RU2820603C2 - Cd33, nkg2d and cd16 binding proteins - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: disclosed are multispecific binding proteins which bind CD33, NKG2D receptor and CD16, as well as pharmaceutical compositions and therapeutic methods useful for treating cancer.

EFFECT: invention provides improved activation of NK-cells as a result of binding of two activating receptors.

17 cl, 57 dwg, 9 tbl, 13 ex

Description

[0001] This application claims the benefit and priority of U.S. Provisional Patent Application No. 62/461,145, filed February 20, 2017, the entire contents of which are incorporated herein by reference in all respects.

LIST OF SEQUENCES

[0002] This application contains a Sequence Listing that has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. The ASCII copy in question, created on February 19, 2018, is named DFY-007PC_SL.txt and is 98304 bytes in size.

FIELD OF TECHNOLOGY TO WHICH THE INVENTION RELATES

[0003] The invention relates to multispecific binding proteins that bind to CD33, NKG2D receptor and CD16.

BACKGROUND OF THE ART

[0004] Cancer remains a major health problem, despite significant research and scientific advances described in the literature in the treatment of this disease. Some of the most commonly diagnosed forms of cancer in adults include prostate cancer, breast cancer, and lung cancer. Hemoblastoses, although less common than solid cancers, have a low survival rate. Current treatment options for these cancers are not effective in all patient populations and/or may have significant unwanted side effects. Other forms of cancer also remain difficult to treat with current treatment regimens.

[0005] Cancer immunotherapy methods are preferred because they are highly specific and can promote the destruction of cancer cells by the patient's own immune system. Fusion proteins, such as bispecific T cell activators, are anticancer immunotherapies described in the literature that bind to tumor cells and T cells, promoting the destruction of tumor cells. Antibodies that bind to certain tumor-associated antigens and certain immune cells have been described in the literature. See, for example, WO 2016/134371 and WO 2015/095412.

[0006] Natural killer cells (NK cells) are a component of the innate immune system and make up approximately 15% of circulating lymphocytes. NK cells infiltrate virtually all tissues and are initially distinguished by their ability to effectively kill tumor cells without the need for prior sensitization. Activated NK cells kill target cells similar to cytotoxic T cells, i.e. through cytolytic granules that contain perforin and granzymes, and through the death receptor pathway. Activated NK cells also secrete inflammatory cytokines, such as IFN-gamma, and chemokines, which cause the recruitment of other leukocytes to the target tissue.

[0007] NK cells respond to signals through a variety of activating and inhibitory receptors on their surface. For example, when NK cells encounter healthy cells, their activity is inhibited as a result of activation of killer cell immunoglobulin-like receptors (KIRs). Alternatively, when NK cells encounter foreign cells or cancer cells, they are activated through their activating receptors (eg, NKG2D, NCR, DNAM1). NK cells are also activated by the constant region of some immunoglobulins through CD16 receptors on their surface. The overall sensitivity of NK cells to activation depends on the sum of stimulatory and inhibitory signals.

[0008] CD33 is a member of the sialic acid-binding immunoglobulin-like lectins. CD33, as a transmembrane receptor expressed primarily on myeloid lineage cells, modulates inflammatory and immune responses through a dampening effect on tyrosine kinase-regulated signaling pathways. For example, CD33 has been shown to constitutively suppress the production of proinflammatory cytokines such as IL-1β, TNF-α, and IL-8 in human monocytes.

[0009] CD33 is associated with hematological malignancies. It is widely expressed in blast cells of almost all forms of acute myeloid leukemia (AML). In addition, hematopoietic cancer stem and/or progenitor cells have been found to be CD33+, suggesting that CD33-targeted therapy could potentially eliminate malignant stem and/or progenitor cells in such cases, sparing normal hematopoietic stem cells . In addition to its expression in AML, CD33 is found on other myeloid tumors (eg, myelodysplastic syndromes and myeloproliferative neoplasias) and on subsets of B-cell and T-cell acute lymphoblastic leukemia (ALL)/lymphoblastic lymphomas. This expression profile has led to the use of CD33-targeted therapy in patients with malignancies including AML, myelodysplastic syndromes, chronic myelomonocytic leukemia, myeloid blast crisis in chronic myeloid leukemia, and various forms of ALL.

SUMMARY OF THE INVENTION

[0010] The invention provides multispecific binding proteins that bind to CD33 on a cancer cell and to the NKG2D receptor and CD16 receptor on NK cells. Such proteins may bind more than one type of NK-activating receptor and may block the binding of natural ligands to NKG2D. In some embodiments, the proteins may have agonistic effects on NK cells in humans as well as other species such as rodents and cynomolgus monkeys. Various aspects and embodiments of the invention are described in more detail below.

[0011] Thus, one aspect of the invention relates to a protein that includes a first antigen binding site that binds NKG2D; a second antigen binding site that binds to CD33; and the Fc domain of the antibody, a portion thereof sufficient to bind CD16, or a third antigen-binding region that binds CD16. Each of the antigen binding regions may include an antibody heavy chain variable domain and an antibody light chain variable domain, for example, located as in an antibody or fused together with a scFv, or one or more antigen binding regions may be a single domain antibody, such as a V _H H antibody such as camelid antibody, or V _NAR antibody, similar to antibodies found in cartilaginous fish.

[0012] The first antigen binding region that binds NKG2D, in one embodiment, may include a heavy chain variable domain related to SEQ ID NO:1, for example in that it has an amino acid sequence that is at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identical to SEQ ID NO:1, and/or includes amino acid sequences identical to CDR1 sequences (SEQ ID NO:54), CDR2 (SEQ ID NO:55) and CDR3 (SEQ ID NO:56) in SEQ ID NO:1. Alternatively, the first antigen binding region may include a heavy chain variable domain related to SEQ ID NO:41 and a light chain variable domain related to SEQ ID NO:42. For example, the heavy chain variable domain of the first antigen binding region may be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identical to SEQ ID NO:41, and/or may include amino acid sequences identical to the sequences CDR1 (SEQ ID NO:57), CDR2 (SEQ ID NO:58) and CDR3 (SEQ ID NO:59) in SEQ ID NO:41. Likewise, the light chain variable domain of the second antigen binding region may be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99 % or 100%) is identical to SEQ ID NO:42, and/or may include amino acid sequences identical to the sequences CDR1 (SEQ ID NO:60), CDR2 (SEQ ID NO:61) and CDR3 (SEQ ID NO:62) in SEQ ID NO:42. In other embodiments, the first antigen binding region may include a heavy chain variable domain related to SEQ ID NO:43 and a light chain variable domain related to SEQ ID NO:44. For example, the heavy chain variable domain of the first antigen binding region may be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identical to SEQ ID NO:43, and/or may include amino acid sequences identical to the sequences CDR1 (SEQ ID NO:63), CDR2 (SEQ ID NO:64), and CDR3 (SEQ ID NO:65) in SEQ ID NO:43. Likewise, the light chain variable domain of the second antigen binding region may be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99 % or 100%) is identical to SEQ ID NO:44, and/or may include amino acid sequences identical to the sequences CDR1 (SEQ ID NO:66), CDR2 (SEQ ID NO:67) and CDR3 (SEQ ID NO:68) in SEQ ID NO:44.

[0013] In some embodiments, the first antigen binding region may include a heavy chain variable domain related to SEQ ID NO:45 and a light chain variable domain related to SEQ ID NO:46, for example in that it has an amino acid sequence that is at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO:45, and at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO:46, respectively . In another embodiment, the first antigen binding region may include a heavy chain variable domain related to SEQ ID NO:47 and a light chain variable domain related to SEQ ID NO:48, for example in that it has an amino acid sequence that is at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO:47 and at least 90 % (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO:48, respectively.

[0014] In some embodiments, the first antigen binding region may include a heavy chain variable domain related to SEQ ID NO:69 and a light chain variable domain related to SEQ ID NO:70. For example, the heavy chain variable domain of the first antigen binding region may be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identical to SEQ ID NO:69, and/or may include amino acid sequences identical to the sequences CDR1 (SEQ ID NO:71), CDR2 (SEQ ID NO:72), and CDR3 (SEQ ID NO:73) in SEQ ID NO:69. Likewise, the light chain variable domain of the second antigen binding region may be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99 % or 100%) is identical to SEQ ID NO:70, and/or may include amino acid sequences identical to the sequences of CDR1 (SEQ ID NO:74), CDR2 (SEQ ID NO:75), and CDR3 (SEQ ID NO:76) in SEQ ID NO:70. In some embodiments, the first antigen binding region may include a heavy chain variable domain related to SEQ ID NO:77 and a light chain variable domain related to SEQ ID NO:78. For example, the heavy chain variable domain of the first antigen binding region may be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identical to SEQ ID NO:77, and/or may include amino acid sequences identical to the sequences CDR1 (SEQ ID NO:69), CDR2 (SEQ ID NO:80) and CDR3 (SEQ ID NO:81) in SEQ ID NO:77. Likewise, the light chain variable domain of the second antigen binding region may be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99 % or 100%) is identical to SEQ ID NO:78, and/or may include amino acid sequences identical to the sequences of CDR1 (SEQ ID NO:82), CDR2 (SEQ ID NO:83), and CDR3 (SEQ ID NO:84) in SEQ ID NO:78.

[0015] In some embodiments, the first antigen binding region may include a heavy chain variable domain related to SEQ ID NO:85 and a light chain variable domain related to SEQ ID NO:86. For example, the heavy chain variable domain of the first antigen binding region may be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identical to SEQ ID NO:85, and/or may include amino acid sequences identical to the sequences CDR1 (SEQ ID NO:87), CDR2 (SEQ ID NO:88), and CDR3 (SEQ ID NO:89) in SEQ ID NO:85. Likewise, the light chain variable domain of the second antigen binding region may be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99 % or 100%) is identical to SEQ ID NO:86, and/or may include amino acid sequences identical to the sequences CDR1 (SEQ ID NO:90), CDR2 (SEQ ID NO:91) and CDR3 (SEQ ID NO:92) in SEQ ID NO:86.

[0016] In some embodiments, the first antigen binding region may include a heavy chain variable domain related to SEQ ID NO:133 and a light chain variable domain related to SEQ ID NO:134. For example, the heavy chain variable domain of the first antigen binding region may be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identical to SEQ ID NO:133, and/or may include amino acid sequences identical to the sequences CDR1 (SEQ ID NO:135), CDR2 (SEQ ID NO:136), and CDR3 (SEQ ID NO:137) in SEQ ID NO:133. Likewise, the light chain variable domain of the second antigen binding region may be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99 % or 100%) is identical to SEQ ID NO:134, and/or may include amino acid sequences identical to the sequences CDR1 (SEQ ID NO:138), CDR2 (SEQ ID NO:139) and CDR3 (SEQ ID NO:140) in SEQ ID NO:134.

[0017] The second antigen binding region may optionally include a heavy chain variable domain related to SEQ ID NO:93 and a light chain variable domain related to SEQ ID NO:94. For example, the heavy chain variable domain of the second antigen binding region may be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identical to SEQ ID NO:93, and/or may include amino acid sequences identical to the sequences CDR1 (SEQ ID NO:95), CDR2 (SEQ ID NO:96) and CDR3 (SEQ ID NO:97) in SEQ ID NO:93. Likewise, the light chain variable domain of the second antigen binding region may be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99 % or 100%) is identical to SEQ ID NO:94, and/or may include amino acid sequences identical to the sequences CDR1 (SEQ ID NO:98), CDR2 (SEQ ID NO:99) and CDR3 (SEQ ID NO:100) in SEQ ID NO:94.

[0018] Alternatively, the second antigen binding region may include a heavy chain variable domain related to SEQ ID NO:101 and a light chain variable domain related to SEQ ID NO:102. For example, the heavy chain variable domain of the second antigen binding region may be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identical to SEQ ID NO:101, and/or may include amino acid sequences identical to the sequences CDR1 (SEQ ID NO:103), CDR2 (SEQ ID NO:104) and CDR3 (SEQ ID NO:105) in SEQ ID NO:101. Likewise, the light chain variable domain of the second antigen binding region may be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99 % or 100%) is identical to SEQ ID NO:58, and/or may include amino acid sequences identical to the sequences CDR1 (SEQ ID NO:106), CDR2 (SEQ ID NO:107) and CDR3 (SEQ ID NO:108) in SEQ ID NO:102.

[0019] In another embodiment, the second antigen binding region may include a heavy chain variable domain related to SEQ ID NO:109 and a light chain variable domain related to SEQ ID NO:110. For example, the heavy chain variable domain of the second antigen binding region may be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identical to SEQ ID NO:59, and/or may include amino acid sequences identical to the sequences CDR1 (SEQ ID NO:111), CDR2 (SEQ ID NO:112) and CDR3 (SEQ ID NO:113) in SEQ ID NO:109. Likewise, the light chain variable domain of the second antigen binding region may be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99 % or 100%) is identical to SEQ ID NO:110, and/or may include amino acid sequences identical to the sequences CDR1 (SEQ ID NO:114), CDR2 (SEQ ID NO:115) and CDR3 (SEQ ID NO:116) in SEQ ID NO:110.

[0020] In another embodiment, the second antigen binding region may include a heavy chain variable domain related to SEQ ID NO:117 and a light chain variable domain related to SEQ ID NO:118. For example, the heavy chain variable domain of the second antigen binding region may be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identical to SEQ ID NO:117, and/or may include amino acid sequences identical to the sequences CDR1 (SEQ ID NO:119), CDR2 (SEQ ID NO:120) and CDR3 (SEQ ID NO:121) in SEQ ID NO:117. Likewise, the light chain variable domain of the second antigen binding region may be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99 % or 100%) is identical to SEQ ID NO:118, and/or may include amino acid sequences identical to the sequences CDR1 (SEQ ID NO:122), CDR2 (SEQ ID NO:123) and CDR3 (SEQ ID NO:124) in SEQ ID NO:118.

[0021] In another embodiment, the second antigen binding region may include a heavy chain variable domain related to SEQ ID NO:125 and a light chain variable domain related to SEQ ID NO:126. For example, the heavy chain variable domain of the second antigen binding region may be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identical to SEQ ID NO:125, and/or may include amino acid sequences identical to the sequences CDR1 (SEQ ID NO:127), CDR2 (SEQ ID NO:128) and CDR3 (SEQ ID NO:129) in SEQ ID NO:125. Likewise, the light chain variable domain of the second antigen binding region may be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99 % or 100%) is identical to SEQ ID NO:126, and/or may include amino acid sequences identical to the sequences CDR1 (SEQ ID NO:130), CDR2 (SEQ ID NO:131) and CDR3 (SEQ ID NO:132) in SEQ ID NO:126.

[0022] In some embodiments, the second antigen binding region includes a light chain variable domain having an amino acid sequence identical to the amino acid sequence of the light chain variable domain present in the first antigen binding region.

[0023] In some embodiments, the protein includes a portion of an antibody Fc domain sufficient to bind CD16, wherein the antibody Fc domain includes hinge and CH2 domains and/or amino acid sequences that are at least 90% identical to amino acid sequences 234-332 of IgG human antibodies.

[0024] Dosage forms containing one of these proteins are also provided; cells containing one or more nucleic acids expressing these proteins, and methods of enhancing tumor cell death using these proteins.

[0025] In another aspect of the invention, there is provided a method of treating cancer in a patient. The method includes administering to a patient in need thereof a therapeutically effective amount of a multispecific binding protein described herein. Examples of cancers for treatment using multispecific binding proteins include, for example, cancers selected from the group consisting of AML, myelodysplastic syndromes, chronic myelomonocytic leukemia, myeloid blast crisis in chronic myeloid leukemia, and various forms of ALL.

BRIEF DESCRIPTION OF THE DRAWINGS

[0026] FIG. 1 is a schematic representation of a heterodimeric multispecific antibody. Each arm can be either an NKG2D binding domain or a CD33 binding domain. In some embodiments, the NKG2D and CD33 binding domains may have the same light chain.

[0027] FIG. 2 is a schematic representation of a heterodimeric multispecific antibody. The NKG2D or CD33 binding domain may be in scFv format (right arm).

[0028] FIG. 3 are line graphs showing the binding affinity of NKG2D binding domains (listed as clones) to recombinant human NKG2D in an ELISA assay.

[0029] FIG. 4 are line graphs showing the binding affinity of NKG2D binding domains (listed as clones) to recombinant cynomolgus NKG2D in an ELISA assay.

[0030] FIG. 5 are line graphs showing the binding affinity of NKG2D binding domains (listed as clones) to recombinant mouse NKG2D in an ELISA assay.

[0031] FIG. 6 are histograms showing the binding of NKG2D binding domains (listed as clones) to EL4 cells expressing human NKG2D by flow cytometry, showing the fold ratio of mean fluorescence intensity (MFI) to background.

[0032] FIG. 7 are histograms showing the binding of NKG2D binding domains (listed as clones) to EL4 cells expressing mouse NKG2D by flow cytometry, showing the fold ratio of mean fluorescence intensity (MFI) to background.

[0033] FIG. 8 are line graphs showing the specific binding affinity of NKG2D binding domains (listed as clones) to recombinant human NKG2D-Fc in competition with the natural ligand ULBP-6.

[0034] FIG. 9 are line graphs showing the specific binding affinity of NKG2D binding domains (listed as clones) to recombinant human NKG2D-Fc when competed with the natural MICA ligand.

[0035] FIG. 10 are line graphs showing the specific binding affinity of NKG2D binding domains (listed as clones) to recombinant mouse NKG2D-Fc when competed with the natural ligand Rae-1 delta.

[0036] FIG. 11 are histograms showing activation of human NKG2D by NKG2D binding domains (listed as clones) by quantifying the percentage of TNF-alpha positive cells that express human NKG2D-CD3 zeta fusion proteins.

[0037] FIG. 12 are histograms showing activation of mouse NKG2D by NKG2D binding domains (listed as clones) by quantifying the percentage of TNF-alpha positive cells that express mouse NKG2D-CD3 zeta fusion proteins.

[0038] FIG. 13 are histograms showing activation of human NK cells by NKG2D binding domains (listed as clones).

[0039] FIG. 14 are histograms showing activation of human NK cells by NKG2D binding domains (listed as clones).

[0040] FIG. 15 are histograms showing activation of murine NK cells by NKG2D binding domains (listed as clones).

[0041] FIG. 16 are histograms showing activation of murine NK cells by NKG2D binding domains (listed as clones).

[0042] FIG. 17 are histograms showing the cytotoxic effect of NKG2D binding domains (listed as clones) on tumor cells.

[0043] FIG. 18 are histograms showing the melting temperature of NKG2D binding domains (listed as clones) measured by differential scanning fluorimetry.

[0044] FIG. 19A-19C are histograms of synergistic NK cell activation using NKG2D and CD16 binding. In FIG. 19A demonstrates CD107a levels; in FIG. 19B demonstrates IFNγ levels; in FIG. 19C demonstrated CD107a and IFNγ levels. Graphs indicate mean (n=2) ±SD. Data are representative of five independent experiments using five different healthy donors.

[0045] FIG. 20 is a schematic representation of TriNKET in the form of Triomab, which is a trifunctional bispecific antibody that retains an IgG-like form. This chimera consists of two half-antibodies, each containing one light and one heavy chain, which are derived from two parent antibodies. The Triomab form can be a _{heterodimeric construct containing 1/2 rat antibody and 1/2} ^{mouse antibody} _.

[0046] FIG. 21 is a schematic representation of TriNKET in the same light chain (LC) KiH form, which incorporates knob-in-valley (KIH) technology. KiH is a heterodimer containing 2 target-binding Fabs 1 and 2, and an Fc stabilized by heterodimerization mutations. TriNKET in KiH format can be a heterodimeric construct with 2 fabs binding to target 1 and target 2, containing two different heavy chains and the same light chain that is paired with both heavy chains.

[0047] FIG. 22 is a schematic representation of TriNKET in the form of dual variable domain immunoglobulin (DVD-Ig ^™ ), which combines the target binding domains of two monoclonal antibodies through flexible natural linkers to produce a tetravalent IgG-like molecule. DVD-Ig ^™ is a homodimeric construct in which the antigen 2 targeting variable domain is fused to the N-terminus of the antigen 1 targeting variable domain Fab, the construct containing a conventional Fc.

[0048] FIG. 23 is a schematic representation of TriNKET in the form of an Orthogonal Fab interface (Ortho-Fab), which is a heterodimeric construct that contains 2 Fabs binding to target 1 and target 2 fused to an Fc. LC-HC pairing is provided by an orthogonal interface. Heterodimerization is ensured by mutations in Fc.

[0049] FIG. 24 is a schematic representation of TrinKET in 2 in 1 Ig format.

[0050] FIG. 25 is a schematic representation of TriNKET in ES form, which is a heterodimeric construct containing two different Fabs binding to target 1 and target 2 fused to an Fc. Heterodimerization is ensured by mutations of the electrostatic direction in Fc.

[0051] FIG. 26 is a schematic representation of TriNKET in the form of Fab Arm Swap: antibodies exchange Fab arms, replacing the heavy chain and attached light chain (half-molecule) with a heavy-light chain pair of another molecule, resulting in bispecific antibodies. The Arm Exchange form of Fab (cFae) is a heterodimer containing 2 target-binding Fabs 1 and 2, and an Fc stabilized by heterodimerization mutations.

[0052] FIG. 27 is a schematic representation of TriNKET in SEED Body form, which is a heterodimer containing 2 target binding Fabs 1 and 2, and an Fc stabilized by heterodimerization mutations.

[0053] FIG. 28 is a schematic representation of TriNKET in the form of LuZ-Y, in which a leucine zipper is used to induce heterodimerization of two different HCs. The LuZ-Y form is a heterodimer containing two different target-binding scFabs 1 and 2 fused to an Fc. Heterodimerization is mediated by leucine zipper motifs fused to the C terminus of the Fc.

[0054] FIG. 29 is a schematic illustration of TriNKET in Cov-X-Body form.

[0055] FIG. 30A-30B are schematic representations of TriNKETs in κλ-Body forms, which are heterodimeric constructs with two different Fabs fused to an Fc stabilized by heterodimerization mutations: Fab1 directed against antigen 1 contains LC kappa, while the second Fab directed against antigen 2 , contains LC lambda. FIG. 30A is an exemplary diagram of one κλ-Body shape; FIG. 30B is an example diagram of another κλ-Body.

[0056] FIG. 31 is a heterodimeric Oasc-Fab construct that includes target-binding Fab 1 and target-binding scFab 2 fused to an Fc. Heterodimerization is ensured by mutations in Fc.

[0057] FIG. 32 - DuetMab, which is a heterodimeric construct containing two different Fabs that bind antigens 1 and 2, and an Fc stabilized by heterodimerization mutations. Fab 1 and 2 contain different S-S bridges that ensure proper connection between the light chain (LC) and the heavy chain (HC).

[0058] FIG. 33 - CrossmAb, which is a heterodimeric construct with two different Fabs binding to targets 1 and 2 fused to an Fc stabilized by heterodimerization. The CL and CH1 domains and the VH and VL domains are rearranged, for example, CH1 is fused in series to VL while CL is fused in series to VH.

[0059] FIG. 34 - Fit-Ig, which is a homodimeric construct in which a Fab that binds antigen 2 is fused to the N-terminus of an HC Fab that binds antigen 1. The construct contains a wild-type Fc.

[0060] FIG. 35A-35B - binding profiles of CD33-targeting TriNKET to NKG2D expressed on EL4 cells. In FIG. 35A shows the binding of TriNKET constructs compared to monoclonal antibodies that contain the corresponding NKG2D binding domain. In FIG. 35B shows the binding profile of CD33 targeting TriNKETs, which include 6 different NKG2D binding domains.

[0061] FIG. 36A and 36B are CD33-targeting TriNKET binding profiles of CD33 expressed on human AML MV4-11 cells. FIG. 36C - binding profile of CD33 targeting TriNKET and anti-CD33 monoclonal antibody to CD33 expressed on Molm-13 human AML cells. FIG. 36D - Binding profile of CD33 targeting TriNKET and anti-CD33 monoclonal antibody to CD33 expressed on the human AML cell line MV4-11.

[0062] FIG. 37A-37B are line graphs demonstrating TriNKET-mediated activation of quiescent or IL-2-activated human NK cells in coculture with the CD33-expressing human AML cell line MV4-11. In FIG. 37A shows TriNKET-mediated activation of resting human NK cells. In FIG. 37B shows TriNKET-mediated activation of IL-2-activated human NK cells from the same donor. Separately cultured NK cells, coculture of NK cells with MV4-11 cells but without TriNKET, and CD20-guided TriNKET were used as controls.

[0063] FIG. 38A-38C are histograms showing the expression of high affinity FcRγI (CD64) in three human AML cell lines: the Molm-13 cell line (FIG. 38A), the MV4-11 cell line (FIG. 38B), and the THP-1 cell line ( FIG. 38C).

[0064] FIG. 39A-39B are line graphs of anti-CD33 monoclonal antibody-mediated human NK cell activation, or TriNKET, in coculture with Molm-13 cells (FIG. 39B) or THP-1 (FIG. 39A). In FIG. 39C shows activation of human NK cells by TriNKET constructs in coculture with the human AML cell line MV4-11. HER2-TriNKET was used as a control.

[0065] FIG. 40A-40C are line graphs of human NK cytotoxicity to three human AML cell lines mediated by CD33-targeting TriNKET and the corresponding anti-CD33 monoclonal antibody. In FIG. 40A shows that the anti-CD33 monoclonal antibody showed reduced efficacy against MV4-11 cells, which express CD64, but at a lower level than THP-1. In FIG. 40B demonstrated that the CD33 monoclonal antibody showed good activity against Molm-13 cells that do not express CD64. In FIG. 40C demonstrated that the anti-CD33 monoclonal antibody had no effect on THP-1 cells.

[0066] FIG. 41 shows TriNKET-mediated cytotoxicity of resting human NK cells against Molm-13 cells.

[0067] FIG. 42A is a bar graph showing that healthy donor B cells are protected from CD33-targeting TriNKET-mediated lysis. FIG. 42B is a bar graph showing that autologous CD33+ myeloid cells were protected from CD33-guided TriNKET-mediated NK cell responses and were thus resistant to TriNKET-mediated lysis.

DETAILED DESCRIPTION

[0068] The invention provides multispecific binding proteins that bind CD33 on a cancer cell and the NKG2D receptor and CD16 receptor on natural killer cells to activate NK cells, pharmaceutical compositions containing such multispecific binding proteins, and therapeutic methods using such multispecific proteins , as well as pharmaceutical compositions, including for the treatment of cancer. Various aspects of the invention are set forth in the sections below; however, aspects of the invention described in one specific section are not intended to be limited to any particular section.

[0069] To facilitate understanding of the present invention, a number of terms and phrases are defined below.

[0070] The singular forms "a" and "an", when used herein, mean "one or more" and include the plural unless inconsistent with context.

[0071] As used herein, the term “antigen-binding site” refers to the portion of the immunoglobulin molecule that is involved in antigen binding. In human antibodies, the antigen-binding region is formed by amino acid residues of the N-terminal variable (“V”) regions of the heavy (“H”) and light (“L”) chains. Three highly heterogeneous fragments within the boundaries of the V regions of the heavy and light chains are called "hypervariable regions", which alternate with more conserved flanking fragments known as "framework regions" or "FR". Thus, the term "FR" refers to amino acid sequences that are typically located between and adjacent to hypervariable regions in immunoglobulins. In a human antibody molecule, three light chain hypervariable regions and three heavy chain hypervariable regions are located relative to each other in three-dimensional space to form an antigen-binding surface. The antigen-binding surface is complementary to the three-dimensional surface of the bound antigen, and the three hypervariable regions of each heavy and light chain are called “complementarity determining regions” or “CDR regions”. In some animals, such as camels and cartilaginous fish, the antigen-binding region is formed by a single chain of antibody, representing a "single domain antibody". Antigen-binding regions may exist in an intact antibody, in an antigen-binding fragment of an antibody that retains an antigen-binding surface, or in a recombinant polypeptide such as a scFv by using a peptide linker to join a heavy chain variable domain to a light chain variable domain in a single polypeptide.

[0072] The term “tumor-associated antigen” as used herein means any antigen, including, but not limited to, protein, glycoprotein, ganglioside, carbohydrate, lipid, that is associated with cancer. Such an antigen may be expressed on malignant cells or in the tumor microenvironment, such as tumor-associated blood vessels, extracellular matrix, mesenchymal stroma, or immune infiltrates.

[0073] As used herein, the terms “subject” and “patient” refer to the organism to be treated using the methods and compositions described herein. Such organisms preferably include, but are not limited to, mammals (eg, mice, apes, horses, cows, pigs, canids, felines, etc.), and more preferably include humans.

[0074] As used herein, the term “effective amount” refers to an amount of a compound (eg, a compound of the present invention) sufficient to obtain the useful or desired results. An effective amount may be administered in one or more administrations, applications or dosages and does not need to be limited to a particular dosage form or route of administration. As used herein, the term “treatment” includes any effect, such as amelioration, reduction, modulation, improvement, or elimination, that results in an improvement, reduction in the severity of a disease, disorder, etc., or attenuation of a symptom thereof.

[0075] As used herein, the term “pharmaceutical composition” refers to the combination of an active substance with a carrier, inert or active, which makes the composition particularly suitable for diagnostic or therapeutic use in vivo or ex vivo.

[0076] As used herein, the term “pharmaceutically acceptable carrier” refers to any of typical pharmaceutical carriers such as phosphate-buffered saline, water, emulsions (for example, such as oil/water or water/oil emulsions), and various types of wetting agents. The compositions may also include stabilizers and preservatives. For examples of carriers, stabilizers and excipients, see, for example, Martin, Remington's Pharmaceutical Sciences, 15th Ed., Mack Publ. Co., Easton, PA [1975].

[0077] As used herein, the term “pharmaceutically acceptable salt” refers to any pharmaceutically acceptable salt (eg, with an acid or base) of a compound of the present invention that, when administered to a subject, is capable of producing the compound of the present invention or an active metabolite or residue thereof. As is known to those skilled in the art, "salts" of the compounds of this invention can be prepared from inorganic or organic acids and bases. Examples of acids include, but are not limited to, hydrochloric, hydrobromic, sulfuric, nitric, perchloric, fumaric, maleic, phosphoric, glycolic, lactic, salicylic, succinic, toluene-p-sulfonic, tartaric, acetic, citric, methanesulfonic, ethanesulfonic, formic, benzoic, malonic, naphthalene-2-sulfonic, benzenesulfonic acid, etc. Other acids, such as oxalic acid, which are not themselves pharmaceutically acceptable, can be used in the preparation of salts suitable as intermediates in the preparation of the compounds of the invention and their pharmaceutically acceptable acid addition salts.

[0078] Examples of bases include, but are not limited to, alkali metal hydroxides (eg, sodium), alkaline earth metal hydroxides (eg, magnesium), ammonia, and compounds of the formula NW ₄ ⁺ where W is C _1-4 alkyl, and the like .

[0079] Examples of salts include, without restrictions on the listed: acetate, adipate, alginate, aspartate, benzoate, gasolusphone, bisulfate, butyrate, citrate, campfire, cyclopentantanpropionate, digluconate, dodecil sulfate, ethangent, fumarat, glucogheptanoate Rofosphate, hemysulfate, heptanoate , hexanoate, hydrochloride, hydro-bromide, hydroeodide, 2-hydroxyetan sulfate, lactate, maleat, methane sulfate, 2-naftalin sulfate, nicotinat, oxalat, palmoate, pectinate, perishefat, picopionate, picrot, pyvales, pioneer, sequicinate, tartraet, tartraet , Tozylate, undkanoat and so on. Other examples of salts include the anions of the compounds of the present invention in combination with a suitable cation such as Na ⁺ , NH ₄ ⁺ and NW ₄ ⁺ (where W is a C _1-4 alkyl group), and the like.

[0080] For therapeutic use, salts of the compounds of the present invention are considered pharmaceutically acceptable. However, salts of acids and bases that are not pharmaceutically acceptable may also find use, for example, in the preparation or purification of a pharmaceutically acceptable compound.

[0081] Throughout the specification, where compositions are described as containing or including specific components, or where processes and methods are described as containing or including specific steps, it is intended that in addition there are compositions of the present invention that consist essentially of or consist of the following components, and that there are processes and methods according to the present invention that consist essentially of or consist of the listed technological steps.

[0082] Typically, in compositions for which a percentage is stated, the percentage is by weight unless otherwise stated. Additionally, if a variable is not accompanied by a definition, then the previous definition of the variable takes precedence.

I. PROTEINS

[0083] The invention provides multispecific binding proteins that bind CD33 on a cancer cell and the NKG2D receptor and CD16 receptor on NK cells, causing activation of the NK cell. Multispecific binding proteins can be used in the pharmaceutical compositions and therapeutic methods described herein. The binding of multispecific binding protein to the NKG2D receptor and the CD16 receptor on the NK cell increases the activity of the NK cell towards destroying the cancer cell. Binding of the multispecific binding protein to CD33 on a cancer cell brings the cancer cell into close proximity to the NK cell, which facilitates direct and indirect destruction of the cancer cell by the NK cell. Additional description of exemplary multispecific binding proteins is provided below.

[0084] The first component of the multispecific binding proteins binds to cells expressing the NKG2D receptor, which may include, but is not limited to, NK cells, γδ T cells, and CD8 ⁺ αβ T cells. When NKG2D binds, multispecific binding proteins can block natural ligands such as ULBP6 and MICA from binding to NKG2D and activating NKG2D receptors.

[0085] The second component of the multispecific binding proteins binds to CD33-expressing cells, which may include, but are not limited to, AML, myelodysplastic syndromes, chronic myelomonocytic leukemia, myeloid blast crisis in chronic myeloid leukemia, and various forms of ALL.

[0086] The third component of multispecific binding proteins binds to cells expressing CD16, an Fc receptor on the surface of leukocytes, including natural killer cells, macrophages, neutrophils, eosinophils, mast cells and follicular dendritic cells.

[0087] The multispecific binding proteins described herein can be in a variety of formats. For example, one format is a heterodimeric, multispecific antibody comprising a first immunoglobulin heavy chain, a first immunoglobulin light chain, a second immunoglobulin heavy chain, and a second immunoglobulin light chain (FIG. 1). The first immunoglobulin heavy chain includes a first Fc domain (CH2-CH3 hinge), a first heavy chain variable domain, and optionally a first heavy chain CH1 domain. The first immunoglobulin light chain includes a first light chain variable domain and a first light chain constant domain. The first immunoglobulin light chain, together with the first immunoglobulin heavy chain, forms the antigen-binding site that binds NKG2D. The second immunoglobulin heavy chain includes a second Fc domain (CH2-CH3 hinge), a second heavy chain variable domain, and optionally a second heavy chain CH1 domain. The second immunoglobulin light chain includes a second light chain variable domain and a second light chain constant domain. The second immunoglobulin light chain, together with the second immunoglobulin heavy chain, forms the antigen-binding site that binds CD33. The first Fc domain and the second Fc domain together are capable of binding to CD16 (FIG. 1). In some embodiments, the first immunoglobulin light chain may be identical to the second immunoglobulin light chain.

[0088] Another exemplary format includes a heterodimeric, multispecific antibody comprising a first immunoglobulin heavy chain, a second immunoglobulin heavy chain, and an immunoglobulin light chain (FIG. 2). The first immunoglobulin heavy chain includes a first Fc domain (hinge-CH2-CH3), fused through either a linker or the hinge region of the antibody to a single chain variable fragment (scFv), consisting of a heavy chain variable domain and a light chain variable domain, which pairs and binds NKG2D or CD33. The second immunoglobulin heavy chain includes a second Fc domain (CH2-CH3 hinge), a second heavy chain variable domain, and optionally a CH1 heavy chain domain. The light chain of an immunoglobulin includes a light chain variable domain and a light chain constant domain. The second immunoglobulin heavy chain pairs with the immunoglobulin light chain and binds to NKG2D or CD33. The first Fc domain and the second Fc domain together are capable of binding to CD16 (FIG. 2).

[0089] One or more additional binding motifs may be fused to the C-terminus of the CH3 domain of the constant region, optionally via a linker sequence. In some embodiments, the antigen binding region may be a single chain or disulfide bond stabilized variable region (scFv) or may form a tetravalent or trivalent molecule.

[0090] In some embodiments, the multispecific binding protein is in the form of Triomab, which is a trifunctional, bispecific antibody that retains an IgG-like form. This chimera consists of two half-antibodies, each containing one light and one heavy chain, which are derived from two parent antibodies.

[0091] In some embodiments, the multispecific binding protein is in the same light chain (LC) KiH form, which includes knobs-in-holes (KIH) technology. KIH involves the design of C _H 3 domains to create a "bump" or a "cavity" in each heavy chain, which promotes heterodimerization. The concept of the Knobs-to-Hawts (KiH) Fc technology was to introduce a knob into one CH3 domain (CH3A) by replacing a small residue with a large one (eg T366W _CH3A according to EU numbering). To place a knob in another CH3 domain (CH3B), a complementary trench surface was created by replacing the closest neighboring residues to the knob with smaller ones (e.g., T366S/L368A/Y407V _CH3B ). The mutation resulting in the dimple was optimized using a phage library structural screen (Atwell S, Ridgway JB, Wells JA, Carter P, Stable heterodimers from remodeling the domain interface of a homodimer using a phage display library, J. Mol Biol (1997) 270(1):26-35). X-ray crystal structures of KiH Fc variants (Elliott JM, Ultsch M, Lee J, Tong R, Takeda K, Spiess C, et al., Antiparallel conformation of knob and hole aglycosylated half-antibody homodimers is mediated by a CH2-CH3 hydrophobic interaction. J. Mol. Biol. (2014) 426(9):1947–57; Mimoto F, Kadono S, Katada H, Igawa T, Kamikawa T, Hattori K. Crystal structure of a novel asymmetrically engineered Fc variant with improved affinity for FcgammaRs Mol. Immunol. (2014) 58(1):132-8) demonstrated that heterodimerization is thermodynamically promoted by hydrophobic interactions due to steric complementarity in the central contact region between the CH3 domains, while the knob-knob and trench-valley contact regions are not. homodimerization due to steric hindrance and blocking of required interactions, respectively.

[0092] In some embodiments, the multispecific binding protein is in the form of a dual variable domain immunoglobulin (DVD-Ig ^™ ), in which the target binding domains of two monoclonal antibodies are combined through flexible natural linkers to produce a tetravalent IgG-like molecule.

[0093] In some embodiments, the multispecific binding protein is in the form of an Orthogonal Fab interface (Ortho-Fab). In the Ortho-Fab IgG method (Lewis SM, Wu X, Pustilnik A, Sereno A, Huang F, Rick HL, et al., Generation of bispecific IgG antibodies by structure-based design of an orthogonal Fab interface. Nat. Biotechnol. ( 2014) 32(2):191-8), using structure-based region engineering, introduces additional mutations in the LC and the HCVH-CH1 interface in only one Fab without introducing any changes in the other Fab.

[0094] In some embodiments, the multispecific binding protein is in the Ig 2 in 1 format. In some embodiments, the multispecific binding protein is in the ES form, which is a heterodimeric construct containing two different Fabs binding to target 1 and target 2 fused to an Fc. Heterodimerization is mediated by electrostatically directed mutations in Fc. In some embodiments, the multispecific binding protein is in the form of a κλ-Body, which is a heterodimeric construct with two different Fabs fused to an Fc stabilized by heterodimerization mutations: Fab1 directed against antigen 1 contains LC kappa, while the second Fab directed against antigen 2, contains LC lambda. FIG. 30A is an illustrative diagram of one form of κλ-Body; FIG. 30B is an illustrative diagram of yet another κλ-Body.

[0095] In some embodiments, the multispecific binding protein is in the form of a Fab Arm Exchange (antibodies that exchange Fab arms by replacing the heavy chain and attached light chain (half-molecule) with a heavy light chain pair from another molecule, resulting in bispecific antibodies). In some embodiments, the multispecific binding protein is in the form of a SEED Body. The strand exchange engineered domain (SEED) platform has been developed to produce asymmetric and bispecific antibody-like molecules whose properties expand the therapeutic applications of natural antibodies. This protein engineering platform is based on the exchange of structurally related immunoglobulin sequences within conserved CH3 domains. The SEED design allows efficient creation of AG/GA heterodimers while eliminating homodimerization of the AG and GA SEED CH3 domains (Muda M. et al., Protein Eng. Des. Sel. (2011, 24(5):447-54)). In some embodiments, the multispecific binding protein is in the form of LuZ-Y, in which a leucine zipper is used to induce heterodimerization of two different HCs (Wranik, BJ. et al., J. Biol. Chem. (2012), 287:43331-9) .

[0096] In some embodiments, the multispecific binding protein is in the form of Cov-X-Body. In bispecific CovX bodies, two different peptides are combined using a branched azetidinone linker and fused to a scaffold antibody under mild conditions in a site-specific manner. Given that pharmacophores are responsible for functional activities, the immunoglobulin scaffold imparts a long half-life and Ig-like distribution. Pharmacophores can be chemically optimized or replaced with other pharmacophores to produce optimized or unique bispecific antibodies (Doppalapudi VR et al., PNAS (2010), 107(52);22611-22616).

[0097] In some embodiments, the multispecific binding protein is in an Oasc-Fab heterodimeric form that includes a target-binding Fab 1 and a target-binding scFab 2 fused to an Fc. Heterodimerization is ensured by mutations in Fc.

[0098] In some embodiments, the multispecific binding protein is in the form of a DuetMab, which is a heterodimeric construct containing two different Fabs that bind antigens 1 and 2, and an Fc stabilized by heterodimerization mutations. Fab 1 and 2 contain different S-S bridges that ensure proper LC and HC connection.

[0099] In some embodiments, the multispecific binding protein is in the form of a CrossmAb, which is a heterodimeric construct with two different Fabs binding to targets 1 and 2 fused to an Fc stabilized by heterodimerization. The CL and CH1 domains and the VH and VL domains are rearranged, for example, CH1 is fused in series to VL while CL is fused in series to VH.

[00100] In some embodiments, the multispecific binding protein is in the form of a Fit-Ig, which is a homodimeric construct in which a Fab that binds antigen 2 is fused to the N-terminus of an HC Fab that binds antigen 1. The construct contains a wild-type Fc .

[00101] Table 1 lists the peptide sequences of heavy chain variable domains and light chain variable domains that, in combination, can bind to NKG2D. NKG2D-binding domains may have different binding affinities for NKG2D, however, they all activate human NKG2D and NK cells.

Table 1 Clones Amino acid sequence of the heavy chain variable region Amino acid sequence of the light chain variable region ADI-27705 QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEIDHSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARARGPWSFDPWGQGTLVTVSS
(SEQ ID NO:1)
CDR1 (SEQ ID NO:54) - GSFSGYYWS
CDR2 (SEQ ID NO:55) -
EIDHSGSTNYNPSLKS
CDR3 (SEQ ID NO:56) -
ARARGPWSFDP DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYKASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYNSYPITFGGGTKVEIK
(SEQ ID NO:2) ADI-27724 QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEIDHSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARARGPWSFDPWGQGTLVTVSS
(SEQ ID NO:3) EIVLTQSPGTLLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPITFGGGTKVEIK
(SEQ ID NO:4) ADI-27740
(A40) QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEIDHSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARARGPWSFDPWGQGTLVTVSS
(SEQ ID NO:5) DIQMTQSPSTLSASVGDRVTITCRASQSIGSWLAWYQQKPGKAPKLLIYKASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYHSFYTFGGGTKVEIK
(SEQ ID NO:6) ADI-27741 QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEIDHSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARARGPWSFDPWGQGTLVTVSS
(SEQ ID NO:7) DIQMTQSPSTLSASVGDRVTITCRASQSIGSWLAWYQQKPGKAPKLLIYKASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQSNSYYTFGGGTKVEIK
(SEQ ID NO:8) ADI-27743 QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEIDHSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARARGPWSFDPWGQGTLVTVSS
(SEQ ID NO:9) DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYKASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYNSYPTFGGGTKVEIK
(SEQ ID NO:10) ADI-28153 QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEIDHSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARARGPWGFDPWGQGTLVTVSS
(SEQ ID NO:11) ELQMTQSPSSLSASVGDRVTITCRTSQSISSYLNWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQPEDSATYYCQQSYDIPYTFGQGTKLEIK
(SEQ ID NO:12) ADI-28226
(C26) QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEIDHSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARARGPWSFDPWGQGTLVTVSS
(SEQ ID NO:13) DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYKASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYGSFPITFGGGTKVEIK
(SEQ ID NO:14) ADI-28154 QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEIDHSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARARGPWSFDPWGQGTLVTVSS
(SEQ ID NO:15) DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYKASSLESGVPSRFSGSGSGTDFTLTISSLQPDDFATYYCQQSKEVPWTFGQGTKVEIK
(SEQ ID NO:16) ADI-29399 QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEIDHSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARARGPWSFDPWGQGTLVTVSS
(SEQ ID NO:17) DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYKASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYNSFPTFGGGTKVEIK
(SEQ ID NO:18) ADI-29401 QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEIDHSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARARGPWSFDPWGQGTLVTVSS
(SEQ ID NO:19) DIQMTQSPSTLSASVGDRVTITCRASQSIGSWLAWYQQKPGKAPKLLIYKASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYDIYPTFGGGTKVEIK
(SEQ ID NO:20) ADI-29403 QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEIDHSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARARGPWSFDPWGQGTLVTVSS
(SEQ ID NO:21) DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYKASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYDSYPTFGGGTKVEIK
(SEQ ID NO:22) ADI-29405 QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEIDHSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARARGPWSFDPWGQGTLVTVSS
(SEQ ID NO:23) DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYKASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYGSFPTFGGGTKVEIK
(SEQ ID NO:24) ADI-29407 QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEIDHSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARARGPWSFDPWGQGTLVTVSS
(SEQ ID NO:25) DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYKASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYQSFPTFGGGTKVEIK
(SEQ ID NO:26) ADI-29419 QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEIDHSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARARGPWSFDPWGQGTLVTVSS
(SEQ ID NO:27) DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYKASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYSSFSTFGGGTKVEIK
(SEQ ID NO:28) ADI-29421 QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEIDHSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARARGPWSFDPWGQGTLVTVSS
(SEQ ID NO:29) DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYKASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYESYSTFGGGTKVEIK
(SEQ ID NO:30) ADI-29424 QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEIDHSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARARGPWSFDPWGQGTLVTVSS
(SEQ ID NO:31) DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYKASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYDSFITFGGGTKVEIK
(SEQ ID NO:32) ADI-29425 QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEIDHSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARARGPWSFDPWGQGTLVTVSS
(SEQ ID NO:33) DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYKASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYQSYPTFGGGTKVEIK
(SEQ ID NO:34) ADI-29426 QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEIDHSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARARGPWSFDPWGQGTLVTVSS
(SEQ ID NO:35) DIQMTQSPSTLSASVGDRVTITCRASQSIGSWLAWYQQKPGKAPKLLIYKASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYHSFPTFGGGTKVEIK
(SEQ ID NO:36) ADI-29429 QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEIDHSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARARGPWSFDPWGQGTLVTVSS
(SEQ ID NO:37) DIQMTQSPSTLSASVGDRVTITCRASQSIGSWLAWYQQKPGKAPKLLIYKASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYELYSYTFGGGTKVEIK
(SEQ ID NO:38) ADI-29447
(F47) QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEIDHSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARARGPWSFDPWGQGTLVTVSS
(SEQ ID NO:39) DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYKASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYDTFITFGGGTKVEIK
(SEQ ID NO:40) ADI-27727 QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARGDSSIRHAYYYYGMDVWGQGTTVTVSS
(SEQ ID NO:41)
CDR1 (SEQ ID NO:57) -
GTFSSYAIS
CDR2 (SEQ ID NO:58) -
GIIPIFGTANYAQKFQG
CDR3 (SEQ ID NO:59) -
ARGDSSIRHAYYYYGMDV DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSTPITFGGGTKVEIK
(SEQ ID NO:42)
CDR1 (SEQ ID NO:60) -
KSSQSVLYSSNNNKNYLA
CDR2 (SEQ ID NO:61) -
WASTRES
CDR3 (SEQ ID NO:62) -
QQYYSTPIT ADI-29443
(F43) QLQLQESGPGLVKPSETLSLTCTVSGGSISSSSYYWGWIRQPPGKGLEWIGSIYYSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARGSDRFHPYFDYWGQGTLVTVSS
(SEQ ID NO:43)
CDR1 (SEQ ID NO:63) -
GSISSSSYYWG
CDR2 (SEQ ID NO:64) -
SIYYSGSTYYNPSLKS
CDR3 (SEQ ID NO:65) -
ARGSDRFHPYFDY EIVLTQSPATLSLSPGERATLSCRASQSVSRYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQFDTWPPTFGGGTKVEIK
(SEQ ID NO:44)
CDR1 (SEQ ID NO:66) -
RASQSVSRYLA
CDR2 (SEQ ID NO:67) -
DASNRAT
CDR3 (SEQ ID NO:68) -
QQFDTWPPT ADI-29404
(F04) QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEIDHSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARARGPWSFDPWGQGTLVTVSS
(SEQ ID NO:45) DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYKASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCEQYDSYPTFGGGTKVEIK
(SEQ ID NO:46) ADI-28200 QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARRGRKASGSFYYYYGMDVWGQGTTVTVSS
(SEQ ID NO:47) DIVMTQSPDSLAVSLGERATINCESSQSLLNSGNQKNYLTWYQQKPGQPPKPLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQNDYSYPYTFGQGTKLEIK
(SEQ ID NO:48) ADI-27744
(A44) EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDGGYYDSGAGDYWGQGTLVTVSS
(SEQ ID NO:69)
CDR1 (SEQ ID NO:71) - FTFSSYAMS
CDR2 (SEQ ID NO:72) - AISGSGGSTYYADSVKG
CDR3 (SEQ ID NO:73) - AKDGGYYDSGAGDY DIQMTQSPSSVSASVGDRVTITCRASQGIDSWLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGVSYPRTFGGGTKVEIK
(SEQ ID NO:70)
CDR1 (SEQ ID NO:74) - RASQGIDSWLA
CDR2 (SEQ ID NO:75) - AASSLQS
CDR3 (SEQ ID NO:76) - QQGVSYPRT ADI-27749
(A49) EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSSISSSSSYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGAPMGAAAGWFDPWGQGTLVTVSS
(SEQ ID NO:77)
CDR1 (SEQ ID NO:79) - FTFSSYSMN
CDR2 (SEQ ID NO:80) - SISSSSSYIYYADSVKG
CDR3 (SEQ ID NO:81) - ARGAPMGAAAGWFDP DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGVSFPRTFGGGTKVEIK
(SEQ ID NO:78)
CDR1 (SEQ ID NO:82) - RASQGISSWLA
CDR2 (SEQ ID NO:83) - AASSLQS
CDR3 (SEQ ID NO:84) - QQGVSFPRT ADI-29463
(F63) QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPNSGGTNYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARDTGEYYDTDDHGMDVWGQGTTVTVSS
(SEQ ID NO:85)
CDR1 (SEQ ID NO:87) - YTFTGYYMH
CDR2 (SEQ ID NO:88) - WINPNSGGTNYAQKFQG
CDR3 (SEQ ID NO:89) - ARDTGEYYDTDDHGMDV EIVLTQSPGTLLSLSPGERATLSCRASQSVSSNLAWYQQKPGQAPRLLIYGASTRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQDDYWPPTFGGGTKVEIK
(SEQ ID NO:86)
CDR1 (SEQ ID NO:90) - RASQSVSSNLA
CDR2 (SEQ ID NO:91) - GASTRAT
CDR3 (SEQ ID NO:92) - QQDDYWPPT ADI-29379
(E79) QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGIINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGAPNYGDTTHDYYYMDVWGKGTTVTVSS
(SEQ ID NO:133)
CDR1 (SEQ ID NO:135) - YTFTSYYMH
CDR2 (SEQ ID NO:136) - IINPSGGSTSYAQKFQG
CDR3 (SEQ ID NO:137) - ARGAPNYGDTTHDYYYMDV EIVMTQSPATLSVSPGERATLLSCRASQSVSSNLAWYQQKPGQAPRLLIYGASTRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYDDWPFTFGGGTKVEIK
(SEQ ID NO:134)
CDR1 (SEQ ID NO:138) - RASQSVSSNLA
CDR2 (SEQ ID NO:139) - GASTRAT
CDR3 (SEQ ID NO:140) - QQYDDWPFT

[00102] Alternatively, the heavy chain variable domain defined by SEQ ID NO:49 can be combined with the light chain variable domain defined by SEQ ID NO:50 to produce an antigen binding region that can bind NKG2D, as shown in US 9,273,136.

QVQLVESGGGLVKPGGSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAFIRYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDRGLGDGTYFDYWGQGTTVTVSS (SEQ ID NO:49)

QSALTQPASVSGSPGQSITISCSGSSSNIGNNAVNWYQQLPGKAPKLLIYYDDLLPSGVSDRFSGSKSGTSAFLAISGLQSEDEADYYCAAWDDSLNGPVFGGGTKLTVL (SEQ ID NO:50)

[00103] Alternatively, the heavy chain variable domain defined by SEQ ID NO:51 can be paired with the light chain variable domain defined by SEQ ID NO:52 to produce an antigen binding region that can bind NKG2D, as shown in US 7,879,985.

QVHLQESGPGLVKPSETLSLTCTVSDDSISSYYWSWIRQPPGKGLEWIGHISYSGSANYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCANWDDAFNIWGQGTMVTVSS (SEQ ID NO:51)

EIVLTQSPGTLLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPWTFGQGTKVEIK (SEQ ID NO:52)

[00104] Table 2 lists the peptide sequences of heavy chain variable domains and light chain variable domains that, in combination, can bind CD33.

table 2 Clones Heavy chain variable region peptide sequence Light chain variable region peptide sequence Lintuzumab QVQLVQSGAEVKKPGSSVKVSCKASGYTFTDYNMHWVRQAPGQGLEWIGYIYPYNGGTGYNQKFKSKATITADESTNTAYMELSSLRSEDTAVYYCARGRPAMDYWGQGTLVTVSS
(SEQ ID NO:93)
CDR1 (SEQ ID NO:95) - GYTFTDY
CDR2 (SEQ ID NO:96) - YIYPYNGGTG
CDR3 (SEQ ID NO:97) - GRPAMDY DIQMTQSPSSLSSASVGDRVTITCRASESVDNYGISFMNWFQQKPGKAPKLLIYAASNQGSGVPSRFSGSGSGTDFTLTISSLQPDDFATYYCQQSKEVPWTFGQGTKVEIK
(SEQ ID NO:94)
CDR1(SEQ ID NO:98) - ESVDNYGISFMN
CDR2 (SEQ ID NO:99) - AASNQGS
CDR3 (SEQ ID NO:100) - QQSKEVPWT Gemtuzumab EVQLVQSGAEVKKPGSSVKVSCKASGYTITDSNIHWVRQAPGQSLEWIGYIYPYNGGTDYNQKFKNRATLTVDNPTNTAYMELSSLRSEDTAFYYCVNGNPWLAYWGQGTLVTVSS
(SEQ ID NO:101)
CDR1 (SEQ ID NO:103) - GYTITDS
CDR2 (SEQ ID NO:104) - YIYPYNGGTD
CDR3 (SEQ ID NO:105) - GNPWLAY DIQLTQSPSTLSASVGDRVTITCRASESLDNYGIRFLTWFQQKPGKAPKLLMYAASNQGSGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQTKEVPWSFGQGTKVEVK
(SEQ ID NO:102)
CDR1 (SEQ ID NO:106) - ESLDNYGIRFLT
CDR2 (SEQ ID NO:107) - AASNQGS
CDR3 (SEQ ID NO:108) - QQTKEVPWS Anti-CD33 antibody (US 7,557,189) QVQLQQPGAEVVKPGASVKMSCKASGYTFTSYYIHWIKQTPGQGLEWVGVIYPGNDDISYNQKFQGKATLTADKSSTTAYMQLSSLTSEDSAVYYCAREVRLRYFDVWGQGTTVTVSS
(SEQ ID NO:109)
CDR1 (SEQ ID NO:111) - GYTFTSY
CDR2 (SEQ ID NO:112) - YPGNDD
CDR3 (SEQ ID NO:113) - EVRLRYFDV EIVLTQSPGSLAVSPGERVTMSCKSSQSVFFSSSQKNYLAWYQQIPGQSPRLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQPEDLAIYYCHQYLSSRTFGQGTKLEIKR
(SEQ ID NO:110)
CDR1 (SEQ ID NO:114) - QSVFFSSSQKNYLA
CDR2 (SEQ ID NO:115) - WASTRES
CDR3 (SEQ ID NO:116) - HQYLSSRT Vadastuximab (US 13/804,227) QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYDINWVRQAPGQGLEWIGWIYPGDGSTKYNEKFKAKATLTADTSTSTAYMELRSLRSDDTAVYYCASGYEDAMDYWGQGTTVTVSSA
(SEQ ID NO:117)
CDR1 (SEQ ID NO:119): GYTFTNY
CDR2 (SEQ ID NO:120): YPGDGS
CDR3 (SEQ ID NO:121): GYEDAMDY DIQMTQSPSSLSSASVGDRVTINCKASQDINSYLSWFQQKPGKAPKTLIYRANRLVDGVPSRFSGSGSGQDYTLTISSLQPEDFATYYCLQYDEFPLTFGGGTKVEIKR
(SEQ ID NO:118)
CDR1 (SEQ ID NO:122): QDINSYLS
CDR2 (SEQ ID NO:123): RANRLVD
CDR3 (SEQ ID NO:124): LQYDEFPLT

[00105] Alternatively, new antigen binding sites that can bind CD33 can be identified by screening for binding to the amino acid sequence defined by SEQ ID NO:53.

SEQ ID NO:53

MPLLLLLPLLWAGALAMDPNFWLQVQESVTVQEGLCVLVPCTFFHPIPYYDKNSPVHGYWFREGAIISRDSPVATNKLDQEVQEETQGRFRLLGDPSRNNCSLSIVDARRRDNGSYFFRMERGSTKYSYKSPQLSVHVTDLTHRPKILIPGTLEPGHSKNLTCSVSWACEQGTPPIFSWLSAAPTSLGPRTTHSSVLIITPRPQ DHGTNLTCQVKFAGAGVTTERTIQLNVTYVPQNPTTGIFPGDGSGKQETRAGVVHGAIGGAGVTALLALCLCLIFFIVKTHRRKAARTAVGRNDTHPTTGSASPKHQKKSKLHGPTETSSCSGAAPTVEMDEELHYASLNFHGMNPSKDTSTEYSEVRTQ

[00106] In the Fc domain, CD16 binding is mediated by the hinge region and the CH2 domain. For example, in human IgG1, interaction with CD16 is primarily directed to the amino acid residues Asp 265 - Glu 269, Asn 297 - Thr 299, Ala 327 - Ile 332, Leu 234 - Ser 239 and the carbohydrate residue N-acetyl-D-glucosamine in CH2 domain (see, Sondermann et al., Nature, 406 (6793):267-273). Based on known domains, mutations can be selected to increase or decrease CD16 binding affinity, for example using phage display libraries or yeast cDNA surface display libraries, or they can be designed based on a known three-dimensional interaction structure.

[00107] Assembly of heterodimeric antibody heavy chains can be accomplished by expressing two different antibody heavy chain sequences in the same cell, which can result in the assembly of homodimers of each antibody heavy chain, as well as the assembly of heterodimers. Ensuring preferential assembly of heterodimers can be achieved by introducing various mutations into the CH3 domain of each constant region of the antibody heavy chain, as shown in US13/494870, US16/028850, US11/533709, US12/875015, US13/289934, US14/773418, US12/ 811207, US13/866756, US14/647480 and US14/830336. For example, mutations may be introduced into the CH3 domain of human IgG1 and involve different pairs of amino acid substitutions in the first polypeptide and the second polypeptide that allow the two chains to selectively heterodimerize with each other. The positions of the amino acid substitutions presented below are numbered according to the EU index, as described in Kabat's publication.

[00108] In one situation, an amino acid substitution in the first polypeptide consists of replacing the parent amino acid with a larger amino acid selected from arginine (R), phenylalanine (F), tyrosine (Y), or tryptophan (W), and at least one amino acid substitution in the second polypeptide consists of replacing the original amino acid(s) with a smaller amino acid(s) selected from alanine (A), serine (S), threonine (T) or valine (V), with the larger amino acid replacement (overhang) placed in surface of a smaller amino acid substitution (hole). For example, one polypeptide may include the T366W substitution, and another may include three substitutions including T366S, L368A, and Y407V.

[00109] The heavy chain variable domain of an antibody of the invention may optionally be linked to an amino acid sequence that is at least 90% identical to an antibody constant region, such as an IgG constant region including the hinge, CH2 and CH3 domains, with or without a CH1 domain. . In some embodiments, the amino acid sequence of the constant region is at least 90% identical to the constant region of a human antibody, such as a human IgG1 constant region, a human IgG2 constant region, a human IgG3 constant region, or a human IgG4 constant region. In some other embodiments, the amino acid sequence of the constant region is at least 90% identical to the constant region of an antibody from another mammal, such as a rabbit, dog, cat, mouse, or horse. One or more mutations may be introduced into the constant region compared to the human IgG1 constant region, for example, Q347, Y349, L351, S354, E356, E357, K360, Q362, S364, T366, L368, K370, N390, K392, T394, D399, S400, D401, F405, Y407, K409, T411 and/or K439. Example replacements include, for example, Q347E, Q347R, Y349S, Y349K, Y349T, Y349D, Y349E, Y349C, T350V, L351K, L351D, L351Y, S354C, E356K, E357Q, E357L, E357W, K360E, K360W, 62E, S364K, S364E, S364H, S364D, T366V, T366I, T366L, T366M, T366K, T366W, T366S, L368E, L368A, L368D, K370S, N390D, N390E, K392L, K392M, K392V, K392F, K392D, 2E, T394F, T394W, D399R, D399K, D399V, S400K, S400R, D401K, F405A, F405T, Y407A, Y407I, Y407V, K409F, K409W, K409D, T411D, T411E, K439D and K439E.

[00110] In some embodiments, mutations that may be introduced into the CH1 constant region of human IgG1 may be at amino acids V125, F126, P127, T135, T139, A140, F170, P171, and/or V173. In some embodiments, mutations that may be introduced into the Cκ constant region of human IgG1 may be at amino acids E123, F116, S176, V163, S174, and/or T164.

[00111] Amino acid substitutions can be selected from the following sets of substitutions shown in Table 3.

Table 3 First polypeptide Second polypeptide Set 1 S364E/F405A Y349K/T394F Set 2 S364H/D401K Y349T/T411E Set 3 S364H/T394F Y349T/F405A Set 4 S364E/T394F Y349K/F405A Set 5 S364E/T411E Y349K/D401K Set 6 S364D/T394F Y349K/F405A Set 7 S364H/F405A Y349T/T394F Set 8 S364K/E357Q L368D/K370S Set 9 L368D/K370S S364K Set 10 L368E/K370S S364K Set 11 K360E/Q362E D401K Set 12 L368D/K370S S364K/E357L Set 13 K370S S364K/E357Q Set 14 F405L K409R Set 15 K409R F405L

[00112] Alternatively, amino acid substitutions may be selected from the following sets of substitutions shown in Table 4.

Table 4 First polypeptide Second polypeptide Set 1 K409W D399V/F405T Set 2 Y349S E357W Set 3 K360E Q347R Set 4 K360E/K409W Q347R/D399V/F405T Set 5 Q347E/K360E/K409W Q347R/D399V/F405T Set 6 Y349S/K409W E357W/D399V/F405T

[00113] Alternatively, amino acid substitutions may be selected from the following sets of substitutions shown in Table 5.

Table 5 First polypeptide Second polypeptide Set 1 T366K/L351K L351D/L368E Set 2 T366K/L351K L351D/Y349E Set 3 T366K/L351K L351D/Y349D Set 4 T366K/L351K L351D/Y349E/L368E Set 5 T366K/L351K L351D/Y349D/L368E Set 6 E356K/D399K K392D/K409D

[00114] Alternatively, at least one amino acid substitution in each polypeptide chain may be selected from Table 6.

Table 6 First polypeptide Second polypeptide L351Y, D399R, D399K, S400K, S400R, Y407A, Y407I, Y407V T366V, T366I, T366L, T366M, N390D, N390E, K392L, K392M, K392V, K392F K392D, K392E, K409F, K409W, T411D and T411E

[00115] Alternatively, the at least one amino acid substitution may be selected from the following set of substitutions in Table 7, wherein the position(s) indicated in the First Polypeptide column is replaced by any known negatively charged amino acid, and the position(s) indicated in the column The second polypeptide is replaced by any known positively charged amino acid.

Table 7 First polypeptide Second polypeptide K392, K370, K409 or K439 D399, E356 or E357

[00116] Alternatively, the at least one amino acid substitution may be selected from the following set in Table 8, wherein the position(s) indicated in the First polypeptide column is replaced by any known positively charged amino acid, and the position(s) indicated in the Second column polypeptide replaced by any known negatively charged amino acid.

Table 8 First polypeptide Second polypeptide D399, E356 or E357 K409, K439, K370 or K392

[00117] Alternatively, amino acid substitutions may be selected from the following set in Table 9.

Table 9 First polypeptide Second polypeptide T350V, L351Y, F405A and Y407V T350V, T366L, K392L and T394W

[00118] Alternatively or additionally, the structural stability of the heteromultimeric protein can be increased by introducing S354C into either the first or second polypeptide chain and Y349C into the opposing polypeptide chain, which forms an artificial disulfide bond at the interface of the two polypeptides.

[00119] The multispecific proteins described above can be produced using recombinant DNA technology known to one skilled in the art. For example, a first nucleic acid sequence encoding a first immunoglobulin heavy chain may be cloned into a first expression vector; a second nucleic acid sequence encoding a second immunoglobulin heavy chain may be cloned into a second expression vector; a third nucleic acid sequence encoding an immunoglobulin light chain may be cloned into a third expression vector; the first, second and third expression vectors can be stably transfected together into host cells to produce multimeric proteins.

[00120] To obtain the highest yield of multispecific protein, various ratios of the first, second, and third expression vectors can be examined to determine the optimal ratio for transfection into host cells. After transfection, individual clones can be isolated to obtain a cell bank using methods known in the art, such as serial dilution, ELISA, FACS, microscopy or Clonepix.

[00121] Clones can be cultured under conditions suitable for bioreactor scale-up and maintenance of multispecific protein expression. Multispecific proteins can be isolated and purified using methods known in the art, including centrifugation, depth filtration, cell lysis, homogenization, freeze-thaw, affinity purification, gel filtration, ion exchange chromatography, hydrophobic interaction exchange chromatography and mixed-mode chromatography.

II. Characteristics of multispecific proteins

[00122] In some embodiments, the multispecific proteins described herein, which include the NKG2D binding domain and the CD33 binding domain, bind to cells expressing human NKG2D. In some embodiments, the multispecific proteins bind to the CD33 tumor-associated antigen at a level comparable to a monoclonal antibody having the same CD33-binding domain. However, the multispecific proteins described herein may be more effective in reducing tumor growth and killing CD33-expressing cancer cells than the corresponding anti-CD33 monoclonal antibodies.

[00123] In some embodiments, the multispecific proteins described herein, which include the NKG2D binding domain and the CD33 binding domain, can activate primary human NK cells when cultured with tumor cells expressing the CD33 antigen. NK cell activation is characterized by increased CD107a degranulation and production of the cytokine IFNγ. In addition, compared with a monoclonal antibody that includes the same CD33-binding domain, the multispecific proteins show superior activation of human NK cells in the presence of tumor cells expressing the CD33 antigen.

[00124] In some embodiments, the multispecific proteins described herein, which include the NKG2D binding domain and the CD33 binding domain, can enhance the activity of resting and IL-2-activated human NK cells in the presence of tumor cells expressing the CD33 antigen.

[00125] In some embodiments, the multispecific proteins described herein, which include the NKG2D-binding domain and the CD33 tumor-associated antigen binding domain, can enhance the cytotoxic activity of resting and IL-2-activated human NK cells in the presence of tumor cells expressing CD33 antigen. In some embodiments, compared to corresponding monoclonal antibodies, multispecific proteins may provide an advantage against tumor cells expressing moderate to low levels of CD33.

[00126] In some embodiments, the multispecific proteins described herein may be useful in the treatment of cancers with high Fc receptor (FcR) expression or cancers located in the tumor microenvironment with high levels of FcR, compared to corresponding monoclonal antibodies to CD33. Monoclonal antibodies exert their effects on tumor growth through multiple mechanisms including, but not limited to, ADCC, CDC, phagocytosis, and signal blockade. Among the various FcγR receptors, CD16 has the lowest affinity for IgG Fc; FcγRI (CD64) is a high-affinity FcR that binds approximately 1000 times more strongly to IgG Fc than CD16. CD64 is commonly expressed on many hematopoietic lineages, such as the myeloid lineage, and can be expressed on tumors derived from these cell types, such as acute myeloid leukemia (AML). Tumor-infiltrating immune cells, such as MDSCs and monocytes, also express CD64 and are known to infiltrate the tumor microenvironment. Expression of CD64 by the tumor or in the tumor microenvironment may have a negative impact on monoclonal antibody therapy. Expression of CD64 in the tumor microenvironment makes it difficult for these antibodies to interact with CD16 on the surface of NK cells, as the antibodies prefer to bind the receptor with high affinity. Multispecific proteins, when acting on two activating receptors on the surface of NK cells, can overcome the negative impact of CD64 expression (either on tumors or in the tumor microenvironment) on monoclonal antibody therapy. Regardless of CD64 expression on tumor cells, multispecific proteins are able to mediate human NK cell responses against all tumor cells because dual interaction with two activating receptors on NK cells allows for stronger NK cell specific binding.

[00127] In some embodiments, the multispecific proteins described herein may provide a better safety profile by reducing targeted non-tumor side effects. Natural killer cells and CD8 T cells are able to directly lyse tumor cells, although the mechanisms by which NK cells and CD8 T cells recognize normal autologous cells, distinguishing them from tumor cells, differ. The activity of NK cells is regulated by the balance of signals from activating (NCR, NKG2D, CD16, etc.) and inhibitory (KIR, NKG2A, etc.) receptors. The balance of these activating and inhibitory signals allows NK cells to distinguish healthy autologous cells from stressed, virus-infected, or degenerated autologous cells. This "built-in" self-tolerance mechanism allows normal healthy tissue to be protected from NK cell responses. To extend this principle, NK cell self-tolerance would allow TriNKET constructs to specifically interact with antigens expressed on both autologous cells and tumors without non-tumor side effects or with an extended therapeutic window. Unlike natural killer cells, T cells require recognition of a specific peptide presented by MHC molecules for activation and effector functions. T cells have been a major target of immunotherapy, and many strategies have been developed to redirect T cell responses against the tumor. Bispecific T cells, checkpoint inhibitors, and CAR T cells have been approved by the FDA, but they often suffer from dose-limiting toxicities. Bispecific T cells and CAR-T cells operate within the TCR-MHC recognition system, using binding domains to target antigens on the surface of tumor cells and using modified signaling domains to transmit activation signals to the effector cell. Although these treatments are effective in inducing an antitumor immune response, they are often associated with cytokine release syndrome (CRS) and nontumor side effects. Multispecific proteins are unique in that they will not “block” the natural NK cell activation and inhibition systems. In contrast, multispecific proteins are designed to maintain balance and provide additional activation signals to NK cells while maintaining NK cell tolerance to healthy autologous cells.

[00128] In some embodiments, the multispecific proteins described herein may delay tumor development more effectively than corresponding anti-CD33 monoclonal antibodies that include the same CD33-binding domain. In some embodiments, the multispecific proteins described herein may be more effective against metastases than corresponding anti-CD33 monoclonal antibodies that include the same CD33-binding domain.

III. THERAPEUTIC APPLICATIONS

[00129] The invention provides methods for treating cancer using a multispecific binding protein described herein and/or a pharmaceutical composition described herein. The methods can be used to treat various forms of cancer that express CD33 by administering to a patient in need thereof a therapeutically effective amount of the multispecific binding protein described herein.

[00130] The therapeutic method may be characterized depending on the cancer being treated. For example, in some embodiments, the cancer is AML, myelodysplastic syndromes, chronic myelomonocytic leukemia, myeloid blast crisis in chronic myeloid leukemia, and various forms of ALL.

[00131] In some other embodiments, the cancer is brain cancer, breast cancer, cervical cancer, colon cancer, colorectal cancer, endometrial cancer, esophageal cancer, leukemia, lung cancer, liver cancer, melanoma, cancer ovarian cancer, pancreatic cancer, colorectal cancer, kidney cancer, stomach cancer, testicular cancer or uterine cancer. In other embodiments, the cancer is squamous cell carcinoma, adenocarcinoma, small cell carcinoma, melanoma, neuroblastoma, sarcoma (e.g., angiosarcoma or chondrosarcoma), laryngeal cancer, parotid cancer, bile duct cancer, thyroid cancer, acral lentiginous melanoma, actinic keratosis, acute lymphocytic leukemia, acute myeloid leukemia, adenoid cystic carcinoma, adenomas, adenosarcoma, adenosquamous carcinoma, anal cancer, astrocytic tumor, Bartholin gland carcinoma, basal cell carcinoma, biliary tract cancer, bone cancer, bone marrow cancer, bronchial cancer, bronchial gland carcinoma, carcinoid, cholangiocarcinoma, chondosarcoma, choroid plexus papilloma/carcinoma, chronic lymphocytic leukemia, chronic myeloid leukemia, clear cell carcinoma, connective tissue cancer, cystadenoma, digestive system cancer, duodenal cancer, endocrine system cancer, endodermal sinus tumor, endometrial hyperplasia, stromal endometrial sarcoma, endometrioid adenocarcinoma, endothelial cell cancer, ependymal cancer, epithelial cancer, Ewing's sarcoma, eye cancer, genital cancer in women, focal nodular hyperplasia, gallbladder cancer, antral cancer, fundus cancer, gastrinoma, glioblastoma, glucagonoma, heart cancer, hemangiblastoma, hemangioendothelioma, hemangiomas, liver adenoma, hepatic adenomatosis, hepatobiliary cancer, hepatocellular cancer, Hodgkin's disease, ileal cancer, insulinoma, intraepithelial neoplasia, interepithelial squamous neoplasia, intrahepatic bile duct cancer, invasive squamous cell carcinoma, jejunal cancer colon, joint cancer, Kaposi's sarcoma, renal pelvis cancer, large cell carcinoma, colon cancer, leiomyosarcoma, lentigo maligna melanoma, lymphoma, male genital cancer, malignant melanoma, malignant mesothelial tumors, medulloblastoma, medulloepithelioma, meningeal cancer, mesothelial cancer , metastatic carcinoma, oral cancer, mucoepidermoid carcinoma, multiple myeloma, muscle cancer, nasal canal cancer, nervous system cancer, neuroepithelial adenocarcinoma, nodular melanoma, nonepithelial skin cancer, non-Hodgkin's lymphoma, oat cell carcinoma, oligodendroglial cancer, oral cancer, osteosarcoma, papillary serous adenocarcinoma, penile cancer, pharyngeal cancer, pituitary tumors, plasmacytoma, pseudosarcoma, lung blastoma, rectal cancer, renal cell cancer, respiratory system cancer, retinoblastoma, rhabdomyosarcoma, sarcoma, serous carcinoma, sinus cancer, skin cancer , skin cancer, small cell carcinoma, small bowel cancer, smooth muscle cancer, soft tissue cancer, somatostatin-secreting tumor, spine cancer, squamous cell carcinoma, striated muscle cancer, submesothelial cancer, superficial spreading melanoma, T-cell leukemia, tongue cancer , undifferentiated carcinoma, ureteral cancer, urethral cancer, bladder cancer, urinary system cancer, cervical cancer, uterine cancer, uveal melanoma, vaginal cancer, verrucous carcinoma, VIPoma, vulvar cancer, well-differentiated carcinoma or Wilms tumor.

[00132] In some other embodiments, the cancer is non-Hodgkin's lymphoma, such as B-cell lymphoma or T-cell lymphoma. In some embodiments, the non-Hodgkin lymphoma is a B-cell lymphoma, such as diffuse large B-cell lymphoma, primary mediastinal B-cell lymphoma, follicular lymphoma, lymphocytic lymphoma, mantle cell lymphoma, marginal zone cell B-cell lymphoma, extranodal B-cell lymphoma marginal zone cell lymphoma, nodal marginal zone B-cell lymphoma, splenic marginal zone B-cell lymphoma, Burkitt's lymphoma, lymphoplasmacytic lymphoma, hairy cell leukemia, or primary central nervous system (CNS) lymphoma. In some other embodiments, the non-Hodgkin's lymphoma is a T-cell lymphoma, such as T-lymphoblastic progenitor cell lymphoma, peripheral T-cell lymphoma, cutaneous T-cell lymphoma, angioimmunoblastic T-cell lymphoma, extranodal NK cell/T-cell lymphoma, enteropathic type T-cell lymphoma, subcutaneous panniculitis-like T-cell lymphoma, anaplastic large cell lymphoma, or peripheral T-cell lymphoma.

[00133] The cancer to be treated can be characterized according to the presence of a specific antigen expressed on the surface of the cancer cell. In some embodiments, the cancer cell may express one or more of the following in addition to CD33: CD2, CD19, CD20, CD30, CD38, CD40, CD52, CD70, EGFR/ERBB1, IGF1R, HER3/ERBB3, HER4/ERBB4, MUC1, cMET, SLAMF7, PSCA, MICA, MICB, TRAILR1, TRAILR2, MAGE-A3, B7.1, B7.2, CTLA4 and PD1.

IV. COMBINATION THERAPY

[00134] In another aspect of the invention, combination therapy is provided. The multispecific binding proteins described herein can be used to treat cancer in combination with additional therapeutic agents.

[00135] Examples of therapeutic agents that may be used as part of combination therapy in the treatment of cancer include, for example, radiation, mitomycin, tretinoin, ribomustine, gemcitabine, vincristine, etoposide, cladribine, mitobronitol, methotrexate, doxorubicin, carboquone, pentostatin, nitracrine , zinostatin, cetrorelix, letrozole, raltitrexed, daunorubicin, fadrozole, fotemustine, thymalfasin, sobuzoxan, nedaplatin, cytarabine, bicalutamide, vinorelbine, vesnarinone, aminoglutethimide, amsacrine, proglumide, elliptinium acetate, ketanserin, doxyfluridine, , isotretinoin, streptozocin, nimustine, vindesine, flutamide, drogenil, butocin, carmofur, razoxane, sisofilan, carboplatin, mitolactol, tegafur, ifosfamide, prednimustine, picibanil, levamisole, teniposide, improsulfan, enocytabine, lisuride, oxymetholone, tamoxifen, progesterone, mepithiostane, epithiostanol, formestane, interferon- 2-alpha, interferon-beta, interferon-gamma, colony-stimulating factor-1, colony-stimulating factor-2, denileukin diftitox, interleukin-2, luteinizing hormone releasing factor, and variations of the above agents that may exhibit differential binding to its cognate receptor, and also increase or decrease the half-life of existence in serum.

[00136] An additional class of agents that may be used as part of combination therapy in the treatment of cancer are immune checkpoint inhibitors. Examples of immune checkpoint inhibitors include agents that inhibit one or more of the following: (i) cytotoxic T-lymphocyte-associated antigen 4 (CTLA4), (ii) programmed cell death protein 1 (PD1), (iii) PDL1, (iv) LAG3, (v) B7-H3, (vi) B7-H4 and (vii) TIM3. The CTLA4 inhibitor ipilimumab has been approved by the US Food and Drug Administration for the treatment of melanoma.

[00137] Other agents that may be used as part of combination therapy in the treatment of cancer are monoclonal antibody agents that target non-checkpoint targets (e.g., Herceptin) and non-cytotoxic agents (e.g., inhibitors tyrosine kinase).

[00138] Other categories of antitumor agents include, for example: (i) an inhibitor selected from ALK inhibitor, ATR inhibitor, A2A antagonist, base excision repair inhibitor, Bcr-Abl tyrosine kinase inhibitor, Bruton's tyrosine kinase inhibitor, CDC7 inhibitor, CHK1 inhibitor, cyclin inhibitor -dependent kinase, DNA-PK inhibitor, DNA-PK and mTOR inhibitor, DNMT1 inhibitor, DNMT1 inhibitor plus 2-chloro-deoxyadenosine, HDAC inhibitor, Hedgehog signaling pathway inhibitor, IDO inhibitor, JAK inhibitor, mTOR inhibitor, MEK inhibitor, MELK inhibitor , MTH1 inhibitor, PARP inhibitor, phosphoinositide 3-kinase inhibitor, PARP1 and DHODH inhibitor, proteasome inhibitor, topoisomerase-II inhibitor, tyrosine kinase inhibitor, VEGFR inhibitor and WEE1 inhibitor; (ii) an agonist for OX40, CD137, CD40, GITR, CD27, HVEM, TNFRSF25, or ICOS; and (iii) a cytokine selected from IL-12, IL-15, GM-CSF and G-CSF

[00139] The proteins of the invention can also be used as an adjuvant in surgical removal of the primary lesion.

[00140] The amount of multispecific binding protein and additional therapeutic agent, as well as the relative timing of application, can be selected to achieve the desired combined therapeutic effect. For example, in the case of administering combination therapy to a patient in need thereof, the therapeutic agents in the combination or the pharmaceutical composition or compositions comprising the therapeutic agents may be administered in any order, for example, sequentially, in parallel, together, simultaneously, and the like. In addition, for example, the multispecific binding protein may be administered during the time that the additional therapeutic agent(s) are exerting their prophylactic or therapeutic effects, or vice versa.

V. PHARMACEUTICAL COMPOSITIONS

[00141] The present invention also provides pharmaceutical compositions that contain a therapeutically effective amount of a protein described herein. The composition can be formulated for use in various drug delivery systems. One or more physiologically acceptable excipients or carriers may also be included in the composition to obtain the appropriate dosage form. Suitable dosage forms for use in the present invention can be found in Remington's Pharmaceutical Sciences, Mack Publishing Company, Philadelphia, Pa., 17th ed., 1985. For a brief overview of drug delivery methods, see, for example, Langer (Science 249: 1527-1533, 1990).

[00142] The intravenous delivery dosage form of the present invention may be contained in a pouch, pen, or syringe. In some embodiments, the package may be connected to a channel containing a tube and/or a needle. In some embodiments, the dosage form may be a lyophilized form or a liquid form. In some embodiments, the dosage form may be freeze-dried (lyophilized) and contained in approximately 12-60 vials. In some embodiments, the dosage form may be lyophilized, and 45 mg of the lyophilized dosage form may be contained in one vial. In some embodiments, approximately 40-100 mg of the lyophilized dosage form may be contained in one vial. In some embodiments, a lyophilized dosage form of 12, 27, or 45 vials is combined to produce a therapeutic dose of protein in an intravenous drug product. In some embodiments, the dosage form may be a liquid form and be stored in an amount of from about 250 mg/vial to about 1000 mg/vial. In some embodiments, the dosage form may be a liquid form and stored in an amount of approximately 600 mg/vial. In some embodiments, the dosage form may be a liquid form and stored in an amount of approximately 250 mg/vial.

[00143] The present disclosure may include a liquid aqueous pharmaceutical composition containing a therapeutically effective amount of protein in a buffer solution constituting the dosage form.

[00144] These compositions can be sterilized using standard sterilization methods or can be sterilized by filtration. The resulting aqueous solutions may be packaged for use as is or lyophilized, the lyophilized preparation being combined with a sterile aqueous vehicle prior to administration. The pH of the preparations is usually from 3 to 11, more preferably from 5 to 9 or from 6 to 8, and most preferably from 7 to 8, for example from 7 to 7.5. The resulting compositions in solid form may be packaged in multiple unit dosage units, each containing a fixed amount of the above agent or agents. The composition in solid form may also be packaged in a container to provide variable quantities.

[00145] Some embodiments of the present invention provide an extended shelf life dosage form comprising a protein of the present invention in combination with mannitol, citric acid monohydrate, sodium citrate, disodium phosphate dihydrate, sodium dihydrogen phosphate dihydrate, sodium chloride, polysorbate 80, water and sodium hydroxide.

[00146] In some embodiments, an aqueous dosage form is formulated comprising a protein of the present invention in a pH buffer solution. The buffer of the present invention may have a pH ranging from about 4 to about 8, such as from about 4.5 to about 6.0 or from about 4.8 to about 5.5, or may have a pH from about 5.0 to approximately 5.2. Intermediate ranges to the above pH values are also included herein. For example, ranges of values should be included using a combination of any of the above values as an upper and/or lower bound. Examples of buffers that will adjust the pH within this range include acetate (such as sodium acetate), succinate (such as sodium succinate), gluconate, histidine, citrate, and other organic acid-based buffers.

[00147] In some embodiments, the dosage form includes a buffer system that contains citrate and phosphate to maintain the pH in the range of about 4 to about 8. In some embodiments, the pH range can be from about 4.5 to about 6.0, or from about pH 4.8 to about 5.5, or in the pH range from about 5.0 to about 5.2. In some embodiments, the buffer system includes citric acid monohydrate, sodium citrate, disodium phosphate dihydrate, and/or sodium dihydrogen phosphate dihydrate. In some embodiments, the buffer system includes about 1.3 mg/mL citric acid (e.g., 1.305 mg/mL), about 0.3 mg/mL sodium citrate (e.g., 0.305 mg/mL), about 1.5 mg/mL disodium phosphate dihydrate (eg, 1.53 mg/ml), approximately 0.9 mg/ml sodium dihydrogen phosphate dihydrate (eg, 0.86), and approximately 6.2 mg/ml sodium chloride (eg, 6.165 mg/ml). In some embodiments, the buffer system includes 1-1.5 mg/ml citric acid, 0.25-0.5 mg/ml sodium citrate, 1.25-1.75 mg/ml disodium phosphate dihydrate, 0.7-1 .1 mg/ml sodium dihydrogen phosphate dihydrate and 6.0-6.4 mg/ml sodium chloride. In some embodiments, the pH of the dosage form is adjusted with sodium hydroxide.

[00148] A polyol that acts as a tonicity regulator and can stabilize the antibody may also be included in the dosage form. The polyol is added to the dosage form in an amount that may vary depending on the desired isotonicity of the dosage form. In some embodiments, the aqueous dosage form may be isotonic. The amount of polyol added can also be changed depending on the molecular weight of the polyol. For example, a smaller amount of a monosaccharide (such as mannitol) may be added compared to a disaccharide (such as trehalose). In some embodiments, the polyol that can be used in the dosage form as a tonicity regulator is mannitol. In some embodiments, the concentration of mannitol may be from about 5 to about 20 mg/ml. In some embodiments, the concentration of mannitol may be approximately 7.5-15 mg/ml. In some embodiments, the concentration of mannitol may be approximately 10-14 mg/ml. In some embodiments, the concentration of mannitol may be approximately 12 mg/ml. In some embodiments, a polyol such as sorbitol may be included in the dosage form.

[00149] A detergent or surfactant may also be added to the dosage form. Examples of detergents include nonionic detergents such as polysorbates (eg, polysorbates 20, 80, etc.) or poloxamers (eg, poloxamer 188). The amount of detergent added is such that it reduces aggregation of the incorporated antibody and/or minimizes particle formation in the dosage form and/or reduces adsorption. In some embodiments, the dosage form may include a surfactant that is a polysorbate. In some embodiments, the dosage form may contain the detergent polysorbate 80 or Tween 80. Tween 80 is the term used to describe polyoxyethylene (20) sorbitan monooleate (see Fiedler, Lexikon der Hifsstoffe, Editio Cantor Verlag Aulendorf, 4th edi., 1996). In some embodiments, the dosage form may contain from about 0.1 to about 10 mg/ml polysorbate 80, or from about 0.5 to about 5 mg/ml. In some embodiments, approximately 0.1% polysorbate 80 may be added to the composition.

[00150] In embodiments, the protein product of the present invention is formulated in a liquid dosage form. The liquid dosage form may be provided at a concentration of 10 mg/ml in a USP/Ph Eur Type I 50R vial, capped with a rubber stopper and sealed with an aluminum crimp cap. The plug can be made of elastomer that meets USP and Ph Eur requirements. In some embodiments, the vials may be filled with 61.2 mL of protein product solution to provide a 60 mL draw volume. In some embodiments, the liquid dosage form may be reconstituted with a 0.9% sodium chloride solution.

[00151] In some embodiments, the liquid dosage form of the invention may be formulated as a solution at a concentration of 10 mg/ml in combination with sugar at stabilizing levels. In some embodiments, the liquid dosage form may be formulated in an aqueous vehicle. In some embodiments, the stabilizer may be added in an amount no greater than an amount that would result in an undesirable or unsuitable viscosity for intravenous administration. In some embodiments, the sugar may be a disaccharide, such as sucrose. In some embodiments, the liquid dosage form may also include one or more of a buffering agent, a surfactant, and a preservative.

[00152] In some embodiments, the pH of the liquid dosage form can be adjusted by adding a pharmaceutically acceptable acid and/or base. In some embodiments, the pharmaceutically acceptable acid may be hydrochloric acid. In some embodiments, the base may be sodium hydroxide.

[00153] In addition to aggregation, deamidation is a common product of peptides and proteins that can be produced during fermentation, cell collection/clarification, purification, drug substance/drug product storage, and during sample analysis. Deamidation is the loss of NH3 by a protein to form a succinimide intermediate that can undergo hydrolysis. The succinimide intermediate reduces the mass of the parent peptide by 17 daltons. Subsequent hydrolysis results in an increase in mass of 18 daltons. Isolation of the succinimide intermediate is difficult due to instability under aqueous conditions. Thus, deamidation is usually defined as an increase in mass of 1 Dalton. Deamidation of asparagine leads to the formation of aspartic or isoaspartic acid. Parameters affecting the rate of deamidation include pH, temperature, solvent dielectric constant, ionic strength, primary sequence, local polypeptide conformation, and tertiary structure. Amino acid residues adjacent to Asn in the peptide chain influence the rate of deamidation. Gly and Ser following Asn in protein sequences result in a higher propensity for deamidation.

[00154] In some embodiments, the liquid dosage form of the present invention may be stored under pH and humidity conditions to prevent deamidation of the protein product.

[00155] The aqueous carrier of interest herein is pharmaceutically acceptable (safe and non-toxic for administration to humans) and suitable for the preparation of a liquid composition. Exemplary vehicles include sterile water for injection (SWFI), bacteriostatic water for injection (BWFI), pH buffer solution (eg, phosphate buffered saline), sterile saline, Ringer's solution, or dextrose solution.

[00156] A preservative may optionally be added to the dosage forms herein to reduce bacterial activity. The addition of a preservative may, for example, facilitate the production of a multi-dose dosage form.

[00157] Dosage forms for intravenous (IV) administration may be the preferred route of administration in specific cases, for example, when a patient is in the hospital after a transplant receiving all medications via the IV route. In some embodiments, the liquid dosage form is diluted with a 0.9% sodium chloride solution prior to administration. In some embodiments, the diluted injectable drug product is isotonic and suitable for administration by intravenous infusion.

[00158] In some embodiments, salt or buffer components may be added in an amount of 10-200 mM. Salts and/or buffers are pharmaceutically acceptable and are derived from various known acids (inorganic and organic) with "base-forming" metals or amines. In some embodiments, the buffer may be a phosphate buffer. In some embodiments, the buffer may be a glycinate, carbonate, citrate buffer, in which case the counter ion may be sodium, potassium, or ammonium ions.

[00159] A preservative may optionally be added to the dosage forms herein to reduce bacterial activity. The addition of a preservative may, for example, facilitate the production of a multi-dose dosage form.

[00160] The aqueous carrier of interest herein is pharmaceutically acceptable (safe and non-toxic for administration to humans) and suitable for the preparation of a liquid composition. Exemplary vehicles include sterile water for injection (SWFI), bacteriostatic water for injection (BWFI), pH buffer solution (eg, phosphate buffered saline), sterile saline, Ringer's solution, or dextrose solution.

[00161] The present invention may include a lyophilized dosage form including proteins and a lyoprotectant. The lyoprotectant may be a sugar, for example a disaccharide. In some embodiments, the lyoprotectant may be sucrose or maltose. The lyophilized dosage form may also include one or more of a buffering agent, a surfactant, a bulking agent, and/or a preservative.

[00162] The amount of sucrose or maltose suitable for stabilizing the lyophilized pharmaceutical product may be in a weight ratio of at least 1:2 protein to sucrose or maltose. In some embodiments, the weight ratio of protein to sucrose or maltose may be from 1:2 to 1:5.

[00163] In some embodiments, the pH of the dosage form prior to lyophilization can be adjusted by adding a pharmaceutically acceptable acid and/or base. In some embodiments, the pharmaceutically acceptable acid may be hydrochloric acid. In some embodiments, the pharmaceutically acceptable base may be sodium hydroxide.

[00164] Before lyophilization, the pH of the solution containing the protein of the present invention can be adjusted to 6-8. In some embodiments, the pH range of the lyophilized pharmaceutical product may be between 7 and 8.

[00165] In some embodiments, salt or buffer components may be added in an amount of 10-200 mM. Salts and/or buffers are pharmaceutically acceptable and are derived from various known acids (inorganic and organic) with "base-forming" metals or amines. In some embodiments, the buffer may be a phosphate buffer. In some embodiments, the buffer may be a glycinate, carbonate, citrate buffer, in which case the counter ion may be sodium, potassium, or ammonium ions.

[00166] In some embodiments, a "bulking agent" may be added. A "bulking agent" is a compound that increases the mass of the lyophilized mixture and improves the physical structure of the lyophilized mixture (eg, facilitates the production of a substantially uniform lyophilized mixture that retains an open-cell structure). Exemplary bulking agents include mannitol, glycine, polyethylene glycol, and sorbitol. The lyophilized dosage forms of the present invention may contain such bulking agents.

[00167] A preservative may optionally be added to the dosage forms herein to reduce bacterial activity. The addition of a preservative may, for example, facilitate the production of a multi-dose dosage form.

[00168] In some embodiments, the lyophilized pharmaceutical product may be formulated with an aqueous carrier. The aqueous carrier of interest herein is pharmaceutically acceptable (eg, safe and non-toxic for administration to humans) and suitable for the preparation of a liquid dosage form after lyophilization. Exemplary vehicles include sterile water for injection (SWFI), bacteriostatic water for injection (BWFI), pH buffer solution (eg, phosphate buffered saline), sterile saline, Ringer's solution, or dextrose solution.

[00169] In some embodiments, the lyophilized pharmaceutical product of the present invention is reconstituted with either Sterile Water for Injection (USP (SWFI)) or 0.9% Sodium Chloride Injection (USP). During reconstitution, the lyophilized powder dissolves into the solution.

[00170] In some embodiments, the lyophilized protein product of the present invention is made up to about 4.5 ml of water for injection and diluted with 0.9% saline (sodium chloride solution).

[00171] The actual dosage levels of the active agents in the pharmaceutical compositions of the present invention may be varied to obtain an amount of the active agent that is effective to achieve the desired therapeutic response in a particular patient, composition, and route of administration, without causing toxicity to the patient.

[00172] The determined dose may be a single dose for each patient, for example, 50-5000 mg of protein. Alternatively, the patient dose may be calculated based on the patient's approximate weight or body surface area. Other factors in determining the appropriate dosage may include the disease or condition being treated or prevented, the severity of the disease, the route of administration, and the age, sex, and medical condition of the patient. Further refinement of the calculations necessary to determine the appropriate dosage for treatment will typically be performed by those skilled in the art, particularly in light of the dosing information and assays disclosed herein. The dose can also be determined using known dose determination assays used in combination with appropriate dose response data. Doses for an individual patient may be adjusted as disease progression is observed. Levels of the target construct or complex in the patient's blood can be measured to determine whether dosage adjustments are required to obtain or maintain an effective concentration. Pharmacogenetic indicators can be used to determine which targeted constructs and/or complexes and their doses are most likely to be effective for a given patient (Schmitz et al., Clinica Chimica Acta 308: 43-53, 2001; Steimer et al., Clinica Chimica Acta 308: 33-41, 2001).

[00173] Typically, dosages per body weight range from about 0.01 μg to about 100 mg per kg body weight, such as from about 0.01 μg to about 100 mg/kg body weight, from about 0.01 mcg to approximately 50 mg/kg body weight, approximately 0.01 mcg to approximately 10 mg/kg body weight, approximately 0.01 mcg to approximately 1 mg/kg body weight, approximately 0.01 mcg to approximately 100 mcg /kg body weight, from about 0.01 μg to about 50 μg/kg body weight, from about 0.01 μg to about 10 μg/kg body weight, from about 0.01 μg to about 1 μg/kg body weight, from about 0.01 μg to about 0.1 μg/kg body weight, from about 0.1 μg to about 100 mg/kg body weight, from about 0.1 μg to about 50 mg/kg body weight, from about 0 .1 μg to approximately 10 mg/kg body weight, approximately 0.1 μg to approximately 1 mg/kg body weight, approximately 0.1 μg to approximately 100 μg/kg body weight, approximately 0.1 μg to approximately 10 mcg/kg body weight, from about 0.1 mcg to about 1 mcg/kg body weight, from about 1 mcg to about 100 mg/kg body weight, from about 1 mcg to about 50 mg/kg body weight, from about 1 mcg to about 10 mg/kg body weight, from about 1 mcg to about 1 mg/kg body weight, from about 1 mcg to about 100 mcg/kg body weight, from about 1 mcg to about 50 mcg/kg body weight, from about 1 μg to about 10 μg/kg body weight, from about 10 μg to about 100 mg/kg body weight, from about 10 μg to about 50 mg/kg body weight, from about 10 μg to about 10 mg/kg body weight body weight, from about 10 μg to about 1 mg/kg body weight, from about 10 μg to about 100 μg/kg body weight, from about 10 μg to about 50 μg/kg body weight, from about 50 μg to about 100 mg/ kg body weight, from about 50 μg to about 50 mg/kg body weight, from about 50 μg to about 10 mg/kg body weight, from about 50 μg to about 1 mg/kg body weight, from about 50 μg to about 100 mcg/kg body weight, from about 100 mcg to about 100 mg/kg body weight, from about 100 mcg to about 50 mg/kg body weight, from about 100 mcg to about 10 mg/kg body weight, from about 100 mcg to about 1 mg/kg body weight, about 1 mg to about 100 mg/kg body weight, about 1 mg to about 50 mg/kg body weight, about 1 mg to about 10 mg/kg body weight, about 10 mg to about 100 mg/kg body weight, from about 10 mg to about 50 mg/kg body weight, from about 50 mg to about 100 mg/kg body weight.

[00174] Doses may be administered one or more times per day, week, month or year, or even once every 2-20 years. Those of ordinary skill in the art can easily estimate the frequency of repeated dosing based on the measured residence times and concentrations of the targeted construct or complex in biological fluids or tissues. Administration according to the present invention may be intravenous, intraarterial, intraperitoneal, intramuscular, subcutaneous, intrapleural, intrathecal, intracavitary, by perfusion through a catheter or direct intralesional injection. Administration may be one or more times per day, one or more times per week, one or more times per month, and one or more times per year.

[00175] The above description describes many aspects and embodiments of the invention. The patent application expressly addresses all combinations and combinations of aspects and embodiments.

EXAMPLES

[00176] The invention, having already been described generally, will now be more readily understood by reference to the following examples, which are included solely for the purpose of illustrating certain aspects and embodiments of the present invention and are not intended to limit the invention.

Example 1 - NKG2D-binding domains bind to NKG2D

NKG2D-binding domains bind to purified recombinant NKG2D

[00177] Human, mouse, or cynomolgus NKG2D ectodomain nucleic acid sequences were fused to nucleic acid sequences encoding human IgG1 Fc domains and introduced into mammalian cells for expression. After purification, NKG2D-Fc fusion proteins were adsorbed into the wells of microplates. After blocking the wells with bovine serum albumin to prevent nonspecific binding, NKG2D-binding domains were titrated and added to wells containing preadsorbed NKG2D-Fc fusion proteins. Primary antibody binding was determined using a secondary antibody that is conjugated to horseradish peroxidase and specifically recognizes human kappa light chain to exclude cross-reactivity with Fc. 3,3',5,5'-tetramethylbenzidine (TMB), a horseradish peroxidase substrate, was added to the wells to visualize the binding signal, the absorbance of which was measured at 450 nM and corrected at 540 nM. An NKG2D binding domain clone, isotype control or positive control (selected from SEQ ID NO: 45-48 or anti-mouse NKG2D, MI-6 and CX-5 clones available from eBioscience) was added to each well.

[00178] The isotype control showed minimal binding to the recombinant NKG2D-Fc proteins, while the positive control bound most strongly to the recombinant antigens. The NKG2D-binding domains produced by all clones showed binding to recombinant human, mouse, and cynomolgus NKG2D-Fc proteins, although with varying affinities depending on the clone. In general, each anti-NKG2D clone bound to recombinant human (FIG. 3) and cynomolgus (FIG. 4) NKG2D-Fc with similar affinity, but bound with lower affinity to recombinant mouse NKG2D-Fc (FIG. 5).

NKG2D-binding domains bind to cells expressing NKG2D

[00179] EL4 murine lymphoma cell lines have been modified to express chimeric antigen human or murine NKG2D receptors with a CD3 zeta signaling domain. NKG2D-binding clone, isotype control, or positive control was used at a concentration of 100 nM to stain extracellular NKG2D expressed on EL4 cells. Antibody binding was determined using fluorophore-conjugated anti-human IgG secondary antibodies. Cells were analyzed by flow cytometry and fold over background (FOB) signal intensity was calculated using the mean fluorescence intensity (MFI) of NKG2D-expressing cells compared to parental EL4 cells.

[00180] NKG2D-binding domains produced by all clones bound to EL4 cells expressing human and mouse NKG2D. Positive control antibodies (selected from SEQ ID NOs: 45-48, or anti-mouse NKG2D, MI-6 and CX-5 clones available from eBioscience) gave the best FOB binding signal. The NKG2D binding affinity of each clone was similar between cells expressing human NKG2D (FIG. 6) and mouse NKG2D (FIG. 7).

Example 2 - NKG2D-binding domains block natural ligand binding to NKG2D

Competition with ULBP-6

[00181] Recombinant human NKG2D-Fc proteins were adsorbed to microplate wells and the wells were blocked with bovine serum albumin to reduce nonspecific binding. A saturating concentration of ULBP-6-His-biotin was added to the wells, followed by the addition of NKG2D-binding domain clones. After a 2-hour incubation, the wells were washed and ULBP-6-His-biotin, which remained bound in the NKG2D-Fc-coated wells, was detected with horseradish peroxidase-conjugated streptavidin and the substrate TMB. Absorbance was measured at 450 nM and corrected at 540 nM. After background subtraction, specific binding of NKG2D-binding domains to NKG2D-Fc proteins was calculated based on the percentage of ULBP-6-His-biotin that was blocked from binding to NKG2D-Fc proteins in wells. A positive control antibody (selected from SEQ ID NO:45-48) and various NKG2D binding domains blocked ULBP-6 binding to NKG2D, while the isotype control showed little competition with ULBP-6 (FIG. 8).

Competition with MICA

[00182] Recombinant human MICA-Fc proteins were adsorbed into microplate wells and the wells were blocked with bovine serum albumin to reduce nonspecific binding. NKG2D-Fc-biotin was added to the wells, followed by the addition of NKG2D-binding domains. After incubation and washing, NKG2D-Fc-biotin, which remained bound in MICA-Fc-coated wells, was detected using streptavidin-HRP and TMB substrate. Absorbance was measured at 450 nM and corrected at 540 nM. After background subtraction, specific binding of NKG2D-binding domains to NKG2D-Fc proteins was calculated based on the percentage of NKG2D-Fc-biotin that was blocked from binding to MICA-Fc-coated wells. The positive control antibody (selected from SEQ ID NO:45-48) and various NKG2D binding domains blocked MICA binding to NKG2D, while the isotype control showed little competition with MICA (FIG. 9).

Competition with Rae-1 delta

[00183] Recombinant mouse Rae-1delta-Fc (purchased from R&D Systems) was adsorbed to microplate wells and the wells were blocked with bovine serum albumin to reduce nonspecific binding. Mouse NKG2D-Fc-biotin was added to the wells, followed by the addition of NKG2D-binding domains. After incubation and washing, NKG2D-Fc-biotin, which remained bound in the Rae-1delta-Fc-coated wells, was detected using streptavidin-HRP and TMB substrate. Absorbance was measured at 450 nM and corrected at 540 nM. After background subtraction, specific binding of NKG2D-binding domains to NKG2D-Fc proteins was calculated based on the percentage of NKG2D-Fc-biotin that was blocked from binding to Rae-1-delta-Fc-coated wells. Positive control (selected from SEQ ID NO:45-48 or the anti-mouse NKG2D, MI-6 and CX-5 clones available from eBioscience) and various NKG2D-binding domain clones blocked Rae-1-delta binding to mouse NKG2D, whereas The isotype control antibody showed low competition with Rae-1delta (FIG. 10).

Example 3 - NKG2D-binding domain clones activate NKG2D

[00184] Human and mouse NKG2D nucleic acid sequences were fused to nucleic acid sequences encoding the CD3 zeta signaling domain to produce chimeric antigen receptor (CAR) constructs. The NKG2D-CAR constructs were then cloned into a retroviral vector using Gibson assembly and transfected into expi293 cells to produce retrovirus. EL4 cells were infected with viruses containing NKG2D-CAR along with 8 μg/ml polybrene. At 24 hours postinfection, NKG2D-CAR expression levels in EL4 cells were examined by flow cytometry, and clones that highly expressed NKG2D-CAR on the cell surface were selected.

[00185] To determine whether NKG2D binding domains activate NKG2D, they were adsorbed to microplate wells and NKG2D-CAR EL4 cells were cultured in antibody fragment-coated wells for 4 hours in the presence of brefeldin-A and monensin. Intracellular production of TNF-alpha, an indicator of NKG2D activation, was examined by flow cytometry. The percentage of TNF-alpha positive cells was normalized to cells treated with the positive control. All NKG2D binding domains activated human NKG2D (FIG. 11) and mouse NKG2D (FIG. 12).

Example 4 - NKG2D-binding domains activate NK cells

Primary human NK cells

[00186] Peripheral blood mononuclear cells (PBMCs) were isolated from the buffy platelet layer of peripheral blood using density gradient centrifugation. NK cells (CD3 ⁻ CD56 ⁺ ) were isolated using negative selection using magnetic beads from PBMCs, and the purity of isolated NK cells was typically >95%. Isolated NK cells were then cultured in a medium containing 100 ng/ml IL-2 for 24-48 hours, after which they were transferred to microplate wells in which NKG2D-binding domains were adsorbed and cultured in a medium containing conjugated fluorophore antibody against CD107a, brefeldin-A and monensin. After culture, NK cells were examined by flow cytometry using fluorophore-conjugated antibodies against CD3, CD56, and IFN-gamma. CD107a and IFN-gamma staining were examined in CD3 ^– CD56 ⁺ cells to assess NK cell activation. The increase in CD107a/IFN-gamma double positive cells indicates better NK cell activation as a result of binding of two activating receptors rather than a single receptor. NKG2D-binding domains and positive control (selected from SEQ ID NO:45-48) showed a higher percentage of CD107a ⁺ and IFN-gamma ⁺ NK cells than isotype control (FIG. 13 and FIG. 14 show data from two independent experiments, in each of which PBMCs from different donors were used to obtain NK cells).

Primary mouse NK cells

[00187] Spleens were obtained from C57Bl/6 mice and passed through a 70 μm cell sieve to obtain a single cell suspension. Cells were pelleted and resuspended in ACK lysis buffer (purchased from Thermo Fisher Scientific, #A1049201; 155 mM ammonium chloride, 10 mM potassium bicarbonate, 0.01 mM EDTA) to remove red blood cells. The remaining cells were cultured with 100 ng/ml hIL-2 for 72 hours, after which they were harvested and prepared for NK cell isolation. NK cells (CD3 ^– NK1.1 ⁺ ) were then isolated from spleen cells using a negative depletion technique using magnetic beads with a purity of >90%. Purified NK cells were cultured in medium containing 100 ng/ml mIL-15 for 48 hours, after which they were transferred to microplate wells in which NKG2D-binding domains were adsorbed and cultured in medium containing a fluorophore-conjugated antibody against CD107a, brefeldin-A and monensin. After culture in NKG2D-binding domain-coated wells, NK cells were analyzed by flow cytometry using fluorophore-conjugated antibodies against CD3, NK1.1, and IFN-gamma. CD107a and IFN-gamma staining were examined in CD3-NK1.1 ⁺ cells to assess NK cell activation. The increase in CD107a/IFN-gamma double positive cells indicates better NK cell activation as a result of binding of two activating receptors rather than a single receptor. NKG2D-binding domains and positive controls (selected from the anti-mouse NKG2D, MI-6, and CX-5 clones available from eBioscience) showed a higher percentage of CD107a ⁺ and IFN-gamma ⁺ NK cells converted than the isotype control (in FIG. 15 and FIG. 16 show data from two independent experiments, in each of which different mice were used to obtain NK cells).

Example 5 - NKG2D Binding Domains Provide Cytotoxicity to Target Tumor Cells

[00188] Primary human and mouse NK cell activation assays demonstrate an increase in cytotoxicity markers on NK cells following incubation with NKG2D binding domains. To determine whether this results in increased tumor cell lysis, a cell-based assay was used in which each NKG2D-binding domain was converted into a monospecific antibody. The Fc region was used as one targeting arm, while the Fab region (NKG2D binding domain) acted as the other targeting arm for NK cell activation. THP-1 cells, which are of human origin and express high levels of Fc receptors, were used as the target tumor and the Perkin Elmer DELFIA Cytotoxicity Kit was used. THP-1 cells were labeled with BATDA reagent and resuspended at a density of 10 ⁵ /ml in culture medium. Labeled THP-1 cells were then combined with NKG2D antibodies and isolated murine NK cells in microtiter plate wells at 37°C for 3 hours. After incubation, 20 μl of the culture supernatant was removed, mixed with 200 μl of europium solution and incubated with shaking for 15 minutes in the dark. Fluorescence was measured as a function of time using a PHERAstar microplate analyzer equipped with a time-resolved fluorescence module (excitation 337 nm, emission 620 nm) and specific lysis was calculated according to the instructions in the kit.

[00189] The positive control, ULBP-6, a natural ligand of NKG2D, showed increased specific lysis of THP-1 target cells by murine NK cells. NKG2D antibodies also increased specific lysis of THP-1 target cells, whereas the isotype control antibody showed reduced specific lysis. The dotted line indicates specific lysis of THP-1 cells by murine NK cells without the addition of antibody (FIG. 17).

Example 6 - antibodies to NKG2D demonstrate high thermal stability

[00190] Melting temperatures of NKG2D binding domains were measured using differential scanning fluorimetry. The extrapolated apparent melting temperatures are relatively high for typical IgG1 antibodies (FIG. 18).

Example 7 - Synergistic activation of human NK cells by cross-linking of NKG2D and CD16

Primary Human NK Cell Activation Assay

[00191] Peripheral blood mononuclear cells (PBMCs) were isolated from the buffy coat of human peripheral blood using density gradient centrifugation. NK cells were purified from PBMCs using negative magnetic beads (StemCell #17955). NK cells were >90% CD3 ^– CD56 ⁺ as determined by flow cytometry. Cells were then cultured for 48 hours in medium containing 100 ng/ml hIL-2 (Peprotech #200-02) before use in activation assays. Antibodies were coated into 96-well flat-bottom plates at a concentration of 2 μg/ml (anti-CD16 antibody, Biolegend #302013) and 5 μg/ml (anti-NKG2D antibody, R&D #MAB139) in 100 μl sterile PBS overnight at 4°C followed by thorough washing of the wells to remove excess antibody. To assess degranulation, IL-2-activated NK cells were resuspended at a density of 5x10 ⁵ cells/ml in culture medium supplemented with 100 ng/ml hIL2 and 1 μg/ml APC-conjugated mAb against CD107a (Biolegend #328619). Then 1×10 ⁵ cells/well were added to the antibody-coated plates. The protein transport inhibitors brefeldin A (BFA, Biolegend #420601) and monensin (Biolegend #420701) were added at final dilutions of 1:1000 and 1:270, respectively. The seeded cells were incubated for 4 hours at 37°C in 5% CO ₂ . For intracellular IFN-γ staining, NK cells were labeled with anti-CD3 (Biolegend #300452) and anti-CD56 (Biolegend #318328) mAbs and then fixed, permeabilized, and labeled with anti-IFN-γ mAb (Biolegend #506507). NK cells were analyzed for CD107a and IFN-γ expression by flow cytometry after gating on live CD56 ⁺ CD3 ⁻ cells.

[00192] To examine the relative activity of a combination of receptors, NKG2D or CD16 was cross-linked and both receptors were cross-linked when stimulated in the bound state on the plate. As shown in Figure 19 (FIG. 19A-19C), combined stimulation of CD16 and NKG2D resulted in highly elevated levels of CD107a production (degranulation) (FIG. 19A) and/or IFN-γ (FIG. 19B). The dotted lines represent the additive effect of individual stimulation of each receptor.

[00193] CD107a levels and intracellular IFN-γ production by IL-2-activated NK cells were analyzed after 4 hours of bound stimulation on an anti-CD16, anti-NKG2D plate, or a combination of both monoclonal antibodies. The graphs show the mean (n=2) ±SD. FIG. 19A shows CD107a levels; FIG. 19B shows IFNγ levels; FIG. 19C shows CD107a and IFNγ levels. The data shown in FIG. 19A-19C are representative of five independent experiments using five different healthy donors.

Example 8 - Multispecific binding proteins bind to NKG2D

[00194] EL4 mouse lymphoma cell lines have been modified to express human NKG2D. Trispecific binding proteins (TriNKET), each of which contained an NKG2D binding domain, a tumor associated antigen binding domain (CD33 binding domain) and an Fc domain that binds CD16, as shown in FIG. 1 were tested for their affinity for extracellular NKG2D expressed on EL4 cells. Binding of multispecific binding proteins to NKG2D was detected using fluorophore-conjugated anti-human IgG secondary antibodies. Cells were examined by flow cytometry and fold over background (FOB) was calculated using the mean fluorescence intensity (MFI) of NKG2D-expressing cells compared to parental EL4 cells.

[00195] TriNKETs tested included CD33-TriNKET-C26 (ADI-28226 and CD33 binding domain), CD33-TriNKET-F04 (ADI-29404 and CD33 binding domain), CD33-TriNKET-A44 (ADI-27744 and CD33 CD33 -binding domain), CD33-TriNKET-F47 (ADI-29447 and CD33-binding domain), CD33-TriNKET-A49 (ADI-27749 and CD33-binding domain) and CD33-TriNKET-F63 (ADI-27463 and CD33-binding domain domain). The CD33 binding domain used in the test molecules consisted of a heavy chain variable domain and a light chain variable domain, as follows.

Heavy chain variable domain to CD33 (SEQ ID NO:125):

Light chain variable domain to CD33 (SEQ ID NO:126):

[00196] Data show that TriNKETs, which include a CD33-binding domain and an NKG2D-binding domain, bind to NKG2D (FIGS. 35A-35B). In FIG. 35A shows the binding of various TriNKETs compared to monoclonal antibodies that contain the corresponding NKG2D binding domain. In FIG. 35B shows the binding profile of CD33 targeting TriNKETs, which include 6 different NKG2D binding domains.

Example 9 - Multispecific binding proteins bind to human tumor antigens

Trispecific binding proteins bind to CD33

[00197] The CD33-expressing human AML cell line MV4-11 and Molm-13 was used to assay the binding of TriNKET to the tumor-associated antigen CD33. TriNKET proteins and optionally the parent anti-CD33 monoclonal antibody were incubated with cells and binding was detected using fluorophore-conjugated anti-human IgG secondary antibodies. Cells were analyzed by flow cytometry and fold over background (FOB) was calculated using mean fluorescence intensity (MFI) for TriNKET proteins and parent anti-CD33 monoclonal antibody normalized to secondary control antibodies. The CD33-targeting TriNKETs show comparable levels of binding to CD33 compared to the parent CD33 antibody (FIG. 36). TriNKETs demonstrate binding to cell surface CD33 on MV4-11 (FIG. 36A, 36B and 36D) and Molm-13 (FIG. 36C) cells. The overall binding signal is comparable among different TriNKETs because they contain the same CD33-binding domain.

Example 10 - Multispecific binding proteins activate NK cells

[00198] Peripheral blood mononuclear cells (PBMCs) were isolated from the buffy platelet layer of human peripheral blood using density gradient centrifugation. NK cells (CD3 ^– CD56 ⁺ ) were isolated using negative selection with magnetic beads from PBMCs, and the purity of isolated NK cells was typically >90%. Isolated NK cells were cultured in media containing 100 ng/ml IL-2 for activation or left overnight without the cytokine. IL-2-activated NK cells were used within 24-48 hours of activation.

[00199] MV4-11 cells expressing CD33 were collected and resuspended in culture media at a density of 2×10 ⁶ /ml. Anti-CD33 monoclonal antibodies or CD33-targeting TriNKET antibodies were diluted in culture media. Activated NK cells were collected, washed and resuspended at a density of 2×10 ⁶ /ml in culture media. Cancer cells were then mixed with monoclonal antibodies/TriNKET and activated NK cells in the presence of IL-2. Brefeldin-A and monensin were also added to the mixed culture to block protein transport out of the cell for intracellular cytokine staining. Fluorophore-conjugated antibody against CD107a was added to the mixed culture and the culture was incubated for 4 hours, after which samples were prepared for FACS analysis using fluorophore-conjugated antibodies against CD3, CD56 and IFN-gamma. CD107a and IFN-gamma staining were examined in CD3 ^– CD56 ⁺ cells to assess NK cell activation. The increase in CD107a/IFN-gamma double positive cells indicates better NK cell activation as a result of binding of two activating receptors rather than a single receptor.

[00200] Co-culture of primary human NK cells with CD33-positive MV4-11 cells resulted in TriNKET-mediated activation of primary human NK cells. CD33-targeting TriNKET mediated the activation of human NK cells co-cultured with MV4-11 cells, as indicated by increased CD107a degranulation and IFNγ cytokine production (FIGS. 37A AND 37B). NK cells alone, NK cells cocultured with MV4-11 cells but without TriNKET, and CD20-guided TriNKET were used as controls. Compared to anti-C33 monoclonal antibody, CD33-targeting TriNKET showed increased NK cell activity (FIGS. 37A and 37B).

Example 11 - Trispecific binding proteins provide cytotoxicity to target cancer cells

[00201] Peripheral blood mononuclear cells (PBMCs) were isolated from the buffy coat of human peripheral blood using density gradient centrifugation. NK cells (CD3 ^– CD56 ⁺ ) were isolated using negative selection with magnetic beads from PBMCs, and the purity of isolated NK cells was typically >90%. Isolated NK cells were cultured in media containing 100 ng/ml IL-2 for activation or left overnight without the cytokine. IL-2-activated or resting NK cells were used the next day in cytotoxicity assays.

DELFIA cytotoxicity assay:

[00202] Human cancer cell lines expressing CD33 were harvested from culture, cells were washed with PBS and resuspended in growth media at a density of 10 ⁶ /ml for labeling with BATDA reagent (Perkin Elmer AD0116). Target cell labeling was performed according to the manufacturer's instructions. After labeling, the cells were washed 3 times with PBS and resuspended at a density of 0.5-1.0×10 ⁵ /ml in culture media. To prepare background wells, an aliquot of labeled cells was removed and cells were pelleted from the medium by centrifugation. 100 μl of medium was carefully added to the wells in triplicate, taking care not to damage the pelleted cells. 100 μl of BATDA-labeled cells was added to each well of a 96-well plate. Wells were maintained for spontaneous release from target cells, and wells were prepared to maximize target cell lysis by adding 1% Triton-X. Monoclonal antibodies or TriNKET against the target tumor of interest were diluted in culture media and 50 μl of the diluted mAb or TriNKET was added to each well. Resting and/or activated NK cells were collected from culture, cells were washed and resuspended at a density of 10 ⁵ -2.0×10 ⁶ /ml in culture media depending on the desired ratio of effector cells to target cells (E:T). 50 μl of NK cells was added to each well of the plate to obtain a total culture volume of 200 μl. The plate was incubated at 37°C in 5% CO ₂ for 2-3 hours, after which the assay was developed.

[00203] After culturing for 2-3 hours, the plate was removed from the thermostat and the cells were pelleted by centrifugation at 200 g for 5 minutes. 20 μL of culture supernatant was transferred into a clean microplate provided by the manufacturer, and 200 μL of europium solution was added to each well at room temperature. The plate was protected from light and incubated on a plate shaker at 250 rpm for 15 minutes. The plate was read using Victor 3 or SpectraMax i3X instruments. % specific lysis was calculated as follows: % specific lysis = ((Experimental release - spontaneous release)/(Maximum release - spontaneous release))*100%.

[00204] TriNKETs mediated the cytotoxicity of human NK cells against CD33-positive human cancer cell lines. Resting human NK cells were mixed with MV4-11 cancer cells (FIG. 40A), resting human NK cells were mixed with Molm-13 cancer cells (FIG. 40B), and resting human NK cells were mixed with THP-1 cancer cells (FIG. .40C). TriNKET constructs (e.g., CD33-TriNKET-C26 and CD33-TriNKET-F04) are capable of dose-dependently enhancing the cytotoxic activity of resting human NK cells against cancer cells. The dotted line indicates the cytotoxic activity of resting NK cells without TriNKET. An anti-CD33 monoclonal antibody showed reduced efficacy against MV4-11 cells, which express CD64, but at a lower level than THP-1 cells. An anti-CD33 monoclonal antibody showed good activity against Molm-13 cells that do not express CD64. Anti-CD33 monoclonal antibody showed no effect on THP-1 cells, which have high levels of CD64.

[00205] TriNKET-mediated lysis of CD33-positive Molm-13 human cancer cells was studied. Resting human NK cells were mixed with Molm-13 cancer cells in a 5:1 ratio (FIG. 41), with TriNKET (e.g. CD33-TriNKET-A49, CD33-TriNKET-A44, CD33-TriNKET-C26 and CD33-TriNKET -E79) could dose-dependently increase the cytotoxic activity of resting human NK cells against cancer cells. Dashed line indicates cytotoxic activity of resting NK cells without TriNKET.

Example 12 - Advantage of TriNKET in the treatment of malignant tumors with high FcR expression or in tumor microenvironments with high FcR levels

[00206] Monoclonal antibody therapy has been approved for the treatment of many types of cancer, including hematologic malignancies and solid tumors. Although the use of monoclonal antibodies in cancer treatment has improved patient outcomes, limitations still exist. Mechanism of action studies have demonstrated that monoclonal antibodies exert their effects on tumor growth through various mechanisms including, but not limited to, ADCC, CDC, phagocytosis, and signal blockade.

[00207] First of all, ADCC is believed to be the primary mechanism by which monoclonal antibodies exert their effect. ADCC is based on the interaction of Fc antibodies with low-affinity FcγRIII (CD16) on the surface of natural killer cells, which mediate direct lysis of the tumor cell. Of all the FcγRs, CD16 has the lowest affinity for IgG Fc, FcγRI (CD64) is a high affinity FcR and binds approximately 1000 times more strongly to IgG Fc than CD16.

[00208] CD64 is commonly expressed on many hematopoietic lineages, such as the myeloid lineage, and can be expressed on tumors derived from these cell types, such as acute myeloid leukemia (AML). Tumor-infiltrating immune cells, such as MDSCs and monocytes, also express CD64 and are known to infiltrate the tumor microenvironment. Expression of CD64 by the tumor or in the tumor microenvironment may have a negative impact on monoclonal antibody therapy. Expression of CD64 in the tumor microenvironment makes it difficult for these antibodies to interact with CD16 on the surface of NK cells, as the antibodies prefer to bind the high-affinity receptor. By targeting two activating receptors on the surface of NK cells, TriNKET constructs can overcome the negative impact of CD64 expression on monoclonal antibody therapy.

Expression of FcRγI (CD64) in three AML cell lines

[00209] An in vitro culture system was developed to test the activity of TriNKET and monoclonal antibodies against tumors with high and low surface expression of CD64. Molm-13 and THP-1 are two human AML cell lines that have similar expression of surface CD33, however Molm-13 cells do not express CD64, whereas THP-1 cells express CD64 on their surface (FIGS. 38A-38C). Using monoclonal antibodies or TriNKET directed against the CD33 target, the effect of tumor CD64 expression on monoclonal antibody or TriNKET therapy was tested. In FIG. 38A-38C show expression of high affinity FcRγI (CD64) on three human AML cell lines, the Molm-13 cell line (FIG. 38A), the MV4-11 cell line (FIG. 38B), and the THP-1 cell line (FIG. 38C). Molm-13 cells do not express CD64, whereas MV4-11 cells have low levels and THP-1 cells have high levels of CD64 on the cell surface.

TriNKET constructs have the advantage of targeting tumor cells with high surface FcR expression

[00210] In FIG. 39A-39B show monoclonal antibody or TriNKET-mediated activation of human NK cells in coculture with Molm-13 cells (FIG. 39B) or with THP-1 (FIG. 39A). Anti-human CD33 monoclonal antibody demonstrated good activation of human NK cells in the Molm-13 co-culture system, as evidenced by increased CD107a degranulation and IFNγ production. The monoclonal antibody had no effect in the THP-1 co-culture system, where high levels of CD64 are present on cancer cells. Notably, TriNKETs were effective against both Molm-13 cells (FIG. 39B) and THP-1 cells (FIG. 39A), indicating that TriNKETs are able to overcome binding to CD64 on tumors and effectively interact with NK -cells for activation. Dual targeting of two activating receptors on NK cells provided stronger specific binding to NK cells. Monoclonal antibodies that interact only with CD16 on NK cells can bind to other high-affinity FcRs and interfere with the binding of CD16 on NK cells. As shown in FIG. 39C, TriNKET also effectively mediated the activation of quiescent human NK cells in coculture with human AML MV4-11 cells.

TriNKETs demonstrate efficacy against AML cell lines independent of FcγRI expression

[00211] In FIG. 40A-40C show human NK cytotoxicity assays using three human AML cell lines as targets. The anti-CD33 monoclonal antibody showed good activity against Molm-13 cells (FIG. 40B), which do not express CD64. MV4-11 cells (FIG. 40A), which express CD64, but at a lower level than THP-1, showed reduced efficacy with the anti-CD33 monoclonal antibody. THP-1 cells (FIG. 40C) showed no effect with anti-CD33 monoclonal antibody alone. Regardless of CD64 expression on tumor cells, TriNKETs were able to mediate human NK cell responses against all tumor cells tested in this example.

[00212] In FIG. 40A-40C show that THP-1 cells were protected from monoclonal antibody treatment due to high levels of expression of a high-affinity FcR on their surface. TriNKETs overcame this defense by targeting two activating receptors on the surface of NK cells. The cytotoxicity data directly correlated with the results observed in co-culture activation experiments. TriNKETs were able to overcome the protection from mAb therapy observed in THP-1 cells and caused NK cell-mediated lysis despite high FcR levels.

Example 13 - Cytolysis of Normal Myeloid and Normal B Cells in PBMC Cultures: TriNKETs Provide a Better Safety Profile as a Result of Reduced Targeted Non-Tumor Side Effects

[00213] Natural killer cells and CD8 T cells are capable of directly causing tumor cell lysis, although the mechanisms by which NK cells and CD8 T cells distinguish normal autologous from tumor cells differ. The activity of NK cells is regulated by the balance of signals from activating (NCR, NKG2D, CD16, etc.) and inhibitory (KIR, NKG2A, etc.) receptors. The balance of these activating and inhibitory signals allows NK cells to distinguish healthy autologous cells from stressed, virus-infected, or degenerated autologous cells. This "built-in" self-tolerance mechanism allows normal healthy tissue to be protected from NK cell responses. To extend this principle, NK cell self-tolerance would allow TriNKET constructs to specifically interact with antigens expressed on both autologous cells and tumors without non-tumor side effects or with an extended therapeutic window.

[00214] Unlike natural killer cells, T cells require recognition of a specific peptide presented by MHC molecules for activation and effector functions. T cells have been a major target of immunotherapy, and many strategies have been developed to redirect T cell responses against the tumor. Bispecific T cells, checkpoint inhibitors, and CAR T cells have been approved by the FDA, but they often suffer from dose-limiting toxicities. Bispecific T cells and CAR-T cells operate within the TCR-MHC recognition system, using binding domains to target antigens on the surface of tumor cells and using modified signaling domains to transmit activation signals to the effector cell. Although these treatments are effective in inducing an antitumor immune response, they are often associated with cytokine release syndrome (CRS) and targeted nontumor side effects. TriNKETs are unique in this regard because they will not “block” the natural NK cell activation and inhibition systems. In contrast, TriNKETs are engineered to maintain balance and provide additional activation signals to NK cells while maintaining NK cell tolerance to healthy autologous cells.

[00215] PBMCs were isolated from whole blood using density gradient centrifugation. The remaining contaminating erythrocytes were lysed by incubation in ACK lysis buffer. PBMCs were washed 3 times in PBS and the total number of PBMCs was counted. PBMCs were adjusted to a density of 10 ⁶ /ml in primary cell culture medium. 1 ml of PBMC was seeded into the wells of a 24-well plate, the indicated TriNKET or mAb was added to the PBMC cultures in an amount of 10 μg/ml. Cells were cultured overnight at 37°C in 5% CO ₂ . The next day (24 hours later), PBMCs were collected from culture and prepared for FACS analysis. CD45+ percentage; CD19+ B cells and CD45+; CD33+; CD11b+ myeloid cells were determined in different treatment groups.

[00216] In FIG. 42A shows that B cells (CD33 negative) from a healthy donor were not exposed to CD33 targeting TriNKET. PBMCs treated with TriNKET, which specifically interacts with CD33, showed no effect on the population of CD45+, CD3-, CD56- lymphocytes. In FIG. 42B shows that autologous CD33+ myeloid cells were protected from CD33-guided TriNKET-mediated NK cell responses and were therefore resistant to TriNKET-mediated lysis. In these cultures, the frequency of CD45+, CD33+, CD11b+ myeloid cells did not change when incubated with CD33-targeting TriNKET.

INCLUSION BY REFERENCE

[00217] The entire description of each of the patent documents and scientific articles referenced herein is incorporated by reference in all respects.

EQUIVALENTS

[00218] The invention may be embodied in other specific forms without departing from its spirit or essential characteristics. Thus, the preceding embodiments are to be considered in all respects as illustrative and not limiting of the invention described herein. Thus, the scope of the invention is determined by the appended claims and not by the preceding description, and all changes that are within the meaning and range of equivalence of the claims are to be included therein.

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LIST OF SEQUENCES

<110> ADIMAB, LLC.

<120> CD33, NKG2D AND CD16 BINDING PROTEINS

<130>DFY-007PC

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Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Gly Ser Trp

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Lys Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Asn Ser Tyr Tyr Thr

85 90 95

Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 9

<211> 117

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polypeptide

<400> 9

Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr

20 25 30

Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Asp His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys

50 55 60

Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Ala Arg Gly Pro Trp Ser Phe Asp Pro Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 10

<211> 106

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polypeptide

<400> 10

Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Trp

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Lys Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser Tyr Pro Thr

85 90 95

Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 11

<211> 117

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polypeptide

<400> 11

Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr

20 25 30

Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Asp His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys

50 55 60

Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Ala Arg Gly Pro Trp Gly Phe Asp Pro Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 12

<211> 107

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polypeptide

<400> 12

Glu Leu Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Thr Ser Gln Ser Ile Ser Ser Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile

35 40 45

Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val Pro Asp Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Ser Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Asp Ile Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 13

<211> 117

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polypeptide

<400> 13

Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr

20 25 30

Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Asp His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys

50 55 60

Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Ala Arg Gly Pro Trp Ser Phe Asp Pro Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 14

<211> 107

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polypeptide

<400> 14

Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Trp

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Lys Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Gly Ser Phe Pro Ile

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 15

<211> 117

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polypeptide

<400> 15

Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr

20 25 30

Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Asp His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys

50 55 60

Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Ala Arg Gly Pro Trp Ser Phe Asp Pro Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 16

<211> 107

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polypeptide

<400> 16

Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Trp

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Lys Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Lys Glu Val Pro Trp

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 17

<211> 117

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polypeptide

<400> 17

Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr

20 25 30

Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Asp His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys

50 55 60

Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Ala Arg Gly Pro Trp Ser Phe Asp Pro Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 18

<211> 106

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polypeptide

<400> 18

Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Trp

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Lys Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser Phe Pro Thr

85 90 95

Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 19

<211> 117

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polypeptide

<400> 19

Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr

20 25 30

Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Asp His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys

50 55 60

Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Ala Arg Gly Pro Trp Ser Phe Asp Pro Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 20

<211> 106

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polypeptide

<400> 20

Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Gly Ser Trp

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Lys Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asp Ile Tyr Pro Thr

85 90 95

Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 21

<211> 117

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polypeptide

<400> 21

Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr

20 25 30

Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Asp His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys

50 55 60

Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Ala Arg Gly Pro Trp Ser Phe Asp Pro Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 22

<211> 106

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polypeptide

<400> 22

Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Trp

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Lys Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asp Ser Tyr Pro Thr

85 90 95

Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 23

<211> 117

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polypeptide

<400> 23

Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr

20 25 30

Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Asp His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys

50 55 60

Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Ala Arg Gly Pro Trp Ser Phe Asp Pro Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 24

<211> 106

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polypeptide

<400> 24

Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Trp

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Lys Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Gly Ser Phe Pro Thr

85 90 95

Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 25

<211> 117

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polypeptide

<400> 25

Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr

20 25 30

Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Asp His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys

50 55 60

Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Ala Arg Gly Pro Trp Ser Phe Asp Pro Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 26

<211> 106

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polypeptide

<400> 26

Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Trp

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Lys Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Gln Ser Phe Pro Thr

85 90 95

Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 27

<211> 117

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polypeptide

<400> 27

Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr

20 25 30

Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Asp His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys

50 55 60

Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Ala Arg Gly Pro Trp Ser Phe Asp Pro Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 28

<211> 106

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polypeptide

<400> 28

Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Trp

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Lys Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Ser Phe Ser Thr

85 90 95

Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 29

<211> 117

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polypeptide

<400> 29

Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr

20 25 30

Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Asp His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys

50 55 60

Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Ala Arg Gly Pro Trp Ser Phe Asp Pro Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 30

<211> 106

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polypeptide

<400> 30

Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Trp

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Lys Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Glu Ser Tyr Ser Thr

85 90 95

Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 31

<211> 117

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polypeptide

<400> 31

Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr

20 25 30

Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Asp His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys

50 55 60

Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Ala Arg Gly Pro Trp Ser Phe Asp Pro Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 32

<211> 106

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polypeptide

<400> 32

Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Trp

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Lys Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asp Ser Phe Ile Thr

85 90 95

Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 33

<211> 117

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polypeptide

<400> 33

Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr

20 25 30

Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Asp His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys

50 55 60

Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Ala Arg Gly Pro Trp Ser Phe Asp Pro Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 34

<211> 106

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polypeptide

<400> 34

Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Trp

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Lys Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Gln Ser Tyr Pro Thr

85 90 95

Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 35

<211> 117

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polypeptide

<400> 35

Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr

20 25 30

Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Asp His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys

50 55 60

Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Ala Arg Gly Pro Trp Ser Phe Asp Pro Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 36

<211> 106

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polypeptide

<400> 36

Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Gly Ser Trp

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Lys Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr His Ser Phe Pro Thr

85 90 95

Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 37

<211> 117

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polypeptide

<400> 37

Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr

20 25 30

Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Asp His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys

50 55 60

Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Ala Arg Gly Pro Trp Ser Phe Asp Pro Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 38

<211> 107

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polypeptide

<400> 38

Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Gly Ser Trp

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Lys Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Glu Leu Tyr Ser Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 39

<211> 117

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polypeptide

<400> 39

Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr

20 25 30

Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Asp His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys

50 55 60

Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Ala Arg Gly Pro Trp Ser Phe Asp Pro Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 40

<211> 106

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polypeptide

<400> 40

Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Trp

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Lys Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asp Thr Phe Ile Thr

85 90 95

Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 41

<211> 125

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polypeptide

<400> 41

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser Tyr

20 25 30

Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Gly Ile Ile Pro Ile Phe Gly Thr Ala Asn Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Asp Ser Ser Ile Arg His Ala Tyr Tyr Tyr Tyr Gly Met

100 105 110

Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser

115 120 125

<210> 42

<211> 113

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polypeptide

<400> 42

Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Val Leu Tyr Ser

20 25 30

Ser Asn Asn Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln

85 90 95

Tyr Tyr Ser Thr Pro Ile Thr Phe Gly Gly Gly Thr Lys Val Glu Ile

100 105 110

Lys

<210> 43

<211> 121

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polypeptide

<400> 43

Gln Leu Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Ser

20 25 30

Ser Tyr Tyr Trp Gly Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu

35 40 45

Trp Ile Gly Ser Ile Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser

50 55 60

Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe

65 70 75 80

Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Arg Gly Ser Asp Arg Phe His Pro Tyr Phe Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 44

<211> 107

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polypeptide

<400> 44

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Arg Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Phe Asp Thr Trp Pro Pro

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 45

<211> 117

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polypeptide

<400> 45

Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr

20 25 30

Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Asp His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys

50 55 60

Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Ala Arg Gly Pro Trp Ser Phe Asp Pro Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 46

<211> 106

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polypeptide

<400> 46

Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Trp

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Lys Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Asp Asp Phe Ala Thr Tyr Tyr Cys Glu Gln Tyr Asp Ser Tyr Pro Thr

85 90 95

Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 47

<211> 126

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polypeptide

<400> 47

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser Tyr

20 25 30

Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Gly Ile Ile Pro Ile Phe Gly Thr Ala Asn Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Arg Gly Arg Lys Ala Ser Gly Ser Phe Tyr Tyr Tyr Tyr Gly

100 105 110

Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser

115 120 125

<210> 48

<211> 113

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polypeptide

<400> 48

Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Glu Ser Ser Gln Ser Leu Leu Asn Ser

20 25 30

Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Pro Pro Lys Pro Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Asn

85 90 95

Asp Tyr Ser Tyr Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile

100 105 110

Lys

<210> 49

<211> 121

<212> PROTEIN

<213> Homo sapiens

<400> 49

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Phe Ile Arg Tyr Asp Gly Ser Asn Lys Tyr 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 Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Lys Asp Arg Gly Leu Gly Asp Gly Thr Tyr Phe Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 50

<211> 110

<212> PROTEIN

<213> Homo sapiens

<400> 50

Gln Ser Ala Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln

1 5 10 15

Ser Ile Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Asn Asn

20 25 30

Ala Val Asn Trp Tyr Gln Gln Leu Pro Gly Lys Ala Pro Lys Leu Leu

35 40 45

Ile Tyr Tyr Asp Asp Leu Leu Pro Ser Gly Val Ser Asp Arg Phe Ser

50 55 60

Gly Ser Lys Ser Gly Thr Ser Ala Phe Leu Ala Ile Ser Gly Leu Gln

65 70 75 80

Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Ser Leu

85 90 95

Asn Gly Pro Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105 110

<210> 51

<211> 115

<212> PROTEIN

<213> Homo sapiens

<400> 51

Gln Val His Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Asp Asp Ser Ile Ser Ser Tyr

20 25 30

Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly His Ile Ser Tyr Ser Gly Ser Ala Asn Tyr Asn Pro Ser Leu Lys

50 55 60

Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Asn Trp Asp Asp Ala Phe Asn Ile Trp Gly Gln Gly Thr Met Val Thr

100 105 110

Val Ser Ser

115

<210> 52

<211> 108

<212> PROTEIN

<213> Homo sapiens

<400> 52

Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser

20 25 30

Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu

35 40 45

Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu

65 70 75 80

Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Pro

85 90 95

Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 53

<211> 364

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polypeptide

<400> 53

Met Pro Leu Leu Leu Leu Leu Pro Leu Leu Trp Ala Gly Ala Leu Ala

1 5 10 15

Met Asp Pro Asn Phe Trp Leu Gln Val Gln Glu Ser Val Thr Val Gln

20 25 30

Glu Gly Leu Cys Val Leu Val Pro Cys Thr Phe Phe His Pro Ile Pro

35 40 45

Tyr Tyr Asp Lys Asn Ser Pro Val His Gly Tyr Trp Phe Arg Glu Gly

50 55 60

Ala Ile Ile Ser Arg Asp Ser Pro Val Ala Thr Asn Lys Leu Asp Gln

65 70 75 80

Glu Val Gln Glu Glu Thr Gln Gly Arg Phe Arg Leu Leu Gly Asp Pro

85 90 95

Ser Arg Asn Asn Cys Ser Leu Ser Ile Val Asp Ala Arg Arg Arg Asp

100 105 110

Asn Gly Ser Tyr Phe Phe Arg Met Glu Arg Gly Ser Thr Lys Tyr Ser

115 120 125

Tyr Lys Ser Pro Gln Leu Ser Val His Val Thr Asp Leu Thr His Arg

130 135 140

Pro Lys Ile Leu Ile Pro Gly Thr Leu Glu Pro Gly His Ser Lys Asn

145 150 155 160

Leu Thr Cys Ser Val Ser Trp Ala Cys Glu Gln Gly Thr Pro Pro Ile

165 170 175

Phe Ser Trp Leu Ser Ala Ala Pro Thr Ser Leu Gly Pro Arg Thr Thr

180 185 190

His Ser Ser Val Leu Ile Ile Thr Pro Arg Pro Gln Asp His Gly Thr

195 200 205

Asn Leu Thr Cys Gln Val Lys Phe Ala Gly Ala Gly Val Thr Thr Glu

210 215 220

Arg Thr Ile Gln Leu Asn Val Thr Tyr Val Pro Gln Asn Pro Thr Thr

225 230 235 240

Gly Ile Phe Pro Gly Asp Gly Ser Gly Lys Gln Glu Thr Arg Ala Gly

245 250 255

Val Val His Gly Ala Ile Gly Gly Ala Gly Val Thr Ala Leu Leu Ala

260 265 270

Leu Cys Leu Cys Leu Ile Phe Phe Ile Val Lys Thr His Arg Arg Lys

275 280 285

Ala Ala Arg Thr Ala Val Gly Arg Asn Asp Thr His Pro Thr Thr Gly

290 295 300

Ser Ala Ser Pro Lys His Gln Lys Lys Ser Lys Leu His Gly Pro Thr

305 310 315 320

Glu Thr Ser Ser Cys Ser Gly Ala Ala Pro Thr Val Glu Met Asp Glu

325 330 335

Glu Leu His Tyr Ala Ser Leu Asn Phe His Gly Met Asn Pro Ser Lys

340 345 350

Asp Thr Ser Thr Glu Tyr Ser Glu Val Arg Thr Gln

355 360

<210> 54

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

peptide

<400> 54

Gly Ser Phe Ser Gly Tyr Tyr Trp Ser

15

<210> 55

<211> 16

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

peptide

<400> 55

Glu Ile Asp His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys Ser

1 5 10 15

<210> 56

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

peptide

<400> 56

Ala Arg Ala Arg Gly Pro Trp Ser Phe Asp Pro

1 5 10

<210> 57

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

peptide

<400> 57

Gly Thr Phe Ser Ser Tyr Ala Ile Ser

15

<210> 58

<211> 17

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

peptide

<400> 58

Gly Ile Ile Pro Ile Phe Gly Thr Ala Asn Tyr Ala Gln Lys Phe Gln

1 5 10 15

Gly

<210> 59

<211> 18

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

peptide

<400> 59

Ala Arg Gly Asp Ser Ser Ile Arg His Ala Tyr Tyr Tyr Tyr Gly Met

1 5 10 15

Asp Val

<210> 60

<211> 17

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

peptide

<400> 60

Lys Ser Ser Gln Ser Val Leu Tyr Ser Ser Asn Asn Lys Asn Tyr Leu

1 5 10 15

Ala

<210> 61

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

peptide

<400> 61

Trp Ala Ser Thr Arg Glu Ser

15

<210> 62

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

peptide

<400> 62

Gln Gln Tyr Tyr Ser Thr Pro Ile Thr

15

<210> 63

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

peptide

<400> 63

Gly Ser Ile Ser Ser Ser Ser Tyr Tyr Trp Gly

1 5 10

<210> 64

<211> 16

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

peptide

<400> 64

Ser Ile Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser Leu Lys Ser

1 5 10 15

<210> 65

<211> 13

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

peptide

<400> 65

Ala Arg Gly Ser Asp Arg Phe His Pro Tyr Phe Asp Tyr

1 5 10

<210> 66

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

peptide

<400> 66

Arg Ala Ser Gln Ser Val Ser Arg Tyr Leu Ala

1 5 10

<210> 67

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

peptide

<400> 67

Asp Ala Ser Asn Arg Ala Thr

15

<210> 68

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

peptide

<400> 68

Gln Gln Phe Asp Thr Trp Pro Pro Thr

15

<210> 69

<211> 121

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polypeptide

<400> 69

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 Ser Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr 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 Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Lys Asp Gly Gly Tyr Tyr Asp Ser Gly Ala Gly Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 70

<211> 107

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polypeptide

<400> 70

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Asp Ser Trp

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Val Ser Tyr Pro Arg

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 71

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

peptide

<400> 71

Phe Thr Phe Ser Ser Tyr Ala Met Ser

15

<210> 72

<211> 17

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

peptide

<400> 72

Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys

1 5 10 15

Gly

<210> 73

<211> 14

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

peptide

<400> 73

Ala Lys Asp Gly Gly Tyr Tyr Asp Ser Gly Ala Gly Asp Tyr

1 5 10

<210> 74

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

peptide

<400> 74

Arg Ala Ser Gln Gly Ile Asp Ser Trp Leu Ala

1 5 10

<210> 75

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

peptide

<400> 75

Ala Ala Ser Ser Leu Gln Ser

15

<210> 76

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

peptide

<400> 76

Gln Gln Gly Val Ser Tyr Pro Arg Thr

15

<210> 77

<211> 122

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polypeptide

<400> 77

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Ser Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Ser Ser Ser Ser Ser Tyr Ile Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Ala Pro Met Gly Ala Ala Ala Gly Trp Phe Asp Pro Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 78

<211> 107

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polypeptide

<400> 78

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Trp

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Val Ser Phe Pro Arg

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 79

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

peptide

<400> 79

Phe Thr Phe Ser Ser Tyr Ser Met Asn

15

<210> 80

<211> 17

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

peptide

<400> 80

Ser Ile Ser Ser Ser Ser Ser Tyr Ile Tyr Tyr Ala Asp Ser Val Lys

1 5 10 15

Gly

<210> 81

<211> 15

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

peptide

<400> 81

Ala Arg Gly Ala Pro Met Gly Ala Ala Ala Gly Trp Phe Asp Pro

1 5 10 15

<210> 82

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

peptide

<400> 82

Arg Ala Ser Gln Gly Ile Ser Ser Trp Leu Ala

1 5 10

<210> 83

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

peptide

<400> 83

Ala Ala Ser Ser Leu Gln Ser

15

<210> 84

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

peptide

<400> 84

Gln Gln Gly Val Ser Phe Pro Arg Thr

15

<210> 85

<211> 124

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polypeptide

<400> 85

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Gly Tyr

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Asn Pro Asn Ser Gly Gly Thr Asn Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Thr Gly Glu Tyr Tyr Asp Thr Asp Asp His Gly Met Asp

100 105 110

Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 86

<211> 107

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polypeptide

<400> 86

Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Asn

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Gly Ala Ser Thr Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Asp Asp Tyr Trp Pro Pro

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 87

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

peptide

<400> 87

Tyr Thr Phe Thr Gly Tyr Tyr Met His

15

<210> 88

<211> 17

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

peptide

<400> 88

Trp Ile Asn Pro Asn Ser Gly Gly Thr Asn Tyr Ala Gln Lys Phe Gln

1 5 10 15

Gly

<210> 89

<211> 17

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

peptide

<400> 89

Ala Arg Asp Thr Gly Glu Tyr Tyr Asp Thr Asp Asp His Gly Met Asp

1 5 10 15

Val

<210> 90

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

peptide

<400> 90

Arg Ala Ser Gln Ser Val Ser Ser Asn Leu Ala

1 5 10

<210> 91

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

peptide

<400> 91

Gly Ala Ser Thr Arg Ala Thr

15

<210> 92

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

peptide

<400> 92

Gln Gln Asp Asp Tyr Trp Pro Pro Thr

15

<210> 93

<211> 116

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polypeptide

<400> 93

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Asn Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Tyr Pro Tyr Asn Gly Gly Thr Gly Tyr Asn Gln Lys Phe

50 55 60

Lys Ser Lys Ala Thr Ile Thr Ala Asp Glu Ser Thr Asn Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Arg Pro Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu Val

100 105 110

Thr Val Ser Ser

115

<210> 94

<211> 111

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polypeptide

<400> 94

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu Ser Val Asp Asn Tyr

20 25 30

Gly Ile Ser Phe Met Asn Trp Phe Gln Gln Lys Pro Gly Lys Ala Pro

35 40 45

Lys Leu Leu Ile Tyr Ala Ala Ser Asn Gln Gly Ser Gly Val Pro Ser

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

Ser Leu Gln Pro Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Lys

85 90 95

Glu Val Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 95

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

peptide

<400> 95

Gly Tyr Thr Phe Thr Asp Tyr

15

<210> 96

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

peptide

<400> 96

Tyr Ile Tyr Pro Tyr Asn Gly Gly Thr Gly

1 5 10

<210> 97

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

peptide

<400> 97

Gly Arg Pro Ala Met Asp Tyr

15

<210> 98

<211> 12

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

peptide

<400> 98

Glu Ser Val Asp Asn Tyr Gly Ile Ser Phe Met Asn

1 5 10

<210> 99

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

peptide

<400> 99

Ala Ala Ser Asn Gln Gly Ser

15

<210> 100

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

peptide

<400> 100

Gln Gln Ser Lys Glu Val Pro Trp Thr

15

<210> 101

<211> 116

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polypeptide

<400> 101

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Ile Thr Asp Ser

20 25 30

Asn Ile His Trp Val Arg Gln Ala Pro Gly Gln Ser Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Tyr Pro Tyr Asn Gly Gly Thr Asp Tyr Asn Gln Lys Phe

50 55 60

Lys Asn Arg Ala Thr Leu Thr Val Asp Asn Pro Thr Asn Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Phe Tyr Tyr Cys

85 90 95

Val Asn Gly Asn Pro Trp Leu Ala Tyr Trp Gly Gln Gly Thr Leu Val

100 105 110

Thr Val Ser Ser

115

<210> 102

<211> 111

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polypeptide

<400> 102

Asp Ile Gln Leu Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu Ser Leu Asp Asn Tyr

20 25 30

Gly Ile Arg Phe Leu Thr Trp Phe Gln Gln Lys Pro Gly Lys Ala Pro

35 40 45

Lys Leu Leu Met Tyr Ala Ala Ser Asn Gln Gly Ser Gly Val Pro Ser

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser

65 70 75 80

Ser Leu Gln Pro Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Thr Lys

85 90 95

Glu Val Pro Trp Ser Phe Gly Gln Gly Thr Lys Val Glu Val Lys

100 105 110

<210> 103

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

peptide

<400> 103

Gly Tyr Thr Ile Thr Asp Ser

15

<210> 104

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

peptide

<400> 104

Tyr Ile Tyr Pro Tyr Asn Gly Gly Thr Asp

1 5 10

<210> 105

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

peptide

<400> 105

Gly Asn Pro Trp Leu Ala Tyr

15

<210> 106

<211> 12

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

peptide

<400> 106

Glu Ser Leu Asp Asn Tyr Gly Ile Arg Phe Leu Thr

1 5 10

<210> 107

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

peptide

<400> 107

Ala Ala Ser Asn Gln Gly Ser

15

<210> 108

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

peptide

<400> 108

Gln Gln Thr Lys Glu Val Pro Trp Ser

15

<210> 109

<211> 118

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polypeptide

<400> 109

Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Val Val Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Tyr Ile His Trp Ile Lys Gln Thr Pro Gly Gln Gly Leu Glu Trp Val

35 40 45

Gly Val Ile Tyr Pro Gly Asn Asp Asp Ile Ser Tyr Asn Gln Lys Phe

50 55 60

Gln Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Thr Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Val Arg Leu Arg Tyr Phe Asp Val Trp Gly Gln Gly Thr

100 105 110

Thr Val Thr Val Ser Ser

115

<210> 110

<211> 113

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polypeptide

<400> 110

Glu Ile Val Leu Thr Gln Ser Pro Gly Ser Leu Ala Val Ser Pro Gly

1 5 10 15

Glu Arg Val Thr Met Ser Cys Lys Ser Ser Gln Ser Val Phe Phe Ser

20 25 30

Ser Ser Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Ile Pro Gly Gln

35 40 45

Ser Pro Arg Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Val Gln Pro Glu Asp Leu Ala Ile Tyr Tyr Cys His Gln

85 90 95

Tyr Leu Ser Ser Arg Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

Arg

<210> 111

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

peptide

<400> 111

Gly Tyr Thr Phe Thr Ser Tyr

15

<210> 112

<211> 6

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

peptide

<400> 112

Tyr Pro Gly Asn Asp Asp

15

<210> 113

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

peptide

<400> 113

Glu Val Arg Leu Arg Tyr Phe Asp Val

15

<210> 114

<211> 14

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

peptide

<400> 114

Gln Ser Val Phe Phe Ser Ser Ser Gln Lys Asn Tyr Leu Ala

1 5 10

<210> 115

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

peptide

<400> 115

Trp Ala Ser Thr Arg Glu Ser

15

<210> 116

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

peptide

<400> 116

His Gln Tyr Leu Ser Ser Arg Thr

15

<210> 117

<211> 118

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polypeptide

<400> 117

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr

20 25 30

Asp Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Trp Ile Tyr Pro Gly Asp Gly Ser Thr Lys Tyr Asn Glu Lys Phe

50 55 60

Lys Ala Lys Ala Thr Leu Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ser Gly Tyr Glu Asp Ala Met Asp Tyr Trp Gly Gln Gly Thr Thr

100 105 110

Val Thr Val Ser Ser Ala

115

<210> 118

<211> 108

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polypeptide

<400> 118

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Asn Cys Lys Ala Ser Gln Asp Ile Asn Ser Tyr

20 25 30

Leu Ser Trp Phe Gln Gln Lys Pro Gly Lys Ala Pro Lys Thr Leu Ile

35 40 45

Tyr Arg Ala Asn Arg Leu Val Asp Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Gln Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Tyr Asp Glu Phe Pro Leu

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg

100 105

<210> 119

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

peptide

<400> 119

Gly Tyr Thr Phe Thr Asn Tyr

15

<210> 120

<211> 6

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

peptide

<400> 120

Tyr Pro Gly Asp Gly Ser

15

<210> 121

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

peptide

<400> 121

Gly Tyr Glu Asp Ala Met Asp Tyr

15

<210> 122

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

peptide

<400> 122

Gln Asp Ile Asn Ser Tyr Leu Ser

15

<210> 123

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

peptide

<400> 123

Arg Ala Asn Arg Leu Val Asp

15

<210> 124

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

peptide

<400> 124

Leu Gln Tyr Asp Glu Phe Pro Leu Thr

15

<210> 125

<211> 120

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polypeptide

<400> 125

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Val Val His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Tyr Ile Asn Pro Tyr Asn Asp Gly Thr Lys Tyr Asn Glu Lys Phe

50 55 60

Lys Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Tyr Arg Tyr Glu Val Tyr Gly Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 126

<211> 106

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polypeptide

<400> 126

Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Pro Gly

1 5 10 15

Gln Arg Ala Thr Ile Thr Cys Thr Ala Ser Ser Ser Val Asn Tyr Ile

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr

35 40 45

Asp Thr Ser Lys Val Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn Pro Val Glu Ala Asn

65 70 75 80

Asp Thr Ala Asn Tyr Tyr Cys Gln Gln Trp Arg Ser Tyr Pro Leu Thr

85 90 95

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 127

<211> 5

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

peptide

<400> 127

Asp Tyr Val Val His

15

<210> 128

<211> 17

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

peptide

<400> 128

Tyr Ile Asn Pro Tyr Asn Asp Gly Thr Lys Tyr Asn Glu Lys Phe Lys

1 5 10 15

Gly

<210> 129

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

peptide

<400> 129

Asp Tyr Arg Tyr Glu Val Tyr Gly Met Asp Tyr

1 5 10

<210> 130

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

peptide

<400> 130

Thr Ala Ser Ser Ser Val Asn Tyr Ile His

1 5 10

<210> 131

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

peptide

<400> 131

Asp Thr Ser Lys Val Ala Ser

15

<210> 132

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

peptide

<400> 132

Gln Gln Trp Arg Ser Tyr Pro Leu Thr

15

<210> 133

<211> 126

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic peptide

<400> 133

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Ile Ile Asn Pro Ser Gly Gly Ser Thr Ser Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Ala Pro Asn Tyr Gly Asp Thr Thr His Asp Tyr Tyr Tyr

100 105 110

Met Asp Val Trp Gly Lys Gly Thr Thr Val Thr Val Ser Ser

115 120 125

<210> 134

<211> 107

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic peptide

<400> 134

Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Asn

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Gly Ala Ser Thr Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Asp Asp Trp Pro Phe

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 135

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic peptide

<400> 135

Tyr Thr Phe Thr Ser Tyr Tyr Met His

15

<210> 136

<211> 17

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic peptide

<400> 136

Ile Ile Asn Pro Ser Gly Gly Ser Thr Ser Tyr Ala Gln Lys Phe Gln

1 5 10 15

Gly

<210> 137

<211> 19

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic peptide

<400> 137

Ala Arg Gly Ala Pro Asn Tyr Gly Asp Thr Thr His Asp Tyr Tyr Tyr

1 5 10 15

Met Asp Val

<210> 138

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic peptide

<400> 138

Arg Ala Ser Gln Ser Val Ser Ser Asn Leu Ala

1 5 10

<210> 139

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic peptide

<400> 139

Gly Ala Ser Thr Arg Ala Thr

15

<210> 140

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic peptide

<400> 140

Gln Gln Tyr Asp Asp Trp Pro Phe Thr

15

<---

Claims (26)

1. Multispecific binding protein that binds to NKG2D, CD33 and CD16, including: (a) a first antigen binding region of an immunoglobulin molecule that binds NKG2D, where the first antigen binding region includes a heavy chain variable domain (VH) and a light chain variable domain (VL); (b) a second antigen binding region of the immunoglobulin molecule that binds CD33, where the second antigen binding region includes VH and VL; And (c) Fc domain of a human IgG1 antibody that binds to CD16, wherein the human IgG1 antibody comprises a first Fc domain of a human IgG1 antibody and a second Fc domain of a human IgG1 antibody, and wherein a VH or VL of a first antigen binding region is linked to a first Fc domain of a human IgG1 antibody, and wherein a VH or VL of a second antigen binding region is linked to a second Fc -domain of human IgG1 antibody; and, wherein the first antigen binding region comprises: (i) VH containing the amino acid sequence of SEQ ID NO:13, and VL containing the amino acid sequence of SEQ ID NO:14; (ii) VH containing the amino acid sequence of SEQ ID NO:45, and VL containing the amino acid sequence of SEQ ID NO:46; (iii) VH CDR1 containing the amino acid sequence of SEQ ID NO:71, VH CDR2 containing the amino acid sequence of SEQ ID NO:72, VH CDR3 containing the amino acid sequence of SEQ ID NO:73, VL CDR1 containing the amino acid sequence of SEQ ID NO:74 , VL CDR2 containing the amino acid sequence of SEQ ID NO:75, and VL CDR3 containing the amino acid sequence of SEQ ID NO:76; (iv) VH CDR1 containing the amino acid sequence of SEQ ID NO:79, VH CDR2 containing the amino acid sequence of SEQ ID NO:80, VH CDR3 containing the amino acid sequence of SEQ ID NO:81, VL CDR1 containing the amino acid sequence of SEQ ID NO:82 , VL CDR2 containing the amino acid sequence of SEQ ID NO:83, and VL CDR3 containing the amino acid sequence of SEQ ID NO:84; or (v) VH CDR1 containing the amino acid sequence of SEQ ID NO:135, VH CDR2 containing the amino acid sequence of SEQ ID NO:136, VH CDR3 containing the amino acid sequence of SEQ ID NO:137, VL CDR1 containing the amino acid sequence of SEQ ID NO:138 , VL CDR2 containing the amino acid sequence of SEQ ID NO:139, and VL CDR3 containing the amino acid sequence of SEQ ID NO:140. 2. The multispecific binding protein of claim 1, wherein the first antigen binding site binds to human and non-human primate NKG2D. 3. The multispecific binding protein according to claim 1, wherein the VH of the first antigen-binding site and the VL of the first antigen-binding site are present on a single polypeptide. 4. The multispecific binding protein according to claim 1, wherein the VH of the second antigen-binding site and the VL of the second antigen-binding site are present on a single polypeptide. 5. The multispecific binding protein according to claim 1, wherein the VH of the second antigen binding region includes the amino acid sequence of SEQ ID NO:93, and the VL of the second antigen binding region includes the amino acid sequence of SEQ ID NO:94. 6. The multispecific binding protein according to claim 1, wherein the VH of the second antigen binding region includes the amino acid sequence of SEQ ID NO:101, and the VL of the second antigen binding region includes the amino acid sequence of SEQ ID NO:102. 7. The multispecific binding protein of claim 1, wherein the VH of the second antigen binding region includes the amino acid sequence of SEQ ID NO:109, and the VL of the second antigen binding region includes the amino acid sequence of SEQ ID NO:110. 8. The multispecific binding protein according to claim 1, wherein the VH of the second antigen binding region includes the amino acid sequence of SEQ ID NO:117, and the VL of the second antigen binding region includes the amino acid sequence of SEQ ID NO:118. 9. The multispecific binding protein according to claim 1, wherein the VH of the second antigen binding region includes the amino acid sequence of SEQ ID NO:125, and the VL of the second antigen binding region includes the amino acid sequence of SEQ ID NO:126. 10. The multispecific binding protein according to claim 1, wherein the Fc domain of the human IgG1 antibody includes a hinge and CH2 domains. 11. The multispecific binding protein of claim 10, wherein the Fc domain of the human IgG1 antibody comprises an amino acid sequence that is at least 90% identical to amino acids 234-332 of the human IgG1 antibody. 12. The multispecific binding protein of claim 10, wherein the Fc domain of the human IgG1 antibody comprises an amino acid sequence that is at least 90% identical to the Fc domain of human IgG1 and differs at one or more positions selected from the group consisting of Q347 , Y349, T350, L351, S354, E356, E357, K360, Q362, S364, T366, L368, K370, N390, K392, T394, D399, S400, D401, F405, Y407, K409, T411 and K439. 13. A dosage form for the treatment of cancer, comprising a multispecific binding protein according to any of the previous claims and a pharmaceutically acceptable carrier. 14. A host cell for producing a multispecific binding protein according to any one of claims. 1-12, including one or more nucleic acids encoding a polypeptide of a multispecific binding protein according to any one of paragraphs. 1-12. 15. A method of directly and/or indirectly enhancing the cytolysis of a tumor cell, including contact of a tumor cell and a natural killer cell with a multispecific binding protein according to any one of claims. 1-12. 16. A method of treating cancer, where the method includes administering to a patient a multispecific binding protein according to any one of claims. 1-12 or dosage form according to paragraph 13. 17. The method of claim 16, wherein the cancer is selected from the group consisting of acute myeloid leukemia (AML), myelodysplastic syndromes, chronic myelomonocytic leukemia, myeloid blast crisis in chronic myeloid leukemia, and various forms of acute lymphoblastic leukemia (ALL).

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