HK1244011B

HK1244011B - Common light chains and methods of use

Info

Publication number: HK1244011B
Application number: HK18103433.9A
Authority: HK
Inventors: Christian Klein; Ekkehard Moessner; Ralf Hosse; Peter Bruenker; Pablo Umana; Christiane Jaeger
Original assignee: F. Hoffmann-La Roche Ag
Priority date: 2014-11-20
Filing date: 2015-11-17
Publication date: 2023-03-10

Description

Common light chains and methods of use

Sequence listing

The present application contains a sequence listing that has been submitted electronically in ASCII format and is incorporated by reference herein in its entirety. The ASCII copy was created at 11, 10, 2015 under the name 32398_ sl.txt and with a size 537,667 bytes.

Technical Field

The present invention relates generally to bispecific antigen binding molecules for activating T cells. Furthermore, the present invention relates to polynucleotides encoding such bispecific antigen binding molecules, as well as vectors and host cells comprising such polynucleotides. The invention also relates to methods for producing the bispecific antigen binding molecules of the invention and methods of using these bispecific antigen binding molecules in the treatment of disease.

Background

In various clinical settings, selective destruction of individual cells or specific cell types is often desired. For example, the main goal of cancer therapy is to specifically destroy tumor cells while leaving healthy cells and tissues intact.

An attractive way to achieve this is to have immune effector cells such as Natural Killer (NK) cells or Cytotoxic T Lymphocytes (CTL) attack and destroy the tumor cells by inducing an immune response against the tumor. CTLs constitute the most efficient effector cells of the immune system, but they cannot be activated by effector mechanisms mediated by the Fc domain of conventional therapeutic antibodies.

In this regard, bispecific antibodies have become of interest in recent years, which are designed to bind one "arm" to a surface antigen on a target cell, and a second "arm" to an activating invariant component of the T Cell Receptor (TCR) complex. This antibody binds to both of its targets simultaneously, forcing a transient interaction between the target cell and the T cell, causing activation of any cytotoxic T cells and subsequent lysis of the target cell. Thus, the immune response is redirected to the target cell and is independent of peptide antigen presentation by the target cell or specificity of the T cell, which is relevant for normal MHC-restricted activation of CTLs. In this case, it is crucial that the CTL is activated when the bispecific antibody binds to the target cell and the CTL (i.e. mimics the immune synapse). Particularly desirable are bispecific antibodies that do not require lymphocyte pretreatment or co-stimulation to elicit effective lysis of target cells.

Several bispecific antibody formats have been developed and investigated for their applicability to T cell-mediated immunotherapy. Among these, the so-called BiTE (bi-specific T-cell engager) molecules have been well characterized and have shown some promise clinically (in Nagorsen andreviewed in Exp Cell Res 317, 1255-. BiTE is a tandem scFv molecule in which two scFv molecules are fused by a flexible linker. Other bispecific formats for assessing T cell engagement include diabodies (Holliger et al, Prot Eng 9,299-305(1996)) and derivatives thereof, such as tandem diabodies (Kipriyanov et al, J Mol Biol 293,41-66 (1999)). A recent development is the so-called DART (dual affinity retargeting) molecule, which is based on a diabody format, but is characterized by a C-terminal disulfide bond for additional stabilization (Moore et al, Blood 117,4542-51 (2011)). The so-called Triomab is a fully heterozygous mouse/rat IgG molecule currently being evaluated in clinical trials, representing a larger size form (see Seimetz et al, Cancer Treat Rev 36, 458-.

The various formats being developed show great potential due to T cell redirection and activation in immunotherapy. However, the task of generating bispecific antibodies suitable therefor is by no means trivial, but involves the necessity to meet a number of challenges related to the efficacy, toxicity, applicability and productivity of the antibodies.

Small constructs, such as BiTE molecules-while effective in cross-linking effector and target cells-have a very short serum half-life, requiring them to be administered to a patient by continuous infusion. On the other hand, IgG-like forms, while having the great benefit of long half-life, suffer from toxicity associated with the natural effector functions inherent to IgG molecules. Their immunogenic potential constitutes another disadvantageous feature of IgG-like bispecific antibodies, particularly non-human forms of antibodies, for successful therapeutic development. Finally, a major challenge in the overall development of bispecific antibodies is to produce bispecific antibody constructs in clinically sufficient amounts and purity, which can be difficult to separate from the desired bispecific antibody due to mismatches in the antibody heavy and light chains of different specificities upon co-expression, reducing the yield of correctly assembled constructs and resulting in many non-functional by-products.

In view of the difficulties and disadvantages associated with bispecific antibodies currently available for T cell mediated immunotherapy, there remains a need for new and improved forms of this molecule.

Summary of The Invention

In a first aspect, the present invention provides a T cell activating bispecific antigen binding molecule comprising a first and a second antigen binding moiety, wherein the first antigen binding moiety comprises a first light chain, and wherein the first antigen binding moiety is capable of specifically binding to an activating T cell antigen and the second antigen binding moiety comprises a second light chain, wherein the second antigen binding moiety is capable of specifically binding to a target cell antigen, wherein the amino acid sequences of the first and second light chains are identical. In one embodiment, the first antigen binding portion is a Fab. In one embodiment, the second antigen-binding moiety is a Fab. In one embodiment, the first and second antigen-binding moieties are Fab.

In one aspect, the invention provides a T cell activating bispecific antigen binding molecule comprising a first and a second antigen binding moiety, one of which is a Fab molecule capable of specific binding to an activating T cell antigen and the other of which is a Fab molecule capable of specific binding to a target cell antigen, wherein the first and the second Fab molecule have the same VLCL light chain.

In one embodiment, the T cell activating bispecific antigen binding molecule further comprises an Fc domain consisting of a first and a second subunit capable of stable binding.

In one embodiment, the T cell activating bispecific antigen binding molecule comprises a light chain comprising the light chain CDRs of SEQ ID NO 32, SEQ ID NO 33 and SEQ ID NO 34.

In one embodiment, the T cell activating bispecific antigen binding molecule comprises a light chain comprising SEQ ID NO. 31.

In one embodiment, a Fab molecule capable of specifically binding an activated T cell antigen comprises a heavy chain comprising the heavy chain CDRs of SEQ ID NO 37, 38 and 39.

In particular embodiments, there is no more than one antigen binding moiety in the T cell activating bispecific antigen binding molecule that is capable of specifically binding to an activating T cell antigen (i.e., the T cell activating bispecific antigen binding molecule provides monovalent binding to an activating T cell antigen).

In some embodiments, the first and second antigen-binding portions of the T cell activating bispecific antigen binding molecule are fused to each other, optionally via a peptide linker. In one such embodiment, the second antigen binding portion is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen binding portion. In another such embodiment, the first antigen-binding portion is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen-binding portion. In another such embodiment, the second antigen binding portion is fused at the C-terminus of the Fab light chain to the N-terminus of the Fab light chain of the first antigen binding portion. In yet another such embodiment, the first antigen binding portion is fused at the C-terminus of the Fab light chain to the N-terminus of the Fab light chain of the second antigen binding portion. In one embodiment, the first antigen binding portion of the T cell activating bispecific antigen binding molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first or second subunit of the Fc domain.

In one embodiment, the second antigen-binding portion of the T cell activating bispecific antigen binding molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first or second subunit of the Fc domain.

In another embodiment, the first antigen binding portion is fused to the N-terminus of the first or second subunit of the Fc domain at the C-terminus of the Fab heavy chain.

In one embodiment, the first and second antigen-binding portions of the T cell activating bispecific antigen binding molecule are each fused at the C-terminus of the Fab heavy chain to the N-terminus of one subunit of the Fc domain.

In certain embodiments, the T cell activating bispecific antigen binding molecule comprises a third antigen binding moiety which is a Fab molecule capable of specifically binding to a target cell antigen. In one embodiment, the third antigen-binding portion is a Fab molecule comprising the same VLCL light chain as the first and second antigen-binding portions.

In one such embodiment, the first, second and third antigen binding portions are each Fab molecules comprising the light chain CDRs of SEQ ID NO:32, SEQ ID NO:33 and SEQ ID NO: 34.

In one such embodiment, the first, second and third antigen binding portions are each a Fab molecule comprising a light chain comprising SEQ ID NO. 31.

In one embodiment, the third antigen binding portion is fused to the N-terminus of the first or second subunit of the Fc domain at the C-terminus of the Fab heavy chain. In a specific embodiment, the second and third antigen binding portions of the T cell activating antigen binding molecule are each fused at the C-terminus of the Fab heavy chain to the N-terminus of one subunit of the Fc domain, and the first antigen binding portion is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen binding portion. In another specific embodiment, the first and third antigen binding portions of the T cell activating antigen binding molecule are each fused at the C-terminus of the Fab heavy chain to the N-terminus of one subunit of the Fc domain, and the second antigen binding portion is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen binding portion. The components of the T cell activating bispecific antigen binding molecule may be fused directly or through a suitable peptide linker. In one embodiment, the second and third antigen binding portions and the Fc domain are part of an immunoglobulin molecule. In one embodiment, the first and third antigen binding portions and the Fc domain are part of an immunoglobulin molecule. In a specific embodiment, the immunoglobulin molecule is an immunoglobulin of the IgG class. In more detail In an embodiment of (2), the immunoglobulin is IgG₁Subclass immunoglobulin. In another embodiment, the immunoglobulin is an IgG₄Subclass immunoglobulin.

In a specific embodiment, the Fc domain is an IgG Fc domain. In a specific embodiment, the Fc domain is IgG₁An Fc domain. In another specific embodiment, the Fc domain is an IgG₄An Fc domain. In a more specific embodiment, the Fc domain is an IgG comprising the amino acid substitution S228P₄An Fc domain. In even more particular embodiments, the Fc domain is an IgG comprising the amino acid substitutions L235E and S228P (SPLE)₄An Fc domain. In particular embodiments, the Fc domain is a human Fc domain.

In particular embodiments, the Fc domain comprises a modification that facilitates association of the first and second Fc domain subunits. In particular such embodiments, amino acid residues in the CH3 domain of the first subunit of the Fc domain are replaced with amino acid residues having a larger side chain volume, thereby creating a protuberance within the CH3 domain of the first subunit that is positionable in a cavity within the CH3 domain of the second subunit, and amino acid residues in the CH3 domain of the second subunit of the Fc domain are replaced with amino acid residues having a smaller side chain volume, thereby creating a cavity within the CH3 domain of the second subunit, and a protuberance within the CH3 domain of the first subunit is positionable in a cavity within the CH3 domain of the second subunit.

In a particular embodiment, the IgG is naturally associated with₁Fc domains exhibit reduced binding affinity to Fc receptors and/or reduced effector function compared to Fc domains. In certain embodiments, the Fc domain is engineered to have reduced binding affinity for an Fc receptor and/or reduced effector function as compared to an unengineered Fc domain. In one embodiment, the Fc domain comprises one or more amino acid substitutions that reduce binding to an Fc receptor and/or effector function. In one embodiment, one or more amino acid substitutions in the Fc domain that reduce binding to an Fc receptor and/or effector function are located in a position selected from the group consisting of L234, L235, and P329 (Kaba)t number) of the display. In a specific embodiment, each subunit of the Fc domain comprises three amino acid substitutions that reduce binding to an Fc receptor and/or effector function, wherein the amino acid substitutions are L234A, L235A, and P329G. In one such embodiment, the Fc domain is IgG₁Fc domain, in particular human IgG₁An Fc domain. In other embodiments, each subunit of the Fc domain comprises two amino acid substitutions that reduce binding to an Fc receptor and/or effector function, wherein the amino acid substitutions are L235E and P329G. In one such embodiment, the Fc domain is an IgG ₄Fc domain, in particular human IgG₄An Fc domain.

In one embodiment, the Fc receptor is an fey receptor. In one embodiment, the Fc receptor is a human Fc receptor. In one embodiment, the Fc receptor is an activating Fc receptor. In particular embodiments, the Fc receptor is human Fc γ RIIa, Fc γ RI and/or Fc γ RIIIa. In one embodiment, the effector function is antibody-dependent cell-mediated cytotoxicity (ADCC).

In a specific embodiment, the activated T cell antigen to which the bispecific antigen binding molecule is capable of binding is CD 3. In other embodiments, the target cell antigen to which the bispecific antigen binding molecule is capable of binding is a tumor cell antigen. In one embodiment, the target cell antigen is selected from the group consisting of folate receptor 1(FolR1), mucin-1 (MUC1-1), and B Cell Maturation Antigen (BCMA). In a particular embodiment, the target cell antigen is not BCMA.

In another aspect, the invention provides a light chain comprising the amino acid sequences of SEQ ID NO 32, SEQ ID NO 33 and SEQ ID NO 34 for a T cell activating bispecific antigen binding molecule. In one embodiment, the light chain comprises the amino acid sequence of SEQ ID NO 31. In one embodiment, the light chain comprises the amino acid sequence of SEQ ID NO 35.

In another aspect, the invention provides light chains comprising the amino acid sequences of SEQ ID NO 32, SEQ ID NO 33 and SEQ ID NO 34 for use in generating a library of T cell activating bispecific antigen binding molecules. In one embodiment, the light chain comprises the amino acid sequence of SEQ ID NO. 31. In one embodiment, the light chain comprises the amino acid sequence of SEQ ID NO 35.

In another aspect, the present invention provides an isolated polypeptide comprising the amino acid sequence of SEQ ID NO. 31.

In another aspect, the present invention provides an isolated polypeptide comprising the amino acid sequence of SEQ ID NO 35.

According to another aspect of the present invention there is provided an isolated polynucleotide encoding a T cell activating bispecific antigen binding molecule of the present invention or a fragment thereof. The invention also includes polypeptides encoded by the polynucleotides of the invention. The invention also provides expression vectors comprising the isolated polynucleotides of the invention and host cells comprising the isolated polynucleotides or expression vectors of the invention. In some embodiments, the host cell is a eukaryotic cell, particularly a mammalian cell.

In another aspect, there is provided a method of making a T cell activating bispecific antigen binding molecule of the invention comprising the steps of a) culturing a host cell of the invention under conditions suitable for expression of the T cell activating bispecific antigen binding molecule, and b) recovering the T cell activating bispecific antigen binding molecule. The invention also includes T cell activating bispecific antigen binding molecules prepared by the methods of the invention.

The invention also provides a pharmaceutical composition comprising a T cell activating bispecific antigen binding molecule of the invention and a pharmaceutically acceptable carrier. The invention also includes methods of using the T cell activating bispecific antigen binding molecules and pharmaceutical compositions of the invention. In one aspect, the invention provides a T cell activating bispecific antigen binding molecule for use as a medicament or a pharmaceutical composition of the invention. In one aspect, there is provided a T cell activating bispecific antigen binding molecule or pharmaceutical composition according to the invention for use in the treatment of a disease in an individual in need thereof. In a specific embodiment, the disease is cancer.

Also provided is the use of a T cell activating bispecific antigen binding molecule of the invention in the manufacture of a medicament for the treatment of a disease in an individual in need thereof; and methods of treating a disease in an individual comprising administering to the individual a therapeutically effective amount of a composition comprising a T cell activating bispecific antigen binding molecule of the invention in a pharmaceutically acceptable form. In a specific embodiment, the disease is cancer. In any of the above embodiments, the individual is preferably a mammal, particularly a human.

The invention also provides a method of inducing lysis of a target cell, particularly a tumor cell, comprising contacting the target cell with a T cell activating bispecific antigen binding molecule of the invention in the presence of a T cell, particularly a cytotoxic T cell.

In another aspect, the invention provides a method for identifying the variable heavy chain of a bispecific antigen binding molecule specific for a T cell activation antigen and a target cell antigen, comprising the step of screening a combinatorial library comprising the variable heavy chain and a light chain comprising the amino acid sequences of SEQ ID NO 32, SEQ ID NO 33 and SEQ ID NO 34. In one embodiment, the light chain comprises the amino acid sequence of SEQ ID NO 31. In one embodiment, the light chain comprises the amino acid sequence of SEQ ID NO 35.

Brief Description of Drawings

Figures 1A-I illustrate exemplary configurations of T cell activating bispecific antigen binding molecules (TCBs) disclosed herein. (FIG. 1I) all constructs except the kappa-lambda form have the P329G LALA mutation and comprise a knob-in-hole Fc fragment with a knob-in-hole (knob-in-hole) modification. (FIG. 1A) schematic representation of "FolR 1 TCB 2+1 inverted (common light chain)". The FolR1 binding agent was fused at the C-terminus of the Fab heavy chain to the N-terminus of the first subunit comprising the knob modified Fc domain. These constructs were not hybrid and had three times the same VLCL light chain. (FIG. 1B) "FolR 1 TCB 1+1 head-to-tail (common light chain)" diagram. These constructs were not hybridized and had twice the same VLCL light chain. (FIG. 1C) schematic representation of "FolR 1 TCB 1+1 classical (common light chain)". These constructs were not hybridized and had twice the same VLCL light chain. (FIG. 1D) schematic representation of "FolR 1 TCB 2+1 classical (common light chain)". The CD3 binding agent was fused at the C-terminus of the Fab heavy chain to the N-terminus of the first subunit comprising the knob modified Fc domain. These constructs were not hybrid and had three times the same VLCL light chain. (FIG. 1E) "FolR 1 TCB 2+1 crossfab classical" representation. These constructs comprise the Ck-VH chain used for CD3 binders, rather than the conventional CH1-VH chain. The CD3 binding agent was fused at the C-terminus of the Fab heavy chain to the N-terminus of the first subunit comprising the knob modified Fc domain. (FIG. 1F) "FolR 1 TCB 2+1 crossfab inverted". These constructs comprise the Ck-VH chain used for CD3 binding agents, rather than the conventional CH1-VH chain. The FolR1 binding agent was fused at the C-terminus of the Fab heavy chain to the N-terminus of the first subunit comprising the knob modified Fc domain. (FIG. 1G) "FolR 1 TCB 1+1 crossfab head-to-tail" is a schematic representation. These constructs comprise the Ck-VH chain used for CD3 binding agents, rather than the conventional CH1-VH chain. (FIG. 1H) "FolR 1 TCB 1+1 crossfab classic" presentation. These constructs comprise the Ck-VH chain used for CD3 binding agents, rather than the conventional CH1-VH chain. FIG. 1I illustrates the CD3/FolR1 kappa-lambda antibody format. These constructs comprise hybrid common light chain VLCH1 and one hybrid VHCL chain specific for CD3 and one hybrid VHCL chain specific for FolR 1.

Fig. 2A-C depict graphs summarizing binding of FoLR1 IgG binding agents to HeLa cells. Binding of the newly generated FolR1 binding agent to FolR1 expressed on HeLa cells was determined by flow cytometry. Bound antibody was detected with fluorescently labeled anti-human secondary antibody.

Fig. 3A-B depict graphs summarizing the specificity of FolR1 binding agents for FolR 1. Binding of FolR1 IgG to HEK cells transiently transfected with FolR1 or FolR2 was analyzed by flow cytometry to identify clones that specifically bound to FolR1 but not FolR 2. The antibody was detected with a fluorescently labeled anti-human secondary antibody.

Fig. 4A-B depict graphs summarizing cross-reactivity of FolR1 binding agents with cyFoLR 1. Cross-reactivity of FolR1 antibody to cyno FolR1 was treated by flow cytometry on HEK cells transiently transfected with cyFolR 1. The antibody was detected with a fluorescently labeled anti-human secondary antibody.

FIG. 5 depicts a graph of internalization of FolR1 TCB upon binding. Internalization of four FolR1 TCBs upon binding to FolR1 was detected on HeLa cells. After a specified time point of incubation at 37 ℃, the remaining FolR1 TCB on the surface was detected with fluorescently labeled anti-human secondary antibody. Percent internalization was calculated.

FIGS. 6A-E depict graphs summarizing FolR1 IgG binding to cells with different levels of FolR1 expression. The binding of 9D11,16D5 and Mov19 IgG to tumor cells with different levels of FolR1 expression was analyzed by flow cytometry. DP47 IgG was included as an isotype control, and MKN-45 was included as a FolR1 negative cell line. The antibody was detected with a fluorescently labeled anti-human secondary antibody.

FIGS. 7A-L depict graphs summarizing T cell-mediated killing of HT-29 and SKOV3 cells. FolR1 TCB was used to test T-cell mediated killing of HT-29 and SKOV3 tumor cells and to activate upregulation of markers on T cells upon killing. (FIGS. 7A-D) T cell mediated killing of HT-29 and SKOV3 cells in the presence of 9D11 FolR1 TCB and 16D5 FolR1 TCB was measured by LDH release after 24h and 48 h. DP47 TCB was included as a negative control. After 48 hours of incubation, upregulation of activation markers CD25 and CD69 on CD 8T cells and CD 4T cells was assessed by flow cytometry after killing SKOV3 (FIG. 7E-H) or HT-29 (FIG. 7I-L) tumor cells.

FIG. 8 shows a graph in which anti-FluR 1 is not present in association with red blood cells. Erythrocytes were gated to the CD235a positive population and binding of 9D11 IgG,16D5 IgG, Mov19 IgG and DP47 IgG to this population was determined by flow cytometry. The antibody was detected with a fluorescently labeled anti-human secondary antibody.

FIGS. 9A-D depict graphs summarizing upregulation of activation markers in whole blood. CD 4T cells and CD 8T cells were analyzed for upregulation of CD25 and CD69 activation markers 24h after addition of 9D11 FolR1 TCB,16D5 FolR1 TCB, Mov19 FolR1 TCB and DP47 TCB1 by flow cytometry.

FIG. 109D 11 binding of TCB a-glyco variants to HeLa cells. Binding of the 9D11 FolR1 TCB a-glyco variant to Hela cells was compared to binding of the original 9D11 TCB on HeLa cells. Antibodies were detected with fluorescently labeled anti-human secondary antibodies and binding was determined by flow cytometry.

FIGS. 11A-F depict graphs summarizing T-cell mediated killing with 9D11 FolR1 TCB a-glyco variants of tumor cells. The 9D11 FolR1 TCB a-glyco variant was used to test T cell mediated killing of SKOV3 (FIGS. 11A-D), MKN-45 (as FolR1 negative control) and (FIGS. 11E-F) HT-29 tumor cells compared to the original 9D11 FolR1 TCB killing. Readout LDH release after 24h and 48h was used.

Fig. 12A-X depict graphs summarizing T cell-mediated killing of primary epithelial cells. Primary epithelial cells with very low levels of FolR1 were used to test T cell mediated killing of 16D5 FolR1 TCB and 9D11 FolR1 TCB, with DP47 TCB as a negative control, including HT29 cells as a positive control. (FIGS. 12A-H) LDH release was measured after 24 hours and 48 hours for Human Retinal Pigment (HRP), Human Renal Cortex (HRC), human bronchial tubes (HB) and HT29 cells. Killing of HRP, (FIG. 12M-P) HRC, (FIG. 12Q-T) HB and (FIG. 12U-X) HT29 was measured by flow cytometry after 48 hours with upregulation of CD25 and CD69 activation markers on CD4T cells and CD8T cells.

FIGS. 13A-C show a comparison of different forms of TCB with 16D 5. The binding of four different TCB forms containing FolR1 binding agent 16D5 to HeLa cells is compared in fig. 13A, T cell mediated killing of SKOV3 cells after 24 hours 48 hours in fig. 14B, and up-regulation of CD25 and CD69 activation markers on CD4T and CD8T cells 48 hours after killing in fig. 14C.

FIGS. 14A-C depict a comparison of different forms of TCB with 9D 11. Three different forms of TCB containing FolR1 binder 9D11 were compared in a) binding to HeLa cells, B) T cell-mediated killing of SKOV3 cells after 24 and 48 hours and C) upregulation of CD25 and CD69 activation markers on CD 4T cells and CD 8T cells 48 hours post-killing.

Figure 15 depicts the PK-profile of FOLR1 TCB at three different doses in NOG mice.

Figure 16 illustrates the experimental protocol for efficacy studies using FOLR1 TCB.

FIGS. 17A-B depict tumor growth curves. (FIG. 17A) mean and SEM of tumor volumes in different treatment groups. (FIG. 17B) tumor growth of individual mice in all treatment groups. TGI (tumor growth inhibition) gives the percentage of the mean tumor volume compared to the vehicle group.

Figure 18 shows tumor weight at study termination.

FIGS. 19A-B show FACS analysis of tumor infiltrating T cells at study day 32. (FIG. 19A) single tumor cell suspensions stained with anti-human CD3/CD4/CD8 and analyzed by flow cytometry. (FIG. 19B) mean and SEM of T cell counts per mg of tumor tissue in the different treatment groups.

FIGS. 20A-B show FACS analysis of T-cell activation/degranulation and cytokine secretion at study day 32. CD4+ (fig. 20A) and CD8+ (fig. 20B) tumor infiltrating T cells stained for cytokines, activation and degranulation markers. Mean and SEM of T cell counts per mg of tumor tissue in different treatment groups are shown.

FIGS. 21A-B show percent tumor lysis. SKOV3 cells were incubated with PBMCs in the presence of either κ λ FoLR1 TCB or DP47 TCB. Killing of tumor cells was determined by measuring LDH release 24 hours (fig. 21A) and 48 hours later (fig. 21B).

FIGS. 22A-D show upregulation of CD25 and CD69 on CD4T cells. SKOV3 cells were incubated with PBMCs in the presence of either κ λ FoLR1 TCB or DP47 TCB. After 48 hours, the upregulation of CD25 and CD69 on CD4T cells (fig. 22A-B) and CD 8T cells (fig. 22C-D) was measured by flow cytometry.

FIGS. 23A-B show percent tumor lysis. T cell killing of SKov-3 cells (culture medium FolR1) induced by 36F2 TCB, Mov19 TCB and 21a5 TCB after 24 hours (fig. 23A) and 48 hours (fig. 23B) incubation (E: T10: 1, effector human PBMC).

FIGS. 24A-C show T cell killing induced by Hela (high FolR1) (FIG. 24A), Skov-3 (medium FolR1) (FIG. 24B) and HT-29 (low FolR1) (FIG. 24C) 36F2 TCB,16D5 TCB,16D5 TCB classical, 16D5 TCB 1+1 and 16D5 TCB HT of human tumor cells (E: T ═ 10:1, effector human PBMC, incubation time 24 hours). DP47 TCB was included as a non-binding control.

FIGS. 25A-C show upregulation of CD25 and CD69 on human CD8+ (FIGS. 25A, B) and CD4+ (FIG. 25C) T cells following T cell-mediated killing by Hela cells (high FolR1) (FIG. 25A), SKov-3 cells (medium FolR1) (FIG. 25B) and HT-29 cells (low FolR1) (FIG. 25C) (E: T ═ 10:1,48 hour incubation) induced by 36F2 TCB,16D5 TCB and DP47 TC (non-binding control).

Fig. 26A-F show T cell killing induced by 36F2 TCB,16D5 TCB, and DP47 TCB of human renal cortical epithelial cells (fig. 26A, B), human retinal pigment epithelial cells (fig. 26C, D), and HT-29 cells (fig. 26E, F) after 24 hours (fig. 26A, C, E) and 48 hours (fig. 26B, D, F) incubation (E: T ═ 10:1, effector human PBMC).

FIG. 27 depicts a table summarizing the quantification of FolR1 binding sites on various normal and cancer cell lines.

FIGS. 28A-B show binding of 16D5 TCB and its corresponding deamidated variant CD3 16D5 TCB N100A and 16D5 TCB S100aA and 9D11 TCB and its deamidated variants 9D11 TCB N100A and 9D11 TCB S100aA to human CD3 expressed on Jurkat cells.

Fig. 29A-B show T cell killing of SKov-3 (medium FolR1) human tumor cells induced by 16D5 TCB and its corresponding CD3 deamidated variant 16D5 TCB N100A and 16D5 TCB S100aA (fig. 29A) and 9D11 TCB and its deamidated variant 9D11 TCB N100A and 9D11 TCB S100aA (fig. 29B) (E: T10: 1, effector human PBMCs, incubation time 24 hours). DP47 TCB was included as a non-binding control.

FIGS. 30A-B show T cell killing of HT-29 (low FolR1) human tumor cells induced by 16D5 TCB and its corresponding CD3 deamidated variant 16D5 TCB N100A and 16D5 TCB S100aA (FIG. 30A) and 9D11 TCB and its deamidated variant 9D11 TCB N100A and 9D11 TCB S100aA (FIG. 30B) (E: T10: 1, effector human PBMCs, incubation time 24 hours). DP47 TCB was included as a non-binding control.

Figure 31 shows a sequence alignment of the VH domains of 3 identified specific binders to MUC 1. All three clones were derivatives of IGHV3-23 germline (SEQ ID NO: 136). Clones 58D6(SEQ ID NO:60) and 110A5(SEQ ID NO:64) were derived from a library randomized in CDR3 only, while clone 106D2(SEQ ID NO:62) was identified from a library randomized in all 3 CDRs. Positions in CDRs 1 and 2 that deviate from the germline sequence are printed in italics.

Fig. 32A-B show the characterization results of CLC binders. (FIG. 32A) SPR analysis. SPR-based kinetic analysis of 3 clones specifically binding to MUC 1. The smooth line represents the global fit of the data to the 1:1 interaction model. (FIG. 32B) summary of kinetic and thermodynamic parameters.

Fig. 33 depicts a schematic of the TCB constructs generated. The CLC TCB construct consists of 3 different immunoglobulin chains 1) an IgG heavy chain containing a "pore mutation" in the Fc portion and containing a target-specific VH domain; 2) ig chains consisting of target-specific VH and CH1 domains, followed by a CD 3-specific VH domain and CH1 domain, followed by an Fc portion containing "knob" mutations; and 3) a common light chain that anneals simultaneously to MUC 1-specific and CD 3-specific sequences.

Figures 34A-B depict purification and analytical characterization of the resulting MUC 1-specific TCB (figures 34A and 34B). The purification procedure involved an affinity step (protein a) followed by size exclusion chromatography (Superdex 200, GE Healthcare). The final product was analyzed and characterized by analytical size exclusion chromatography (Superdex 200 column) and capillary electrophoresis.

Figure 35 depicts SPR analysis of TCB format MUC1 specific binding agents. The binding of two MUC 1-specific TCBs at different concentrations (see text) to MUC1 or unrelated antigens is shown. The smooth line represents the global fit of the data to the 1:1 interaction model.

FIGS. 36A-G depict bispecific bivalent and trivalent antibodies comprising only Fab fragments (specific for CD3 and BCMA) with or without an Fc portion as described below (A) Fab BCMA-Fc-Fab CD 3; (B) fab BCMA-Fc-Fab CD3-Fab BCMA; (C) fab BCMA-Fc-Fab BCMA-Fab CD 3; (D) Fc-Fab CD3-Fab BCMA; (E) Fc-Fab BCMA-Fab CD 3; (F) fab CD3-Fab BCMA-Fab BCMA; (G) fab CD3-Fab BCMA. Preferably, the LC of Fab CD3 and Fab BCMA are identical (common LC) to avoid LC mismatches and reduce by-products. Fab CD3 and Fab BCMA were linked to each other by a flexible linker.

Figure 37 depicts the lack of BCMA IgG antibody binding to TACI receptor detected by Surface Plasmon Resonance (SPR). Curve 1 corresponds to the signal on the reference channel, curve 2 corresponds to the channel where binding occurs (binding channel), and curve 2-1 is the subtracted signal (binding channel-reference channel), meaning that this is due to the signal of the binding event. The SPR binding assay clearly demonstrated that pSCHLI372 IgG did not bind to human TACI receptors.

FIGS. 38A-C show the production and purification of BCMA-TCB CLC. CE-SDS patterns (non-reduced (top) and reduced (bottom)) of final protein preparations after Protein A (PA) affinity chromatography and Size Exclusion Chromatography (SEC) purification steps applied to (A) pSCHLI333-TCB CLC, (B) pSCHLI372-TCB CLC, (C) pSCHLI373-TCB CLC. All three molecules are in molecular form as depicted in fig. 36B.

FIGS. 39A-B show BCMA-TCB CLC antibodies in BCMA by flow cytometry^hiBinding on positive H929 cells. Plotting the median fluorescence intensity of the BCMA-TCB CLC antibodies as a function of antibody concentration (0.12 to 500 nM); (A) pSCHLI372-TCB CLC and pSCHLI373-TCB CLC on H929 cells (A) and MKN45 cells (B). DP47-TCB is a negative control TCB that does not bind BCMA below 100nM (see example 7).

FIGS. 40A-B show BCMA-TCB CLC antibody binding on CD3 positive Jurkat T cells as measured by flow cytometry. Median fluorescence intensity of BCMA-TCB CLC antibodies (pSCHLI372-TCB CLC and pSCHLI373-TCB CLC) binding to Jurkat T cells was plotted as a function of antibody concentration. No binding to BCMA negative and CD3 negative MKN45 cells at concentrations below 100 nM.

FIG. 41 shows BCMA-TCB CLC antibody-mediated T cell activation in the presence of H929 cells as detected by flow cytometry. CD4 after 48 hours of incubation ⁺And CD8⁺Expression levels of the early activation marker CD69 and the late activation marker CD25 on T cells. pSCHLI372-TCB CLC and pSCHLI373-TCB CLC antibodies induced upregulation of CD69 and CD25 activation markers in a concentration-dependent and specific manner in the presence of BCMA positive target cells. The ratio of E to T was used as 10 PBMCs to 1H 929 cell; cells were incubated for 48 hours before measuring up-regulation of CD69 and CD 25. DP47-TCB as a negative control TCB did not induce T cell activation. Representative results were from two independent experiments.

FIGS. 42A-B show that BCMA-TCB CLC antibodies induce BCMA as detected by a colorimetric LDH release assay^hi-T cell redirected killing of positive H929 myeloma cells. BCMA-TCB CLC antibodies pSCHLI372-TCB CLC (A, B) and pSCHLI373-TCB CLC (A) induced BCMA as measured by LDH release^hiConcentration-dependent killing of positive H929 myeloma cells. DP47-TCB as a negative control TCB that binds not BCMA but only CD3 did not induceLead to killing of H929 cells. The E: T ratio was used as 10 PBMCs: 1H 929 cells; cells were incubated for 24 hours before measuring LDH release. Representative results are from three independent experiments.

FIG. 43 shows BCMA-TCB CLC antibody induces BCMA as detected by a colorimetric LDH Release assay ^med/loT cell redirected killing of positive U266 myeloma cells. BCMA-TCB CLC antibodies pSCHLI372-TCB CLC and pSCHLI373-TCB CLC induce BCMA as measured by LDH release^med/loConcentration-dependent killing of positive U266 myeloma cells. DP47-TCB as a negative control TCB that did not bind BCMA but only CD3 did not induce H929 cell killing. The E: T ratio was used as 10 PBMCs to 1U 266 cell; cells were incubated for 24 hours before measuring LDH release. Representative results were from two independent experiments.

Detailed Description

Definition of

Terms are used herein as they are commonly used in the art, unless otherwise defined below.

As used herein, the term "antigen binding molecule" refers in its broadest sense to a molecule that specifically binds to an antigenic determinant. Examples of antigen binding molecules are immunoglobulins and derivatives, e.g., fragments, thereof.

The term "bispecific" refers to an antigen-binding molecule capable of specifically binding at least two different antigenic determinants. Typically, bispecific antigen binding molecules comprise two antigen binding sites, each of which is specific for a different antigenic determinant. In certain embodiments, the bispecific antigen binding molecule is capable of binding two antigenic determinants simultaneously, in particular two antigenic determinants expressed on two different cells.

The term "valency" as used herein means the presence of a specified number of antigen binding sites in an antigen binding molecule. Thus, the term "monovalent binding to an antigen" means that there is one (and no more than one) antigen-specific antigen binding site in the antigen binding molecule.

An "antigen binding site" refers to a site, i.e., one or more amino acid residues, that provides an antigen binding molecule that interacts with an antigen. For example, the antigen binding site of an antibody comprises amino acid residues from a Complementarity Determining Region (CDR). Natural immunoglobulin molecules typically have two antigen binding sites, and Fab molecules typically have a single antigen binding site.

As used herein, the term "antigen-binding portion" refers to a polypeptide molecule that specifically binds to an antigenic determinant. In one embodiment, the antigen-binding portion is capable of directing the entity to which it is attached (e.g., the second antigen-binding portion) to a target site, e.g., to a particular type of tumor cell or tumor stroma having an antigenic determinant. In another embodiment, the antigen binding portion can activate signaling through its target antigen (e.g., a T cell receptor complex antigen). Antigen binding portions include antibodies and fragments thereof as further defined herein. Specific antigen-binding portions include the antigen-binding domain of an antibody, which comprises an antibody heavy chain variable region and an antibody light chain variable region. In certain embodiments, the antigen-binding portion may comprise an antibody constant region as further defined herein and known in the art. Useful heavy chain constant regions include any of the five isoforms α, δ, ε, γ or μ. Useful light chain constant regions include either of the following two isoforms, kappa and lambda.

As used herein, the term "antigenic determinant" is synonymous with "antigen" and "epitope" and refers to a site (e.g., a contiguous stretch of amino acids or a conformational configuration composed of different regions of non-contiguous amino acids) on a polypeptide macromolecule to which an antigen-binding portion binds, forming an antigen-binding portion-antigen complex. Useful antigenic determinants can be, for example, on the surface of tumor cells, virus-infected cells, other diseased cells, immune cells, free in serum and/or in the extracellular matrix (ECM). The protein referred to herein as an antigen (e.g., MCSP, FAP, CEA, EGFR, CD33, CD3) can be any native form of protein from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise indicated. In a specific embodiment, the antigen is a human protein. Where reference is made herein to a particular protein, the term includes "full-length," unprocessed protein as well as any form of protein that is produced by processing in a cell. The term also includes naturally occurring variants of the protein, e.g., splice variants or allelic variants. Exemplary human proteins that can be used as antigens include, but are not limited to, folate receptor 1(FolR1, Folate Receptor Alpha (FRA); Folate Binding Protein (FBP); human FolR1 UniProt No.: P15328; mouse FolR1 UniProt No.: P35846; cynomolgus monkey FolR1 UniProt No.: G7PR14), mucin-1 (MUC1), and B Cell Maturation Antigen (BCMA) and CD3, particularly the epsilon subunit of CD3 (see UniProt No. P07766 (version 130) for human sequences), NCBI Refseq No. NP-000724.1; or for cynomolgus monkey [ Macaca fascicularis ] sequences see UniProt No. Q95LI5 (version 49), NCBI GenBank No. BAB71849.1).

In certain embodiments, the T cell activating bispecific antigen binding molecules of the invention bind to an epitope of an activated T cell antigen or target cell antigen that is conserved between activated T cell antigens or target antigens from different species.

By "specific binding" is meant binding that is selective for the antigen and can be distinguished from unwanted or non-specific interactions. The ability of an antigen-binding moiety to bind a specific epitope can be measured by enzyme-linked immunosorbent assays (ELISAs) or other techniques familiar to those skilled in the art, for example, Surface Plasmon Resonance (SPR) techniques (analyzed on a BIAcore instrument) (Liljeblad et al, Glyco J17, 323-. In one embodiment, the extent of binding of the antigen-binding portion to an unrelated protein is less than about 10% of the binding of the antigen-binding portion to the antigen, as measured by SPR. In certain embodiments, an antigen-binding portion that binds an antigen or an antigen-binding molecule comprising the antigen-binding portion has a dissociation constant (K) of less than or equal to 1 μ M, less than or equal to 100nM, less than or equal to 10nM, less than or equal to 1nM, less than or equal to 0.1nM, less than or equal to 0.01nM, or less than or equal to 0.001nM_D) (e.g., 10) ^-8M or less, e.g. 10^-8M to 10^-13M, e.g. 10^-9M to 10^-13M)。

"affinity" refers to a molecule(e.g., receptor) and its binding partner (e.g., ligand). As used herein, unless otherwise specified, "binding affinity" refers to an intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antigen-binding portion and an antigen, or a receptor and its ligand). The affinity of a molecule X for its partner Y can generally be determined by the dissociation constant (K)_D) To show the dissociation constant (K)_D) Is the ratio of the dissociation and association rate constants (koff and kon, respectively). Thus, equivalent affinities may include different rate constants as long as the ratio of rate constants remains the same. Affinity can be measured by established methods known in the art, including the methods described herein. A particular method for measuring affinity is Surface Plasmon Resonance (SPR).

"reduced binding", e.g., reduced binding to Fc receptors, refers to a reduction in affinity for the corresponding interaction, e.g., as measured by SPR. For the sake of clarity, the term also includes reducing the affinity to zero (or below the detection limit of the analytical method), i.e. eliminating the interaction completely. Conversely, "increased binding" refers to an increase in the binding affinity of the corresponding interaction.

As used herein, an "activated T cell antigen" refers to an antigenic determinant expressed on the surface of a T lymphocyte, particularly a cytotoxic T lymphocyte, that is capable of inducing T cell activation upon interaction with an antigen binding molecule. Specifically, the interaction of antigen binding molecules with activated T cell antigens can induce T cell activation by triggering a signaling cascade of the T cell receptor complex. In a specific embodiment, the activated T cell antigen is CD 3.

As used herein, "T cell activation" refers to one or more cellular responses of T lymphocytes, particularly cytotoxic T lymphocytes, selected from the group consisting of proliferation, differentiation, cytokine secretion, release of cytotoxic effector molecules, cytotoxic activity and expression of activation markers. The T cell activating bispecific antigen binding molecules of the invention are capable of inducing T cell activation. Suitable assays for measuring T cell activation are known in the art described herein.

As used herein, a "target cell antigen" refers to an antigenic determinant present on the surface of a target cell, for example, a cell in a tumor, such as a cancer cell, or tumor stroma.

As used herein, with respect to antigen binding moieties and the like, the terms "first" and "second" are used to facilitate distinction in the case of more than one moiety of each type. The use of these terms is not intended to confer a particular order or orientation to the T cell activating bispecific antigen binding molecules unless explicitly stated as such.

The term "BCMA" as used herein relates to the human B cell maturation target, also known as BCMA; TR17_ HUMAN, TNFRSF17(UniProt Q02223), which is a member of the tumor necrosis receptor superfamily that is preferentially expressed in differentiated plasma cells. The extracellular domain of BCMA consists of amino acids 1-54 (or 5-51) according to UniProt. The term "antibody against BCMA, anti-BCMA antibody" as used herein refers to an antibody that specifically binds to BCMA.

The term "CD 3 epsilon or CD 3" as used herein relates to HUMAN CD3 epsilon described under UniProt P07766(CD3E _ HUMAN). The term "antibody against CD3, anti-CD 3 antibody" relates to an antibody that binds to CD3 epsilon.

By "Fab molecule" is meant a protein consisting of the VH and CH1 domains of the heavy chain of an immunoglobulin ("Fab heavy chain") and the VL and CL domains of the light chain ("Fab light chain"). The term "Fab molecules with the same VLCL light chain" refers to binding agents that share a light chain but still have individual specificity. The T cell activating bispecific molecule of the invention comprises at least two Fab molecules with the same VLCL light chain. The corresponding heavy chains are remodeled and confer specific binding to T cell activating bispecific antigen and target cell antigen, respectively.

By "fused" is meant that the components (e.g., Fab molecule and Fc domain subunit) are linked by a peptide bond, either directly or through one or more peptide linkers.

The term "common light chain" as used herein refers to a light chain paired with more than one heavy chain or fragment thereof to form at least first and second antigen binding sites, e.g., fabs, each specific for a different antigen within a bispecific or multispecific molecule. For example, the common light chain pairs with a first heavy chain or fragment thereof within the antigen binding molecule to form a first binding site specific for a tumor antigen, and another copy of the common light chain pairs with a second heavy chain or fragment thereof within the antigen binding molecule to form a second binding site specific for a T cell activation antigen (e.g., CD 3).

The term "immunoglobulin molecule" refers to a protein having the structure of a naturally occurring antibody. For example, immunoglobulins of the IgG class are heterotetrameric glycoproteins of about 150,000 daltons, composed of two light chains and two heavy chains joined by disulfide bonds. From N to C-terminus, each heavy chain has a variable region (VH), also known as a variable heavy domain or heavy chain variable domain, followed by three constant domains (CH1, CH2 and CH3), also weighed as chain constant regions. Similarly, from N to C-terminus, each light chain has a variable region (VL), also known as a variable light domain or light chain variable domain, followed by a Constant Light (CL) domain, also known as a light chain constant region. The heavy chains of immunoglobulins can be assigned to one of five types, called α (IgA), δ (IgD), ε (IgE), γ (IgG), or μ (IgM), some of which can be further divided into subtypes, e.g., γ (IgM) ₁(IgG₁),γ₂(IgG₂),γ₃(IgG₃),γ₄(IgG₄),α₁(IgA₁) And alpha₂(IgA₂). Based on the amino acid sequence of its constant domain, the light chain of an immunoglobulin can be assigned to one of two types, called kappa and lambda. An immunoglobulin essentially consists of two Fab molecules and an Fc domain connected by an immunoglobulin hinge region.

The term "antibody" is used herein in the broadest sense and encompasses a variety of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies and antibody fragments, so long as they exhibit the desired antigen binding activity.

An "antibody fragment" refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds to an antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab ', Fab ' -SH, F (ab ')₂Diabodies, linear antibodies, single chain antibodiesBody molecules (e.g., scFv) and single domain antibodies. For a review of certain antibody fragments, see Hudson et al, Nat Med 9, 129-. For reviews on scFv fragments, see Pl ü ckthun, in The Pharmacology of Monoclonal Antibodies, vol.113, Rosenburg and Moore eds., Springer-Verlag, New York, pp.269-315 (1994); see also WO 93/16185; and U.S. patent nos. 5,571,894 and 5,587,458. With respect to Fab and F (ab') containing salvage receptor binding epitope residues and having increased in vivo half-life ₂See U.S. Pat. No. 5,869,046 for a discussion of fragments. Diabodies are antibody fragments with two antigen binding sites that may be bivalent or bispecific. See, e.g., EP 404,097; WO 1993/01161; hudson et al, Nat Med 9, 129-; and Hollinger et al, Proc Natl Acad Sci USA 90, 6444-. Tri-and tetraantibodies are also described in Hudson et al, Nat Med 9,129-134 (2003). A single domain antibody is an antibody fragment comprising all or part of the heavy chain variable domain of an antibody or all or part of the light chain variable domain of an antibody. In certain embodiments, the single domain antibody is a human single domain antibody (Domantis, Inc., Waltham, MA; see, e.g., U.S. Pat. No. 6,248,516B 1). Antibody fragments can be prepared by a variety of techniques, including but not limited to proteolytic digestion of intact antibodies and production by recombinant host cells (e.g., E.coli or phage), as described herein.

The term "antigen binding domain" refers to the portion of an antibody that comprises a region that specifically binds to and is complementary to part or all of an antigen. The antigen binding domain may be provided by, for example, one or more antibody variable domains (also referred to as antibody variable regions). In particular, the antigen binding domain comprises an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH).

The term "variable region" or "variable domain" refers to a domain of an antibody heavy or light chain that involves binding of the antibody to an antigen. The variable domains of the heavy and light chains of natural antibodies (VH and VL, respectively) are generally of similar structure, each domain comprising four conserved Framework Regions (FR) and three hypervariable regions (HVRs). See, e.g., Kindt et al, Kuby Immunology,6^th ed.,W.H.Freeman and Co.Page 91 (2007). A single VH or VL domain may be sufficient to confer antigen binding specificity.

As used herein, the term "hypervariable region" or "HVR" refers to each region of an antibody variable domain which is hypervariable in sequence and/or forms structurally defined loops ("hypervariable loops"). Typically, a native four-chain antibody comprises six HVRs; three in VH (H1, H2, H3), three in VL (L1, L2, L3). HVRs typically contain amino acid residues from hypervariable loops and/or Complementarity Determining Regions (CDRs) that have the highest sequence variability and/or are involved in antigen recognition. In addition to CDR1 in VH, the CDRs typically comprise amino acid residues that form hypervariable loops. Hypervariable regions (HVRs) are also referred to as "complementarity determining regions" (CDRs) and these terms are used interchangeably herein to refer to the portions of the variable regions that form the antigen-binding regions. This particular region is described in Kabat et al, U.S. Dept. of Health and Human Services, Sequences of Proteins of Immunological Interest (1983) and Chothia et al, J Mol Biol 196:901-917(1987), wherein when compared to each other an overlap or subset of amino acid residues is defined. However, any definition applied to refer to a CDR of an antibody, or variant thereof, is intended to be within the scope of the terms defined and used herein. For comparison, suitable amino acid residues comprising the CDRs defined in each of the above-cited references are listed in table a below. The exact number of residues covering a particular CDR will vary depending on the sequence and size of the CDR. Given the variable region amino acid sequence of an antibody, one skilled in the art can routinely determine which residues comprise a particular CDR.

CDR definitions¹

CDR	Kabat	Chothia	AbM²
				V_H CDR1	31-35	26-32	26-35
V_H CDR2	50-65	52-58	50-58
				V_H CDR3	95-102	95-102	95-102
V_L CDR1	24-34	26-32	24-34
				V_L CDR2	50-56	50-52	50-56
V_L CDR3	89-97	91-96	89-97

¹The numbering of all CDR definitions in table a is according to the numbering convention set forth by Kabat et al (see below).

²The "AbM" with lower case "b" used in table a refers to the CDR defined by Oxford Molecular's "AbM" antibody modeling software.

Kabat et al also define a numbering system applicable to the variable region sequences of any antibody. One of ordinary skill in the art can unambiguously assign this "Kabat numbering" system to any variable region sequence, without relying on any experimental data beyond the sequence itself. As used herein, "Kabat numbering" refers to the numbering system set forth by Kabat et al, U.S. Dept.of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983). Reference to numbering of particular amino acid residue positions in the variable region of an antibody is according to the Kabat numbering system unless otherwise indicated.

The polypeptide sequences of the sequence listing are not numbered according to the Kabat numbering system. However, one skilled in the art would be able to convert the sequence numbering of the sequence listing to the Kabat numbering.

"framework" or "FR" refers to variable domain residues other than hypervariable region (HVR) residues. The FRs of the variable domains typically consist of four FR domains, FR1, FR2, FR3 and FR 4. Thus, the HVR and FR sequences typically occur in VH (or VL) in the order FR1-H1(L1) -FR2-H2(L2) -FR3-H3(L3) -FR 4.

The "class" of antibodies or immunoglobulins refers to the type of constant domain or constant region that the heavy chain has. There are five major classes of antibodies, IgA, IgD, IgE, IgG and IgM, and several of these may be further divided into subclasses (isotypes), e.g. IgG₁,IgG₂,IgG₃,IgG₄,IgA₁And IgA₂. The heavy chain constant domains corresponding to different classes of immunoglobulins are called α, δ, ε, γ and μ, respectively.

The term "Fc domain" or "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain that contains at least a portion of a constant region. The term includes native sequence Fc regions and variant Fc regions. Although the boundaries of the Fc region of IgG heavy chains may vary slightly, the human IgG heavy chain Fc region is generally defined as extending from Cys226 or from Pro230 to the carboxy terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. Unless otherwise indicated, the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also known as EU index, as described in Kabat et al, Sequences of Proteins of Immunological Interest,5th ed. As used herein, a "subunit" of an Fc domain refers to one of two polypeptides that form a dimeric Fc domain, i.e., a polypeptide comprising the C-terminal constant region of an immunoglobulin heavy chain, which is capable of stable self-association. For example, subunits of the IgG Fc domain comprise IgG CH2 and IgG CH3 constant domains.

A "modification that facilitates binding of the first and second subunits of an Fc domain" is manipulation of the peptide backbone or post-translational modification of the Fc domain subunits that reduces or prevents binding of a polypeptide comprising an Fc domain subunit to the same polypeptide to form a homodimer. As used herein, a modification that facilitates binding specifically includes making a separate modification to each of the two Fc domain subunits (i.e., the first and second subunits of the Fc domain) for which binding is desired, wherein the modifications are complementary to each other to facilitate binding of the two Fc domain subunits. For example, modifications that promote binding may alter the structure or charge of one or both Fc domain subunits such that binding thereof is sterically or electrostatically favorable, respectively. Thus, (hetero) dimerization occurs between a polypeptide comprising a first Fc domain subunit and a polypeptide comprising a second Fc domain subunit, which may not be identical in the sense that the other components fused to each subunit (e.g., antigen binding portion) are not identical. In some embodiments, the modification that facilitates binding comprises an amino acid mutation, particularly an amino acid substitution, in the Fc domain. In a specific embodiment, the modification that facilitates binding comprises a separate amino acid mutation, in particular an amino acid substitution, in each of the two subunits of the Fc domain.

The term "effector functions" refers to those biological activities attributed to the Fc region of an antibody, which vary with antibody isotype. Examples of antibody effector functions include C1q binding and Complement Dependent Cytotoxicity (CDC), Fc receptor binding, antibody dependent cell mediated cytotoxicity (ADCC), Antibody Dependent Cellular Phagocytosis (ADCP), cytokine secretion, immune complex mediated antigen uptake by antigen presenting cells, down-regulation of cell surface receptors (e.g., B cell receptors), and B cell activation.

As used herein, the term "engineered" is considered to include any manipulation of the peptide backbone or post-translational modification of naturally occurring or recombinant polypeptides or fragments thereof. Engineering includes modification of the amino acid sequence, glycosylation pattern or side chain groups of individual amino acids, as well as combinations of these methods.

The term "amino acid mutation" as used herein is intended to include amino acid substitutions, deletions, insertions and modifications. Any combination of substitutions, deletions, insertions and modifications can be made to arrive at the final construct, so long as the final construct possesses the desired characteristics, e.g., reduced binding to an Fc receptor, or increased binding to another peptide. Amino acid sequence deletions and insertions include amino and/or carboxy terminal deletions and amino acid insertions. Particular amino acid mutations are amino acid substitutions. In order to alter the binding characteristics of, for example, the Fc region, non-conservative amino acid substitutions, i.e., substitution of one amino acid with another having different structural and/or chemical properties, are particularly preferred. Amino acid substitutions include substitutions by non-naturally occurring amino acids or naturally occurring amino acid derivatives of twenty standard amino acids (e.g., 4-hydroxyproline, 3-methylhistidine, ornithine, homoserine, 5-hydroxylysine). Amino acid mutations can be generated using genetic or chemical methods well known in the art. Genetic methods may include site-directed mutagenesis, PCR, gene synthesis, and the like. It is expected that a method of changing the side chain group of an amino acid by a method other than genetic engineering, such as chemical modification, is also useful. Various designs may be used herein to represent the same amino acid mutation. For example, substitution of proline to glycine at position 329 of the Fc domain can be represented as 329G, G329, G ₃₂₉P329G, or Pro329 Gly.

As used herein, the term "polypeptide" refers to a molecule composed of monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds). The term "polypeptide" refers to any chain of two or more amino acids and does not refer to a product of a particular length. Thus, included within the definition of "polypeptide" are peptides, dipeptides, tripeptides, oligopeptides, "proteins," "amino acid chains," or any other term used to refer to a chain of two or more amino acids, and the term "polypeptide" may be used in place of, or interchangeably with, any of these terms. The term "polypeptide" is also intended to mean the product of post-expression modification of the polypeptide, including but not limited to glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, or modification by non-naturally occurring amino acids. Polypeptides may be derived from natural biological sources or produced by recombinant techniques, but are not necessarily translated from a specified nucleic acid sequence. It may be produced in any manner, including by chemical synthesis. The polypeptides of the invention can be about 3 or more, 5 or more, 10 or more, 20 or more, 25 or more, 50 or more, 75 or more, 100 or more, 200 or more, 500 or more, 1,000 or more, or 2,000 or more amino acids in size. Polypeptides may have a defined three-dimensional structure, although they do not necessarily have such a structure. Polypeptides having a defined three-dimensional structure are referred to as folded, and polypeptides that do not have a defined three-dimensional structure but can adopt a large number of different conformations are referred to as unfolded.

An "isolated" polypeptide or variant or derivative thereof refers to a polypeptide that is not in its natural environment. No specific level of purification is required. For example, an isolated polypeptide may be removed from its natural or native environment. For the purposes of the present invention, recombinantly produced polypeptides and proteins expressed in host cells are considered isolated, have been isolated by any suitable technique, and naturally occurring or recombinant polypeptides, fractionated or partially or substantially purified, are also considered isolated.

"percent (%) amino acid sequence identity" with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and without considering any conservative substitutions as part of the sequence identity. Alignment for the purpose of determining percent amino acid sequence identity can be accomplished in a variety of ways within the skill in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or megalign (dnastar) software. One skilled in the art can determine appropriate parameters for aligning the sequences, including any algorithms necessary to achieve maximum alignment over the full length of the sequences being compared. However, for purposes herein,% amino acid sequence identity values are generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program is written by Genentech, Inc. and the source code has submitted a user document, registered with U.S. copyright registration number TXU510087, at the U.S. copyright office, Washington, D.C. 20559. The ALIGN-2 program is publicly available from Genentech, Inc. of south san Francisco, Calif., or may be compiled from source code. The ALIGN-2 program should be compiled for use on a UNIX operating system, including the digital UNIX V4.0D. All sequence comparison parameters were set by the ALIGN-2 program and were not changed. In the case of using ALIGN-2 for amino acid sequence comparisons, the% amino acid sequence identity of a given amino acid sequence a with or against a given amino acid sequence B (or alternatively expressed as a given amino acid sequence a with or comprising a certain% amino acid sequence identity with or against a given amino acid sequence B) is calculated as follows:

100 times a fraction X/Y

Wherein X is the number of amino acid residues scored as identical matches in the alignment of A and B by the sequence alignment program ALIGN-2, and wherein Y is the total number of amino acid residues in B. It will be understood that where the length of amino acid sequence A is not equal to the length of amino acid sequence B, the% amino acid sequence identity of A to B will not be equal to the% amino acid sequence identity of B to A. Unless otherwise indicated, all% amino acid sequence identity values used herein were obtained as described in the preceding paragraph using the ALIGN-2 computer program.

The term "polynucleotide" refers to an isolated nucleic acid molecule or construct, e.g., messenger RNA (mrna), virus-derived RNA or plasmid dna (pdna). Polynucleotides may comprise conventional phosphodiester bonds or unconventional bonds (e.g., amide bonds, such as found in Peptide Nucleic Acids (PNAs)). The term "nucleic acid molecule" refers to any one or more nucleic acid fragments, such as DNA or RNA fragments, present in a polynucleotide.

An "isolated" nucleic acid molecule or polynucleotide means a nucleic acid molecule, DNA or RNA, that is removed from its natural environment. For example, for the purposes of the present invention, a recombinant polynucleotide encoding a polypeptide contained in a vector is considered isolated. Other examples of isolated polynucleotides include recombinant polynucleotides maintained in a heterologous host cell or purified (partially or substantially) polynucleotides in solution. An isolated polynucleotide includes a polynucleotide molecule contained in a cell that normally contains the polynucleotide molecule, but the polynucleotide molecule exists extrachromosomally or at a chromosomal location different from its native chromosomal location. Isolated RNA molecules include RNA transcripts of the invention, either in vivo or in vitro, as well as both positive and negative stranded forms, and double stranded forms. Isolated polynucleotides or nucleic acids according to the invention also include synthetically produced such molecules. In addition, the polynucleotide or nucleic acid may be or may include regulatory elements such as a promoter, ribosome binding site or transcription terminator.

By a nucleic acid or polynucleotide having a nucleotide sequence that is at least 95% "identical" to a reference nucleotide sequence of the present invention, the nucleotide sequence of the polynucleotide is intended to be identical to the reference sequence, except that the polynucleotide sequence may include up to 5 point mutations per 100 nucleotides of the reference nucleotide sequence. In other words, in order to obtain a polynucleotide having a nucleotide sequence at least 95% identical to a reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be deleted or substituted with additional nucleotides or up to 5% of the total nucleotides in the reference sequence over the number of nucleotides may be inserted into the reference sequence. These changes to the reference sequence may occur at the 5 'or 3' terminal positions of the reference nucleotide sequence or anywhere between these terminal positions interspersed among residues of the reference sequence or one or more contiguous groups in the reference sequence, respectively. Indeed, whether any particular polynucleotide sequence is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to a nucleotide sequence of the present invention can be determined routinely using known computer programs, such as those discussed above for polypeptides (e.g., ALIGN-2).

The term "expression cassette" refers to a recombinantly or synthetically produced polynucleotide having a series of specialized nucleic acid elements that permit transcription of a specialized nucleic acid in a target cell. The recombinant expression cassette may be incorporated into a plasmid, chromosome, mitochondrial DNA, plastid DNA, virus, or nucleic acid fragment. Typically, the recombinant expression cassette portion of the expression vector includes the nucleic acid sequence to be transcribed, a promoter, and the like. In certain embodiments, the expression cassette of the invention comprises a polynucleotide sequence encoding a bispecific antigen binding molecule of the invention or a fragment thereof.

The term "vector" or "expression vector" is synonymous with "expression construct" and refers to a DNA molecule for introducing and directing the expression of a particular gene to which it is operatively associated in a target cell. The term includes vectors which are self-replicating nucleic acid structures as well as vectors which are incorporated into the genome of a host cell into which they have been introduced. The expression vector of the present invention comprises an expression cassette. Expression vectors allow transcription of large amounts of stable mRNA. Once the expression vector is in the target cell, the ribonucleic acid molecule or protein encoded by the gene is produced by the cellular transcription and/or translation machinery. In one embodiment, the expression vector of the invention comprises an expression cassette comprising a polynucleotide sequence encoding the bispecific antigen binding molecule of the invention or a fragment thereof.

The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to a cell into which an exogenous nucleic acid is introduced, including the progeny of such a cell. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and progeny derived therefrom, regardless of the number of passages. The nucleic acid content of the progeny may not be identical to that of the parent cell, but may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein. The host cell is any type of cellular system that can be used to produce the bispecific antigen binding molecules of the present invention. Host cells include cultured cells, such as mammalian cultured cells, e.g., CHO cells, BHK cells, NS0 cells, SP2/0 cells, YO myeloma cells, P3X63 mouse myeloma cells, PER cells, per.c6 cells or hybridoma cells, yeast cells, insect cells and plant cells, to name a few, also including cells within transgenic animals, transgenic plants or cultured plant or animal tissues.

An "activating Fc receptor" is an Fc receptor that, when engaged by the Fc domain of an antibody, initiates signaling events that stimulate receptor-bearing cells to perform effector functions. Human activating Fc receptors include Fc γ RIIIa (CD16a), Fc γ RI (CD64), Fc γ RIIa (CD32) and Fc α RI (CD 89).

Antibody-dependent cell-mediated cytotoxicity (ADCC) is an immune mechanism that results in the lysis of immune effector cells from antibody-coated target cells. The target cell is a cell to which an antibody or derivative thereof comprising an Fc region specifically binds, typically via a protein moiety at the N-terminus of the Fc region. As used herein, the term "reduced ADCC" is defined as a reduction in the number of target cells lysed by the mechanism of ADCC as defined above at a given antibody concentration in the medium surrounding the target cells in a given time and/or an increase in antibody concentration in the medium surrounding the target cells required to achieve lysis of a given number of target cells in a given time by the ADCC mechanism. The reduction in ADCC is relative to the ADCC mediated by the same but not engineered antibody produced by the same type of host cell using the same standard production, purification, formulation and storage methods (known to those skilled in the art). For example, the reduction in ADCC mediated by an antibody comprising an amino acid substitution in the Fc domain that reduces ADCC is relative to the ADCC mediated by the same antibody without such amino acid substitution in the Fc domain. Suitable assays for measuring ADCC are well known in the art (see, e.g., PCT publication No. WO 2006/082515 or PCT patent application No. PCT/EP 2012/055393).

An "effective amount" of an active agent is that amount necessary to cause a physiological change in the cell or tissue to which it is administered.

A "therapeutically effective amount" of an active agent, e.g., a pharmaceutical composition, refers to an amount that is effective, at dosages and for periods of time necessary to achieve the desired therapeutic or prophylactic result. A therapeutically effective amount of an active agent, for example, eliminates, reduces, delays, minimizes or prevents the adverse effects of the disease.

An "individual" or "subject" is a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In particular, the individual or subject is a human.

The term "pharmaceutical composition" refers to a formulation in a form that is effective in allowing the biological activity of the active ingredient contained therein, and that does not contain additional components with unacceptable toxicity to the subject to whom the formulation is to be administered.

By "pharmaceutically acceptable carrier" is meant an ingredient of the pharmaceutical composition other than the active ingredient that is not toxic to the subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers or preservatives.

As used herein, "treatment" (and grammatical variations thereof such as "treating" or "treatment") refers to clinical intervention in an attempt to alter the natural course of a disease in the individual being treated, and may be performed prophylactically or during clinical pathology. Desirable effects of treatment include, but are not limited to, preventing occurrence or recurrence of disease, alleviation of symptoms, alleviation of any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, amelioration or palliation of the disease state, remission or improved prognosis. In some embodiments, the T cell activating bispecific antigen binding molecules of the invention are used to delay the progression of a disease or slow the progression of a disease.

The term "package insert" is used to refer to instructions typically included in commercial packages of therapeutic products that contain information regarding the indications, usage, dosage, administration, combination therapy, contraindications and/or warnings for use of such therapeutic products.

Detailed Description

The present invention provides bispecific antigen binding molecules designed for T cell activation and redirection that combine good efficacy and productivity as well as methods of making and using them. In particular, the present invention relates to bispecific molecules wherein at least two binding moieties have the same light chain, and in some embodiments, corresponding reconstituted heavy chains, which confer specific binding to a T cell activating bispecific antigen and a target cell antigen, respectively. Light chain mismatches are prevented using the so-called "common light chain" principle, i.e. combining two binders that share a light chain but still have individual specificities. Thus, fewer by-products are produced during the manufacturing process, which facilitates the uniform preparation of the T cell activating bispecific antigen binding molecule.

The invention particularly relates to predefined rare light chains that significantly contribute to antigen binding and heteromultimeric pairing with different binding partners (e.g., heavy chains and fragments thereof). Thus, this common light chain is suitable for use in libraries where novel bispecific or multispecific antigen-binding molecules may be prepared. Advantageously, the common light chain associated with the antigen binding molecules and methods disclosed herein can be used to form antigen binding molecules useful for T cell activation. One skilled in the art will recognize the advantageous efficiency of having a light chain that can function in both T cell activating antigen binding moieties and target antigen binding moieties. This allows for efficient production of T cell activating bispecific antigen binding molecules comprising a T cell activating component and a target antigen binding component. In a specific embodiment, the common light chain is a lambda constant light chain domain. In a specific embodiment, the common light chain is a human or humanized lambda light chain. In a specific embodiment, the common light chain is a rare human λ 7 family light chain. The use of lambda, and particularly the rare lambda 7 family of light chains, is a rare approach that is expected to reduce the likelihood of identifying suitable heavy chain binding partners to generate antigen binding molecules specific for various targets. Thus, prior to the work of the inventors disclosed herein, it was not known that λ 7 light chains with suitable properties could be developed against a variety of different unrelated target antigens (e.g., FolR1, MUC1, and BCMA). It is not known that lambda light chains can be developed to generate stable, functional high affinity binders to improve the production of T cell activating bispecific antigen binding molecules with CD3 specificity and target antigen specificity, where the target antigens are unrelated, e.g., FolR1, MUC1 and BCMA. In one embodiment, as described below, this common light chain can be used to construct a Common Light Chain (CLC) library based on a specific CD3 binding agent rather than a germline antibody to generate a specific CD3 binding agent that contributes to the binding agent. The advantage of this approach is that it allows the maintenance of previously identified and validated CD3 binding agents such that only new target antigen binding agents that activate the target antigen binding portion of the bispecific antigen binding molecule based on the heavy chain have to be identified. This allows a modular approach to generate different T cell activating bispecific antigen binding molecules with identical or highly homologous chains. Although the light chains are identical within a given T cell activating antigen binding molecule, the light chains of different T cell activating bispecific antigen binding molecules may be identical or highly homologous. By "highly homologous" is meant that the light chain of the different T cell activating bispecific antigen binding molecules produced by this modular approach comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98% or 99% identical. Preferably, the highly homologous light chains of the present invention have identical variable light chain regions and differ only in their constant regions. For example, in some embodiments, the amino acid variation is limited to the linker region. In some embodiments, the common light chain comprises a kappa constant light chain domain.

In addition to the advantages described above, as described above, since the light chains used within the antigen-binding portion of the T cell activating bispecific antigen binding molecule are identical, the yield of correctly paired heteromultimeric molecules using this method is enhanced. In other words, the use of a common light chain facilitates the production of these molecules, as any mismatch of the light chain to the incorrect heavy chain is eliminated. Thus, the isolation of high purity T cell activating bispecific antigen binding molecule species is facilitated. In a specific embodiment, the T cell activating bispecific antigen binding molecule uses Fab as a building block. Compared to other formats, the use of Fab fragments as building blocks rather than, for example, scFv fragments, leads to higher thermostability and lack of scFv aggregation and intermolecular scFv formation.

Prior to the work of the inventors described herein, it was not known that a common light chain could be generated which could not only be used as a common light chain in bispecific or multispecific molecules, but which also supported the functional properties of T cell activation of the T cell activating bispecific antigen binding molecules. Furthermore, prior to the work of the present inventors described herein, it was not known that a common light chain could be generated that significantly promoted the antigen binding properties of the antigen binding portion within the T cell activating bispecific antigen binding molecule. The strategy identified was to identify heavy chains or fragments thereof that contributed to most of the binding properties (e.g., strength and specificity). According to the present invention, the common light chain contributes significantly to the binding properties.

Accordingly, in a first aspect, the present invention provides a T cell activating bispecific antigen binding molecule comprising a first and a second antigen binding moiety, wherein the first antigen binding moiety comprises a first light chain, and wherein the first antigen binding moiety is capable of specifically binding to an activated T cell antigen, wherein the second antigen binding moiety comprises a second light chain, and wherein the second antigen binding moiety is capable of specifically binding to a target cell antigen, wherein the amino acid sequences of the first and second light chains are identical. In one embodiment, the first antigen binding portion is a Fab. In one embodiment, the second antigen-binding moiety is a Fab. In one aspect, the invention provides a T cell activating bispecific antigen binding molecule comprising a first and a second antigen binding moiety, one of which is a Fab molecule capable of specifically binding to an activating T cell antigen and the other of which is a Fab molecule capable of specifically binding to a target cell antigen, wherein the first and the second Fab molecule have the same light chain (variable light chain and constant light chain region, VLCL). In one embodiment, the light chain (VLCL) comprises the light chain CDRs of SEQ ID NO:32, SEQ ID NO:33, and SEQ ID NO: 34. In one embodiment, the same light chain (VLCL) comprises SEQ ID NO 35.

T cell activating bispecific antigen binding molecule forms

The components of the T cell activating bispecific antigen binding molecule can be fused to each other in various configurations. Exemplary configurations include, but are not limited to, the configurations shown in fig. 1A-D.

In some embodiments, the T cell activating bispecific antigen binding molecule further comprises an Fc domain consisting of a first and a second subunit capable of stable association. Exemplary embodiments of T cell activating bispecific antigen binding molecules comprising an Fc domain are described. All these T cell activating bispecific antigen binding molecules comprise at least two Fab fragments having the same light chain (VLCL) and having different heavy chains (VHCL) conferring two different antigen specificities, i.e. one Fab fragment is capable of specifically binding to a T cell activating antigen and the other Fab fragment is capable of specifically binding to a target cell antigen.

In some embodiments, the first and second antigen-binding portions of the T cell activating bispecific antigen binding molecule are fused to each other, optionally via a peptide linker. In one such embodiment, the second antigen binding portion is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen binding portion. In another such embodiment, the first antigen-binding portion is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen-binding portion. In another such embodiment, the second antigen binding portion is fused at the C-terminus of the Fab light chain to the N-terminus of the Fab light chain of the first antigen binding portion. In yet another such embodiment, the first antigen binding portion is fused at the C-terminus of the Fab light chain to the N-terminus of the Fab light chain of the second antigen binding portion.

In one embodiment, the second antigen-binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first or second subunit of the Fc domain.

In certain such embodiments, the first antigen-binding portion is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen-binding portion. In a particular such embodiment, the T cell activating bispecific antigen binding molecule consists essentially of a first and a second antigen binding portion comprising the same (VLCL) light chain, an Fc domain consisting of a first and a second subunit, and optionally one or more peptide linkers, wherein the first antigen binding portion is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen binding portion, and the second antigen binding portion is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first or second subunit of the Fc domain.

In another such embodiment, the first antigen binding portion is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first or second subunit of the Fc domain. In particular such embodiments, the T cell activating bispecific antigen binding molecule consists essentially of a first and a second antigen binding portion comprising the same (VLCL) light chain, an Fc domain consisting of a first and a second subunit, and optionally one or more peptide linkers, wherein each of the first and second antigen binding portions is fused at the C-terminus of the Fab heavy chain to the N-terminus of one subunit of the Fc domain.

In another such embodiment, the second antigen binding portion is fused at the C-terminus of the Fab light chain to the N-terminus of the Fab light chain of the first antigen binding portion. In particular such embodiments, the T cell activating bispecific antigen binding molecule consists essentially of a first and a second antigen binding portion comprising the same (VLCL) light chain, an Fc domain consisting of a first and a second subunit, and optionally one or more peptide linkers, wherein the first antigen binding portion is fused at the N-terminus of the Fab light chain to the C-terminus of the Fab light chain of the second antigen binding portion, and the second antigen binding portion is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first or second subunit of the Fc domain.

In other embodiments, the first antigen binding portion is fused to the N-terminus of the first or second subunit of the Fc domain at the C-terminus of the Fab heavy chain.

In certain such embodiments, the second antigen-binding portion is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen-binding portion. In particular such embodiments, the T cell activating bispecific antigen binding molecule consists essentially of a first and a second antigen binding portion comprising the same (VLCL) light chain, an Fc domain consisting of a first and a second subunit, and optionally one or more peptide linkers, wherein the second antigen binding portion is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen binding portion, and the first antigen binding portion is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first or second subunit of the Fc domain.

In particular, in these embodiments, the first antigen binding moiety is capable of specifically binding to an activated T cell antigen. In other embodiments, the first antigen binding moiety is capable of specifically binding to a target cell antigen.

The antigen binding portions may be fused to the Fc domain or to each other, either directly or through a peptide linker comprising one or more amino acids, typically about 2-20 amino acids. Peptide linkers are known in the art and described herein. Suitable non-immunogenic peptide linkers include, for example, (G)₄S)_n(SEQ ID NO:41),(SG₄)_n(SEQ ID NO:42),(G₄S)_n(SEQ ID NO:41)or G₄(SG₄)_n(SEQ ID NO:43) peptide linker. "n" is typically between 1 and 10, typically between 2 and 4. A particularly suitable peptide linker for fusing the Fab light chains of the first and second antigen-binding portions to each other is (G)₄S)₂(SEQ ID NO: 44). Additionally, the linker may comprise (a part of) an immunoglobulin hinge region. Particularly when the antigen binding portion is fused to the N-terminus of an Fc domain subunit, it may be fused via an immunoglobulin hinge region or portion thereof, with or without additional peptide linkers.

T cell activating bispecific antigen binding molecules having a single antigen binding moiety capable of specific binding to a target cell antigen are useful, particularly where target cell antigen internalization is desired following binding of a high affinity antigen binding moiety. In this case, the presence of more than one antigen binding moiety specific for a target cell antigen may enhance internalization of the target cell antigen, thereby reducing its availability.

However, in many other cases, it is advantageous to have a T cell activating bispecific antigen binding molecule comprising two or more antigen binding moieties specific for a target cell antigen, e.g. to optimize targeting of the target or to allow cross-linking of the target cell antigen.

Thus, in certain embodiments, the T cell activating bispecific antigen binding molecules of the invention further comprise a third antigen binding moiety which is a Fab molecule capable of specifically binding to a target cell antigen. In one embodiment, the third antigen-binding portion is capable of specifically binding to the same target cell antigen as the first or second antigen-binding portion. In a specific embodiment, the first antigen binding moiety is capable of specifically binding to an activated T cell antigen, and the second and third antigen binding moieties are capable of specifically binding to a target cell antigen. In a preferred embodiment, the first, second and third antigen binding portions comprise the same (VLCL) light chain.

In one embodiment, the third antigen binding portion is fused to the N-terminus of the first or second subunit of the Fc domain at the C-terminus of the Fab heavy chain.

In one embodiment, the first and third antigen binding portions are each fused at the C-terminus of the Fab heavy chain to the N-terminus of one subunit of the Fc domain, and the second antigen binding portion is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen binding portion. In a particular such embodiment, the T cell activating bispecific antigen binding molecule consists essentially of a first, a second and a third antigen binding portion (Fab fragment), an Fc domain consisting of a first and a second subunit, and optionally one or more peptide linkers, wherein the second antigen binding portion is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen binding portion, and the first antigen binding portion is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first subunit of the Fc domain, and wherein the third antigen binding portion is fused at the C-terminus of the Fab heavy chain to the N-terminus of the second subunit of the Fc domain. Preferably, in such embodiments, the first antigen binding moiety is capable of specifically binding an activated T cell antigen and the second and third antigen binding moieties are capable of specifically binding a target cell antigen, wherein the first, second and third antigen binding moieties are Fab fragments comprising the same (VLCL) light chain.

In one embodiment, the second and third antigen binding portions are each fused at the C-terminus of the Fab heavy chain to the N-terminus of one subunit of the Fc domain, and the first antigen binding portion is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen binding portion.

In one embodiment, the T cell activating bispecific antigen binding molecule consists essentially of an immunoglobulin molecule capable of specific binding to a target cell antigen and a Fab molecule capable of specific binding to an activating T cell antigen fused to the N-terminus of one of the immunoglobulin heavy chains, optionally via a peptide linker. Preferably, in such embodiments, the immunoglobulin molecule capable of specifically binding to the target cell antigen and the Fab molecule capable of specifically binding to the activating T cell antigen comprise the same (VLCL) light chain.

The first and third antigen binding portions (or second and third antigen binding portions, respectively) may be fused to the Fc domain directly or through a peptide linker. In a specific embodiment, the first and third antigen-binding portions (or second and third antigen-binding portions, respectively) are fused to the Fc domain by an immunoglobulin hinge region, respectively.

In a specific embodiment, the immunoglobulin hinge region is a human IgG₁A hinge region. In one embodiment, the first and third antigen binding portions (or the second and third antigen binding portions, respectively) and the Fc domain are part of an immunoglobulin molecule. In a specific embodiment, the immunoglobulin molecule is an immunoglobulin of the IgG class. In even more particular embodiments, the immunoglobulin is an IgG₁Subclass immunoglobulin. In another embodiment, the immunoglobulin is an IgG₄Subclass immunoglobulin. In another specific embodiment, the immunoglobulin is a human immunoglobulin. In other embodiments, the immunoglobulin is a chimeric immunoglobulin or a humanized immunoglobulin.

Fc domains

The Fc domain of the T cell activating bispecific antigen binding molecule consists of a pair of polypeptide chains comprising the heavy chain domain of an immunoglobulin molecule. For example, the Fc domain of an immunoglobulin g (IgG) molecule is a dimer, each subunit of which comprises CH2 and a CH3IgG heavy chain constant domain. The two subunits of the Fc domain are capable of stably binding to each other. In one embodiment, the T cell activating bispecific antigen binding molecule of the invention comprises no more than one Fc domain.

In one embodiment of the invention, the Fc domain of the T cell activating bispecific antigen binding molecule is an IgG Fc domain. In particular embodiments, the Fc domain is an IgG₁An Fc domain. In another embodiment, the Fc domain is an IgG₄An Fc domain. In a more specific embodiment, the Fc domain is an IgG comprising an amino acid substitution at position S228 (Kabat numbering), in particular the amino acid substitution S228P₄An Fc domain. The amino acid substitution reduces IgG₄In vivo Fab arm exchange of antibodies (see Stubenrauch et al, Drug Metabolism and Disposition 38,84-91 (2010)). In another specific embodiment, the Fc domain is human.

Fc domain modification to promote heterodimerization

The T cell activating bispecific antigen binding molecules according to the invention comprise different antigen binding portions fused to one or the other of the two subunits of the Fc domain, thus the two subunits of the Fc domain are typically comprised in two non-identical polypeptide chains. Recombinant co-expression and subsequent dimerization of these polypeptides results in several possible combinations of the two polypeptides. In order to increase the yield and purity of the T cell activating bispecific antigen binding molecule in recombinant production, it would therefore be advantageous to introduce modifications in the Fc domain of the T cell activating bispecific antigen binding molecule that facilitate the association of the desired polypeptide.

Thus, in a specific embodiment, the Fc domain of the T cell activating bispecific antigen binding molecule according to the invention comprises a modification that facilitates association of the first and second subunits of the Fc domain. The site of the most extensive protein-protein interaction between the two subunits of the human IgG Fc domain is in the CH3 domain of the Fc domain. Thus, in one embodiment, the modification is in the CH3 domain of the Fc domain.

In a specific embodiment, the modification is a so-called "knob-into-hole" modification, which includes a "knob" modification in one of the two subunits of the Fc domain and a "hole" modification in the other of the two subunits of the Fc domain.

Knob access hole techniques are described, for example, in US 5,731,168; US 7,695,936; ridgway et al, Prot Eng 9, 617. sup. 621(1996) and Carter, J Immunol Meth 248,7-15 (2001). In general, the method involves introducing a protrusion ("knob") in the interface of the first polypeptide and a corresponding cavity ("hole") in the interface of the second polypeptide such that the protrusion can be positioned in the cavity so as to promote heterodimer formation and hinder homodimer formation. The protuberance is constructed by replacing a small amino acid side chain from the interface of the first polypeptide with a larger side chain (e.g., tyrosine or tryptophan). By replacing large amino acid side chains with smaller amino acids (e.g., alanine or threonine), complementary cavities of the same or similar size as the protrusions are created in the interface of the second polypeptide.

Thus, in a specific embodiment, in the CH3 domain of the first subunit of the Fc domain of the T cell activating bispecific antigen binding molecule, the amino acid residue is replaced with an amino acid residue having a larger side chain volume, thereby generating a protuberance within the CH3 domain of the first subunit that can be located in a cavity within the CH3 domain of the second subunit, in the CH3 domain of the second subunit of the Fc domain, the amino acid residue is replaced with an amino acid residue having a smaller side chain volume, thereby generating a cavity within the CH3 domain of the second subunit in which the protuberance within the CH3 domain of the first subunit can be located.

The protuberances and cavities can be made by altering the nucleic acid encoding the polypeptide, for example by site-specific mutagenesis, or by peptide synthesis.

In a specific embodiment, the threonine residue at position 366 is replaced by a tryptophan residue (T366W) in the CH3 domain of the first subunit of the Fc domain and the tyrosine residue at position 407 is replaced by a valine residue (Y407V) in the CH3 domain of the second subunit of the Fc domain. In one embodiment, additionally in the second subunit of the Fc domain, the threonine residue at position 366 is replaced with a serine residue (T366S) and the leucine residue at position 368 is replaced with an alanine residue (L368A).

In another embodiment, in the first subunit of the Fc domain, the serine residue at position 354 is additionally replaced with a cysteine residue (S354C), and in addition in the second subunit of the Fc domain, the tyrosine residue at position 349 is replaced with a cysteine residue (Y349C). The introduction of these two cysteine residues results in the formation of disulfide bonds between the two subunits of the Fc domain, further stabilizing the dimer (Carter, J immunological Methods 248,7-15 (2001)).

In a specific embodiment, an antigen binding moiety capable of binding an activated T cell antigen is fused (optionally via an antigen binding moiety capable of binding a target cell antigen) to the first subunit of the Fc domain (including "knob" modifications). Without wishing to be bound by theory, fusion of an antigen binding moiety capable of binding an activated T cell antigen with a knob-containing subunit of an Fc domain will (further) minimize the production of an antigen binding molecule comprising two antigen binding moieties capable of binding an activated T cell antigen (spatial collision of two knob-containing polypeptides).

In alternative embodiments, the modifications that facilitate binding of the first and second subunits of the Fc domain comprise modifications that mediate electrostatic steering effects, for example, as described in PCT publication WO 2009/089004. Typically, the method comprises replacing one or more amino acid residues at the interface of two Fc domain subunits by charged amino acid residues such that homodimer formation becomes electrostatically unfavorable, but heterodimerization is electrostatically favorable.

Fc domain modifications that reduce Fc receptor binding and/or effector function

The Fc domain confers advantageous pharmacokinetic properties to the T cell activating bispecific antigen binding molecule, including long serum half-life, which contributes to good accumulation in the target tissue and favorable tissue-to-blood distribution ratio. However, it is also possible that the T cell activating bispecific antigen binding molecule does not favour targeting Fc receptor expressing cells rather than the preferred antigen carrying cells. Furthermore, co-activation of the Fc receptor signaling pathway may lead to cytokine release, which in combination with the T cell activating properties and long half-life of the antigen binding molecule, leads to over-activation of cytokine receptors and serious side effects upon systemic administration. Activation of immune cells other than T cells (carrying Fc receptors) may even reduce the efficacy of the T cells to activate the bispecific antigen binding molecule due to potential destruction of the T cells (e.g., by NK cells).

Thus, in particular embodiments, the IgG is naturally associated with₁The Fc domain of the T cell activating bispecific antigen binding molecule according to the invention exhibits reduced binding affinity to Fc receptors and/or reduced effector function compared to the Fc domain. In one such embodiment, the IgG is naturally associated with ₁Fc domain (or comprises native IgG)₁Fc domain of a T cell activating bispecific antigen binding molecule) exhibits a binding affinity to an Fc receptor of less than 50%, preferably less than 20%, more preferably less than 10%, most preferably less than 5%, and/or to a native IgG₁Fc domain (or comprising native IgG)₁Fc domain T cell activating bispecific antigen binding molecules) less than 50%, preferably less than 20%, more preferably 10%, most preferably less than 5% of the effector function. In one embodiment, the Fc domain (or the T cell activating bispecific antigen binding molecule comprising said Fc domain) does not substantially bind Fc receptors and/or induce effector functions. In a specific embodiment, the Fc receptor is an fey receptor. In one embodiment, the Fc receptor is a human Fc receptor. In one embodiment, the Fc receptor is an activating Fc receptor. In a specific embodiment, the Fc receptor is an activating human Fc γ receptor, more specifically human Fc γ RIIIa, Fc γ RI or Fc γ RIIa, most specifically human Fc γ RIIIa. In one embodiment, the effector function is one or more selected from the group consisting of CDC, ADCC, ADCP and cytokine secretion. In a particular embodiment, the effector function is ADCC. In one embodiment, the IgG is naturally associated with ₁Fc Domain in contrast, Fc domain showed with neonatal Fc receptor (FcRn)Substantially similar binding affinities. When the Fc domain (or a T cell activating bispecific antigen binding molecule comprising said Fc domain) exhibits more than about 70%, particularly more than about 80%, more particularly more than about 90% of native IgG₁Fc domain (or comprising native IgG)₁A T cell activating bispecific antigen binding molecule for an Fc domain) to FcRn, substantially similar binding to FcRn is achieved.

In certain embodiments, the Fc domain is engineered to have reduced binding affinity to an Fc receptor and/or reduced effector function as compared to an unengineered Fc domain. In particular embodiments, the Fc domain of the T cell activating bispecific antigen binding molecule comprises one or more amino acid mutations that reduce the binding affinity of the Fc domain to Fc receptors and/or effector functions. Typically, the same one or more amino acid mutations are present in each of the two subunits of the Fc domain. In one embodiment, the amino acid mutation reduces the binding affinity of the Fc domain to an Fc receptor. In one embodiment, the amino acid mutation reduces the binding affinity of the Fc domain to an Fc receptor by at least 2 fold, at least 5 fold, or at least 10 fold. In embodiments where there is more than one amino acid mutation that reduces the binding affinity of the Fc domain to the Fc receptor, the combination of these amino acid mutations can reduce the binding affinity of the Fc domain to the Fc receptor by at least 10-fold, at least 20-fold, or even at least 50-fold. In one embodiment, the T cell activating bispecific antigen binding molecule comprising an engineered Fc domain exhibits a binding affinity to an Fc receptor of less than 20%, particularly less than 10%, more particularly less than 5% compared to a T cell of the T cell activating bispecific antigen binding molecule comprising a non-engineered Fc domain. In a specific embodiment, the Fc receptor is an fey receptor. In some embodiments, the Fc receptor is a human Fc receptor. In some embodiments, the Fc receptor is an activating Fc receptor. In a specific embodiment, the Fc receptor is an activating human Fc γ receptor, more specifically human Fc γ RIIIa, Fc γ RI or Fc γ RIIa, most specifically human Fc γ RIIIa. Preferably, binding to each of these receptors is reduced. In some embodiments, the binding affinity to complement components, particularly to C1q, is also reduced. In one embodiment, the binding affinity for neonatal Fc receptor (FcRn) is not reduced. Substantially similar binding to FcRn is achieved when the Fc domain (or T cell activating bispecific antigen binding molecule comprising said Fc domain) exhibits an affinity binding to FcRn of greater than about 70% of the unengineered form of the Fc domain (or said unengineered form of the T cell activating bispecific antigen binding molecule comprising the Fc domain), i.e. retains the binding affinity of the Fc domain to said receptor. The Fc domain or T cell activating bispecific antigen binding molecule of the invention comprising said Fc domain may exhibit such affinity of more than about 80%, even more than about 90%. In certain embodiments, the Fc domain of the T cell activating bispecific antigen binding molecule is engineered to have reduced effector function compared to a non-engineered Fc domain. Reduced effector function may include, but is not limited to, one or more of reduced Complement Dependent Cytotoxicity (CDC), reduced antibody dependent cell mediated cytotoxicity (ADCC), reduced Antibody Dependent Cellular Phagocytosis (ADCP), reduced cytokine secretion, reduced immune complex mediated antigen uptake by antigen presenting cells, reduced binding to NK cells, reduced binding to macrophages, reduced binding to monocytes, reduced binding to polymorphonuclear cells, reduced direct signaling induced apoptosis, reduced cross-linking of target-binding antibodies, reduced dendritic cell maturation or reduced T-cell priming. In one embodiment, the reduced effector function is one or more selected from the group consisting of reduced CDC, reduced ADCC, reduced ADCP and reduced cytokine secretion. In a specific embodiment, the reduced effector function is reduced ADCC. In one embodiment, the reduced ADCC is less than 20% of the ADCC induced by the non-engineered Fc domain (or the T-cell activating bispecific antigen binding molecule comprising a non-engineered Fc domain).

In one embodiment, the amino acid mutation that reduces the binding affinity of the Fc domain to an Fc receptor and/or effector function is an amino acid substitution. In a fruitIn embodiments, the Fc domain comprises an amino acid substitution at a position selected from the group consisting of E233, L234, L235, N297, P331 and P329. In a more specific embodiment, the Fc domain comprises an amino acid substitution at a position selected from the group consisting of L234, L235, and P329. In some embodiments, the Fc domain comprises the amino acid substitutions L234A and L235A. In one such embodiment, the Fc domain is an IgG₁Fc domain, in particular human IgG₁An Fc domain. In one embodiment, the Fc domain comprises an amino acid substitution at position P329. In more specific embodiments, the amino acid substitution is P329A or P329G, in particular P329G. In one embodiment, the Fc domain comprises an amino acid substitution at position P329 and a further amino acid substitution at a position selected from the group consisting of E233, L234, L235, N297 and P331. In more specific embodiments, the further amino acid substitution is E233P, L234A, L235A, L235E, N297A, N297D or P331S. In particular embodiments, the Fc domain comprises amino acid substitutions at positions P329, L234 and L235. In a more specific embodiment, the Fc domain comprises the amino acid mutations L234A, L235A, and P329G ("P329G LALA"). In one such embodiment, the Fc domain is an IgG ₁Fc domain, in particular human IgG₁An Fc domain. Combination of amino acid substitutions "P329G LALA" almost completely abolished human IgG₁Fc domain Fc γ receptor binding as described in PCT patent application No. PCT/EP2012/055393, the entire contents of which are incorporated herein by reference. PCT/EP2012/055393 also describes methods for preparing such mutant Fc domains and methods for determining properties thereof, such as Fc receptor binding or effector function.

And IgG₁Antibody vs. IgG₄Antibodies show reduced binding affinity to Fc receptors and reduced effector function. Thus, in some embodiments, the Fc domain of the T cell activating bispecific antigen binding molecules of the invention is an IgG₄Fc domain, in particular human IgG₄An Fc domain. In one embodiment, the IgG is₄The Fc domain comprises an amino acid substitution at position S228, in particular the amino acid substitution S228P. In order to further reduce its binding affinity to Fc receptors and/or its effectorsFunctional, in one embodiment, IgG₄The Fc domain comprises an amino acid substitution at position L235, in particular the amino acid substitution L235E. In another embodiment, the IgG is₄The Fc domain comprises an amino acid substitution at position P329, in particular the amino acid substitution P329G. In a specific embodiment, the IgG is ₄The Fc domain comprises amino acid substitutions at positions S228, L235 and P329, in particular amino acid substitutions S228P, L235E and P329G. Such IgG₄Fc domain mutants and their Fc γ receptor binding properties are described in PCT patent application No. PCT/EP2012/055393, the entire contents of which are incorporated herein by reference.

In particular embodiments, the IgG is naturally associated with₁Fc domain exhibiting reduced binding affinity to Fc receptor and/or reduced effector function compared to the Fc domain is a human IgG comprising the amino acid substitutions L234A, L235A and optionally P329G₁Fc domain, or a human IgG comprising the amino acid substitutions S228P, L235E and optionally P329G₄An Fc domain.

In certain embodiments, N-glycosylation of the Fc domain has been eliminated. In one such embodiment, the Fc domain comprises an amino acid mutation at position N297, in particular an amino acid substitution replacing asparagine with alanine (N297A) or aspartic acid (N297D).

In addition to the Fc domains described above and PCT patent application No. PCT/EP2012/055393, Fc domains with reduced Fc receptor binding and/or effector function also include substituted Fc domains with one or more of Fc domain residues 238,265,269,270,297,327 and 329 (U.S. Pat. No. 6,737,056). Such Fc mutants include Fc mutants having substitutions at two or more amino acid positions 265,269,270,297 and 327, including so-called "DANA" Fc mutants in which residues 265 and 297 are substituted with alanine (U.S. Pat. No. 7,332,581).

The mutant Fc domain can be prepared by amino acid deletion, substitution, insertion or modification by genetic or chemical methods well known in the art. Genetic methods may include site-specific mutagenesis of the encoding DNA sequence, PCR, gene synthesis, and the like. The correct nucleotide changes can be verified by sequencing.

Binding to Fc receptors, which can be obtained by recombinant expression, can be readily determined, for example, by ELISA, or by Surface Plasmon Resonance (SPR) using standard instruments such as BIAcore instruments (GE Healthcare). Such suitable binding assays are described herein. Alternatively, cell lines known to express specific Fc receptors (e.g., human NK cells expressing Fc γ IIIa receptors) can be used to assess the binding affinity of the Fc domain or Fc domain-containing cell activating bispecific antigen binding molecule to an Fc receptor.

Effector function of an Fc domain or a T cell activating bispecific antigen binding molecule comprising an Fc domain can be measured by methods known in the art. Suitable assays for measuring ADCC are described herein. Other examples of in vitro assays to assess ADCC activity of molecules of interest are described in U.S. Pat. nos. 5,500,362; hellstrom et al, Proc Natl Acad Sci USA 83, 7059-; U.S. Pat. nos. 5,821,337; bruggemann et al, J Exp Med 166, 1351-. Alternatively, non-radioactive assay methods can be used (see, e.g., ACTI for flow cytometry) ^TMNon-radioactive cytotoxicity assay (CellTechnology, inc. gampton View, CA); and CytotoxNon-radioactive cytotoxicity assay (Promega, Madison, WI)). Useful effector cells for such assays include Peripheral Blood Mononuclear Cells (PBMC) and Natural Killer (NK) cells. Alternatively or additionally, the ADCC activity of the molecule of interest can be assessed in vivo, for example, in an animal model disclosed in Clynes et al, Proc Natl Acad Sci USA 95, 652-.

In some embodiments, Fc domain binding to complement components (particularly C1q) is reduced. Thus, in some embodiments wherein the Fc domain is engineered to have reduced effector function, said reduced effector function comprises reduced CDC. A C1q binding assay can be performed to determine whether a T cell activating bispecific antigen binding molecule is capable of binding to C1q and therefore has CDC activity. See, e.g., WO 2006/029879 and WO 2005/100402 for C1q and C3C binding ELISAs. To assess complement activation, CDC assays may be performed (see, e.g., Gazzano-Santoro et al, J Immunol Methods 202,163 (1996); Cragg et al, Blood 101,1045-1052 (2003); and Cragg and Glennie, Blood 103,2738-2743 (2004)).

Antigen binding moieties

The antigen binding molecule of the present invention is bispecific, i.e. it comprises at least two antigen binding portions capable of specifically binding two different antigenic determinants. According to one embodiment of the invention, the antigen binding portion is a Fab molecule (i.e. an antigen binding domain consisting of a heavy chain and a light chain, each comprising a variable region and a constant region), wherein the light chains (VLCL) of the at least two Fab molecules comprise the same sequence. In one embodiment, the VLCL light chains of the Fab molecules capable of specific binding to the target cell antigen and the T cell activation antigen, respectively, comprise the light chain CDRs of SEQ ID NO:32, SEQ ID NO:33 and SEQ ID NO:34, respectively.

In one embodiment, the VLCL light chain of the Fab molecule capable of specifically binding to the target cell antigen and the T cell activation antigen, respectively, comprises SEQ ID NO 31.

In one embodiment, the Fab molecule is human. In another embodiment, the Fab molecule is humanized. In another embodiment, the Fab molecule comprises human heavy and light chain constant regions.

In a particular embodiment according to the present invention, the T cell activating bispecific antigen binding molecule is capable of binding to a target cell antigen, in particular a tumor cell antigen, and an activated T cell antigen simultaneously. In one embodiment, the T cell activating bispecific antigen binding molecule is capable of cross-linking a T cell and a target cell by simultaneously binding to the target cell antigen and an activating T cell antigen. In even more specific embodiments, such simultaneous binding results in lysis of target cells, particularly tumor cells. In one embodiment, such simultaneous binding results in activation of T cells. In other embodiments, such simultaneous binding results in a cellular response of the T-lymphocytes, in particular cytotoxic T-lymphocytes, selected from the group consisting of proliferation, differentiation, cytokine secretion, release of cytotoxic effector molecules, cytotoxic activity and expression of activation markers. In one embodiment, binding of the T cell activating bispecific antigen binding molecule to an activating T cell antigen without simultaneous binding to a target cell antigen does not result in T cell activation.

In one embodiment, the T cell activating bispecific antigen binding molecule is capable of redirecting the cytotoxic activity of the T cell to a target cell. In a specific embodiment, the redirecting is independent of MHC-mediated peptide antigen presentation of the target cell and/or specificity of the T cell.

In particular, the T cell according to any embodiment of the invention is a cytotoxic T cell. In some embodiments, the T cell is CD4⁺Or CD8⁺T cells, in particular CD8⁺T cells.

Activated T cell antigen binding moieties

The T cell activating bispecific antigen binding molecules of the invention comprise at least one antigen binding moiety capable of binding an activating T cell antigen (also referred to herein as "activating T cell antigen binding moiety"). In a specific embodiment, the T cell activating bispecific antigen binding molecule comprises no more than one antigen binding moiety capable of specifically binding to an activated T cell antigen. In one embodiment, the T cell activating bispecific antigen binding molecule provides monovalent binding to an activating T cell antigen. The activating T cell antigen-binding portion is a Fab molecule and comprises the same VLCL light chain as the antigen-binding portion capable of specifically binding to the target cell antigen.

In a particular embodiment, the activated T cell antigen is CD3, particularly human CD3 or cynomolgus monkey CD3, most particularly human CD 3. In a specific embodiment, the activated T cell antigen-binding moiety cross-reacts with (i.e., specifically binds to) human and cynomolgus monkey CD 3. In some embodiments, the activated T cell antigen is the epsilon subunit of CD3 (SEQ ID NO: 56).

In one embodiment, the activated T cell antigen binding portion can compete with monoclonal antibody H2C (described in PCT publication No. WO2008/119567) for binding to an epitope of CD 3. In another embodiment, the activated T cell antigen binding portion can compete with monoclonal antibody V9 (described in Rodrigues et al, Int J Cancer Suppl 7,45-50(1992) and U.S. Pat. No. 6,054,297) for binding to an epitope of CD 3. In another embodiment, the activated T cell antigen-binding portion may compete with monoclonal antibody FN18 (described in Nooij et al, Eur J Immunol 19,981-984 (1986)) for binding to an epitope of CD 3. In a particular embodiment, the activated T cell antigen-binding portion may compete with monoclonal antibody SP34(Pessano et al, EMBO J4,337-340 (1985)) for binding to an epitope of CD 3. In one embodiment, the activated T cell antigen binding portion binds to the same epitope of CD3 as monoclonal antibody SP 34.

In one embodiment, the activated T cell antigen-binding portion comprises the heavy chain CDR1 of SEQ ID NO. 14, the heavy chain CDR2 of SEQ ID NO. 15, the heavy chain CDR3 of SEQ ID NO. 16, the light chain CDR1 of SEQ ID NO. 32, the light chain CDR2 of SEQ ID NO. 33 and the light chain CDR3 of SEQ ID NO. 34. In another embodiment, the activated T cell antigen binding portion comprises a heavy chain variable region sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:36, or a variant thereof that retains functionality.

In another embodiment, the activated T cell antigen binding portion comprises a light chain variable region sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:31, or a functionally retained variant thereof.

In one embodiment, the activated T cell antigen-binding portion comprises a heavy chain comprising the sequence of SEQ ID NO. 36 and a light chain comprising the sequence of SEQ ID NO. 31.

In one embodiment, the activated T cell antigen-binding portion comprises the heavy chain of SEQ ID NO. 40 and the light chain comprising SEQ ID NO. 35.

Target cell antigen binding moieties

The T cell activating bispecific antigen binding molecules of the invention comprise at least one antigen binding moiety (also referred to herein as "target cell antigen binding moiety") capable of binding a target cell antigen. In certain embodiments, the T cell activating bispecific antigen binding molecule comprises two antigen binding moieties capable of binding to a target cell antigen. In certain such embodiments, each of these antigen binding portions specifically binds to the same antigenic determinant. In one embodiment, the T cell activating bispecific antigen binding molecule comprises an immunoglobulin molecule capable of specific binding to a target cell antigen. In one embodiment, the T cell activating bispecific antigen binding molecule comprises no more than two antigen binding moieties capable of binding to a target cell antigen. The target cell antigen binding portion is typically a Fab molecule that binds to a specific antigenic determinant and is capable of directing the T cell activated bispecific antigen binding molecule to a target site, e.g. to a specific type of tumor cell having an antigenic determinant. The Fab molecule has the same VLCL light chain as a Fab molecule capable of specifically binding to a T cell activation antigen. In a preferred embodiment, the VLCL light chain of a Fab molecule capable of specifically binding to a target cell antigen and the Fab molecule capable of specifically binding to a T cell activation antigen comprise the light chain CDR1 of SEQ ID NO:32, the light chain CDR2 of SEQ ID NO:33 and the light chain CDR3 of SEQ ID NO: 34. In a preferred embodiment, the VLCL light chain of the Fab molecule capable of specifically binding to a target cell antigen and the Fab molecule capable of specifically binding to a T cell activation antigen comprise the VLCL light chain of SEQ ID NO 31.

In another embodiment, the target cell antigen-binding portion comprises a light chain variable region sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:31 or a functionally-retained variant thereof.

In another embodiment, the target cell antigen-binding portion comprises a light chain sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO 35 or a functionally-retained variant thereof.

In certain embodiments, the target cell antigen-binding portion targets an antigen associated with a pathological condition, such as an antigen present on a tumor cell or a virus-infected cell. Suitable antigens are cell surface antigens such as, but not limited to, cell surface receptors. In a specific embodiment, the antigen is a human antigen. In a specific embodiment, the target cell antigen is selected from the group consisting of folate receptor 1(FolR1), mucin-1 (MUC1), and B Cell Maturation Antigen (BCMA).

In a specific embodiment, the T cell activating bispecific antigen binding molecule comprises a polypeptide sequence encoded by a polynucleotide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to a sequence selected from the group consisting of SEQ ID NO:183, SEQ ID NO:193, SEQ ID NO:263, SEQ ID NO:264, SEQ ID NO:191, SEQ ID NO:198, SEQ ID NO:267, and SEQ ID NO:272, including functional fragments or variants thereof.

Polynucleotide

The invention also provides an isolated polynucleotide encoding a T cell activating bispecific antigen binding molecule or fragment thereof as described herein.

Polynucleotides of the invention include those polynucleotides, including functional fragments or variants thereof, that are at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequences set forth in SEQ ID NO 183, 193, 263, 264, 191, 198, 267, and 272.

The polynucleotide encoding the T cell activation bispecific antigen binding molecule of the invention can be expressed as a single polynucleotide encoding the entire T cell activation bispecific antigen binding molecule or as multiple (e.g., two or more) polynucleotides that are co-expressed. Polypeptides encoded by the co-expressed polynucleotides may associate through, for example, disulfide bonds or other means to form a functional T cell activating bispecific antigen binding molecule. For example, the light chain portion of the antigen-binding portion may be encoded by a polynucleotide separate from the portion of the T cell activating bispecific antigen-binding molecule comprising the heavy chain portion of the antigen-binding portion, the Fc domain subunit and optionally (part of) another antigen-binding portion. When co-expressed, the heavy chain polypeptide will associate with the light chain polypeptide to form an antigen-binding portion. In another example, the portion of the T cell activating bispecific antigen binding molecule comprising one of the two Fc domain subunits and optionally (part of) the one or more antigen binding moieties can be encoded by a polynucleotide that is isolated from the portion of the T cell activating bispecific antigen binding molecule comprising the other of the two Fc domain subunits and optionally (part of) the antigen binding moiety. When co-expressed, the Fc domain subunits will associate to form an Fc domain.

In certain embodiments, the polynucleotide or nucleic acid is DNA. In other embodiments, the polynucleotide of the invention is RNA, for example in the form of messenger RNA (mrna). The RNA of the present invention may be single-stranded or double-stranded.

Recombination method

The T cell activating bispecific antigen binding molecules of the invention may be obtained, for example, by solid phase peptide synthesis (e.g. Merrifield solid phase synthesis) or recombinant production. For recombinant production, one or more polynucleotides encoding T cell activating bispecific antigen binding molecules (fragments) as described above are isolated and inserted into one or more vectors for further cloning and/or expression in a host cell. Such polynucleotides can be readily isolated and sequenced using conventional procedures. In one embodiment, a vector, preferably an expression vector, comprising one or more polynucleotides of the invention is provided. Methods well known to those skilled in the art can be used to construct expression vectors containing the coding sequence for the T cell activating bispecific antigen binding molecules (fragments) and appropriate transcription/translation control signals. These methods include in vitro recombinant DNA techniques, synthetic techniques and in vivo recombination/genetic recombination. See, e.g., Maniatis et al, MOLECULAR CLONING: A LABORATORY MANUAL, Cold Spring Harbor LABORATORY, N.Y. (1989); and Ausubel et al, Current PROTOCOLS IN MOLECULAR BIOLOGY, Greene Publishing Associates and Wiley Interscience, N.Y. (1989). The expression vector may be a plasmid, part of a virus, or may be a nucleic acid fragment. Expression vectors include expression cassettes in which a polynucleotide encoding a T cell activating bispecific antigen binding molecule (fragment) (i.e., coding region) is operably linked to a promoter and/or other transcriptional or translational control elements. As used herein, a "coding region" is the portion of a nucleic acid that consists of codons that are translated into amino acids. Although the "stop codon" (TAG, TGA or TAA) is not translated into an amino acid, it may be considered part of the coding region if present, but any flanking sequences, such as promoters, ribosome binding sites, transcription terminators, introns, 5 'and 3' untranslated regions, etc., are not part of the coding region. The two or more coding regions may be present in a single polynucleotide construct, e.g., on a single vector, or in separate polynucleotide constructs, e.g., on separate (different) vectors. In addition, any vector may contain a single coding region, or may contain two or more coding regions, e.g., a vector of the invention may encode one or more polypeptides which are separated post-translationally or concurrently with translation into the final protein by proteolytic cleavage. Furthermore, the vector, polynucleotide or nucleic acid of the invention may encode a heterologous coding region fused or unfused to a polynucleotide encoding a T cell activating bispecific antigen binding molecule (fragment) of the invention or a variant or derivative thereof. Heterologous coding regions include, but are not limited to, specialized elements or motifs, such as secretory signal peptides or heterologous functional domains. An effective combination is when the coding region of the gene product, e.g., a polypeptide, is combined with one or more regulatory sequences in such a way that expression of the gene product is under the influence or control of the regulatory sequences. Two DNA fragments (e.g., a polypeptide coding region and a promoter associated therewith) are "operably associated" if induction of promoter function results in transcription of mRNA encoding the desired gene product, and if the nature of the linkage between the two DNA fragments does not interfere with the ability of the expression control sequences to direct expression of the gene product or interfere with the transcription of the DNA template. Thus, if a promoter is capable of affecting transcription of the nucleic acid, the promoter region will be operably associated with the nucleic acid encoding the polypeptide. The promoter may be a cell-specific promoter that directs substantial transcription of DNA only in predetermined cells. In addition to promoters, other transcriptional control elements, such as enhancers, operators, repressors, and transcriptional termination signals, may be operably associated with the polynucleotide to direct cell-specific transcription. Suitable promoters and other transcriptional control regions are disclosed herein. Various transcriptional control regions are known to those skilled in the art. These include, but are not limited to, transcriptional control regions that function in vertebrate cells, such as, but not limited to, cytomegalovirus promoter and enhancer segments (e.g., immediate early promoter, linked to intron-a), simian virus 40 (e.g., early promoter), and retroviruses (e.g., such as, for example, rous sarcoma virus). Other transcriptional control regions include those derived from vertebrate genes such as actin, heat shock proteins, bovine growth hormone and rabbit α -globin, as well as other sequences capable of controlling gene expression in eukaryotic cells. Additional suitable transcriptional control regions include tissue-specific promoters and enhancers and inducible promoters (e.g., promoter-inducible tetracycline). Similarly, various translational control elements are known to those of ordinary skill in the art. These include, but are not limited to, ribosome binding sites, translation initiation and termination codons, and elements derived from viral systems (in particular internal ribosome entry sites or IRES, also known as CITE sequences). The expression cassette may also include other features, such as an origin of replication, and/or a chromosomal integration element such as a retroviral Long Terminal Repeat (LTR) or an adeno-associated virus (AAV) Inverted Terminal Repeat (ITR).

The polynucleotide and nucleic acid coding regions of the present invention may be combined with additional coding regions that encode secretion or signal peptides that direct the secretion of the polypeptide encoded by the polynucleotide of the present invention. For example, if secretion of a T cell activating bispecific antigen binding molecule is desired, a DNA encoding a signal sequence can be placed upstream of a nucleic acid encoding a T cell activating bispecific antigen binding molecule of the invention or a fragment thereof. According to the signal hypothesis, proteins secreted by mammalian cells have a signal peptide or secretory leader sequence that is cleaved from the mature protein once the export of the growing protein chain from the rough endoplasmic reticulum has begun. One of ordinary skill in the art will recognize that polypeptides secreted by vertebrate cells typically have a signal peptide fused to the N-terminus of the polypeptide, which is cleaved from the translated polypeptide to yield a secreted or "mature" form of the polypeptide. In certain embodiments, a native signal peptide, such as an immunoglobulin heavy or light chain signal peptide, or a functional derivative of such a sequence, is used that retains the ability to direct secretion of the polypeptide to which it is operatively bound. Alternatively, a heterologous mammalian signal peptide or functional derivative thereof may be used. For example, the wild-type leader sequence may be replaced with the leader sequence of human Tissue Plasminogen Activator (TPA) or mouse β -glucuronidase.

DNA encoding a short protein sequence that can be used to facilitate subsequent purification (e.g., histidine tag) or to aid in labeling of the T cell activation bispecific antigen binding molecule can be included within or at the end of the polynucleotide encoding the T cell activation bispecific antigen binding molecule (fragment).

In another embodiment, a host cell comprising one or more polynucleotides of the invention is provided. In certain embodiments, host cells comprising one or more vectors of the invention are provided. The polynucleotide and vector may incorporate any of the features described herein, either alone or in combination, with respect to the polynucleotide and vector, respectively. In one such embodiment, the host cell comprises (e.g. has been transformed or transfected) a vector comprising a polynucleotide encoding (part of) a T cell activating bispecific antigen binding molecule of the invention. As used herein, the term "host cell" refers to any kind of cellular system that can be engineered to produce the T cell activating bispecific antigen binding molecules of the invention or fragments thereof. Host cells suitable for replicating and supporting expression of T cell activating bispecific antigen binding molecules are well known in the art. Such cells can be suitably transfected or transduced with a particular expression vector, and large numbers of vector-containing cells can be grown for seeding large-scale fermentors to obtain sufficient quantities of T cell activating bispecific antigen binding molecules for clinical use. Suitable host cells include prokaryotic microorganisms such as E.coli, or various eukaryotic cells such as Chinese Hamster Ovary (CHO) cells, insect cells, and the like. For example, the polypeptide may be produced in bacteria, particularly when glycosylation is not required. After expression, the polypeptide may be removed from the bacterial cell paste in the soluble fraction Isolated and may be further purified. In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast are suitable cloning or expression hosts for polypeptide-encoding vectors, including fungal and yeast strains, whose glycosylation pathways have been "humanized" resulting in the production of polypeptides with partially or fully human glycosylation patterns. See Gerngross, Nat Biotech 22, 1409-. Suitable host cells for the expression of (glycosylated) polypeptides are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. A number of baculovirus strains have been identified which can be used in conjunction with insect cells, particularly for transfecting Spodoptera frugiperda (Spodoptera frugiperda) cells. Plant cell cultures may also be used as hosts. See, e.g., U.S. Pat. Nos. 5,959,177,6,040,498,6,420,548,7,125,978, and 6,417,429 (describing PLANTIBODIIES for antibody production in transgenic plants^TMA technique). Vertebrate cells can also be used as hosts. For example, mammalian cell lines suitable for growth in suspension may be useful. Other examples of useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7); human embryonic kidney cell lines (e.g., 293 or 293T cells described in Graham et al, J Gen Virol 36,59(1977), baby hamster Kidney cells (BHK), mouse support cells (TM 4 cells as described in Mather, Biol Reprod 23,243- & 251 (1980)), monkey Kidney cells (CV1), African Green monkey Kidney cells (VERO-76), human cervical carcinoma cells (HELA), Canine Kidney cells (MDCK), buffalo rat liver cells (BRL 3A), human Lung cells (W138), human liver cells (Hep G2), mouse mammary tumor cells (MMT 060562), TRI cells (as described in Mather et al, Annals N.Y. Acad Sci 383,44-68 (1982)), MRC 5 cells and FS4 mammalian host cell lines other useful mammalian host cell lines include hamster ovary (CHO) cells including hamster dhdhdhcho, including hamster kidney (CHO) ^-CHO cells (Urlaub et al, Proc Natl Acad Sci USA 77,4216 (1980)); and myeloma cell lines such as YO, NS0, P3X63, and Sp 2/0. For a review of certain mammalian host cell lines suitable for protein production, see, e.g., Yazaki and Wu, Methods in Molecular Biology, vol.248(b.k.c.lo, e)d., Humana Press, Totowa, NJ), pp.255-268 (2003). Host cells include cultured cells, such as mammalian cultured cells, yeast cells, insect cells, bacterial cells and plant cells, to name a few, and include cells within transgenic animals, transgenic plants or cultured plant or animal tissues. In one embodiment, the host cell is a eukaryotic cell, preferably a mammalian cell, such as a Chinese Hamster Ovary (CHO) cell, a Human Embryonic Kidney (HEK) cell or a lymphocyte (e.g., Y0, NS0, Sp20 cell).

Standard techniques are known in the art to express foreign genes in these systems. A cell expressing a polypeptide comprising a heavy or light chain of an antigen binding domain (e.g., an antibody) can be engineered to also express another antibody chain, such that the expressed product is an antibody having a heavy chain and a light chain.

In one embodiment, a method of producing a T cell activating bispecific antigen binding molecule according to the invention is provided, wherein the method comprises culturing a host cell comprising a polynucleotide encoding a T cell activating bispecific antigen binding molecule provided herein under conditions suitable for expression of the T cell activating bispecific antigen binding molecule, and recovering the T cell activating bispecific antigen binding molecule from the host cell (or host cell culture medium).

The components of the T cell activating bispecific antigen binding molecule are genetically fused to each other. T cell activating bispecific antigen binding molecules can be designed such that their components are fused to each other directly or indirectly through linker sequences. The composition and length of the linker can be determined according to methods well known in the art, and its efficacy can be tested. Examples of linker sequences between the different components of the T cell activating bispecific antigen binding molecule are found in the sequences provided herein. Additional sequences can also be included to incorporate cleavage sites to isolate individual components of the fusion, such as endopeptidase recognition sequences, if desired.

In certain embodiments, one or more antigen binding portions of a T cell activating bispecific antigen binding molecule comprise at least an antibody variable region capable of binding an antigenic determinant. The variable regions may form part of and be derived from naturally or non-naturally occurring antibodies and fragments thereof. Methods for producing polyclonal and monoclonal Antibodies are well known in the art (see, e.g., Harlow and Lane, "Antibodies, a Laboratory", Cold Spring Harbor Laboratory, 1988). Non-naturally occurring antibodies can be constructed using solid phase peptide synthesis, can be recombinantly produced (e.g., as described in U.S. patent No. 4,186,567), or can be obtained, for example, by screening combinatorial libraries comprising variable heavy and variable light chains (see U.S. patent No. 5,969,108 to McCafferty).

Any animal species of antibody, antibody fragment, antigen binding domain or variable region can be used for the T cell activating bispecific antigen binding molecules of the invention. Non-limiting antibodies, antibody fragments, antigen binding domains or variable regions useful in the invention can be of murine, primate, or human origin. If the T cell activating bispecific antigen binding molecule is for human use, a chimeric form of the antibody may be used in which the constant region of the antibody is from a human. Humanized or fully human forms of antibodies can also be prepared according to methods well known in the art (see, e.g., U.S. Pat. No. 5,565,332 to Winter). Humanization can be achieved by a variety of methods, including but not limited to (a) grafting non-human (e.g., donor antibody) CDRs to human (e.g., acceptor antibody) frameworks and retained constant regions with or without critical framework residues (e.g., those residues important for maintaining good antigen binding affinity or antibody function), (b) grafting only non-human specificity determining regions (SDRs or a-CDRs; residues critical for antibody-antigen interaction) to human frameworks and constant regions, or (c) grafting entire non-human variable domains, but "masking" them from human-like moieties by replacing surface residues. Humanized antibodies and methods for their preparation are reviewed, for example, in Almagro and Fransson, Front Biosci 13,1619-1633(2008), and further described, for example, in Riechmann et al, Nature 332,323-329 (1988); queen et al, Proc Natl Acad Sci USA 86, 10029-; U.S. Pat. nos. 5,821,337,7,527,791,6,982,321, and 7,087,409; jones et al, Nature 321,522-525 (1986); morrison et al, Proc Natl Acad Sci 81,6851-6855 (1984); morrison and Oi, Adv Immunol 44,65-92 (1988); verhoeyen et al, Science 239, 1534-; padlan, Molec Immun 31(3),169-217 (1994); kashmiri et al, Methods 36,25-34(2005) (describing SDR (a-CDR) grafting); padlan, Mol Immunol 28,489-498(1991) (description "resurfacing"); dall' Acqua et al, Methods 36,43-60(2005) (description "FR shuffling"); and Osbourn et al, Methods 36,61-68(2005) and Klimka et al, Br J Cancer 83, 252-. Various techniques known in the art can be used to produce human antibodies and human variable regions. Human antibodies are generally described in van Dijk and van de Winkel, Curr Opin Pharmacol 5,368-74(2001) and Lonberg, Curr Opin Immunol 20, 450-. The human variable region may form part of, and be derived from, human Monoclonal antibodies produced by the hybridoma method (see, e.g., Monoclonal Antibody Production Techniques and Applications, pp.51-63(Marcel Dekker, Inc., New York, 1987)). Human antibodies and Human variable regions can also be prepared by administering immunogens to transgenic animals that have been modified to produce either fully Human antibodies or fully antibodies with Human variable regions in response to antigenic challenge (see, e.g., Lonberg, Nat Biotech 23,1117-1125 (2005). Human antibodies and Human variable regions can also be generated by isolating Fv clone variable region sequences selected from Human-derived phage display libraries (see, e.g., Hoogenboom et al. in Methods in Molecular Biology 178,1-37 (O' Brien et al., ed., Human Press, Totowa, NJ, 2001); and McCafferty et al., Nature 348, 552-554; Clackson et al., Nature 352,624-628 (1991)). phage typically display antibody fragments as single chain Fv fragments (scFv) or Fab fragments.

In certain embodiments, antigen binding moieties useful in the invention are engineered to have enhanced binding affinity according to, for example, the methods disclosed in U.S. patent application publication No. 2004/0132066 (which is incorporated herein by reference in its entirety). The ability of the T cell activating bispecific antigen binding molecules of the invention to bind to specific antigenic determinants can be measured by enzyme-linked immunosorbent assays (ELISA) or other techniques familiar to the person skilled in the art, such as the surface plasmon resonance technique (analyzed on the BIACORE T100 system) (Liljeblad, et al, Glyco J17, 323-. Competition assays can be used to identify antibodies, antibody fragments, antigen binding domains or variable domains that compete with a reference antibody for binding to a particular antigen, e.g., antibodies that compete with the V9 antibody for binding to CD 3. In certain embodiments, such a competing antibody binds to the same epitope (e.g., a linear or conformational epitope) that is bound by the reference antibody. Detailed exemplary Methods for Mapping epitopes bound by antibodies are provided in Morris (1996) "Epitope Mapping Protocols," in Methods in Molecular Biology vol.66(Humana Press, Totowa, NJ). In an exemplary competition assay, an immobilized antigen (e.g., CD3) is incubated in a solution comprising a first labeled antibody (e.g., V9 antibody) that binds to the antigen, and a second unlabeled antibody, which is being tested for its ability to compete with the first antibody for binding to the antigen. The second antibody may be present in the hybridoma supernatant. As a control, the immobilized antigen was incubated in a solution containing the first labeled antibody but no second unlabeled antibody. After incubation under conditions that allow the first antibody to bind to the antigen, excess unbound antibody is removed and the amount of label bound to the immobilized antigen is measured. If the amount of label bound to the immobilized antigen is significantly reduced in the test sample relative to the control sample, it is indicative that the second antibody competes with the first antibody for binding to the antigen. See Harlow and Lane (1988) Antibodies: A Laboratory Manual ch.14(Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.).

The T cell activating bispecific antigen binding molecules prepared as described herein can be purified by techniques known in the art such as high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography, size exclusion chromatography, and the like. The actual conditions used to purify a particular protein will depend in part on factors such as net charge, hydrophobicity, hydrophilicity, and the like, and will be apparent to those skilled in the art. For affinity chromatography purification, antibodies, ligands, receptors or antigens to which the T cell activating bispecific antigen binding molecule binds may be used. For example, for affinity chromatography purification of the T cell activating bispecific antigen binding molecules of the invention, a matrix with protein a or protein G may be used. Essentially as described in the examples, sequential protein a or G affinity chromatography and size exclusion chromatography can be used to isolate T cell activating bispecific antigen binding molecules. The purity of the T cell activating bispecific antigen binding molecule can be determined by any of a variety of well known analytical methods, including gel electrophoresis, high pressure liquid chromatography, and the like. For example, heavy chain fusion proteins expressed as described in the examples proved to be intact and correctly assembled as demonstrated by reducing SDS-PAGE. Three bands resolved at about 25,000, 50,000 and mr75,000, corresponding to predicted molecular weights of T cell activating bispecific antigen binding molecule light chain, heavy chain and heavy chain/light chain fusion protein.

Assay method

The T cell activating bispecific antigen binding molecules provided herein can be identified, screened for, or characterized for their physical/chemical properties and/or biological activity by various assays known in the art.

Affinity assay

The affinity of the T cell activating bispecific antigen binding molecule for the Fc receptor or target antigen can be determined by Surface Plasmon Resonance (SPR) using standard instruments such as BIAcore instruments (GE Healthcare) according to the methods set forth in the examples, and the receptor or target protein can be obtained by recombinant expression. Alternatively, cell lines expressing a particular receptor or target antigen can be used to assess the binding of the T cell activating bispecific antigen binding molecule to a different receptor or target antigen, for example by flow cytometry (FACS). The following and following examples describe specific illustrative and exemplary embodiments for measuring binding affinity.

According to one embodiment, use is made ofK is measured by surface plasmon resonance at 25 ℃ by a T100 machine (GE Healthcare)_D。

To analyze the interaction between the Fc portion and the Fc receptor, the His-tagged recombinant Fc receptor was captured by an anti-Penta His antibody (Qiagen) immobilized on a CM5 chip ("Penta His" is disclosed as SEQ ID NO:45), and the bispecific construct was used as the analyte. Briefly, carboxymethylated dextran biosensor chips (CM5, GE Healthcare) were activated with N-ethyl-N '- (3-dimethylaminopropyl) -carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. An anti-Penta-His antibody ("Penta His" as disclosed in SEQ ID NO:45) was diluted to 40. mu.g/ml with 10mM sodium acetate (pH5.0) and then injected at a flow rate of 5. mu.l/min to achieve about 6500 Reaction Units (RU) of the coupled protein. After ligand injection, 1M ethanolamine was injected to block unreacted groups. Subsequently, the Fc receptor was captured at 4 or 10nM for 60 seconds. For kinetic measurements, four-fold serial dilutions of the bispecific construct (range between 500nM and 4000 nM) were injected in HBS-EP (GE Healthcare, 10mM HEPES, 150mM NaCl, 3mM EDTA, 0.05% surfactant P20, pH 7.4) at 25 ℃ at a flow rate of 30 μ l/min for 120 seconds.

To determine affinity for the target antigen, the bispecific construct was captured by an anti-human Fab specific antibody (GE Healthcare) immobilized on the surface of an activated CM 5-sensor chip, as described for an anti-Penta-His antibody ("Penta His" as disclosed in SEQ ID NO: 45). The final amount of coupled protein was about 12000 RU. Bispecific construct 90s were captured at 300 nM. The target antigen was passed through the flow cell at a concentration range of 250 to 1000nM and a flow rate of 30. mu.l/min for 180 seconds. Dissociation was monitored for 180 seconds.

Bulk refractive index differences were corrected by subtracting the response obtained on the reference flow cell. Steady-state response is used to derive the dissociation constant K by nonlinear curve fitting of Langmuir binding isotherms_D. (ii) use of a simple one-to-one Langmuir binding model by simultaneous fitting of binding and dissociation sensorgrams: (T100 evaluation software version 1.1.1) calculation of the binding Rate (k)_on) And dissociation Rate (k)_off). Equilibrium dissociation constant (K)_D) Is calculated as k_off/k_oAnd (4) the ratio. Ginseng radixSee, e.g., Chen et al, J Mol Biol 293, 865-.

Activity assay

The biological activity of the T cell activating bispecific antigen binding molecules of the invention can be measured by various assays described in the examples. Biological activity may for example include induction of proliferation of T cells, induction of signaling in T cells, induction of expression of activation markers in T cells, induction of cytokine secretion by T cells, induction of lysis of target cells, such as tumor cells, and induction of tumor regression and/or improvement of survival.

Compositions, formulations and routes of administration

In another aspect, the invention provides a pharmaceutical composition comprising any of the T cell activating bispecific antigen binding molecules provided herein, e.g. for use in any of the following methods of treatment. In one embodiment, the pharmaceutical composition comprises any of the T cell activating bispecific antigen binding molecules provided herein and a pharmaceutically acceptable carrier. In another embodiment, the pharmaceutical composition comprises any of the T cell activating bispecific antigen binding molecules provided herein and at least one additional therapeutic agent, e.g., as described below.

Also provided is a method of making a T cell activating bispecific antigen binding molecule of the invention in a form suitable for in vivo administration, the method comprising (a) obtaining a T cell activating bispecific antigen binding molecule of the invention, and (b) formulating the T cell activating bispecific antigen binding molecule with at least one pharmaceutically acceptable carrier, thereby formulating the T cell activating bispecific antigen binding molecule for in vivo administration.

The pharmaceutical compositions of the present invention comprise a therapeutically effective amount of one or more T cell activating bispecific antigen binding molecules dissolved or dispersed in a pharmaceutically acceptable carrier. The phrase "pharmaceutically or pharmacologically acceptable" refers to molecular entities and compositions that are generally non-toxic to the recipient at the dosages and concentrations employed, i.e., do not produce an adverse, allergic, or other untoward reaction when administered to an animal, e.g., a human (where appropriate). In light of the present disclosure, the skilled person will know the preparation of a Pharmaceutical composition comprising at least one T cell activating bispecific antigen binding molecule and optionally further active ingredients as exemplified by Remington's Pharmaceutical Sciences,18th ed. In addition, for animal (e.g., human) administration, it is understood that the formulation should meet sterility, pyrogenicity, general safety and purity standards as required by the FDA office of biological standards or corresponding departments in other countries. Preferred compositions are lyophilized formulations or aqueous solutions. As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, buffers, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, antioxidants, proteins, drugs, drug stabilizers, polymers, gels, binders, excipients, disintegrants, lubricants, sweeteners, flavoring agents, dyes, such materials, and combinations thereof, as known to those of ordinary skill in the art (see, e.g., Remington's Pharmaceutical Sciences,18th ed. mach Printing Company,1990, pp.1289-1329, incorporated herein by reference). Except insofar as any conventional carrier is incompatible with the active ingredient, its use in therapeutic or pharmaceutical compositions is contemplated.

The composition may contain different types of carriers depending on whether it is to be administered in solid, liquid or aerosol form and whether it needs to be sterile for administration, e.g. by injection. The T cell activating bispecific antigen binding molecules of the invention (and any additional therapeutic agent) may be administered intravenously, intradermally, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostaticaly, intrasplenically, intrarenally, intrapleurally, intratracheally, intranasally, intravitreally, intravaginally, intrarectally, intratumorally, intramuscularly, intraperitoneally, subcutaneously, subconjunctival, intravesicularly, mucosa, intrapericardially, intraumbilically, intraocularly, buccally, topically (locally), locally (locally), by inhalation (e.g., aerosol inhalation), injection, infusion, continuous infusion, direct local perfusion washing of target cells, by catheter, by lavage, in emulsion, with lipid compositions (e.g., liposomes) or other methods or any combination as would be known to one of ordinary skill in the art (see, e.g., Remington's Pharmaceutical Sciences,18th ed. Incorporated herein by reference). Parenteral administration, particularly intravenous injection, is most commonly used to administer polypeptide molecules, such as the T cell activating bispecific antigen binding molecules of the invention.

Parenteral compositions include those designed for administration by injection, for example, subcutaneous, intradermal, intralesional, intravenous, intraarterial, intramuscular, intrathecal or intraperitoneal injection. For injection, the T cell activating bispecific antigen binding molecules of the invention may be formulated in aqueous solution, preferably in a physiologically compatible buffer such as hanks 'solution, ringer's solution or physiological saline buffer. The solution may contain formulating agents, such as suspending, stabilizing and/or dispersing agents. Alternatively, the T cell activating bispecific antigen binding molecule may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use. Sterile injectable solutions are prepared by incorporating the T cell-activating bispecific antigen binding molecules of the present invention in the required amount in an appropriate solvent with various other ingredients as required, which are enumerated below. Sterility can be readily achieved, for example, by filtration through sterile filtration membranes. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains an alkaline dispersing medium and/or other ingredients. In the case of sterile powders for the preparation of sterile injectable solutions, suspensions or emulsions, the preferred methods of preparation are vacuum drying or freeze-drying techniques which yield a powder of the active ingredient plus additional desired ingredient from a previously sterile-filtered liquid medium thereof. The liquid medium should be suitably buffered, if necessary, and the liquid diluent first rendered isotonic prior to injection of sufficient saline or glucose. The compositions must be stable under the conditions of manufacture and storage and prevent the contaminating action of microorganisms such as bacteria and fungi. It will be appreciated that endotoxin contamination should be kept to a minimum at safe levels, for example less than 0.5ng/mg protein. Suitable pharmaceutically acceptable carriers include, but are not limited to, buffers such as phosphate, citrate and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (such as octadecyl dimethyl benzyl ammonium chloride, hexamethyl ammonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butanol or benzyl alcohol, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol, m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and/or a non-ionic surfactant such as polyethylene glycol (PEG). Aqueous injection suspensions may contain compounds which increase the viscosity of the suspension, for example sodium carboxymethyl cellulose, sorbitol, dextran and the like. Optionally, the suspension may also contain suitable stabilizers or agents that increase the solubility of the compound to allow for the preparation of highly concentrated solutions. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil or synthetic fatty acid esters, for example ethyl lipids or triglycerides, or liposomes.

The active ingredient may be embedded in microcapsules prepared, for example, by coacervation techniques or interfacial polymerization, such as hydroxymethylcellulose or gelatin microcapsules and poly (methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in macroemulsions. This technique is disclosed in Remington's Pharmaceutical Sciences (18 th edition, Mack Printing Company, 1990). Sustained release formulations can be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the polypeptide, which matrices are in the form of shaped articles, e.g., films, or microcapsules. In particular embodiments, prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate, gelatin or combinations thereof.

In addition to the compositions previously described, the T cell activating bispecific antigen binding molecule may also be formulated as a depot formulation. Such long acting formulations may be administered by implantation (e.g. subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the T cell activating bispecific antigen binding molecule may be formulated with suitable polymeric or hydrophobic materials (e.g. as an emulsion in an acceptable oil) or ion exchange resins or as sparingly soluble derivatives, e.g. as a sparingly soluble salt.

Pharmaceutical compositions comprising the T cell activating bispecific antigen binding molecules of the invention may be prepared by conventional mixing, dissolving, emulsifying, encapsulating, entrapping or lyophilizing processes. The pharmaceutical compositions may be formulated in conventional manner using one or more physiologically acceptable carriers, diluents, excipients or auxiliaries, which facilitate processing of the proteins into preparations which can be used pharmaceutically. The appropriate formulation will depend on the route of administration chosen.

The T cell activating bispecific antigen binding molecule may be formulated as a composition in free acid or base, neutral or salt form. Pharmaceutically acceptable salts are salts that substantially retain the biological activity of the free acid or base. These include acid addition salts, for example with the free amino groups of the protein composition, or salts formed with inorganic acids, for example hydrochloric or phosphoric acids, or organic acids such as acetic, oxalic, tartaric or mandelic acid. Salts formed from free carboxyl groups may also be derived from inorganic bases such as sodium hydroxide, potassium, ammonium, calcium or iron; or an organic base such as isopropylamine, trimethylamine, histidine or procaine. The drug salts have a higher solubility in aqueous solutions and other protic solvents than the corresponding free base forms.

Therapeutic methods and compositions

Any of the T cell activating bispecific antigen binding molecules provided herein can be used in a method of treatment. The T cell activating bispecific antigen binding molecules of the invention are useful as immunotherapeutic agents, e.g. for the treatment of cancer.

For use in a method of treatment, the T cell activating bispecific antigen binding molecules of the invention will be formulated, dosed and administered in a manner consistent with good medical practice. Factors to be considered in this context include the particular condition being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the condition, the site of delivery of the agent, the method of administration, the regimen of administration, and other factors known to practitioners.

In one aspect, there is provided a T cell activating bispecific antigen binding molecule of the invention for use as a medicament. In a further aspect, there is provided a T cell activating bispecific antigen binding molecule of the invention for use in the treatment of a disease. In certain embodiments, the T cell activating bispecific antigen binding molecules of the invention are provided for use in a method of treatment. In one embodiment, the invention provides a T cell activating bispecific antigen binding molecule as described herein for use in the treatment of a disease in an individual in need thereof. In certain embodiments, the present invention provides a T cell activating bispecific antigen binding molecule for use in a method of treating an individual having a disease, the method comprising administering to the individual a therapeutically effective amount of a T cell activating bispecific antigen binding molecule. In certain embodiments, the disease to be treated is a proliferative disease. In a particular embodiment, the disease is cancer. In certain embodiments, the method further comprises administering to the individual a therapeutically effective amount of at least one additional therapeutic agent, such as an anti-cancer agent (if the disease to be treated is cancer). In further embodiments, the invention provides the T cell activating bispecific antigen binding molecules described herein for use in inducing lysis of a target cell, in particular a tumor cell. In certain embodiments, the present invention provides a T cell activating bispecific antigen binding molecule for use in a method of inducing lysis of a target cell, in particular a tumor cell, in an individual, the method comprising administering to the individual an effective amount of the T cell activating bispecific antigen binding molecule to induce lysis of the target cell. An "individual" according to any of the above embodiments is a mammal, preferably a human.

In another aspect, the invention provides the use of a T cell activating bispecific antigen binding molecule of the invention in the manufacture or preparation of a medicament. In one embodiment, the medicament is for treating a disease in an individual in need thereof. In another embodiment, the medicament is for use in a method of treating a disease comprising administering to an individual having the disease a therapeutically effective amount of the medicament. In certain embodiments, the disease to be treated is a proliferative disease. In a particular embodiment, the disease is cancer. In one embodiment, the method further comprises administering to the individual a therapeutically effective amount of at least one additional therapeutic agent, such as an anti-cancer agent (if the disease to be treated is cancer). In another embodiment, the medicament is for inducing lysis of a target cell, particularly a tumor cell. In yet another embodiment, the medicament is for use in a method of inducing lysis of a target cell, particularly a tumor cell, in an individual, the method comprising administering to the individual an effective amount of the medicament to induce lysis of the target cell. An "individual" according to any of the above embodiments may be a mammal, preferably a human.

In another aspect, the invention provides methods of treating diseases. In one embodiment, the method comprises administering to an individual having such a disease a therapeutically effective amount of a T cell activating bispecific antigen binding molecule of the invention. In one embodiment, a composition comprising a T cell activating bispecific antigen binding molecule of the invention in a pharmaceutically acceptable form is administered to the individual. In certain embodiments, the disease to be treated is a proliferative disease. In a particular embodiment, the disease is cancer. In certain embodiments, the method further comprises administering to the individual a therapeutically effective amount of at least one additional therapeutic agent, such as an anti-cancer agent (if the disease to be treated is cancer). An "individual" according to any of the above embodiments may be a mammal, preferably a human.

In another aspect, the invention provides methods of inducing lysis of target cells, particularly tumor cells. In one embodiment, the method comprises contacting a target cell with a T cell activating bispecific antigen binding molecule of the invention in the presence of a T cell, in particular a cytotoxic T cell. In another aspect, methods for inducing lysis of a target cell, particularly a tumor cell, in an individual are provided. In one such embodiment, the method comprises administering to the individual an effective amount of a T cell activating bispecific antigen binding molecule to induce lysis of the target cells. In one embodiment, an "individual" is a human.

In certain embodiments, the disease to be treated is a proliferative disease, particularly cancer. Non-limiting examples of cancer include bladder cancer, brain cancer, head and neck cancer, pancreatic cancer, lung cancer, breast cancer, ovarian cancer, uterine cancer, cervical cancer, endometrial cancer, esophageal cancer, colon cancer, colorectal cancer, rectal cancer, gastric cancer, prostate cancer, blood cancer, skin cancer, squamous cell carcinoma, bone cancer, and kidney cancer. Other cell proliferative disorders that can be treated using the T cell activating bispecific antigen binding molecules of the invention include, but are not limited to, neoplasms located in the abdomen, bones, mammary glands, digestive system, liver, pancreas, peritoneum, endocrine glands (adrenal, parathyroid, pituitary, testis, ovary, thymus, thyroid), eye, head and neck, nervous system (central and peripheral nervous system), lymphatic system, pelvis, skin, soft tissues, spleen, thoracic region and urogenital system. Also included are precancerous conditions or lesions and cancer metastases. In certain embodiments, the cancer is selected from renal cell carcinoma, skin cancer, lung cancer, colorectal cancer, breast cancer, brain cancer, head and neck cancer. One skilled in the art will readily recognize that in many cases, T cell activating bispecific antigen binding molecules may not provide a cure, but may only provide partial benefit. In some embodiments, physiological changes with certain benefits are also considered therapeutically beneficial. Thus, in some embodiments, the amount of T cell activating bispecific antigen binding molecule that provides a physiological change is considered an "effective amount" or a "therapeutically effective amount". The subject, patient or individual in need of treatment is typically a mammal, more particularly a human.

In some embodiments, an effective amount of a T cell activating bispecific antigen binding molecule of the invention is administered to a cell. In other embodiments, a therapeutically effective amount of a T cell activating bispecific antigen binding molecule of the invention is administered to an individual to treat a disease.

For the prevention or treatment of disease, the appropriate dosage of the T cell activating bispecific antigen binding molecule of the invention (when used alone or in combination with one or more other additional therapeutic agents) will depend on the type of disease to be treated, the route of administration, the weight of the patient, the type of T cell activating bispecific antigen binding molecule, the severity and course of the disease, whether the T cell activating bispecific antigen binding molecule is for prophylactic or therapeutic purposes, previous or concurrent therapeutic interventions, the clinical history and response of the patient to the T cell activating bispecific antigen binding molecule, and the discretion of the attending physician. In any event, the practitioner responsible for administration will determine the concentration of the active ingredient in the composition and the appropriate dosage for the individual subject. Various dosing regimens are contemplated herein, including but not limited to single or multiple administrations over various time points, bolus administrations and pulsed infusions.

The T cell activating bispecific antigen binding molecule is suitably administered to the patient in one or a series of treatments. Depending on the type and severity of the disease, about 1 μ g/kg to 15mg/kg (e.g., 0.1mg/kg-10mg/kg) of the T cell activating bispecific antigen binding molecule may be the initial candidate dose for administration to the patient (whether by one or more separate administrations, or by continuous infusion). Depending on the factors mentioned above, a typical daily dose may be in the range of about 1. mu.g/kg to 100mg/kg or higher. For repeated administrations over several days or longer, depending on the condition, the treatment will generally continue until the desired suppression of disease symptoms occurs. An exemplary dose of T cell activating bispecific antigen binding molecule will range from about 0.005mg/kg to about 10 mg/kg. In other non-limiting examples, the dose may further comprise about 1 microgram/kg body weight, about 5 microgram/kg body weight, about 10 microgram/kg body weight, about 50 microgram/kg body weight, about 100 microgram/kg body weight, about 200 microgram/kg body weight, about 350 microgram/kg body weight, about 500 microgram/kg body weight, about 1 milligram/kg body weight, about 5 milligrams/kg body weight, about 10 milligrams/kg body weight, about 50 milligrams/kg body weight, about 100 milligrams/kg body weight, about 200 milligrams/kg body weight, about 350 milligrams/kg body weight, about 500 milligrams/kg body weight, to about 1000 milligrams/kg body weight or higher per administration, and any range derivable therein. In non-limiting examples of derivable ranges for the amounts listed herein, a range of about 5mg/kg body weight to about 100mg/kg body weight, about 5 μ g/kg body weight to about 500mg/kg body weight, and the like can be administered based on the amounts listed above. Thus, one or more doses of about 0.5mg/kg, 2.0mg/kg, 5.0mg/kg or 10mg/kg (or any combination thereof) may be administered to the patient. Such a dose may be administered intermittently, e.g., weekly or every three weeks (e.g., such that the patient receives about 2 to about 20, or, e.g., about 6 doses of the T cell activating bispecific antigen binding molecule). An initial higher loading dose may be administered followed by one or more lower doses. However, other dosage regimens may be useful. The progress of the therapy is readily monitored by conventional techniques and assays.

The T cell activating bispecific antigen binding molecules of the invention are typically used in an amount effective to achieve the intended purpose. For use in treating or preventing a disease condition, the T cell activating bispecific antigen binding molecules of the invention or pharmaceutical compositions thereof are administered or applied in a therapeutically effective amount. Determination of a therapeutically effective amount is within the ability of those skilled in the art, particularly in light of the detailed disclosure provided herein.

For systemic administration, the therapeutically effective dose can be estimated initially from in vitro assays (e.g., cell culture assays). The dose can then be formulated in animal models to achieve a circulating concentration range that includes the IC determined in cell culture₅₀. This information can be used to more accurately determine useful doses in humans.

Initial doses can also be estimated from in vivo data (e.g., animal models) using techniques well known in the art. Administration to humans can be readily optimized by one of ordinary skill in the art based on animal data.

The dosage and interval can be adjusted individually to provide plasma levels of the T cell activating bispecific antigen binding molecule sufficient to maintain a therapeutic effect. Typical patient doses for administration by injection are about 0.1 to 50 mg/kg/day, usually about 0.5 to 1 mg/kg/day. Therapeutically effective plasma levels can be achieved by administering multiple doses per day. The level in plasma can be measured, for example, by HPLC.

In the case of local administration or selective absorption, the effective local concentration of the T cell activating bispecific antigen binding molecule may not be related to the plasma concentration. One skilled in the art will be able to optimize therapeutically effective local dosages without undue experimentation.

A therapeutically effective dose of the T cell activating bispecific antigen binding molecules described herein will generally provide therapeutic benefit without causing substantial toxicity. Toxicity and therapeutic efficacy of T cell activating bispecific antigen binding molecules can be determined by standard pharmaceutical procedures in cell cultures or experimental animals. Cell culture assays and animal studies can be used to determine LD₅₀(dose lethal to 50% of the population) and ED₅₀(a therapeutically effective dose in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index, which can be expressed as LD₅₀/ED₅₀The ratio of (a) to (b). T cell activating bispecific antigen binding molecules exhibiting a large therapeutic index are preferred. In one embodiment, the T cell activating bispecific antigen binding molecule according to the invention exhibits a high therapeutic index. The data obtained from cell culture assays and animal studies can be used to formulate a range of dosages suitable for use in humans. The dose is preferably within the circulating concentration range, which includes ED with little or no toxicity ₅₀. The dosage may vary within this range depending on a variety of factors, such as the dosage form employed, the route of administration employed, the condition of the subject, and the like. The exact formulation, route of administration and dosage may be selected by The individual physician in view of The condition of The patient (see, e.g., Fingl et al, 1975, in: The Pharmacological Basis of Therapeutics, Ch.1, p.1, incorporated herein by reference in its entirety).

The attending physician of a patient treated with a T cell activating bispecific antigen binding molecule of the invention will know how and when to terminate, discontinue or adjust dosing due to toxicity, organ dysfunction, etc. Conversely, if the clinical response is inadequate (to rule out toxicity), the attending physician will also know to adjust the treatment to a higher level. The amount of administered dose in managing the disorder of interest will vary with the severity of the condition to be treated, the route of administration, and the like. The severity of the condition can be assessed, for example, in part, by standard prognostic evaluation methods. In addition, the dose and possibly the frequency of dosing will also vary according to the age, weight and response of the individual patient.

Other active agents and treatments

The T cell activating bispecific antigen binding molecules of the invention may be administered in combination with one or more other active agents in therapy. For example, the T cell activating bispecific antigen binding molecules of the invention may be co-administered with at least one additional therapeutic agent. The term "therapeutic agent" includes any therapeutic agent used to treat a symptom or disease in an individual in need of such treatment. Such additional therapeutic agents may comprise any active ingredient suitable for the particular indication being treated, preferably those having complementary activities that do not adversely affect each other. In certain embodiments, the additional therapeutic agent is an immunomodulator, a cytostatic agent, a cell adhesion inhibitor, a cytotoxic agent, an apoptosis activator, or a therapeutic agent that increases the sensitivity of a cell to an apoptosis-inducing agent. In a particular embodiment, the additional therapeutic agent is an anti-cancer agent, such as a microtubule disrupting agent, an anti-metabolite, a topoisomerase inhibitor, a DNA intercalating agent, an alkylating agent, a hormone therapy, a kinase inhibitor, a receptor antagonist, an activator of tumor cell apoptosis, or an anti-angiogenic agent.

Such other active agents are suitably present in combination in an amount effective for the intended purpose. The effective amount of these other agents will depend on the amount of T cell activating bispecific antigen binding molecule used, the type of disorder or treatment, and other factors discussed above. T cell activating bispecific antigen binding molecules are typically used at the same dosages and routes of administration as described herein or at dosages of about 1 to 99% as described herein, or at any dosage and any route that is empirically/clinically determined to be appropriate.

The above-described combination therapies include both combined administration (where two or more therapeutic agents are included in the same or separate compositions) and separate administration, in which case the administration of the T cell activating bispecific antigen binding molecules of the invention may occur prior to, simultaneously with and/or subsequently to the administration of additional therapeutic agents and/or adjuvants. The T cell activating bispecific antigen binding molecules of the invention may also be used in combination with radiotherapy.

Article of manufacture

In another aspect of the invention, articles of manufacture containing materials useful for the treatment, prevention and/or diagnosis of the above-mentioned conditions are provided. The article comprises a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, and the like. The container may be formed from a variety of materials such as glass or plastic. The container contains the composition, either by itself or in combination with another composition effective for treating, preventing and/or diagnosing a condition, and may have a sterile access port (e.g., the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). At least one active agent in the composition is a T cell activating bispecific antigen binding molecule of the invention. The label or package insert indicates that the composition is for use in treating the selected condition. Furthermore, the article of manufacture may comprise (a) a first container having a composition therein, wherein the composition comprises a T cell activating bispecific antigen binding molecule of the invention; and (b) a second container having a composition therein, wherein the composition comprises an additional cytotoxic agent or other therapeutic agent. The article of manufacture in this embodiment of the invention may further comprise a package insert indicating that the composition is useful for treating a particular condition. Alternatively or additionally, the article of manufacture may further comprise a second (or third) container comprising a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate buffered saline, ringer's solution, and dextrose solution. It may also include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles and syringes.

Screening method

Described herein is the advantageous efficiency of a method of using a single light chain or highly homologous variant thereof to identify suitable heavy chain variable regions to construct a T cell activating bispecific antigen binding molecule. To construct these binders, the light chain must not only be able to pair with the various heavy chains to produce binding moieties of different specificity, but also retain the ability to form binding moieties that can activate the T cells to which it binds.

The light chains described herein may be used as Common Light Chains (CLC) for identifying suitable heavy chain variable regions, for example by screening a library of heavy chain variable regions. This allows the retention of previously identified and validated CD3 binding agents such that only new target antigen binding agents need to be identified for the target antigen binding portion of the T cell activating bispecific antigen binding molecule.

Thus, in another aspect, the present invention provides a method for identifying the variable heavy chain of a bispecific antigen binding molecule specific for a T cell activation antigen and a target cell antigen, comprising the step of screening a combinatorial library comprising the variable heavy chain and a light chain comprising the amino acid sequences of SEQ ID NO 32, SEQ ID NO 33 and SEQ ID NO 34. In one embodiment, the light chain comprises the amino acid sequence of SEQ ID NO 31. In one embodiment, the light chain comprises the amino acid sequence of SEQ ID NO 35. This approach can be used to develop stable, functional high affinity binders to improve the production of T cell activating bispecific antigen binding molecules, e.g., having CD3 specificity and target antigen specificity, where the target antigen is irrelevant, e.g., FolR1, MUC1 and BCMA.

Examples

The following are examples of the methods and compositions of the present invention. It is to be understood that various other embodiments may be implemented in accordance with the general description provided above.

General procedure

Recombinant DNA technology

DNA is manipulated using standard methods, such as Sambrook et al, Molecular cloning: A laboratory manual; cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989. Molecular biological reagents were used according to the manufacturer's instructions. General information on the nucleotide Sequences of human immunoglobulin light and heavy chains is given in Kabat, E.A. et al, (1991) Sequences of Proteins of Immunological Interest,5^th ed.,NIH Publication No.91-3242。

DNA sequencing

DNA sequence was determined by standard double-stranded sequencing on Synergene (Schlieren).

Gene synthesis

If desired, the desired gene fragments are generated by PCR using suitable templates or are synthesized by Geneart AG (Regensburg, Germany) from synthetic oligonucleotides and PCR products by automated gene synthesis. Without the exact gene sequence, oligonucleotide primers were designed based on the sequence from the nearest homolog and the gene was isolated by RT-PCR from RNA derived from appropriate tissues. Gene segments flanked by single restriction endonuclease cleavage sites were cloned into standard cloning/sequencing vectors. Plasmid DNA was purified from transformed bacteria and the concentration was determined by UV spectroscopy. The DNA sequence of the subcloned gene fragments was confirmed by DNA sequencing. Gene segments with appropriate restriction sites were designed to allow subcloning into the corresponding expression vectors. All constructs were designed with a 5' DNA sequence encoding a leader peptide that targets the protein for secretion in eukaryotic cells.

Isolation of Primary human pan T cells from PBMCs

Peripheral Blood Mononuclear Cells (PBMCs) were prepared from enriched lymphocyte preparations (buffy coats) prepared from local blood banks or fresh blood from healthy human donors by Histopaque density centrifugation. Briefly, blood was diluted with sterile PBS and carefully layered on a Histopaque gradient (Sigma, H8889). After centrifugation at 450 Xg for 30 min at room temperature (brake off), the portion of plasma above PBMC containing the phases (interphase) was discarded. The PBMCs were transferred to a new 50ml Falcon tube and the tube was filled with PBS to a total volume of 50 ml. The mixture was centrifuged at 400 Xg for 10 minutes at room temperature (brake on). The supernatant was discarded and the PBMC pellet was washed twice with sterile PBS (centrifugation step at 350Xg for 10 min at 4 ℃). The resulting PBMC population (ViCell) was counted automatically and treated at 37 ℃ with 5% CO₂Was stored in RPMI1640 medium containing 10% FCS and 1% L-alanyl-L-glutamine (Biochrom, K0302) until the start of the assay.

Pan T was used according to the manufacturer's instructionsCell isolation kit II (Miltenyi Biotec # 130-. Briefly, the cell pellet was diluted every 1000 ten thousand cells into 40 μ l of cold buffer (PBS containing 0.5% BSA, 2mM EDTA, sterile filtered) and incubated with 10 μ l of biotin-antibody mixture per 1000 ten thousand cells for 10 minutes at 4 ℃. 30. mu.l of cold buffer and 20. mu.l of avidin beads were added per 1000 ten thousand cells and the mixture was incubated at 4 ℃ for a further 15 minutes. The cells were washed by adding 10-20 times the current volume and a subsequent centrifugation step at 300 Xg for 10 minutes. Up to 1 hundred million cells were resuspended in 500. mu.l buffer. Magnetic separation of unlabeled human pan T cells was performed using an LS column (Miltenyi Biotec # 130-. The resulting T cell population (ViCell) was counted automatically and at 37 ℃ with 5% CO ₂Until the start of the assay (no more than 24 hours).

Isolation of Primary human Primary T cells from PBMCs

Peripheral Blood Mononuclear Cells (PBMCs) were prepared from enriched lymphocyte preparations (buffy coats) prepared from local blood banks or fresh blood from healthy human donors by Histopaque density centrifugation. Initial CD8 from Miltenyi Biotec (#130-⁺T cell isolation kit (#130-093-244), T cell enrichment of PBMCs was performed according to the manufacturer's instructions, but CD8 was skipped⁺The last isolation step of T cells (see also the description of the isolation of primary human T cells).

Isolation of murine pan T cells from splenocytes

Spleens were isolated from C57BL/6 mice, transferred to GentlemACS C-Tube (Miltenyi Biotech # 130-. The cell suspension is passed through a pre-separation filter to remove remaining undissociated tissue particles. After centrifugation at 400 Xg for 4 minutes at 4 ℃ ACK lysis buffer was added to lyse the erythrocytes (5 min incubation at room temperature). The remaining cells were washed twice with MACS buffer, counted and used to isolate murine pan T cells. Negative (magnetic) selection was performed using the Pan T cell isolation kit from Miltenyi Biotec (# 130-. The resulting T cell population (ViCell) was counted automatically and immediately used for further assays.

Isolation of Primary cynomolgus PBMCs from heparinized blood

Peripheral Blood Mononuclear Cells (PBMC) were prepared by density centrifugation of fresh blood from healthy crab donors by diluting heparinized blood 1:3 with sterile PBS and diluting Lymphoprep medium (Axon Lab #1114545) to 90% with sterile PBS. Two volumes of diluted blood were layered on one volume dilution density gradient and PBMC fractions were isolated by centrifugation at 520 × g for 30 min at room temperature without braking. The PBMC bands were transferred to fresh 50ml Falcon tubes and washed with sterile PBS by centrifugation at 400 Xg for 10 min at 4 ℃. One low speed centrifugation was performed to remove platelets (15 min at 150 Xg, 4 ℃) and the resulting PBMC population (ViCell) was counted automatically and immediately used for further assays.

Exemplary antigen Generation

The antigen is expressed in two different forms. For non-Fc containing constructs, the extracellular domain is fused to an avi-His tag attached to its C-terminus. For Fc-containing antigens, Fc fusion to the N-terminus of the heterodimeric Fc portion using knob-in-hole technology (Merchant et al) was applied so that each Fc dimer has only one molecule of the protein of interest. This is done to avoid the formation of any artificial dimeric structure of the protein of interest. The avi tag here is attached to the C-terminus of the Fc. These proteins can be readily transiently expressed in mammalian cells such as HEK or CHO, purified by protein a chromatography, biotinylated, and can be attached to streptavidin beads for phage selection via biotinylated avi tags according to standard methods.

Affinity maturation

Affinity maturation of Fab fragments from the CLC library must be limited to the heavy chain only to keep the light chain in a constant way to retain CD3e binding affinity. Since our initial library was randomized only in CDR3 of the heavy chain, we focused on the maturation steps of CDR1 and 2. To this end, we designed PCR primers for each framework that introduced randomization in CDR1 and CDR2, respectively. Each primer was designed to bind to one of the six heavy chain frameworks and can be used in a universal fashion for each antibody clone from this particular phage library. The maturation process was according to Knappik or Steidl (Knappik et al, j.mol.biol. (2000)296, 57-86.) s.steidl et al; molecular Immunology 46(2008) 135-144). Phage panning was performed as described above, except that the concentration of soluble antigen was used at 2x10^ -8M and the final concentration was reduced to 2x10^ -10M.

Cloning, production, purification and Biochemical characterization of CLC TCB

The variable regions of the resulting heavy and light chain DNA sequences were subcloned using either constant heavy chains or constant light chains pre-inserted into the corresponding recipient mammalian expression vectors. Antibody expression is driven by the MPSV promoter and carries a synthetic polyA signal sequence at the 3' end of the CDS. In addition, each vector contained the EBV OriP sequence for transient expression in HEK293-EBNA cells. IgG 1P 329G LALA or IgG4 SPLE PG were used as antibody isotypes.

CLC TCB was produced by co-transfecting HEK293-EBNA cells with mammalian expression vectors using polyethylenimine. Cells were transfected with the corresponding expression vectors at a ratio of 1:1.3 ("vector heavy chain Fc (well)": vector heavy chain Fc (knob) -fabcussfab ": vector common light chain".

For transfection, HEK293 EBNA cells were cultured in CD CHO medium in suspension and serum free. For production in 500ml shake flasks, 4 hundred million HEK293 EBNA cells were inoculated 24 hours prior to transfection. For transfection, cells were centrifuged at 210 × g for 5 min, and the supernatant was replaced with pre-warmed 20ml CD CHO medium. The expression vector was mixed in 20ml of CD CHO medium to a final amount of 200g DNA. After addition of 540. mu.l PEI, the solution was vortexed for 15 seconds and then incubated at room temperature for 10 minutes. The cells were then mixed with the DNA/PEI solution, transferred to 500ml shake flasks and incubated for 3 hours at 37 ℃ in an incubator with an atmosphere of 5% CO 2. After incubation, 160ml of F17 medium was added and the cells were cultured for 24 hours. 1 day after transfection, 1mM valproic acid and 7% Charge 1 were added. After 7 days, the culture supernatant was collected, purified by centrifugation at 210 Xg for 15 minutes, the solution was sterile filtered (0.22 μm filter) and sodium azide was added to a final concentration of 0.01% w/v and maintained at 4 ℃.

Secreted proteins were purified from cell culture supernatants by affinity chromatography using ProteinA. The supernatant was loaded onto a HiTrap protein a HP column (CV 5mL, GE Healthcare) equilibrated with 40mL of 20mM sodium phosphate, 20mM sodium citrate, 0.5M sodium chloride, ph 7.5. Unbound protein was removed by washing with at least 10 column volumes of 20mM sodium phosphate, 20mM sodium citrate, 0.5M sodium chloride, pH 7.5. The target protein was eluted in a gradient from 20mM sodium citrate, 0.5M sodium chloride, pH7.5 to 20mM sodium citrate, 0.5M sodium chloride, pH2.5 in more than 20 column volumes. The protein solution was neutralized by the addition of 1/10 of 0.5M sodium phosphate (pH 8). The target protein was concentrated and filtered, then loaded onto a HiLoad Superdex 200 column (GE Healthcare) equilibrated with 20mM histidine, 140mM sodium chloride solution, pH 6.0.

The protein concentration of the purified protein sample was determined by measuring the Optical Density (OD) at 280nm by using the molar extinction coefficient calculated based on the amino acid sequence.

The purity and molecular weight of the molecules were analyzed by CE-SDS analysis in the presence and absence of reducing agents. The Caliper LabChip gxi system (Caliper Lifescience) was used according to the manufacturer's instructions. 2ug of sample was used for analysis.

Size exclusion columns (Tosoh) were analyzed using TSKgel G3000SW XL to analyze the aggregate content of antibody samples at 25mM K2HPO4,125mM NaCl,200mM L-arginine monohydrochloride, 0.02% (w/v) NaN3, pH 6.7 running buffer at 25 ℃.

CLC TCB characterization in cell-based assays

Binding of CLC TCB to ECD tumor antigen and CD3 expressing cells

CLC TCB binding was tested using tumor cells expressing antigen X of interest and an immortalized T lymphocyte cell line (Jurkat) expressing CD3 e. Briefly, cells were collected, counted, checked for viability, and tested at 2 × 10⁶Individual cells/ml were resuspended in FACS buffer (100. mu.l PBS 0.1% BSA). Mu.l of cell suspension (containing 0.2X 10)⁶Cells) inIncubation at 4 ℃ for 30 min in round bottom 96-well plates with increasing concentrations of CLC TCB (3pM-200nM), washing twice with cold PBS 0.1% BSA, further incubation at 4 ℃ with PE conjugated AffiniPure F (ab') 2 fragment goat anti-human IgG Fcg fragment specific secondary antibody (Jackson Immuno Research Lab PE # 109-. Binding curves were obtained using GraphPadPrism 5.

Example 1

Purification of biotinylated folate receptor-Fc fusions

To generate new antibodies against human FolR1, the following antigens and screening tools were generated as monovalent Fc fusion proteins (extracellular domain of antigen linked to the hinge region of Fc-knob, which is co-expressed with Fc-knob molecule). Antigenic genes (Geneart, Regensburg, Germany) were synthesized based on sequences obtained from GenBank or SwissProt and inserted into expression vectors to generate fusion proteins with Fc-knobs with C-terminal Avi markers for biotinylation in vivo or in vitro. In vivo biotinylation was achieved by co-expression of the bacterial birA gene encoding bacterial biotin ligase during production. Expression of all genes was performed under the control of the chimeric MPSV promoter on a plasmid containing the oriP element for stable maintenance of the plasmid in EBNA-containing cell lines.

To prepare biotinylated monomeric antigen/Fc fusion molecules, exponentially growing suspension HEK293 EBNA cells were co-transfected with three vectors encoding the two components of the fusion protein (knob and pore chain) and the enzyme BirA necessary for the biotinylation reaction. The corresponding vectors were used at a ratio of 9.5:9.5:1 ("antigen ECD-Fc knob-avi label": "Fc well": "BirA").

For protein production in 500ml shake flasks, 4 hundred million HEK293 EBNA cells were seeded 24 hours prior to transfection. For transfection, cells were centrifuged at 210g for 5 minutes and the supernatant was replaced with pre-warmed CD CHO medium. The expression vector was resuspended in 20mL of CD CHO medium containing 200. mu.g of vector DNA. After addition of 540. mu.L of Polyethyleneimine (PEI), the solution was mixed for 15 seconds and incubated at room temperatureFor 10 minutes. The cells were then mixed with DNA/PEI solution, transferred to 500mL shake flasks and incubated with 5% CO₂The incubation was carried out at 37 ℃ for 3 hours in an atmosphere incubator. After incubation, 160mL F17 medium was added and the cells were cultured for 24 hours. 1 day after transfection, 1mM valproic acid and 7% feed 1(Lonza) were added to the culture. The production medium was also supplemented with 100. mu.M biotin. After 7 days of culture, cell supernatants were collected by centrifugation of the cells at 210g for 15 minutes. The solution was sterile filtered (0.22 μm filter), supplemented with sodium azide to a final concentration of 0.01% (w/v), and maintained at 4 ℃.

Secreted proteins were purified from cell culture supernatants by affinity chromatography using protein a, followed by size exclusion chromatography. For affinity chromatography, the supernatant was loaded on a HiTrap protein a HP column (CV 5mL, GE Healthcare) equilibrated with 40mL of 20mM sodium phosphate, 20mM sodium citrate, ph 7.5. Unbound protein was removed by washing with at least 10 column volumes of 20mM sodium phosphate, 20mM sodium citrate pH 7.5. Bound protein was eluted using a linear pH gradient generated over 20 column volumes of 20mM sodium citrate, 100mM sodium chloride, 100mM glycine, pH 3.0. The column was then washed with 10 column volumes of 20mM sodium citrate, 100mM sodium chloride, 100mM glycine, ph 3.0.

The pH of the collected fractions was adjusted by the addition of 1/10(v/v)0.5M sodium phosphate, pH 8.0. The protein was concentrated and filtered, then loaded onto a HiLoad Superdex 200 column (GE Healthcare) equilibrated with 20mM histidine, 140mM sodium chloride, pH 6.0.

The protein concentration was determined by measuring the Optical Density (OD) at 280nm by using the molar extinction coefficient calculated based on the amino acid sequence. The purity and molecular weight of the FolR 1-Fc-fusion were analyzed by SDS capillary electrophoresis in the presence and absence of reducing agents according to the manufacturer's instructions (Instrument Caliper LabChipGX, Perkin Elmer). The samples were analyzed for aggregate content using a TSKgel G3000 SW XL analytical size exclusion column (Tosoh) equilibrated at 25 ℃ in 25mM K2HPO4,125mM NaCl,200mM L-arginine monohydrochloride, 0.02% (w/v) NaN3, pH 6.7 running buffer.

The purified antigen-Fc-fusion protein was analyzed by surface plasmon resonance assay using commercially available antibodies to confirm the correct and natural conformation of the antigen (data not shown).

TABLE 1 antigens generated by isolation, selection and counter-selection of human FolR1 antibodies

TABLE 2 summary of yield and final monomer content of FolR-Fc fusions

Example 2

Generation of a common light chain with specificity for CD3 epsilon

The T cell activating bispecific molecules described herein comprise at least one CD3 binding moiety. The moiety may be generated by immunizing an experimental animal, screening a phage library or using a known anti-CD 3 antibody. A common light chain with specificity for CD3 epsilon was generated by humanizing the light chain of the mouse parent anti-CD 3 epsilon antibody (CH 2527). For humanization of non-human antibodies, CDR residues from the non-human antibody (donor) must be grafted onto the framework of the human (acceptor) antibody. Typically, acceptor framework sequences are selected by aligning the sequence of the donor with a collection of potential acceptor sequences and selecting acceptor sequences that have reasonable homology to the donor or exhibit similar amino acids at certain positions critical to structure and activity. In the present case, a search for antibody acceptor frameworks is performed by aligning the mouse VL-domain sequence of the parent antibody with a collection of human germline sequences and selecting human sequences showing high sequence identity. Surprisingly, a good match in framework sequence homology was found in the rather rare human light chain belonging to V domain family 7 of the lambda type, more precisely in hVL _7_46(IMGT nomenclature, GenBank Acc No. z73674). This rare human light chain was then selected as the acceptor framework for humanization of the CH2527 light chain. Three Complementarity Determining Regions (CDRs) of the mouse light chain variable domain were grafted onto the acceptor framework. Since the framework 4 region is not part of the variable region of the germline V gene, alignment of this region (J-element) was performed separately. Thus, the IGLJ3-02 sequence was selected for humanization of the light chain.

13 humanized variants were generated (CH2527-VL7_46-1 to VL7_46-10, VL7_46-12 to VL7_ 46-14). These variants differ in framework residues (and combinations thereof) that are back-mutated to the murine V-domain sequence, or in CDR residues (Kabat definition) that may remain the same as the human germline sequence. The following framework residues outside the CDRs were back-mutated in the final humanized VL domain variant VL7_46-13 to murine residues (murine residues are listed), V36, E38, F44, G46, G49 and G57. Human J-element IGLJ3-02 is 100% identical to the J-element of the murine parent antibody.

Example 3

SPR assessment of humanized variants with specificity for CD3 epsilon

The humanized VL variants were evaluated as chimeras in the 2+1 canonical form (fig. 1D), i.e., the humanized light chain V-domain paired with the murine heavy chain V-domain. SPR assessment was performed on a ProteOn XPR36 instrument (Bio-Rad). More precisely, in vertical orientation, variants were captured directly from the culture supernatant on an Anti-Fab derivatized GLM sensor chip ((coat Anti-Human IgG, F (ab') 2 Fragment specificity, Jackson ImmunoResearch.) the following analytes were then injected at single concentration levels in order to assess binding to Human and cynomolgus monkey CD3 epsilon: 3 μ M hu CD3 epsilon (-1-26) -fc (knob) -Avi (ID807) and 2.5 μ M cy CD3 epsilon- (-1-26) -fc (knob) -Avi-fc (Avi) Avi-fc (Avi 873) the binding reactions to the murine control construct were qualitatively compared and graded + (comparable binding observed), +/- (reduced binding observed) and- (no binding observed) — capture antibodies were regenerated after each cycle of ligand capture and analyte binding, and re-injection of the mouse construct at the end of the study to confirm the activity of the capture surface. The results are summarized in table 3.

Humanized VL variants	Binding of CD3 epsilon
		murine_CH2527-VL	+
CH2527-VL7_46-1	-
		CH2527-VL7_46-2	-
CH2527-VL7_46-3	-
		CH2527-VL7_46-4	-
CH2527-VL7_46-5	-
		CH2527-VL7_46-6	-
CH2527-VL7_46-7	-
		CH2527-VL7_46-8	-
CH2527-VL7_46-9	-
		CH2527-VL7_46-10	-
CH2527-VL7_46-12	+/-
		CH2527-VL7_46-13	+
CH2527-VL7_46-14	-

Table 3 qualitative binding assessment of humanized light chain variants in combination with murine heavy chain of CH2527 based on SPR. Only the finally selected humanized light chain variant CH2527-VL7_46-13 is highlighted in bold font, showing comparable binding to human and cynomolgus monkey CD3 epsilon.

Example 4

Properties of humanized common light chain with specificity for CD3 epsilon

The light chain V-domain variant selected for the humanized leader molecule was VL 7-46-13. The degree of humanization, i.e., the sequence homology of the humanized V-domain to the human germline V-domain sequence, was determined. For VL7_46-13, the overall sequence identity to the closest human germline homologue before humanization was 65% and 80% after humanization. The CDR regions were omitted and the sequence identity with the closest human germline homologue was 92%. As can be seen from table 3, VL7_46-13 is the only humanized VL variant in the set of 13 variants that showed comparable binding to the parent murine antibody and also retained its cross-reactivity to cynomolgus monkey CD3 epsilon. This result indicates that the humanized murine VL domain is not trivial without losing binding affinity to CD3 epsilon, requiring some back mutations in murine framework residues (particularly G46) while retaining G24 in CD 1. Furthermore, the results indicate that the VL domain plays a key role in target recognition. Importantly, the humanized VL domain VL7_46-13, which is based on the rare human germline belonging to V domain family 7 of the lambda type and retains affinity and specificity for CD3 epsilon, is also suitable for use as a common light chain in a phage-displayed antibody library in Fab-format, and enables successful selection of new specificities, which greatly facilitates the production and production of bispecific molecules that bind CD3 epsilon and e.g. tumor targets and share the same "common" light chain.

Example 5

Generation of phage display antibody libraries using human germline common light chains derived from HVK1-39

Several methods of generating bispecific antibodies similar to full length human IgG utilize modifications in the Fc region that induce heterodimerization of two different heavy chains. Examples include knob access holes (Merchant et al, Nat Biotechnol.1998Jul; 16(7):677-81), SEED (Davis et al, Protein Eng Des Sel.2010Apr; 23(4): 195) 202) and electrostatic steering technology (Gunasekaran et al, J Biol chem.2010Jun 18; 285(25): 19637-46). Although these methods are effective in heterodimerizing two different heavy chains, proper pairing of the cognate light and heavy chains remains a problem. The use of a common Light Chain (LC) can solve this problem (Merchant, et al. Nat Biotech 16, 677-.

Here, we describe the generation of antibody libraries displayed on M13 phage. In essence, we designed a multi-framework library for heavy chains with one constant (or "common") light chain. The library is designed for the generation of multispecific antibodies without the need to use complex techniques to avoid light chain mismatches.

By using a common light chain, the production of these molecules can be facilitated, since no more mismatches occur, and the isolation of high purity bispecific antibodies is facilitated. Compared to other formats, the use of Fab fragments as building blocks rather than, for example, scFv fragments, leads to higher thermostability and lack of scFv aggregation and intermolecular scFv formation.

Library generation

The generation of antibody libraries for display on M13 phage is described below. Basically, we designed a multi-framework library for heavy chains and a constant (or "common") light chain.

We used these heavy chains in the library (GenBank accession numbers in parentheses):

IGHV1-46*01(X92343)(SEQ ID NO:104),

IGHV1-69*06(L22583),(SEQ ID NO:105)

IGHV3-15*01(X92216),(SEQ ID NO:106)

IGHV3-23*01(M99660),(SEQ ID NO:107)

IGHV4-59*01(AB019438),(SEQ ID NO:108)

IGHV5-51*01(M99686),(SEQ ID NO:109)

all heavy chains used IGHJ2 as J elements, except IGHV1-69 x 06 using the IGHJ6 sequence. Randomized designs include CDR-H1, CDR-H2, and CDR-H3. For CDR-H1 and CDR-H2, a "soft" randomization strategy was chosen, randomizing the oligonucleotides so that codons for amino acids of the germline sequence are present at 50%. With the exception of cysteine, all other amino acids make up the remaining 50%. In CDR-H3, where no germline amino acid is present due to the presence of a genetic D element, oligonucleotides were designed that allow the use of random inserts between the V element and the J element that are 4 to 9 amino acids in length. Those oligonucleotides comprised in their randomized part, e.g. the three amino acids G/Y/S are each present at 15%, those amino acids A/D/T/R/P/L/V/N/W/F/I/E are each present at 4, 6%.

In Hoogenboom et al, Nucleic Acids Res.1991,19, 4133-; an exemplary method for generating antibody libraries is described in Lee et., al J.mol.biol. (2004)340, 1073-. The light chain is derived from human sequence hVK1-39 and is used in an unmodified and non-randomized fashion. This will ensure that the same light chain can be used for other projects without additional modification.

Exemplary library selection:

selection of all affinity maturation libraries was performed in solution according to the following procedure using monomers of the target antigen X and biotinylated extracellular domain.

1. 10^12 phagemid particles per library bound 100nM biotinylated soluble antigen for 0.5 hours in a total volume of 1 ml. 2. Biotinylated antigen was captured and specifically bound phage particles were isolated by adding approximately-5X 10^7 streptavidin coated magnetic beads for 10 minutes. 3. The beads were washed with 5-10X 1ml PBS/Tween20 and 5-10X 1ml PBS. 4. Elution of the phage particles was performed by adding 1ml 100mM TEA (triethylamine) for 10 min and neutralization by adding 500ul 1M Tris/HCl pH 7.4, and 5. exponentially growing E.coli TG1 bacteria were reinfected, infected with the helper phage VCSM13 and applied to subsequent PEG/NaCl precipitation of the phage particles in a subsequent selection round. Selection was performed for 3-5 rounds using constant or decreasing (from 10-7M to 2X 10-9M) antigen concentrations. In round 2, capture of antigen/phage complexes was performed using neutravidin (neutravidin) plates instead of streptavidin beads. All binding reactions were supplemented with 100nM bovine serum albumin or nonfat dry milk in order to compete for unwanted clones resulting from pure adhesive binding of the antibody to the plastic support.

Three or four rounds of selection were performed using decreasing antigen concentrations starting at 100nM and decreasing to 5nM in the last round of selection. Specific binders were defined as approximately 5-fold higher signal than background and identified by ELISA. Specific binders were identified by ELISA by coating 100. mu.l biotinylated antigen at 10nM per well on neutravidin plates. Fab-containing bacterial supernatants were added and bound Fab was detected by its Flag label using anti-Flag/HRP secondary antibody. ELISA positive clones were expressed by bacteria as soluble Fab fragments in 96-well format and supernatants were subjected to kinetic screening experiments by SPR analysis using ProteOn XPR36 (BioRad). Clones expressing the Fab with the highest affinity constant were identified and the corresponding phagemids were sequenced. For further characterization, Fab sequences were amplified from phagemids by PCR and cloned into the human IgG1 expression vector for mammalian production via appropriate restriction sites.

Generation of phage-displayed antibody libraries using humanized CD3 epsilon-specific common light chains

Described herein is the generation of antibody libraries for display on the M13 phage. Basically, we designed a multi-framework library for heavy chains and a constant (or "common") light chain. The library was designed to generate Fc-containing but FcgR-binding inactive T cell bispecific antibodies of the IgG 1P 329G LALA or IgG4 SPLE PG isotype, in which one or both fabs recognize tumor surface antigens expressed on tumor cells, while the remaining Fab arms of the antibody recognize CD3e on T cells.

Library generation

The generation of antibody libraries for display on M13 phage is described below. Basically, we designed a multi-framework library for heavy chains and a constant (or "common") light chain. This library was designed only for the generation of Fc-containing but FcgR-binding inactive T cell bispecific antibodies of the IgG 1P 329G LALA or IgG4 SPLE PG isotype.

Diversity was introduced only in the CDR3 of the different heavy chains by randomizing the oligonucleotides. Methods for generating antibody libraries are well known in the art and are described in (Hoogenboom et al, Nucleic Acids Res.1991,19, 4133-109413; or in Lee et., al J.mol.biol. (2004)340, 1073-1093).

We used these heavy chains in the library:

IGHV1-46*01(X92343),(SEQ ID NO:104)

IGHV1-69*06(L22583),(SEQ ID NO:105)

IGHV3-15*01(X92216),(SEQ ID NO:106)

IGHV3-23*01(M99660),(SEQ ID NO:107)

IGHV4-59*01(AB019438),(SEQ ID NO:108)

IGHV5-51*01(M99686),(SEQ ID NO:109)

we used light chains derived from humanized human and cynomolgus monkey CD3 epsilon specific antibody CH2527 in the library (VL 7-46-13; SEQ ID NO: 112). The light chain was not randomized and used without any further modification to ensure compatibility with different bispecific binders.

All heavy chains used IGHJ2 as the J element, except IGHV1-69 x 06 using the IGHJ6 sequence. Randomized design focused only on CDR-H3, a PCR oligonucleotide was designed that allowed the use of random inserts between the V element and the J element that were 4 to 9 amino acids in length.

Example 6

Selection of antibody fragments from a common light chain library (comprising light chains with specificity of CD3 epsilon) against FolR1

Antibodies 16A3,15a1,18D3,19E5,19a4,15H7,15B6,16D5,15E12,21D1,16F12,21a5,21G8,19H3,20G6 and 20H7 comprising a common light chain VL7_46-13 with CD3 epsilon specificity were obtained by phage display selection of different species (human, cynomolgus monkey and mouse) of FolR 1. Clones 16A3,15a1,18D3,19E5,19a4,15H7,15B6,21D1,16F12,19H3,20G6 and 20H7 were selected from subfluids in which the common light chain was paired with a human germline VH1_46 based heavy chain repertoire. In this sub-library, CDR3 of VH1_46 has been randomized based on 6 different CDR3 lengths. Clones 16D5,15E12,21a5 and 21G8 were selected from a sub-library in which the common light chain was paired with a human germline VH3_15 based heavy chain repertoire. In this sub-library, CDR3 of VH3_15 has been randomized based on 6 different CDR3 lengths. To obtain species cross-reactive (or murine FolR 1-reactive) antibodies, different species of FolR1 were alternated (or held constant) in different ways in the following 3 rounds of biopanning 16A3 and 15a1 (human-cynomolgus-human FolR 1); 18D3 (cynomolgus monkey-human-mouse FolR 1); 19E5 and 19a4 (3 rounds on mouse FolR 1); 15H7,15B6,16D5,15E12,21D1,16F12,21a5,21G8 (human-cynomolgus-human FolR 1); 19H3,20G6 and 20H7 (3 rounds on mouse FolR 1).

As a human for phage display selection and ELISA and SPR-based screening of antigens, mice and cynomolgus monkey FolR1 were transiently expressed as N-terminal monomeric Fc-fusions in HEK EBNA cells and site-specifically biotinylated in vivo by co-expressing the BirA biotin ligase at the avi tag recognition sequence located at the C-terminus of the Fc portion carrying the receptor chain (Fc knob chain). To assess specificity for FolR1, two related receptors, human FolR2 and FolR3, were generated in the same manner.

Selection rounds (biopanning) were performed in solution according to the following pattern:

1. pre-clearing on 10. mu.g/ml irrelevant human IgG coated maxisorp plates～10¹²Phagemid particles to consume the antibody library recognizing the Fc portion of the antigen.

2. non-Fc binding phagemid particles were incubated with 100nM biotinylated human, cynomolgus monkey or mouse FolR1 for 0.5 hours in the presence of 100nM of an unrelated, non-biotinylated Fc knob-into-hole construct to further deplete the Fc-binding agent in a total volume of 1 ml.

3. Biotinylated FolR1 and attached specifically binding phage were captured by transfer to 4 wells of a neutravidin pre-coated microtiter plate for 10 minutes (in rounds 1 and 3).

4. Wells were washed with 5x PBS/Tween20 and 5x PBS.

5. The phage particles were eluted by adding 250. mu.l of 100mM TEA (triethylamine) per well for 10 minutes and neutralized by adding 500ul of 1M Tris/HCl pH 7.4 to the pooled eluates from 4 wells.

6. Post-clearing of the neutralized eluate was performed by incubation on neutravidin pre-coated microtiter plates containing 100nM biotin-captured FolR2 or FolR3 for final removal of Fc binding agents and non-specific binding agents.

7. Coli TG1 cells were reinfected in log phase with the supernatant of the eluted phage particles, infected with the helper phage VCSM13, incubated overnight on a shaker at 30 ℃, and then PEG/NaCl precipitated the phagemid particles to be used in the next selection round.

3 rounds of selection were performed using a constant antigen concentration of 100 nM. In round 2, to avoid enrichment of the neutravidin binding agent, the concentration was adjusted by adding 5.4X 10⁷A streptavidin-coated magnetic bead was used to capture the antigen-phage complex. Specific binders were identified by ELISA by coating 100ul of 25nM biotinylated human, cynomolgus or mouse FolR1 and 10. mu.g/ml human IgG on neutral avidin and maxisorp plates, respectively. Fab-containing bacterial supernatants were added and bound fabs were detected by their Flag labels using anti-Flag/HRP secondary antibodies. Clones showing signal on human FolR1 and negative for human IgG were pooled for further analysis, and the remaining two FolR1 were pooled in a similar manner The test was performed. They were expressed by bacteria in 0.5 liter culture volumes, affinity purified and further characterized by SPR analysis using the ProteOn XPR36 biosensor from BioRad.

The affinity of selected clones was measured by Surface Plasmon Resonance (SPR) using a ProteOn XPR36 instrument (Biorad) to capture human FolR2 and FolR3 immobilized on NLC chips with biotinylated human, cynomolgus monkey and mouse FolR1 and neutravidin (negative control) at 25 ℃ (K_D). Immobilization of antigen (ligand) recombinant antigen was diluted to 10. mu.g/ml with PBST (10mM phosphate, 150mM sodium chloride, pH 7.4, 0.005% Tween 20) and then injected at 30. mu.l/min in the vertical direction. Injection of analyte for "one-shot kinetics" measurements, the direction of injection was changed to horizontal, and a two-fold dilution series (different concentration ranges) of purified Fab was injected simultaneously along separate channels 1-5, with an association time of 200 seconds and a dissociation time of 600 seconds. Buffer (PBST) was injected along the sixth channel to provide an "in-line" blank for reference. The association rate constant (k) was calculated in the ProteOn Manager v3.1 software using a simple one-to-one Langmuir binding model by simultaneous fitting of the association and dissociation sensorgrams _on) And dissociation rate constant (k)_off). Equilibrium dissociation constant (K)_D) Is calculated as k_off/k_onAnd (4) the ratio. Table 4 lists the equilibrium dissociation constants (K) for selected clones specific for FolR1_D)。

Table 4: the equilibrium dissociation constant (KD) of an anti-FolR 1 antibody (Fab-form) selected by phage display from a common light chain sublibrary comprising a humanized light chain VL7_46-13 specific for CD3 epsilon. KD is in nM.

Clone	huFolR1[nM]	cyFolR1[nM]	muFolR1[nM]	huFolR2[nM]	huFolR3[nM]
						16A3	21.7	18	very weak	no binding	no binding
15A1	30.9	17.3	very weak	no binding	no binding
						18D3	93.6	40.2	very weak	no binding	no binding
19E5	522	276	19.4	no binding	no binding
						19A4	2050	4250	43.1	no binding	no binding
15H7	13.4	72.5	no binding	no binding	no binding
						15B6	19.1	13.9	no binding	no binding	no binding
16D5	39.5	114	no binding	no binding	no binding
						15E12	55.7	137	no binding	no binding	no binding
21D1	62.6	32.1	no binding	no binding	no binding
						16F12	68	90.9	no binding	no binding	no binding
21A5	68.8	131	no binding	no binding	no binding
						21G8	130	261	no binding	no binding	no binding
19H3	no binding	no binding	89.7	no binding	no binding
						20G6	no binding	no binding	78.5	no binding	no binding

Example 7

Selection of antibody fragments from a universal multi-framework library against FolR1

Antibodies 11F8, 36F2, 9D11, 5D9, 6B6 and 14E4 were obtained by phage display selection based on a universal multi-framework sublibrary of FolR1 for different species (human, cynomolgus monkey and mouse). In these multi-framework sublibraries, different VL domains with randomized CDRs 3(3 different lengths) were paired with different VH domains with randomized CDRs 3(6 different lengths). Selected clones had the following VL/VH pairing: 11F8(Vk _1_5/VH _1_69), 36F2(Vk _3_20/VH _1_46), 9D11(Vk2D _28/VH1_46), 5D9(Vk3_20/VH1_46), 6B6(Vk3_20/VH1_46), and 14E4(Vk3_20/VH3_ 23). To obtain species cross-reactive (or murine FolR 1-reactive) antibodies, FolR1 of different species were alternated (or held constant) in different ways by 3 or 4 rounds of biopanning: 11F8 (cynomolgus monkey-mouse-human FolR 1); 36F2 (human-mouse-cynomolgus monkey-mouse FolR 1); 9D11 (cynomolgus monkey-human-cynomolgus monkey FolR 1); 5D9 (human-cynomolgus monkey-human FolR 1); 6B6 (human-cynomolgus monkey-human FolR1) and 14E4 (3 rounds against mouse FolR 1).

1. pre-clearing on 10. mu.g/ml irrelevant human IgG-coated maxisorp plates-10¹²Phagemid particles to consume the antibody library recognizing the Fc portion of the antigen.

4. Wells were washed with 5x PBS/Tween20 and 5x PBS.

A constant antigen concentration of 100nM was used for 3 rounds of selection. In rounds 2 and 4, to avoid enrichment with neutravidin binders, the concentration of the neutral avidin binders was adjusted by adding 5.4X 10⁷Streptavidin-coated magnetic beads were used for antigen-phage complex capture. Specific binders were identified by ELISA by coating 100ul of 25nM biotinylated human, cynomolgus or mouse FolR1 and 10. mu.g/ml human IgG on a neutravidin plate and a maxisorp plate, respectively. Fab-containing bacterial supernatants were added and bound fabs were detected by their Flag labels using anti-Flag/HRP secondary antibodies. Clones showing signal on human FolR1 and negative for human IgG were included for further analysis and the remaining two FolR1 were tested in a similar manner. They were expressed by bacteria in a culture volume of 0.5 liter, affinity purified and Further characterization was by SPR analysis using the ProteOn XPR36 biosensor from BioRad.

The affinity of selected clones was measured by Surface Plasmon Resonance (SPR) using a ProteOn XPR36 instrument (Biorad) to capture human FolR2 and FolR3 immobilized on NLC chips with biotinylated human, cynomolgus monkey and mouse FolR1 and neutravidin (negative control) at 25 ℃ (K_D). Immobilization of antigen (ligand) recombinant antigen was diluted to 10. mu.g/ml with PBST (10mM phosphate, 150mM sodium chloride, pH 7.4, 0.005% Tween 20) and then injected at 30. mu.l/min in the vertical direction. Injection of analyte for "one-shot kinetics" measurements, the direction of injection was changed to horizontal, and a two-fold dilution series (different concentration ranges) of purified Fab was injected simultaneously along separate channels 1-5 with an association time of 150 or 200 seconds, respectively, and a dissociation time of 200 or 600 seconds, respectively. Buffer (PBST) was injected along the sixth channel to provide an "in-line" blank for reference. The association rate constant (k) was calculated in the ProteOn Manager v3.1 software using a simple one-to-one Langmuir binding model by simultaneous fitting of the association and dissociation sensorgrams_on) And dissociation rate constant (k)_off). Equilibrium dissociation constant (K) _D) Is calculated as k_off/k_onAnd (4) a ratio. Table 5 lists the equilibrium dissociation constants (K) for selected clones specific for FolR1_D)。

TABLE 5 equilibrium dissociation constant (K) of anti-FolR 1 antibodies (Fab-form) selected by phage display from the Universal Multi-framework library_D)。K_DIn nM.

Example 8

Production and purification of novel FolR1 binding agents in IgG and T cell bispecific format

To identify FolR1 binding agents capable of inducing T cell-dependent killing of selected target cells, antibodies isolated from a common light chain or Fab library were converted to the corresponding human IgG1 format. Briefly, variable heavy and variable light chains from phage-displayed unique FolR1 binding agents were amplified by standard PCR reactions using Fab clones as templates. The PCR products were purified and inserted (by restriction endonuclease and ligase-based cloning, or by "recombination" using the InFusion kit from Invitrogen) into suitable expression vectors where they were fused to appropriate human constant heavy or constant light chains. The expression cassette in these vectors consists of a chimeric MPSV promoter and a synthetic polyadenylation site. In addition, the plasmid contains the oriP region from the EB virus for stable maintenance of the plasmid in HEK293 cells carrying the EBV nuclear antigen (EBNA). Following PEI mediated transfection, antibodies were transiently produced in HEK293 EBNA cells and purified by standard protein a affinity chromatography followed by size exclusion chromatography as follows:

Transient transfection and production

All (bispecific) antibodies used herein were transiently produced in HEK293 EBNA cells (if not obtained from commercial sources) using the PEI mediated transfection method of the required vector as described below. HEK293 EBNA cells were cultured in CD CHO medium in suspension without serum. For production in 500ml shake flasks, 4 hundred million HEK293 EBNA cells were seeded 24 hours prior to transfection (for alternate scales, all amounts were adjusted accordingly). For transfection, cells were centrifuged at 210 × g for 5 min, and the supernatant was replaced with pre-warmed 20ml CD CHO medium. The expression vector was mixed in 20ml of CD CHO medium to a final amount of 200. mu.g DNA. After addition of 540. mu.l PEI, the solution was vortexed for 15 seconds and subsequently incubated at room temperature for 10 minutes. The cells were then mixed with the DNA/PEI solution, transferred to 500ml shake flasks and incubated for 3 hours at 37 ℃ in an incubator with an atmosphere of 5% CO 2. After incubation, 160ml of F17 medium was added and the cells were cultured for 24 hours. 1 day after transfection, 1mM valproic acid and 7% feed 1 were added. After 7 days, the supernatant was collected, purified by centrifugation at 210 Xg for 15 minutes, the solution was sterile filtered (0.22 μm filter) and sodium azide was added to a final concentration of 0.01% w/v and maintained at 4 ℃. After production, the supernatant was collected, and the antibody-containing supernatant was filtered through a 0.22 μm sterile filter and stored at 4 ℃ until purification.

Antibody purification

Two-step purification of all molecules using standard methods, e.g.protein A affinity purification: (Explorer) and size exclusion chromatography. The supernatant obtained from the transient production was adjusted to pH8.0 (using 2M TRIS pH 8.0) and applied to HiTrap PA FF (GE Healthcare, column volume (cv) ═ 5ml) equilibrated with 8 column volumes (cv) of buffer a (20mM sodium phosphate, 20mM sodium citrate, pH 7.5). After washing with 10cv buffer A, the protein was eluted in 12cv to buffer B (20mM sodium citrate pH3,100mM NaCl, 100mM glycine) using a pH gradient. Fractions containing the protein of interest were combined and the pH of the solution was gently adjusted to pH6.0 (using 0.5M Na)₂HPO₄pH 8.0)。

The samples were concentrated to 2ml using a super concentrator (Vivaspin 15R 30.000MWCO HY, Sartorius) and subsequently applied to HiLoad equilibrated with 20mM histidine, pH6.0,140 mM NaCl, 0.01% Tween-20^TM 16/60Superdex^TM200 preparative grade (GE Healthcare). The eluted fractions were analyzed for aggregate content by analytical size exclusion chromatography. Thus, 30. mu.l fractions were applied at 25mM K₂HPO₄125mM NaCl,200mM L-arginine monohydrochloride, 0.02% (w/v) NaN₃TSKgel G3000 SW XL equilibrated at 25 ℃ in running buffer pH 6.7 was analyzed for size exclusion column (Tosoh). Fractions containing less than 2% oligomers were combined and concentrated to a final concentration of 1-1.5mg/ml using a super concentrator (Vivaspin 15R 30.000MWCO HY, Sartorius). Protein concentration was determined by measuring the Optical Density (OD) at 280nm using the molar extinction coefficient calculated based on the amino acid sequence. The purity and molecular weight of the constructs were analyzed by SDS capillary electrophoresis in the presence and absence of reducing agents according to the manufacturer's instructions (apparatus Caliper LabChipGX, Perkin Elmer). The purified protein was frozen in liquid nitrogen and stored at-80 ℃. Based on in vitro characterization results, the selected binding agent was converted to a T cell bispecific format. In these molecules, FolR1: CD3 binding moieties were arranged in a 2:1 order with FolR1 Fab at the N-terminus. For clones isolated from standard Fab libraries, the CD3 binding moiety Separately produced as CrossFab (CH1C κ hybrid), whereas for clones from a common light chain library, no hybridization was required. These bispecific molecules were produced and purified similarly to IgG.

TABLE 6 yield and monomer content of the novel FolR1 binding agent in IgG and TCB form, respectively.

CLC common light chain

Example 9

2+1 and 1+ 1T-cell bispecific formats

Four different T cell bispecific formats were prepared for one common light chain binder (16D5) and three formats were prepared for one binder from the Fab library (9D11) to compare their killing properties in vitro.

The standard format is the 2+1 inverted format already described (FolR1: CD3 binding moieties are arranged in 2:1 order, with FolR1 Fab at the N-terminus). In the 2+1 canonical form, the FolR1: CD3 binding moieties are arranged in a 2:1 order with the CD3 Fab at the N-terminus. Two monovalent forms were also prepared. 1+1 head to tail has FolR1: CD3 binding moieties arranged in a 1:1 order on the same arm of the molecule, with FolR1 Fab at the N-terminus. In the 1+1 canonical form, the FolR1: CD3 binding moieties are each present once on one arm of the molecule. For the 9D11 clone isolated from the standard Fab library, the CD3 binding moiety was generated as CrossFab (CH1C κ hybrid), whereas for the 16D5 from the common light chain library, no hybridization was required. These bispecific molecules were produced and purified similar to the standard inverted T cell bispecific format.

TABLE 7 summary of the yields and final monomer content of different T cell bispecific formats.

Example 10

Biochemical characterization of FolR1 binding agents by surface plasmon resonance

Binding of the FolR1 binders to different recombinant folate receptors (human FolR1,2 and 3, mouse FolR1 and cynomolgus monkey FolR 1; all Fc fusions) was assessed by Surface Plasmon Resonance (SPR) as IgG or in a T-cell bispecific format. All SPR experiments were performed at 25 ℃ on a Biacore T200 using HBS-EP as running buffer (0.01M HEPES pH 7.4,0.15M NaCl,3mM EDTA, 0.005% surfactant P20, Biacore, Freiburg/Germany).

Single injection

anti-FolR 1 IgGs were first analyzed by single injection (table 1) to characterize their cross-reactivity (to human, mouse and cynomolgus monkey FolR1) and specificity (to human FolR1, human FolR2, human FolR 3). Recombinant biotinylated monomeric Fc fusions to human, cynomolgus and mouse folate receptor 1(FolR1-Fc) or human folate receptors 2 and 3(FolR2-Fc, FolR3-Fc) were coupled directly onto SA chips using standard coupling protocols (Biacore, frieburg/germany). The immobilization level was about 300-400 RU. IgG was injected at a concentration of 500nM for 60 seconds. IgG binding to huFolR2 and huFolR3 was rejected due to lack of specificity. Most binders were only cross-reactive between human and cynomolgus monkey FolR1, with the additional cross-reactivity of mouse FolR1 being most of the time associated with loss of specificity.

TABLE 8 Cross-reactivity and specificity of 25 novel folate receptor 1 binding agents (as IgG) and two control IgGs (Mov19 and Farletuzumab). + means binding, -means no binding, +/-means weak binding.

Affinity for folate receptor 1

The affinity of the interaction between anti-FolR 1 IgG or T cell bispecific molecules and recombinant folate receptors was determined as described below (table 9).

Recombinant biotinylated monomeric Fc fusions of human, cynomolgus and mouse folate receptor 1(FolR1-Fc) were directly coupled to SA chips using standard coupling protocols (Biacore, Freiburg/Germany). The immobilization level was about 300-400 RU. anti-FolR 1 IgG or T cell bispecific molecules were passed through the flow cell at a concentration ranging from 2.1 to 500nM and a flow rate of 30. mu.L/min for 180 seconds. Dissociation was monitored for 600 seconds. Bulk refractive index differences were corrected by subtracting the responses obtained on a reference flow cell immobilized with recombinant biotinylated IL2 receptor Fc fusions. To analyze the interaction of 19H3 IgG and mouse folate receptor 1, folate (Sigma F7876) was added to HBS-EP running buffer at a concentration of 2.3. mu.M. The binding curve resulting from bivalent binding of IgG or T cell bispecific molecules was approximated as 1:1 Langmuir binding and fitted with the model (this is incorrect, but gives an idea of avidity). The apparent affinity constant for the interaction was derived from the fitted rate constant using Bia Evaluation software (GE Healthcare).

TABLE 9 selected FolR1 binding agents as IgG or as bivalent binding (avidity versus apparent KD) of T-cell bispecific molecules (TCBs) on human and cynomolgus monkey FolR 1.

Affinity for folate receptor 1

The affinity of the interaction between the anti-FolR 1 IgG or T cell bispecific molecule and the recombinant folate receptor was determined as described below (table 10).

For affinity measurements, direct coupling of approximately 6000 and 7000 Resonance Units (RU) of anti-human Fab specific antibody (Fab capture kit, GE Healthcare) was performed on CM5 chips at pH5.0 using a standard amine coupling kit (GE Healthcare). The anti-FolR 1 IgGs or T-cell bispecific molecules were captured at 20nM for 20 or 40 seconds at a flow rate of 10. mu.l/min, the reference flow cell was not captured. Human or cynomolgus folate receptor 1Fc fusions in dilution series (6.17 to 500nM or 12.35 to 3000nM) were passed through all flow cells at 30 μ Ι/min for 120 or 240 seconds to record the binding period. The dissociation period was monitored for 240 seconds and switched from the sample solution to HBS-EP priming. After each cycle, the chip surface was regenerated using a 60 second double injection of 10mM glycine-hydrochloric acid pH2.1 or pH 1.5. The bulk refractive index difference is corrected by subtracting the response obtained on the reference flow cell 1. The affinity constants for the interactions were derived from the rate constants by fitting 1:1Langmuir binding using Bia Evaluation software (GE Healthcare).

TABLE 10 monovalent binding (affinity) of selected FolR1 binding agents on human and cynomolgus monkey FolR1 as IgG or as T cell bispecific molecules (TCB).

Affinity for CD3

The affinity of the interaction between the anti-FolR 1T cell bispecific molecule and recombinant human CD3 epsilon delta-Fc was determined as described below (table 11).

For affinity measurements, direct coupling of approximately 9000 Resonance Units (RU) of anti-human Fab specific antibodies (Fab capture kit, GE Healthcare) was performed on a CM5 chip at ph5.0 using a standard amine coupling kit (GE Healthcare). The anti-FolR 1T cell bispecific molecules were captured at 20nM for 40 sec at a flow rate of 10. mu.l/min, with the reference flow cell uncaptured. A dilution series (6.17 to 500nM) of human CD3 ε δ -Fc fusion was passed through all flow cells at 30 μ l/min for 240 seconds to record the binding period. The dissociation period was monitored for 240 seconds and initiated by switching from the sample solution to HBS-EP. After each cycle, the chip surface was regenerated using a 60 second double injection of 10mM glycine-hydrochloric acid, pH 2.1. The bulk refractive index difference is corrected by subtracting the response obtained on the reference flow cell 1. The affinity constant for the interaction was derived from the rate constant by fitting 1:1 Langmuir binding using Bia Evaluation software (GE Healthcare).

TABLE 11 monovalent binding (affinity) of selected FolR1T cell bispecific molecules (TCBs) on human CD 3-Fc.

Ligands	Analyte	ka(1/Ms)	kd(1/s)	KD(M)
					16D5 TCB	huCD3	4.25E+04	3.46E-03	8.14E-08
21A5 TCB	huCD3	3.72E+04	3.29E-03	8.8E-08

The CD3 binding moiety was the same for all constructs and the affinities were similar for the measured T cell bispecific molecules (KD range between 60 and 90 nM).

Example 11

Bispecific molecules that simultaneously bind T cells at folate receptor 1 and CD3

Simultaneous binding of the anti-FolR 1T cell bispecific molecule on recombinant folate receptor 1 and recombinant human CD3 epsilon delta-Fc was determined by surface plasmon resonance as described below. Recombinant biotinylated monomeric Fc fusions of human, cynomolgus and mouse folate receptor 1(FolR1-Fc) were directly coupled to SA chips using standard coupling protocols (Biacore, Freiburg/Germany). The fixed level was about 300 and 400 RU. anti-FolR 1T cell bispecific molecules were injected through the flow cell at a flow rate of 30. mu.L/min at 500nM for 60 seconds, followed by hu CD ε δ -Fc at 500nM for 60 seconds. Bulk refractive index differences were corrected by subtracting the response obtained with a reference flow cell immobilized with recombinant biotinylated IL2 receptor Fc fusion. The four T cell bispecific molecules tested (16D5 TCB,21a5 TCB,51C7 TCB and 45D2 TCB) were able to bind folate receptor 1 and human CD3 simultaneously as expected.

Example 12

Epitope grouping (epise binding)

For epitope grouping, anti-FolR 1 IgG or T cell bispecific molecules were immobilized directly on CM5 chips at ph5.0 using a standard amine coupling kit (GE Healthcare) with a final response of approximately 700 RU. 500nM huFolR1-Fc was then captured for 60 seconds followed by 500nM of the different binders for 30 seconds. Surfaces were regenerated with two injections of 10mM glycine pH 2 for 30 seconds each. It was assessed whether different binders could bind to huFolR1 captured on the immobilized binder (table 12).

TABLE 12 characterization of selected FolR1 binding agents as IgG or as epitopes for T-cell bispecific molecules (TCBs) on human FolR 1. + means binding, -means no binding, +/-means weak binding

Based on these results and additional data for simultaneous binding on immobilized huFolR1, the binders were divided into three groups. It is unclear whether 9D11 has a separate epitope because it replaces all other binders. 16D5 and 21A5 appeared in the same group, Mov19, Farletuzumab (Coney et al, Cancer Res.1991 Nov 15; 51(22): 6125-32; Kalli et al, Curr Opin Investig drugs.2007 Dec; 8(12):1067-73) and 36F2 in the other group (Table 13). However, 36F2 binds a different epitope than Mov19 and farlettuzumab because it binds human, cynomolgus monkey and mouse FolR 1.

TABLE 13 epitope grouping of selected FolR1 binding agents on human FolR1 as IgG or as T-cell bispecific molecules (TCBs)

Epitope 1	Epitope 2	Epitope 3
			16D5	9D11	Mov19
21A5		Farletuzumab
					36F2

Example 13

Selection of binding Agents

The IgG format of FolR1 binding agents was screened by Surface Plasmon Resonance (SPR) and in vitro assays on cells to select the best candidate.

anti-FolR 1 IgGs were analyzed by SPR to characterize their cross-reactivity (to human, mouse and cynomolgus FolR1) and specificity (to human FolR1, human FolR2, human FolR 3). Non-specific binding to human FolR2 and 3 was considered to be an exclusion factor. Binding and specificity to human FolR1 was demonstrated on cells. Some binding agents did not bind on cells expressing FolR1 even though they recognized recombinant human FolR1 in SPR. The aggregation temperature is determined, but not exclusively, because the selected binders are stable. Selected binders were tested in a multireactive ELISA to check for non-specific binding, which resulted in the exclusion of four binders. This approach led to the initial selection of three binders, 36F2(Fab library), 9D11(Fab library) and 16D5 (common light chain). 36F2 was rapidly dissociated from huFolR1 and was therefore initially not favoured.

Example 14

Specific binding of newly generated FolR1 binding agents to human FolR1 positive tumor cells

Novel FolR1 binding agents were generated by phage display using a Fab library or a common light chain library using CD3 light chain. The identified binding agents were converted to human IgG1 form and treated for binding to HeLa cells highly expressing FolR 1. As reference molecules, human FolR1 binder Mov19 was included. Most of the binders tested in this assay showed moderate to good binding to FolR1, with some clones binding equally well to Mov19 (see figure 2). Clones 16a3,18D3,15H7,15B6,21D1,14E4 and 16F12 were excluded because binding to FolR1 on the cells could not be confirmed by flow cytometry. In the next step, selected clones were tested for specificity to human FolR1 by excluding binding to closely related human FolR 2. HEK cells were transiently transfected with human FolR1 or human FolR2 to resolve specificity. Clones 36F2 and 9D11 derived from the Fab library and clones 16D5 and 21a5 derived from the CLC library specifically bound human FolR1 but not human FolR2 (see fig. 3A-B). All other clones tested showed at least some binding to human FolR2 (see fig. 3A-B). Therefore, these clones were excluded from further characterization. Parallel cross-reactivity of the FolR1 clone with cynomolgus monkey FolR1 was resolved by binding studies on HEK cells transiently transfected with cynomolgus monkey FolR 1. All clones tested were able to bind cynomolgus monkey FolR1, and the four selected human FolR1 specific clones 36F2,9D11,16D5 and 21a5 bound both human and cynomolgus monkey FolR1 quite well (fig. 4). Three human FolR 1-specific cynomolgus monkey cross-reactive binders were subsequently converted to the TCB form and tested for induction of T cell killing and T cell activation. These clones were 9D11 from the Fab library and 16D5 and 21a5 from the CLC library. As a reference molecule, Mov19 FolR1 TCB was included in all studies. These FolR1 TCBs were then used to compare the induction of internalization upon binding to FolR1 on HeLa cells. All three clones tested were internalized upon binding to FolR1, comparable to that upon binding to Mov19 FolR1 TCB (fig. 5). 21A5 FolR1 TCB was discontinued due to evidence of polyreactivity.

Example 15

T cell mediated killing of FolR1 expressing tumor target cells induced by FolR1 TCB antibody

FolR1 TCB was used to determine T cell mediated killing of tumor cells expressing FolR 1. The FoLR1 binding site was determined by a Qifikit analysis using a panel of potential target cell lines.

The tumor cell set used comprised FolR1 high-expressing, medium-expressing and low-expressing tumor cells and a FolR1 negative cell line.

TABLE 14 FolR1 binding site on tumor cells

Cell lines	Source	FolR1 binding site
			Hela	Adenocarcinoma of cervix	2’240’716
Skov3	Ovarian adenocarcinoma	91’510
			OVCAR5	Ovarian adenocarcinoma	22’077
HT29	Colorectal adenocarcinoma	10’135
			MKN45	Gastric adenocarcinoma	54

Binding of three different FoLR1 TCBs (containing 9D11,16D5 and Mov19 binding agents) to this group of tumor cell lines was determined, indicating that FoLR1 TCB specifically binds to FoLR 1-expressing tumor cells and not to FoLR 1-negative tumor cell lines. The amount of construct bound was proportional to the level of FolR1 expression and the construct still bound well to the detectable FolR1 low cell line HT-29. Additionally, there was no binding of the negative control DP47 TCB to any of the used cell lines (FIGS. 6A-E).

The intermediate expression cell line SKOV3 and the low expression cell line HT-29 were further used to test T cell mediated killing and T cell activation using 16D5 TCB and 9D11 TCB; DP47 TCB was included as a negative control. In the presence of already very low levels of 16D5 TCB and 9D11 TCB, both cell lines were killed, and even though 9D11 TCB bound more strongly to FolR1 than 16D5 TCB, there was no difference in activity between the two TCBs. The overall killing rate was higher for SKOV3 cells compared to HT-29, which reflects a higher expression level of FolR1 on SKOV3 cells (fig. 7A-D). In line with this, CD4 was detected ⁺T cells and CD8⁺Strong upregulation of activation markers CD25 and CD69 on T cells. The activation of T cells was very similar in the presence of SKOV3 cells and HT-29 cells. The negative control DP47 TCB did not induce any killing at the concentrations used, and there was no significant upregulation of CD25 and CD69 on T cells.

TABLE 15 EC50 values for tumor cell killing and T cell activation using SKOV3 cells

TABLE 16 EC50 values for tumor cell killing and T cell activation using HT-29 cells

Example 16

Combined erythrocyte and T cell activation in whole blood

To demonstrate that there was no spontaneous activation in the absence of tumor cells expressing FoLR1, we tested whether there was binding of FoLR1 clone to erythrocytes that may potentially express FoLR 1. We could not observe any specific binding of 9D11 IgG, 16D5 IgG and Mov19 IgG to erythrocytes because the negative control DP47 IgG was included (fig. 8).

To exclude any further non-specific binding or non-specific activation of blood cells by FoLR1 TCB, 9D11 TCB,16D5 TCB and Mov19 TCB were added to whole blood and CD4 was analyzed by flow cytometry⁺T cells and CD8⁺Upregulation of CD25 and CD69 on T cells. DP47 TCB was included as a negative control. By analysis of CD4 ⁺T cells and CD8⁺Upregulation of CD25 and CD69 on T cells, it was observed that no T cell activation occurred with any of the constructs tested (fig. 9).

Example 17

Removal of the N-glycosylation site in the 9D11 light chain

In analyzing the different FolR1 binding agents to identify potential sequence hotspots, putative N-glycosylation sites were identified at the end of CDR L3 of clone 9D 11. In general, the consensus motif for N-glycosylation is defined as N-X-S/T-X (where X is not P). Sequence of CDR L3(MQASIMNRT(SEQ ID NO:46) ) perfectly matched with a consensus motif having the sequence N-R-T. Since glycosylation may not be fully reproducible in different production batches, if glycosylation in CDR L3 contributes to antigen binding, this may affect FolR1 binding. To assess whether this N-glycosylation site is important for FolR1 binding, or can be replaced without compromising binding, different variants of 9D11 light chain were generated in which the N-glycosylation site was exchanged by site-specific mutagenesis.

1. Transient transfection and production

Four T cell bispecific molecules were transiently produced in HEK293 EBNA cells using PEI mediated transfection methods for the required vectors as described below. HEK293 EBNA cells were cultured in CD CHO medium without serum suspension. For production in 500ml shake flasks, 4 hundred million HEK293 EBNA cells were seeded 24 hours prior to transfection (all amounts adjusted accordingly for alternative scales). For transfection, cells were centrifuged at 210 × g for 5 min, and the supernatant was replaced with pre-warmed 20ml of CD CHO medium. The expression vector was mixed in 20ml of CD CHO medium to a final amount of 200. mu.g DNA. After addition of 540. mu.l PEI, the solution was vortexed for 15 seconds and subsequently incubated at room temperature for 10 minutes. The cells were then mixed with the DNA/PEI solution, transferred to 500ml shake flasks and incubated for 3 hours at 37 ℃ in an incubator with an atmosphere of 5% CO 2. After incubation, 160ml of F17 medium was added and the cells were cultured for 24 hours. 1 day after transfection, 1mM valproic acid and 7% Charge 1 were added. After 7 days, the culture supernatant was collected, purified by centrifugation at 210 Xg for 15 minutes, the solution was sterile filtered (0.22 μm filter) and sodium azide was added to a final concentration of 0.01% w/v and maintained at 4 ℃. After production, the supernatant was harvested, and the antibody-containing supernatant was filtered through a 0.22 μm sterile filter and stored at 4 ℃ until purification.

2. Antibody purification

Using standard methods, e.g. protein A affinity purification: (Explorer) and size exclusion chromatography. The supernatant obtained from the transient production was adjusted to pH 8.0 (using 2M TRIS pH 8.0) and applied to HiTrap PA HP (GE Healthcare, column volume (cv) ═ 5ml) equilibrated with 8 column volumes (cv) of buffer a (20mM sodium phosphate, 20mM sodium citrate, 0.5M NaCl, 0.01% Tween-20, pH 7.5). After washing with 10cv buffer A, the protein was eluted in 20cv to buffer B (20mM sodium citrate pH2.5,0.5M NaCl, 0.01% Tween-20) using a pH gradient. Fractions containing the protein of interest were pooled and the pH of the solution was gently adjusted to pH 6.0 (using 2M Tris pH 8.0). The samples were concentrated to 1ml using a super concentrator (Vivaspin15R 30.000 MWCO HY, Sartorius) and subsequently applied to Superdex equilibrated with 20mM histidine, pH 6.0,140mM NaCl, 0.01% Tween-20^TM20010/300 GL (GE healthcare). The eluted fractions were analyzed for aggregate content by analytical size exclusion chromatography. Thus, 30. mu.l fractions were applied at 25mM K₂HPO₄125mM NaCl,200mM L-arginine monohydrochloride, 0.02% (w/v) NaN₃pH 6.7 running buffer TSKgel G3000 SW XL equilibrated at 25 ℃ assay size exclusion column (Tosoh). Fractions containing less than 2% oligomers were combined and concentrated to a final concentration of 1-1.5mg/ml using a super concentrator (Vivaspin15R 30.000.000 MWCO HY, Sartorius). Protein concentration was determined by measuring the Optical Density (OD) at 280nm using the molar extinction coefficient calculated based on the amino acid sequence. The purity and molecular weight of the constructs were analyzed by SDS capillary electrophoresis in the presence and absence of reducing agents according to the manufacturer's instructions (apparatus Caliper LabChipGX, Perkin Elmer). The purified protein was frozen in liquid nitrogen and stored at-80 ℃.

3. Temperature of aggregation

The stability of the four constructs was tested on Optim1000(Avacta, PALL Corporation) by heating at a gradient from 25 ℃ to 80 ℃ at 0.1 ℃/min. The temperature at which aggregation started was recorded.

TABLE 34 production, monomer content and aggregation temperature of four N-glycosylation site knockout mutants of 9D11 binding agents in 2+1 inverted T cell bispecific format. All four mutants performed similarly to the wild-type 9D11 binding agent.

Cloning	Mutations	Yield [ mg/L }	Monomer [% ]]	Temperature of aggregation
					9D11	T102N	1.34	97	56°
9D11	T102A	1.29	100	56°
					9D11	N100Q	2.5	100	56°
9D11	N100S	2.05	100	56°
					9D11	-	2.6	100	57°

The following variants were generated, N100S (N95S); N100Q (N95Q), T102A (T97A) and T102N (T97N) (Kabat numbering indicated in parentheses) and converted to T cell bispecific format. After transient production and purification of HEK293 EBNA cells, different variants were analyzed for target binding and cell killing activity compared to the original 9D11 clone.

TABLE 17 primers for removal of the N-glycosylation site in CDR L3 of 9D11 (see sequence below)

Example 18

Binding of 9D11 a-sugar variants and T cell mediated killing

Due to the glycosylation sites in the CDRs, four different variants of 9D11 were generated with mutations that remove the glycosylation sites (example 17). The four variants were tested for binding to FolR1 on HeLa cells (FIG. 10) and induction of tumor cell killing on SKOV3 and HT-29 (FIGS. 11A-B, E-F) compared to the original 9D 11. None of the variants showed differences in the induction of binding or tumor cell killing. Non-specific killing of the FolR1 negative cell line MKN-45 was resolved in parallel (FIGS. 11C-D). In addition, differences between the variants and the original binding agent can be observed. None of the constructs induced non-specific killing of FoLR1 negative tumor cells.

Example 19

FolR1 expression on primary epithelial cells

FolR1 was expressed at low levels on primary epithelial cells. Here we wanted to test whether these levels were sufficient to induce T cell mediated killing in the presence of FolR1 TCB. For testing, we used primary human bronchial epithelial cells, primary human choroid plexus epithelial cells, primary human renal cortical epithelial cells, and primary human retinal pigment epithelial cells. FolR1 positive SKOV3 cells or HT-29 cells were included as positive controls. First, we validated the expression of FolR1 on the primary cells used and determined the amount of FolR1 binding site on these cells. Bronchial epithelial cells, renal cortical epithelial cells and retinal pigment epithelial cells express very low but significant levels of FolR1 compared to the levels expressed on tumor cells. Choroid plexus neuroepithelial cells do not express significant levels of FolR 1.

TABLE 18 FolR1 binding sites on primary epithelial cells

Cell lines	Binding sites
		Bronchial epithelium	492
Choroid plexus epithelium	104
		Renal cortical epithelium	312
Retinal pigment epithelium	822
		Skov3	69’890

Primary epithelial cells that show FolR1 expression on their surface were used to solve the problem of whether these cells could be killed by T cells in the presence of FolR1 TCB. No significant level of killing was measured, but induction of T cell activation in the presence of retinal pigment epithelial cells, bronchial epithelial cells and renal cortex cells was detected, resulting in upregulation of CD25 and CD 69. The strongest activation was observed with retinal pigment epithelial cells, resulting in CD4 ⁺T cells and CD8⁺Upregulation of CD25 and CD69 on T cells. CD4 in the presence of bronchial epithelial cells⁺T cells and CD8⁺Upregulation of CD69 on T cells induced lower activation of T cells, but only on CD4⁺T cells other than CD8⁺The up-regulation of CD25 on T cells is very low. Minimal activation of T cells was obtained in the presence of renal epithelial cells, at CD4⁺T cells and CD8⁺There was no upregulation of CD25 on T cells, and CD69 was only on CD8⁺T cells were upregulated (FIGS. 12A-X).

Example 20

Comparison of different TCB forms containing 16D5 or 9D11 binding agents

To determine whether the TCB 2+1 inverted form is the most active form using the selected FolR1 adhesive, different forms were generated comprising 16D5 or 9D11 and compared in target cell binding, T cell mediated killing, and T cell activation. 16D5 binding agents were tested in TCB 2+1 reverse (fig. 1A), TCB 2+1 classical (fig. 1D), TCB 1+1 classical (fig. 1C) and TCB 1+1 head-to-tail (fig. 1B) formats; the 9D11 binding agent was tested in TCB 2+1 reverse (fig. 1A), TCB 1+1 classical (fig. 1C) and TCB 1+1 head-to-tail (fig. 1B) formats.

All constructs were tested for binding to FolR1 on HeLa cells. Bivalent molecules that bind FolR1 are stronger due to avidity compared to monovalent constructs. The difference between bivalent versus monovalent constructs was more pronounced for 16D 5. The reason may be that the avidity effect of the binding agent is stronger due to the lower affinity of 16D 5. There was no significant difference in binding between the two 1+1 TCBs, but there was a difference between the two 2+1 constructs. The reverse 2+1 construct binds more strongly to FolR1 than the classical 2+1 construct. This indicates that in the classical 2+1 construct, binding to FoLR1 was affected by the presence of CD3 Fab, whereas in the reverse construct binding was less affected.

By testing T cell mediated killing with these constructs, we can show that stronger binding of 2+1 reverse TCB translates into stronger tumor cell killing and T cell activation compared to 2+1 classical TCB. The 16D5 FolR1 TCB 2+1 canonical was only a little more active than the respective 1+1 head-to-tail constructs. The 1+1 head-to-tail construct was significantly more active than the 1+1 canonical construct. This does not reflect what is seen in binding and may be due to better cross-linking with the head-to-tail construct. Overall tumor cell killing and T cell activation were comparable to all tested constructs, with the only difference in potency seen being the EC50 value. In general, it can be concluded that FolR1 TCB 2+1 reversal is a preferred form of inducing T cell-mediated tumor cell killing and T cell activation, independent of the binding agent used (see fig. 13A-C and fig. 14A-C).

TABLE 19 EC50 values for target cell binding and T cell-mediated killing of different TCB forms

TABLE 20 EC50 values for T cell activation in the presence of SKOV3 cells with different TCB patterns

Example 21

Tumor cell lines and primary cells

HeLa cells (CCL-2) were obtained from ATCC and cultured in DMEM containing 10% FCS and 2mM glutamine, SKOV3(HTB-77) was obtained from ATCC and cultured in RPMI containing 10% FCS and 2mM glutamine, OVCAR5 was obtained from NCI and cultured in RPMI containing 10% FCS and 2mM glutamine, HT-29(ACC-299) was obtained from DSMZ and cultured in McCoy's 5A medium containing 10% FCS and 2mM glutamine, MKN-45(ACC-409) was obtained from DSMZ and cultured in RPMI containing 10% FCS and 2mM glutamine.

All primary epithelial cells tested were obtained from ScienCell Research Laboratories. Human bronchial epithelial cells (HBEpiC, catalog No. 3210) were cultured in bronchial epithelial cell culture medium (BEpiCM, catalog No. 3211, ScienCell). Human colonic epithelial cells (HCoEpiC), Cat No. 2950 were cultured in colonic epithelial cell culture medium ((CoEpiCM, Cat.No.2951, ScienCell.) human retinal pigment epithelial cells (HRPEiC), Cat No. 6540 were cultured in epithelial cell culture medium (EpiCM, Cat No. 4101, ScienCell.) human renal cortical epithelial cells (HRCEPIC), Cat No. 4110 were cultured in epithelial cell culture medium (EpiCM, Cat No. 4101, ScienCell.) human choroid plexus epithelial cells (HCPEiC), Cat No. 1310 were cultured in epithelial cell culture medium (EpiCM, Cat No. 1, ScienCell).

Example 22

Targeted binding by flow cytometry

Target cells as indicated were harvested with cell dissociation buffer, washed with PBS and resuspended in FACS buffer. Antibody staining was performed in 96-well round bottom plates. Thus, 200,000 cells were seeded per well. The plate was centrifuged at 400g for 4 minutes and the supernatant removed. The test antibody was diluted in FACS buffer and 20 μ l of antibody solution was added to the cells for 30 minutes at 4 ℃. To remove unbound antibody, two washes with FACS buffer were performed before addition of diluted secondary antibody (FITC-conjugated affinipur F (ab ') 2 fragment goat anti-human IgG, Fcg fragment, Jackson ImmunoResearch #109-096-098 or PE-conjugated affinipur F (ab') 2 fragment goat anti-human IgG Fcg fragment specific, Jackson ImmunoResearch # 109-116-170). After incubation at 4 ℃ for 30 minutes, unbound secondary antibody is washed away. Cells were resuspended in 200 μ l FACS buffer prior to measurement and analyzed by flow cytometry using BD Canto II or BD Fortessa.

Example 23

Internalization

Cells were harvested and assayed for viability. Cells were resuspended in fresh cold medium at 2Mio cells per ml and the cell suspension was transferred to 15ml falcon tubes for each antibody. The cells were added with the antibody to be tested for internalization at a final concentration of 20 μ g/ml. The tubes were incubated in a cold room on a shaker for 45 minutes. After incubation, cells were washed three times with cold PBS to remove unbound antibody. 0.2Mio cells/well were transferred to FACS plates as time point 0. The labeled cells were resuspended in warm medium and incubated at 37 ℃. At the indicated time points, 0.2Mio cells/well were transferred to cold PBS and washed on FACS plates. To detect the constructs remaining on the surface, cells were stained with PE-labeled anti-human Fc secondary antibody. Therefore, 20. mu.l of diluted antibody was added to each well and the plates were incubated at 4 ℃ for 30 minutes. Cells were then washed twice to remove unbound antibody and then fixed with 1% PFA to prevent any further internalization. Fluorescence was measured using a BD FACS cantonii.

Example 24

Analysis of

Contains a series of beads 10 μm in diameter and coated with different but defined amounts of mouse Mab molecules (high affinity anti-human CD5, clone CRIS-1, isotype IgG2 a). The beads mimic cells with different antigen densities, which have been labeled with a primary mouse Mab, isotype IgG. Briefly, cells are labeled with a primary mouse monoclonal antibody directed against an antigen of interest. In separate test wells, cells were labeled with an irrelevant mouse monoclonal antibody (isotype control). Then conjugated with fluorescein contained in the kit Anti-mouse secondary antibody labeled cells, set beads and calibration beads. The primary antibody used to label the cells must be used at saturating concentrations. The primary antibody may be of any mouse IgG isotype. Under these conditions, the number of primary antibody molecules bound corresponds to the number of antigen sites present on the cell surface. Secondary antibodies were also used at saturating concentrations. Thus, fluorescence is related to the number of primary antibody molecules bound on the cells and on the beads.

Example 25

T cell mediated tumor cell killing and T cell activation

Target cells were harvested with trypsin/EDTA, counted and examined for cell viability. The cells were resuspended in the respective medium to a final concentration of 300,000 cells per ml. Then 100. mu.l of the target cell suspension was transferred to each well of a 96-flat bottom plate. The plates were incubated overnight in an incubator at 37 ℃ to allow the cells to adhere to the plates. PBMCs were isolated the following day from whole blood of healthy donors. Blood was diluted with PBS 2:1 and overlaid on 15ml Histopaque-1077(#10771, Sigma-Aldrich) in a Leucosep tube and centrifuged at 450g without interruption for 30 minutes. After centrifugation, the cell-containing band was collected with a 10ml pipette and transferred to a 50ml tube. The tube was filled with PBS to 50ml and centrifuged (400g, 10 min, room temperature). The supernatant was removed and the pellet was resuspended in PBS. After centrifugation (300g, 10 min, room temperature), the supernatant was discarded, the 2 tubes were combined and the washing step was repeated (this time centrifugation at 350Xg, room temperature for 10 min). The cells were then resuspended and the pellet was pooled in 50ml PBS for cell counting. After counting, the cells were centrifuged (350g, 10 min, rt) and resuspended at 6Mio cells/ml in RPMI containing 2% FCS and 2nM glutamine. The medium was removed from the plated target cells and test antibody diluted with RPMI with 2% FCS and 2nM glutamine was added. 300,000 cells of the effector cell solution were transferred to each well to give an E: T ratio of 10: 1. To determine the maximum release, target cells were lysed with Triton X-100. LDH release was measured after 24 hours and 48 hours using a cytotoxicity detection kit (#1644793, Roche Applied Science). Upregulation of activation markers on T cells after tumor cell killing was measured by flow cytometry. Briefly, PBMCs were collected, transferred to 96-well round bottom plates, stained with CD4 PE-Cy7(#3557852, BD biosciences), CD8 FITC (#555634, BD biosciences), CD25 APC (#555434, BD biosciences), CD69 PE (#310906, BioLegend) antibodies diluted in FACS buffer. After incubation for 30 min at 4 ℃, the cells were washed twice with FACS buffer. Cells were resuspended in 200 μ l FACS buffer before fluorescence was measured using BD Canto II.

Example 26

T cell activation in whole blood

280 μ l of fresh blood was added to a 96-well conical deep-well plate. Then 20. mu.l of diluted TCB was added to the blood and mixed well by shaking the plate. After incubation for 24 hours at 37 ℃ in an incubator, the blood was mixed and 35. mu.l was transferred to a 96-well round bottom plate. Then 20 μ l of an antibody staining mixture consisting of CD4 PE-Cy7(#3557852, BD Bioscience), CD8 FITC (#555634, BD Bioscience), CD25 APC (#555434, BD Bioscience), CD69 PE (#310906, BioLegend) and CD 45V 500(#560777, BD Horizon) was added and incubated for 15 minutes at room temperature in the dark. To the blood was added 200 μ l of freshly prepared BD FACS lysis solution (#349202, BD FCAS) prior to measurement. After 15 min incubation at room temperature, cells were measured with BD Fortessa.

Example 27

SDPK (single dose pharmacokinetics) study of humanized FOLR1 TCB (clone 16D5) in immunodeficient NOD/Shi-scid/IL-2R γ Null (NOG) mice

6-7 week old female NOD/Shi-scid/IL-2R γ Null (NOG) mice (bred at Taconic, Denmark) at the start of the experiment were kept under specific pathogen-free conditions according to the promised guidelines (GV-Solas; Felasa; TierschG) with 12 hours light/12 hours dark cycles daily. Experimental study protocol was approved by local government review (P2011/128). Upon arrival, the animals were maintained for one week to become accustomed to the new environment and for observation. Continuous health monitoring was performed periodically.

Mice were injected intravenously with 10/1/0.1 μ g/mouse of FOLR1 TCB, and 3 mice were bled per group and time point. All mice were injected with a total volume of 200. mu.l of the appropriate solution. To obtain the appropriate amount of FOLR1 TCB per 200 μ l, the stock solution was diluted with PBS as necessary. Serum samples were collected 5 minutes, 1 hour, 3 hours, 8 hours, 24 hours, 48 hours, 72 hours, 96 hours and 168 hours after the treatment injection.

Figure 15 shows that 16D5 FOLR1 TCB shows IgG-like PK properties at typical and dose ratios in NOG mice with slow clearance.

TABLE 21

Example 28

In vivo efficacy of FOLR1 TCB (clone 16D5) following transfer of human PBMC in NOG mice harboring Skov3

FOLR1 TCB was tested in the human ovarian cancer cell line Skov3, injected subcutaneously into PBMC-transplanted NOG mice.

Skov3 ovarian cancer cells were obtained from ATCC (HTB-77). Tumor cell lines in RPMI containing 10% FCS (Gibco), 5% CO at 37 ℃₂The culture is carried out in a water-saturated atmosphere. Passage 35 for transplantation, survival>95 percent. Each animal was injected subcutaneously 5X10 in a total of 100. mu.l of RPMI cell culture medium (Gibco)⁶Individual cells reached the right flank of the animal.

6-7 week old female NOD/Shi-scid/IL-2R γ Null (NOG) mice (bred at Taconic, Denmark) at the start of the experiment were kept under specific pathogen-free conditions according to the promised guidelines (GV-Solas; Felasa; TierschG) with 12 hours light/12 hours dark cycles daily. The experimental study protocol was approved by local government review (P2011/128). Upon arrival, the animals were maintained for one week to become accustomed to the new environment and for observation. Continuous health monitoring was performed periodically.

Mice were injected subcutaneously with 5x10 on study day 0 according to protocol (fig. 16)⁶Skov 3. On study day 21, human PBMCs from healthy donors were isolated by Ficoll method and injected intraperitoneally with 10X10⁶One cell to tumor bearing mice. Two days later, mice were randomized and evenly distributed over 5 treatment groups (n-12) followed by intravenous injection of 10/1/FOLR 1 TCB at 0.1 μ g/mouse or DP47 control TCB at 10 μ g/mouse, once a weekNext, for three weeks. All mice were injected intravenously with 200. mu.l of the appropriate solution. Mice in the vehicle group were injected with PBS. To obtain the appropriate amount of TCB per 200. mu.l, the stock solution was diluted with PBS, if necessary. Tumor growth was measured once a week using calipers (fig. 17) and tumor volume was calculated as follows:

T_v:(W²/2) x L (W: width, L: length)

Once weekly injection of FOLR1 TCB resulted in a dose-dependent anti-tumor effect. Whereas doses of 10. mu.g/mouse and 1. mu.g/mouse induced tumor shrinkage and tumor arrest of 0.1. mu.g/mouse (FIG. 17, Table 22). Maximal tumor shrinkage was achieved at a dose of 10 μ g/mouse compared to the non-targeted control DP47 TCB.

TABLE 22

Compound (I)	Dosage form	Tumor growth inhibition
			DP47 TCB control TCB	10 μ g (corresponding to about 0.5mg/kg)	7％
FOLR1 TCB(16D5)	10 μ g (corresponding to about 0.5mg/kg)	90％
			FOLR1 TCB(16D5)	1 μ g (corresponding to about 0.05mg/kg)	74％
FOLR1 TCB(16D5)	0.1. mu.g (corresponding to about 0.005mg/kg)	56％

For PD readings, on study day 32, 3 mice were sacrificed per treatment group, tumors removed, and single cell suspensions prepared by enzymatic digestion with collagenase V, Dispase II and DNAse for subsequent FACS analysis (fig. 19 and 20). Single cells were used directly for extracellular antigen staining and marker activation or restimulation with 5ng/ml PMA and 500ng/ml ionomycin in the presence of the protein transport inhibitor monensin for 5 hours in normal medium. After restimulation, the cells are stained for surface antigens, followed by a fixation and permeabilization step. The fixed samples were then stained intracellularly for TNF-. alpha.IFN-. gamma.IL-10 and IL-2 and analyzed by flow cytometry. The same procedure was used for cell degranulation, but with the addition of anti-CD 107a antibody during restimulation, the fixed samples were stained for intracellular perforin and granzyme B content. FACS analysis showed infiltrative CD4 in tumor tissue after treatment with FOLR1 TCB compared to vehicle and non-targeted control TCB⁺And CD8⁺The number of T cells was statistically higher. In addition, a greater number of TNF-alpha, IFN-gamma and IL-2 production and perforin were detected in FOLR1 TCB treated tumors ⁺granzyme-B⁺CD4⁺And CD8⁺T cells. Tumor infiltrating T cells treated with FOLR1 TCB also showed higher degranulation rates compared to the control group.

At study termination on day 38, all animals were sacrificed; the tumors were removed and weighed (fig. 18). The weights of tumors treated with 10 and 1 μ g/mouse FOLR1 TCB showed statistically significant differences compared to the control group.

TABLE 23

Example 29

Generation of bispecific FolR1/CD 3-kappa-Lambda antibodies

In order to generate bispecific antibodies (monovalent for each antigen) that can bind both human CD3 and human folate receptor alpha (FolR1) without using any heterodimerization method (e.g. knob-in-hole technology), a combination of a common light chain library and the so-called CrossMab technology was applied, the variable region of a humanized CD3 binding agent (CH2527_ VL7_46/13) fused to the CH1 domain of a standard human IgG1 antibody, forming a VLVH hybrid molecule (fused to Fc), which is common for both specificities. To generate the hybrid counterpart (VHCL), the CD 3-specific variable heavy domain (CH2527_ VH _23/12) was fused to a constant human λ light chain, while the variable heavy domain specific for human FolR1 (clone 16D5, isolated from a common light chain library) was fused to a constant human κ light chain. This enables the desired bispecific antibody to be purified by a subsequent purification step using kappa-select and LambdaFabSelect columns (GE Healthcare) to remove unwanted homodimeric antibodies.

All antibody expression vectors are used, for example, in Sambrook, J.et al, Molecular cloning: A laboratory Manual; color Spring Harbor Laboratory Press, color Spring Harbor, New York, 1989. The molecular biological reagents were used according to the manufacturer's recommendations. The genes or gene fragments were amplified by Polymerase Chain Reaction (PCR) or generated from synthetic oligonucleotides by automated gene synthesis in Geneart AG (Regensburg, Germany). The PCR amplified or subcloned DNA fragments were confirmed by DNA sequencing (Synergene GmbH, Switzerland). Plasmid DNA was transformed and amplified in the appropriate E.coli host strain and transfection grade plasmid DNA was prepared using a standard Maxiprep kit (Qiagen). To produce bispecific molecules, HEK293 EBNA cells were transfected with plasmids encoding the respective genes using a standard Polyethyleneimine (PEI) based approach. The plasmid ratio for the three expression vectors was 1:1: 1. Transfected cells were cultured for 7 days, and then the supernatant was harvested for purification. Bispecific FolR1/CD 3-kappa-lambda antibodies were prepared and purified as follows.

1. Transient transfection and production

The kappa-lambda bispecific antibody was transiently produced in HEK293 EBNA cells using PEI mediated transfection method on the required vector as described below. HEK293 EBNA cells were cultured in CD CHO medium without serum suspension. For production in 500ml shake flasks, 4 hundred million HEK293 EBNA cells were seeded 24 hours prior to transfection (all amounts adjusted accordingly for alternative scales). For transfection, cells were centrifuged at 210 × g for 5 min and the supernatant was replaced with pre-warmed 20ml CD CHO media. The expression vector was mixed in 20ml of CD CHO medium to a final amount of 200. mu.g DNA. After addition of 540. mu.l PEI, the solution was vortexed for 15 seconds and subsequently incubated at room temperature for 10 minutes. The cells were then mixed with the DNA/PEI solution, transferred to 500ml shake flasks and incubated for 3 hours at 37 ℃ in an incubator with an atmosphere of 5% CO 2. After incubation, 160ml of F17 medium was added and the cells were cultured for 24 hours. 1 day after transfection, 1mM valproic acid and 7% feed 1 were added. After 7 days, the culture supernatant was collected, purified by centrifugation at 210 Xg for 15 minutes, the solution was sterile filtered (0.22 μm filter), and sodium azide was added to a final concentration of 0.01% w/v and maintained at 4 ℃.

2. Purification of

Kappa-lambda bispecific antibodies were purified in three steps using an affinity step specific for the kappa light chain followed by an affinity step specific for the lambda light chain and finally used to remove aggregates by size exclusion chromatography. The supernatant obtained from transient production was adjusted to pH8.0 (using 2M TRIS pH8.0) and applied to capture selection of kappa affinity matrix or HiTrap kappasselect, GE Healthcare with 5 column volumes (cv) of buffer a (50mM TRIS, 100mM glycine, 150mM NaCl, pH8.0) equilibrated with 1ml column volume (cv). After washing with 15cv buffer a, the protein was eluted in 25cv using a pH gradient to buffer B (50mM Tris, 100mM glycine, 150mM NaCl, pH 2.0). Fractions containing the protein of interest were pooled and the pH of the solution was adjusted to pH8.0 (2M Tris pH8.0 was used). The neutralized pooled fractions were applied to a capture selection lambda affinity matrix (now: HiTrap Lambda Fabselect, GE Healthcare, column volume; Capture) equilibrated with 5 column volumes (cv) of buffer A (50mM Tris, 100mM glycine, 150mM NaCl, pH8.0)(cv) ═ 1 ml). After washing with 15cv buffer A, the protein was eluted in 25cv to buffer B (50mM Tris, 100mM glycine, 150mM NaCl, pH2.0) using a pH gradient. Fractions containing the protein of interest were pooled and the pH of the solution was adjusted to pH8.0 (2M Tris pH8.0 was used). The solution was concentrated using a super concentrator (Vivaspin 15R 30.000 MWCO HY, Sartorius) and subsequently applied to Superdex equilibrated with 20mM histidine, pH 6.0,140mM NaCl, 0.01% Tween-20 ^TM20010/300 GL (GE healthcare). The combined fractions after size exclusion were again concentrated using a super concentrator (Vivaspin 15R 30.000 MWCO HY, Sartorius).

The protein concentration was determined by measuring the Optical Density (OD) at 280nm using the molar extinction coefficient calculated based on the amino acid sequence. The purity and molecular weight of the constructs were analyzed by SDS capillary electrophoresis in the presence and absence of reducing agents according to the manufacturer's instructions (apparatus Caliper LabChipGX, Perkin Elmer). Only a small amount of protein could be purified, with a final yield of 0.17 mg/L.

Example 30

T cell mediated killing of bispecific FolR1/CD 3-kappa-lambda antibodies

The activity of κ λ FolR1 TCB was tested on SKOV3 cells in the presence of freshly isolated PBMC. DP47 TCB was included as a negative control. T cell mediated killing of SKOV3 cells was determined by LDH release after 24 hours and 48 hours. After 48 hours, T cells were harvested and measured by flow cytometry for CD69 and CD25 upregulation on CD 4T cells and CD 8T cells.

The κ λ FolR1 construct induced killing of SKOV3 cells in a concentration-dependent manner, which was accompanied by upregulation of CD69 and CD25 on CD 4T cells and CD 8T cells.

SKOV3 cells were incubated with PBMCs in the presence of either κ λ FoLR1 TCB or DP47 TCB. Tumor cell killing was determined by measuring LDH release after 24 hours and 48 hours (fig. 21). SKOV3 cells were incubated with PBMCs in the presence of either κ λ FoLR1 TCB or DP47 TCB. After 48 hours, upregulation of CD25 and CD69 on CD 4T cells and CD 8T cells was measured by flow cytometry (figure 22).

Example 31

Biochemical characterization of 16D5 and 36F2 FolR1 binding agents by surface plasmon resonance

The binding of anti-FolR 116D 5 and anti-FolR 136F 2 as IgG or T cell bispecific molecules to recombinant human, cynomolgus monkey and mouse folate receptor 1 (both Fc fusions) in different monovalent or bivalent T cell bispecific formats was assessed by Surface Plasmon Resonance (SPR). All SPR experiments were performed at 25 ℃ on a Biacore T200 using HBS-EP as running buffer (0.01M HEPES pH 7.4,0.15M NaCl,3mM EDTA, 0.005% surfactant P20, Biacore, GE Healthcare).

1. Test molecules

The molecules used for the affinity and avidity assays are described in table 24.

TABLE 24 name and description of the 6 constructs used in SPR analysis

Name (R)	Description of the invention
		16D5 TCB	2+ 1T-cell bispecific, reverse format (common light chain)
16D5 TCB classic	2+ 1T-cell bispecific, classical format (common light chain)
		16D5 TCB 1+1	1+ 1T-cell bispecific (common light chain)
16D5 TCB 1+1HT	1+ 1T-cell bispecific head-to-tail (common light chain)
		36F2 IgG	Human IgG1 with P329G LALA
36F2 TCB	2+ 1T-cell bispecific, inverted form, crossfab

2. Affinity for folate receptor 1

The affinity of the interaction between anti-FolR 1 IgG or T cell bispecific molecules and recombinant folate receptors was determined as described below (table 25).

Recombinant biotinylated monomeric Fc fusions of human, cynomolgus and mouse folate receptor 1(FolR1-Fc) were directly conjugated to SA chips using standard conjugation instructions (Biacore, GE Healthcare). The immobilization level was about 300-400 RU. anti-FL 1R1 IgG or T cell bispecific molecules were passed through the flow cell at a flow rate of 30 μ L/min for 180 seconds at a concentration in the range of 3.7 to 900 nM. Dissociation was monitored for 240 or 600 seconds. The chip surface was regenerated after each cycle using a 30 second double injection of 10mM glycine-HCl pH 2. Bulk refractive index differences were corrected by subtracting the response obtained on a reference flow cell immobilized with recombinant biotinylated murine CD134 Fc fusion. The binding curve resulting from bivalent binding of IgG or T cell bispecific molecules was approximated as 1:1 Langmuir binding (even though it was a 1:2 binding) and fitted to this model to give an apparent KD which represents the avidity of the bivalent binding. The apparent affinity constant for the interaction was derived from the fitted rate constant using Bia Evaluation software (GE Healthcare). For the 1+ 1T cell bispecific format, the interaction is actually 1:1, KD indicates affinity, since there is only one FolR1 binding agent in the construct.

TABLE 25 bivalent binding (with apparent KD avidity) of anti-FolR 116D 5 and 36F2 as IgG or as T cell bispecific molecule (TCB) on human, cynomolgus monkey and mouse FolR 1.

3. Affinity for folate receptor 1

The affinity of the interaction between anti-FolR 1IgG or T cell bispecific molecules and recombinant folate receptors was determined as described below (table 26).

For affinity measurements, direct coupling of approximately 12000 Resonance Units (RU) of anti-human Fab specific antibody (Fab capture kit, GE Healthcare) was performed on a CM5 chip at ph5.0 using a standard amine coupling kit (GE Healthcare). The anti-FolR 1IgG or T-cell bispecific molecules were captured at 20nM for 40 sec at a flow rate of 10. mu.l/min, with no capture of the reference flow cell. Dilution series (12.3 to 3000nM) of human, cynomolgus or mouse folate receptor 1Fc fusions were passed over all flow cells at 30. mu.l/min for 240 seconds to record binding periods. The dissociation period was monitored for 300 seconds and initiated by switching from the sample solution to HBS-EP. After each cycle, the chip surface was regenerated using a 60 second double injection of 10mM glycine-HCl pH 1.5. Bulk refractive index differences were corrected by subtracting the response obtained on the reference flow cell 1. Affinity constants for the interaction were derived from the rate constants by fitting 1:1 Langmuir binding using the Bia Evaluation software (GE Healthcare).

TABLE 26 monovalent binding (affinity) of anti-FolR 116D 5 and 36F2 on human, cynomolgus monkey and mouse FolR1 as IgG or as T cell bispecific molecule (TCB).

36F2 TCB had similar affinities (monovalent binding) for human and cynomolgus monkey FolR1-Fc, about 1000nM for both, while the affinity for mouse FolR1-Fc was slightly better and about 300 nM. 36F2 can be used in murine and primate models without the need for alternatives.

36F2 TCB had an affinity (apparent KD) for human FolR1 that was about 30-fold lower than 16D5 TCB for human FolR 1. In the bivalent form, 36F2 TCB was in the low nanomolar range, while 16D5 TCB was in the low picomolar range (1000 fold difference).

FolR1 is expressed on tumor cells, over-expressed at moderate and high levels on the surface of cancer cells in a variety of epithelial malignancies (including ovarian, breast, kidney, colorectal, lung and other solid cancers), and also expressed on the apical surface of a limited subset of polarized epithelial cells in normal tissues. These non-tumor normal cells express FolR1 only at low levels and include, for example, bronchiolar epithelial cells on the alveolar surface of the cells, the renal cortical luminal border of the renal tubule cells, the retinal pigment epithelium (basolateral membrane) and the choroid plexus.

16D5 TCB bound to normal tissue cells expressing low amounts of FolR1, resulting in T cell mediated killing thereof. This may at least partially explain the limited tolerance observed at 10 μ g/kg in cynomolgus monkeys. The inventors wanted to determine if decreasing the affinity of T cell bispecific molecules could increase differentiation between high and low target density tissues, thereby reducing toxicity by exploiting bivalent binding and avidity. Low affinity binders are generally not selected as suitable candidates for further analysis, as low affinity is often associated with low potency and efficacy. However, the low affinity FolR1 binding agent 36F2 was developed in several forms, characterized by its biological properties. For 36F2 used in the bivalent T cell bispecific format, the avidity effect (difference between monovalent and bivalent binding) was about 250-fold (1000nM versus 4 nM). At low target densities, affinity defines the interaction and results in low potency of TCB at 1000 nM. However, at high target densities, the avidity of the molecules worked and 4nM resulted in high activity of TCB (see example 32).

In an alternative approach, the inventors generated the 16D5 monovalent form and a low affinity variant of 16D5 (with an affinity of about 10-40nM) in a bivalent format. The 16D5 binding agent used in monovalent form (1+1) had an affinity of about 50 nM. Differentiation between high and low target density tissues can be better achieved by exploiting avidity effects.

Example 32

T cell killing of SKov-3 cells induced by 36F2 TCB, Mov19 TCB and 21A5 TCB

T cell killing mediated by 36F2 TCB, Mov19 TCB and 21A5 TCB was assessed on SKov-3 cells (FolR1 medium). Human PBMC were used as effectors and killing was detected at 24 and 48 hours of incubation with bispecific antibody. Briefly, target cells were harvested with trypsin/EDTA, washed and plated with flat-bottom 96-well plates at a density of 25000 cells/well. Cells were left adherent overnight. Peripheral Blood Mononuclear Cells (PBMCs) were prepared by Histopaque density centrifugation of enriched lymphocyte preparations (buffy coats) obtained from healthy human donors. Fresh blood was diluted with sterile PBS and layered on a Histopaque gradient (Sigma, # H8889). After centrifugation (450 Xg, 30 min, room temperature), plasma above the interface containing PBMC was discarded, PBMC transferred to a new falcon tube and subsequently filled with 50ml PBS. The mixture was centrifuged (400 Xg, 10 min, room temperature), the supernatant discarded, and the PBMC pellet washed twice with sterile PBS (centrifugation step 350 Xg, 10 min). The resulting PBMC population (ViCell) was counted automatically and stored in RPMI1640 medium containing 10% FCS and 1% L-alanyl-L-glutamine (Biochrom, K0302) at 37 ℃ in a cell culture incubator with 5% CO2 until further use (no longer than 24 hours). For the killing assay, antibodies were added at the indicated concentrations (range of 0.005pM-5nM, in triplicate). PBMCs were added to the target cells at a final E: T ratio of 10: 1. By quantifying LDH released into the cell supernatant by apoptotic/necrotic cells (LDH detection kit, Roche Applied Science, # 11644793001), at 37 ℃, 5% CO ₂Target cell killing was assessed after 24 and 48 hours of incubation. Maximal lysis of target cells was achieved by incubating the target cells with 1% Triton X-100 (═ 100%). Minimal lysis (═ 0%) refers to target cells co-incubated with effector cells without bispecific constructs.

The results show that killing by 36F2 was greatly reduced compared to Mov19 TCB and 21a5 TCB (fig. 23A-B). EC50 values calculated using GraphPadPrism6 in relation to the killing test are summarized in table 27.

TABLE 27 EC50 values (pM) for T cell-mediated killing of FolR 1-expressing SKov-3 cells induced by 36F2 TCB, Mov19 TCB and 21A5 TCB.

Curve does not reach saturation, values are assumed

Example 33

T cell killing induced by different monovalent and bivalent T cell bispecific versions of 36F2 TCB and 16D5 TCB

T cell killing mediated by human tumor cells was evaluated by 36F2 TCB, 16D5 TCB, 16D5 TCB classical, HeLa 16D5 TCB 1+1 and 16D5 TCB HT antibodies (high FolR1, about 200 ten thousand copies, Table 14, FIG. 27), Skov-3 (medium FolR1, about 70000 90000 copies, Table 14, FIG. 27) and HT-29 (low FolR1, about 10000, Table 14, FIG. 27). DP47 TCB antibody was included as a negative control. Human PBMC were used as effectors and killing was detected upon incubation with bispecific antibody for 24 hours. Briefly, target cells were harvested with trypsin/EDTA, washed and plated with flat-bottom 96-well plates at a density of 25000 cells/well. Cells were left adherent overnight. Peripheral Blood Mononuclear Cells (PBMCs) were prepared by Histopaque density centrifugation of enriched lymphocyte preparations (buffy coats) obtained from healthy human donors. Fresh blood was diluted with sterile PBS and layered on a Histopaque gradient (Sigma, # H8889). After centrifugation (450 Xg, 30 min, room temperature), plasma above the interface containing PBMC was discarded, PBMC transferred to a new falcon tube and subsequently filled with 50ml PBS. The mixture was centrifuged (400 Xg, 10 min, room temperature), the supernatant discarded, and the PBMC pellet washed twice with sterile PBS (centrifugation step 350 Xg, 10 min). The resulting PBMC population was counted automatically (ViCell) and stored in RPMI1640 medium containing 10% FCS and 1% L-alanyl-L-glutamine (Biochrom, K0302) at 37 ℃ in a cell culture incubator with 5% CO2 until further use (no longer than 24 hours). For the killing assay, antibodies were added at the indicated concentrations (range 0.01pM to 100nM, in triplicate). PBMCs were added to the target cells at a final E: T ratio of 10: 1. By quantifying release into cell supernatant by apoptotic/necrotic cells LDH (LDH detection kit, Roche Applied Science, # 11644793001), 5% CO at 37 ℃₂Target cell killing was assessed after 24 hours incubation. Maximal lysis of target cells was achieved by incubating the target cells with 1% Triton X-100 (═ 100%). Minimal lysis (═ 0%) refers to target cells co-incubated with effector cells without bispecific constructs.

The results showed that target-specific killing was much weaker for all three FolR1+ target cell lines induced by 36F2 TCB compared to 16D5 TCB-induced killing (fig. 24A-C, table 29). Target-specific killing induced by monovalent 16D5 TCB (16D5 HT and 16D 51 +1) was poor compared to bivalent 16D5 TCB (16D5 TCB and 16D5 TCB classical). EC50 values calculated using GraphPadPrism6 associated with killing assays are summarized in table 28. Importantly, this data shows that the use of a bivalent 2+1 TCB form of 36F2 FOLR1 binding agent expanded the therapeutic window compared to 16D5 FOLR1 TCB (fig. 24A-C). Whereas the decrease in potency between 16D5 and 36F2 FOLR1 TCB was about 45-fold for Hela cells (high FOLR1 expression, see table 28:16D5 TCB ═ 0.8 versus 36F2 TCB 36.0), about 297-fold for Skov3 cells (moderate FOLR1 expression, see table 28:16D5 TCB ═ 0.6 versus 36F2 TCB 178.4), and almost 7000-fold for HT29 with low FOLR1 expression (see table 28:16D5 TCB ═ 5.7 versus 36F2 TCB 39573). Thus, 36F2 FOLR1 TCB distinguishes between high-and low-expressing cells, which is particularly important for reducing toxicity, since some normal non-tumor tissue cells express very low levels of FOLR1 (approximately less than 1000 copies per cell). Consistent with this observation, the results discussed in example 35 below show that 36F2 TCB did not induce T cell killing of primary cells (fig. 26A-D), whereas some killing was observed on hrceipic and HRPEpiC cells after 48 hours of incubation for 16D5 TCB (fig. 26B and C). This important feature of 36F2 TCB allows for administration for the treatment of FolR1 positive tumors so that it mediates effective killing of tumor tissues with high or moderate FolR1 expression rather than normal tissues with low (partially polarized) expression. Notably, this feature appears to be mediated by the avidity of the bivalent 2+1 inverted form of 36F2 TCB, as it was not observed when using the 1+1 monovalent form carrying the same low affinity 36F2 binding agent.

In other words, the bivalent 2+1 form of 36F2 TCB included a relatively low affinity FolR1 binding moiety, but it had an avidity effect that allowed differentiation between high and low FolR1 expressing cells. Because tumor cells express FolR1 at high or moderate levels, the TCB selectively binds to tumor cells, but not to normal, non-cancerous cells that express low levels of FolR1 or do not express FolR1 at all

In addition to the advantageous features described above, the divalent 2+1 inverted form of 36F2 TCB has the advantage of not requiring chemical crosslinking or other mixing methods. This makes it suitable for the preparation of a medicament for the treatment of patients, for example patients with FolR1 positive cancerous tumours. The divalent 2+1 inverted form of 36F2 TCB can be prepared using standard CHO methods with oligomeric aggregates. Furthermore, 36F2 TCB in bivalent 2+1 contains human and humanized sequences, making it superior to molecules using rat and mouse polypeptides, which are highly immunogenic when administered to humans. In addition, the divalent 2+1 form of 36F2 TCB was engineered to eliminate FcgR binding, thus not causing FcgR cross-linking and infusion reactions, further enhancing its safety when administered to patients.

As demonstrated by the above results, its head-to-tail geometry makes the bivalent 2+1 inverted form of 36F2 TCB a highly potent molecule for inducing absolute target cell killing. Its bivalent character enhances avidity and potency, but also allows differentiation between high-and low-expressing cells. Due to its avidity, its preference for high-or medium-target expressing cells reduces the toxicity of T cell-mediated killing of normal cells that express FolR1 at low levels.

Another advantage of the bivalent 2+1 form and the 36F2 TCB in other embodiments disclosed herein is that their clinical development does not require the use of alternative molecules, as they bind to human, cynomolgus monkey and mouse FolR 1. Thus, the molecules disclosed herein recognize a different epitope than the previously described antibodies to FolR1, which do not recognize FolR1 from all three species.

TABLE 28 EC50 values (pM) for T cell-mediated killing of FolR 1-expressing tumor cells induced by different monovalent and bivalent T cell bispecific versions of 36F2 TCB and 16D5 TCB after 24 h incubation.

Curve does not reach saturation, only assumed values

Table 29 shows a comparison of EC50 values for 16D5 TCB and 36F2 TCB on the different cell lines tested. In addition to the EC50 values obtained, Δ (EC 50 for 16D5 TCB minus EC50 for 36F2 TCB) and x-fold difference (EC 50 for 16D5 TCB divided by EC50 for 36F2 TCB) were calculated.

TABLE 29 comparison of EC50 values for 16D5 TCB and 36F2 TCB.

Curves do not reach saturation, only assumed values

The calculated EC50 values clearly show that the greater the difference between 36F2 TCB and 16D5 TCB, the lower the FolR1 expression on the target cells.

The same calculations were made for the comparison of EC50 values for 16D5 TCB and 36F2 TCB versus 16D5 TCB and two monovalent 16D5 TCBs (16D5 TCB HT and 16D 51 + 1). Tables 30 and 31 show a comparison of EC50 values for 16D5 TCB versus 16D5 TCB HT (table 30) and 16D5 TCB versus 16D5 TCB 1+1 (table 31) and the corresponding Δ (EC 50 for 16D5 TCB minus EC50 for 16D5 TCB HT/1+ 1) and x fold difference (EC 50 for 16D5 TCB divided by EC50 for 16D5 TCB HT/1+ 1).

TABLE 30 comparison of EC50 values for 16D5 TCB (2+1 inverse) and 16D5 TCB HT.

TABLE 31 comparison of EC50 values for 16D5 TCB and 16D5 TCB 1+ 1.

Curves do not reach saturation, only assumed values

Comparison of EC50 values for 16D5 TCB and 36F2 TCB (table 29) shows that the greater the difference in EC50 values, the lower the expression of FolR1 on target cells. This effect was not seen in the comparison of 16D5 TCB and monovalent 16D5 TCB (tables 29 and 30). For 16D5 TCB 1+1 (table 31), the difference between EC50 and 16D5 TCB 1+1 for 16D5 TCB increased slightly, while FolR1 expression decreased, but was much less pronounced than seen in the comparison of 16D5 TCB and 36F2 TCB.

Example 34

CD8 following T-cell killing of FolR 1-expressing tumor cells induced by the 36F2 TCB and 16D5 TCB antibodies⁺And CD4⁺Upregulation of CD25 and CD69 on effector cells

CD8 after T cell killing of FolR 1-expressing Hela, SKov-3 and HT-29 tumor cells mediated by 36F2 TCB and 16D5 TCB was assessed by FACS analysis using antibodies recognizing the T cell activation markers CD25 (late activation marker) and CD69 (early activation marker)⁺And CD4⁺Activation of T cells. DP47 TCB was included as a non-binding control. Antibody and killing assay conditions were essentially as described above (example 32), using the same antibody concentration range (0.01pM to 100nM, in triplicate), an E: T ratio of 10:1, and an incubation time of 48 hours.

After incubation, PBMCs were transferred to round bottom 96 well plates, centrifuged at 400 × g for 4 minutes, and washed twice with PBS containing 0.1% BSA. CD8(PE anti-human CD8, BD #555635), CD4(Brilliant Violet 421)^TMSurface staining of anti-human CD4, Biolegend #300532), CD69(FITC anti-human CD69, BD #555530) and CD25 (APC-anti-human CD25BD #555434) was performed according to the manufacturer's instructions. Cells were washed twice with 150. mu.l/well PBS containing 0.1% BSA. After centrifugation, the samples were resuspended in 200. mu.l/well PBS 0.1% for FACS measurement. Samples were analyzed in a BD FACS Canto II.

36F2 TCB induced CD8 after killing Hela (FIG. 25A) and SKov-3 (FIG. 25B) cells⁺And CD4⁺Target-specific upregulation of activation markers (CD25, CD69) on T cells. And 16D5 TCB in contrast, CD8 induced by 36F2⁺And CD4⁺Upregulation of CD25 and CD69 on T cells was much weaker.

On HT-29 (low FolR1), upregulation of the activation marker was only seen at the highest concentration of 36F2 TCB. In contrast, 16D5 TCB upregulation of CD25 and CD69 has been seen at much lower antibody concentrations (fig. 25C).

It can also be seen in tumor lysis experiments that post-killer T cells (CD 4)⁺And CD8⁺) The analysis of the activation markers (CD25 and CD69) above clearly showed that the greater the difference between 16D5 TCB and 36F2 TCB becomes, the lower the level of FolR1 expression on the target cells.

Example 35

T cell killing of primary cells induced by 36F2 TCB and 16D5 TCB

T cell killing mediated by 36F2 TCB and 16D5 TCB was assessed on primary cells (human renal cortical epithelial cells (HRCEPIC) (science cell Research Laboratories; Cat No 4110) and human retinal pigment epithelial cells (HRPEPIC) (science cell Research Laboratories; Cat No 6540)). HT-29 cells (low FolR1) were included as a control cell line. DP47 TCB served as a non-binding control. Human PBMC were used as effectors and killing was detected at 24 and 48 hours of incubation with bispecific antibody. Briefly, target cells were harvested with trypsin/EDTA, washed and plated with flat-bottom 96-well plates at a density of 25000 cells/well. Cells were left attached overnight. Peripheral Blood Mononuclear Cells (PBMCs) were prepared by Histopaque density centrifugation of enriched lymphocyte preparations (buffy coats) obtained from healthy human donors. Fresh blood was diluted with sterile PBS and layered on a Histopaque gradient (Sigma, # H8889). After centrifugation (450 Xg, 30 min, room temperature), plasma above the interface containing PBMC was discarded, and PBMC were transferred to a new falcon tube, followed by filling with 50ml of PBS. The mixture was centrifuged (400 Xg, 10 min, room temperature), the supernatant discarded and the PBMC pellet washed twice with sterile PBS (centrifugation step 350 Xg, 10 min). The resulting PBMC populations (ViCell) were counted automatically and stored in RPMI1640 medium containing 10% FCS and 1% L-alanyl-L-glutamine (Biochrom, K0302) at 37 ℃ in a cell incubator with 5% CO2 until further Used (no longer than 24 hours). For the killing assay, antibodies were added at the indicated concentrations (range 0.01pM-10nM, in triplicate). PBMC were added to the target cells at a final E: T ratio of 10: 1. By quantifying LDH released into the cell supernatant by apoptotic/necrotic cells (LDH detection kit, Roche Applied Science, # 11644793001), at 37 ℃, 5% CO₂Target cell killing was assessed after 24 and 48 hours of incubation. Maximal lysis of target cells was achieved by incubating the target cells with 1% Triton X-100 (═ 100%). Minimal lysis (═ 0%) refers to target cells co-incubated with effector cells without bispecific constructs.

The results show that 36F2 TCB did not induce T cell killing of primary cells (fig. 26A-D), whereas some killing could be observed on hrceipic and HRPEpiC cells after 48 hours of incubation for 16D5 TCB (fig. 26B and D). As described above, a strong difference in T cell killing between HT-29 cells was observed between 16D5 TCB and 36F2 TCB (fig. 26E, F).

Example 36

Preparation of DP47 GS TCB

(2+1 Crossfab-IgG P329G LALA reverse ═ non-targeting TCB')

The "non-targeted TCB" was used as a control in the above experiments. Bispecific antibodies engage CD3e but do not bind to any other antigen and therefore are unable to cross-link T cells to any target cell (and subsequently are unable to induce any killing). Thus, it was used as a negative control in the assay to monitor any non-specific T cell activation. The untargeted TCB was prepared as described in WO 2014/131712. Briefly, the variable regions of the heavy and light chain DNA sequences have been subcloned in frame with the constant heavy chain or constant light chain previously inserted into the corresponding recipient mammalian expression vector. Antibody expression is driven by the MPSV promoter and carries a synthetic poly a signal sequence at the 3' end of the CDS. In addition, each vector contains the EBV OriP sequence.

The molecule was produced by co-transfecting HEK293-EBNA cells with mammalian expression vectors using polyethylenimine. Cells were transfected with the corresponding expression vectors at a ratio of 1:2:1:1 ("vector heavy chain Fc (pore):" vector light chain ":" vector light chain Crossfab ":" vector heavy chain Fc (knob) -fabrosfab ").

For transfection, HEK293 EBNA cells were cultured in CD CHO media in suspension and serum free. For production in 500ml shake flasks, 4 hundred million HEK293 EBNA cells were seeded 24 hours prior to transfection. For transfection, cells were centrifuged at 210 × g for 5 minutes and the supernatant was replaced with pre-warmed 20ml CD CHO medium. The expression vector was mixed in 20ml of CD CHO medium to a final amount of 200. mu.g DNA. After addition of 540. mu.l PEI, the solution was vortexed for 15 seconds and subsequently incubated at room temperature for 10 minutes. The cells were then mixed with the DNA/PEI solution, transferred to 500ml shake flasks and incubated for 3 hours at 37 ℃ in an incubator with an atmosphere of 5% CO 2. After incubation, 160ml of F17 medium was added and the cells were cultured for 24 hours. 1 day after transfection, 1mM valproic acid and 7% feed 1 were added. After 7 days of incubation, the supernatant was collected, purified by centrifugation at 210 Xg for 15 minutes, the solution was sterile filtered (0.22 μm filter), and sodium azide was added to a final concentration of 0.01% w/v and maintained at 4 ℃.

Secreted proteins were purified from cell culture supernatants by affinity chromatography using protein a. The supernatant was loaded onto a HiTrap protein a HP column (CV 5mL, GE Healthcare) equilibrated with 40mL of 20mM sodium phosphate, 20mM sodium citrate, 0.5M sodium chloride, ph 7.5. Unbound protein was removed by washing with at least 10 column volumes of 20mM sodium phosphate, 20mM sodium citrate, 0.5M sodium chloride, pH 7.5. The target protein was eluted in a gradient from 20mM sodium citrate, 0.5M sodium chloride, pH7.5 to 20mM sodium citrate, 0.5M sodium chloride, pH2.5 in 20 column volumes. The protein solution was neutralized by the addition of 1/10 of 0.5M sodium phosphate (pH 8). The target protein was concentrated and filtered and then loaded on a HiLoad Superdex 200 column (GE Healthcare) equilibrated with 20mM histidine, 140mM sodium chloride solution, pH 6.0.

The protein concentration of the purified protein sample was determined by measuring the Optical Density (OD) at 280nm using the molar extinction coefficient calculated based on the amino acid sequence.

Size exclusion columns (Tosoh) were analyzed using TSKgel G3000 SW XL to analyze the aggregate content of antibody samples at 25 ℃ in 25mM K2HPO4,125mM NaCl,200mM L-arginine monohydrochloride, 0.02% (w/v) NaN3, pH 6.7 running buffer.

TABLE 32 summary of DP47 GS TCB production and purification.

TABLE 33 CE-SDS analysis of DP47 GS TCB.

Example 37

Binding of 16D5 TCB and 9D11 TCB and their corresponding CD3 deamidation variants N100A and S100aA to CD 3-expressing Jurkat cells

Binding of 16D5 TCB and the corresponding deamidated variants of CD3 16D5 TCB N100A and 16D5 TCB S100aA and 9D11 TCB and deamidated variants 9D11 TCB N100A and 9D11 TCB S100aA to human CD3 was assessed on an immortalized T lymphocyte cell line (Jurkat) expressing CD 3. Briefly, cells were collected, counted, examined for viability, and tested at 2 × 10⁶Individual cells/ml were resuspended in FACS buffer (100. mu.l PBS 0.1% BSA). Mu.l of cell suspension (containing 0.2X 10) at different concentrations of bispecific antibody (686pM-500nM)⁶Individual cells) were incubated in a round bottom 96 well plate at 4 ℃ for 30 minutes. After two washes with cold PBS 0.1% BSA, the samples were incubated with PE-conjugated AffiniPure F (ab') 2 fragment goat anti-human IgG Fcg fragment-specific secondary antibody (Jackson Immuno Research Lab # 109-. After washing the samples twice with cold PBS 0.1% BSA, FACS CantoII (Software FACS Diva) was used Samples were analyzed immediately by FACS. Binding curves were obtained using GraphPadPrism6 (fig. 28A-B).

The results showed reduced binding of deamidated variants N100A and S100aA to CD3 compared to the parent antibodies 16D5 TCB (fig. 28A) and 9D11 TCB (fig. 28B).

Example 38

T cell killing of SKov-3 and HT-29 cells induced by 16D5 TCB and 9D11 TCB and their CD3 deamidation variants N100A and S100aA

T cell killing mediated by 16D5 TCB and the corresponding CD3 deamidated variants 16D5 TCB N100A and 16D5 TCB S100aA and 9D11 TCB and deamidated variants 9D11 TCB N100A and 9D11 TCB S100aA were evaluated on SKov-3 (medium FolR1) and HT-29 (low FolR1) cells. Human PBMC were used as effectors and killing was detected when incubated with bispecific antibodies for 24 hours. Briefly, target cells were harvested with trypsin/EDTA, washed and plated with flat-bottom 96-well plates at a density of 25000 cells/well. Cells were left attached overnight. Peripheral Blood Mononuclear Cells (PBMCs) were prepared by Histopaque density centrifugation of enriched lymphocyte preparations (buffy coats) obtained from healthy human donors. Fresh blood was diluted with sterile PBS and layered on a Histopaque gradient (Sigma, # H8889). After centrifugation (450 Xg, 30 min, room temperature), plasma above the interface containing PBMC was discarded, and PBMC were transferred to a new falcon tube, followed by filling with 50ml of PBS. The mixture was centrifuged (400 Xg, 10 min, room temperature), the supernatant discarded, and the PBMC pellet washed twice with sterile PBS (centrifugation step 350 Xg, 10 min). The resulting PBMC population (ViCell) was counted automatically and stored in RPMI1640 medium containing 10% FCS and 1% L-alanyl-L-glutamine (Biochrom, K0302) at 37 ℃, 5% CO2 until further use (no longer than 24 hours). For the killing assay, antibodies were added at the indicated concentrations (range 0.01pM to 10nM, in triplicate). PBMC were added to the target cells at a final E: T ratio of 10: 1. By quantifying LDH released into the cell supernatant by apoptotic/necrotic cells (LDH detection kit, Roche Applied Science, # 11644793001), at 37 ℃, 5% CO ₂Target cell killing was assessed after 24 hours of incubation. Maximization of target cells by incubating target cells with 1% Triton X-100Cleavage (═ 100%). Minimal lysis (═ 0%) refers to target cells co-incubated with effector cells without bispecific constructs.

The results show that CD3 deamidated variants 16D5 TCB N100A and 16D 5S 100aA induced killing on SKov-3 cells comparable to 16D5 TCB induced killing (fig. 29A). The same is true for 9D11 TCB and its variants 9D11 TCB N100A and 9D11 TCB S100aA (fig. 29B). On low-expressing HT-29 cells of FolR1, the S100aA variant showed impaired killing efficiency, for 16D5 TCB (fig. 30A) and for 9D11 TCB (fig. 30B). EC50 values calculated using GraphPadPrism6 associated with killing assays are given in table 35.

TABLE 35 EC50 values (pM) for T-cell mediated killing of FolR 1-expressing SKov-3 and HT-29 cells induced by 16D5 TCB and 9D11 TCB and their deamidated variants N100A and A100 aA.

Left undetermined

Example 39

Generation of mucin-1T cell bispecific constructs containing a common light chain

Gene synthesis

The desired gene fragment was synthesized by Geneart AG (Regensburg, Germany) from synthetic oligonucleotides and PCR products by an automated gene synthesis method.

Production and purification of MUC1 antigen

To generate a Common Light Chain (CLC) antibody to the "Hemicentrotus sperm protein, enterokinase and agrin" (SEA) domain of human mucin-1 (MUC1), a DNA fragment encoding the SEA domain was synthesized (Uniprot P15941, amino acids 1041-. To prevent autologous proteolysis of SEA between positions G1097 and S1098, the above method is described by Ligtenber et al (1992), Cell-associated epitalin a complex linking two proteins derived from a common precorsor J Biol Chem 267,6171-7. Parry et al, Identification of MUC1Proteolytic Cleavage Sites in vivo. biochem Biophys Res Commun 283,715-20, inserted four additional glycine residues between G1097 and S1098. This insertion results in a reduction of conformational stress and the protein remains intact. The DNA fragment was inserted into an inducible bacterial expression vector, pETXX. The resulting plasmid expresses an SEA domain with a C-terminal avi-tag and a His 6-tag (SEQ ID NO: 47). Purification was performed using the His6 tag (SEQ ID NO:47) when the avi tag was used for BirA-mediated in vivo biotinylation.

500ml of culture were inoculated with the bacterial strain BL21D3, transformed with the corresponding plasmid and the BirA-expressing plasmid, and OD with 1mM IPTG ₆₀₀Induction at 0.8. The culture was then incubated overnight at 25 ℃ and harvested by centrifugation. The bacterial pellet was taken up in 25mlProtein Extraction Reagent (Millipore) was resuspended and incubated at room temperature for 20 minutes as described in the protocol. After centrifugation at 16000Xg for 20 min, the supernatant was filtered and loaded onto an IMAC column (His gravitrap, GE Healthcare). The column was washed with 40ml of washing buffer (500mM NaCl, 20mM imidazole, 20mM NaH)₂PO₄pH 7.4) washing. From column (500mM NaCl, 500mM imidazole, 20mM NaH)₂PO₄pH 7.4), the eluate was re-buffered using a PD10 column (GE Healthcare).

Selection of anti-human MUC1 SEA domain binders from CLC Fab libraries

Selection for the SEA domain of human MUC1 was performed using e.coli derived and in vivo biotinylated MUC 1. The antigen was enzymatically biotinylated by biotin ligase BirA co-expressed via a C-terminal avi tag. The panning wheel was performed in solution according to the following pattern: 1. phagemid particles of the CLC library were bound to 100nM biotinylated antigenic protein for 0.5 h in a total volume of 1ml, 2. by adding 5.4X 10⁷Each streptavidin-coated magnetic bead captured biotinylated antigen and specifically attached bound phage for 10 minutes, 3. Wash with 5X1ml PBS/Tween20 and 5X1ml PBS Beads, 4. elution of phage particles by addition of 1ml 100mM Triethylamine (TEA) for 10 min and neutralization by addition of 500ul 1M Tris/HCl pH 7.4, 5. reinfection of log phase E.coli TG1 cells with phage particles in supernatant, infection with helper phage VCSM13 and subsequent PEG/NaCl precipitation of the phagemid particles to be used in subsequent selection rounds. Using a constant or decreasing (10)^- ⁷M to 2x10^-9M) antigen concentration for 3 rounds of selection. In round 2, antigen-phage complex capture was performed using neutravidin plates instead of streptavidin beads. Specific binders were identified by ELISA by coating 100. mu.l of biotinylated antigen 50nM per well on a neutravidin (neutravidin) plate. Fab-containing bacterial supernatants were added and bound Fab was detected by its Flag tag using anti-Flag/HRP secondary antibody. The VH domains of clones showing significant signal in the background were included for sequencing (58D6 VH,106D2 VH,110a5 VH) and further analysis. All clones were derived from IGHV3-23 germline sequence (FIG. 31). Notably, clone 58D6 and 110a5 were from libraries randomized in CDR3 only, while clone 106D2 was identified from libraries randomized in all 3 CDRs. Positions in CDRs 1 and 2 that deviate from the germline sequence are printed in italics. All VH variants were expressed in combination with the same common light chain (common light chain VL).

Purification of Fab

Fab from bacterial cultures (protein sequences of the variable heavy domain of 58D6VH, 106D2VH, 110A5VH, all clones expressing the same CLC variable domain as listed in SEQ ID NO: 31) were purified to accurately analyze kinetic parameters. For each clone, 500ml of culture was inoculated with bacteria containing the corresponding phagemid and treated with 1mM IPTG at OD₆₀₀Induction at 0.9. The cultures were then incubated overnight at 25 ℃ and harvested by centrifugation. After incubating the resuspended pellet in 25ml of PPB buffer (30mM Tris-HCl pH8,1mM EDTA, 20% sucrose) for 20 minutes, the bacteria were centrifuged again and the supernatant harvested. This incubation step was performed with 25ml of 5mM MgSO₄The solution was repeated once. The supernatants from both incubation steps were combined, filtered and loaded onto an IMAC column (His gravitrap, GE Healthcare). Subsequently, 40ml of washing buffer were used(500mM NaCl, 20mM imidazole, 20mM NaH)₂PO₄pH 7.4) washing the column. Elution (500mM NaCl, 500mM imidazole, 20mM NaH)₂PO₄pH 7.4), the eluate was re-buffered with a PD10 column (GE Healthcare). The kinetic parameters of the purified Fab were then studied by SPR analysis (Proteon XPR36, Biorad) in dilution lines ranging from 100nM to 6.25 nM.

Affinity assay by SPR Using ProteOn XPR36 biosensor from BioRad

The affinity (K) of selected Fab clones was measured by surface plasmon resonance at 25 ℃ using a ProteOn XPR36 instrument (Biorad)_D) And capture of biotinylated MUC1 antigen immobilized on NLC chip by neutravidin. Immobilization of recombinant antigen (ligand) antigen was diluted to 10. mu.g/ml with PBST (10mM phosphate, 150mM sodium chloride, pH 7.4, 0.005% Tween 20) and then injected at 30. mu.l/min at different contact times to achieve an immobilization level of about 200 Response Units (RU) in the vertical direction. Injection of analytes for a single kinetic measurement, the direction of injection was changed to horizontal, and a double dilution series of purified Fab (varying concentrations ranging between 100 and 6.25 nM) was injected simultaneously at 50 μ l/min along channels 1-5, respectively, with an association time of 250 or 300s and a dissociation time of 300 s. Buffer (PBST) was injected along the sixth channel to provide an "in-line" blank for reference. The association rate constant (k) was calculated in the ProteOn Manager v3.1 software using a simple one-to-one Langmuir binding model by simultaneous fitting of the association and dissociation sensorgrams _on) And dissociation rate constant (k)_off). Equilibrium dissociation constant (K)_D) Is calculated as k_off/k_onAnd (4) the ratio. Regeneration was carried out using 10mM glycine (pH 1.5) at a flow rate of 100ul/min in the horizontal direction with a contact time of 30 s. 3 clones bound specifically to MUC1 (fig. 32A), but did not bind specifically to the "in-line" blank, indicating the specificity of these binders. The kinetic and thermodynamic data for all measurements are summarized in fig. 32B.

MUC 1-based cloning, expression and characterization

All fabs that showed specific binding to MUC1 by SPR were converted to T Cell Bispecific (TCB) form. To do this, PCR-amplified DNA fragments of the heavy and light chain VH domains were inserted in-frame into the Fc-containing Ig chain required for TCB production (fig. 33). Antibody chain expression is driven by the MPSV promoter and transcription is terminated by a synthetic poly a signal sequence located downstream of the CDS. In addition to the expression cassette, each vector contains an EBV oriP sequence for autonomous replication in an EBV-EBNA expressing cell line. The resulting DNA construct was co-expressed in combination with CLC (clone 58D 6; clone 110A5) and purified from mammalian-derived cell culture supernatant (clone 58D6: clone 110A 5). A summary of the analytical data for the two bispecific antibodies is shown in fig. 34A and B.

Binding analysis of MUC1 specific TCB using a ProteOn XPR36 biosensor from BioRad

The binding of the generated MUC1 specific TCB was measured by surface plasmon resonance at 25 ℃ using a ProteOn XPR36 instrument (Biorad). Biotinylated MUC1 antigen and irrelevant biotinylated antigen were immobilized on NLC chips by neutravidin capture. Immobilization of the antigen was performed as described previously. For single kinetic measurements, the direction of injection was changed to horizontal, and two-fold dilution series of purified constructs (varying concentrations ranging between 30 and 1.88nM or between 100 and 6.25 nM) were injected simultaneously at 50 μ l/min along separate channels 1-5 with an association time of 120s and an dissociation time of up to 600 s. Buffer (PBST) was injected along the sixth channel to provide an "in-line" blank for reference.

Regeneration was carried out horizontally using 10mM glycine (pH 1.5) at a flow rate of 100ul/min for a contact time of 30 s. 3 clones bound specifically to MUC1 (FIG. 35), but failed to bind specifically to the "in-line" blank, indicating the specificity of these binders. Due to the affinity effect of the bivalent TCB form, very strong binding to MUC1 was observed. In contrast, no binding to an unrelated antigen was detected, indicating specific binding of the TCB construct.

Example 40

Generation of anti-BCMA antibodies

1.1 production of antigens and tool reagents

1.1.1 recombinant, soluble, human BCMA extracellular Domain

The extracellular domains of human, cynomolgus and mouse BCMA used as phage display selection antigens were transiently expressed as N-terminal monomeric Fc fusions in HEK EBNA cells and site-specifically biotinylated in vivo by co-expression of BirA biotin ligase at the avi tag recognition sequence located C-terminal to the Fc portion carrying the receptor chain (Fc knob chain). The extracellular domains of human, cynomolgus monkey and mouse BCMA comprise methionine 4 to asparagine 53, methionine 4 to asparagine 52 and alanine 2 to threonine 49, respectively. These were fused to the hinge N-terminus of human IgG1, and allowed to dimerize with the unfused human IgG1 Fc portion (pore chain) by knob-into-hole techniques.

To recover the extracellular domain of BCMA, the following primers were used:

AAGCTTGGATCCATGTTGCAGATGGCTGGGCAGTGCTCC-3(SEQ ID NO:48), which incorporates the BamH1 site (bold, underlined) and

reverse primer

5-GAATTCGCGGCCGCTCATCCTTTCACTGAATTGGTCACACTTGCATTAC-3(SEQ ID NO:49)

Primer 5-ACGTTAGATCTCCACTCAGTCCTGCATCTTGTTCCAGTTAAC-3(SEQ ID NO:50) and reverse primer 5-AACGTTGCGGCCGCTAGTTTCACAAACCCCAGG-3(SEQ ID NO:51)

GAATTCAAGCTTGCCACCATGTTGCAGATGGCTGGGCAGTGCTCC-3(SEQ ID NO:52) including a HindIII restriction site (bold, underlined) and a Kozak consensus sequence

And

reverse primer

5-GAATTCTCTAGATTACCTAGCAGAAATTGATTTCTCTATCTCCGTAGC-3(SEQ ID NO:53)

Gene synthesis can also be used to obtain the extracellular domain of BCMA.

1.2 BCMA expressing cells as tools

1.2.1 human myeloma cell line expressing BCMA on its surface

BCMA expression was assessed on four human myeloma cell lines (NCI-H929, RPMI-8226, U266B1 and L-363) by flow cytometry. NCI-H929 cells ((H929)CRL-9068^TM) Cultured in 80-90% RPMI 1640 with 10-20% heat-inactivated FCS and which may contain 2mM L-glutamine, 1mM sodium pyruvate and 50. mu.M mercaptoethanol. RPMI-8226 cells ((RPMI)CCL-155^TM) Cultured in medium containing 90% RPMI 1640 and 10% heat-inactivated FCS. Mixing U266B1((U266)TIB-196^TM) Cells were cultured in RPMI-1640 medium modified to contain 2mM L-glutamine, 10mM HEPES, 1mM sodium pyruvate, 4500mg/L glucose and 1500mg/L sodium bicarbonate and 15% heat-inactivated FCS. The L-363 cell line (Leibniz Institute DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH; DSMZ No. ACC49) was cultured in 85% RPMI 1640 and 15% heat-inactivated FCS. Briefly, cells were harvested, washed, counted for viability, resuspended at 50,000 cells/well in a 96-well round bottom plate, and incubated with 10 μ g/ml of anti-human BCMA antibody (Abcam, # ab54834, mouse IgG1) for 30min at 4 ℃ (to prevent internalization). Mouse IgG1 was used as isotype control (BD Biosciences, # 554121). The cells were then centrifuged (350Xg for 5 min), washed twice, and incubated with FITC-conjugated anti-mouse secondary antibody for 30min at 4 ℃. At the end of the incubation time, the cells were centrifuged (350xg 5 min), washed twice with FACS buffer, resuspended in 100 μ l FACS buffer, and analyzed on a cantonii device running FACS Diva software. Relative quantification of the number of BCMA receptors on surface membranes of H929, U266B1, RPMI-8226 and L-363 myeloma cell lines was assessed by QIFIKIT analysis (Dako, # K0078, according to the manufacturer's instructions). H929 cells express the highest density of human BCMA, 3.8-27 times higher than other myeloma cell lines. And as BCMA ^med/loU266 expressing myeloma cells and as BCMA^loH929 is considered BCMA in comparison to L363 and RPMI-8226 expressing myeloma cells^hi-expressing a myeloma cell line. Table 36 summarizes the cell surface expression of human multiple myeloma cell linesRelative BCMA receptor number.

TABLE 36 quantification of BCMA receptor number on cell surface of NCI-H929, U266B1, RPMI-8226 and L-363 human myeloma cell lines

1.3 obtaining BCMA binding Agents from in vitro recombinant libraries

1.3.1 construction of common light chain Fab-libraries

A Fab-format antibody library was constructed based on the humanized anti-CD 3 antibody light chain. Diversity is introduced only in the heavy chain. Six different heavy chain frameworks were used, VH1-46, VH1-69, VH3-15, VH3-23, VH4-59 and VH 5-1. CDRs 1,2 and 3 were randomized, each heavy chain library in combination with a non-randomized light chain. Such as Nissim et al EMBO J.1994 Feb 1; 13(3) 692-8, and Silaci et al. proteomics.2005Jun; 5(9) 2340-50 the library was generated as described.

1.3.2 selection of anti-BCMA Fab clones

Establishment of anti-BCMA Fab from a synthetic Fab library consisting of one constant VL and six different human VH sequences by phage display

TABLE 37 anti-BCMA clones and respective VL/VH pairings

Selection rounds (biopanning) were performed in solution according to 1) pre-clearing of each library pool in immune tubes coated with 10ug/ml of irrelevant human IgG-10 ¹²Phagemid particles to consume a library of antibodies recognizing the Fc-portion of the antigen; 2) non-Fc binding phagemid particles were incubated with 100nM biotinylated BCMA for 0.5 hours in the presence of 100nM of an unrelated non-biotinylated Fc knob-into-hole construct to further deplete Fc binding agent in a total volume of 2 ml; 3) capture was performed on a shaker by shunting and transferring the panning reactants to 16 wells of a neutravidin or streptavidin pre-coated microtiter plate for 20 minutesBiotinylated BCMA and specifically binding phage; 4) washing each well 10-30 times with PBS/Tween20 and 10-30 times with PBS using a plate washer; 5) a non-competitive wash step of selecting APRIL non-competitive phage antibodies by adding 230nM murine APRIL instead of Fab clones recognizing the natural ligand binding site; 6) the phage particles were eluted by adding 125ul 100mM TEA (triethylamine) per well for 5-10 minutes and neutralized by adding an equal volume of 1M Tris/HCl pH 7.4; 7) coli TG1 cells were re-infected at log phase with the eluted phage particles, infected with the helper phage VCSM13, incubated overnight at 30 ℃ on a shaker, followed by PEG/NaCl precipitation of the phagemid particles to be used in the next selection round.

3 to 5 rounds of selection were performed using a constant antigen concentration of 100 nM. In addition to selection exercises using only human BCMA as an antigen, further selection exercises were performed during which cynomolgus monkey or mouse BCMA was used alternately with human BCMA to select cross-reactive antibodies. Furthermore, as an alternative to streptavidin plate-based capture, by combining 5.4 × 10⁷A streptavidin-coated magnetic bead was added to the panning reaction, followed by a washing step using the respective magnet under the conditions described above, to capture the antigen-phage complex.

Fab-containing bacterial culture supernatants were screened for identification of specific binding agents by surface plasmon resonance using a BioRad ProteOn XPR36 biosensor. Briefly, after infection of log phase e.coli TG1 cells with eluted phage particles, single colony forming units (cfu) were plated and picked for inoculation of 1ml expression culture in 96-deep well plates. Fab was captured from the supernatant on a ProteOn GLM chip derivatized in the vertical direction with 8.000-10.000RU goat anti-human IgG, F (ab') 2 fragment specific polyclonal antibody (Jackson ImmunoResearch, # 109-. Subsequently, human, cynomolgus and mouse BCMA and an unrelated Fc knob-into-hole construct were injected in a horizontal direction as analyte. Clones that showed significant binding reactions to BCMA and did not bind the Fc portion of the antigen were expressed bacterially in 0.5 liter culture volumes, affinity purified and characterized kinetically by SPR analysis using a single kinetic protocol on the ProteOn XPR36 biosensor from BioRad.

Example 41

BCMA binding assay surface plasmon resonance

Binding of anti-BCMA antibodies to recombinant BCMA was assessed by Surface Plasmon Resonance (SPR) as follows. All SPR experiments were performed at 25 ℃ on a Biacore T200 using HBS-EP as running buffer (0.01M HEPES pH 7.4,0.15M NaCl,3mM EDTA, 0.005% surfactant P20, Biacore, Freiburg/Germany). The affinity of the interaction between anti-BCMA antibodies and recombinant BCMA Fc (kih) (human, cynomolgus monkey and mouse) was determined (tables 38-40). Biotinylated recombinant human, cynomolgus and mouse BCMA Fc (kih) were coupled directly onto SA chips according to the instructions (Biacore, Freiburg/Germany). The immobilization level ranged from 80 to 120 RU. anti-BCMA antibody was passed through the flow cell at a flow rate of 30 μ L/min at a 4-fold concentration range (1.95 to 500nM) over 120 seconds. Dissociation was monitored for 180 seconds. Bulk refractive index differences were corrected by subtracting the response obtained on the reference flow cell. Here, anti-BCMA antibody was run over previously activated empty surfaces and deactivated as described in standard amine conjugate kit. Despite the bivalent nature of the interaction for comparison purposes, the Biacore T200 evaluation software (v 1.0, Biacore AB, Uppsala/Sweden) was used to obtain the apparent kinetic constants in order to fit the rate equation for 1:1 Langmuir binding by numerical integration.

TABLE 38 affinity values for comparison only (experiment 1)

TABLE 39 affinity values for comparative purposes only (experiment 2)

TABLE 40 affinity values for comparative purposes only (experiment 3)

The affinity of the interaction between the anti-BCMA antibody or BCMA-TCB CLC antibody and recombinant human BCMA Fc (kih) was also determined. Anti-human Fab antibodies (GE Healthcare) were directly coupled to CM5 chips at pH5.0 using a standard amine coupling kit (Biacore, Freiburg/Germany). The fixed level was about 6500 RU. The anti-BCMA antibody or BCMA-TCB CLC antibody was captured at 25nM for 90 seconds. Recombinant human BCMA Fc (kih) was passed through the flow cell at a flow rate of 30 μ L/min at a 4-fold concentration range (1.95 to 500nM) over 120 or 180 seconds. Dissociation was monitored for 120 or 400 seconds. The bulk refractive index difference is corrected by subtracting the response obtained on the reference flow cell. Here, recombinant BCMA was flowed over the surface of the immobilized anti-human Fab antibody, and HBS-EP was injected thereon instead of the anti-BCMA antibody or BCMA-TCB CLC antibody. Kinetic constants were obtained using Biacore T200 evaluation software (v 1.0, Biacore AB, Uppsala/Sweden) to fit the rate equation for 1:1 Langmuir binding by numerical integration (table 41).

TABLE 41 affinity constants determined by the fitting Rate equation for 1:1 Langmuir binding

Example 42

Generation of bivalent anti-BCMA IgG antibodies with a common light chain

To verify the hypothesis that the use of a common light chain can be applied to any BCMA antibody, a 83a10-CLC bivalent BCMA antibody was generated by replacing the native light chain of bivalent BCMA antibody 83a10 previously described in WO/2014/122143 with a common light chain (CD3LC (CLC)). anti-BCMA antibodies comprising two heavy chains of 83a10 and two common light chains were prepared using the general techniques for generating bivalent IgG antibodies as described in the materials and general methods section. The affinity of 83a10-CLC IgG antibody to human BCMA was measured by SPR by a similar method to that described in example 41. Table 42 describes the binding of recombinant human BCMA Fc (kih) to 83a10-CLC BCMA IgG antibody.

TABLE 42 determination of 1:1Langmuir binding affinity constants by fitting the Rate equation binding of recombinant BCMA Fc (kih) to 83A10-CLC anti-BCMA antibody

Example 43

Specific detection of huTACI-R and huBAFF-R by anti-BCMA IgG antibodies

As a member of the TNF-TNF-R superfamily, the TACI and BAFF receptors are related to the BCMA receptor with 22% and 18.5% homology in the extracellular domain, respectively. Therefore, Surface Plasmon Resonance (SPR) binding experiments were performed to detect the specificity of anti-BCMA IgG antibodies. All SPR experiments were performed at 25 ℃ on Biacore T200(GE Healthcare) with HBS-EP as running buffer (0.01M HEPES pH 7.4,0.15M NaCl,3mM EDTA, 0.005% surfactant P20). Fc fused huBCMA, huBAFF-R and huTACI-R were immobilized at high levels at pH 5.0 on different flow channels of a Biacore CM5 sensor chip using a standard amine coupling kit (GE Healthcare) 5000UU) were chemically fixed. A high concentration solution (3. mu.M in HBS-EP) of anti-BCMA IgG pSCHLI372 and either a-huTACI-R IgG or a-huBAFF-R IgG was first injected as a positive control (binding time: 80s, dissociation time: 600s, flow rate: 30. mu.l/min) to examine whether binding occurred. Positive binding events of a-huTACI-R IgG to huTACI-R and hu-PAH-IgG to huBCFF-R and anti-BCMA IgG antibodies to huBCMA indicated that all receptors remained recognized after immobilization. For anti-BCMA IgG antibodies that bind to huBAFF-R and/or huTACI-R with a fast kinetic rate constant, at a novel CM5 sensorA closer examination of kinetic parameters with a low fixed level (300RU) was performed on the chip. Dilutions of anti-BCMA IgG antibody (binding time: 80s, dissociation time: 300s, flow rate: 30. mu.l/min) were injected at a concentration of 3000-93.75nM (2-fold dilution in HBS-EP) and the samples were tested in duplicate. Regeneration, i.e., without rapid and complete dissociation, may also be performed when applicable. Kinetic assessment of the interaction between anti-BCMA IgG antibodies and huBAFF-R or huTACI-R was performed by global fitting of the data to a 1:1 interaction model including mass transport terms (Biacore T200 assessment version 1.0). Steady state analysis was also performed. As shown in fig. 37, curve 1 corresponds to the signal on the reference channel, curve 2 corresponds to the channel where binding occurs (binding channel), and curve 2-1 is the subtracted signal (binding channel-reference channel), meaning that this is the signal due to the binding event. As shown in fig. 37, the SPR binding assay clearly demonstrated that pSCHLI372 IgG does not bind to human TACI receptors. As a positive control for binding, another BCMA IgG known to bind slightly to TACI receptor but with very fast binding and dissociation rates was used (data not shown).

Example 44

Production and purification of antibodies containing BCMA-TCB CLC Fc (2+1)

For the production of bispecific antibodies, bispecific antibodies were expressed by polymer-based transient co-transfection of the respective mammalian expression vectors in HEK293-EBNA cells, which were cultured in suspension. One day prior to transfection, HEK293-EBNA cells were seeded at 1.5Mo viable cells/mL in Ex-Cell medium supplemented with 6mM L-glutamine. For the final production volume of each ml, 2.0Mio viable cells were centrifuged (5 min at 210 Xg). The supernatant was aspirated and the cells were resuspended in 100. mu.L of CD CHO medium. DNA per mL final production volume was prepared by mixing 1 μ g of DNA (bispecific antibody production ratio: heavy chain: modified heavy chain: light chain: modified light chain ═ 1:1:2: 1; standard antibody ratio: heavy chain: light chain ═ 1:1 ratio) in 100 μ L of CD CHO medium. After addition of 0.27. mu.L PEI solution (1mg/mL), the mixture was vortexed for 15 seconds and allowed to stand at room temperature for 10 minutes. After 10 minutes, the resuspended cells and DNA/PEI mixture were brought together and transferred to a suitable container, which was then removedPut into a shaking apparatus (37 ℃, 5% CO)₂) In (1). After 3 hours incubation time, 800. mu.L of Ex-Cell medium supplemented with 6mM L-glutamine, 1.25mM valproic acid and 12.5% Pepsoy (50g/L) was added per ml of final production volume. After 24 hours, 70 μ L of feed solution was added per ml of final production. After 7 days or when the cell viability was equal to or lower than 70%, the cells were separated from the supernatant by centrifugation and sterile filtration. The antibody is purified by size exclusion chromatography using an affinity step and a refining step.

For the affinity step, the supernatant was loaded onto a Protein a column (HiTrap Protein a FF, 5mL, GE Healthcare) equilibrated with 6CV 20mM sodium phosphate, 20mM sodium citrate, ph 7.5. After a washing step with the same buffer, the antibody was eluted from the column with a stepwise elution of 20mM sodium phosphate, 100mM sodium chloride, 100mM glycine, pH 3.0. Fractions of the desired antibody were immediately neutralized with 0.5M sodium phosphate, pH8.0(1:10), combined and concentrated by centrifugation. The concentrate was sterile filtered and further processed by size exclusion chromatography.

For the size exclusion step, the concentrated protein was injected onto an XK16/60 HiLoad Superdex 200 column (GE Healthcare) and 20mM histidine, 140mM sodium chloride, pH 6.0, with or without Tween20 as formulation buffer. The fractions containing the monomer were combined, concentrated by centrifugation, sterile filtered into sterile vials.

The antibody concentration was determined by measuring the absorbance at 280nm using the theoretical value of the absorbance of a 0.1% antibody solution. The values are based on amino acid sequence and calculated by GPMAW software (Lighthouse data).

The purity and monomer content of the final protein preparation was determined by CE-SDS (Caliper LabChip GXII system (Caliper Life Sciences)) and HPLC (TSKgel G3000 SW XL analytical size exclusion column (Tosoh)) in 25mM potassium phosphate, 125mM sodium chloride, 200mM L-arginine monohydrochloride, 0.02% (w/v) sodium azide, pH6.7 buffer, respectively. FIGS. 38A-C depict CE-SDS images (unreduced (top) and reduced (bottom)) of final protein preparations after Protein A (PA) affinity chromatography and Size Exclusion Chromatography (SEC) purification steps. (FIG. 38B) pSCHLI333-TCB CLC (FIG. 38B) pSCHLI372-TCB CLC (FIG. 38C) pSCHLI373-TCB CLC. The PA affinity chromatography and SEC purification steps applied to the pSCHLI333-TCB CLC antibody resulted in monomer contents of 89.2% and 100% purity (FIG. 38A), the pSCHLI372-TCB CLC antibody was 88.5% and 100% purity (FIG. 38B), and the pSCHLI372-TCB CLC antibody was 91.8% and 100% purity (FIG. 38C). Table 43 summarizes the properties of the pSCHLI333-TCB CLC, pSCHLI372-TCB CLC, and pSCHLI373-TCB CLC antibodies after PA affinity chromatography and SEC purification steps. In all BCMA-TCB CLC antibodies > 88% purity and 100% monomer content was consistently achieved. The overall results clearly show that the use of a Common Light Chain (CLC) for TCB antibodies can achieve the advantages of production/purification features and that only two purification steps (i.e. PA affinity chromatography and SEC) are required to achieve an already high quality protein preparation, which has very good exploitability.

TABLE 43 production/purification Profile of BCMA-TCB CLC antibodies after protein A affinity chromatography and size exclusion chromatography purification steps

Example 44

Binding of BCMA-TCB CLC antibodies to BCMA positive multiple myeloma cell lines

Analysis of BCMA-TCB CLC antibody (pSCHLI333, pSCHLI372, pSCHLI373) expression by flow cytometry^hiBinding of human BCMA on H929 cells of (1). MKN45 (human gastric adenocarcinoma cell line that does not express BCMA) was used as a negative control. Briefly, cultured cells were harvested, counted and assessed for cell viability using ViCell. Viable cells were then adjusted to 2x10 per ml in FACS staining buffer (BD Biosciences) containing BSA⁶And (4) one cell. 100 μ l of this cell suspension was further aliquoted per well into round bottom 96 well plates and incubated with 30 μ l of BCMA-TCB CLC antibody or corresponding TCB control for 30 minutes at 4 ℃. All BCMA-TCB CLC antibodies (and TCB controls) were titrated and analyzed at a final concentration range of 0.12-500 nM. The cells were then centrifuged (5 min, 350 Xg), washed with 120. mu.l/well FACS staining buffer (BD Biosciences), resuspended, and conjugated with a fluorescent dye at 4 ℃The PE-conjugated AffiniPure F (ab') 2 fragment goat anti-human IgG Fc fragment-specific molecule (Jackson immune Research Lab; 109-116-170) was incubated for another 30 minutes. The cells were then washed twice with staining Buffer (Stain Buffer) (BD Biosciences), fixed using 100 μ l of BD fixation Buffer (# BD Biosciences, 554655) per well at 4 ℃ for 20 minutes, resuspended in 120 μ l FACS Buffer, and analyzed using BD FACS cantonii. As shown in FIGS. 39A-B, the mean fluorescence intensity of the BCMA-TCB CLC antibodies was plotted as a function of antibody concentration; (FIG. 39A) pSCHLI372-TCB CLC and pSCHLI373-TCB CLC on H929 cells; (FIG. 39B) pSCHLI372-TCB CLC and pSCHLI373-TCB CLC on MKN45 cells. BCMA-TCB CLC antibody (pSCHLI333, pSCHLI372, pSCHLI373) did not bind to BCMA-negative and CD 3-negative MKN45 cells at concentrations below 100 nM. EC50 was calculated using Prism GraphPad (LaJolla, CA, USA) where applicable, and EC50 values, which represent the antibody concentration required to achieve 50% of the maximum binding of anti-BCMA/anti-CD 3 TCB antibody to H929 cells, are summarized in table 44.

TABLE 44 EC50 values for BCMA-TCB CLC antibody binding to H929 cells

Example 45

Binding of BCMA-TCB CLC antibody to CD3 Positive Jurkat T cell line (flow cytometry)

The binding properties of BCMA-TCB CLC antibodies (pSCHLI333, pSCHLI372, pSCHLI373) to human CD3 expressed on human leukemia T cells Jurkat (ATCC TIB-152) were also analyzed by flow cytometry. Jurkat T cells were cultured in RPMI supplemented with 10% heat inactivated FCS. Briefly, cultured cells were harvested, counted and cell viability assessed using ViCell. Viable cells were then adjusted to 2x10 per ml in FACS staining buffer (BD Biosciences) containing 0.1% BSA⁶And (4) cells. 100. mu.l of this cell suspension was added per wellFurther aliquoting into round bottom 96-well plates. Mu.l of BCMA-TCB CLC antibody or the corresponding TCB control was added to the wells containing cells to obtain a final concentration of 0.12nM to 500 nM. BCMA-TCB CLC antibody and control IgG were used at the same molar concentration. After incubation at 4 ℃ for 30 min, the cells were centrifuged (5 min, 350 Xg), washed twice with FACS staining buffer (BD Biosciences) containing 150. mu.l/well BSA, then 100. mu.l BD immobilization buffer (# BD Biosciences, 554655) was used per well to immobilize the cells at 4 ℃ for 20 min, resuspended in 120. mu.l FACS buffer, and analyzed using BD FACS CantoII. The binding of BCMA-TCB CLC antibodies to T cells was evaluated and the median gated fluorescence intensity on Jurkat T cells expressing CD3 was determined and plotted as a histogram or dot plot. FIGS. 40A-B show median fluorescence intensity of BCMA-TCB CLC antibodies (pSCHLI333, pSCHLI372, pSCHLI373) bound to Jurkat T cells (FIG. 40A) or MKN45 cells (FIG. 40B) plotted as a function of antibody concentration. The EC50 values and maximum binding of the anti-BCMA/anti-CD 3 TCB antibody to CD3 positive Jurkat T cells was not achieved. The isotype control antibody did not bind to Jurkat T cells, BCMA-TCB CLC antibody (pSCHLI333, pSCHLI372, pSCHLI373) did not bind to BCMA negative and CD3 negative MKN45 cells at concentrations below 100 nM.

Example 46

Activation of human T cells when BCMA-TCB CLC antibodies bind to CD 3-positive T cells and BCMA-positive multiple myeloma cells

Evaluation of CD4 by flow cytometry in the presence or absence of MM cells expressing human BCMA⁺And CD8⁺Surface expression of the early activation marker CD69 or the late activation marker CD25 on T cells, the ability of BCMA-TCB CLC antibodies (pSCHLI372, pSCHLI373) to induce T cell activation was analyzed. Briefly, BCMA positive H929 cells were harvested with cell dissociation buffer, counted and checked for viability. Cells were conditioned to 0.3X10 in modified RPMI-1640 medium⁶(surviving) cells/ml, 100. mu.l of this cell suspension was transferred per well into a round bottom 96 well plate (as shown). Mu.l (diluted) BCMA-TCB CLC antibody was added to the wells containing the cells to obtain a final concentration of 0.012pM-100 nM. Isolation of human PBMC effector cells from fresh blood of healthy donors and use of modified RPMI-1640 medium adjusted to 6x10 per ml⁶(surviving) cells. 50 μ l of this cell suspension was added to each well of the assay plate to obtain a final E: T ratio of 10:1 PBMC to myeloma tumor cells. To analyze whether BCMA-TCB CLC antibodies were able to specifically activate T cells in the presence of target cells expressing human BCMA, wells containing 3 to 10nM of the respective BCMA-TCB CLC antibody molecule and PBMCs but no target cells were included. At 37 ℃ 5% CO ₂After 48 hours of incubation, cells were centrifuged (5 min, 350 Xg) and washed twice with 150. mu.l/well PBS containing 0.1% BSA. Surface staining of CD4 (mouse IgGl, K; clone RPA-T4), CD8 (mouse IgGl, K; clone HIT8 a; BD #555635), CD69 (mouse IgGl; clone L78; BD #340560) and CD25 (mouse IgGl, K; clone M-A251; BD #555434) was carried out at 4 ℃ for 30 min according to the supplier's recommendations. Cells were washed twice with 150. mu.l/well PBS containing 0.1% BSA and fixed for 15 min at 4 ℃ using 100. mu.l/well fixing buffer (BD # 554655). After centrifugation, samples were resuspended in 200 μ l/well PBS containing 0.1% BSA and analyzed using a FACS CantoII machine (Software FACS Diva). FIG. 41 depicts CD4 after 48 hours of incubation⁺And CD8⁺Expression levels of the early activation marker CD69 and the late activation marker CD25 on T cells (representative results from two independent experiments). pSCHLI372-TCB CLC and pSCHLI373-TCB CLC antibodies induced upregulation of CD69 and CD25 activation markers in a concentration-dependent and specific manner in the presence of BCMA positive target cells. No CD4 was observed when human PBMC were treated with DP47-TCB control antibody⁺And CD8⁺Activation of T cells, which indicates that despite binding to CD3 on T cells, T cell activation does not occur when TCB antibodies do not bind to BCMA positive target cells.

Example 47

BCMA expression induced by anti-BCMA/anti-CD 3T cell bispecific antibody^hiRedirected T cell toxicity of H929 myeloma cells (colorimetric LDH Release assay)

The potential of BCMA-TCB CLC antibodies (pSCHLI372, pSCHLI373) to induce T cell-mediated apoptosis in BCMA-high expressing MM cells when cross-linked constructs were bound to BCMA on the cells via antigen binding moieties was also analyzed. Briefly, human BCMA-expressing H929 multiple myeloma target cells were harvested with Cell Dissociation Buffer (Cell Dissociation Buffer), washed and resuspended in RPMI supplemented with 10% fetal bovine serum (Invitrogen). About 30,000 cells/well were seeded in round bottom 96-well plates and corresponding dilutions of the constructs were added to the desired final concentration (in triplicate); the final concentration ranged from 0.12pM to 100 nM. For proper comparison, all TCB constructs and controls were adjusted to the same molar concentration. Human total T cells (effectors) were added to the wells to obtain a final E: T ratio of 5: 1. When human PBMC were used as effector cells, a final E: T ratio of 10:1 was used. The negative control group is represented by effector cells or target cells only. As a positive control for human pan T cell activation, 1. mu.g/ml PHA-M (Sigma # L8902) was used. For normalization, cell death was induced by incubating target cells with a final concentration of 1% Triton X-100, and the maximal lysis of H929MM target cells was determined (═ 100%). Minimal lysis (═ 0%) was indicated by target cells incubated with effector cells only, i.e. without any T cell bispecific antibody. At 37 deg.C, 5% CO ₂Following 20-24 hours or 48 hours of incubation, LDH released into the supernatant from apoptotic/necrotic MM target cells was determined with an LDH detection kit (Roche Applied Science) according to the manufacturer's instructions. The percentage of LDH release was plotted against the concentration of BCMA-TCB CLC antibody in a concentration-response curve. EC 50 values were measured using Prism software (GraphPad) and determined as the TCB antibody concentration that resulted in 50% of the maximum LDH release. As shown in fig. 42A-B, BCMA-TCB CLC antibody, pSCHLI372-TCB CLC (fig. 42A, B) and pSCHLI373-TCB CLC (fig. 42B) induced concentration-dependent killing of BCMA-positive H929 myeloma cells as measured by LDH release. Killing of H929 cells was specific because the DP47-TCB control antibody that did not bind to BCMA positive target cells did not induce LDH release even at the highest tested concentration of 100 nM. Table 45 summarizes the EC 50 values for redirected T cell killing of BCMA positive H929 cells induced by BCMA-TCB CLC antibody.

TABLE 45 EC 50 values for redirected T cell killing of H929 cells induced by BCMA-TCB CLC antibody

anti-BCMA/anti-CD 3 TCB molecules	EC50(pM)	EC50(μg/ml)
			pSCHLI333-TCB CLC	980	0.19
pSCHLI372-TCB CLC (experiment 1)	450	0.08
			pSCHLI373-TCB CLC	1590	0.31
pSCHLI372-TCB CLC (experiment 2)	900	0.17

Example 48

BCMA expression induced by BCMA-TCB CLC antibodies ^med/loRedirected T cell toxicity (LDH release assay) of U266 myeloma cells of (1)

Analysis of BCMA-TCB CLC antibodies (pSCHLI372, pSCHLI373) when the crosslinked construct is bound to BCMA on a cell by an antigen-binding moiety^med/loThe ability to induce T cell-mediated apoptosis in MM cells. Briefly, human BCMA expressed by Cell Dissociation Buffer (Cell Dissociation Buffer) was harvested^med/loOf U266 multiple myelomaTarget cells were washed and resuspended in RPMI supplemented with 10% fetal bovine serum (Invitrogen). About 30,000 cells/well were seeded in round bottom 96-well plates and corresponding dilutions of the constructs were added to the desired final concentration (in triplicate); the final concentration ranged from 0.12pM to 100 nM. For proper comparison, all TCB constructs and controls were adjusted to the same molar concentration. Human total T cells (effectors) were added to the wells to obtain a final E: T ratio of 5: 1. When human PBMC were used as effector cells, a final E: T ratio of 10:1 was used. The negative control group is represented by effector cells or target cells only. As a positive control for human T cell activation, 1. mu.g/ml PHA-M (Sigma # L8902) was used. For normalization, the maximal lysis of MM target cells (═ 100%) was determined by inducing cell death by incubating the target cells with Triton X-100 at a final concentration of 1%. Minimal lysis (═ 0%) was indicated by target cells incubated with effector cells only, i.e. without any T cell bispecific antibody. At 37 ℃ 5% CO ₂Following 20-24 hours of incubation, LDH released into the supernatant from apoptotic/necrotic MM target cells was determined with an LDH detection kit (Roche Applied Science) according to the manufacturer's instructions. The percentage of LDH release was plotted against the concentration of BCMA-TCB CLC antibody in a concentration-response curve. EC 50 values were measured using Prism software (GraphPad) and determined as the TCB antibody concentration that resulted in 50% of the maximum LDH release. Table 46 summarizes the EC 50 values for redirected T cell killing of BCMA positive U266 cells induced by BCMA-TCB CLC antibody.

TABLE 46 EC 50 values for redirected T cell killing of U266 cells induced by BCMA-TCB CLC antibody

anti-BCMA/anti-CD 3 TCB molecules	EC50(pM)	EC50(μg/ml)
			pSCHLI372-TCB CLC	5700	1.1

Amino acid sequences of the exemplary embodiments

1) For binders used in common light chain, variable heavy chain

2) CD3 binder Common Light Chain (CLC)

3) CD3 binding agent, heavy chain

4) MUC1 Binder in common light chain form, variable heavy chain

5) BCMA binding agents used in common light chain form, variable heavy chain

6) Untargeted DP47

7) Exemplary target sequences

8) Nucleotide sequences of the exemplary embodiments

9) Additional common light chain proteins and nucleotide sequences

10) DNA sequence humanized CD3_CH2527(CDR/VH/VL)

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, the illustration and example should not be construed as limiting the scope of the invention. The disclosures of all patent and scientific literature cited herein are expressly incorporated by reference in their entirety.

Claims

1. A T cell activating bispecific antigen binding molecule comprising a first antigen binding moiety and a second antigen binding moiety, wherein the first antigen binding moiety comprises a first light chain, and wherein the first antigen binding moiety is capable of specifically binding an activating T cell antigen, wherein the activating T cell antigen is CD3, wherein the second antigen binding moiety comprises a second light chain, and wherein the second antigen binding moiety is capable of specifically binding a target cell antigen, wherein the target cell antigen is folate receptor 1(FolR1), wherein the amino acid sequences of the first and second light chains are identical, wherein the first light chain and the second light chain comprise the light chain CDR1 of SEQ ID NO:32, the light chain CDR2 of SEQ ID NO:33 and the light chain CDR3 of SEQ ID NO:34,

wherein the first antigen binding portion is Fab comprising the heavy chain CDR1 of SEQ ID NO. 37, the heavy chain CDR2 of SEQ ID NO. 38 and the heavy chain CDR3 of SEQ ID NO. 39; and is

Wherein the second antigen-binding moiety is a Fab and comprises:

-heavy chain CDR1 of SEQ ID No. 8, heavy chain CDR2 of SEQ ID No. 9, and heavy chain CDR3 selected from: NYYAGVTPFDY, NYYTGGSSAFDY the flow of the air in the air conditioner,

NYYLFSTSFDY,NYYIGIVPFDY,NYYVGVSPFDY,NFTVLRVPFDY,NYYIGVVTFDY,GEWRRYTSFDY,GGWIRWEHFDY,

TGWSRWGYMDY, GEWIRYYHFDY, VGWYRWGYMDY, or

-heavy chain CDR1 of SEQ ID No. 16, heavy chain CDR2 of SEQ ID No. 17, and heavy chain CDR3 selected from: PWEWSWYDY, PWEWSYFDY, PWEWAWFDY the flow of the air in the air conditioner,

PWEWAYFDY。

2. The T cell activating bispecific antigen binding molecule of claim 1, further comprising a third antigen binding moiety capable of specifically binding to a target cell antigen.

3. The T cell activating bispecific antigen binding molecule of claim 1, further comprising an Fc domain consisting of a first and a second subunit capable of stable binding.

4. The T cell activating bispecific antigen binding molecule of claim 1, wherein the first and second light chains comprise a kappa constant light chain domain.

5. The T cell activating bispecific antigen binding molecule of claim 1, wherein the first and second light chains comprise lambda constant light chain domains.

6. The T cell activating bispecific antigen binding molecule of claim 5, wherein the first and second light chains are human or humanized λ light chains.

7. The T cell activating bispecific antigen binding molecule of claim 6, wherein the first and second light chains are human or humanized λ 7 light chains.

8. The T cell activating bispecific antigen binding molecule of claim 1, wherein the first and second light chains comprise the amino acid sequence of SEQ ID No. 31.

9. The T cell activating bispecific antigen binding molecule of claim 8, wherein the first and second light chains comprise the amino acid sequence of SEQ ID No. 35.

10. The T cell activating bispecific antigen binding molecule of claim 1, wherein the antigen binding moiety capable of specifically binding FolR1 comprises a heavy chain comprising heavy chain CDR1 of SEQ ID NO. 16, heavy chain CDR2 of SEQ ID NO. 17 and heavy chain CDR3 of SEQ ID NO. 18.

11. The T cell activating bispecific antigen binding molecule of claim 1 comprising not more than one antigen binding moiety capable of specifically binding to an activating T cell antigen.

12. The T cell activating bispecific antigen binding molecule of claim 1, wherein the first and second antigen binding moieties are fused to each other, optionally via a peptide linker.

13. The T cell activating bispecific antigen binding molecule of claim 1, wherein the second antigen binding moiety is fused at the C-terminus of the heavy chain of the Fab to the N-terminus of the heavy chain of the Fab of the first antigen binding moiety.

14. The T cell activating bispecific antigen binding molecule of claim 1, wherein the first antigen binding moiety is fused at the C-terminus of the heavy chain of the Fab to the N-terminus of the heavy chain of the Fab of the second antigen binding moiety.

15. The T cell activating bispecific antigen binding molecule of claim 1 or 6, wherein the molecule further comprises an Fc domain consisting of a first and a second subunit capable of stable binding, and wherein the second antigen binding moiety is fused at the C-terminus of the heavy chain of the Fab to the N-terminus of the first or second subunit of the Fc domain.

16. The T cell activating bispecific antigen binding molecule of claim 1, wherein the molecule further comprises an Fc domain consisting of a first and a second subunit capable of stable binding, and wherein the first antigen binding moiety is fused at the C-terminus of the heavy chain of the Fab to the N-terminus of the first or second subunit of the Fc domain.

17. The T cell activating bispecific antigen binding molecule of claim 1, wherein the molecule further comprises an Fc domain consisting of a first and a second subunit capable of stable binding, and wherein the first antigen binding portion and the second antigen binding portion are each fused at the C-terminus of the heavy chain of the Fab to the N-terminus of one subunit of the Fc domain.

18. The T cell activating bispecific antigen binding molecule of claim 2, wherein the first, second and third antigen binding moiety are each Fab molecules comprising the same VLCL light chain.

19. The T cell activating bispecific antigen binding molecule of claim 2, wherein the first, second and third antigen binding moiety are each a Fab molecule comprising the light chain CDR1 of SEQ ID NO. 32, the light chain CDR2 of SEQ ID NO. 33 and the light chain CDR3 of SEQ ID NO. 34.

20. The T cell activating bispecific antigen binding molecule of claim 2, wherein the first, second and third antigen binding moiety are each a Fab molecule comprising a light chain comprising the amino acid sequence of SEQ ID No. 31.

21. The T cell activating bispecific antigen binding molecule of claim 2, wherein the molecule further comprises an Fc domain consisting of a first and a second subunit capable of stable binding, and wherein the third antigen binding moiety is fused at the C-terminus of the heavy chain of the Fab to the N-terminus of the first or second subunit of the Fc domain.

22. The T cell activating bispecific antigen binding molecule of claim 2, wherein the molecule further comprises an Fc domain consisting of a first and a second subunit capable of stable binding, and wherein the second and third antigen binding portion are each fused at the C-terminus of the heavy chain of the Fab to the N-terminus of one subunit of the Fc domain, and the first antigen binding portion is fused at the C-terminus of the heavy chain of the Fab to the N-terminus of the heavy chain of the Fab.

23. The T cell activating bispecific antigen binding molecule of claim 22, wherein the second and third antigen binding portion and Fc domain are part of an immunoglobulin molecule.

24. The T cell activating bispecific antigen binding molecule of claim 23, wherein the second and third antigen binding portion and Fc domain are part of an IgG class immunoglobulin.

25. The T cell activating bispecific antigen binding molecule of claim 2, wherein the first and third antigen binding moiety are each fused at the C-terminus of the heavy chain of the Fab to the N-terminus of one subunit of the Fc domain and the second antigen binding moiety is fused at the C-terminus of the heavy chain of the Fab to the N-terminus of the heavy chain of the Fab of the first antigen binding moiety.

26. The T cell activating bispecific antigen binding molecule of claim 25, wherein the first and third antigen binding portion and Fc domain are part of an immunoglobulin molecule.

27. The T cell activating bispecific antigen binding molecule of claim 25, wherein the first and third antigen binding portions and Fc domain are part of an IgG class immunoglobulin.

28. The T cell activating bispecific antigen binding molecule of claim 3, wherein the Fc domain is an IgG Fc domain.

29. The T cell activating bispecific antigen binding molecule of claim 28, wherein the Fc domain is IgG ₁Or IgG₄An Fc domain.

30. The T cell activating bispecific antigen binding molecule of claim 3, wherein the Fc domain is a human Fc domain.

31. The T cell activating bispecific antigen binding molecule of claim 3, wherein the Fc domain comprises a modification that facilitates binding of the first and second subunits of the Fc domain.

32. The T cell activating bispecific antigen binding molecule of claim 31, wherein in the CH3 domain of the first subunit of the Fc domain an amino acid residue is substituted with an amino acid residue having a larger side chain volume, thereby generating a protuberance within the CH3 domain of the first subunit that is positionable in a cavity within the CH3 domain of the second subunit, and an amino acid residue in the CH3 domain of the second subunit of the Fc domain is substituted with an amino acid residue having a smaller side chain volume, thereby generating a cavity within the CH3 domain of the second subunit, a protuberance within the CH3 domain of the first subunit being positionable in a cavity within the CH3 domain of the second subunit.

33. The T cell activating bispecific antigen binding molecule of claim 3, wherein native IgG is conjugated₁The Fc domain exhibits reduced binding affinity to an Fc receptor and/or reduced effector function as compared to the Fc domain.

34. The T cell activating bispecific antigen binding molecule of claim 3, wherein the Fc domain comprises one or more amino acid substitutions that reduce binding to an Fc receptor and/or effector function.

35. The T cell activating bispecific antigen binding molecule of claim 34, wherein the one or more amino acid substitutions are at one or more positions selected from the group consisting of L234, L235 and P329.

36. The T cell activating bispecific antigen binding molecule of claim 34, wherein each subunit of the Fc domain comprises three amino acid substitutions that reduce binding to an activating Fc receptor and/or effector function, wherein the amino acid substitutions are L234A, L235A and P329G.

37. The T cell activating bispecific antigen binding molecule of any one of claims 33 to 36, wherein the Fc receptor is an fey receptor.

38. The T cell activating bispecific antigen binding molecule of any one of claims 33 to 36, wherein the effector function is antibody dependent cell mediated cytotoxicity (ADCC).

39. The T cell activating bispecific antigen binding molecule of claim 1, wherein the first and second light chains are humanized light chains.

40. The T cell activating bispecific antigen binding molecule of claim 1, wherein the first and second light chain are capable of being used for more than two antigen binding moieties with different antigen specificities.

41. An isolated polynucleotide encoding the T cell activating bispecific antigen binding molecule of any one of claims 1 to 40 or a fragment thereof.

42. A polypeptide encoded by the isolated polynucleotide of claim 41.

43. A vector comprising the isolated polynucleotide of claim 41.

44. The vector of claim 43, wherein said vector is an expression vector.

45. A host cell comprising the isolated polynucleotide of claim 41 or the vector of claim 43 or 44.

46. A method of producing a T cell activating bispecific antigen binding molecule of any one of claims 1 to 40, comprising the steps of a) culturing a host cell of claim 45 under conditions suitable for expression of the T cell activating bispecific antigen binding molecule, and b) recovering the T cell activating bispecific antigen binding molecule.

47. A T cell activating bispecific antigen binding molecule prepared by the method of claim 46.

48. A pharmaceutical composition comprising the T cell activating bispecific antigen binding molecule of any one of claims 1 to 40 and a pharmaceutically acceptable carrier.

49. Use of the T cell activating bispecific antigen binding molecule of any one of claims 1 to 40 for the manufacture of a medicament for the treatment of a disease in an individual in need thereof, wherein the disease is a cancer selected from ovarian cancer, uterine cancer, non-small cell lung cancer, gastric cancer, breast cancer and renal cancer.