly

305 310 315 320

Gly Gly Leu Val Gln Pro Gly Asn Ser Leu Arg Leu Ser Cys Ala Ala

325 330 335

Ser Gly Phe Thr Phe Ser Ser Phe Gly Met Ser Trp Val Arg Gln Ala

340 345 350

Pro Gly Lys Gly Leu Glu Trp Val Ser Ser Ile Ser Gly Ser Gly Ser

355 360 365

Asp Thr Leu Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg

370 375 380

Asp Asn Ala Lys Thr Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Pro

385 390 395 400

Glu Asp Thr Ala Val Tyr Tyr Cys Thr Ile Gly Gly Ser Leu Ser Arg

405 410 415

Ser Ser Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser

420 425 430

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

435 440 445

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val

450 455 460

Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly Ser Leu

465 470 475 480

Arg Leu Ser Cys Ala Ala Ser Gly Ile Thr Ser Lys Ile Asn Asp Met

485 490 495

Gly Trp Tyr Arg Gln Thr Pro Gly Asn Tyr Arg Glu Trp Val Ala Ser

500 505 510

Ile Thr Ala Thr Gly Thr Thr Asn Tyr Arg Asp Ser Val Lys Gly Arg

515 520 525

Phe Thr Ile Ser Arg Asp Asn Ala Lys Ser Thr Val Tyr Leu Gln Met

530 535 540

Asn Ser Leu Lys Pro Glu Asp Thr Thr Val Tyr Tyr Cys Asn Thr Phe

545 550 555 560

Pro Pro Ile Ser Asn Phe Trp Gly Gln Gly Thr Leu Val Thr Val Ser

565 570 575

Ser

<210> 68

<211> 378

<212> PRT

<213> Homo sapiens

<400> 68

Met Val Leu Leu Trp Leu Thr Leu Leu Leu Ile Ala Leu Pro Cys Leu

1 5 10 15

Leu Gln Thr Lys Glu Asp Pro Asn Pro Pro Ile Thr Asn Leu Arg Met

20 25 30

Lys Ala Lys Ala Gln Gln Leu Thr Trp Asp Leu Asn Arg Asn Val Thr

35 40 45

Asp Ile Glu Cys Val Lys Asp Ala Asp Tyr Ser Met Pro Ala Val Asn

50 55 60

Asn Ser Tyr Cys Gln Phe Gly Ala Ile Ser Leu Cys Glu Val Thr Asn

65 70 75 80

Tyr Thr Val Arg Val Ala Asn Pro Pro Phe Ser Thr Trp Ile Leu Phe

85 90 95

Pro Glu Asn Ser Gly Lys Pro Trp Ala Gly Ala Glu Asn Leu Thr Cys

100 105 110

Trp Ile His Asp Val Asp Phe Leu Ser Cys Ser Trp Ala Val Gly Pro

115 120 125

Gly Ala Pro Ala Asp Val Gln Tyr Asp Leu Tyr Leu Asn Val Ala Asn

130 135 140

Arg Arg Gln Gln Tyr Glu Cys Leu His Tyr Lys Thr Asp Ala Gln Gly

145 150 155 160

Thr Arg Ile Gly Cys Arg Phe Asp Asp Ile Ser Arg Leu Ser Ser Gly

165 170 175

Ser Gln Ser Ser His Ile Leu Val Arg Gly Arg Ser Ala Ala Phe Gly

180 185 190

Ile Pro Cys Thr Asp Lys Phe Val Val Phe Ser Gln Ile Glu Ile Leu

195 200 205

Thr Pro Pro Asn Met Thr Ala Lys Cys Asn Lys Thr His Ser Phe Met

210 215 220

His Trp Lys Met Arg Ser His Phe Asn Arg Lys Phe Arg Tyr Glu Leu

225 230 235 240

Gln Ile Gln Lys Arg Met Gln Pro Val Ile Thr Glu Gln Val Arg Asp

245 250 255

Arg Thr Ser Phe Gln Leu Leu Asn Pro Gly Thr Tyr Thr Val Gln Ile

260 265 270

Arg Ala Arg Glu Arg Val Tyr Glu Phe Leu Ser Ala Trp Ser Thr Pro

275 280 285

Gln Arg Phe Glu Cys Asp Gln Glu Glu Gly Ala Asn Thr Arg Ala Trp

290 295 300

Arg Thr Ser Leu Leu Ile Ala Leu Gly Thr Leu Leu Ala Leu Val Cys

305 310 315 320

Val Phe Val Ile Cys Arg Arg Tyr Leu Val Met Gln Arg Leu Phe Pro

325 330 335

Arg Ile Pro His Met Lys Asp Pro Ile Gly Asp Ser Phe Gln Asn Asp

340 345 350

Lys Leu Val Val Trp Glu Ala Gly Lys Ala Gly Leu Glu Glu Cys Leu

355 360 365

Val Thr Glu Val Gln Val Val Gln Lys Thr

370 375

<210> 69

<211> 378

<212> PRT

<213> Macaca fascicularis

<400> 69

Met Thr Leu Leu Trp Leu Thr Leu Leu Leu Val Ala Thr Pro Cys Leu

1 5 10 15

Leu Arg Thr Lys Glu Asp Pro Asn Ala Pro Ile Arg Asn Leu Arg Met

20 25 30

Lys Glu Lys Ala Gln Gln Leu Met Trp Asp Leu Asn Arg Asn Val Thr

35 40 45

Asp Val Glu Cys Ile Lys Gly Thr Asp Tyr Ser Met Pro Ala Met Asn

50 55 60

Asn Ser Tyr Cys Gln Phe Gly Ala Ile Ser Leu Cys Glu Val Thr Asn

65 70 75 80

Tyr Thr Val Arg Val Ala Ser Pro Pro Phe Ser Thr Trp Ile Leu Phe

85 90 95

Pro Glu Asn Ser Gly Thr Pro Arg Ala Gly Ala Glu Asn Leu Thr Cys

100 105 110

Trp Val His Asp Val Asp Phe Leu Ser Cys Ser Trp Val Val Gly Pro

115 120 125

Ala Ala Pro Ala Asp Val Gln Tyr Asp Leu Tyr Leu Asn Asn Pro Asn

130 135 140

Ser His Glu Gln Tyr Arg Cys Leu His Tyr Lys Thr Asp Ala Arg Gly

145 150 155 160

Thr Gln Ile Gly Cys Arg Phe Asp Asp Ile Ala Pro Leu Ser Arg Gly

165 170 175

Ser Gln Ser Ser His Ile Leu Val Arg Gly Arg Ser Ala Ala Val Ser

180 185 190

Ile Pro Cys Thr Asp Lys Phe Val Phe Phe Ser Gln Ile Glu Arg Leu

195 200 205

Thr Pro Pro Asn Met Thr Gly Glu Cys Asn Glu Thr His Ser Phe Met

210 215 220

His Trp Lys Met Lys Ser His Phe Asn Arg Lys Phe Arg Tyr Glu Leu

225 230 235 240

Arg Ile Gln Lys Arg Met Gln Pro Val Arg Thr Glu Gln Val Arg Asp

245 250 255

Thr Thr Ser Phe Gln Leu Pro Asn Pro Gly Thr Tyr Thr Val Gln Ile

260 265 270

Arg Ala Arg Glu Thr Val Tyr Glu Phe Leu Ser Ala Trp Ser Thr Pro

275 280 285

Gln Arg Phe Glu Cys Asp Gln Glu Glu Gly Ala Ser Ser Arg Ala Trp

290 295 300

Arg Thr Ser Leu Leu Ile Ala Leu Gly Thr Leu Leu Ala Leu Leu Cys

305 310 315 320

Val Phe Leu Ile Cys Arg Arg Tyr Leu Val Met Gln Arg Leu Phe Pro

325 330 335

Arg Ile Pro His Met Lys Asp Pro Ile Gly Asp Thr Phe Gln Gln Asp

340 345 350

Lys Leu Val Val Trp Glu Ala Gly Lys Ala Gly Leu Glu Glu Cys Leu

355 360 365

Val Ser Glu Val Gln Val Val Glu Lys Thr

370 375

<210> 70

<211> 171

<212> PRT

<213> Homo sapiens

<400> 70

Met Glu His Ser Thr Phe Leu Ser Gly Leu Val Leu Ala Thr Leu Leu

1 5 10 15

Ser Gln Val Ser Pro Phe Lys Ile Pro Ile Glu Glu Leu Glu Asp Arg

20 25 30

Val Phe Val Asn Cys Asn Thr Ser Ile Thr Trp Val Glu Gly Thr Val

35 40 45

Gly Thr Leu Leu Ser Asp Ile Thr Arg Leu Asp Leu Gly Lys Arg Ile

50 55 60

Leu Asp Pro Arg Gly Ile Tyr Arg Cys Asn Gly Thr Asp Ile Tyr Lys

65 70 75 80

Asp Lys Glu Ser Thr Val Gln Val His Tyr Arg Met Cys Gln Ser Cys

85 90 95

Val Glu Leu Asp Pro Ala Thr Val Ala Gly Ile Ile Val Thr Asp Val

100 105 110

Ile Ala Thr Leu Leu Leu Ala Leu Gly Val Phe Cys Phe Ala Gly His

115 120 125

Glu Thr Gly Arg Leu Ser Gly Ala Ala Asp Thr Gln Ala Leu Leu Arg

130 135 140

Asn Asp Gln Val Tyr Gln Pro Leu Arg Asp Arg Asp Asp Ala Gln Tyr

145 150 155 160

Ser His Leu Gly Gly Asn Trp Ala Arg Asn Lys

165 170

<210> 71

<211> 182

<212> PRT

<213> Homo sapiens

<400> 71

Met Glu Gln Gly Lys Gly Leu Ala Val Leu Ile Leu Ala Ile Ile Leu

1 5 10 15

Leu Gln Gly Thr Leu Ala Gln Ser Ile Lys Gly Asn His Leu Val Lys

20 25 30

Val Tyr Asp Tyr Gln Glu Asp Gly Ser Val Leu Leu Thr Cys Asp Ala

35 40 45

Glu Ala Lys Asn Ile Thr Trp Phe Lys Asp Gly Lys Met Ile Gly Phe

50 55 60

Leu Thr Glu Asp Lys Lys Lys Trp Asn Leu Gly Ser Asn Ala Lys Asp

65 70 75 80

Pro Arg Gly Met Tyr Gln Cys Lys Gly Ser Gln Asn Lys Ser Lys Pro

85 90 95

Leu Gln Val Tyr Tyr Arg Met Cys Gln Asn Cys Ile Glu Leu Asn Ala

100 105 110

Ala Thr Ile Ser Gly Phe Leu Phe Ala Glu Ile Val Ser Ile Phe Val

115 120 125

Leu Ala Val Gly Val Tyr Phe Ile Ala Gly Gln Asp Gly Val Arg Gln

130 135 140

Ser Arg Ala Ser Asp Lys Gln Thr Leu Leu Pro Asn Asp Gln Leu Tyr

145 150 155 160

Gln Pro Leu Lys Asp Arg Glu Asp Asp Gln Tyr Ser His Leu Gln Gly

165 170 175

Asn Gln Leu Arg Arg Asn

180

<210> 72

<211> 207

<212> PRT

<213> Homo sapiens

<400> 72

Met Gln Ser Gly Thr His Trp Arg Val Leu Gly Leu Cys Leu Leu Ser

1 5 10 15

Val Gly Val Trp Gly Gln Asp Gly Asn Glu Glu Met Gly Gly Ile Thr

20 25 30

Gln Thr Pro Tyr Lys Val Ser Ile Ser Gly Thr Thr Val Ile Leu Thr

35 40 45

Cys Pro Gln Tyr Pro Gly Ser Glu Ile Leu Trp Gln His Asn Asp Lys

50 55 60

Asn Ile Gly Gly Asp Glu Asp Asp Lys Asn Ile Gly Ser Asp Glu Asp

65 70 75 80

His Leu Ser Leu Lys Glu Phe Ser Glu Leu Glu Gln Ser Gly Tyr Tyr

85 90 95

Val Cys Tyr Pro Arg Gly Ser Lys Pro Glu Asp Ala Asn Phe Tyr Leu

100 105 110

Tyr Leu Arg Ala Arg Val Cys Glu Asn Cys Met Glu Met Asp Val Met

115 120 125

Ser Val Ala Thr Ile Val Ile Val Asp Ile Cys Ile Thr Gly Gly Leu

130 135 140

Leu Leu Leu Val Tyr Tyr Trp Ser Lys Asn Arg Lys Ala Lys Ala Lys

145 150 155 160

Pro Val Thr Arg Gly Ala Gly Ala Gly Gly Arg Gln Arg Gly Gln Asn

165 170 175

Lys Glu Arg Pro Pro Pro Val Pro Asn Pro Asp Tyr Glu Pro Ile Arg

180 185 190

Lys Gly Gln Arg Asp Leu Tyr Ser Gly Leu Asn Gln Arg Arg Ile

195 200 205

<210> 73

<211> 164

<212> PRT

<213> Homo sapiens

<400> 73

Met Lys Trp Lys Ala Leu Phe Thr Ala Ala Ile Leu Gln Ala Gln Leu

1 5 10 15

Pro Ile Thr Glu Ala Gln Ser Phe Gly Leu Leu Asp Pro Lys Leu Cys

20 25 30

Tyr Leu Leu Asp Gly Ile Leu Phe Ile Tyr Gly Val Ile Leu Thr Ala

35 40 45

Leu Phe Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr

50 55 60

Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg

65 70 75 80

Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met

85 90 95

Gly Gly Lys Pro Gln Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn

100 105 110

Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met

115 120 125

Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly

130 135 140

Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala

145 150 155 160

Leu Pro Pro Arg

<210> 74

<211> 142

<212> PRT

<213> Homo sapiens

<400> 74

Pro Asn Ile Gln Asn Pro Asp Pro Ala Val Tyr Gln Leu Arg Asp Ser

1 5 10 15

Lys Ser Ser Asp Lys Ser Val Cys Leu Phe Thr Asp Phe Asp Ser Gln

20 25 30

Thr Asn Val Ser Gln Ser Lys Asp Ser Asp Val Tyr Ile Thr Asp Lys

35 40 45

Thr Val Leu Asp Met Arg Ser Met Asp Phe Lys Ser Asn Ser Ala Val

50 55 60

Ala Trp Ser Asn Lys Ser Asp Phe Ala Cys Ala Asn Ala Phe Asn Asn

65 70 75 80

Ser Ile Ile Pro Glu Asp Thr Phe Phe Pro Ser Pro Glu Ser Ser Cys

85 90 95

Asp Val Lys Leu Val Glu Lys Ser Phe Glu Thr Asp Thr Asn Leu Asn

100 105 110

Phe Gln Asn Leu Ser Val Ile Gly Phe Arg Ile Leu Leu Leu Lys Val

115 120 125

Ala Gly Phe Asn Leu Leu Met Thr Leu Arg Leu Trp Ser Ser

130 135 140

<210> 75

<211> 177

<212> PRT

<213> Homo sapiens

<400> 75

Glu Asp Leu Asn Lys Val Phe Pro Glu Val Ala Val Phe Glu Pro

1 5 10 15

Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala Thr Leu Val Cys Leu

20 25 30

Ala Thr Gly Phe Phe Pro Asp His Val Glu Leu Ser Trp Trp Val Asn

35 40 45

Gly Lys Glu Val His Ser Gly Val Ser Thr Asp Pro Gln Pro Leu Lys

50 55 60

Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Cys Leu Ser Ser Arg Leu

65 70 75 80

Arg Val Ser Ala Thr Phe Trp Gln Asn Pro Arg Asn His Phe Arg Cys

85 90 95

Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu Trp Thr Gln Asp

100 105 110

Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu Ala Trp Gly Arg

115 120 125

Ala Asp Cys Gly Phe Thr Ser Val Ser Tyr Gln Gln Gly Val Leu Ser

130 135 140

Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly Lys Ala Thr Leu Tyr Ala

145 150 155 160

Val Leu Val Ser Ala Leu Val Leu Met Ala Met Val Lys Arg Lys Asp

165 170 175

Phe

<210> 76

<211> 108

<212> PRT

<213> Homo sapiens

<400> 76

Gln Leu Leu Glu Gln Ser Pro Gln Phe Leu Ser Ile Gln Glu Gly Glu

1 5 10 15

Asn Leu Thr Val Tyr Cys Asn Ser Ser Ser Val Phe Ser Ser Leu Gln

20 25 30

Trp Tyr Arg Gln Glu Pro Gly Glu Gly Pro Val Leu Leu Val Thr Val

35 40 45

Val Thr Gly Gly Glu Val Lys Lys Leu Lys Arg Leu Thr Phe Gln Phe

50 55 60

Gly Asp Ala Arg Lys Asp Ser Ser Leu His Ile Thr Ala Ala Gln Pro

65 70 75 80

Gly Asp Thr Gly Leu Tyr Leu Cys Ala Gly Ala Gly Ser Gln Gly Asn

85 90 95

Leu Ile Phe Gly Lys Gly Thr Lys Leu Ser Val Lys

100 105

<210> 77

<211> 112

<212> PRT

<213> Homo sapiens

<400> 77

Asp Gly Gly Ile Thr Gln Ser Pro Lys Tyr Leu Phe Arg Lys Glu Gly

1 5 10 15

Gln Asn Val Thr Leu Ser Cys Glu Gln Asn Leu Asn His Asp Ala Met

20 25 30

Tyr Trp Tyr Arg Gln Asp Pro Gly Gln Gly Leu Arg Leu Ile Tyr Tyr

35 40 45

Ser Gln Ile Val Asn Asp Phe Gln Lys Gly Asp Ile Ala Glu Gly Tyr

50 55 60

Ser Val Ser Arg Glu Lys Lys Glu Ser Phe Pro Leu Thr Val Thr Ser

65 70 75 80

Ala Gln Lys Asn Pro Thr Ala Phe Tyr Leu Cys Ala Ser Ser Ser Arg

85 90 95

Ser Ser Tyr Glu Gln Tyr Phe Gly Pro Gly Thr Arg Leu Thr Val Thr

100 105 110

<210> 78

<211> 117

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 78

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Val His Lys Ile Asn

20 25 30

Phe Leu Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Leu Val

35 40 45

Ala Thr Ile Thr Ile Gly Asp Thr Thr Asp Tyr Ala Asp Ser Ala Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala Lys Asn Met Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val His Phe Cys Arg

85 90 95

Ala Gly Ser Arg Leu Tyr Pro Tyr Asn Tyr Trp Gly Gln Gly Thr Gln

100 105 110

Val Thr Val Ser Ser

115

<210> 79

<211> 117

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 79

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Val His Lys Ile Asn

20 25 30

Phe Leu Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Gly Leu Val

35 40 45

Ala Thr Ile Thr Ile Gly Asp Thr Thr Asp Tyr Ala Asp Tyr Ala Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala Arg Asn Met Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Phe Cys Arg

85 90 95

Ala Gly Ser Arg Leu Tyr Pro Tyr Asn Tyr Trp Gly Gln Gly Thr Gln

100 105 110

Val Thr Val Ser Ser

115

<210> 80

<211> 117

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 80

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Pro Cys Ala Ala Ser Gly Ser Val His Lys Ile Asn

20 25 30

Phe Leu Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Met Val

35 40 45

Ala Thr Ile Thr Ile Gly Asp Ala Thr Asp Tyr Ala Asp Ser Ala Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala Lys Asn Met Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Phe Cys Arg

85 90 95

Ala Gly Ser Arg Leu Tyr Pro Tyr Asn Tyr Trp Gly Gln Gly Thr Gln

100 105 110

Val Thr Val Ser Ser

115

<210> 81

<211> 117

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 81

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Val His Lys Ile Asn

20 25 30

Phe Leu Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Leu Val

35 40 45

Ala Thr Ile Thr Ile Gly Asp Thr Thr Asp Tyr Ala Asp Tyr Ala Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala Arg Asn Met Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Phe Cys Arg

85 90 95

Ala Gly Ser Arg Leu Tyr Pro Tyr Asn Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 82

<211> 117

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 82

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Val His Lys Ile Asn

20 25 30

Phe Leu Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Met Val

35 40 45

Ala Thr Ile Thr Ile Gly Asp Ala Thr Asp Tyr Ala Asp Ser Ala Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala Lys Asn Met Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Phe Cys Arg

85 90 95

Ala Gly Ser Arg Leu Tyr Pro Tyr Asn Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 83

<211> 117

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 83

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Val His Lys Ile Asn

20 25 30

Phe Leu Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Leu Val

35 40 45

Ala Thr Ile Thr Ile Gly Asp Thr Thr Asp Tyr Ala Asp Ser Ala Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala Lys Asn Met Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Phe Cys Arg

85 90 95

Ala Gly Ser Arg Leu Tyr Pro Tyr Asn Tyr Trp Gly Gln Gly Thr Gln

100 105 110

Val Thr Val Ser Ser

115

<210> 84

<211> 117

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 84

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Ser Val His Leu Leu Asn

20 25 30

Phe Leu Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Met Val

35 40 45

Ala His Ile Thr Ile Ala Asp Ala Thr Asp Tyr Ser His Phe Ala Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala Lys Asn Met Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Phe Cys Arg

85 90 95

Ala Gly Ser Arg Ile Tyr Pro Tyr Asp Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 85

<211> 117

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 85

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Ala Val His Lys Ile Asn

20 25 30

Phe Leu Gly Trp Tyr Arg Gln Thr Pro Glu Lys Glu Arg Glu Met Val

35 40 45

Ala Thr Ile Thr Ile Gly Asp Asp Val Asp Tyr Ala Asp Ser Ala Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala Lys Asn Met Val Tyr Leu

65 70 75 80

Gln Met Thr Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Val Cys Arg

85 90 95

Ala Gly Ser Arg Leu Tyr Pro Tyr Asn Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 86

<211> 117

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 86

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Arg Ala Ser Gly Asp Val His Lys Ile Asn

20 25 30

Ile Leu Gly Trp Tyr Arg Gln Ala Pro Ala Lys Glu Arg Glu Met Val

35 40 45

Ala His Ile Thr Ile Gly Asp Ala Thr Asp Tyr Ala Glu Ser Ala Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala Lys Asn Met Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Phe Cys Arg

85 90 95

Ala Tyr Ser Arg Ile Tyr Pro Tyr Asn Tyr Trp Gly Gln Gly Thr Gln

100 105 110

Val Thr Val Ser Ser

115

<210> 87

<211> 117

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 87

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Arg Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Asp Val His Lys Ile Asn

20 25 30

Ile Leu Gly Trp Tyr Arg Gln Ala Pro Ala Lys Glu Arg Glu Met Val

35 40 45

Ala His Ile Thr Ile Gly Asp Ala Thr Asp Tyr Ala Asp Ser Ala Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala Lys Asn Met Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Phe Cys Arg

85 90 95

Ala Tyr Ser Arg Ile Tyr Pro Tyr Asn Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 88

<211> 117

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 88

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Asp Val His Lys Ile Asn

20 25 30

Ile Leu Gly Trp Tyr Arg Gln Ala Pro Ala Lys Glu Arg Glu Met Val

35 40 45

Ala His Ile Thr Ile Gly Asp Ala Thr Asp Tyr Ala Asp Ser Ala Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala Lys Asn Met Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Phe Cys Arg

85 90 95

Ala Tyr Ser Arg Ile Tyr Pro Tyr Asn Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 89

<211> 117

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 89

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Asp Val His Lys Ile Asn

20 25 30

Phe Leu Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Lys Val

35 40 45

Ala His Ile Thr Ile Gly Asp Gln Ala Asp Tyr Ala Asp Ser Ala Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala Lys Asn Met Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Phe Cys Arg

85 90 95

Ala Gly Ser Arg Ile Tyr Pro Tyr Asp Tyr Trp Gly Gln Gly Thr Gln

100 105 110

Val Thr Val Ser Ser

115

<210> 90

<211> 117

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 90

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Val His Lys Ile Asn

20 25 30

Phe Leu Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Met Val

35 40 45

Ala Thr Ile Thr Ile Gly Asp Ala Thr Asp Tyr Ala Asp Ser Ala Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala Lys Asn Met Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Ser Pro Glu Asp Thr Ala Val Tyr Phe Cys Arg

85 90 95

Ala Gly Ser Arg Leu Tyr Pro Tyr Asn Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 91

<211> 117

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 91

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Val His Leu Leu Asn

20 25 30

Phe Leu Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Met Val

35 40 45

Ala His Ile Ser Ile Ala Asp Ala Thr Asp Tyr Ala His Phe Ala Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala Lys Asn Met Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Phe Cys Arg

85 90 95

Ala Gly Ser Arg Ile Tyr Pro Tyr Asp Tyr Trp Gly Arg Gly Thr Gln

100 105 110

Val Thr Val Ser Ser

115

<210> 92

<211> 117

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 92

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Arg Ala Ser Gly Asp Val His Lys Ile Asn

20 25 30

Ile Leu Gly Trp Tyr Arg Gln Ala Pro Ala Lys Glu Arg Glu Met Val

35 40 45

Ala His Ile Thr Ile Gly Asp Ala Thr Val Tyr Ala Glu Ser Ala Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala Lys Asn Met Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Phe Cys Arg

85 90 95

Ala Tyr Ser Arg Ile Tyr Pro Tyr Asn Tyr Trp Gly Gln Gly Thr Gln

100 105 110

Val Thr Val Ser Ser

115

<210> 93

<211> 117

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 93

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Val His Leu Leu Asn

20 25 30

Phe Leu Gly Trp Tyr Arg Gln Cys Pro Gly Lys Glu Arg Glu Met Val

35 40 45

Ala His Ile Thr Ile Ala Asp Ala Thr Asp Tyr Ser His Phe Ala Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala Lys Asn Met Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Phe Cys Arg

85 90 95

Ala Gly Ser Arg Ile Tyr Pro Tyr Asp Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 94

<211> 117

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 94

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Pro Ser Cys Ala Ala Ser Gly Ser Val His Leu Leu Asn

20 25 30

Phe Leu Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Met Val

35 40 45

Ala His Ile Thr Ile Ala Asp Ala Thr Asp Tyr Ala His Phe Ala Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala Lys Asn Met Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Phe Cys Arg

85 90 95

Ala Gly Ser Arg Ile Tyr Pro Tyr Asp Tyr Trp Gly Gln Gly Thr Gln

100 105 110

Val Thr Val Ser Ser

115

<210> 95

<211> 117

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 95

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Val His Leu Leu Asn

20 25 30

Phe Leu Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Met Val

35 40 45

Ala His Ile Thr Ile Ala Asp Ala Thr Asp Tyr Ala His Phe Ala Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala Lys Asn Met Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Phe Cys Arg

85 90 95

Ala Gly Ser Arg Ile Tyr Pro Tyr Asp Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 96

<211> 117

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 96

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Ala Val His Lys Ile Asn

20 25 30

Phe Leu Gly Trp Tyr Arg Gln Thr Pro Glu Lys Glu Arg Glu Met Val

35 40 45

Ala His Ile Thr Ile Gly Asp Glu Val Asp Tyr Ala Asp Ser Ala Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala Lys Asn Met Val Tyr Leu

65 70 75 80

Gln Met Thr Ser Leu Thr Pro Glu Asp Thr Ala Val Tyr Val Cys Arg

85 90 95

Ala Gly Ser Arg Leu Tyr Pro Tyr Asn Tyr Trp Gly Gln Gly Thr Gln

100 105 110

Val Thr Val Ser Ser

115

<210> 97

<211> 117

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 97

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val His Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Asp Val His Lys Ile Asn

20 25 30

Phe Leu Gly Trp His Arg Gln Pro Pro Gly Lys Glu Arg Glu Lys Val

35 40 45

Ala His Ile Thr Ile Gly Asp Val Thr Asp Tyr Ala Asp Ser Ala Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala Lys Asn Met Val Tyr Leu

65 70 75 80

Gln Met Asn Asn Leu Lys Pro Glu Asp Thr Ala Val Tyr Phe Cys Arg

85 90 95

Ala Gly Ser Arg Ile Tyr Pro Tyr Asp Tyr Trp Gly Gln Gly Thr Gln

100 105 110

Val Thr Val Ser Ser

115

<210> 98

<211> 117

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 98

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Asp Val His Lys Ile Asn

20 25 30

Val Leu Gly Trp Tyr Arg Gln Ala Pro Ala Lys Glu Arg Glu Met Val

35 40 45

Ala His Ile Thr Ile Gly Asp Ala Thr Asp Tyr Ala Asp Ser Ala Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala Lys Asn Met Val His Leu

65 70 75 80

Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Phe Cys Arg

85 90 95

Ala Tyr Ser Arg Ile Tyr Pro Tyr Asn Tyr Trp Gly Gln Gly Thr Gln

100 105 110

Val Thr Val Ser Ser

115

<210> 99

<211> 117

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 99

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Asp Val His Lys Ile Asn

20 25 30

Ile Leu Gly Trp Tyr Arg Gln Ala Pro Ala Lys Glu Arg Glu Met Val

35 40 45

Ala His Ile Thr Ile Gly Asp Ala Thr Asn Tyr Ala Asp Ser Ala Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala Lys Asn Met Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Phe Cys Arg

85 90 95

Ala Tyr Ser Arg Ile Tyr Pro Tyr Asn Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 100

<211> 117

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 100

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Asp Val His Lys Ile Asn

20 25 30

Phe Leu Gly Trp His Arg Gln Ala Pro Gly Lys Glu Arg Glu Lys Val

35 40 45

Ala His Ile Thr Ile Gly Asp Val Thr Asp Tyr Ala Asp Ser Ala Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala Lys Asn Met Val Phe Leu

65 70 75 80

Gln Met Asn Asn Leu Lys Pro Glu Asp Thr Ala Val Tyr Phe Cys Arg

85 90 95

Ala Gly Ser Arg Ile Tyr Pro Tyr Asp Tyr Trp Gly Gln Gly Thr Gln

100 105 110

Val Thr Val Ser Ser

115

<210> 101

<211> 117

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 101

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Asp Val His Lys Ile Asn

20 25 30

Ile Leu Gly Trp Tyr Arg Gln Ala Pro Ala Lys Glu Arg Glu Met Val

35 40 45

Ala His Ile Thr Ile Gly Asp Ala Thr Asp Tyr Ala Gly Ser Ala Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala Lys Asn Met Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Phe Cys Arg

85 90 95

Ala Tyr Ser Arg Ile Tyr Pro Tyr Asn Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 102

<211> 117

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 102

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Glu Val Tyr Lys Ile Asn

20 25 30

Phe Leu Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Lys Val

35 40 45

Ala His Ile Thr Ile Ala Asp Val Ala Asp Tyr Ala Asp Phe Ala Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala Lys Asn Met Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Phe Cys Arg

85 90 95

Ala Gly Ser Arg Ile Trp Pro Tyr Asp Tyr Trp Gly Gln Gly Thr Gln

100 105 110

Val Thr Val Ser Ser

115

<210> 103

<211> 117

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 103

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Ala Val His Lys Ile Asn

20 25 30

Phe Leu Gly Trp Tyr Arg Gln Ala Pro Glu Lys Glu Arg Glu Met Val

35 40 45

Ala Thr Ile Thr Ile Gly Asp Glu Val Asp Tyr Ala Asp Ser Ala Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala Lys Asn Met Val Tyr Leu

65 70 75 80

Gln Met Thr Ser Leu Lys Pro Glu Asp Thr Thr Val Tyr Val Cys Arg

85 90 95

Ala Gly Ser Arg Leu Tyr Pro Tyr Asn Tyr Trp Gly Gln Gly Thr Gln

100 105 110

Val Thr Val Ser Ser

115

<210> 104

<211> 117

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 104

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Glu Val Tyr Lys Ile Asn

20 25 30

Phe Leu Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Lys Val

35 40 45

Ala His Ile Thr Ile Ala Asp Val Ala Asp Tyr Ala Asp Phe Ala Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala Lys Asn Met Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Phe Cys Arg

85 90 95

Ala Gly Ser Arg Ile Trp Pro Tyr Asp Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 105

<211> 117

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 105

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Asp Val His Lys Ile Asn

20 25 30

Ile Leu Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Lys Val

35 40 45

Ala His Ile Ser Ile Ser Asp Gln Thr Asp Tyr Ala Glu Ser Ala Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Glu Ser Lys Asn Met Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Leu Cys Arg

85 90 95

Ala Phe Ser Arg Ile Tyr Pro Tyr Asp Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 106

<211> 117

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 106

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Val His Lys Ile Asn

20 25 30

Phe Leu Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Leu Val

35 40 45

Ala Thr Ile Thr Ile Gly Asp Ala Thr Asp Tyr Ala Asp Tyr Ala Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala Arg Asn Met Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Phe Cys Arg

85 90 95

Ala Gly Ser Arg Leu Tyr Pro Tyr Asn Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 107

<211> 117

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 107

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Asp Val His Lys Ile Asn

20 25 30

Ile Leu Gly Trp Tyr Arg Gln Ala Pro Ala Lys Glu Arg Glu Met Val

35 40 45

Ala His Ile Thr Ile Gly Asp Ala Thr Asp Tyr Ala Asp Ser Ala Lys

50 55 60

Gly Arg Phe Ala Ile Ser Arg Asp Glu Ala Lys Asn Met Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Phe Cys Arg

85 90 95

Ala Tyr Ser Arg Ile Tyr Pro Tyr Asn Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 108

<211> 117

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 108

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Asp Val His Lys Ile Asn

20 25 30

Phe Leu Gly Trp His Arg Gln Ala Pro Gly Lys Glu Arg Glu Lys Val

35 40 45

Ala His Ile Thr Ile Gly Asp Ala Thr Asp Tyr Ala Asp Ser Ala Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala Lys Asn Met Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Phe Cys Arg

85 90 95

Ala Gly Ser Arg Ile Tyr Pro Tyr Asp Tyr Trp Gly Gln Gly Thr Gln

100 105 110

Val Thr Val Ser Ser

115

<210> 109

<211> 117

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 109

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Val His Lys Ile Asn

20 25 30

Phe Leu Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Val Val

35 40 45

Ala Thr Ile Thr Ile Gly Asp Ala Thr Asp Tyr Ala Asp Ser Ala Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala Lys Asn Met Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Ser Pro Glu Asp Thr Ala Val Tyr Phe Cys Arg

85 90 95

Ala Gly Ser Arg Leu Tyr Pro Tyr Asn Tyr Trp Gly Gln Gly Thr Gln

100 105 110

Val Thr Val Ser Ser

115

<210> 110

<211> 117

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 110

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Ala Arg Leu Ser Cys Val Ala Ser Gly Asp Val His Lys Ile Asn

20 25 30

Phe Leu Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Lys Val

35 40 45

Ala His Ile Thr Ile Gly Asp Gln Thr Asp Tyr Ala Asp Ser Ala Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala Lys Asn Met Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Phe Cys Arg

85 90 95

Ala Gly Ser Arg Ile Trp Pro Tyr Asp Tyr Trp Gly Gln Gly Thr Gln

100 105 110

Val Thr Val Ser Ser

115

<210> 111

<211> 117

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 111

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Val His Leu Leu Asn

20 25 30

Phe Leu Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Met Val

35 40 45

Ala His Ile Thr Ile Ala Asp Ala Thr Asp Tyr Ser His Phe Ala Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala Lys Asn Met Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Phe Cys Arg

85 90 95

Ala Gly Ser Arg Ile Tyr Pro Tyr Asp Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 112

<211> 117

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 112

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Ser Val His Leu Leu Asn

20 25 30

Phe Leu Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Gly Val Val

35 40 45

Ala His Ile Thr Ile Ala Asp Ala Thr Asp Tyr Ser His Phe Ala Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala Lys Asn Met Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Phe Cys Arg

85 90 95

Ala Gly Ser Arg Ile Tyr Pro Tyr Asp Tyr Trp Gly Gln Gly Thr Gln

100 105 110

Val Thr Val Ser Ser

115

<210> 113

<211> 117

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 113

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Thr Leu Ser Cys Ala Ala Ser Gly Asp Val His Lys Ile Asn

20 25 30

Ile Leu Gly Trp Tyr Arg Gln Ala Pro Ala Lys Glu Arg Glu Met Val

35 40 45

Ala His Ile Thr Ile Gly Asp Ala Thr Asp Tyr Ala Asp Ser Ala Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala Lys Asn Met Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Phe Cys Arg

85 90 95

Ala Tyr Ser Arg Ile Tyr Pro Tyr Asn Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 114

<211> 117

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 114

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Gly Val His Lys Ile Asn

20 25 30

Ile Leu Gly Trp Tyr Arg Gln Ala Pro Ala Lys Glu Arg Glu Met Val

35 40 45

Ala His Ile Thr Ile Gly Asp Ala Thr Asp Tyr Ala Asp Ser Ala Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala Lys Asn Met Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Phe Cys Arg

85 90 95

Ala Tyr Ser Arg Ile Tyr Pro Tyr Asn Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 115

<211> 117

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 115

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Glu Val Tyr Lys Ile Asn

20 25 30

Phe Leu Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Lys Val

35 40 45

Ala His Ile Thr Ile Ala Asp Val Ala Asp Tyr Ala Asp Phe Ala Gln

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala Lys Asn Met Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Phe Cys Arg

85 90 95

Ala Gly Ser Arg Ile Trp Pro Tyr Asp Tyr Trp Gly Gln Gly Thr Gln

100 105 110

Val Thr Val Ser Ser

115

<210> 116

<211> 117

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 116

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Asp Val His Lys Ile Asn

20 25 30

Ile Leu Gly Trp Tyr Arg Gln Ala Pro Ala Lys Glu Arg Glu Met Val

35 40 45

Ala His Ile Thr Ile Gly Asp Thr Thr Asp Tyr Ala Asp Ser Ala Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala Lys Asn Met Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Phe Cys Arg

85 90 95

Ala Tyr Ser Arg Ile Tyr Pro Tyr Asn Tyr Trp Gly Gln Gly Thr Gln

100 105 110

Val Thr Val Ser Ser

115

<210> 117

<211> 117

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 117

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Val His Leu Leu Asn

20 25 30

Phe Leu Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Met Val

35 40 45

Ala His Ile Thr Ile Ala Asp Ala Thr Asp Tyr Ser His Phe Ala Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala Lys Asn Met Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Phe Cys Arg

85 90 95

Ala Gly Ser Arg Ile Tyr Pro Tyr Asp Tyr Trp Gly His Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 118

<211> 117

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 118

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Asp Val His Lys Ile Asn

20 25 30

Phe Leu Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Lys Val

35 40 45

Ala His Ile Thr Ile Gly Asp Gln Ala Asp Tyr Ala Asp Ser Ala Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala Lys Asn Met Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Phe Cys Arg

85 90 95

Ala Gly Ser Arg Ile Tyr Pro Tyr Asp Tyr Trp Gly Arg Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 119

<211> 117

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 119

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Ser Val His Leu Leu Asn

20 25 30

Phe Leu Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Met Val

35 40 45

Thr His Ile Thr Ile Ala Asp Ala Thr Asp Tyr Ser His Phe Ala Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala Lys Asn Met Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Phe Cys Arg

85 90 95

Ala Gly Ser Arg Ile Tyr Pro Tyr Asp Tyr Trp Gly Gln Gly Thr Gln

100 105 110

Val Thr Val Ser Ser

115

<210> 120

<211> 117

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 120

Glu Val Gln Leu Val Glu Ser Gly Gly Asp Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Asp Val His Lys Ile Asn

20 25 30

Phe Leu Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Met Val

35 40 45

Ala His Ile Thr Ile Ala Asp Ala Thr Asp Tyr Ala Glu Phe Ala Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Glu Pro Lys Asn Met Val His Leu

65 70 75 80

Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Leu Cys Arg

85 90 95

Ala Gly Ser Arg Ile Tyr Pro Tyr Asn Tyr Trp Gly Gln Gly Thr Gln

100 105 110

Val Thr Val Ser Ser

115

<210> 121

<211> 117

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 121

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Asp Val His Lys Ile Asn

20 25 30

Ile Leu Gly Trp Tyr Arg Gln Ala Pro Ala Lys Glu Arg Glu Met Val

35 40 45

Ala His Ile Thr Ile Gly Asp Ala Thr Asp Tyr Ala Asp Ser Ala Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala Lys Asn Met Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Phe Cys Arg

85 90 95

Ala Tyr Ser Arg Ile Tyr Pro Tyr Asn Tyr Trp Gly Arg Gly Thr Gln

100 105 110

Val Thr Val Ser Ser

115

<210> 122

<211> 117

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 122

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Asp Val His Lys Ile Asn

20 25 30

Ile Leu Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Lys Val

35 40 45

Ala Arg Ile Ser Ile Ser Asp Gln Thr Asp Tyr Ala Glu Ser Ala Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Glu Ser Lys Asn Met Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Leu Cys Arg

85 90 95

Ala Phe Ser Arg Ile Tyr Pro Tyr Asp Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 123

<211> 117

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 123

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Glu Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Glu Val Tyr Lys Ile Asn

20 25 30

Phe Leu Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Lys Val

35 40 45

Ala His Ile Thr Ile Ala Asp Val Ala Asp Tyr Ala Asp Phe Ala Lys

50 55 60

Gly Arg Leu Thr Ile Ser Arg Asp Glu Ala Lys Asn Met Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Phe Cys Arg

85 90 95

Ala Gly Ser Arg Ile Trp Pro Tyr Asp Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 124

<211> 117

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 124

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Lys Leu Pro Cys Ala Ala Ser Gly Ala Val His Lys Ile Asn

20 25 30

Phe Leu Gly Trp Tyr Arg Gln Thr Pro Glu Lys Glu Arg Glu Met Val

35 40 45

Ala Thr Ile Thr Ile Gly Asp Glu Val Asp Tyr Ala Asp Ser Ala Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala Lys Asn Met Val Tyr Leu

65 70 75 80

Gln Met Thr Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Val Cys Arg

85 90 95

Ala Gly Ser Arg Leu Tyr Pro Tyr Asn Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 125

<211> 117

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 125

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Gly Leu Ser Cys Ala Ala Ser Gly Ser Val His Leu Leu Asn

20 25 30

Phe Leu Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Met Val

35 40 45

Ala His Ile Thr Ile Ala Asp Ala Thr Asp Tyr Ala His Phe Ala Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala Lys Asn Met Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Phe Cys Arg

85 90 95

Ala Gly Ser Arg Ile Tyr Pro Tyr Asp Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 126

<211> 117

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 126

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Ala Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Asp Val His Lys Ile Asn

20 25 30

Ile Leu Gly Trp Tyr Arg Gln Ala Pro Ala Lys Glu Arg Glu Met Val

35 40 45

Ala His Ile Thr Ile Gly Asp Ala Thr Asp Tyr Ala Asp Ser Ala Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala Lys Asn Met Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Phe Cys Arg

85 90 95

Ala Tyr Ser Arg Ile Tyr Pro Tyr Asn Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 127

<211> 117

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 127

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Glu Val Tyr Lys Ile Asn

20 25 30

Phe Leu Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Lys Val

35 40 45

Ala His Ile Thr Ile Ala Asp Ala Ala Asp Tyr Ala Asp Phe Ala Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Gly Ala Lys Asn Met Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Phe Cys Arg

85 90 95

Ala Gly Ser Arg Ile Trp Pro Tyr Asp Tyr Trp Gly Gln Gly Thr Gln

100 105 110

Val Thr Val Ser Ser

115

<210> 128

<211> 117

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 128

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Trp Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Val Tyr Lys Ile Asn

20 25 30

Phe Leu Ser Trp Tyr Arg Gln Ala Pro Gly His Glu Arg Glu Leu Val

35 40 45

Ala Thr Ile Thr Ile Gly Asp Ala Ala Asp Tyr Ala Asp Ser Ala Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala Arg Asn Met Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Leu Tyr Phe Cys His

85 90 95

Ala Gly Ser Arg Leu Tyr Pro Tyr Asn Tyr Trp Gly Gln Gly Thr Gln

100 105 110

Val Thr Val Ser Ser

115

<210> 129

<211> 117

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 129

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Val His Leu Leu Asn

20 25 30

Phe Leu Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Met Val

35 40 45

Ala His Ile Thr Ile Ala Asp Ala Thr Asp Tyr Ser His Phe Ala Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala Lys Asn Met Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Phe Cys Arg

85 90 95

Ala Gly Ser Arg Ile Tyr Pro Tyr Asp Tyr Trp Gly Gln Gly Thr Gln

100 105 110

Val Thr Val Ser Ser

115

<210> 130

<211> 117

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 130

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Asp Val His Lys Ile Asn

20 25 30

Ile Leu Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Lys Val

35 40 45

Ala His Ile Ala Ile Ser Asp Gln Thr Asp Tyr Ala Glu Ser Ala Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Glu Ser Lys Asn Met Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Leu Cys Arg

85 90 95

Ala Phe Ser Arg Ile Tyr Pro Tyr Asp Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 131

<211> 117

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 131

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Asp Val His Lys Ile Asn

20 25 30

Ile Leu Gly Trp Tyr Arg Gln Ala Pro Ala Lys Glu Arg Glu Met Val

35 40 45

Ala His Ile Thr Ile Gly Asp Ala Thr Asp Tyr Ala Asp Ser Ala Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala Lys Asn Met Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Ala Tyr Phe Cys Arg

85 90 95

Ala Tyr Ser Arg Ile Tyr Pro Tyr Asn Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 132

<211> 117

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 132

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Asp Val His Lys Ile Asn

20 25 30

Ile Leu Gly Trp Tyr Arg Gln Ala Pro Ala Lys Glu Arg Gly Met Val

35 40 45

Ala His Ile Thr Ile Gly Asp Ala Thr Asp Tyr Ala Asp Ser Ala Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala Lys Asn Met Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Phe Cys Arg

85 90 95

Ala Tyr Ser Arg Ile Tyr Pro Tyr Asn Tyr Trp Gly Gln Gly Thr Gln

100 105 110

Val Thr Val Ser Ser

115

<210> 133

<211> 117

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 133

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Asp Val His Lys Ile Asn

20 25 30

Ile Leu Gly Trp Tyr Arg Gln Ala Pro Ala Lys Glu His Glu Met Val

35 40 45

Ala His Ile Thr Ile Gly Asp Ala Thr Asp Tyr Ala Asp Ser Ala Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala Lys Asn Met Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Phe Cys Arg

85 90 95

Ala Tyr Ser Arg Ile Tyr Pro Tyr Asn Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 134

<211> 117

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 134

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Asp Val His Lys Ile Asn

20 25 30

Ile Leu Gly Trp Tyr Arg Gln Ala Pro Ala Arg Glu Arg Glu Met Val

35 40 45

Ala His Ile Thr Ile Gly Asp Ala Thr Asp Tyr Ala Asp Ser Ala Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala Lys Asn Met Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Phe Cys Arg

85 90 95

Ala Tyr Ser Arg Ile Tyr Pro Tyr Asn Tyr Trp Gly Gln Gly Thr Gln

100 105 110

Val Thr Val Ser Ser

115

<210> 135

<211> 117

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 135

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Asp Val His Lys Ile Asn

20 25 30

Ile Leu Gly Trp Tyr Arg Gln Ala Pro Ala Lys Glu Arg Glu Met Val

35 40 45

Ala His Ile Thr Ile Gly Asp Ala Thr Asp Tyr Ala Asp Ser Ala Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala Glu Asn Met Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Phe Cys Arg

85 90 95

Ala Tyr Ser Arg Ile Tyr Pro Tyr Asn Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 136

<211> 117

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 136

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Ala Val His Lys Ile Asn

20 25 30

Phe Leu Gly Trp Tyr Arg Gln Thr Pro Glu Lys Glu Arg Glu Met Val

35 40 45

Ala Thr Ile Thr Ile Gly Asp Glu Val Asp Tyr Ala Asp Ser Ala Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala Lys Asn Met Val Tyr Leu

65 70 75 80

Gln Met Thr Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Val Cys Arg

85 90 95

Ala Gly Ser Arg Leu Tyr Pro Tyr Asn Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 137

<211> 117

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 137

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Ala Val His Lys Ile Asn

20 25 30

Phe Leu Gly Trp Tyr Arg Gln Ala Pro Glu Lys Glu Arg Glu Met Val

35 40 45

Ala Thr Ile Thr Ile Gly Asp Glu Val Asp Tyr Ala Asp Ser Ala Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala Thr Asn Met Val Tyr Leu

65 70 75 80

Gln Met Thr Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Phe Cys Arg

85 90 95

Ala Gly Ser Arg Ile Tyr Pro Tyr Asn Tyr Trp Gly Gln Gly Thr Gln

100 105 110

Val Thr Val Ser Ser

115

<210> 138

<211> 117

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 138

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Asp Val His Lys Ile Asn

20 25 30

Ile Leu Gly Trp Tyr Arg Gln Ala Pro Ala Lys Glu Arg Glu Met Val

35 40 45

Ala His Ile Thr Ile Gly Asp Ala Thr Ser Tyr Ala Gly Ser Ala Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala Lys Asn Met Val Tyr Leu

65 70 75 80

Gln Leu Asn Asn Leu Lys Pro Glu Asp Thr Ala Val Tyr Phe Cys Arg

85 90 95

Ala Tyr Ser Arg Ile Tyr Pro Tyr Asn Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 139

<211> 117

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 139

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Asp Val His Lys Ile Asn

20 25 30

Ile Leu Gly Trp Tyr Arg Gln Ala Pro Ala Lys Glu Arg Glu Met Val

35 40 45

Ala His Ile Thr Ile Gly Asp Ala Thr Ser Tyr Ala Asp Ser Ala Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala Lys Asn Met Val Tyr Leu

65 70 75 80

Gln Leu Asn Asn Leu Lys Pro Glu Asp Thr Ala Val Tyr Phe Cys Arg

85 90 95

Ala Tyr Ser Arg Ile Tyr Pro Tyr Asn Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 140

<211> 117

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 140

Glu Val Gln Leu Val Glu Ser Gly Gly Asp Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Asp Val His Lys Ile Asn

20 25 30

Phe Leu Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Met Val

35 40 45

Ala His Ile Thr Ile Ala Asp Ala Thr Asp Tyr Ala Glu Phe Ala Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Glu Pro Lys Asn Met Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Leu Cys Arg

85 90 95

Ala Gly Ser Arg Ile Tyr Pro Tyr Asn Tyr Trp Gly Gln Gly Thr Gln

100 105 110

Val Thr Val Ser Ser

115

<210> 141

<211> 117

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 141

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Ala Val His Lys Ile Asn

20 25 30

Phe Leu Gly Trp Tyr Arg Gln Thr Pro Glu Lys Glu Arg Glu Met Val

35 40 45

Ala Thr Ile Thr Ile Gly Asp Glu Val Asp Tyr Ala His Ser Ala Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala Lys Asn Met Val Tyr Leu

65 70 75 80

Gln Met Thr Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Val Cys Arg

85 90 95

Ala Gly Ser Arg Leu Tyr Pro Tyr Asn Tyr Trp Gly Gln Gly Thr Gln

100 105 110

Val Thr Val Ser Ser

115

<210> 142

<211> 117

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 142

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Ala Val His Lys Ile Asn

20 25 30

Phe Leu Gly Trp Tyr Arg Gln Thr Pro Glu Lys Glu Arg Glu Met Val

35 40 45

Ala Thr Ile Thr Ile Gly Asp Glu Val Ala Tyr Ala Asp Ser Ala Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala Lys Asn Met Val Tyr Leu

65 70 75 80

Gln Met Thr Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Val Cys Arg

85 90 95

Ala Gly Ser Arg Leu Tyr Pro Tyr Asn Tyr Trp Gly Gln Gly Thr Gln

100 105 110

Val Thr Val Ser Ser

115

<210> 143

<211> 117

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 143

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Glu Val Tyr Lys Ile Asn

20 25 30

Phe Leu Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Lys Val

35 40 45

Ala His Ile Thr Ile Ala Asp Ala Ala Asp Tyr Ala Asp Phe Ala Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala Lys Asn Met Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Phe Cys Arg

85 90 95

Ala Gly Ser Arg Ile Trp Pro Tyr Asp Tyr Trp Gly Gln Gly Thr Gln

100 105 110

Val Thr Val Ser Ser

115

<210> 144

<211> 117

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 144

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Glu Val Tyr Lys Ile Asn

20 25 30

Phe Leu Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Lys Val

35 40 45

Ala His Ile Thr Ile Ala Asp Ala Ala Asp Tyr Ala Asp Phe Ala Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala Lys Asn Met Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Phe Cys Arg

85 90 95

Ala Gly Ser Arg Ile Tyr Pro Tyr Asp Tyr Trp Gly Gln Gly Thr Gln

100 105 110

Val Thr Val Ser Ser

115

<210> 145

<211> 117

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 145

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Pro Ser Cys Ala Ala Ser Gly Ser Val His Lys Ile Asn

20 25 30

Phe Leu Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Met Val

35 40 45

Ala Thr Ile Thr Ile Gly Asp Ala Thr Asp Tyr Ala Asp Ser Ala Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala Lys Asn Met Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Ser Pro Glu Asp Thr Ala Val Tyr Phe Cys Arg

85 90 95

Ala Gly Ser Arg Leu Tyr Pro Tyr Asn Tyr Trp Gly Gln Gly Thr Gln

100 105 110

Val Thr Val Ser Ser

115

<210> 146

<211> 117

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 146

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Glu Val Tyr Lys Ile Asn

20 25 30

Phe Leu Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Lys Val

35 40 45

Ala His Ile Thr Ile Ala Asp Val Ala Asp Tyr Ala Asp Phe Ala Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Glu Val Lys Asn Met Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Phe Cys Arg

85 90 95

Ala Gly Ser Arg Ile Trp Pro Tyr Asp Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 147

<211> 117

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 147

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Glu

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Asp Val His Lys Ile Asn

20 25 30

Ile Leu Gly Trp Tyr Arg Gln Ala Pro Ala Lys Glu Arg Glu Met Val

35 40 45

Ala His Ile Thr Ile Gly Asp Ala Thr Asp Tyr Ala Asp Ser Ala Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala Lys Asn Met Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Phe Cys Arg

85 90 95

Ala Tyr Ser Arg Ile Tyr Pro Tyr Asn Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 148

<211> 117

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 148

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Glu Val Tyr Lys Ile Asn

20 25 30

Phe Leu Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Lys Val

35 40 45

Ala His Ile Thr Ile Ala Asp Ala Ala Asp Tyr Ala Asp Phe Ala Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala Lys Asn Met Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Phe Cys Arg

85 90 95

Ala Gly Ser Arg Ile Trp Pro Tyr Asp Tyr Trp Gly Gln Gly Thr Gln

100 105 110

Val Thr Val Ser Ser

115

<210> 149

<211> 117

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 149

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Val His Leu Leu Asn

20 25 30

Phe Leu Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Met Val

35 40 45

Ala His Ile Thr Ile Ala Asp Val Thr Asp Tyr Ser Tyr Phe Ala Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala Lys Asn Met Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Phe Cys Arg

85 90 95

Ala Gly Ser Arg Ile Tyr Pro Tyr Asp Tyr Trp Gly Gln Gly Thr Gln

100 105 110

Val Thr Val Ser Ser

115

<210> 150

<211> 117

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 150

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Asp Val His Lys Ile Asn

20 25 30

Ile Leu Gly Trp Tyr Arg Gln Ala Pro Ala Lys Glu Arg Glu Met Val

35 40 45

Ala His Ile Thr Ile Gly Asp Ala Thr Asp Tyr Ala Asp Ser Ala Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala Lys Asn Val Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Phe Cys Arg

85 90 95

Ala Tyr Ser Arg Ile Tyr Pro Tyr Asn Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 151

<211> 117

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 151

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Val His Lys Ile Asn

20 25 30

Phe Leu Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Gly Met Val

35 40 45

Ala Thr Ile Thr Ile Gly Asp Ala Thr Asp Tyr Ala Asp Ser Ala Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala Lys Asn Met Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Ser Pro Glu Asp Thr Ala Val Tyr Phe Cys Arg

85 90 95

Ala Gly Ser Arg Leu Tyr Pro Tyr Asn Tyr Trp Gly Gln Gly Thr Gln

100 105 110

Val Thr Val Ser Ser

115

<210> 152

<211> 117

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 152

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Ala Val His Lys Ile Asn

20 25 30

Phe Leu Gly Trp Tyr Arg Gln Thr Pro Glu Lys Glu Arg Glu Met Val

35 40 45

Ala Thr Ile Thr Ile Gly Asp Glu Val Asp Tyr Glu Asp Ser Ala Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala Lys Asn Met Val Tyr Leu

65 70 75 80

Gln Met Thr Gly Leu Lys Pro Glu Asp Thr Ala Val Tyr Val Cys Arg

85 90 95

Ala Gly Ser Arg Leu Tyr Pro Tyr Asn Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 153

<211> 117

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 153

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Arg Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Asp Val His Lys Ile Asn

20 25 30

Ile Leu Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Lys Val

35 40 45

Ala His Ile Ser Ile Ser Asp Gln Thr Asp Tyr Ala Glu Ser Ala Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Glu Ser Lys Asn Met Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Leu Cys Arg

85 90 95

Ala Phe Ser Arg Ile Tyr Pro Tyr Asp Tyr Trp Gly Gln Gly Thr Gln

100 105 110

Val Thr Val Ser Ser

115

<210> 154

<211> 117

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 154

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Asp Val Tyr Lys Ile Asn

20 25 30

Phe Leu Gly Trp His Arg Gln Ala Pro Gly Lys Glu Arg Glu Lys Val

35 40 45

Ala His Ile Thr Ile Gly Asp Ala Thr Asp Tyr Ala Asp Ser Ala Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala Lys Asn Met Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Phe Cys Arg

85 90 95

Ala Gly Ser Arg Ile Tyr Pro Tyr Asp Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 155

<211> 117

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 155

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Arg Ala Ser Gly Asp Val His Lys Ile Asn

20 25 30

Ile Leu Gly Trp Tyr Arg Gln Ala Pro Ala Lys Glu Arg Glu Met Ile

35 40 45

Ala His Ile Thr Ile Gly Asp Ala Thr Asp Tyr Ala Glu Ser Ala Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala Lys Asn Met Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Phe Cys Arg

85 90 95

Ala Tyr Ser Arg Ile Tyr Pro Tyr Asn Tyr Trp Gly Gln Gly Thr Gln

100 105 110

Val Thr Val Ser Ser

115

<210> 156

<211> 117

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 156

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Asp Val His Lys Ile Asn

20 25 30

Ile Leu Gly Trp Tyr Arg Gln Ala Pro Ala Lys Gly Arg Glu Met Val

35 40 45

Ala His Ile Thr Ile Gly Asp Ala Thr Asp Tyr Ala Asp Ser Ala Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala Lys Asn Met Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Phe Cys Arg

85 90 95

Ala Tyr Ser Arg Ile Tyr Pro Tyr Asn Tyr Trp Gly Gln Gly Thr Gln

100 105 110

Val Thr Val Ser Ser

115

<210> 157

<211> 117

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 157

Glu Val Gln Leu Val Glu Ser Gly Gly Asp Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Asp Val His Lys Ile Asn

20 25 30

Phe Leu Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Met Val

35 40 45

Ala His Ile Thr Ile Ala Asp Ala Thr Asp Tyr Ala Glu Phe Ala Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Glu Pro Lys Asn Met Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Leu Cys Arg

85 90 95

Ala Gly Ser Arg Ile Tyr Pro Tyr Ser Tyr Trp Gly Gln Gly Thr Gln

100 105 110

Val Thr Val Ser Ser

115

<210> 158

<211> 117

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 158

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Met Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Asp Val His Lys Ile Asn

20 25 30

Ile Leu Gly Trp Tyr Arg Gln Ala Pro Ala Lys Glu Arg Glu Met Val

35 40 45

Ala His Ile Thr Ile Gly Asp Ala Thr Asp Tyr Ala Asp Ser Ala Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala Lys Asn Met Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Phe Cys Arg

85 90 95

Ala Tyr Ser Arg Ile Tyr Pro Tyr Asn Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 159

<211> 117

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 159

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Asp Val His Lys Ile Asn

20 25 30

Ile Leu Gly Trp Tyr Arg Gln Val Pro Ala Lys Glu Arg Glu Met Val

35 40 45

Ala His Ile Thr Ile Gly Asp Ala Thr Asp Tyr Ala Asp Ser Ala Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala Lys Asn Met Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Ala Tyr Phe Cys Arg

85 90 95

Ala Tyr Ser Arg Ile Tyr Pro Tyr Asn Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 160

<211> 117

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 160

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Glu Val Tyr Lys Ile Asn

20 25 30

Phe Leu Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Lys Val

35 40 45

Ala His Ile Thr Ile Ala Asp Ala Ala Asp Tyr Ala Asp Phe Ala Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala Lys Asn Met Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Phe Cys Arg

85 90 95

Ala Gly Ser Arg Ile Trp Pro Tyr Asp Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 161

<211> 117

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 161

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Val His Leu Leu Asn

20 25 30

Phe Leu Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Met Val

35 40 45

Ala His Ile Thr Ile Ala Asp Ala Thr Asp Tyr Ser His Phe Ala Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala Lys Asn Met Val Tyr Leu

65 70 75 80

Gln Met Asn Gly Leu Arg Pro Glu Asp Thr Ala Val Tyr Phe Cys Arg

85 90 95

Ala Gly Ser Arg Ile Tyr Pro Tyr Asp Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 162

<211> 117

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 162

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Asp Val His Lys Ile Asn

20 25 30

Phe Leu Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Met Val

35 40 45

Ala His Ile Thr Ile Ala Asp Ala Thr Asp Tyr Ala Glu Phe Ala Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Glu Pro Lys Asn Met Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Leu Cys Arg

85 90 95

Ala Gly Ser Arg Ile Tyr Pro Tyr Asn Tyr Trp Gly Gln Gly Thr Gln

100 105 110

Val Thr Val Ser Ser

115

<210> 163

<211> 117

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 163

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Glu Val His Lys Ile Asn

20 25 30

Ile Leu Gly Trp Tyr Arg Gln Cys Pro Gly Lys Glu Arg Asp Met Val

35 40 45

Ala Thr Ile Thr Ile Gly Asp Ala Thr Asp Tyr Ala Asp Ser Ala Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala Lys Asn Met Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Phe Cys Arg

85 90 95

Ala Leu Ser Arg Leu Tyr Pro Tyr Asn Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 164

<211> 117

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 164

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Asp Val His Lys Ile Asn

20 25 30

Ile Leu Gly Trp Tyr Arg Gln Ala Pro Ala Lys Glu Arg Glu Met Val

35 40 45

Ala His Ile Thr Ile Gly Asp Ala Thr Asp Tyr Ala Asp Ser Ala Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala Lys Asn Met Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Leu Cys Arg

85 90 95

Ala Tyr Ser Arg Ile Tyr Pro Tyr Asn Tyr Trp Gly Gln Gly Thr Gln

100 105 110

Val Thr Val Ser Ser

115

<210> 165

<211> 117

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 165

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Glu Val Tyr Lys Ile Asn

20 25 30

Phe Leu Gly Trp Gln Arg Gln Ala Pro Gly Lys Glu Arg Glu Lys Val

35 40 45

Ala His Ile Thr Ile Ala Asp Val Ala Asp Tyr Ala Asp Phe Ala Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala Lys Asn Met Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Phe Cys Arg

85 90 95

Ala Gly Ser Arg Ile Trp Pro Tyr Asp Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 166

<211> 117

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 166

Glu Val Gln Leu Val Glu Ser Gly Gly Asp Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Asp Val His Lys Ile Asn

20 25 30

Phe Leu Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Met Val

35 40 45

Ala His Ile Thr Ile Ala Asp Ala Thr Asp Tyr Ala Glu Phe Ala Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Glu Pro Lys Asn Met Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Lys Pro Val Asp Thr Ala Val Tyr Leu Cys Arg

85 90 95

Ala Gly Ser Arg Ile Tyr Pro Tyr Asn Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 167

<211> 117

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 167

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Val His Leu Leu Asn

20 25 30

Phe Leu Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Lys Val

35 40 45

Ala His Ile Thr Ile Ala Asp Ala Thr Asp Tyr Ser His Phe Ala Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala Lys Asn Met Val Tyr Leu

65 70 75 80

Gln Met Asn Asn Leu Arg Pro Glu Asp Thr Ala Val Tyr Phe Cys Arg

85 90 95

Ala Gly Ser Arg Ile Tyr Pro Tyr Asp Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 168

<211> 117

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 168

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Pro Arg Leu Ser Cys Val Ala Ser Gly Asp Val His Lys Ile Asn

20 25 30

Phe Leu Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Lys Val

35 40 45

Ala His Ile Thr Ile Gly Asp Gln Ala Asp Tyr Ala Asp Ser Ala Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala Lys Asn Met Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Phe Cys Arg

85 90 95

Ala Gly Ser Arg Ile Tyr Pro Tyr Asp Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 169

<211> 117

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 169

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Asp Val His Lys Ile Asn

20 25 30

Ile Leu Gly Trp Tyr Arg Gln Ala Pro Ala Lys Glu Arg Glu Met Val

35 40 45

Ala His Ile Thr Ile Gly Asp Ala Thr Asp Tyr Ala Asp Ser Ala Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala Lys Asn Met Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Lys Pro Glu Asp Ala Ala Val Tyr Phe Cys Arg

85 90 95

Ala Tyr Ser Arg Ile Tyr Pro Tyr Asn Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 170

<211> 117

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 170

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Glu Val Tyr Lys Ile Asn

20 25 30

Phe Leu Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Lys Val

35 40 45

Ala His Ile Thr Ile Ala Asp Val Ala Asp Tyr Ala Asp Phe Ala Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Gly Ala Lys Asn Met Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Phe Cys Arg

85 90 95

Ala Gly Ser Arg Ile Trp Pro Tyr Asp Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 171

<211> 117

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 171

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Val His Lys Ile Asn

20 25 30

Phe Leu Gly Trp Tyr Cys Gln Ala Pro Gly Lys Glu Arg Glu Met Val

35 40 45

Ala Thr Ile Thr Ile Gly Asp Ala Thr Asp Tyr Ala Asp Ser Ala Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala Lys Asn Met Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Ser Pro Glu Asp Thr Ala Val Tyr Phe Cys Arg

85 90 95

Ala Gly Ser Arg Leu Tyr Pro Tyr Asn Tyr Trp Gly Gln Gly Thr Gln

100 105 110

Val Thr Val Ser Ser

115

<210> 172

<211> 117

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 172

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Asp Val His Lys Ile Asn

20 25 30

Ile Leu Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Lys Val

35 40 45

Ala His Ile Ser Ile Ser Asp Gln Thr Asp Tyr Ala Glu Ser Ala Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Glu Ser Lys Asn Met Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Lys Pro Glu Asp Ala Ala Val Tyr Leu Cys Arg

85 90 95

Ala Phe Ser Arg Ile Tyr Pro Tyr Asp Tyr Trp Gly Gln Gly Thr Gln

100 105 110

Val Thr Val Ser Ser

115

<210> 173

<211> 117

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 173

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Pro Ser Cys Ala Ala Ser Gly Glu Val Tyr Lys Ile Asn

20 25 30

Phe Leu Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Lys Val

35 40 45

Ala His Ile Thr Ile Ala Asp Val Ala Asp Tyr Ala Asp Phe Ala Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala Lys Asn Met Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Phe Cys Arg

85 90 95

Ala Gly Ser Arg Ile Trp Pro Tyr Asp Tyr Trp Gly Gln Gly Thr Gln

100 105 110

Val Thr Val Ser Ser

115

<210> 174

<211> 117

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 174

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Pro Ser Cys Val Ala Ser Gly Asp Val His Lys Ile Asn

20 25 30

Phe Leu Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Lys Val

35 40 45

Ala His Ile Thr Ile Ala Asp Gln Ala Asp Tyr Ala Asp Ser Ala Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala Lys Asn Met Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Phe Cys Arg

85 90 95

Ala Gly Ser Arg Ile Tyr Pro Tyr Asp Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 175

<211> 117

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 175

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Glu Val His Lys Ile Asn

20 25 30

Ile Leu Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Asp Met Val

35 40 45

Ala Thr Ile Thr Ile Gly Asp Glu Thr Gln Tyr Ala Asp Ser Ala Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala Lys Asn Met Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Phe Cys Arg

85 90 95

Ala Leu Ser Arg Leu Tyr Pro Tyr Asn Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 176

<211> 117

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 176

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Asp Val His Lys Ile Asn

20 25 30

Ile Leu Gly Trp Tyr Arg Gln Ala Pro Ala Lys Glu Arg Glu Met Val

35 40 45

Ala His Ile Thr Ile Gly Asp Ala Thr Asp Tyr Ala Asp Ser Ala Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala Lys Asn Met Val Tyr Leu

65 70 75 80

Gln Met Asp Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Phe Cys Arg

85 90 95

Ala Tyr Ser Arg Ile Tyr Pro Tyr Asn Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 177

<211> 117

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 177

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Asp Val His Lys Ile Asn

20 25 30

Ile Leu Gly Trp Tyr Arg Gln Ala Pro Ala Lys Glu Arg Glu Met Val

35 40 45

Ala His Ile Thr Ile Gly Asp Ala Thr Asp Tyr Ala Asp Ser Ala Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala Lys Asn Met Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Lys Pro Gly Asp Thr Ala Val Tyr Phe Cys Arg

85 90 95

Ala Tyr Ser Arg Ile Tyr Pro Tyr Asn Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 178

<211> 117

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 178

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Asp Val His Lys Ile Asn

20 25 30

Ile Leu Gly Trp His Arg Gln Ala Pro Ala Lys Glu Arg Glu Met Val

35 40 45

Ala His Ile Thr Ile Gly Asp Ala Thr Asp Tyr Ala Asp Ser Ala Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala Lys Asn Met Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Phe Cys Arg

85 90 95

Ala Tyr Ser Arg Ile Tyr Pro Tyr Asn Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 179

<211> 117

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 179

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Asp Val His Lys Ile Asn

20 25 30

Ile Leu Gly Trp His Arg Gln Ala Pro Ala Lys Glu Arg Glu Met Val

35 40 45

Ala His Ile Thr Ile Gly Asp Ala Thr Asp Tyr Ala Asp Ser Ala Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala Lys Asn Met Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Ala Tyr Phe Cys Arg

85 90 95

Ala Tyr Ser Arg Ile Tyr Pro Tyr Asn Tyr Trp Gly Gln Gly Thr Gln

100 105 110

Val Thr Val Ser Ser

115

<210> 180

<211> 117

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 180

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Trp Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Asp Val His Lys Ile Asn

20 25 30

Ile Leu Gly Trp Tyr Arg Gln Ala Pro Ala Lys Glu Arg Glu Met Val

35 40 45

Ala His Ile Thr Ile Gly Asp Ala Thr Asp Tyr Ala Asp Ser Ala Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala Lys Asn Met Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Phe Cys Arg

85 90 95

Ala Tyr Ser Arg Ile Tyr Pro Tyr Asn Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 181

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR1

<400> 181

Gly Asp Val His Lys Ile Asn Phe Leu Gly

1 5 10

<210> 182

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR1

<400> 182

Gly Ser Val His Lys Ile Asn Phe Leu Gly

1 5 10

<210> 183

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR1

<400> 183

Gly Ser Val His Leu Leu Asn Phe Leu Gly

1 5 10

<210> 184

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR1

<400> 184

Gly Ala Val His Lys Ile Asn Phe Leu Gly

1 5 10

<210> 185

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR1

<400> 185

Gly Asp Val His Lys Ile Asn Ile Leu Gly

1 5 10

<210> 186

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR1

<400> 186

Gly Asp Val His Lys Ile Asn Val Leu Gly

1 5 10

<210> 187

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR1

<400> 187

Gly Glu Val Tyr Lys Ile Asn Phe Leu Gly

1 5 10

<210> 188

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR1

<400> 188

Gly Gly Val His Lys Ile Asn Ile Leu Gly

1 5 10

<210> 189

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR1

<400> 189

Gly Ser Val Tyr Lys Ile Asn Phe Leu Ser

1 5 10

<210> 190

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR1

<400> 190

Gly Asp Val Tyr Lys Ile Asn Phe Leu Gly

1 5 10

<210> 191

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR1

<400> 191

Gly Glu Val His Lys Ile Asn Ile Leu Gly

1 5 10

<210> 192

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR2

<400> 192

His Ile Ser Ile Gly Asp Gln Thr Asp

fifteen

<210> 193

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR2

<400> 193

Thr Ile Thr Ile Gly Asp Thr Thr Asp

fifteen

<210> 194

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR2

<400> 194

Thr Ile Thr Ile Gly Asp Ala Thr Asp

fifteen

<210> 195

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR2

<400> 195

His Ile Thr Ile Ala Asp Ala Thr Asp

fifteen

<210> 196

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR2

<400> 196

Thr Ile Thr Ile Gly Asp Asp Val Asp

fifteen

<210> 197

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR2

<400> 197

His Ile Thr Ile Gly Asp Ala Thr Asp

fifteen

<210> 198

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR2

<400> 198

His Ile Thr Ile Gly Asp Gln Ala Asp

fifteen

<210> 199

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR2

<400> 199

His Ile Ser Ile Ala Asp Ala Thr Asp

fifteen

<210> 200

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR2

<400> 200

His Ile Thr Ile Gly Asp Ala Thr Val

fifteen

<210> 201

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR2

<400> 201

His Ile Thr Ile Gly Asp Glu Val Asp

fifteen

<210> 202

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR2

<400> 202

His Ile Thr Ile Gly Asp Val Thr Asp

fifteen

<210> 203

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR2

<400> 203

His Ile Thr Ile Gly Asp Ala Thr Asn

fifteen

<210> 204

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR2

<400> 204

His Ile Thr Ile Ala Asp Val Ala Asp

fifteen

<210> 205

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR2

<400> 205

Thr Ile Thr Ile Gly Asp Glu Val Asp

fifteen

<210> 206

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR2

<400> 206

His Ile Ser Ile Ser Asp Gln Thr Asp

fifteen

<210> 207

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR2

<400> 207

His Ile Thr Ile Gly Asp Gln Thr Asp

fifteen

<210> 208

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR2

<400> 208

His Ile Thr Ile Gly Asp Thr Thr Asp

fifteen

<210> 209

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR2

<400> 209

Arg Ile Ser Ile Ser Asp Gln Thr Asp

fifteen

<210> 210

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR2

<400> 210

His Ile Thr Ile Ala Asp Ala Ala Asp

fifteen

<210> 211

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR2

<400> 211

Thr Ile Thr Ile Gly Asp Ala Ala Asp

fifteen

<210> 212

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR2

<400> 212

His Ile Ala Ile Ser Asp Gln Thr Asp

fifteen

<210> 213

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR2

<400> 213

His Ile Thr Ile Gly Asp Ala Thr Ser

fifteen

<210> 214

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR2

<400> 214

Thr Ile Thr Ile Gly Asp Glu Val Ala

fifteen

<210> 215

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR2

<400> 215

His Ile Thr Ile Ala Asp Val Thr Asp

fifteen

<210> 216

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR2

<400> 216

His Ile Thr Ile Ala Asp Gln Ala Asp

fifteen

<210> 217

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR2

<400> 217

Thr Ile Thr Ile Gly Asp Glu Thr Gln

fifteen

<210> 218

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR3

<400> 218

Phe Ser Arg Ile Tyr Pro Tyr Asp Tyr

fifteen

<210> 219

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR3

<400> 219

Gly Ser Arg Leu Tyr Pro Tyr Asn Tyr

fifteen

<210> 220

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR3

<400> 220

Gly Ser Arg Ile Tyr Pro Tyr Asp Tyr

fifteen

<210> 221

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR3

<400> 221

Tyr Ser Arg Ile Tyr Pro Tyr Asn Tyr

fifteen

<210> 222

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR3

<400> 222

Gly Ser Arg Ile Trp Pro Tyr Asp Tyr

fifteen

<210> 223

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR3

<400> 223

Gly Ser Arg Ile Tyr Pro Tyr Asn Tyr

fifteen

<210> 224

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR3

<400> 224

Gly Ser Arg Ile Tyr Pro Tyr Ser Tyr

fifteen

<210> 225

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR3

<400> 225

Leu Ser Arg Leu Tyr Pro Tyr Asn Tyr

fifteen

<210> 226

<211> 25

<212> PRT

<213> Artificial Sequence

<220>

<223> FR1

<400> 226

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Val Ala Ser

20 25

<210> 227

<211> 25

<212> PRT

<213> Artificial Sequence

<220>

<223> FR1

<400> 227

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser

20 25

<210> 228

<211> 25

<212> PRT

<213> Artificial Sequence

<220>

<223> FR1

<400> 228

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Pro Cys Ala Ala Ser

20 25

<210> 229

<211> 25

<212> PRT

<213> Artificial Sequence

<220>

<223> FR1

<400> 229

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Val Ser

20 25

<210> 230

<211> 25

<212> PRT

<213> Artificial Sequence

<220>

<223> FR1

<400> 230

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Lys Leu Ser Cys Ala Ala Ser

20 25

<210> 231

<211> 25

<212> PRT

<213> Artificial Sequence

<220>

<223> FR1

<400> 231

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Arg Ala Ser

20 25

<210> 232

<211> 25

<212> PRT

<213> Artificial Sequence

<220>

<223> FR1

<400> 232

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Arg Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser

20 25

<210> 233

<211> 25

<212> PRT

<213> Artificial Sequence

<220>

<223> FR1

<400> 233

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser

20 25

<210> 234

<211> 25

<212> PRT

<213> Artificial Sequence

<220>

<223> FR1

<400> 234

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Pro Ser Cys Ala Ala Ser

20 25

<210> 235

<211> 25

<212> PRT

<213> Artificial Sequence

<220>

<223> FR1

<400> 235

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val His Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser

20 25

<210> 236

<211> 25

<212> PRT

<213> Artificial Sequence

<220>

<223> FR1

<400> 236

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Ala Arg Leu Ser Cys Val Ala Ser

20 25

<210> 237

<211> 25

<212> PRT

<213> Artificial Sequence

<220>

<223> FR1

<400> 237

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Thr Leu Ser Cys Ala Ala Ser

20 25

<210> 238

<211> 25

<212> PRT

<213> Artificial Sequence

<220>

<223> FR1

<400> 238

Glu Val Gln Leu Val Glu Ser Gly Gly Asp Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser

20 25

<210> 239

<211> 25

<212> PRT

<213> Artificial Sequence

<220>

<223> FR1

<400> 239

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Glu Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser

20 25

<210> 240

<211> 25

<212> PRT

<213> Artificial Sequence

<220>

<223> FR1

<400> 240

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Lys Leu Pro Cys Ala Ala Ser

20 25

<210> 241

<211> 25

<212> PRT

<213> Artificial Sequence

<220>

<223> FR1

<400> 241

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Gly Leu Ser Cys Ala Ala Ser

20 25

<210> 242

<211> 25

<212> PRT

<213> Artificial Sequence

<220>

<223> FR1

<400> 242

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Ala Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser

20 25

<210> 243

<211> 25

<212> PRT

<213> Artificial Sequence

<220>

<223> FR1

<400> 243

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Trp Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser

20 25

<210> 244

<211> 25

<212> PRT

<213> Artificial Sequence

<220>

<223> FR1

<400> 244

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Pro Ser Cys Ala Ala Ser

20 25

<210> 245

<211> 25

<212> PRT

<213> Artificial Sequence

<220>

<223> FR1

<400> 245

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Glu

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser

20 25

<210> 246

<211> 25

<212> PRT

<213> Artificial Sequence

<220>

<223> FR1

<400> 246

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Arg Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Val Ala Ser

20 25

<210> 247

<211> 25

<212> PRT

<213> Artificial Sequence

<220>

<223> FR1

<400> 247

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Met Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser

20 25

<210> 248

<211> 25

<212> PRT

<213> Artificial Sequence

<220>

<223> FR1

<400> 248

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Pro Arg Leu Ser Cys Val Ala Ser

20 25

<210> 249

<211> 25

<212> PRT

<213> Artificial Sequence

<220>

<223> FR1

<400> 249

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Pro Ser Cys Val Ala Ser

20 25

<210> 250

<211> 25

<212> PRT

<213> Artificial Sequence

<220>

<223> FR1

<400> 250

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Trp Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser

20 25

<210> 251

<211> 14

<212> PRT

<213> Artificial Sequence

<220>

<223> FR2

<400> 251

Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Lys Val Ala

1 5 10

<210> 252

<211> 14

<212> PRT

<213> Artificial Sequence

<220>

<223> FR2

<400> 252

Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Leu Val Ala

1 5 10

<210> 253

<211> 14

<212> PRT

<213> Artificial Sequence

<220>

<223> FR2

<400> 253

Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Gly Leu Val Ala

1 5 10

<210> 254

<211> 14

<212> PRT

<213> Artificial Sequence

<220>

<223> FR2

<400> 254

Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Met Val Ala

1 5 10

<210> 255

<211> 14

<212> PRT

<213> Artificial Sequence

<220>

<223> FR2

<400> 255

Trp Tyr Arg Gln Thr Pro Glu Lys Glu Arg Glu Met Val Ala

1 5 10

<210> 256

<211> 14

<212> PRT

<213> Artificial Sequence

<220>

<223> FR2

<400> 256

Trp Tyr Arg Gln Ala Pro Ala Lys Glu Arg Glu Met Val Ala

1 5 10

<210> 257

<211> 14

<212> PRT

<213> Artificial Sequence

<220>

<223> FR2

<400> 257

Trp Tyr Arg Gln Cys Pro Gly Lys Glu Arg Glu Met Val Ala

1 5 10

<210> 258

<211> 14

<212> PRT

<213> Artificial Sequence

<220>

<223> FR2

<400> 258

Trp His Arg Gln Pro Pro Gly Lys Glu Arg Glu Lys Val Ala

1 5 10

<210> 259

<211> 14

<212> PRT

<213> Artificial Sequence

<220>

<223> FR2

<400> 259

Trp His Arg Gln Ala Pro Gly Lys Glu Arg Glu Lys Val Ala

1 5 10

<210> 260

<211> 14

<212> PRT

<213> Artificial Sequence

<220>

<223> FR2

<400> 260

Trp Tyr Arg Gln Ala Pro Glu Lys Glu Arg Glu Met Val Ala

1 5 10

<210> 261

<211> 14

<212> PRT

<213> Artificial Sequence

<220>

<223> FR2

<400> 261

Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Val Val Ala

1 5 10

<210> 262

<211> 14

<212> PRT

<213> Artificial Sequence

<220>

<223> FR2

<400> 262

Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Gly Val Val Ala

1 5 10

<210> 263

<211> 14

<212> PRT

<213> Artificial Sequence

<220>

<223> FR2

<400> 263

Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Met Val Thr

1 5 10

<210> 264

<211> 14

<212> PRT

<213> Artificial Sequence

<220>

<223> FR2

<400> 264

Trp Tyr Arg Gln Ala Pro Gly His Glu Arg Glu Leu Val Ala

1 5 10

<210> 265

<211> 14

<212> PRT

<213> Artificial Sequence

<220>

<223> FR2

<400> 265

Trp Tyr Arg Gln Ala Pro Ala Lys Glu Arg Gly Met Val Ala

1 5 10

<210> 266

<211> 14

<212> PRT

<213> Artificial Sequence

<220>

<223> FR2

<400> 266

Trp Tyr Arg Gln Ala Pro Ala Lys Glu His Glu Met Val Ala

1 5 10

<210> 267

<211> 14

<212> PRT

<213> Artificial Sequence

<220>

<223> FR2

<400> 267

Trp Tyr Arg Gln Ala Pro Ala Arg Glu Arg Glu Met Val Ala

1 5 10

<210> 268

<211> 14

<212> PRT

<213> Artificial Sequence

<220>

<223> FR2

<400> 268

Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Gly Met Val Ala

1 5 10

<210> 269

<211> 14

<212> PRT

<213> Artificial Sequence

<220>

<223> FR2

<400> 269

Trp Tyr Arg Gln Ala Pro Ala Lys Glu Arg Glu Met Ile Ala

1 5 10

<210> 270

<211> 14

<212> PRT

<213> Artificial Sequence

<220>

<223> FR2

<400> 270

Trp Tyr Arg Gln Ala Pro Ala Lys Gly Arg Glu Met Val Ala

1 5 10

<210> 271

<211> 14

<212> PRT

<213> Artificial Sequence

<220>

<223> FR2

<400> 271

Trp Tyr Arg Gln Val Pro Ala Lys Glu Arg Glu Met Val Ala

1 5 10

<210> 272

<211> 14

<212> PRT

<213> Artificial Sequence

<220>

<223> FR2

<400> 272

Trp Tyr Arg Gln Cys Pro Gly Lys Glu Arg Asp Met Val Ala

1 5 10

<210> 273

<211> 14

<212> PRT

<213> Artificial Sequence

<220>

<223> FR2

<400> 273

Trp Gln Arg Gln Ala Pro Gly Lys Glu Arg Glu Lys Val Ala

1 5 10

<210> 274

<211> 14

<212> PRT

<213> Artificial Sequence

<220>

<223> FR2

<400> 274

Trp Tyr Cys Gln Ala Pro Gly Lys Glu Arg Glu Met Val Ala

1 5 10

<210> 275

<211> 14

<212> PRT

<213> Artificial Sequence

<220>

<223> FR2

<400> 275

Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Asp Met Val Ala

1 5 10

<210> 276

<211> 14

<212> PRT

<213> Artificial Sequence

<220>

<223> FR2

<400> 276

Trp His Arg Gln Ala Pro Ala Lys Glu Arg Glu Met Val Ala

1 5 10

<210> 277

<211> 39

<212> PRT

<213> Artificial Sequence

<220>

<223> FR3

<400> 277

Tyr Ala Asp Ser Ala Lys Gly Arg Phe Thr Ile Ser Arg Asp Glu Ser

1 5 10 15

Lys Asn Met Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr

20 25 30

Ala Val Tyr Phe Cys Arg Ala

35

<210> 278

<211> 39

<212> PRT

<213> Artificial Sequence

<220>

<223> FR3

<400> 278

Tyr Ala Asp Ser Ala Lys Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala

1 5 10 15

Lys Asn Met Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr

20 25 30

Ala Val His Phe Cys Arg Ala

35

<210> 279

<211> 39

<212> PRT

<213> Artificial Sequence

<220>

<223> FR3

<400> 279

Tyr Ala Asp Tyr Ala Lys Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala

1 5 10 15

Arg Asn Met Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr

20 25 30

Ala Val Tyr Phe Cys Arg Ala

35

<210> 280

<211> 39

<212> PRT

<213> Artificial Sequence

<220>

<223> FR3

<400> 280

Tyr Ala Asp Ser Ala Lys Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala

1 5 10 15

Lys Asn Met Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr

20 25 30

Ala Val Tyr Phe Cys Arg Ala

35

<210> 281

<211> 39

<212> PRT

<213> Artificial Sequence

<220>

<223> FR3

<400> 281

Tyr Ser His Phe Ala Lys Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala

1 5 10 15

Lys Asn Met Val Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr

20 25 30

Ala Val Tyr Phe Cys Arg Ala

35

<210> 282

<211> 39

<212> PRT

<213> Artificial Sequence

<220>

<223> FR3

<400> 282

Tyr Ala Asp Ser Ala Lys Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala

1 5 10 15

Lys Asn Met Val Tyr Leu Gln Met Thr Ser Leu Lys Pro Glu Asp Thr

20 25 30

Ala Val Tyr Val Cys Arg Ala

35

<210> 283

<211> 39

<212> PRT

<213> Artificial Sequence

<220>

<223> FR3

<400> 283

Tyr Ala Glu Ser Ala Lys Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala

1 5 10 15

Lys Asn Met Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr

20 25 30

Ala Val Tyr Phe Cys Arg Ala

35

<210> 284

<211> 39

<212> PRT

<213> Artificial Sequence

<220>

<223> FR3

<400> 284

Tyr Ala Asp Ser Ala Lys Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala

1 5 10 15

Lys Asn Met Val Tyr Leu Gln Met Asn Ser Leu Ser Pro Glu Asp Thr

20 25 30

Ala Val Tyr Phe Cys Arg Ala

35

<210> 285

<211> 39

<212> PRT

<213> Artificial Sequence

<220>

<223> FR3

<400> 285

Tyr Ala His Phe Ala Lys Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala

1 5 10 15

Lys Asn Met Val Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr

20 25 30

Ala Val Tyr Phe Cys Arg Ala

35

<210> 286

<211> 39

<212> PRT

<213> Artificial Sequence

<220>

<223> FR3

<400> 286

Tyr Ala Asp Ser Ala Lys Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala

1 5 10 15

Lys Asn Met Val Tyr Leu Gln Met Thr Ser Leu Thr Pro Glu Asp Thr

20 25 30

Ala Val Tyr Val Cys Arg Ala

35

<210> 287

<211> 39

<212> PRT

<213> Artificial Sequence

<220>

<223> FR3

<400> 287

Tyr Ala Asp Ser Ala Lys Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala

1 5 10 15

Lys Asn Met Val Tyr Leu Gln Met Asn Asn Leu Lys Pro Glu Asp Thr

20 25 30

Ala Val Tyr Phe Cys Arg Ala

35

<210> 288

<211> 39

<212> PRT

<213> Artificial Sequence

<220>

<223> FR3

<400> 288

Tyr Ala Asp Ser Ala Lys Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala

1 5 10 15

Lys Asn Met Val His Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr

20 25 30

Ala Val Tyr Phe Cys Arg Ala

35

<210> 289

<211> 39

<212> PRT

<213> Artificial Sequence

<220>

<223> FR3

<400> 289

Tyr Ala Asp Ser Ala Lys Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala

1 5 10 15

Lys Asn Met Val Phe Leu Gln Met Asn Asn Leu Lys Pro Glu Asp Thr

20 25 30

Ala Val Tyr Phe Cys Arg Ala

35

<210> 290

<211> 39

<212> PRT

<213> Artificial Sequence

<220>

<223> FR3

<400> 290

Tyr Ala Gly Ser Ala Lys Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala

1 5 10 15

Lys Asn Met Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr

20 25 30

Ala Val Tyr Phe Cys Arg Ala

35

<210> 291

<211> 39

<212> PRT

<213> Artificial Sequence

<220>

<223> FR3

<400> 291

Tyr Ala Asp Phe Ala Lys Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala

1 5 10 15

Lys Asn Met Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr

20 25 30

Ala Val Tyr Phe Cys Arg Ala

35

<210> 292

<211> 39

<212> PRT

<213> Artificial Sequence

<220>

<223> FR3

<400> 292

Tyr Ala Asp Ser Ala Lys Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala

1 5 10 15

Lys Asn Met Val Tyr Leu Gln Met Thr Ser Leu Lys Pro Glu Asp Thr

20 25 30

Thr Val Tyr Val Cys Arg Ala

35

<210> 293

<211> 39

<212> PRT

<213> Artificial Sequence

<220>

<223> FR3

<400> 293

Tyr Ala Glu Ser Ala Lys Gly Arg Phe Thr Ile Ser Arg Asp Glu Ser

1 5 10 15

Lys Asn Met Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr

20 25 30

Ala Val Tyr Leu Cys Arg Ala

35

<210> 294

<211> 39

<212> PRT

<213> Artificial Sequence

<220>

<223> FR3

<400> 294

Tyr Ala Asp Ser Ala Lys Gly Arg Phe Ala Ile Ser Arg Asp Glu Ala

1 5 10 15

Lys Asn Met Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr

20 25 30

Ala Val Tyr Phe Cys Arg Ala

35

<210> 295

<211> 39

<212> PRT

<213> Artificial Sequence

<220>

<223> FR3

<400> 295

Tyr Ala Asp Phe Ala Gln Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala

1 5 10 15

Lys Asn Met Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr

20 25 30

Ala Val Tyr Phe Cys Arg Ala

35

<210> 296

<211> 39

<212> PRT

<213> Artificial Sequence

<220>

<223> FR3

<400> 296

Tyr Ala Glu Phe Ala Lys Gly Arg Phe Thr Ile Ser Arg Asp Glu Pro

1 5 10 15

Lys Asn Met Val His Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr

20 25 30

Ala Val Tyr Leu Cys Arg Ala

35

<210> 297

<211> 39

<212> PRT

<213> Artificial Sequence

<220>

<223> FR3

<400> 297

Tyr Ala Asp Phe Ala Lys Gly Arg Leu Thr Ile Ser Arg Asp Glu Ala

1 5 10 15

Lys Asn Met Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr

20 25 30

Ala Val Tyr Phe Cys Arg Ala

35

<210> 298

<211> 39

<212> PRT

<213> Artificial Sequence

<220>

<223> FR3

<400> 298

Tyr Ala Asp Phe Ala Lys Gly Arg Phe Thr Ile Ser Arg Asp Gly Ala

1 5 10 15

Lys Asn Met Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr

20 25 30

Ala Val Tyr Phe Cys Arg Ala

35

<210> 299

<211> 39

<212> PRT

<213> Artificial Sequence

<220>

<223> FR3

<400> 299

Tyr Ala Asp Ser Ala Lys Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala

1 5 10 15

Arg Asn Met Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr

20 25 30

Ala Leu Tyr Phe Cys His Ala

35

<210> 300

<211> 39

<212> PRT

<213> Artificial Sequence

<220>

<223> FR3

<400> 300

Tyr Ala Asp Ser Ala Lys Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala

1 5 10 15

Lys Asn Met Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr

20 25 30

Ala Ala Tyr Phe Cys Arg Ala

35

<210> 301

<211> 39

<212> PRT

<213> Artificial Sequence

<220>

<223> FR3

<400> 301

Tyr Ala Asp Ser Ala Lys Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala

1 5 10 15

Glu Asn Met Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr

20 25 30

Ala Val Tyr Phe Cys Arg Ala

35

<210> 302

<211> 39

<212> PRT

<213> Artificial Sequence

<220>

<223> FR3

<400> 302

Tyr Ala Asp Ser Ala Lys Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala

1 5 10 15

Thr Asn Met Val Tyr Leu Gln Met Thr Ser Leu Lys Pro Glu Asp Thr

20 25 30

Ala Val Tyr Phe Cys Arg Ala

35

<210> 303

<211> 39

<212> PRT

<213> Artificial Sequence

<220>

<223> FR3

<400> 303

Tyr Ala Gly Ser Ala Lys Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala

1 5 10 15

Lys Asn Met Val Tyr Leu Gln Leu Asn Asn Leu Lys Pro Glu Asp Thr

20 25 30

Ala Val Tyr Phe Cys Arg Ala

35

<210> 304

<211> 39

<212> PRT

<213> Artificial Sequence

<220>

<223> FR3

<400> 304

Tyr Ala Asp Ser Ala Lys Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala

1 5 10 15

Lys Asn Met Val Tyr Leu Gln Leu Asn Asn Leu Lys Pro Glu Asp Thr

20 25 30

Ala Val Tyr Phe Cys Arg Ala

35

<210> 305

<211> 39

<212> PRT

<213> Artificial Sequence

<220>

<223> FR3

<400> 305

Tyr Ala Glu Phe Ala Lys Gly Arg Phe Thr Ile Ser Arg Asp Glu Pro

1 5 10 15

Lys Asn Met Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr

20 25 30

Ala Val Tyr Leu Cys Arg Ala

35

<210> 306

<211> 39

<212> PRT

<213> Artificial Sequence

<220>

<223> FR3

<400> 306

Tyr Ala His Ser Ala Lys Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala

1 5 10 15

Lys Asn Met Val Tyr Leu Gln Met Thr Ser Leu Lys Pro Glu Asp Thr

20 25 30

Ala Val Tyr Val Cys Arg Ala

35

<210> 307

<211> 39

<212> PRT

<213> Artificial Sequence

<220>

<223> FR3

<400> 307

Tyr Ala Asp Phe Ala Lys Gly Arg Phe Thr Ile Ser Arg Asp Glu Val

1 5 10 15

Lys Asn Met Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr

20 25 30

Ala Val Tyr Phe Cys Arg Ala

35

<210> 308

<211> 39

<212> PRT

<213> Artificial Sequence

<220>

<223> FR3

<400> 308

Tyr Ala Asp Phe Ala Lys Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala

1 5 10 15

Lys Asn Met Val Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr

20 25 30

Ala Val Tyr Phe Cys Arg Ala

35

<210> 309

<211> 39

<212> PRT

<213> Artificial Sequence

<220>

<223> FR3

<400> 309

Tyr Ser Tyr Phe Ala Lys Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala

1 5 10 15

Lys Asn Met Val Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr

20 25 30

Ala Val Tyr Phe Cys Arg Ala

35

<210> 310

<211> 39

<212> PRT

<213> Artificial Sequence

<220>

<223> FR3

<400> 310

Tyr Ala Asp Ser Ala Lys Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala

1 5 10 15

Lys Asn Val Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr

20 25 30

Ala Val Tyr Phe Cys Arg Ala

35

<210> 311

<211> 39

<212> PRT

<213> Artificial Sequence

<220>

<223> FR3

<400> 311

Tyr Glu Asp Ser Ala Lys Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala

1 5 10 15

Lys Asn Met Val Tyr Leu Gln Met Thr Gly Leu Lys Pro Glu Asp Thr

20 25 30

Ala Val Tyr Val Cys Arg Ala

35

<210> 312

<211> 39

<212> PRT

<213> Artificial Sequence

<220>

<223> FR3

<400> 312

Tyr Ser His Phe Ala Lys Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala

1 5 10 15

Lys Asn Met Val Tyr Leu Gln Met Asn Gly Leu Arg Pro Glu Asp Thr

20 25 30

Ala Val Tyr Phe Cys Arg Ala

35

<210> 313

<211> 39

<212> PRT

<213> Artificial Sequence

<220>

<223> FR3

<400> 313

Tyr Ala Asp Ser Ala Lys Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala

1 5 10 15

Lys Asn Met Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr

20 25 30

Ala Val Tyr Leu Cys Arg Ala

35

<210> 314

<211> 39

<212> PRT

<213> Artificial Sequence

<220>

<223> FR3

<400> 314

Tyr Ala Glu Phe Ala Lys Gly Arg Phe Thr Ile Ser Arg Asp Glu Pro

1 5 10 15

Lys Asn Met Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Val Asp Thr

20 25 30

Ala Val Tyr Leu Cys Arg Ala

35

<210> 315

<211> 39

<212> PRT

<213> Artificial Sequence

<220>

<223> FR3

<400> 315

Tyr Ser His Phe Ala Lys Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala

1 5 10 15

Lys Asn Met Val Tyr Leu Gln Met Asn Asn Leu Arg Pro Glu Asp Thr

20 25 30

Ala Val Tyr Phe Cys Arg Ala

35

<210> 316

<211> 39

<212> PRT

<213> Artificial Sequence

<220>

<223> FR3

<400> 316

Tyr Ala Asp Ser Ala Lys Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala

1 5 10 15

Lys Asn Met Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Ala

20 25 30

Ala Val Tyr Phe Cys Arg Ala

35

<210> 317

<211> 39

<212> PRT

<213> Artificial Sequence

<220>

<223> FR3

<400> 317

Tyr Ala Glu Ser Ala Lys Gly Arg Phe Thr Ile Ser Arg Asp Glu Ser

1 5 10 15

Lys Asn Met Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Ala

20 25 30

Ala Val Tyr Leu Cys Arg Ala

35

<210> 318

<211> 39

<212> PRT

<213> Artificial Sequence

<220>

<223> FR3

<400> 318

Tyr Ala Asp Ser Ala Lys Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala

1 5 10 15

Lys Asn Met Val Tyr Leu Gln Met Asp Ser Leu Lys Pro Glu Asp Thr

20 25 30

Ala Val Tyr Phe Cys Arg Ala

35

<210> 319

<211> 39

<212> PRT

<213> Artificial Sequence

<220>

<223> FR3

<400> 319

Tyr Ala Asp Ser Ala Lys Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala

1 5 10 15

Lys Asn Met Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Gly Asp Thr

20 25 30

Ala Val Tyr Phe Cys Arg Ala

35

<210> 320

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<223> FR4

<400> 320

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

1 5 10

<210> 321

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<223> FR4

<400> 321

Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

1 5 10

<210> 322

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<223> FR4

<400> 322

Trp Gly Arg Gly Thr Gln Val Thr Val Ser Ser

1 5 10

<210> 323

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<223> FR4

<400> 323

Trp Gly His Gly Thr Leu Val Thr Val Ser Ser

1 5 10

<210> 324

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<223> FR4

<400> 324

Trp Gly Arg Gly Thr Leu Val Thr Val Ser Ser

1 5 10

<210> 325

<211> 5

<212> PRT

<213> Artificial Sequence

<220>

<223> Link sequence

<400> 325

Gly Gly Gly Gly Ser

fifteen

<210> 326

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> Link sequence

<400> 326

Ser Gly Gly Ser Gly Gly Ser

fifteen

<210> 327

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> Link sequence

<400> 327

Gly Gly Gly Gly Ser Gly Gly Gly Ser

fifteen

<210> 328

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> Link sequence

<400> 328

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10

<210> 329

<211> 15

<212> PRT

<213> Artificial Sequence

<220>

<223> Link sequence

<400> 329

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10 15

<210> 330

<211> 18

<212> PRT

<213> Artificial Sequence

<220>

<223> Link sequence

<400> 330

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Gly

1 5 10 15

Gly Ser

<210> 331

<211> 20

<212> PRT

<213> Artificial Sequence

<220>

<223> Link sequence

<400> 331

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

1 5 10 15

Gly Gly Gly Ser

twenty

<210> 332

<211> 25

<212> PRT

<213> Artificial Sequence

<220>

<223> Link sequence

<400> 332

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

1 5 10 15

Gly Gly Gly Ser Gly Gly Gly Gly Ser

20 25

<210> 333

<211> 30

<212> PRT

<213> Artificial Sequence

<220>

<223> Link sequence

<400> 333

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

1 5 10 15

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

20 25 30

<210> 334

<211> 35

<212> PRT

<213> Artificial Sequence

<220>

<223> Link sequence

<400> 334

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

1 5 10 15

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

20 25 30

Gly Gly Ser

35

<210> 335

<211> 40

<212> PRT

<213> Artificial Sequence

<220>

<223> Link sequence

<400> 335

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

1 5 10 15

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

20 25 30

Gly Gly Ser Gly Gly Gly Gly Ser

35 40

<210> 336

<211> 3

<212> PRT

<213> Artificial Sequence

<220>

<223> Linker Sequence

<400> 336

Ala Ala Ala

one

<210> 337

<211> 303

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 337

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Asp Val His Lys Ile Asn

20 25 30

Phe Leu Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Lys Val

35 40 45

Ala His Ile Ser Ile Gly Asp Gln Thr Asp Tyr Ala Asp Ser Ala Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Glu Ser Lys Asn Met Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Phe Cys Arg

85 90 95

Ala Phe Ser Arg Ile Tyr Pro Tyr Asp Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

115 120 125

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

130 135 140

Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly

145 150 155 160

Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Val Ala

165 170 175

Ser Gly Asp Val His Lys Ile Asn Phe Leu Gly Trp Tyr Arg Gln Ala

180 185 190

Pro Gly Lys Glu Arg Glu Lys Val Ala His Ile Ser Ile Gly Asp Gln

195 200 205

Thr Asp Tyr Ala Asp Ser Ala Lys Gly Arg Phe Thr Ile Ser Arg Asp

210 215 220

Glu Ser Lys Asn Met Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu

225 230 235 240

Asp Thr Ala Val Tyr Phe Cys Arg Ala Phe Ser Arg Ile Tyr Pro Tyr

245 250 255

Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Ala Ala

260 265 270

Asp Tyr Lys Asp His Asp Gly Asp Tyr Lys Asp His Asp Ile Asp Tyr

275 280 285

Lys Asp Asp Asp Asp Lys Gly Ala Ala His His His His His His His

290 295 300

<210> 338

<211> 418

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 338

Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Asp Val His Lys Ile Asn

20 25 30

Phe Leu Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Lys Val

35 40 45

Ala His Ile Ser Ile Gly Asp Gln Thr Asp Tyr Ala Asp Ser Ala Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Glu Ser Lys Asn Met Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Phe Cys Arg

85 90 95

Ala Phe Ser Arg Ile Tyr Pro Tyr Asp Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

115 120 125

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

130 135 140

Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly

145 150 155 160

Gly Gly Leu Val Gln Ala Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala

165 170 175

Ser Gly Ile Thr Ser Lys Ile Asn Asp Met Gly Trp Tyr Arg Gln Thr

180 185 190

Pro Gly Asn Tyr Arg Glu Trp Val Ala Ser Ile Thr Ala Thr Gly Thr

195 200 205

Thr Asn Tyr Arg Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp

210 215 220

Asn Ala Lys Ser Thr Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu

225 230 235 240

Asp Thr Thr Val Tyr Tyr Cys Asn Thr Phe Pro Pro Ile Ser Asn Phe

245 250 255

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser

260 265 270

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

275 280 285

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val

290 295 300

Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Asn Ser Leu

305 310 315 320

Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe Gly Met

325 330 335

Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Ser

340 345 350

Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val Lys Gly

355 360 365

Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Thr Thr Leu Tyr Leu Gln

370 375 380

Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys Thr Ile

385 390 395 400

Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Leu Val Thr Val Ser

405 410 415

Ser Ala

<210> 339

<211> 578

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 339

Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Asp Val His Lys Ile Asn

20 25 30

Phe Leu Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Lys Val

35 40 45

Ala His Ile Ser Ile Gly Asp Gln Thr Asp Tyr Ala Asp Ser Ala Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Glu Ser Lys Asn Met Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Phe Cys Arg

85 90 95

Ala Phe Ser Arg Ile Tyr Pro Tyr Asp Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

115 120 125

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

130 135 140

Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly

145 150 155 160

Gly Gly Leu Val Gln Ala Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala

165 170 175

Ser Gly Ile Thr Ser Lys Ile Asn Asp Met Gly Trp Tyr Arg Gln Thr

180 185 190

Pro Gly Asn Tyr Arg Glu Trp Val Ala Ser Ile Thr Ala Thr Gly Thr

195 200 205

Thr Asn Tyr Arg Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp

210 215 220

Asn Ala Lys Ser Thr Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu

225 230 235 240

Asp Thr Thr Val Tyr Tyr Cys Asn Thr Phe Pro Pro Ile Ser Asn Phe

245 250 255

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser

260 265 270

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

275 280 285

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val

290 295 300

Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly Pro Leu

305 310 315 320

Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Ser Tyr Val Met

325 330 335

Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val Ala Ala

340 345 350

Ile Tyr Trp Ser Asn Gly Lys Thr Gln Tyr Thr Asp Ser Val Lys Gly

355 360 365

Arg Phe Thr Ile Ser Gly Asp Asn Ala Lys Asn Thr Val Tyr Leu Gln

370 375 380

Met Asn Ser Leu Asn Pro Glu Asp Thr Ala Val Tyr Tyr Cys Val Ala

385 390 395 400

Asp Lys Asp Glu Thr Gly Phe Arg Thr Leu Pro Ile Ala Tyr Asp Tyr

405 410 415

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser

420 425 430

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

435 440 445

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val

450 455 460

Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Asn Ser Leu

465 470 475 480

Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe Gly Met

485 490 495

Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Ser

500 505 510

Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val Lys Gly

515 520 525

Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Thr Thr Leu Tyr Leu Gln

530 535 540

Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys Thr Ile

545 550 555 560

Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Leu Val Thr Val Ser

565 570 575

Ser Ala

<210> 340

<211> 578

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 340

Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Asp Val His Lys Ile Asn

20 25 30

Phe Leu Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Lys Val

35 40 45

Ala His Ile Ser Ile Gly Asp Gln Thr Asp Tyr Ala Asp Ser Ala Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Glu Ser Lys Asn Met Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Phe Cys Arg

85 90 95

Ala Phe Ser Arg Ile Tyr Pro Tyr Asp Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

115 120 125

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

130 135 140

Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly

145 150 155 160

Gly Gly Leu Val Gln Ala Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala

165 170 175

Ser Gly Ile Thr Ser Lys Ile Asn Asp Met Gly Trp Tyr Arg Gln Thr

180 185 190

Pro Gly Asn Tyr Arg Glu Trp Val Ala Ser Ile Thr Ala Thr Gly Thr

195 200 205

Thr Asn Tyr Arg Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp

210 215 220

Asn Ala Lys Ser Thr Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu

225 230 235 240

Asp Thr Thr Val Tyr Tyr Cys Asn Thr Phe Pro Pro Ile Ser Asn Phe

245 250 255

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser

260 265 270

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

275 280 285

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val

290 295 300

Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Asn Ser Leu

305 310 315 320

Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe Gly Met

325 330 335

Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Ser

340 345 350

Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val Lys Gly

355 360 365

Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Thr Thr Leu Tyr Leu Gln

370 375 380

Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys Thr Ile

385 390 395 400

Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Leu Val Thr Val Ser

405 410 415

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

420 425 430

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

435 440 445

Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val

450 455 460

Gln Ala Gly Gly Pro Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr

465 470 475 480

Phe Ser Ser Tyr Val Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu

485 490 495

Arg Glu Phe Val Ala Ala Ile Tyr Trp Ser Asn Gly Lys Thr Gln Tyr

500 505 510

Thr Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Gly Asp Asn Ala Lys

515 520 525

Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Asn Pro Glu Asp Thr Ala

530 535 540

Val Tyr Tyr Cys Val Ala Asp Lys Asp Glu Thr Gly Phe Arg Thr Leu

545 550 555 560

Pro Ile Ala Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser

565 570 575

Ser Ala

<210> 341

<211> 578

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 341

Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Asp Val His Lys Ile Asn

20 25 30

Phe Leu Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Lys Val

35 40 45

Ala His Ile Ser Ile Gly Asp Gln Thr Asp Tyr Ala Asp Ser Ala Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Glu Ser Lys Asn Met Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Phe Cys Arg

85 90 95

Ala Phe Ser Arg Ile Tyr Pro Tyr Asp Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

115 120 125

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

130 135 140

Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly

145 150 155 160

Gly Gly Leu Val Gln Ala Gly Gly Pro Leu Arg Leu Ser Cys Ala Ala

165 170 175

Ser Gly Arg Thr Phe Ser Ser Tyr Val Met Gly Trp Phe Arg Gln Ala

180 185 190

Pro Gly Lys Glu Arg Glu Phe Val Ala Ala Ile Tyr Trp Ser Asn Gly

195 200 205

Lys Thr Gln Tyr Thr Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Gly

210 215 220

Asp Asn Ala Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Asn Pro

225 230 235 240

Glu Asp Thr Ala Val Tyr Tyr Cys Val Ala Asp Lys Asp Glu Thr Gly

245 250 255

Phe Arg Thr Leu Pro Ile Ala Tyr Asp Tyr Trp Gly Gln Gly Thr Leu

260 265 270

Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

275 280 285

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

290 295 300

Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly

305 310 315 320

Gly Gly Leu Val Gln Ala Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala

325 330 335

Ser Gly Ile Thr Ser Lys Ile Asn Asp Met Gly Trp Tyr Arg Gln Thr

340 345 350

Pro Gly Asn Tyr Arg Glu Trp Val Ala Ser Ile Thr Ala Thr Gly Thr

355 360 365

Thr Asn Tyr Arg Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp

370 375 380

Asn Ala Lys Ser Thr Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu

385 390 395 400

Asp Thr Thr Val Tyr Tyr Cys Asn Thr Phe Pro Pro Ile Ser Asn Phe

405 410 415

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser

420 425 430

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

435 440 445

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val

450 455 460

Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Asn Ser Leu

465 470 475 480

Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe Gly Met

485 490 495

Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Ser

500 505 510

Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val Lys Gly

515 520 525

Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Thr Thr Leu Tyr Leu Gln

530 535 540

Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys Thr Ile

545 550 555 560

Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Leu Val Thr Val Ser

565 570 575

Ser Ala

<210> 342

<211> 578

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 342

Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Asp Val His Lys Ile Asn

20 25 30

Phe Leu Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Lys Val

35 40 45

Ala His Ile Ser Ile Gly Asp Gln Thr Asp Tyr Ala Asp Ser Ala Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Glu Ser Lys Asn Met Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Phe Cys Arg

85 90 95

Ala Phe Ser Arg Ile Tyr Pro Tyr Asp Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

115 120 125

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

130 135 140

Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly

145 150 155 160

Gly Gly Leu Val Gln Ala Gly Gly Pro Leu Arg Leu Ser Cys Ala Ala

165 170 175

Ser Gly Arg Thr Phe Ser Ser Tyr Val Met Gly Trp Phe Arg Gln Ala

180 185 190

Pro Gly Lys Glu Arg Glu Phe Val Ala Ala Ile Tyr Trp Ser Asn Gly

195 200 205

Lys Thr Gln Tyr Thr Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Gly

210 215 220

Asp Asn Ala Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Asn Pro

225 230 235 240

Glu Asp Thr Ala Val Tyr Tyr Cys Val Ala Asp Lys Asp Glu Thr Gly

245 250 255

Phe Arg Thr Leu Pro Ile Ala Tyr Asp Tyr Trp Gly Gln Gly Thr Leu

260 265 270

Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

275 280 285

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

290 295 300

Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly

305 310 315 320

Gly Gly Leu Val Gln Pro Gly Asn Ser Leu Arg Leu Ser Cys Ala Ala

325 330 335

Ser Gly Phe Thr Phe Ser Ser Phe Gly Met Ser Trp Val Arg Gln Ala

340 345 350

Pro Gly Lys Gly Leu Glu Trp Val Ser Ser Ile Ser Gly Ser Gly Ser

355 360 365

Asp Thr Leu Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg

370 375 380

Asp Asn Ala Lys Thr Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Pro

385 390 395 400

Glu Asp Thr Ala Val Tyr Tyr Cys Thr Ile Gly Gly Ser Leu Ser Arg

405 410 415

Ser Ser Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser

420 425 430

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

435 440 445

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val

450 455 460

Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly Ser Leu

465 470 475 480

Arg Leu Ser Cys Ala Ala Ser Gly Ile Thr Ser Lys Ile Asn Asp Met

485 490 495

Gly Trp Tyr Arg Gln Thr Pro Gly Asn Tyr Arg Glu Trp Val Ala Ser

500 505 510

Ile Thr Ala Thr Gly Thr Thr Asn Tyr Arg Asp Ser Val Lys Gly Arg

515 520 525

Phe Thr Ile Ser Arg Asp Asn Ala Lys Ser Thr Val Tyr Leu Gln Met

530 535 540

Asn Ser Leu Lys Pro Glu Asp Thr Thr Val Tyr Tyr Cys Asn Thr Phe

545 550 555 560

Pro Pro Ile Ser Asn Phe Trp Gly Gln Gly Thr Leu Val Thr Val Ser

565 570 575

Ser Ala

<210> 343

<211> 107

<212> PRT

<213> Homo sapiens

<400> 343

Ile Gln Val Glu Gln Ser Pro Asp Leu Ile Leu Gln Glu Gly Ala

1 5 10 15

Asn Ser Thr Leu Arg Cys Asn Phe Ser Asp Ser Val Asn Asn Leu Gln

20 25 30

Trp Phe His Gln Asn Pro Trp Gly Gln Leu Ile Asn Leu Phe Tyr Ile

35 40 45

Pro Ser Gly Thr Lys Gln Asn Gly Arg Leu Ser Ala Thr Thr Val Ala

50 55 60

Thr Glu Arg Tyr Ser Leu Leu Tyr Ile Ser Ser Ser Gln Thr Thr Asp

65 70 75 80

Ser Gly Val Tyr Phe Cys Ala Ala Leu Ile Gln Gly Ala Gln Lys Leu

85 90 95

Val Phe Gly Glyn Gly Thr Arg Leu Thr Ile Asn

100 105

<210> 344

<211> 110

<212> PRT

<213> Homo sapiens

<400> 344

Asn Ala Gly Val Thr Gln Thr Pro Lys Phe Arg Ile Leu Lys Ile Gly

1 5 10 15

Gln Ser Met Thr Leu Gln Cys Thr Gln Asp Met Asn His Asn Tyr Met

20 25 30

Tyr Trp Tyr Arg Gln Asp Pro Gly Met Gly Leu Lys Leu Ile Tyr Tyr

35 40 45

Ser Val Gly Ala Gly Ile Thr Asp Lys Gly Glu Val Pro Asn Gly Tyr

50 55 60

Asn Val Ser Arg Ser Thr Thr Glu Asp Phe Pro Leu Arg Leu Glu Leu

65 70 75 80

Ala Ala Pro Ser Gln Thr Ser Val Tyr Phe Cys Ala Ser Thr Tyr His

85 90 95

Gly Thr Gly Tyr Phe Gly Glu Gly Ser Trp Leu Thr Val Val

100 105 110

<210> 345

<211> 110

<212> PRT

<213> Homo sapiens

<400> 345

Gly Asp Ala Lys Thr Thr Gln Pro Asn Ser Met Glu Ser Asn Glu Glu

1 5 10 15

Glu Pro Val His Leu Pro Cys Asn His Ser Thr Ile Ser Gly Thr Asp

20 25 30

Tyr Ile His Trp Tyr Arg Gln Leu Pro Ser Gln Gly Pro Glu Tyr Val

35 40 45

Ile His Gly Leu Thr Ser Asn Val Asn Asn Arg Met Ala Ser Leu Ala

50 55 60

Ile Ala Glu Asp Arg Lys Ser Ser Thr Leu Ile Leu His Arg Ala Thr

65 70 75 80

Leu Arg Asp Ala Ala Val Tyr Tyr Cys Thr Val Tyr Gly Gly Ala Thr

85 90 95

Asn Lys Leu Ile Phe Gly Thr Gly Thr Leu Leu Ala Val Gln

100 105 110

<210> 346

<211> 114

<212> PRT

<213> Homo sapiens

<400> 346

Val Val Ser Gln His Pro Ser Trp Val Ile Ala Lys Ser Gly Thr Ser

1 5 10 15

Val Lys Ile Glu Cys Arg Ser Leu Asp Phe Gln Ala Thr Thr Met Phe

20 25 30

Trp Tyr Arg Gln Phe Pro Lys Gln Ser Leu Met Leu Met Ala Thr Ser

35 40 45

Asn Glu Gly Ser Lys Ala Thr Tyr Glu Gln Gly Val Glu Lys Asp Lys

50 55 60

Phe Leu Ile Asn His Ala Ser Leu Thr Leu Ser Thr Leu Thr Val Thr

65 70 75 80

Ser Ala His Pro Glu Asp Ser Ser Phe Tyr Ile Cys Ser Ala Arg Gly

85 90 95

Gly Ser Tyr Asn Ser Pro Leu His Phe Gly Asn Gly Thr Arg Leu Thr

100 105 110

Val Thr

<210> 347

<211> 96

<212> PRT

<213> Macaca fascicularis

<400> 347

Pro Tyr Ile Gln Asn Pro Asp Pro Ala Val Tyr Gln Leu Arg Gly Ser

1 5 10 15

Lys Ser Asn Asp Thr Ser Val Cys Leu Phe Thr Asp Phe Asp Ser Val

20 25 30

Met Asn Val Ser Gln Ser Lys Asp Ser Asp Val His Ile Thr Asp Lys

35 40 45

Thr Val Leu Asp Met Arg Ser Met Asp Phe Lys Ser Asn Gly Ala Val

50 55 60

Ala Trp Ser Asn Lys Ser Asp Phe Ala Cys Thr Ser Ala Phe Lys Asp

65 70 75 80

Ser Val Ile Pro Ala Asp Thr Phe Phe Pro Ser Pro Glu Ser Ser Cys

85 90 95

<210> 348

<211> 131

<212> PRT

<213> Macaca mulatta

<400> 348

Glu Asp Leu Lys Lys Val Phe Pro Pro Lys Val Ala Val Phe Glu Pro

1 5 10 15

Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala Thr Leu Val Cys Leu

20 25 30

Ala Thr Gly Phe Tyr Pro Asp His Val Glu Leu Ser Trp Trp Val Asn

35 40 45

Gly Lys Glu Val His Ser Gly Val Ser Thr Asp Pro Gln Pro Leu Lys

50 55 60

Glu Gln Pro Ala Leu Glu Asp Ser Arg Tyr Ser Leu Ser Ser Arg Leu

65 70 75 80

Arg Val Ser Ala Thr Phe Trp His Asn Pro Arg Asn His Phe Arg Cys

85 90 95

Gln Val Gln Phe Tyr Gly Leu Ser Glu Asp Asp Glu Trp Thr Glu Asp

100 105 110

Arg Asp Lys Pro Ile Thr Gln Lys Ile Ser Ala Glu Ala Trp Gly Arg

115 120 125

Ala Asp Cys

130

<210> 349

<211> 107

<212> PRT

<213> Macaca mulatta

<400> 349

Gln Gln Ile Met Gln Ile Pro Gln Tyr Gln His Val Gln Glu Gly Glu

1 5 10 15

Asp Phe Thr Thr Tyr Cys Asn Ser Ser Thr Thr Leu Ser Asn Ile Gln

20 25 30

Trp Tyr Lys Gln Arg Pro Gly Gly His Pro Val Phe Leu Ile Met Leu

35 40 45

Val Lys Ser Gly Glu Val Lys Lys Gln Lys Arg Leu Ile Phe Gln Phe

50 55 60

Gly Glu Ala Lys Lys Asn Ser Ser Leu His Ile Thr Ala Thr Gln Thr

65 70 75 80

Thr Asp Val Gly Thr Tyr Phe Cys Ala Thr Thr Gly Val Asn Asn Leu

85 90 95

Phe Phe Gly Thr Gly Thr Arg Leu Thr Val Leu

100 105

<210> 350

<211> 118

<212> PRT

<213> Macaca mulatta

<400> 350

Ala Gly Pro Val Asn Ala Gly Val Thr Gln Thr Pro Lys Phe Gln Val

1 5 10 15

Leu Lys Thr Gly Gln Ser Met Thr Leu Gln Cys Ala Gln Asp Met Asn

20 25 30

His Asp Tyr Met Tyr Trp Tyr Arg Gln Asp Pro Gly Met Gly Leu Arg

35 40 45

Leu Ile His Tyr Ser Val Gly Glu Gly Ser Thr Glu Lys Gly Glu Val

50 55 60

Pro Asp Gly Tyr Asn Val Thr Arg Ser Asn Thr Glu Asp Phe Pro Leu

65 70 75 80

Arg Leu Glu Ser Ala Ala Pro Ser Gln Thr Ser Val Tyr Phe Cys Ala

85 90 95

Ser Ser Tyr Trp Thr Gly Arg Ser Tyr Glu Gln Tyr Phe Gly Pro Gly

100 105 110

Thr Arg Leu Thr Val Ile

115

<210> 351

<211> 115

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 351

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Ser Phe

20 25 30

Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Pro Glu Trp Val

35 40 45

Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ser

115

<210> 352

<211> 116

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 352

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Asn

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe

20 25 30

Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Thr Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Thr Tyr Tyr Cys

85 90 95

Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ser Ala

115

<210> 353

<211> 116

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 353

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Ser Phe

20 25 30

Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Pro Glu Trp Val

35 40 45

Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Thr Tyr Tyr Cys

85 90 95

Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ser Ala

115

<210> 354

<211> 115

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 354

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Asn

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe

20 25 30

Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Thr Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ser

115

<210> 355

<211> 116

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 355

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Asn

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe

20 25 30

Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Thr Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Leu Val Lys

100 105 110

Val Ser Ser Ala

115

<210> 356

<211> 115

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 356

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Asn

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe

20 25 30

Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Thr Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Leu Tyr Tyr Cys

85 90 95

Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ser

115

<210> 357

<211> 116

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 357

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Asn

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe

20 25 30

Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Thr Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Leu Tyr Tyr Cys

85 90 95

Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ser Ala

115

<210> 358

<211> 117

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 358

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Asn

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe

20 25 30

Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Thr Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Leu Tyr Tyr Cys

85 90 95

Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ser Ala Ala

115

<210> 359

<211> 118

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 359

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Asn

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe

20 25 30

Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Thr Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Leu Tyr Tyr Cys

85 90 95

Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ser Ala Ala Ala

115

<210> 360

<211> 116

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 360

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Asn

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe

20 25 30

Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Thr Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Leu Tyr Tyr Cys

85 90 95

Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ser Gly

115

<210> 361

<211> 117

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 361

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Asn

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe

20 25 30

Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Thr Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Leu Tyr Tyr Cys

85 90 95

Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ser Gly Gly

115

<210> 362

<211> 118

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 362

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Asn

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe

20 25 30

Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Thr Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Leu Tyr Tyr Cys

85 90 95

Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ser Gly Gly Gly

115

<210> 363

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR1

<400> 363

Gly Phe Thr Phe Ser Ser Phe Gly Met Ser

1 5 10

<210> 364

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR1

<400> 364

Gly Phe Thr Phe Arg Ser Phe Gly Met Ser

1 5 10

<210> 365

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR2

<400> 365

Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu

1 5 10

<210> 366

<211> 6

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR3

<400> 366

Gly Gly Ser Leu Ser Arg

fifteen

<210> 367

<211> 17

<212> PRT

<213> Artificial Sequence

<220>

<223> Myc-Tag

<400> 367

Ala Ala Ala Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu Asn Gly Ala

1 5 10 15

Ala

<210> 368

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR1

<220>

<221> VARIANT

<222> 2

<223> Xaa is D, A, S, E or G

<220>

<221> VARIANT

<222> 4

<223> Xaa is H or Y

<220>

<221> VARIANT

<222> 5

<223> Xaa is K or L

<220>

<221> VARIANT

<222> 6

<223> Xaa is I or L

<220>

<221> VARIANT

<222> 8

<223> Xaa is F, I or V

<220>

<221> VARIANT

<222> 10

<223> Xaa is G or S

<400> 368

Gly Xaa Val Xaa Xaa Xaa Asn Xaa Leu Xaa

1 5 10

<210> 369

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR2

<220>

<221> VARIANT

<222> 1

<223> Xaa is H, T or R

<220>

<221> VARIANT

<222> 3

<223> Xaa is S, T or A

<220>

<221> VARIANT

<222> 5

<223> G, S or A

<220>

<221> VARIANT

<222> 7

<223> Xaa is Q, D, E, T, A or V

<220>

<221> VARIANT

<222> 8

<223> Xaa is T, A or V

<220>

<221> VARIANT

<222> 9

<223> Xaa is D, A, Q, N, V or S

<400> 369

Xaa Ile Xaa Ile Xaa Asp Xaa Xaa Xaa

fifteen

<210> 370

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR3

<220>

<221> VARIANT

<222> 1

<223> Xaa is F, Y, G, L or K

<220>

<221> VARIANT

<222> 4

<223> Xaa is I or L

<220>

<221> VARIANT

<222> 5

<223> Xaa is Y or W

<220>

<221> VARIANT

<222> 8

<223> Xaa is D, N or S

<400> 370

Xaa Ser Arg Xaa Xaa Pro Tyr Xaa Tyr

fifteen

<---

Claims (84)

1. A polypeptide that redirects T cells to kill CD123-expressing cells, comprising an immunoglobulin single variable domain (ISV) that specifically binds a T cell receptor (TCR) and one or more ISVs that specifically bind CD123, wherein ISV, which specifically binds TCR, consists of 4 framework regions (FR1-FR4, respectively) and 3 complementarity-determining regions (CDR1-CDR3, respectively), in which: i) CDR1 is selected from the group consisting of: a) SEQ ID NO: 181; or b) amino acid sequences with a difference of 4, 3, 2 or 1 amino acid from the amino acid sequence of SEQ ID NO: 181, where - in position 2, D has been replaced by A, S, E or G; - in position 4 H has been replaced by Y; - in position 5 K has been replaced by L; - in position 6 I was replaced by L; - in position 8, F has been replaced by I or V; and/or - in position 10 G has been replaced by S; provided that an ISV comprising a CDR1 with a 4, 3, 2, or 1 amino acid difference binds the TCR with the same or higher affinity as compared to binding to an ISV comprising a CDR1 with no 4, 3, 2, or 1 amino acid difference, wherein the specified affinity is measured using surface plasmon resonance; and ii) CDR2 is selected from the group consisting of: c) SEQ ID NO: 192; or d) amino acid sequences with a difference of 4, 3, 2 or 1 amino acid from the amino acid sequence of SEQ ID NO: 192, where - in position 1 H has been replaced by T or R; - in position 3 S has been replaced by T or A; - in position 5 G has been replaced by S or A; - in position 7, Q has been replaced by D, E, T, A or V; - in position 8 T has been replaced by A or V; and/or - in position 9, D has been replaced by A, Q, N, V or S; provided that an ISV comprising a CDR2 with a 4, 3, 2, or 1 amino acid difference binds the TCR with the same or higher affinity as compared to binding of an ISV comprising a CDR2 with no 4, 3, 2, or 1 amino acid difference, wherein: affinity measured using surface plasmon resonance; and iii) CDR3 is selected from the group consisting of: e) SEQ ID NO: 218; or f) amino acid sequences with a difference of 4, 3, 2 or 1 amino acid from the amino acid sequence of SEQ ID NO: 218, where - in position 1, F has been replaced by Y, L or G; - in position 4 I was replaced by L; - in position 5 Y has been replaced by W; and/or - in position 8, D has been replaced by N or S; provided that an ISV comprising a CDR3 with a 4, 3, 2, or 1 amino acid difference binds the TCR with the same or higher affinity as compared to binding of an ISV comprising a CDR3 with no 4, 3, 2, or 1 amino acid difference, wherein: affinity measured using surface plasmon resonance; and where one or more ISVs that specifically bind CD123 consist of 4 framework regions (FR1-FR4, respectively) and 3 complementarity-determining regions (CDR1-CDR3, respectively), in which: i) CDR1 is selected from the group consisting of: a) SEQ ID NO: 11; or b) amino acid sequences with a difference of 4, 3, 2 or 1 amino acid with the amino acid sequence of one of SEQ ID NO: 11, where - in position 3, T has been replaced by S or P; - in position 6 I was replaced by S; - in position 7, N has been replaced by D; and/or - in position 8, D has been replaced by V or A; provided that an ISV comprising a CDR1 with a 4, 3, 2, or 1 amino acid difference binds CD123 with the same or higher affinity as compared to binding to an ISV comprising a CDR1 with no 4, 3, 2, or 1 amino acid difference, wherein the specified affinity is measured using surface plasmon resonance; and ii) CDR2 is c) SEQ ID NO: 17 and iii) CDR3 is selected from the group consisting of: e) SEQ ID NO: 21; or f) amino acid sequences with a difference of 1 amino acid from the amino acid sequence of SEQ ID NO: 21, where - in position 3 P has been replaced by A; provided that an ISV comprising a CDR3 with a 1 amino acid difference binds CD123 with the same or higher affinity as compared to binding to an ISV comprising a CDR3 without a 4, 3, 2, or 1 amino acid difference, wherein said affinity is measured by surface plasmon resonance. 2. The polypeptide according to claim 1, characterized in that the ISV that specifically binds the TCR consists of 4 framework regions (FR1-FR4, respectively) and 3 complementarity-determining regions (CDR1-CDR3, respectively), in which CDR1 is SEQ ID NO: 181, CDR2 is SEQ ID NO: 192, and CDR3 is SEQ ID NO: 218. 3. The polypeptide according to any one of paragraphs. 1 or 2, characterized in that the ISV that specifically binds the TCR is selected from the group consisting of SEQ ID NOs: 42 and 78-180, or from ISVs that have a sequence identity of more than 80%, more than 85%, more than 90% , more than 95% or even more than 99% with one of SEQ ID NO: 42 and 78-180. 4. The polypeptide according to any one of paragraphs. 1-3, characterized in that one or more ISVs that specifically bind CD123 consists of 4 framework regions (FR1-FR4, respectively) and 3 complementarity-determining regions (CDR1-CDR3, respectively), in which CDR1 is SEQ ID NO: 11, CDR2 is SEQ ID NO: 17 and CDR3 is SEQ ID NO: 21. 5. The polypeptide according to any one of paragraphs. 1-4, characterized in that one or more ISVs that specifically bind CD123 are selected from the group consisting of SEQ ID NO: 1-6 or ISVs that have a sequence identity of more than 80%, more than 85%, more than 90%, more than 95% or even more than 99% with one of SEQ ID NOs: 1-6. 6. The polypeptide according to any one of paragraphs. 1-5 containing an additional ISV that specifically binds CD123 and essentially consists of 4 framework regions (FR1-FR4, respectively) and 3 complementarity-determining regions (CDR1-CDR3, respectively), in which: i) CDR1 is SEQ ID NO: 16; and ii) CDR2 is selected from the group consisting of: a) SEQ ID NO: 18; or b) amino acid sequences with a difference of 3, 2 or 1 amino acid from the amino acid sequence of SEQ ID NO: 18, where - in position 3 Y has been replaced by W; - in position 6, N was replaced by S; and/or - in position 10 Q has been replaced by E; provided that a polypeptide containing a CDR2 with a difference of 3, 2, or 1 amino acid(s) binds CD123 with the same or higher affinity compared to binding by a polypeptide containing a CDR2 with no difference of 3, 2, or 1 amino acid, where the specified affinity is measured using surface plasmon resonance; and iii) CDR3 is selected from the group consisting of: c) SEQ ID NO: 23; or d) amino acid sequences with a difference of 2 or 1 amino acid from the amino acid sequence of SEQ ID NO: 23, where - in position 4, E was replaced by R; and/or - in position 5, T has been replaced by D or Y; provided that a polypeptide containing CDR3 with a difference of 2 or 1 amino acid binds CD123 with the same or higher affinity compared to the binding of a polypeptide containing CDR3 with no difference of 3, 2 or 1 amino acid, where the specified affinity is measured using surface plasmon resonance. 7. The polypeptide according to claim 6, characterized in that the additional ISV that specifically binds CD123 is selected from the group consisting of SEQ ID NOs: 7-10, or from ISVs that have sequence identity of more than 80%, more than 85%, more than 90%, more than 95% or even more than 99% with one of SEQ ID NO: 7-10. 8. The polypeptide according to any one of paragraphs. 1-7, comprising two or more ISVs that specifically bind CD123, wherein two or more ISVs that specifically bind CD123 are biparatopic, including a first ISV and a second ISV, where the first ISV binds to an epitope on CD123 that is different from an epitope on CD123 bound by a second ISV. 9. The polypeptide according to any one of paragraphs. 1-8, characterized in that the ISV that specifically binds TCR and one or more ISVs that specifically bind CD123 consist of a single domain antibody, dAb, nanobody, VHH, humanized VHH, overblown VH, or VHH that was obtained by maturation affinity. 10. The polypeptide according to any one of paragraphs. 1-9, characterized in that the specified polypeptide is selected from the group consisting of SEQ ID NO: 47, 49, 52, 53, 55, 56 and 58-61, or from polypeptides that have a sequence identity of more than 80%, more than 85 %, more than 90%, more than 95% or even more than 99% with one of SEQ ID NOs: 47, 49, 52, 53, 55, 56 and 58-61. 11. Design for redirecting T cells to destroy CD123-expressing cells with an increased half-life, containing a polypeptide according to any one of paragraphs. 1-10 and one or more binding units that can bind to serum albumin or serum immunoglobulin, in which these one or more binding units provide the construct with an increased half-life compared to the polypeptide according to any one of paragraphs. 1-10. 12. The construct of claim 11 wherein one or more binding units bind to human serum albumin or IgG. 13. Nucleic acid encoding a polypeptide according to any one of paragraphs. 1-10. 14. Nucleic acid encoding the construct according to claim 11 or 12. 15. An expression vector comprising a nucleic acid according to claim 13 or 14. 16. The host cell to obtain the polypeptide according to any one of paragraphs. 1-10 or a construct according to claim 11 or 12 containing a nucleic acid according to claim 13 or 14 or an expression vector according to claim 15. 17. The method of obtaining a polypeptide according to any one of paragraphs. 1-10 or designs according to claim 11 or 12, including the steps: a) expression in a suitable host cell or host organism or other suitable expression system for a nucleic acid according to claim 13 or 14 or an expression vector according to claim 15, and b) isolation and/or purification of the polypeptide according to any one of paragraphs. 1-10 or designs according to item 11 or 12. 18. Composition for redirecting T cells to destroy CD123-expressing cells, containing a polypeptide according to any one of paragraphs. 1-10, or a construct according to claim 11 or 12, or a nucleic acid according to claim 13 or 14, or an expression vector according to claim 15, and a pharmaceutically acceptable carrier, diluent, or excipient. 19. The polypeptide according to any one of paragraphs. 1-10, a construct according to claim 11 or 12, or a composition according to claim 18 for use as a drug. 20. The polypeptide according to any one of paragraphs. 1-10, a construct according to claim 11 or 12 or a composition according to claim 18 for use in the prevention, treatment and/or alleviation of a disease or condition associated with CD123.

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