Multifunctional molecules that bind to t cells, and uses thereof for treating autoimmune diseases

Abstract

To provide improved therapies against autoimmune diseases that may be caused by autoreactive T cell receptor (TCR) clones.SOLUTION: Disclosed herein is a multifunctional molecule comprising one, two or all of: (i) an antigen binding domain that binds to a TCR variable beta chain (TCRBV) antigen; and (ii) an immune cell engager selected from e.g., NK cell engager, T cell engager, B cell engager, dendritic cell engager or macrophage engager); (iii) a cytokine molecule or a cytokine inhibitor molecule; and / or (iv) a cell death receptor signal enhancer. Also disclosed are nucleic acids encoding it, a method for producing the disclosed molecule and a method for treating autoimmune disease using the disclosed molecule.SELECTED DRAWING: None

Description

Technical Field

[0001] Related Applications This application claims priority to U.S. Provisional Application No. 62 / 808,713, filed Feb. 21, 2019, and U.S. Provisional Application No. 62 / 957,045, filed Jan. 3, 2020, the entire contents of each of which are incorporated herein by reference. Sequence Listing This application is electronically filed in ASCII format and includes a sequence listing that is incorporated herein by reference in its entirety. The ASCII copy, created on Feb. 19, 2020, is named E2070-7024WO SL.txt and is 1,519,578 bytes in size.

Background Art

[0002] Antigen recognition via T cells depends on the interaction between the T cell receptor (TCR) and the major histocompatibility complex (MHC). The heterodimeric TCR consists of a combination of an α-chain and a β-chain (αβTCR) expressed by most T cells, i.e., the γδ-chain (γδTCR) is present in only about 1-5% of T cells. A highly diverse TCR repertoire is a fundamental property of an effective immune system. However, the immune repertoire can change significantly with the onset and progression of diseases such as cancer, autoimmunity, inflammation, and infectious diseases.

[0003] Autoimmunity can result from abnormal regulation of the immune system. This can be caused by autoreactive TCR clones that attack the patient's own cells. Improved therapies for autoimmune diseases are needed.

Summary of the Invention

Means for Solving the Problems

[0004] The present disclosure relates in particular to novel multispecific or multifunctional molecules comprising (i) an antigen-binding domain that binds to a TCR variable beta chain (TCRBV) antigen on a T cell (e.g., a TCRBV antigen corresponding to a biased TCRBV clonotype); (ii) an immune cell engager (e.g., selected from an NK cell engager, a T cell engager, a B cell engager, a dendritic cell engager, or a macrophage cell engager); (iii) a cytokine molecule; and / or (iv) one, two or all of a stromal modifying moiety. The terms “multispecific” or “multifunctional” are used interchangeably herein.

[0005] Without being bound by theory, TCR bias may be present in autoimmune diseases. This bias may be associated with dominant autoreactive TCR clones that cause or are associated with the symptoms of the disease. For example, rebalancing the TCR repertoire by removing or depleting T cells that contain autoreactive clonotypes can treat the associated autoimmune disease and / or reduce the symptoms of the autoimmune disease. Thus, the multispecific or multifunctional molecules disclosed herein are expected to target (e.g., localize, crosslink and / or activate) immune effector cells (e.g., selected from NK cells, T cells, B cells, dendritic cells or macrophages) in target cells (e.g., T cells that contain a biased TCRBV clonotype or that contain a TCRBV antigen corresponding to a biased TCRBV clonotype). Increasing the proximity and / or activity of immune cells using the multispecific molecules described herein can target cells (e.g., TCRBV, e.g., TCRBV antigen (e.g., bi It is expected to enhance the immune response against T cells containing TCRBV antigens corresponding to the skewed TCRBV clone types, thereby providing a more effective therapy (e.g., a more effective autoimmune disease therapy). Without being bound by theory, a targeted local immune response against target cells (e.g., T cells containing skewed TCRBV clone types, e.g., T cells not containing skewed TCRBV clone types) is thought to reduce the systemic toxicity effects of the multispecific molecules described herein. A targeted immune response against a population of autoreactive T cells that targets non-autoreactive T cells to a lesser extent (e.g., does not target non-autoreactive T cells) is thought to have fewer adverse effects than a global ablation of all T cells.

[0006] Accordingly, provided herein are, inter alia, multispecific molecules (e.g., multispecific or multifunctional antibody molecules) including the foregoing moieties, nucleic acids encoding such molecules, methods of producing the foregoing molecules, and methods of using the foregoing molecules to treat autoimmune diseases. Also provided herein are anti-TCRβV antibody molecules, nucleic acids encoding such molecules, methods of producing the foregoing molecules, and methods of using anti-TCRβV antibody molecules to treat autoimmune diseases.

[0007] Furthermore, provided, for example, are methods of depleting (e.g., in vivo depleting) skewed TCRBV clone types in the context of an autoimmune disease using a multispecific molecule or an anti-TCRβV antibody molecule. In some embodiments, the method involves identifying in a patient a clone bias in TCRBV usage, e.g., one associated with an autoreactive subpopulation, and in response to this analysis, administering a multifunctional molecule targeted to a TCRBV antigen corresponding to the skewed TCRBV clone type to reduce, e.g., eliminate, the clone bias and promote, e.g., establish, a normal TCRBV distribution, by administering a multifunctional molecule targeted to a TCRBV antigen corresponding to the skewed TCRBV clone type.

[0008] Accordingly, in one aspect, the disclosure is (i) A first antigen-binding domain that binds to a T cell receptor variable beta (TCRBV), for example, binds to a TCRBV antigen, for example, selectively binds, and (ii) (a) An immunocyte engager selected from an NK cell engager, a T cell engager, a B cell engager, a dendritic cell engager, or a macrophage cell engager; (b) A cytokine molecule or a cytokine inhibitor molecule; and (c) A cell death receptor signal engager One, two, or all of characterized by a multifunctional molecule.

[0009] In some embodiments, the first antigen-binding domain comprises an anti-TCRβV antibody molecule, for example, an anti-TCRβV antibody molecule described herein. In another aspect, the present disclosure features a nucleic acid molecule encoding a multifunctional molecule disclosed herein.

[0010] In another aspect, the present disclosure features a vector, for example, an expression vector, comprising a nucleic acid molecule disclosed herein. In another aspect, the present disclosure features a host cell comprising a nucleic acid molecule or a vector disclosed herein.

[0011] In another aspect, the present disclosure features a method of making, for example, producing a multifunctional molecule disclosed herein, comprising culturing a host cell disclosed herein under suitable conditions, for example, conditions suitable for gene expression and / or homo- or hetero-dimerization. characterized by a method.

[0012] In another aspect, the present disclosure features a pharmaceutical composition comprising a multifunctional molecule disclosed herein. In another aspect, the present disclosure features a method of treating TCR bias, the method comprising administering to a subject in need thereof a multifunctional molecule disclosed herein, wherein the multifunctional molecule is administered in an amount effective to treat the TCR bias.

[0013] In another aspect, the present disclosure features a method of treating an autoimmune disease (e.g., an autoimmune disease associated with TCR bias), the method comprising administering to a subject in need thereof a multifunctional molecule disclosed herein, wherein the multifunctional molecule is administered in an amount effective to treat the autoimmune disease.

[0014] In another aspect, the present disclosure features a method of identifying a subject in need of treatment of TCR bias or an autoimmune disease (e.g., associated with TCR bias) using a multifunctional molecule disclosed herein, the method comprising determining whether the subject has TCR bias (e.g., a biased TCRBV clonotype) and / or an autoimmune disease associated with said bias (e.g., directly or indirectly determining it, e.g., obtaining information regarding it), and identifying the subject as a candidate for treatment with a multifunctional molecule comprising an antigen-binding domain that binds to a TCRBV antigen in response to determining that the subject has TCR bias (e.g., a biased TCRBV clonotype) and / or an autoimmune disease associated with said bias.

[0015] In another aspect, the present disclosure features a method of assessing a subject in need of treatment of TCR bias (e.g., a biased TCRBV clonotype) and / or an autoimmune disease associated with said bias, the method comprising determining whether the subject has TCR bias (e.g., a biased TCRBV clonotype) (e.g., directly or indirectly determining it, e.g., obtaining information regarding it).

[0016] In yet another aspect, provided herein is a method of treating an autoimmune disease (e.g., an autoimmune disease associated with TCR bias) in a subject in need thereof, the method comprising administering to the subject an effective amount, e.g., a therapeutically effective amount, of an antibody molecule that binds (e.g., specifically binds) to the T cell receptor beta variable region (“anti-TCRβV antibody molecule”), thereby treating the disorder.

[0017] In another aspect, the present disclosure provides a method of depleting a T cell population in a subject having an autoimmune disorder (e.g., an autoimmune disease associated with TCR bias), the method comprising contacting the T cell population with an effective amount of an antibody molecule that binds (e.g., specifically binds) to the T cell receptor beta variable region (TCRβV) (“anti-TCRβV antibody molecule”).

[0018] In some embodiments, the contacting step occurs in vivo or in vitro. In some embodiments, the anti-TCRβV antibody molecule is not the antibody molecule disclosed in U.S. Patent No. 5,861,155.

[0019] In some embodiments, the anti-TCRβV antibody molecule binds to TCRβV12 with an affinity and / or binding specificity that is lower (e.g., about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or less than about one-half, one-fifth, or one-tenth) than the affinity and / or binding specificity of the 16G8 mouse antibody or its humanized version as described in U.S. Patent No. 5,861,155. In some embodiments, the anti-TCRβV antibody molecule binds to TCRβV12 with an affinity and / or binding specificity that is higher (e.g., about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or about 2-fold, 5-fold, or 10-fold higher) than the affinity and / or binding specificity of the 16G8 mouse antibody or its humanized version as described in U.S. Patent No. 5,861,155.

[0020] In some embodiments, the anti-TCRβV antibody molecule binds to TCRβV12 with an affinity and / or binding specificity that is higher (e.g., about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or about 2-fold, 5-fold, or 10-fold higher) than the affinity and / or binding specificity of the 16G8 mouse antibody or its humanized version as described in U.S. Patent No. 5,861,155.

[0021] In some embodiments, the anti-TCRβV antibody molecule has an affinity and / or binding specificity greater than (e.g., about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or greater than about 2, 5, or 10-fold) the affinity and / or binding specificity of the TM23 mouse antibody or its humanized version, as described in U.S. Patent No. 5,861,155, for TCRβ V5-5 * 01 or TCRβ V5-1 * and binds to 01.

[0022] In some embodiments, the anti-TCRβV antibody molecule has an affinity and / or binding specificity greater than (e.g., about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or greater than about 2, 5, or 10-fold) the affinity and / or binding specificity of the TM23 mouse antibody or its humanized version, as described in U.S. Patent No. 5,861,155, for TCRβ V5-5 * 01 or TCRβ V5-1 * and binds to 01.

[0023] In some embodiments, the anti-TCRβV antibody molecule comprises an Fc region, e.g., an Fc region having effector functions, e.g., an Fc region having antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell phagocytosis (ADCP) and / or complement-dependent cytotoxicity (CDC).

[0024] In some embodiments, the anti-TCRβV antibody molecule comprises an Fc region with enhanced effector function compared to, e.g., the wild-type Fc region. In some embodiments, the anti-TCRβV antibody molecule comprises a human IgG1 region or a human IgG4 region.

[0025] In another aspect, the disclosure features a nucleic acid molecule encoding an anti-TCRβV antibody molecule disclosed herein. In another aspect, the disclosure features a vector, e.g., an expression vector, comprising a nucleic acid molecule disclosed herein.

[0026] In another aspect, the disclosure features a host cell comprising a nucleic acid molecule or vector disclosed herein. In another aspect, the disclosure features a method of making, e.g., producing, an anti-TCRβV antibody molecule disclosed herein, the method comprising culturing a host cell disclosed herein under conditions suitable for, e.g., gene expression and / or homodimerization or heterodimerization.

[0027] In another aspect, the disclosure features a pharmaceutical composition comprising an anti-TCRβV antibody molecule disclosed herein. Any further features of the multifunctional molecule, nucleic acid, vector, host cell, or method described above include one or more of the following listed embodiments.

[0028] One of ordinary skill in the art can recognize or confirm numerous equivalents to the specific embodiments of the invention described herein using only routine experimentation. Such equivalents are intended to be encompassed by the following listed embodiments.

[0029] The listed embodiments 1. (i) A first antigen-binding domain that binds, e.g., selectively binds, to a T cell receptor variable beta (TCRBV), e.g., a TCRBV antigen, and (ii) (a) An immune cell engager selected from an NK cell engager, a T cell engager, a B cell engager, a dendritic cell engager, or a macrophage cell engager; (b) A cytokine molecule or a cytokine inhibitor molecule; and (c) A cell death receptor signal engager of one, two, or all of comprising a multifunctional molecule.

[0030] 2. The TCRBV antigen corresponds to a biased TCRBV clone type and is, for example, the multifunctional molecule of Embodiment 1 present in a subject, for example, a patient, for example, a subject or patient having an autoimmune disease.

[0031] 3. The multifunctional molecule (i) specifically binds to a TCRBV antigen, for example, an epitope identical or similar to the epitope recognized by an anti-TCRBV antibody molecule as described herein; (ii) exhibits a binding affinity and / or specificity identical or similar to that of an anti-TCRBV antibody molecule as described herein, or both; (iii) inhibits the binding of an anti-TCRBV antibody molecule as described herein, for example, competitively inhibits it; (iv) binds to an epitope identical or overlapping with that of an anti-TCRBV antibody molecule as described herein; or (v) is a multifunctional molecule of either Embodiment 1 or 2 that competes with the binding to an anti-TCRBV antibody molecule and / or binds to the same epitope.

[0032] 4. The antigen-binding domain of the multifunctional molecule of Embodiment 3 comprises one or more CDRs, framework regions, variable domains, heavy or light chains, or antigen-binding domains selected from Table 13 or 14, or sequences substantially identical thereto.

[0033] 5. The multifunctional molecule of any one of Embodiments 1 to 4, wherein the antigen-binding domain specifically binds to TCRβ V6 (e.g., TCRβ V6-5 * 01). 6. The pluripotent cell molecule of embodiment 5, wherein the antigen-binding domain is derived from antibody A-H.1 or antibody A-H.2, or is described in Table 1A, or is substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical) to the nucleotide sequence of Table 1A, or any of the foregoing sequences, and comprises at least one (e.g., 1, 2, 3, or 4) variable region or an antigen-binding fragment thereof.

[0034] 7. The multifunctional molecule of any of embodiments 5 or 6, wherein the antigen-binding domain comprises at least one, two, or three CDRs (or all of the CDRs collectively) derived from a heavy chain variable region that is shown in Table 1A or is encoded by the nucleotide sequence shown in Table 1A (or has one, two, three, four, five, six or more changes, e.g., amino acid substitutions or deletions, relative to the amino acid sequence shown in Table 1A, or is a sequence encoded by the nucleotide sequence shown in Table 1A).

[0035] 8. The multifunctional molecule of any of embodiments 5 to 7, wherein the antigen-binding domain comprises at least one, two, or three CDRs (or all of the CDRs collectively) derived from a light chain variable region that is shown in Table 1A or is encoded by the nucleotide sequence shown in Table 1A (or has one, two, three, four, five, six or more changes, e.g., amino acid substitutions or deletions, relative to the amino acid sequence shown in Table 1A, or is a sequence encoded by the nucleotide sequence shown in Table 1A).

[0036] 9. The antigen-binding domain is (i) one, two or all of the light chain complementarity determining region 1 (LC CDR1), light chain complementarity determining region 2 (LC CDR2), and light chain complementarity determining region 3 (LC CDR3) of SEQ ID NO: 2, SEQ ID NO: 10 or SEQ ID NO: 11, and / or (ii) One, two, or all of the heavy chain complementarity determining region 1 (HC CDR1), heavy chain complementarity determining region 2 (HC CDR2), and heavy chain complementarity determining region 3 (HC CDR3) of SEQ ID NO: 1 or SEQ ID NO: 9 A multifunctional molecule according to any one of embodiments 5 to 8, comprising

[0037] 10. A multifunctional molecule according to any one of embodiments 5 to 8, wherein the antigen-binding domain comprises the LC CDR1, LC CDR2, and LC CDR3 of SEQ ID NO: 2, and the HC CDR1, HC CDR2, and HC CDR3 of SEQ ID NO: 1.

[0038] 11. A multifunctional molecule according to any one of embodiments 5 to 8, wherein the antigen-binding domain comprises the LC CDR1, LC CDR2, and LC CDR3 of SEQ ID NO: 10, and the HC CDR1, HC CDR2, and HC CDR3 of SEQ ID NO: 9.

[0039] 12. A multifunctional molecule according to any one of embodiments 5 to 8, wherein the antigen-binding domain comprises the LC CDR1, LC CDR2, and LC CDR3 of SEQ ID NO: 11, and the HC CDR1, HC CDR2, and HC CDR3 of SEQ ID NO: 9.

[0040] 13. The antigen-binding domain is (i) The LC CDR1 amino acid sequence of SEQ ID NO: 6, the LC CDR2 amino acid sequence of SEQ ID NO: 7, or the LC CDR3 amino acid sequence of SEQ ID NO: 8; and / or (ii) The HC CDR1 amino acid sequence of SEQ ID NO: 3, the HC CDR2 amino acid sequence of SEQ ID NO: 4, or the HC CDR3 amino acid sequence of SEQ ID NO: 5 A multifunctional molecule according to any one of embodiments 5 to 8, comprising

[0041] 14. The antigen-binding domain is (i) A light chain variable region (VL) comprising the LC CDR1 amino acid sequence of SEQ ID NO: 6, the LC CDR2 amino acid sequence of SEQ ID NO: 7, or the LC CDR3 amino acid sequence of SEQ ID NO: 8; and / or (ii) A heavy chain variable region (VH) comprising the HC CDR1 amino acid sequence of SEQ ID NO: 3, the HC CDR2 amino acid sequence of SEQ ID NO: 4, or the HC CDR3 amino acid sequence of SEQ ID NO: 5 A multifunctional molecule according to any one of embodiments 5 to 8, comprising

[0042] 15. The antigen-binding domain is (i) The LC CDR1 amino acid sequence of SEQ ID NO: 51, the LC CDR2 amino acid sequence of SEQ ID NO: 52, or the LC CDR3 amino acid sequence of SEQ ID NO: 53; and / or (ii) The HC CDR1 amino acid sequence of SEQ ID NO: 45, the HC CDR2 amino acid sequence of SEQ ID NO: 46, or the HC CDR3 amino acid sequence of SEQ ID NO: 47 A multifunctional molecule according to any one of embodiments 5 to 8, comprising

[0043] 16. The antigen-binding domain is (i) A light chain variable region (VL) comprising the LC CDR1 amino acid sequence of SEQ ID NO: 51, the LC CDR2 amino acid sequence of SEQ ID NO: 52, or the LC CDR3 amino acid sequence of SEQ ID NO: 53 and / or (ii) A heavy chain variable region (VH) comprising the HC CDR1 amino acid sequence of SEQ ID NO: 45, the HC CDR2 amino acid sequence of SEQ ID NO: 46, or the HC CDR3 amino acid sequence of SEQ ID NO: 47 A multifunctional molecule according to any one of embodiments 5 to 8, comprising

[0044] 17. The antigen-binding domain is (i) The LC CDR1 amino acid sequence of SEQ ID NO: 54, the LC CDR2 amino acid sequence of SEQ ID NO: 55, or the LC CDR3 amino acid sequence of SEQ ID NO: 56; and / or (ii) The HC CDR1 amino acid sequence of SEQ ID NO: 48, the HC CDR2 amino acid sequence of SEQ ID NO: 49, or the HC CDR3 amino acid sequence of SEQ ID NO: 50 A multifunctional molecule according to any one of Embodiments 5 to 8, comprising

[0045] 18. The antigen-binding domain is (i) a light chain variable region (VL) comprising the LC CDR1 amino acid sequence of SEQ ID NO: 54, the LC CDR2 amino acid sequence of SEQ ID NO: 55, or the LC CDR3 amino acid sequence of SEQ ID NO: 56 and / or (ii) a heavy chain variable region (VH) comprising the HC CDR1 amino acid sequence of SEQ ID NO: 48, the HC CDR2 amino acid sequence of SEQ ID NO: 49, or the HC CDR3 amino acid sequence of SEQ ID NO: 50 A multifunctional molecule according to any one of Embodiments 5 to 8, comprising

[0046] 19. The antigen-binding domain is, for example, as shown in FIG. 1B, a light chain variable region (VL) comprising, for example, the light chain framework region 1 (VLFWR1) of antibody A-H.1 or antibody A-H.2 of SEQ ID NO: 2, 10, or 11, a multifunctional molecule according to any one of Embodiments 5 to 18.

[0047] 20. The antigen-binding domain is, for example, as shown in FIG. 1B, a light chain variable region (VL) comprising, for example, the light chain framework region 2 (VLFWR2) of antibody A-H.1 or antibody A-H.2 of SEQ ID NO: 2, 10, or 11, a multifunctional molecule according to any one of Embodiments 5 to 19.

[0048] 21. The antigen-binding domain is, for example, as shown in FIG. 1B, a light chain variable region (VL) comprising, for example, the light chain framework region 3 (VLFWR3) of antibody A-H.1 or antibody A-H.2 of SEQ ID NO: 2, 10, or 11, a multifunctional molecule according to any one of Embodiments 5 to 20.

[0049] 22. The antigen-binding domain is, for example, as shown in FIG. 1B, a light chain variable region (VL) comprising, for example, the light chain framework region 4 (VLFWR4) of antibody A-H.1 or antibody A-H.2 of SEQ ID NO: 2, 10, or 11, a multifunctional molecule according to any one of Embodiments 5 to 21.

[0050] 23. The antigen-binding domain comprises a variable light chain (VL) containing the variable light chain framework region 1 (VLFWR1), variable light chain framework region 2 (VLFWR2), variable light chain framework region 3 (VLFWR3), and variable light chain framework region 4 (VLFWR4) of SEQ ID NO: 2 The multifunctional molecule according to any one of embodiments 5 to 22, which comprises

[0051] 24. The antigen-binding domain comprises a variable light chain (VL) containing the variable light chain framework region 1 (VLFWR1), variable light chain framework region 2 (VLFWR2), variable light chain framework region 3 (VLFWR3), and variable light chain framework region 4 (VLFWR4) of SEQ ID NO: 10, The multifunctional molecule according to any one of embodiments 5 to 22, which comprises

[0052] 25. The antigen-binding domain comprises the variable light chain framework region 1 (VLFWR1), variable light chain framework region 2 (VLFWR2), variable light chain framework region 3 (VLFWR3), and variable light chain framework region 4 (VLFWR4) of SEQ ID NO: 11, The multifunctional molecule according to any one of embodiments 5 to 22, which comprises

[0053] 26. The antigen-binding domain comprises a variable light chain domain containing a framework region with a change, substitution (e.g., conservative substitution) at position 10 according to Kabat numbering, e.g., the variable light chain framework region 1 (VLFWR1), and the change at position 10 is phenylalanine, e.g., a substitution from serine to phenylalanine, The multifunctional molecule according to any one of embodiments 5 to 25, which comprises

[0054] 27. A multifunctional molecule according to any one of embodiments 5 to 26, wherein the antigen-binding domain comprises a light chain variable domain comprising a framework region, such as framework region 2 (VLFWR2), comprising one or more (e.g., one or two) changes, such as substitutions (e.g., conservative substitutions), at positions selected from 36 and 46 according to Kabat numbering, wherein the change at position 36 is a substitution from tyrosine to histidine, and the change at position 46 is a substitution from arginine to alanine.

[0055] 28. A multifunctional molecule according to any one of embodiments 5 to 27, wherein the antigen-binding domain comprises a light chain variable domain comprising a framework region, such as framework region 3 (VLFWR3), comprising a change at position 87 according to Kabat numbering, such as a substitution (e.g., conservative substitution), wherein the change at position 87 is a substitution from tyrosine to phenylalanine.

[0056] 29. A multifunctional molecule according to any one of embodiments 5 to 28, wherein the antigen-binding domain comprises a light chain variable domain comprising: (a) framework region 1 (VLFWR1) comprising a phenylalanine at position 10, such as a substitution at position 10 according to Kabat numbering, such as a substitution from serine to phenylalanine; (b) framework region 2 (VLFWR2) comprising a histidine at position 36, such as a substitution at position 36 according to Kabat numbering, such as a substitution from tyrosine to histidine, and an alanine at position 46, such as a substitution at position 46 according to Kabat numbering, such as a substitution from arginine to alanine; and (c) framework region 3 (VLFWR3) comprising a phenylalanine at position 87, such as a substitution at position 87 according to Kabat numbering, such as a substitution from tyrosine to phenylalanine, as shown in the amino acid sequence of SEQ ID NO: 10.

[0057] 30. The antigen-binding domain includes a light chain variable domain that includes, for example, as shown in the amino acid sequence of SEQ ID NO: 11, (a) a histidine at position 36, such as a substitution at position 36 according to Kabat numbering, such as a substitution from tyrosine to histidine, and an alanine at position 46, such as a substitution at position 46 according to Kabat numbering, such as a substitution from arginine to alanine, in framework region 2 (FR2); and (b) a phenylalanine at position 87, such as a substitution at position 87 according to Kabat numbering, such as a substitution from tyrosine to phenylalanine, in framework region 3 (FR3), of any of the multifunctional molecules of embodiments 5 to 28.

[0058] 31. The antigen-binding domain includes a heavy chain variable region (VH) that includes, for example, as shown in FIG. 1A, a heavy chain framework region 1 (VHFWR1) of antibody A-H.1 or antibody A-H.2, such as SEQ ID NO: 1 or 9, of any of the multifunctional molecules of embodiments 5 to 30.

[0059] 32. The antigen-binding domain includes a heavy chain variable region (VH) that includes, for example, as shown in FIG. 1A, a heavy chain framework region 2 (VHFWR2) of antibody A-H.1 or antibody A-H.2, such as SEQ ID NO: 1 or 9, of any of the multifunctional molecules of embodiments 5 to 31.

[0060] 33. The antigen-binding domain includes a heavy chain variable region (VH) that includes, for example, as shown in FIG. 1A, a heavy chain framework region 3 (VHFWR3) of antibody A-H.1 or antibody A-H.2, such as SEQ ID NO: 1 or 9, of any of the multifunctional molecules of embodiments 5 to 32.

[0061] 34. The antigen-binding domain includes a heavy chain variable region (VH) that includes, for example, as shown in FIG. 1A, a heavy chain framework region 4 (VHFWR4) of antibody A-H.1 or antibody A-H.2, such as SEQ ID NO: 1 or 9, of any of the multifunctional molecules of embodiments 5 to 33.

[0062] 35. A multifunctional molecule according to any one of embodiments 5 to 34, wherein the antigen-binding domain comprises heavy chain framework region 1 (VHFWR1), heavy chain framework region 2 (VHFWR2), heavy chain framework region 3 (VHFWR3), and heavy chain framework region 4 (VHFWR4) of SEQ ID NO: 1.

[0063] 36. A multifunctional molecule according to any one of embodiments 5 to 34, wherein the antigen-binding domain comprises heavy chain framework region 1 (VHFWR1), heavy chain framework region 2 (VHFWR2), heavy chain framework region 3 (VHFWR3), and heavy chain framework region 4 (VHFWR4) of SEQ ID NO: 9.

[0064] 37. A multifunctional molecule according to any one of embodiments 5 to 36, wherein the antigen-binding domain comprises a heavy chain variable domain comprising a framework region, for example, framework region 3 (VHFWR3), having one or more (e.g., one or two) changes, such as substitutions (e.g., conservative substitutions), at positions selected from 73 and 94 according to Kabat numbering, wherein the change at position 73 is a substitution from threonine, e.g., glutamic acid to threonine, and the change at position 94 is a substitution from glycine, e.g., arginine to glycine.

[0065] 38. A multifunctional molecule according to any one of embodiments 5 to 37, wherein the antigen-binding domain comprises a heavy chain variable domain comprising framework region 3 (FR3) having a threonine at position 73, e.g., a substitution at position 73 according to Kabat numbering, e.g., a substitution from glutamic acid to threonine, and a glycine at position 94, e.g., a substitution at position 94 according to Kabat numbering, e.g., a substitution from arginine to glycine, as shown in the amino acid sequence of SEQ ID NO: 10, for example.

[0066] 39. A multifunctional molecule according to any one of embodiments 5 to 18, wherein the antigen-binding domain comprises heavy chain framework regions 1 to 4 of antibody A-H.1, e.g., SEQ ID NO: 9; and light chain framework regions 1 to 4 of antibody A-H.1, e.g., SEQ ID NO: 10, or those shown in FIGS. 1A and 1B.

[0067] 40. The antigen-binding domain includes the heavy chain framework regions 1 to 4 of antibody A-H.2, for example, SEQ ID NO: 9; and the light chain framework regions 1 to 4 of antibody A-H.2, for example, SEQ ID NO: 11, or those shown in FIGS. 1A and 1B, and is a multifunctional molecule according to any one of embodiments 5 to 18.

[0068] 41. The antigen-binding domain contains a VH domain having the amino acid sequence of SEQ ID NO: 9, an amino acid sequence that is at least about 85%, 90%, 95%, 99% or more identical to the amino acid sequence of SEQ ID NO: 9, or an amino acid sequence that differs from the amino acid sequence of SEQ ID NO: 9 by 1, 2, 5, 10 or 15 or fewer amino acid residues; and / or contains a VL domain having the amino acid sequence of SEQ ID NO: 10, an amino acid sequence that is at least about 85%, 90%, 95%, 99% or more identical to the amino acid sequence of SEQ ID NO: 10, or an amino acid sequence that differs from the amino acid sequence of SEQ ID NO: 10 by 1, 2, 5, 10 or 15 or fewer amino acid residues and is a multifunctional molecule according to any one of embodiments 5 to 18.

[0069] 42. The antigen-binding domain contains a VH domain having the amino acid sequence of SEQ ID NO: 9, an amino acid sequence that is at least about 85%, 90%, 95%, 99% or more identical to the amino acid sequence of SEQ ID NO: 9, or an amino acid sequence that differs from the amino acid sequence of SEQ ID NO: 9 by 1, 2, 5, 10 or 15 or fewer amino acid residues; and / or contains a VL domain having the amino acid sequence of SEQ ID NO: 11, an amino acid sequence that is at least about 85%, 90%, 95%, 99% or more identical to the amino acid sequence of SEQ ID NO: 11, or an amino acid sequence that differs from the amino acid sequence of SEQ ID NO: 11 by 1, 2, 5, 10 or 15 or fewer amino acid residues and is a multifunctional molecule according to any one of embodiments 5 to 18.

[0070] 43. The antigen-binding domain is TCRβ V12 (e.g., TCRβ V12-3* A multifunctional molecule according to any one of Embodiments 1 to 4 that specifically binds to * . 44. The pluripotent stem cell molecule of Embodiment 43, wherein the antigen-binding domain is described in Table 2A or is encoded by a nucleotide sequence that is substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical) to the nucleotide sequence of Table 2A or any of the foregoing sequences, and includes at least one (e.g., 1, 2, 3, or 4) variable region or an antigen-binding fragment thereof.

[0071] 45. The multifunctional molecule according to any one of Embodiments 43 or 44, wherein the antigen-binding domain includes at least one, two, or three CDRs (or all of the CDRs collectively) derived from a heavy chain variable region that includes an amino acid sequence shown in Table 2A or encoded by the nucleotide sequence shown in Table 2A (or having one, two, three, four, five, six or more changes, e.g., amino acid substitutions or deletions, relative to the amino acid sequence shown in Table 2A, or a sequence encoded by the nucleotide sequence shown in Table 2A).

[0072] 46. The multifunctional molecule according to any one of Embodiments 43 to 45, wherein the antigen-binding domain includes at least one, two, or three CDRs (or all of the CDRs collectively) derived from a light chain variable region that includes an amino acid sequence shown in Table 2A or encoded by the nucleotide sequence shown in Table 2A (or having one, two, three, four, five, six or more changes, e.g., amino acid substitutions or deletions, relative to the amino acid sequence shown in Table 2A, or a sequence encoded by the nucleotide sequence shown in Table 2A).

[0073] 47. The antigen-binding domain is (i) One, two, or all of the light chain complementarity determining region 1 (LC CDR1), light chain complementarity determining region 2 (LC CDR2), and light chain complementarity determining region 3 (LCCDR3) of SEQ ID NO: 16, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, or SEQ ID NO: 30; and / or (ii) One, two, or all of the heavy chain complementarity determining region 1 (HC CDR1), heavy chain complementarity determining region 2 (HC CDR2), and heavy chain complementarity determining region 3 (HC CDR3) of SEQ ID NO: 15, SEQ ID NO: 23, SEQ ID NO: 24, or SEQ ID NO: 25 A multifunctional molecule according to any one of embodiments 43 to 46, comprising

[0074] 48. The antigen-binding domain is (i) The LC CDR1 amino acid sequence of SEQ ID NO: 20, the LC CDR2 amino acid sequence of SEQ ID NO: 21, or the LC CDR3 amino acid sequence of SEQ ID NO: 22; and / or (ii) The HC CDR1 amino acid sequence of SEQ ID NO: 17, the HC CDR2 amino acid sequence of SEQ ID NO: 18, or the HC CDR3 amino acid sequence of SEQ ID NO: 19 A multifunctional molecule according to any one of embodiments 43 to 47, comprising

[0075] 49. The antigen-binding domain is (i) A light chain variable region (VL) comprising the LC CDR1 amino acid sequence of SEQ ID NO: 20, the LC CDR2 amino acid sequence of SEQ ID NO: 21, and the LC CDR3 amino acid sequence of SEQ ID NO: 2; and / or (ii) A heavy chain variable region (VH) comprising the HC CDR1 amino acid sequence of SEQ ID NO: 17, the HC CDR2 amino acid sequence of SEQ ID NO: 18, and the HC CDR3 amino acid sequence of SEQ ID NO: 19 A multifunctional molecule according to any one of embodiments 43 to 47, comprising

[0076] 50. The antigen-binding domain is (i) The LC CDR1 amino acid sequence of SEQ ID NO: 63, the LC CDR2 amino acid sequence of SEQ ID NO: 64, or the LC CDR3 amino acid sequence of SEQ ID NO: 65; and / or (ii) The HC CDR1 amino acid sequence of SEQ ID NO: 57, the HC CDR2 amino acid sequence of SEQ ID NO: 58, or the HC CDR3 amino acid sequence of SEQ ID NO: 59 A multifunctional molecule according to any one of embodiments 43 to 47, comprising

[0077] 51. The antigen-binding domain is (i) A light chain variable region (VL) comprising the LC CDR1 amino acid sequence of SEQ ID NO: 63, the LC CDR2 amino acid sequence of SEQ ID NO: 64, or the LC CDR3 amino acid sequence of SEQ ID NO: 65 and / or (ii) A heavy chain variable region (VH) comprising the HC CDR1 amino acid sequence of SEQ ID NO: 57, the HC CDR2 amino acid sequence of SEQ ID NO: 58, or the HC CDR3 amino acid sequence of SEQ ID NO: 59 A multifunctional molecule according to any one of embodiments 43 to 47, comprising

[0078] 52. The antigen-binding domain is (i) The LC CDR1 amino acid sequence of SEQ ID NO: 66, the LC CDR2 amino acid sequence of SEQ ID NO: 67, or the LC CDR3 amino acid sequence of SEQ ID NO: 68; and / or (ii) The HC CDR1 amino acid sequence of SEQ ID NO: 60, the HC CDR2 amino acid sequence of SEQ ID NO: 61, or the HC CDR3 amino acid sequence of SEQ ID NO: 62 A multifunctional molecule according to any one of embodiments 43 to 47, comprising

[0079] 53. The antigen-binding domain is (i) A light chain variable region (VL) comprising the LC CDR1 amino acid sequence of SEQ ID NO: 63, the LC CDR2 amino acid sequence of SEQ ID NO: 64, or the LC CDR3 amino acid sequence of SEQ ID NO: 65 and / or (ii) A heavy chain variable region (VH) comprising the HC CDR1 amino acid sequence of SEQ ID NO: 57, the HC CDR2 amino acid sequence of SEQ ID NO: 58, or the HC CDR3 amino acid sequence of SEQ ID NO: 59 A multifunctional molecule according to any one of embodiments 43 to 47, comprising

[0080] 54. The multifunctional molecule according to any one of embodiments 43 to 53, wherein the antigen-binding domain comprises a variable light chain region (VL) comprising a variable light chain framework region 1 (VLFWR1) of, for example, SEQ ID NO: 16 or 26 to 30, as shown in, for example, FIG. 2B.

[0081] 55. The multifunctional molecule according to any one of embodiments 43 to 54, wherein the antigen-binding domain comprises a variable light chain region (VL) comprising a variable light chain framework region 2 (VLFWR2) of, for example, SEQ ID NO: 16 or 26 to 30, as shown in, for example, FIG. 2B.

[0082] 56. The multifunctional molecule according to any one of embodiments 43 to 55, wherein the antigen-binding domain comprises a variable light chain region (VL) comprising a variable light chain framework region 3 (VLFWR3) of, for example, SEQ ID NO: 16 or 26 to 30, as shown in, for example, FIG. 2B.

[0083] 57. The multifunctional molecule according to any one of embodiments 43 to 56, wherein the antigen-binding domain comprises a variable light chain region (VL) comprising a variable light chain framework region 4 (VLFWR4) of, for example, SEQ ID NO: 16 or 26 to 30, as shown in, for example, FIG. 2B.

[0084] 58. The multifunctional molecule according to any one of embodiments 43 to 57, wherein the antigen-binding domain comprises a variable light chain region (VL) comprising variable light chain framework region 1 (VLFWR1), variable light chain framework region 2 (VLFWR2), variable light chain framework region 3 (VLFWR3), and variable light chain framework region 4 (VLFWR4) of SEQ ID NO: 16.

[0085] 59. The multifunctional molecule according to any one of embodiments 43 to 57, wherein the antigen-binding domain comprises a variable light chain region (VL) comprising variable light chain framework region 1 (VLFWR1), variable light chain framework region 2 (VLFWR2), variable light chain framework region 3 (VLFWR3), and variable light chain framework region 4 (VLFWR4) of SEQ ID NO: 26.

[0086] 60. A multifunctional molecule according to any one of embodiments 43 to 57, wherein the antigen-binding domain comprises a variable light chain region (VL) comprising variable light chain framework region 1 (VLFWR1), variable light chain framework region 2 (VLFWR2), variable light chain framework region 3 (VLFWR3), and variable light chain framework region 4 (VLFWR4) of SEQ ID NO: 27.

[0087] 61. A multifunctional molecule according to any one of embodiments 43 to 57, wherein the antigen-binding domain comprises a variable light chain region (VL) comprising variable light chain framework region 1 (VLFWR1), variable light chain framework region 2 (VLFWR2), variable light chain framework region 3 (VLFWR3), and variable light chain framework region 4 (VLFWR4) of SEQ ID NO: 28.

[0088] 62. A multifunctional molecule according to any one of embodiments 43 to 57, wherein the antigen-binding domain comprises a variable light chain region (VL) comprising variable light chain framework region 1 (VLFWR1), variable light chain framework region 2 (VLFWR2), variable light chain framework region 3 (VLFWR3), and variable light chain framework region 4 (VLFWR4) of SEQ ID NO: 29.

[0089] 63. A multifunctional molecule according to any one of embodiments 43 to 57, wherein the antigen-binding domain comprises a variable light chain region (VL) comprising variable light chain framework region 1 (VLFWR1), variable light chain framework region 2 (VLFWR2), variable light chain framework region 3 (VLFWR3), and variable light chain framework region 4 (VLFWR4) of SEQ ID NO: 30.

[0090] 64. A multifunctional molecule according to any of embodiments 43 to 57, wherein the antigen-binding domain comprises a light chain variable domain comprising a framework region, such as framework region 1 (VLFWR1), having one or more (e.g., one, two, or three) changes, e.g., substitutions (e.g., conservative substitutions), at positions selected from 1, 2, and 4 according to Kabat numbering, wherein the change at position 1 is a substitution from alanine, e.g., aspartic acid, to asparagine, the change at position 2 is a substitution from isoleucine, e.g., asparagine, to asparagine, and the change at position 4 is a substitution from methionine, e.g., leucine, to leucine.

[0091] 65. A multifunctional molecule according to any of embodiments 43 to 57 or 64, wherein the antigen-binding domain comprises a light chain variable domain comprising a framework region, such as framework region 3 (VLFWR3), having one or more (e.g., one, two, or three) changes, e.g., substitutions (e.g., conservative substitutions), at positions selected from 66, 69, and 71 according to Kabat numbering, wherein the change at position 66 is a substitution from lysine, e.g., glycine, to glycine, the change at position 69 is a substitution from tyrosine, e.g., asparagine, to asparagine, and the change at position 71 is a substitution from phenylalanine, e.g., tyrosine, to tyrosine.

[0092] 66. A multifunctional molecule according to any of embodiments 43 to 57, 64, or 65, wherein the antigen-binding domain comprises a light chain comprising a framework region 1 (FR1) having a substitution at position 2 according to Kabat numbering, e.g., a substitution from isoleucine to aparagine, as shown in the amino acid sequence of SEQ ID NO: 26, and a framework region 3 (FR3) having a substitution at position 69 according to Kabat numbering, e.g., a substitution from threonine to aparagine, and a substitution at position 71 according to Kabat numbering, e.g., a substitution from phenylalanine to tyrosine.

[0093] 67. An embodiment 43 to 57, 64 or 65 multifunctional molecule, wherein the antigen-binding domain comprises a light chain comprising (a) a framework region 1 (FR1) comprising a substitution at position 1 according to Kabat numbering, for example a substitution from alanine to aspartic acid, and a substitution at position 2 according to Kabat numbering, for example a substitution from isoleucine to asparagine, as shown in the amino acid sequence of SEQ ID NO: 27; and (b) a framework region 3 (FR3) comprising a substitution at position 69 according to Kabat numbering, for example a substitution from threonine to asparagine, and a substitution at position 71 according to Kabat numbering, for example a substitution from phenylalanine to tyrosine.

[0094] 68. An embodiment 43 to 57, 64 or 65 multifunctional molecule, wherein the antigen-binding domain comprises a light chain comprising (a) a framework region 1 (FR1) comprising a substitution at position 2 according to Kabat numbering, for example a substitution from serine to asparagine; and a substitution at position 4 according to Kabat numbering, for example a substitution from methionine to leucine, as shown in the amino acid sequence of SEQ ID NO: 28; and (b) a framework region 3 (FR3) comprising a substitution at position 69 according to Kabat numbering, for example a substitution from threonine to asparagine, and a substitution at position 71 according to Kabat numbering, for example a substitution from phenylalanine to tyrosine.

[0095] 69. The antigen-binding domain is, for example, as shown in the amino acid sequence of SEQ ID NO: 29 , an embodiment 43 to 57, 64 or 65 multifunctional molecule, wherein the antigen-binding domain comprises a light chain comprising (a) a framework region 1 (FR1) comprising a substitution at position 2 according to Kabat numbering, for example a substitution from serine to asparagine; and (b) a framework region 3 (FR3) comprising a substitution at position 66 according to Kabat numbering, for example a substitution from lysine to glycine; a substitution at position 69 according to Kabat numbering, for example a substitution from threonine to asparagine; and a substitution at position 71 according to Kabat numbering, for example a substitution from alanine to tyrosine.

[0096] 70. The antigen-binding domain comprises, for example, a heavy-chain variable region (VH) containing a heavy-chain framework region 1 (VHFWR1) of, for example, SEQ ID NO: 15 or 23-25, as shown in FIG. 2A, and any of the multifunctional molecules of embodiments 43 to 69.

[0097] 71. The antigen-binding domain comprises, for example, a heavy-chain variable region (VH) containing a heavy-chain framework region 2 (VHFWR2) of, for example, SEQ ID NO: 15 or 23-25, as shown in FIG. 2A, and any of the multifunctional molecules of embodiments 43 to 70.

[0098] 72. The antigen-binding domain comprises, for example, a heavy-chain variable region (VH) containing a heavy-chain framework region 3 (VHFWR3) of, for example, SEQ ID NO: 15 or 23-25, as shown in FIG. 2A, and any of the multifunctional molecules of embodiments 43 to 71.

[0099] 73. The antigen-binding domain comprises, for example, a heavy-chain variable region (VH) containing a heavy-chain framework region 4 (VHFWR4) of, for example, SEQ ID NO: 15 or 23-25, as shown in FIG. 2A, and any of the multifunctional molecules of embodiments 43 to 72.

[0100] 74. The antigen-binding domain comprises a heavy-chain framework region 1 (VHFWR1), a heavy-chain framework region 2 (VHFWR2), a heavy-chain framework region 3 (VHFWR3), and a heavy-chain framework region 4 (VHFWR4) of SEQ ID NO: 23, and any of the multifunctional molecules of embodiments 43 to 73.

[0101] 75. The antigen-binding domain comprises a heavy-chain framework region 1 (VHFWR1), a heavy-chain framework region 2 (VHFWR2), a heavy-chain framework region 3 (VHFWR3), and a heavy-chain framework region 4 (VHFWR4) of SEQ ID NO: 24, and any of the multifunctional molecules of embodiments 43 to 73.

[0102] 76. A multifunctional molecule according to any one of embodiments 43 to 73, wherein the antigen-binding domain comprises heavy chain framework region 1 (VHFWR1), heavy chain framework region 2 (VHFWR2), heavy chain framework region 3 (VHFWR3), and heavy chain framework region 4 (VHFWR4) of SEQ ID NO: 25.

[0103] 77. A multifunctional molecule according to any one of embodiments 43 to 73, wherein the antigen-binding domain comprises a heavy chain comprising heavy chain framework regions 1 to 4 of SEQ ID NO: 23, 24, or 25; and a light chain comprising light chain framework regions 1 to 4 of SEQ ID NO: 26, 27, 28, 29, or 30.

[0104] 78. The antigen-binding domain is an amino acid sequence selected from the amino acid sequences of SEQ ID NO: 23, SEQ ID NO: 24, or SEQ ID NO: 25, an amino acid sequence that is at least about 85%, 90%, 95%, 99% or more identical to the amino acid sequence of SEQ ID NO: 23, SEQ ID NO: 24, or SEQ ID NO: 25, or an amino acid sequence that differs from the amino acid sequence of SEQ ID NO: 23, SEQ ID NO: 24, or SEQ ID NO: 25 by 1, 2, 5, 10 or 15 or fewer amino acid residues, and comprises a VH domain; and / or is an amino acid sequence selected from the amino acid sequences of SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, or SEQ ID NO: 30, an amino acid sequence that is at least about 85%, 90%, 95%, 99% or more identical to the amino acid sequence of SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, or SEQ ID NO: 30, an amino acid sequence that differs from the amino acid sequence of SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, or SEQ ID NO: 30 by 1, 2, 5, 10 or 15 or fewer amino acid residues, and comprises a VL domain A multifunctional molecule according to any one of embodiments 43 to 73.

[0105] 79. The antigen-binding domain is The VH domain comprising the amino acid sequence of SEQ ID NO: 23, an amino acid sequence that is at least about 85%, 90%, 95%, 99% or more identical to the amino acid sequence of SEQ ID NO: 23, or an amino acid sequence that differs from the amino acid sequence of SEQ ID NO: 23 by 1, 2, 5, 10, or 15 or fewer amino acid residues; and The VL domain comprising the amino acid sequence of SEQ ID NO: 26, an amino acid sequence that is at least about 85%, 90%, 95%, 99% or more identical to the amino acid sequence of SEQ ID NO: 26, or an amino acid sequence that differs from the amino acid sequence of SEQ ID NO: 26 by 1, 2, 5, 10, or 15 or fewer amino acid residues A multifunctional molecule according to any one of embodiments 43 to 73, comprising

[0106] 80. The antigen-binding domain is The VH domain comprising the amino acid sequence of SEQ ID NO: 23, an amino acid sequence that is at least about 85%, 90%, 95%, 99% or more identical to the amino acid sequence of SEQ ID NO: 23, or an amino acid sequence that differs from the amino acid sequence of SEQ ID NO: 23 by 1, 2, 5, 10, or 15 or fewer amino acid residues; and The VL domain comprising the amino acid sequence of SEQ ID NO: 27, an amino acid sequence that is at least about 85%, 90%, 95%, 99% or more identical to the amino acid sequence of SEQ ID NO: 27, or an amino acid sequence that differs from the amino acid sequence of SEQ ID NO: 27 by 1, 2, 5, 10, or 15 or fewer amino acid residues A multifunctional molecule according to any one of embodiments 43 to 73, comprising

[0107] 81. The antigen-binding domain is The VH domain comprising the amino acid sequence of SEQ ID NO: 23, an amino acid sequence that is at least about 85%, 90%, 95%, 99% or more identical to the amino acid sequence of SEQ ID NO: 23, or an amino acid sequence that differs from the amino acid sequence of SEQ ID NO: 23 by 1, 2, 5, 10, or 15 or fewer amino acid residues; and The VL domain comprising the amino acid sequence of SEQ ID NO: 28, an amino acid sequence that is at least about 85%, 90%, 95%, 99% or more identical to the amino acid sequence of SEQ ID NO: 28, or an amino acid sequence that differs from the amino acid sequence of SEQ ID NO: 28 by 1, 2, 5, 10, or 15 or fewer amino acid residues A multifunctional molecule according to any one of embodiments 43 to 73, comprising

[0108] 82. The antigen-binding domain is The VH domain comprising the amino acid sequence of SEQ ID NO: 23, an amino acid sequence that is at least about 85%, 90%, 95%, 99% or more identical to the amino acid sequence of SEQ ID NO: 23, or an amino acid sequence that differs from the amino acid sequence of SEQ ID NO: 23 by 1, 2, 5, 10, or 15 or fewer amino acid residues; and The VL domain comprising the amino acid sequence of SEQ ID NO: 29, an amino acid sequence that is at least about 85%, 90%, 95%, 99% or more identical to the amino acid sequence of SEQ ID NO: 29, or an amino acid sequence that differs from the amino acid sequence of SEQ ID NO: 29 by 1, 2, 5, 10, or 15 or fewer amino acid residues A multifunctional molecule according to any one of embodiments 43 to 73, comprising A multifunctional molecule according to any one of embodiments 43 to 73, comprising

[0109] 83. The antigen-binding domain is The VH domain comprising the amino acid sequence of SEQ ID NO: 23, an amino acid sequence that is at least about 85%, 90%, 95%, 99% or more identical to the amino acid sequence of SEQ ID NO: 23, or an amino acid sequence that differs from the amino acid sequence of SEQ ID NO: 23 by 1, 2, 5, 10, or 15 or fewer amino acid residues; and The VL domain comprising the amino acid sequence of SEQ ID NO: 30, an amino acid sequence that is at least about 85%, 90%, 95%, 99% or more identical to the amino acid sequence of SEQ ID NO: 30, or an amino acid sequence that differs from the amino acid sequence of SEQ ID NO: 30 by 1, 2, 5, 10, or 15 or fewer amino acid residues A multifunctional molecule according to any one of embodiments 43 to 73, comprising

[0110] 84. The antigen-binding domain is The VH domain comprising the amino acid sequence of SEQ ID NO: 24, an amino acid sequence that is at least about 85%, 90%, 95%, 99% or more identical to the amino acid sequence of SEQ ID NO: 24, or an amino acid sequence that differs from the amino acid sequence of SEQ ID NO: 24 by 1, 2, 5, 10, or 15 or fewer amino acid residues; and The VL domain comprising the amino acid sequence of SEQ ID NO: 26, an amino acid sequence that is at least about 85%, 90%, 95%, 99% or more identical to the amino acid sequence of SEQ ID NO: 26, or an amino acid sequence that differs from the amino acid sequence of SEQ ID NO: 26 by 1, 2, 5, 10, or 15 or fewer amino acid residues A multifunctional molecule according to any one of embodiments 43 to 73, comprising

[0111] 85. The antigen-binding domain is The VH domain comprising the amino acid sequence of SEQ ID NO: 24, an amino acid sequence that is at least about 85%, 90%, 95%, 99% or more identical to the amino acid sequence of SEQ ID NO: 24, or an amino acid sequence that differs from the amino acid sequence of SEQ ID NO: 24 by 1, 2, 5, 10, or 15 or fewer amino acid residues; and The VL domain comprising the amino acid sequence of SEQ ID NO: 27, an amino acid sequence that is at least about 85%, 90%, 95%, 99% or more identical to the amino acid sequence of SEQ ID NO: 27, or an amino acid sequence that differs from the amino acid sequence of SEQ ID NO: 27 by 1, 2, 5, 10, or 15 or fewer amino acid residues A multifunctional molecule according to any one of embodiments 43 to 73, comprising

[0112] 86. The antigen-binding domain is The VH domain comprising the amino acid sequence of SEQ ID NO: 24, an amino acid sequence that is at least about 85%, 90%, 95%, 99% or more identical to the amino acid sequence of SEQ ID NO: 24, or an amino acid sequence that differs from the amino acid sequence of SEQ ID NO: 24 by 1, 2, 5, 10, or 15 or fewer amino acid residues; and The VL domain comprising the amino acid sequence of SEQ ID NO: 28, an amino acid sequence that is at least about 85%, 90%, 95%, 99% or more identical to the amino acid sequence of SEQ ID NO: 28, or an amino acid sequence that differs from the amino acid sequence of SEQ ID NO: 28 by 1, 2, 5, 10, or 15 or fewer amino acid residues A multifunctional molecule according to any one of embodiments 43 to 73, comprising

[0113] 87. The antigen-binding domain is The VH domain comprising the amino acid sequence of SEQ ID NO: 24, an amino acid sequence that is at least about 85%, 90%, 95%, 99% or more identical to the amino acid sequence of SEQ ID NO: 24, or an amino acid sequence that differs from the amino acid sequence of SEQ ID NO: 24 by 1, 2, 5, 10, or 15 or fewer amino acid residues; and The VL domain comprising the amino acid sequence of SEQ ID NO: 29, an amino acid sequence that is at least about 85%, 90%, 95%, 99% or more identical to the amino acid sequence of SEQ ID NO: 29, or an amino acid sequence that differs from the amino acid sequence of SEQ ID NO: 29 by 1, 2, 5, 10, or 15 or fewer amino acid residues A multifunctional molecule according to any one of embodiments 43 to 73, comprising A multifunctional molecule according to any one of embodiments 43 to 73, comprising

[0114] 88. The antigen-binding domain is The VH domain comprising the amino acid sequence of SEQ ID NO: 24, an amino acid sequence that is at least about 85%, 90%, 95%, 99% or more identical to the amino acid sequence of SEQ ID NO: 24, or an amino acid sequence that differs from the amino acid sequence of SEQ ID NO: 24 by 1, 2, 5, 10, or 15 or fewer amino acid residues; and The VL domain comprising the amino acid sequence of SEQ ID NO: 30, an amino acid sequence that is at least about 85%, 90%, 95%, 99% or more identical to the amino acid sequence of SEQ ID NO: 30, or an amino acid sequence that differs from the amino acid sequence of SEQ ID NO: 30 by 1, 2, 5, 10, or 15 or fewer amino acid residues A multifunctional molecule according to any one of embodiments 43 to 73, comprising

[0115] 89. The antigen-binding domain is A VH domain comprising the amino acid sequence of SEQ ID NO: 25, an amino acid sequence that is at least about 85%, 90%, 95%, 99% or more identical to the amino acid sequence of SEQ ID NO: 25, or an amino acid sequence that differs from the amino acid sequence of SEQ ID NO: 25 by 1, 2, 5, 10, or 15 or fewer amino acid residues; and A VL domain comprising the amino acid sequence of SEQ ID NO: 26, an amino acid sequence that is at least about 85%, 90%, 95%, 99% or more identical to the amino acid sequence of SEQ ID NO: 26, or an amino acid sequence that differs from the amino acid sequence of SEQ ID NO: 26 by 1, 2, 5, 10, or 15 or fewer amino acid residues A multifunctional molecule according to any of embodiments 43 to 73, comprising the same.

[0116] 90. The antigen-binding domain is A VH domain comprising the amino acid sequence of SEQ ID NO: 25, an amino acid sequence that is at least about 85%, 90%, 95%, 99% or more identical to the amino acid sequence of SEQ ID NO: 25, or an amino acid sequence that differs from the amino acid sequence of SEQ ID NO: 25 by 1, 2, 5, 10, or 15 or fewer amino acid residues; and A VL domain comprising the amino acid sequence of SEQ ID NO: 27, an amino acid sequence that is at least about 85%, 90%, 95%, 99% or more identical to the amino acid sequence of SEQ ID NO: 27, or an amino acid sequence that differs from the amino acid sequence of SEQ ID NO: 27 by 1, 2, 5, 10, or 15 or fewer amino acid residues A multifunctional molecule according to any of embodiments 43 to 73, comprising the same.

[0117] 91. The antigen-binding domain is A VH domain comprising the amino acid sequence of SEQ ID NO: 25, an amino acid sequence that is at least about 85%, 90%, 95%, 99% or more identical to the amino acid sequence of SEQ ID NO: 25, or an amino acid sequence that differs from the amino acid sequence of SEQ ID NO: 25 by 1, 2, 5, 10, or 15 or fewer amino acid residues; and The VL domain comprising the amino acid sequence of SEQ ID NO: 28, an amino acid sequence that is at least about 85%, 90%, 95%, 99% or more identical to the amino acid sequence of SEQ ID NO: 28, or an amino acid sequence that differs from the amino acid sequence of SEQ ID NO: 28 by 1, 2, 5, 10, or 15 or fewer amino acid residues A multifunctional molecule according to any one of embodiments 43 to 73, comprising

[0118] 92. The antigen-binding domain is The VH domain comprising the amino acid sequence of SEQ ID NO: 25, an amino acid sequence that is at least about 85%, 90%, 95%, 99% or more identical to the amino acid sequence of SEQ ID NO: 25, or an amino acid sequence that differs from the amino acid sequence of SEQ ID NO: 25 by 1, 2, 5, 10, or 15 or fewer amino acid residues; and The VL domain comprising the amino acid sequence of SEQ ID NO: 29, an amino acid sequence that is at least about 85%, 90%, 95%, 99% or more identical to the amino acid sequence of SEQ ID NO: 29, or an amino acid sequence that differs from the amino acid sequence of SEQ ID NO: 29 by 1, 2, 5, 10, or 15 or fewer amino acid residues A multifunctional molecule according to any one of embodiments 43 to 73, comprising

[0119] 93. The antigen-binding domain is The VH domain comprising the amino acid sequence of SEQ ID NO: 25, an amino acid sequence that is at least about 85%, 90%, 95%, 99% or more identical to the amino acid sequence of SEQ ID NO: 25, or an amino acid sequence that differs from the amino acid sequence of SEQ ID NO: 25 by 1, 2, 5, 10, or 15 or fewer amino acid residues; and The VL domain comprising the amino acid sequence of SEQ ID NO: 30, an amino acid sequence that is at least about 85%, 90%, 95%, 99% or more identical to the amino acid sequence of SEQ ID NO: 30, or an amino acid sequence that differs from the amino acid sequence of SEQ ID NO: 30 by 1, 2, 5, 10, or 15 or fewer amino acid residues A multifunctional molecule according to any one of embodiments 43 to 73, comprising

[0120] 94. The first antigen-binding domain has a higher affinity for a T cell receptor containing a TCRBV antigen, and optionally the K for the binding between the first antigen-binding domain and a T cell receptor containing a TCRBV antigen D is less than 40%, 30%, 20%, 10%, 1%, 0.1%, or 0.01% of the K for the binding between the first antigen-binding domain and a T cell receptor not containing a TCRBV antigen, a multifunctional molecule according to any one of embodiments 1 to 93. D

[0121] 95. A multifunctional molecule according to any of the preceding embodiments, wherein, for example, the binding of the first antigen-binding domain to the TCRBV antigen on a lymphocyte (e.g., a T cell) does not activate the lymphocyte, e.g., the T cell.

[0122] 96. A multifunctional molecule according to any of the preceding embodiments, wherein, for example, the binding of the first antigen-binding domain to the TCRBV antigen on a lymphocyte (e.g., a T cell) hardly activates the lymphocyte (e.g., as measured by T cell proliferation, expression of T cell activation markers (e.g., CD69 or CD25), and / or expression of cytokines (e.g., TNFα and IFNγ)).

[0123] 97. A multifunctional molecule according to any of the preceding embodiments, wherein the multifunctional molecule preferentially binds to lymphocytes containing a TCRBV antigen over lymphocytes not containing a TCRBV antigen, and optionally the binding between the multifunctional molecule and lymphocytes containing a TCRBV antigen is greater than 10, 20, 30, 40, or 50-fold greater than the binding between the multifunctional molecule and lymphocytes not containing a TCRBV antigen.

[0124] 98. A multifunctional molecule according to any one of embodiments 1 to 97, wherein the multifunctional molecule comprises an immune cell engager selected from an NK cell engager, a T cell engager, a B cell engager, a dendritic cell engager, or a macrophage cell engager.

[0125] ​​99. The multifunctional molecule of embodiment 98, wherein the immune cell engager binds to and activates an immune cell, such as an effector cell. 100. The multifunctional molecule of embodiment 98, wherein the immune cell engager binds to an immune cell, such as an effector cell, but does not activate it.

[0126] 101. The multifunctional molecule according to any one of embodiments 98 to 100, wherein the immune cell engager is a T cell engager, such as a T cell engager that mediates binding and activation to T cells, or a T cell engager that mediates binding to T cells but does not mediate activation.

[0127] 102. The multifunctional molecule of embodiment 101, wherein the T cell engager binds to TCRα, TCRγ, TCRζ, ICOS, CD28, CD27, HVEM, LIGHT, CD40, 4-1BB, OX40, DR3, GITR, CD30, TIM1, SLAM, CD2, CD3, or CD226, for example, the T cell engager is an anti-CD3 antibody molecule.

[0128] 103. The multifunctional molecule according to any one of embodiments 98 to 100, wherein the immune cell engager is an NK cell engager, such as an NK cell engager that mediates binding and activation to NK cells, or an NK cell engager that mediates binding to NK cells but does not mediate activation.

[0129] 104. The NK cell engager binds to (e.g., activates) an antibody molecule, such as NKp30, NKp40, NKp44, NKp46, NKG2D, DNAM1, DAP10, CD16 (e.g., CD16a, CD16b, or both), CRTAM, CD27, PSGL1, CD96, CD100 (SEMA4D), NKp80, CD244 (also known as SLAMF4 or 2B4), SLAMF6, SLAMF7, KIR2DS2, KIR2DS4, KIR3DS1, KIR2DS3, KIR2DS5, KIR2DS1, CD94, NKG2C, NKG2E, or CD160, and is selected from, for example, an antibody molecule or a ligand. For example, the NK cell engager is an antibody molecule or a ligand that binds to (e.g., activates) NKp30, which is the multifunctional molecule of embodiment 103.

[0130] 105. The NK cell engager is an antibody molecule, such as an antigen-binding domain, which is the multifunctional molecule of embodiment 103. 106. The NK cell engager is a multifunctional molecule according to any one of embodiments 104 or 105 that can engage with NK cells.

[0131] 107. The NK cell engager is an antibody molecule that binds to NKp30, NKp46, NKG2D, or CD16, such as an antigen-binding domain, which is the multifunctional molecule according to any one of embodiments 103 to 106.

[0132] 108. The multifunctional molecule (i) specifically binds to an epitope of NKp30, NKp46, NKG2D, or CD16, such as an epitope identical or similar to the epitope recognized by an anti-NKp30, anti-NKp46, anti-NKG2D, or anti-CD16 antibody molecule as described herein; (ii) exhibits a binding affinity or specificity identical or similar to that of an anti-NKp30, anti-NKp46, anti-NKG2D, or anti-CD16 antibody molecule as described herein, or both thereof; (iii) As described herein, inhibit the binding of an anti-NKp30, anti-NKp46, anti-NKG2D, or anti-CD16 antibody molecule, e.g., competitively inhibit; (iv) As described herein, bind to an epitope that is identical or overlapping with an anti-NKp30, anti-NKp46, anti-NKG2D, or anti-CD16 antibody molecule; or (v) A multifunctional molecule of any of the preceding embodiments that competes with the binding to an anti-NKp30, anti-NKp46, anti-NKG2D, or anti-CD16 antibody molecule and / or binds to the same epitope.

[0133] 109. A multifunctional molecule of any of embodiments 103 to 108, wherein the anti-NKp30 or anti-NKp46 antibody molecule comprises one or more CDRs, framework regions, variable domains, heavy or light chains, or antigen-binding domains selected from Tables 7 to 10, or sequences substantially identical thereto.

[0134] 110. A multifunctional molecule of any of embodiments 103 to 109, wherein the NK cell engager is an antibody molecule that binds to NKp30, e.g., an antigen-binding domain. 111. A multifunctional molecule of any of embodiments 103 to 110, wherein the lysis of lymphocytes, e.g., lymphocytes containing a TCRBV antigen corresponding to a biased TCRBV clone type, is mediated by NKp30.

[0135] 112. A multifunctional molecule of any of embodiments 103 to 111, wherein the multifunctional molecule does not activate NK cells when incubated with NK cells in the absence of a TCRBV antigen. 113. A multifunctional molecule of any of embodiments 103 to 112, wherein the NK cells are NK cells expressing NKp30 and the multifunctional molecule activates the NK cells when a TCRBV antigen is also present.

[0136] 114. When the NK cell is not an NK cell expressing NKp30 and the TCRBV antigen is also present, a multifunctional molecule that does not activate the NK cell, a multifunctional molecule according to any of embodiments 103 to 113.

[0137] 115. The NK cell engager is (i) a heavy chain variable region (VH) comprising the amino acid sequence of the heavy chain complementarity determining region 1 (VHCDR1) of SEQ ID NO: 6000 (or a sequence having 1, 2, 3, or 4 or fewer mutations, such as substitutions, additions, or deletions), the amino acid sequence of VHCDR2 of SEQ ID NO: 6001 (or a sequence having 1, 2, 3, or 4 or fewer mutations, such as substitutions, additions, or deletions), and / or the amino acid sequence of VHCDR3 of SEQ ID NO: 6002 (or a sequence having 1, 2, 3, or 4 or fewer mutations, such as substitutions, additions, or deletions); and (ii) a light chain variable region (VL) comprising the amino acid sequence of the light chain complementarity determining region 1 (VLCDR1) of SEQ ID NO: 6063 (or a sequence having 1, 2, 3, or 4 or fewer mutations, such as substitutions, additions, or deletions), the amino acid sequence of VLCDR2 of SEQ ID NO: 6064 (or a sequence having 1, 2, 3, or 4 or fewer mutations, such as substitutions, additions, or deletions), and / or the amino acid sequence of VLCDR3 of SEQ ID NO: 6065 (or a sequence having 1, 2, 3, or 4 or fewer mutations, such as substitutions, additions, or deletions) comprising a multifunctional molecule according to any of embodiments 103 to 113.

[0138] 116. The NK cell engager is (i) a heavy chain variable region (VH) comprising the amino acid sequence of the heavy chain complementarity determining region 1 (VHCDR1) of SEQ ID NO: 6000, the amino acid sequence of VHCDR2 of SEQ ID NO: 6001, and / or the VHC DR3 amino acid sequence of SEQ ID NO: 6002, and (ii) A variable light chain (VL) comprising the amino acid sequence of the variable light chain complementarity determining region 1 (VLCDR1) of SEQ ID NO: 6063, the amino acid sequence of VLCDR2 of SEQ ID NO: 6064, and / or the amino acid sequence of VLCDR3 of SEQ ID NO: 6065 The multifunctional molecule of embodiment 115, comprising

[0139] 117. The NK cell engager is (1) A variable heavy chain (VH) comprising the amino acid sequence of the variable heavy chain framework region 1 (VHFWR1) of SEQ ID NO: 6003 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), the amino acid sequence of VHFWR2 of SEQ ID NO: 6004 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), the amino acid sequence of VHFWR3 of SEQ ID NO: 6005 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), or the amino acid sequence of VHFWR4 of SEQ ID NO: 6006 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions); and / or (2) A variable light chain (VL) comprising the amino acid sequence of the variable light chain framework region 1 (VLFWR1) of SEQ ID NO: 6066 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), the amino acid sequence of VLFWR2 of SEQ ID NO: 6067 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), the amino acid sequence of VLFWR3 of SEQ ID NO: 6068 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), or the amino acid sequence of VLFWR4 of SEQ ID NO: 6069 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions) The multifunctional molecule of any one of embodiments 103 to 116, comprising

[0140] 118. The NK cell engager is (1) A heavy chain variable region (VH) comprising the amino acid sequence of the heavy chain framework region 1 (VHFWR1) of SEQ ID NO: 6003, the amino acid sequence of VHFWR2 of SEQ ID NO: 6004, the amino acid sequence of VHFWR3 of SEQ ID NO: 6005, or the amino acid sequence of VHFWR4 of SEQ ID NO: 6006; and (2) A light chain variable region (VL) comprising the amino acid sequence of the light chain framework region 1 (VLFWR1) of SEQ ID NO: 6066, the amino acid sequence of VLFWR2 of SEQ ID NO: 6067, the amino acid sequence of VLFWR3 of SEQ ID NO: 6068, or the amino acid sequence of VLFWR4 of SEQ ID NO: 6069 The multifunctional molecule of embodiment 117, comprising

[0141] 119. The NK cell engager is (i) A VH comprising the amino acid sequence of SEQ ID NO: 6121 (or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with SEQ ID NO: 6121), and / or (ii) A VL comprising the amino acid sequence of SEQ ID NO: 6135 (or an amino acid sequence having at least about 93%, 95%, or 99% sequence identity with SEQ ID NO: 6135) The multifunctional molecule of any one of embodiments 103 to 118, comprising

[0142] 120. The NK cell engager comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 6148 or 6149 (or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with SEQ ID NO: 6148 or 6149), the multifunctional molecule of any one of embodiments 103 to 119.

[0143] 121. The NK cell engager comprises a light chain comprising the amino acid sequence of SEQ ID NO: 6150 (or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with SEQ ID NO: 6150), the multifunctional molecule of any one of embodiments 103 to 120.

[0144] 122. A multifunctional molecule according to any one of embodiments 103 to 121, wherein the NK cell engager comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 6148 or 6149 (or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with SEQ ID NO: 6148 or 6149), and a light chain comprising the amino acid sequence of SEQ ID NO: 6150 (or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with SEQ ID NO: 6150).

[0145] 123. A multifunctional molecule according to any one of embodiments 103 to 116, wherein the NK cell engager comprises a variable heavy chain region (VH) comprising the amino acid sequence of SEQ ID NO: 6014 of the heavy chain framework region 1 (VHFWR1) (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), the amino acid sequence of SEQ ID NO: 6015 of VHFWR2 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), the amino acid sequence of SEQ ID NO: 6016 of VHFWR3 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), or the amino acid sequence of SEQ ID NO: 6017 of VHFWR4 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions).

[0146] 124. A multifunctional molecule according to embodiment 123, wherein the NK cell engager comprises a variable heavy chain region (VH) comprising the amino acid sequence of SEQ ID NO: 6014 of the heavy chain framework region 1 (VHFWR1), the amino acid sequence of SEQ ID NO: 6015 of VHFWR2, the amino acid sequence of SEQ ID NO: 6016 of VHFWR3, or the amino acid sequence of SEQ ID NO: 6017 of VHFWR4.

[0147] 125. A multifunctional molecule according to embodiment 124, wherein the NK cell engager comprises a VH comprising the amino acid sequence of SEQ ID NO: 6123 (or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with SEQ ID NO: 6123).

[0148] 126. An NK cell engager comprising a heavy chain variable region (VH) comprising the amino acid sequence of the heavy chain framework region 1 (VHFWR1) of SEQ ID NO: 6018 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), the amino acid sequence of VHFWR2 of SEQ ID NO: 6019 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), the amino acid sequence of VHFWR3 of SEQ ID NO: 6020 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), or the amino acid sequence of VHFWR4 of SEQ ID NO: 6021 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), a multifunctional molecule of any of embodiments 103 to 116.

[0149] 127. An NK cell engager comprising a heavy chain variable region (VH) comprising the amino acid sequence of the heavy chain framework region 1 (VHFWR1) of SEQ ID NO: 6018, the amino acid sequence of VHFWR2 of SEQ ID NO: 6019, the amino acid sequence of VHFWR3 of SEQ ID NO: 6020, or the amino acid sequence of VHFWR4 of SEQ ID NO: 6021, a multifunctional molecule of embodiment 126.

[0150] 128. An NK cell engager comprising a VH comprising the amino acid sequence of SEQ ID NO: 6124 (or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with SEQ ID NO: 6124), a multifunctional molecule of embodiment 127. number 6124 and at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with SEQ ID NO: 6124), a multifunctional molecule of embodiment 127.

[0151] 129. An NK cell engager, which is a multifunctional molecule of any one of embodiments 103 to 116, comprising a heavy chain variable region (VH) containing the amino acid sequence of the heavy chain framework region 1 (VHFWR1) of SEQ ID NO: 6022 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), the amino acid sequence of VHFWR2 of SEQ ID NO: 6023 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), the amino acid sequence of VHFWR3 of SEQ ID NO: 6024 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), or the amino acid sequence of VHFWR4 of SEQ ID NO: 6025 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions).

[0152] 130. An NK cell engager, which is a multifunctional molecule of embodiment 129, comprising a heavy chain variable region (VH) containing the amino acid sequence of the heavy chain framework region 1 (VHFWR1) of SEQ ID NO: 6022, the amino acid sequence of VHFWR2 of SEQ ID NO: 6023, the amino acid sequence of VHFWR3 of SEQ ID NO: 6024, or the amino acid sequence of VHFWR4 of SEQ ID NO: 6025.

[0153] 131. An NK cell engager, which is a multifunctional molecule of embodiment 130, comprising a VH containing the amino acid sequence of SEQ ID NO: 6125 (or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with SEQ ID NO: 6125).

[0154] 132. An NK cell engager comprising a heavy chain variable region (VH) comprising the amino acid sequence of the heavy chain framework region 1 (VHFWR1) of SEQ ID NO: 6026 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), the amino acid sequence of VHFWR2 of SEQ ID NO: 6027 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), the amino acid sequence of VHFWR3 of SEQ ID NO: 6028 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), or the amino acid sequence of VHFWR4 of SEQ ID NO: 6029 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), a multifunctional molecule of any of embodiments 103 to 116.

[0155] 133. An NK cell engager comprising a heavy chain variable region (VH) comprising the amino acid sequence of the heavy chain framework region 1 (VHFWR1) of SEQ ID NO: 6026, the amino acid sequence of VHFWR2 of SEQ ID NO: 6027, the amino acid sequence of VHFWR3 of SEQ ID NO: 6028, or the amino acid sequence of VHFWR4 of SEQ ID NO: 6029, a multifunctional molecule of embodiment 132.

[0156] 134. An NK cell engager comprising a VH comprising the amino acid sequence of SEQ ID NO: 6126 (or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 6126), a multifunctional molecule of embodiment 133.

[0157] 135. An NK cell engager comprising a heavy chain variable region (VH) comprising the amino acid sequence of the heavy chain framework region 1 (VHFWR1) of SEQ ID NO: 6030 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), the amino acid sequence of VHFWR2 of SEQ ID NO: 6032 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), the amino acid sequence of VHFWR3 of SEQ ID NO: 6033 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), or the amino acid sequence of VHFWR4 of SEQ ID NO: 6034 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), a multifunctional molecule of any of embodiments 103 to 116. A multifunctional molecule of any of embodiments 103 to 116, comprising a heavy chain variable region (VH) comprising the amino acid sequence of the heavy chain framework region 1 (VHFWR1) of SEQ ID NO: 6030, the amino acid sequence of VHFWR2 of SEQ ID NO: 6032, the amino acid sequence of VHFWR3 of SEQ ID NO: 6033, or the amino acid sequence of VHFWR4 of SEQ ID NO: 6034.

[0158] 136. An NK cell engager, a multifunctional molecule of embodiment 135, comprising a heavy chain variable region (VH) comprising the amino acid sequence of the heavy chain framework region 1 (VHFWR1) of SEQ ID NO: 6030, the amino acid sequence of VHFWR2 of SEQ ID NO: 6032, the amino acid sequence of VHFWR3 of SEQ ID NO: 6033, or the amino acid sequence of VHFWR4 of SEQ ID NO: 6034.

[0159] 137. An NK cell engager, a multifunctional molecule of embodiment 136, comprising a VH comprising the amino acid sequence of SEQ ID NO: 6127 (or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 6127).

[0160] 138. An NK cell engager comprising a heavy chain variable region (VH) comprising the amino acid sequence of the heavy chain framework region 1 (VHFWR1) of SEQ ID NO: 6035 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), the amino acid sequence of VHFWR2 of SEQ ID NO: 6036 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), the amino acid sequence of VHFWR3 of SEQ ID NO: 6037 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), or the amino acid sequence of VHFWR4 of SEQ ID NO: 6038 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), a multifunctional molecule of any of embodiments 103 to 116.

[0161] 139. An NK cell engager comprising a heavy chain variable region (VH) comprising the amino acid sequence of the heavy chain framework region 1 (VHFWR1) of SEQ ID NO: 6035, the amino acid sequence of VHFWR2 of SEQ ID NO: 6036, the amino acid sequence of VHFWR3 of SEQ ID NO: 6037, or the amino acid sequence of VHFWR4 of SEQ ID NO: 6038, a multifunctional molecule of embodiment 138.

[0162] 140. An NK cell engager comprising a VH comprising the amino acid sequence of SEQ ID NO: 6128 (or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 6128), a multifunctional molecule of embodiment 139.

[0163] 141. An NK cell engager comprising a variable light chain (VL) region comprising the amino acid sequence of the variable light chain framework region 1 (VLFWR1) of SEQ ID NO: 6077 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), the amino acid sequence of VLFWR2 of SEQ ID NO: 6078 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), the amino acid sequence of VLFWR3 of SEQ ID NO: 6079 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), or the amino acid sequence of VLFWR4 of SEQ ID NO: 6080 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), a multifunctional molecule of any of embodiments 103 to 116 or 123 to 140.

[0164] 142. An NK cell engager comprising a variable light chain (VL) region comprising the amino acid sequence of the variable light chain framework region 1 (VLFWR1) of SEQ ID NO: 6077, the amino acid sequence of VLFWR2 of SEQ ID NO: 6078, the amino acid sequence of VLFWR3 of SEQ ID NO: 6079, or the amino acid sequence of VLFWR4 of SEQ ID NO: 6080, a multifunctional molecule of embodiment 141.

[0165] 143. An NK cell engager comprising a VL region comprising the amino acid sequence of SEQ ID NO: 6137 (or an amino acid sequence having at least about 93%, 95%, or 99% sequence identity with SEQ ID NO: 6137), a multifunctional molecule of embodiment 142. No. 6137 and at least about 93%, 95%, or 99% sequence identity with SEQ ID NO: 6137), a multifunctional molecule of embodiment 142.

[0166] 144. An NK cell engager, which is a multifunctional molecule of any one of embodiments 103 to 116 or 123 to 140, comprising a variable light chain region (VL) that includes the amino acid sequence of the variable light chain framework region 1 (VLFWR1) of SEQ ID NO: 6081 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), the amino acid sequence of VLFWR2 of SEQ ID NO: 6082 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), the amino acid sequence of VLFWR3 of SEQ ID NO: 6083 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), or the amino acid sequence of VLFWR4 of SEQ ID NO: 6084 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions).

[0167] 145. An NK cell engager, which is a multifunctional molecule of embodiment 144, comprising a variable light chain region (VL) that includes the amino acid sequence of the variable light chain framework region 1 (VLFWR1) of SEQ ID NO: 6081, the amino acid sequence of VLFWR2 of SEQ ID NO: 6082, the amino acid sequence of VLFWR3 of SEQ ID NO: 6083, or the amino acid sequence of VLFWR4 of SEQ ID NO: 6084.

[0168] 146. An NK cell engager, which is a multifunctional molecule of embodiment 145, comprising a VL that includes the amino acid sequence of SEQ ID NO: 6138 (or an amino acid sequence having at least about 93%, 95%, or 99% sequence identity with SEQ ID NO: 6138).

[0169] 147. An NK cell engager, which is a multifunctional molecule according to any one of embodiments 103 to 116 or 123 to 140, comprising a variable light chain (VL) region containing the amino acid sequence of the variable light chain framework region 1 (VLFWR1) of SEQ ID NO: 6085 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), the amino acid sequence of VLFWR2 of SEQ ID NO: 6086 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), the amino acid sequence of VLFWR3 of SEQ ID NO: 6087 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), or the amino acid sequence of VLFWR4 of SEQ ID NO: 6088 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions).

[0170] 148. A multifunctional molecule according to embodiment 147, wherein the NK cell engager comprises a variable light chain (VL) region containing the amino acid sequence of the variable light chain framework region 1 (VLFWR1) of SEQ ID NO: 6085, the amino acid sequence of VLFWR2 of SEQ ID NO: 6086, the amino acid sequence of VLFWR3 of SEQ ID NO: 6087, or the amino acid sequence of VLFWR4 of SEQ ID NO: 6088.

[0171] 149. A multifunctional molecule according to embodiment 148, wherein the NK cell engager comprises a VL region containing the amino acid sequence of SEQ ID NO: 6139 (or an amino acid sequence having at least about 93%, 95%, or 99% sequence identity with SEQ ID NO: 6139).

[0172] 150. An NK cell engager comprising a variable light chain (VL) region comprising the amino acid sequence of the light chain framework region 1 (VLFWR1) of SEQ ID NO: 6089 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), the amino acid sequence of VLFWR2 of SEQ ID NO: 6090 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), the amino acid sequence of VLFWR3 of SEQ ID NO: 6091 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), or the amino acid sequence of VLFWR4 of SEQ ID NO: 6092 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), a multifunctional molecule of any of embodiments 103 to 116 or 123 to 140. A multifunctional molecule of any of embodiments 103 to 116 or 123 to 140, wherein the NK cell engager comprises a variable light chain (VL) region comprising the amino acid sequence of the light chain framework region 1 (VLFWR1) of SEQ ID NO: 6089, the amino acid sequence of VLFWR2 of SEQ ID NO: 6090, the amino acid sequence of VLFWR3 of SEQ ID NO: 6091, or the amino acid sequence of VLFWR4 of SEQ ID NO: 6092.

[0173] 151. A multifunctional molecule of embodiment 150, wherein the NK cell engager comprises a variable light chain (VL) region comprising the amino acid sequence of the light chain framework region 1 (VLFWR1) of SEQ ID NO: 6089, the amino acid sequence of VLFWR2 of SEQ ID NO: 6090, the amino acid sequence of VLFWR3 of SEQ ID NO: 6091, or the amino acid sequence of VLFWR4 of SEQ ID NO: 6092.

[0174] 152. A multifunctional molecule of embodiment 151, wherein the NK cell engager comprises a VL region comprising the amino acid sequence of SEQ ID NO: 6140 (or an amino acid sequence having at least about 93%, 95%, or 99% sequence identity with SEQ ID NO: 6140).

[0175] 153. An NK cell engager comprising a variable light chain region (VL) comprising the amino acid sequence of the variable light chain framework region 1 (VLFWR1) of SEQ ID NO: 6093 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), the amino acid sequence of VLFWR2 of SEQ ID NO: 6094 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), the amino acid sequence of VLFWR3 of SEQ ID NO: 6095 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), or the amino acid sequence of VLFWR4 of SEQ ID NO: 6096 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), a multifunctional molecule of any of embodiments 103 to 116 or 123 to 140.

[0176] 154. The multifunctional molecule of embodiment 153, wherein the NK cell engager comprises the amino acid sequence of the variable light chain framework region 1 (VLFWR1) of SEQ ID NO: 6093, the amino acid sequence of VLFWR2 of SEQ ID NO: 6094, the amino acid sequence of VLFWR3 of SEQ ID NO: 6095, or the amino acid sequence of VLFWR4 of SEQ ID NO: 6096.

[0177] 155. The multifunctional molecule of embodiment 154, wherein the NK cell engager comprises a VL comprising the amino acid sequence of SEQ ID NO: 6141 (or an amino acid sequence having at least about 93%, 95%, or 99% sequence identity to SEQ ID NO: 6141).

[0178] 156. The NK cell engager is (i) A heavy chain variable region (VH) comprising the amino acid sequence of the heavy chain complementarity determining region 1 (VHCDR1) of SEQ ID NO: 6007 (or a sequence having 1, 2, 3, or 4 or fewer mutations, such as substitutions, additions, or deletions), the amino acid sequence of VHCDR2 of SEQ ID NO: 6008 (or a sequence having 1, 2, 3, or 4 or fewer mutations, such as substitutions, additions, or deletions), and / or the amino acid sequence of VHCDR3 of SEQ ID NO: 6009 (or a sequence having 1, 2, 3, or 4 or fewer mutations, such as substitutions, additions, or deletions); and (ii) A light chain variable region (VL) comprising the amino acid sequence of the light chain complementarity determining region 1 (VLCDR1) of SEQ ID NO: 6070 (or a sequence having 1, 2, 3, or 4 or fewer mutations, such as substitutions, additions, or deletions), the amino acid sequence of VLCDR2 of SEQ ID NO: 6071 (or a sequence having 1, 2, 3, or 4 or fewer mutations, such as substitutions, additions, or deletions), and / or the amino acid sequence of VLCDR3 of SEQ ID NO: 6072 (or a sequence having 1, 2, 3, or 4 or fewer mutations, such as substitutions, additions, or deletions) A multifunctional molecule according to any one of embodiments 103 to 114, comprising

[0179] 157. The NK cell engager is (i) A heavy chain variable region (VH) comprising the amino acid sequence of the heavy chain complementarity determining region 1 (VHCDR1) of SEQ ID NO: 6007, the amino acid sequence of VHCDR2 of SEQ ID NO: 6008, and / or the amino acid sequence of VHCDR3 of SEQ ID NO: 6009; and (ii) A light chain variable region (VL) comprising the amino acid sequence of the light chain complementarity determining region 1 (VLCDR1) of SEQ ID NO: 6070, the amino acid sequence of VLCDR2 of SEQ ID NO: 6071, and / or the amino acid sequence of VLCDR3 of SEQ ID NO: 6072 A multifunctional molecule according to embodiment 156, comprising

[0180] 158. The NK cell engager is (1) A heavy chain variable region (VH) comprising the amino acid sequence of heavy chain framework region 1 (VHFWR1) of SEQ ID NO: 6010 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), the amino acid sequence of VHFWR2 of SEQ ID NO: 6011 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), the amino acid sequence of VHFWR3 of SEQ ID NO: 6012 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), or the amino acid sequence of VHFWR4 of SEQ ID NO: 6013 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), and / or (2) A light chain variable region (VL) comprising the amino acid sequence of light chain framework region 1 (VLFWR1) of SEQ ID NO: 6073 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), the amino acid sequence of VLFWR2 of SEQ ID NO: 6074 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), the amino acid sequence of VLFWR3 of SEQ ID NO: 6075 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), or the amino acid sequence of VLFWR4 of SEQ ID NO: 6076 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), A multifunctional molecule according to any one of embodiments 103 to 114, 156, or 157, comprising

[0181] 159. The NK cell engager is (1) A heavy chain variable region (VH) comprising the amino acid sequence of heavy chain framework region 1 (VHFWR1) of SEQ ID NO: 6010, the amino acid sequence of VHFWR2 of SEQ ID NO: 6011, the amino acid sequence of VHFWR3 of SEQ ID NO: 6012, or the amino acid sequence of VHFWR4 of SEQ ID NO: 6013, and (3) A light chain variable region (VL) comprising the amino acid sequence of light chain framework region 1 (VLFWR1) of SEQ ID NO: 6073, the amino acid sequence of VLFWR2 of SEQ ID NO: 6074, the amino acid sequence of VLFWR3 of SEQ ID NO: 6075, or the amino acid sequence of VLFWR4 of SEQ ID NO: 6076 The multifunctional molecule of Embodiment 158, comprising

[0182] 160. The NK cell engager is (i) a VH comprising the amino acid sequence of SEQ ID NO: 6122 (or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with SEQ ID NO: 6122), and / or (ii) a VL comprising the amino acid sequence of SEQ ID NO: 6136 (or an amino acid sequence having at least about 93%, 95%, or 99% sequence identity with SEQ ID NO: 6136) The multifunctional molecule of any one of Embodiments 103 to 114 or 156 to 159, comprising

[0183] 161. The NK cell engager is a heavy chain comprising the amino acid sequence of SEQ ID NO: 6151 or 6152 (or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with SEQ ID NO: 6151 or 6152), the multifunctional molecule of any one of Embodiments 103 to 114 or 156 to 160.

[0184] 162. The NK cell engager is a light chain comprising the amino acid sequence of SEQ ID NO: 6153 (or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with SEQ ID NO: 6153), the multifunctional molecule of any one of Embodiments 103 to 114 or 156 to 161.

[0185] 163. A multifunctional molecule according to any one of embodiments 103 to 114 or 156 to 162, wherein the NK cell engager comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 6151 or 6152 (or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 6151 or 6152), and a light chain comprising the amino acid sequence of SEQ ID NO: 6153 (or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 6153).

[0186] 164. A multifunctional molecule according to any one of embodiments 103 to 114, 156, or 157, wherein the NK cell engager comprises a variable heavy chain region (VH) comprising the amino acid sequence of SEQ ID NO: 6039 for heavy chain framework region 1 (VHFWR1) (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), the amino acid sequence of SEQ ID NO: 6040 for VHFWR2 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), the amino acid sequence of SEQ ID NO: 6041 for VHFWR3 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), or the amino acid sequence of SEQ ID NO: 6042 for VHFWR4 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions).

[0187] 165. A multifunctional molecule according to embodiment 164, wherein the NK cell engager comprises a variable heavy chain region (VH) comprising the amino acid sequence of SEQ ID NO: 6039 for heavy chain framework region 1 (VHFWR1), the amino acid sequence of SEQ ID NO: 6040 for VHFWR2, the amino acid sequence of SEQ ID NO: 6041 for VHFWR3, or the amino acid sequence of SEQ ID NO: 6042 for VHFWR4.

[0188] 166. The multifunctional molecule of embodiment 165, wherein the NK cell engager comprises a VH comprising the amino acid sequence of SEQ ID NO: 6129 (or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with SEQ ID NO: 6129).

[0189] 167. The multifunctional molecule of any one of embodiments 103 to 114, 156, or 157, wherein the NK cell engager comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 6043 (heavy chain variable region 1 (VHFWR1)) (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), the amino acid sequence of SEQ ID NO: 6044 (VHFWR2) (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), the amino acid sequence of SEQ ID NO: 6045 (VHFWR3) (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), or the amino acid sequence of SEQ ID NO: 6046 (VHFWR4) (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions).

[0190] 168. The NK cell engager comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 6043 ( VHFWR1), the amino acid sequence of SEQ ID NO: 6044 (VHFWR2), the amino acid sequence of SEQ ID NO: 6045 (VHFWR3), or the amino acid sequence of SEQ ID NO: 6046 (VHFWR4), and the multifunctional molecule of embodiment 167.

[0191] 169. The multifunctional molecule of embodiment 168, wherein the NK cell engager comprises a VH comprising the amino acid sequence of SEQ ID NO: 6130 (or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with SEQ ID NO: 6130).

[0192] 170. An NK cell engager, which is a multifunctional molecule of any of embodiments 103 to 114, 156, or 157, comprising a heavy chain variable region (VH) that includes the amino acid sequence of the heavy chain framework region 1 (VHFWR1) of SEQ ID NO: 6047 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), the amino acid sequence of VHFWR2 of SEQ ID NO: 6048 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), the amino acid sequence of VHFWR3 of SEQ ID NO: 6049 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), or the amino acid sequence of VHFWR4 of SEQ ID NO: 6050 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions).

[0193] 171. An NK cell engager, which is a multifunctional molecule of embodiment 170, comprising a heavy chain variable region (VH) that includes the amino acid sequence of the heavy chain framework region 1 (VHFWR1) of SEQ ID NO: 6047, the amino acid sequence of VHFWR2 of SEQ ID NO: 6048, the amino acid sequence of VHFWR3 of SEQ ID NO: 6049, or the amino acid sequence of VHFWR4 of SEQ ID NO: 6050.

[0194] 172. An NK cell engager, which is a multifunctional molecule of embodiment 171, comprising a VH that includes the amino acid sequence of SEQ ID NO: 6131 (or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 6131).

[0195] 173. An NK cell engager comprising a heavy chain variable region (VH) comprising the amino acid sequence of heavy chain framework region 1 (VHFWR1) of SEQ ID NO: 6051 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), the amino acid sequence of VHFWR2 of SEQ ID NO: 6052 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), the amino acid sequence of VHFWR3 of SEQ ID NO: 6053 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), or the amino acid sequence of VHFWR4 of SEQ ID NO: 6054 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), a multifunctional molecule of any of embodiments 103 to 114, 156, or 157.

[0196] 174. An NK cell engager comprising a heavy chain variable region (VH) comprising the amino acid sequence of heavy chain framework region 1 (VHFWR1) of SEQ ID NO: 6051, the amino acid sequence of VHFWR2 of SEQ ID NO: 6052, the amino acid sequence of VHFWR3 of SEQ ID NO: 6053, or the amino acid sequence of VHFWR4 of SEQ ID NO: 6054, a multifunctional molecule of embodiment 173.

[0197] 175. An NK cell engager comprising a VH comprising the amino acid sequence of SEQ ID NO: 6132 (or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with SEQ ID NO: 6132), a multifunctional molecule of embodiment 174.

[0198] 176. An NK cell engager comprising the amino acid sequence of heavy chain framework region 1 (VHFWR1) of SEQ ID NO: 6055 (or 1, 2, 3, 4, 5, or A multifunctional molecule of any of embodiments 103 to 114, 156, or 157, comprising a heavy chain variable region (VH) comprising the amino acid sequence of heavy chain framework region 1 (VHFWR1) of SEQ ID NO: 6055, the amino acid sequence of VHFWR2 of SEQ ID NO: 6056 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), the amino acid sequence of VHFWR3 of SEQ ID NO: 6057 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), or the amino acid sequence of VHFWR4 of SEQ ID NO: 6058 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions).

[0199] 177. A multifunctional molecule of embodiment 176, wherein the NK cell engager comprises a heavy chain variable region (VH) comprising the amino acid sequence of heavy chain framework region 1 (VHFWR1) of SEQ ID NO: 6055, the amino acid sequence of VHFWR2 of SEQ ID NO: 6056, the amino acid sequence of VHFWR3 of SEQ ID NO: 6057, or the amino acid sequence of VHFWR4 of SEQ ID NO: 6058.

[0200] 178. A multifunctional molecule of embodiment 177, wherein the NK cell engager comprises a VH comprising the amino acid sequence of SEQ ID NO: 6133 (or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 6133).

[0201] 179. An NK cell engager, which is a multifunctional molecule of any of embodiments 103 to 114, 156, or 157, comprising a heavy chain variable region (VH) that includes the amino acid sequence of the heavy chain framework region 1 (VHFWR1) of SEQ ID NO: 6059 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), the amino acid sequence of VHFWR2 of SEQ ID NO: 6060 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), the amino acid sequence of VHFWR3 of SEQ ID NO: 6061 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), or the amino acid sequence of VHFWR4 of SEQ ID NO: 6062 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions).

[0202] 180. An NK cell engager, which is a multifunctional molecule of embodiment 179, comprising a heavy chain variable region (VH) that includes the amino acid sequence of the heavy chain framework region 1 (VHFWR1) of SEQ ID NO: 6059, the amino acid sequence of VHFWR2 of SEQ ID NO: 6060, the amino acid sequence of VHFWR3 of SEQ ID NO: 6061, or the amino acid sequence of VHFWR4 of SEQ ID NO: 6062.

[0203] 181. An NK cell engager, which is a multifunctional molecule of embodiment 180, comprising a VH that includes the amino acid sequence of SEQ ID NO: 6134 (or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 6134).

[0204] 182. An NK cell engager, which is a multifunctional molecule of any one of embodiments 103 to 114, 156, 157, or 164 to 181, comprising a variable light chain (VL) region that includes the amino acid sequence of the variable light chain framework region 1 (VLFWR1) of SEQ ID NO: 6097 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), the amino acid sequence of VLFWR2 of SEQ ID NO: 6098 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), the amino acid sequence of VLFWR3 of SEQ ID NO: 6099 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), or the amino acid sequence of VLFWR4 of SEQ ID NO: 6100 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions).

[0205] 183. An NK cell engager, which is a multifunctional molecule of embodiment 182, comprising a variable light chain (VL) region that includes the amino acid sequence of the variable light chain framework region 1 (VLFWR1) of SEQ ID NO: 6097, the amino acid sequence of VLFWR2 of SEQ ID NO: 6098, the amino acid sequence of VLFWR3 of SEQ ID NO: 6099, or the amino acid sequence of VLFWR4 of SEQ ID NO: 6100.

[0206] 184. An NK cell engager, which is a multifunctional molecule of embodiment 183, comprising a VL that includes the amino acid sequence of SEQ ID NO: 6142 (or an amino acid sequence having at least about 93%, 95%, or 99% sequence identity with SEQ ID NO: 6142).

[0207] 185. An NK cell engager comprising a variable light chain region (VL) comprising the amino acid sequence of the variable light chain framework region 1 (VLFWR1) of SEQ ID NO: 6101 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), the amino acid sequence of VLFWR2 of SEQ ID NO: 6102 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), the amino acid sequence of VLFWR3 of SEQ ID NO: 6103 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), or the amino acid sequence of VLFWR4 of SEQ ID NO: 6104 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), a multifunctional molecule of any of embodiments 103 to 114, 156, 157, or 164 to 181.

[0208] 186. An NK cell engager comprising a variable light chain region (VL) comprising the amino acid sequence of the variable light chain framework region 1 (VLFWR1) of SEQ ID NO: 6101, the amino acid sequence of VLFWR2 of SEQ ID NO: 6102, the amino acid sequence of VLFWR3 of SEQ ID NO: 6103, or the amino acid sequence of VLFWR4 of SEQ ID NO: 6104, a multifunctional molecule of embodiment 185.

[0209] 187. An NK cell engager comprising a VL comprising the amino acid sequence of SEQ ID NO: 6143 (or an amino acid sequence having at least about 93%, 95%, or 99% sequence identity with SEQ ID NO: 6143), a multifunctional molecule of embodiment 186.

[0210] 188. An NK cell engager, which is a multifunctional molecule of any one of embodiments 103 to 114, 156, 157, or 164 to 181, comprising a variable light chain region (VL) that includes the amino acid sequence of the variable light chain framework region 1 (VLFWR1) of SEQ ID NO: 6105 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), the amino acid sequence of VLFWR2 of SEQ ID NO: 6106 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), the amino acid sequence of VLFWR3 of SEQ ID NO: 6107 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), or the amino acid sequence of VLFWR4 of SEQ ID NO: 6108 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions).

[0211] 189. An NK cell engager, which is a multifunctional molecule of embodiment 188, comprising a variable light chain region (VL) that includes the amino acid sequence of the variable light chain framework region 1 (VLFWR1) of SEQ ID NO: 6105, the amino acid sequence of VLFWR2 of SEQ ID NO: 6106, the amino acid sequence of VLFWR3 of SEQ ID NO: 6107, or the amino acid sequence of VLFWR4 of SEQ ID NO: 6108.

[0212] 190. An NK cell engager, which is a multifunctional molecule of embodiment 189, comprising a VL that includes the amino acid sequence of SEQ ID NO: 6144 (or an amino acid sequence having at least about 93%, 95%, or 99% sequence identity to SEQ ID NO: 6144).

[0213] 191. An NK cell engager, which is a multifunctional molecule of any one of embodiments 103 to 114, 156, 157, or 164 to 181, comprising a variable light chain region (VL) that includes the amino acid sequence of the variable light chain framework region 1 (VLFWR1) of SEQ ID NO: 6109 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), the amino acid sequence of VLFWR2 of SEQ ID NO: 6110 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), the amino acid sequence of VLFWR3 of SEQ ID NO: 6111 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), or the amino acid sequence of VLFWR4 of SEQ ID NO: 6112 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions).

[0214] 192. An NK cell engager, which is a multifunctional molecule of embodiment 191, comprising a variable light chain region (VL) that includes the amino acid sequence of the variable light chain framework region 1 (VLFWR1) of SEQ ID NO: 6109, the amino acid sequence of VLFWR2 of SEQ ID NO: 6110, the amino acid sequence of VLFWR3 of SEQ ID NO: 6111, or the amino acid sequence of VLFWR4 of SEQ ID NO: 6112.

[0215] 193. An NK cell engager, which is a multifunctional molecule of embodiment 192, comprising a VL that includes the amino acid sequence of SEQ ID NO: 6145 (or an amino acid sequence having at least about 93%, 95%, or 99% sequence identity with SEQ ID NO: 6145).

[0216] 194. An NK cell engager, which is a multifunctional molecule of any one of embodiments 103 to 114, 156, 157, or 164 to 181, comprising a variable light chain region (VL) that includes the amino acid sequence of the variable light chain framework region 1 (VLFWR1) of SEQ ID NO: 6113 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), the amino acid sequence of VLFWR2 of SEQ ID NO: 6114 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), the amino acid sequence of VLFWR3 of SEQ ID NO: 6115 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), or the amino acid sequence of VLFWR4 of SEQ ID NO: 6116 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions).

[0217] 195. An NK cell engager, which is a multifunctional molecule of embodiment 194, comprising a variable light chain region (VL) that includes the amino acid sequence of the variable light chain framework region 1 (VLFWR1) of SEQ ID NO: 6113, the amino acid sequence of VLFWR2 of SEQ ID NO: 6114, the amino acid sequence of VLFWR3 of SEQ ID NO: 6115, or the amino acid sequence of VLFWR4 of SEQ ID NO: 6116.

[0218] 196. An NK cell engager, which is a multifunctional molecule of embodiment 195, comprising a VL that includes the amino acid sequence of SEQ ID NO: 6146 (or an amino acid sequence having at least about 93%, 95%, or 99% sequence identity with SEQ ID NO: 6146).

[0219] 197. The NK cell engager is selected from the group consisting of a light chain framework region 1 (VLFWR1) amino acid sequence of SEQ ID NO: 6117 (or a sequence having 1, 2, 3, 4, 5, or 6 or less mutations, e.g., substitutions, additions, or deletions therefrom), a VLFWR2 amino acid sequence of SEQ ID NO: 6118 (or a sequence having 1, 2, 3, 4, 5, or 6 or less mutations, e.g., substitutions, additions, or deletions therefrom), a VLFWR3 amino acid sequence of SEQ ID NO: 6119 (or a sequence having 1, 2, 3, 4, 5, or 6 or less mutations, e.g., substitutions, additions, or deletions therefrom), or a VL The multifunctional molecule of any of embodiments 103 to 114, 156, 157, or 164 to 181, comprising a light chain variable region (VL) comprising a FWR4 amino acid sequence (or a sequence having no more than 1, 2, 3, 4, 5, or 6 mutations therein, e.g., substitutions, additions, or deletions).

[0220] 198. The multifunctional molecule of embodiment 197, wherein the NK cell engager comprises a light chain variable region (VL) comprising the light chain framework region 1 (VLFWR1) amino acid sequence of SEQ ID NO: 6117, the VLFWR2 amino acid sequence of SEQ ID NO: 6118, the VLFWR3 amino acid sequence of SEQ ID NO: 6119, or the VLFWR4 amino acid sequence of SEQ ID NO: 6120.

[0221] 199. The multifunctional molecule of embodiment 198, wherein the NK cell engager comprises a VL comprising the amino acid sequence of SEQ ID NO: 6147 (or an amino acid sequence having at least about 93%, 95%, or 99% sequence identity to SEQ ID NO: 6147).

[0222] 200. The multifunctional molecule of any of embodiments 103 to 106, wherein the NK cell engager is an antibody molecule, e.g., an antigen-binding domain, that binds to NKp46. 201. The multifunctional molecule of embodiment 200, wherein the lysis of lymphoma cells is mediated by NKp46.

[0223] When incubated with NK cells in the absence of a 202.TCRBV antigen (e.g., a TCRBV antigen corresponding to a biased TCRBV clonotype), a multifunctional molecule of any of embodiments 200 or 201 does not activate the NK cells.

[0224] 203.A multifunctional molecule of any one of embodiments 200 to 202, wherein the NK cell is an NK cell expressing NKp46 and a TCRBV antigen (e.g., a TCRBV antigen corresponding to a biased TCRBV clonotype) is also present, and the multifunctional molecule activates the NK cells.

[0225] 204.A multifunctional molecule of any one of embodiments 200 to 203, wherein the NK cell is not an NK cell expressing NKp46 and a TCRBV antigen (e.g., a TCRBV antigen corresponding to a biased TCRBV clonotype) is also present, and the multifunctional molecule does not activate the NK cells.

[0226] 205.A multifunctional molecule of any one of embodiments 200 to 204, wherein the NK cell engager comprises a VH comprising the amino acid sequence of SEQ ID NO: 6182 (or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with SEQ ID NO: 6182).

[0227] 206.A multifunctional molecule of any one of embodiments 200 to 205, wherein the NK cell engager comprises a VL comprising the amino acid sequence of SEQ ID NO: 6183 (or an amino acid sequence having at least about 93%, 95%, or 99% sequence identity with SEQ ID NO: 6183).

[0228] 207.A multifunctional molecule of embodiments 200 to 205, wherein the NK cell engager comprises an scFV comprising the amino acid sequence of SEQ ID NO: 6181 (or an amino acid sequence having at least about 93%, 95%, or 99% sequence identity with SEQ ID NO: 6181).

[0229] 208. An NK cell engager that is an antibody molecule that binds to NKG2D, for example, a multifunctional molecule of any of embodiments 103 to 106 that is an antigen-binding domain. 209. The multifunctional molecule of embodiment 208, wherein lysis of lymphoma cells is mediated by NKG2D.

[0230] 210. The multifunctional molecule of either embodiment 208 or 209, wherein the multifunctional molecule does not activate NK cells when incubated with NK cells in the absence of a TCRBV antigen (e.g., a TCRBV antigen corresponding to a biased TCRBV clone type).

[0231] 211. The multifunctional molecule of any one of embodiments 208 to 210, wherein the NK cell is an NK cell that expresses NKG2D, and when a TCRBV antigen (e.g., a TCRBV antigen corresponding to a biased TCRBV clone type) is also present, the multifunctional molecule activates the NK cell.

[0232] 212. The multifunctional molecule of any one of embodiments 208 to 211, wherein the NK cell is not an NK cell that expresses NKG2D, and when a TCRBV antigen (e.g., a TCRBV antigen corresponding to a biased TCRBV clone type) is also present, the multifunctional molecule does not activate the NK cell.

[0233] 213. The multifunctional molecule of any one of embodiments 208 to 212, wherein the NK cell engager comprises a VH comprising the amino acid sequence of SEQ ID NO: 6176 (or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 6176).

[0234] 214. The multifunctional molecule of any one of embodiments 208 to 213, wherein the NK cell engager comprises a VL comprising the amino acid sequence of SEQ ID NO: 6177 (or an amino acid sequence having at least about 93%, 95%, or 99% sequence identity to SEQ ID NO: 6177).

[0235] 215. A multifunctional molecule according to any one of embodiments 208 to 214, wherein the NK cell engager comprises a scFV comprising the amino acid sequence of SEQ ID NO: 6175 (or an amino acid sequence having at least about 93%, 95%, or 99% sequence identity with SEQ ID NO: 6175).

[0236] 216. A multifunctional molecule according to any one of embodiments 208 to 212, wherein the NK cell engager comprises a VH comprising the amino acid sequence of SEQ ID NO: 6179 (or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with SEQ ID NO: 6179).

[0237] 217. A multifunctional molecule according to any one of embodiments 208 to 212 or 216, wherein the NK cell engager comprises a VL comprising the amino acid sequence of SEQ ID NO: 6180 (or an amino acid sequence having at least about 93%, 95%, or 99% sequence identity with SEQ ID NO: 6180).

[0238] 218. A multifunctional molecule according to any one of embodiments 208 to 212, 216 or 217, wherein the NK cell engager comprises a scFV comprising the amino acid sequence of SEQ ID NO: 6178 (or an amino acid sequence having at least about 93%, 95%, or 99% sequence identity with SEQ ID NO: 6178).

[0239] 219. A multifunctional molecule according to any one of embodiments 103 to 106, wherein the NK cell engager is an antibody molecule that binds to CD16, for example, an antigen-binding domain. 220. The multifunctional molecule of embodiment 219, wherein lysis of lymphoma cells is mediated by CD16.

[0240] 221. When incubated with NK cells in the absence of a TCRBV antigen (for example, a TCRBV antigen corresponding to a biased TCRBV clone type), the multifunctional molecule does not activate NK cells, which is a multifunctional molecule according to any one of embodiments 219 or 220.

[0241] 222. When the NK cell is an NK cell expressing CD16 and a TCRBV antigen (for example, a TCRBV antigen corresponding to a biased TCRBV clone type) also exists, a multifunctional molecule according to any one of Embodiments 219 to 221 that activates the NK cell.

[0242] 223. When the NK cell is not an NK cell expressing CD16 and a TCRBV antigen (for example, a TCRBV antigen corresponding to a biased TCRBV clone type) also exists, a multifunctional molecule according to any one of Embodiments 219 to 222 that does not activate the NK cell.

[0243] 224. A multifunctional molecule according to any one of Embodiments 219 to 223, wherein the NK cell engager comprises a VH comprising the amino acid sequence of SEQ ID NO: 6185 (or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with SEQ ID NO: 6185).

[0244] 225. A multifunctional molecule according to any one of Embodiments 219 to 224, wherein the NK cell engager comprises a VL comprising the amino acid sequence of SEQ ID NO: 6186 (or an amino acid sequence having at least about 93%, 95%, or 99% sequence identity with SEQ ID NO: 6186).

[0245] 226. A multifunctional molecule according to any of Embodiments 219 to 225, wherein the NK cell engager comprises an scFV comprising the amino acid sequence of SEQ ID NO: 6184 (or an amino acid sequence having at least about 93%, 95%, or 99% sequence identity with SEQ ID NO: 6184).

[0246] 227. A multifunctional molecule according to Embodiment 103, wherein the NK cell engager is a ligand and, optionally, the ligand further comprises an immunoglobulin constant region, for example, an Fc region. 228. A multifunctional molecule according to Embodiment 227, wherein the NK cell engager is a ligand for NKp44 or NKp46, for example, viral HA.

[0247] 229. The NK cell engager of the multifunctional molecule of embodiment 227, which is a ligand of DAP10, for example, a co-receptor of NKG2D. 230. The NK cell engager of the multifunctional molecule of embodiment 227, which is a ligand of CD16, for example, a CD16a / b ligand, for example, a CD16a / b ligand further comprising an antibody Fc region.

[0248] 231. The immune cell engager of any one of embodiments 98 to 100, which mediates binding to, activation of, or both of one or more of B cells, macrophages, and / or dendritic cells.

[0249] 232. The immune cell engager of embodiment 231, which comprises a B cell, macrophage, and / or dendritic cell engager selected from one or more of CD40 ligand (CD40L) or CD70 ligand; an antibody molecule that binds to CD40 or CD70; an antibody molecule against OX40; OX40 ligand (OX40L); an agonist of Toll-like receptor (e.g., TLR4, e.g., constitutively active TLR4 (caTLR4) or TLR9 agonist); 41BB; a CD2 agonist; CD47; or a STING agonist, or a combination thereof.

[0250] 233. The immune cell engager of any one of embodiments 98 to 100, which is a B cell engager, for example, CD40L, OX40L, or CD70 ligand, or an antibody molecule that binds to OX40, CD40, or CD70. 234. The immune cell engager of any one of embodiments 98 to 100, which is a macrophage engager, for example, a TLR4 agonist (e.g., caTLR4) or a TLR9 agonist.

[0251] 234. The immune cell engager is a multifunctional molecule according to any one of embodiments 98 to 100, which is a macrophage cell engager, such as a CD2 agonist; CD40L; OX40L; an antibody molecule that binds to OX40, CD40 or CD70; an agonist of Toll-like receptor (TLR) (e.g., TLR4, e.g., constitutively active TLR4 (caTLR4) or TLR9 agonist); CD47; or a STING agonist.

[0252] 235. The immune cell engager is a multifunctional molecule according to any one of embodiments 98 to 100, which is a dendritic cell engager, such as a CD2 agonist, OX40 antibody, OX40L, 41BB agonist, a Toll-like receptor agonist or a fragment thereof (e.g., TLR4, e.g., constitutively active TLR4 (caTLR4)), a CD47 agonist, or a STING agonist.

[0253] 236. The STING agonist includes cyclic dinucleotides, such as cyclic di-GMP (cdGMP), cyclic di-AMP (cdAMP), or a combination thereof, and optionally has a 2’,5’ or 3’,5’ phosphate linkage. For example, the STING agonist is covalently coupled to the multifunctional molecule. The multifunctional molecule of embodiment 234 or 235.

[0254] 237. The multifunctional molecule according to any one of embodiments 1 to 97 includes a cytokine molecule. 238. The cytokine molecule is selected from interleukin-2 (IL-2), interleukin-7 (IL-7), interleukin-12 (IL-12), interleukin-15 (IL-15), interleukin-18 (IL-18), interleukin-21 (IL-21), or interferon γ, or a fragment or variant thereof, or a combination of any of the aforementioned cytokines. The multifunctional molecule of embodiment 237.

[0255] The multifunctional molecule of embodiment 237 or 238, wherein the cytokine molecule is a monomer or a dimer. The multifunctional molecule of any one of embodiments 237 to 239, wherein the cytokine molecule further comprises a receptor dimerization domain, for example, an IL15R alpha dimerization domain.

[0256] The multifunctional molecule of embodiment 240, wherein the cytokine molecule (for example, IL-15) and the receptor dimerization domain (for example, the IL15R alpha dimerization domain) are not covalently linked, for example, they are non-covalently associated.

[0257] The multifunctional molecule of any one of embodiments 1 to 97, wherein the multifunctional molecule comprises a cytokine inhibitor molecule. The multifunctional molecule of embodiment 242, wherein the cytokine inhibitor molecule is a TGF-beta inhibitor.

[0258] The multifunctional molecule of any one of embodiments 242 or 243, wherein the TGF-beta inhibitor inhibits (for example, reduces its activity) (i) TGF-beta 1; (ii) TGF-beta 2; (iii) TGF-beta 3; (iv) (i) and (ii); (v) (i) and (iii); (vi) (ii) and (iii); or (vii) (i), (ii), and (iii).

[0259] The multifunctional molecule of any one of embodiments 242 to 244, wherein the TGF-beta inhibitor comprises a part of a TGF-beta receptor (for example, the extracellular domain of a TGF-beta receptor) that can inhibit (for example, reduce its activity) TGF-beta, or a functional fragment or variant thereof.

[0260] The multifunctional molecule of embodiment 245, wherein the TGF-beta inhibitor comprises a part of (i) TGFBR1; (ii) TGFBR2; (iii) TGFBR3; (iv) (i) and (ii); (v) (i) and (iii); (vi) (ii) and (iii); or (vii) (i), (ii), and (iii).

[0261] 247. A multifunctional molecule according to any of embodiments 242 to 246, wherein the TGF-beta inhibitor comprises an amino acid sequence selected from Table 16, or an amino acid sequence having at least about 93%, 95%, or 99% sequence identity thereto.

[0262] 248. A multifunctional molecule according to any of embodiments 1 to 97, wherein the multifunctional molecule comprises a cell death receptor signal engager selected from a TNF-related apoptosis-inducing ligand (TRAIL) molecule, a cell death receptor molecule, or an antigen-binding domain that specifically binds to a cell death receptor.

[0263] 249. The multifunctional molecule of embodiment 248, wherein the cell death receptor signal engager activates cell death receptor signaling in lymphocytes (e.g., T cells) comprising, for example, a TCRBV antigen, and induces apoptosis or cell death in said cells, for example.

[0264] 250. The multifunctional molecule according to any of embodiments 248 or 249, wherein the cell death receptor signal engager does not activate cell death receptor signaling on cells other than lymphocytes comprising a TCRBV antigen.

[0265] 251. The multifunctional molecule according to any of embodiments 248 to 250, wherein the cell death receptor signal engager comprises a TRAIL molecule, for example, one or more TRAIL polypeptides or fragments thereof.

[0266] 252. The multifunctional molecule of embodiment 251, wherein the TRAIL molecule specifically binds to death receptor 4 (DR4) or death receptor 5 (DR5). 253. The multifunctional molecule according to any of embodiments 251 or 252, wherein the TRAIL molecule comprises a truncated TRAIL polypeptide, for example, relative to a wild-type TRAIL polypeptide.

[0267] The multifunctional molecule of embodiment 253, wherein the TRAIL molecule comprises at least a residue corresponding to amino acids 95-281 of human TRAIL, for example, a truncated TRAIL molecule comprising a residue corresponding to amino acids 95-281 of human TRAIL.

[0268] The multifunctional molecule of embodiment 254, wherein the TRAIL molecule comprises amino acids 95-281 of human TRAIL, for example, a truncated TRAIL polypeptide that does not include amino acids 1-94 of human TRAIL.

[0269] The multifunctional molecule of embodiment 253, wherein the TRAIL molecule comprises at least a residue corresponding to amino acids 122-281 of human TRAIL, for example, a truncated TRAIL molecule comprising a residue corresponding to amino acids 122-281 of human TRAIL.

[0270] 257. The TRAIL molecule comprises amino acids 122-281 of human TRAIL, for example and does not include amino acids 1-121 of human TRAIL, and the multifunctional molecule of embodiment 256 comprises a truncated TRAIL polypeptide.

[0271] The multifunctional molecule of any one of embodiments 251-257, wherein the cell death receptor signal engager comprises one, two, or three TRAIL molecules. The multifunctional molecule of any one of embodiments 248-250, wherein the cell death receptor signal engager comprises an antigen-binding domain that specifically binds to a cell death receptor, for example, cell death receptor 4 (DR4) or cell death receptor 5 (DR5).

[0272] The multifunctional molecule of embodiment 259, wherein the cell death receptor signal engager comprises one, two, or three antigen-binding domains that specifically bind to a cell death receptor. The multifunctional molecule of any one of embodiments 259 or 260, wherein the antigen-binding domain that specifically binds to a cell death receptor binds to DR5.

[0273] A multifunctional molecule according to any of embodiments 259 to 261, wherein the antigen-binding domain that specifically binds to a cell death receptor comprises chigatumumab, drozitumab, or conatumumab.

[0274] A multifunctional molecule according to any of embodiments 248 to 262, wherein the cell death receptor signal engager comprises an amino acid sequence selected from Table 11, or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto.

[0275] A multifunctional molecule according to any of embodiments 248 to 263, wherein the cell death receptor signal engager comprises the amino acid sequence of SEQ ID NO: 6157, or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto.

[0276] A multifunctional molecule according to any of embodiments 248 to 263, wherein the cell death receptor signal engager comprises the amino acid sequence of SEQ ID NO: 6158, or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto.

[0277] A multifunctional molecule according to any of embodiments 248 to 263, wherein the cell death receptor signal engager comprises the amino acid sequence of SEQ ID NO: 6159, or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto.

[0278] A multifunctional molecule according to any of embodiments 248 to 263, wherein the cell death receptor signal engager comprises the amino acid sequence of SEQ ID NO: 6160, or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto.

[0279] 268. A multifunctional molecule according to any of embodiments 248 to 263, wherein the cell death receptor signal engager comprises the amino acid sequence of SEQ ID NO: 6161, or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto.

[0280] 269. A multifunctional molecule according to any of embodiments 248 to 263, wherein the cell death receptor signal engager comprises the amino acid sequence of SEQ ID NO: 6162, or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto.

[0281] 270. A multifunctional molecule according to any of embodiments 248 to 263, wherein the cell death receptor signal engager comprises the amino acid sequence of SEQ ID NO: 6163, or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto.

[0282] 271. A multifunctional molecule according to any of embodiments 248 to 263, wherein the cell death receptor signal engager comprises the amino acid sequence of SEQ ID NO: 6164, or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto.

[0283] 272. A multifunctional molecule according to any of embodiments 248 to 263, wherein the cell death receptor signal engager comprises the amino acid sequence of SEQ ID NO: 6165, or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto.

[0284] 273. A multifunctional molecule according to embodiment 102, wherein the T cell engager binds to TCRβ. 274. A multifunctional molecule according to embodiment 273, wherein the T cell engager comprises an antigen-binding domain (e.g., an antibody molecule or a fragment thereof) that binds to (e.g., and in some embodiments, activates) CD3.

[0285] 275. The multifunctional molecule of any one of embodiments 273 or 274, wherein the T cell engager does not bind to lymphocytes containing TCRBV antigen. 276. The multifunctional molecule of any one of embodiments 273 to 275, wherein the T cell engager does not activate lymphocytes containing TCRBV.

[0286] 277. The multifunctional molecule (i) an immune cell engager (e.g., a T cell engager, an NK cell engager, a B cell engager, a dendritic cell engager, or a macrophage cell engager) and a cytokine molecule, (ii) an immune cell engager (e.g., a T cell engager, an NK cell engager, a B cell engager, a dendritic cell engager, or a macrophage cell engager) and a cytokine inhibitor molecule, (iii) an immune cell engager (e.g., a T cell engager, an NK cell engager, a B cell engager, a dendritic cell engager, or a macrophage cell engager) and a cell death receptor signal engager, (iv) a cytokine molecule and a cell death receptor signal engager, (v) a cytokine inhibitor molecule and a cell death receptor signal engager, (vi) an immune cell engager (e.g., a T cell engager, an NK cell engager, a B cell engager, a dendritic cell engager, or a macrophage cell engager), a cytokine molecule, and a cell death receptor signal engager, or (vii) an immune cell engager (e.g., a T cell engager, an NK cell engager, a B cell engager, a dendritic cell engager, or a macrophage cell engager), a cytokine inhibitor molecule, and a cell death receptor signal engager comprising the multifunctional molecule of any one of embodiments 1 to 276.

[0287] 278. The multifunctional molecule has the following structure: A, B - [Dimerization module] - C, - D wherein (a) the dimerization module comprises a constant domain of an immunoglobulin, such as a heavy chain constant domain (e.g., a homodimeric or heterodimeric heavy chain constant region, e.g., the Fc region), or a constant domain of an immunoglobulin variable region (e.g., the Fab region); (b) A, B, C, and D are independently absent or are (i) an antigen - binding domain that selectively binds to a TCRBV antigen; (ii) an immune cell engager selected from a T - cell engager, an NK - cell engager, a B - cell engager, a dendritic cell engager, or a macrophage cell engager; (iii) a cytokine molecule or a cytokine inhibitor molecule; (iv) a cell - death receptor signal engager; or (v) a stromal modification moiety, provided that at least one, two, or three of A, B, C, and D comprise an antigen - binding domain that selectively binds to a TCRBV antigen, and any of the remaining A, B, C, and D is absent or comprises one of an immune cell engager, a cytokine molecule, a cytokine inhibitor molecule, a cell - death receptor signal engager, or a stromal modification moiety, a multifunctional molecule according to any one of embodiments 1 to 277.

[0288] 279. (1) A comprises an antigen - binding domain that selectively binds to a TCRBV antigen, and B, C, or D comprises an immune cell engager, such as a T - cell engager, such as an anti - CD3 antibody molecule; (2) A comprises an antigen - binding domain that selectively binds to a TCRBV antigen, and B, C, or D comprises an immune cell engager, such as an NK - cell engager, such as an anti - NKp30, anti - NKp46, anti - NKG2D, or anti - CD16 antibody molecule; (3) A comprises an antigen - binding domain that selectively binds to a TCRBV antigen, and B, C, or D comprises a cytokine molecule; (4) A comprises an antigen-binding domain that selectively binds to a TCRBV antigen, and B, C, or D comprises a cytokine inhibitor molecule; (5) A comprises an antigen-binding domain that selectively binds to a TCRBV antigen, and B, C, or D comprises a cell death receptor signal engager; (6) A comprises a first antigen-binding domain that selectively binds to a TCRBV antigen, B comprises a second antigen-binding domain that selectively binds to a TCRBV antigen, and C or D comprises an immune cell engager, such as a T cell engager, such as an anti-CD3 antibody molecule; (7) A comprises a first antigen-binding domain that selectively binds to a TCRBV antigen, B comprises a second antigen-binding domain that selectively binds to a TCRBV antigen, and C or D comprises an immune cell engager, such as an NK cell engager, such as an anti-NKp30, anti-NKp46, anti-NKG2D, or anti-CD16 antibody molecule; (8) A comprises a first antigen-binding domain that selectively binds to a TCRBV antigen, B comprises a second antigen-binding domain that selectively binds to a TCRBV antigen, and C or D comprises a cytokine molecule; (9) A comprises a first antigen-binding domain that selectively binds to a TCRBV antigen, B comprises a second antigen-binding domain that selectively binds to a TCRBV antigen, and C or D comprises a cytokine inhibitor molecule; (10) A comprises a first antigen-binding domain that selectively binds to a TCRBV antigen, B comprises a second antigen-binding domain that selectively binds to a TCRBV antigen, and C or D comprises a cell death receptor signal engager; (11) A comprises a first antigen-binding domain that selectively binds to a TCRBV antigen, C comprises a second antigen-binding domain that selectively binds to a TCRBV antigen, and B or D comprises an immune cell engager, such as a T cell engager, such as an anti-CD3 antibody molecule; (12) A comprises a first antigen-binding domain that selectively binds to a TCRBV antigen, C comprises a second antigen-binding domain that selectively binds to a TCRBV antigen, and B or D comprises an immune cell engager, e.g., an NK cell engager, e.g., an anti-NKp30, anti-NKp46, anti-NKG2D, or anti-CD16 antibody molecule; (13) A comprises a first antigen-binding domain that selectively binds to a TCRBV antigen, C comprises a second antigen-binding domain that selectively binds to a TCRBV antigen, and B or D comprises a cytokine molecule; (14) A comprises a first antigen-binding domain that selectively binds to a TCRBV antigen, C comprises a second antigen-binding domain that selectively binds to a TCRBV antigen, and B or D comprises a cytokine inhibitor molecule; (15) A comprises a first antigen-binding domain that selectively binds to a TCRBV antigen, C comprises a second antigen-binding domain that selectively binds to a TCRBV antigen, and B or D comprises a cell death receptor signal engager; (16) A comprises a first antigen-binding domain that selectively binds to a TCRBV antigen, and B, C, or D comprises (a) an immune cell engager, e.g., an NK cell engager, e.g., an anti-NKp30, anti-NKp46, anti-NKG2D, or anti-CD16 antibody molecule, and (b) a cytokine molecule; (17) A comprises a first antigen-binding domain that selectively binds to a TCRBV antigen, and B, C, or D comprises (a) an immune cell engager, e.g., an NK cell engager, e.g., an anti-NKp30, anti-NKp46, anti-NKG2D, or anti-CD16 antibody molecule, and (b) a cytokine inhibitor molecule; (18) A comprises a first antigen-binding domain that selectively binds to a TCRBV antigen, and B, C, or D comprises (a) an immune cell engager, e.g., an NK cell engager, e.g., an anti-NKp30, anti-NKp46, anti-NKG2D, or anti-CD16 antibody molecule, and (b) a cell death receptor signal engager; (19)A comprises a first antigen-binding domain that selectively binds to a TCRBV antigen, and B, C, or D comprises (a) an immune cell engager, such as a T cell engager, such as an anti-CD3 antibody molecule, and (b) a cytokine molecule; (20)A comprises a first antigen-binding domain that selectively binds to a TCRBV antigen, and B, C, or D comprises (a) an immune cell engager, such as a T cell engager, such as an anti-CD3 antibody molecule, and (b) a cytokine inhibitor molecule; (21)A comprises a first antigen-binding domain that selectively binds to a TCRBV antigen, and B, C, or D comprises (a) an immune cell engager, such as a T cell engager, such as an anti-CD3 antibody molecule, and (b) a cell death receptor signal engager; (22)A comprises a first antigen-binding domain that selectively binds to a TCRBV antigen, and B, C, or D comprises (a) a cytokine molecule and (b) a cell death receptor signal engager; (23)A comprises a first antigen-binding domain that selectively binds to a TCRBV antigen, and B, C, or D comprises (a) a cytokine inhibitor molecule and (b) a cell death receptor signal engager; (24)A comprises a first antigen-binding domain that selectively binds to a TCRBV antigen, B comprises a second antigen-binding domain that selectively binds to a TCRBV antigen, and C or D comprises (a) an immune cell engager, such as an NK cell engager, such as an anti-NKp30, anti-NKp46, anti-NKG2D, or anti-CD16 antibody molecule, and (b) a cytokine molecule; (25)A comprises a first antigen-binding domain that selectively binds to a TCRBV antigen, B comprises a second antigen-binding domain that selectively binds to a TCRBV antigen, and C or D comprises (a) an immune cell engager, such as an NK cell engager, such as an anti-NKp30, anti-NKp46, anti-NKG2D, or anti-CD16 antibody molecule, and (b) a cytokine inhibitor molecule; (26) A comprises a first antigen-binding domain that selectively binds to a TCRBV antigen, B comprises a second antigen-binding domain that selectively binds to a TCRBV antigen, and C or D comprises (a) an immune cell engager, such as an NK cell engager, such as an anti-NKp30, anti-NKp46, anti-NKG2D, or anti-CD16 antibody molecule, and (b) a cell death receptor signal engager; (27) A comprises a first antigen-binding domain that selectively binds to a TCRBV antigen, B comprises a second antigen-binding domain that selectively binds to a TCRBV antigen, and C or D comprises (a) an immune cell engager, such as an NK cell engager, such as an anti-NKp30, anti-NKp46, anti-NKG2D, or anti-CD16 antibody molecule, and (b) a stromal modification moiety; (28) A comprises a first antigen-binding domain that selectively binds to a TCRBV antigen, B comprises a second antigen-binding domain that selectively binds to a TCRBV antigen, and C or D comprises (a) an immune cell engager, such as a T cell engager, such as an anti-CD3 antibody molecule, and (b) a cytokine molecule; (29) A comprises a first antigen-binding domain that selectively binds to a TCRBV antigen, B comprises a second antigen-binding domain that selectively binds to a TCRBV antigen, and C or D comprises (a) an immune cell engager, such as a T cell engager, such as an anti-CD3 antibody molecule, and (b) a cytokine inhibitor molecule; (30) A comprises a first antigen-binding domain that selectively binds to a TCRBV antigen, B comprises a second antigen-binding domain that selectively binds to a TCRBV antigen, and C or D comprises (a) an immune cell engager, such as a T cell engager, such as an anti-CD3 antibody molecule, and (b) a cell death receptor signal engager; (31) A comprises a first antigen-binding domain that selectively binds to a TCRBV antigen, B comprises a second antigen-binding domain that selectively binds to a TCRBV antigen, and C or D comprises (a) a cytokine molecule and (b) a cell death receptor signal engager; (32) A comprises a first antigen-binding domain that selectively binds to a TCRBV antigen, B comprises a second antigen-binding domain that selectively binds to a TCRBV antigen, and C or D comprises (a) a cytokine inhibitor molecule and (b) a cell death receptor signal engager; (33) A comprises a first antigen-binding domain that selectively binds to a TCRBV antigen, C comprises a second antigen-binding domain that selectively binds to a TCRBV antigen, and B or D comprises (a) an immune cell engager, such as an NK cell engager, such as an anti-NKp30, anti-NKp46, anti-NKG2D, or anti-CD16 antibody molecule, and (b) a cytokine molecule; (34) A comprises a first antigen-binding domain that selectively binds to a TCRBV antigen, C comprises a second antigen-binding domain that selectively binds to a TCRBV antigen, and B or D comprises (a) an immune cell engager, such as an NK cell engager, such as an anti-NKp30, anti-NKp46, anti-NKG2D, or anti-CD16 antibody molecule, and (b) a cytokine inhibitor molecule; (35) A comprises a first antigen-binding domain that selectively binds to a TCRBV antigen, C comprises a second antigen-binding domain that selectively binds to a TCRBV antigen, and B or D comprises (a) an immune cell engager, such as an NK cell engager, such as an anti-NKp30, anti-NKp46, anti-NKG2D, or anti-CD16 antibody molecule, and (b) a cell death receptor signal engager; (36) A comprises a first antigen-binding domain that selectively binds to a TCRBV antigen, C comprises a second antigen-binding domain that selectively binds to a TCRBV antigen, and B or D comprises (a) an immune cell engager, such as a T cell engager, such as an anti-CD3 antibody molecule, and (b) a cytokine molecule; (37) A comprises a first antigen-binding domain that selectively binds to a TCRBV antigen, C comprises a second antigen-binding domain that selectively binds to a TCRBV antigen, and B or D comprises (a) an immune cell engager, such as a T cell engager, such as an anti-CD3 antibody molecule, and (b) a cytokine inhibitor molecule; (38) A comprises a first antigen-binding domain that selectively binds to a TCRBV antigen, C comprises a second antigen-binding domain that selectively binds to a TCRBV antigen, and B or D comprises (a) an immune cell engager, such as a T cell engager, such as an anti-CD3 antibody molecule, and (b) a cell death receptor signal engager; (39) A comprises a first antigen-binding domain that selectively binds to a TCRBV antigen, C comprises a second antigen-binding domain that selectively binds to a TCRBV antigen, and B or D comprises (a) a cytokine molecule and (b) a cell death receptor signal engager; (40) A comprises a first antigen-binding domain that selectively binds to a TCRBV antigen, C comprises a second antigen-binding domain that selectively binds to a TCRBV antigen, and B or D comprises (a) a cytokine inhibitor molecule and (b) a cell death receptor signal engager, the multifunctional molecule of embodiment 278.

[0289] 280. The multifunctional molecule of embodiment 278 or 279, wherein the dimerization module comprises one or more immunoglobulin chain constant regions (e.g., Fc region) comprising one or more of paired holes and protrusions (“knob-in-hole”), electrostatic interactions, or strand exchange.

[0290] 281. One or more immunoglobulin heavy chain constant regions (e.g., Fc regions) contain amino acid substitutions at positions selected from one or more of 347, 349, 350, 351, 366, 368, 370, 392, 394, 395, 397, 398, 399, 405, 407, or 409 of, for example, the Fc region of human IgG1, and optionally one or more immunoglobulin heavy chain constant regions (e.g., Fc regions) contain amino acid substitutions selected from T366S, L368A, or Y407V (e.g., corresponding to a pore or hole), or T366W (e.g., corresponding to a protrusion or knob), or combinations thereof, the multifunctional molecule of embodiment 280.

[0291] 282. The multifunctional molecule of embodiments 1 to 281 further comprising a linker, for example, between one or more of an antigen-binding domain and an immune cell engager, an antigen-binding domain and a cytokine molecule, an antigen-binding domain and a stromal modifying moiety, an immune cell engager and a cytokine molecule, an immune cell engager and a cytokine molecule, an immune cell engager and a stromal modifying moiety, a cytokine molecule and a stromal modifying moiety, a cytokine molecule and a stromal modifying moiety, an antigen-binding domain and a dimerization module, an immune cell engager and a dimerization module, a cytokine molecule and a dimerization module or a stromal modifying moiety and a dimerization module.

[0292] 283. The multifunctional molecule of embodiment 282, wherein the linker is selected from a cleavable linker, a non-cleavable linker, a peptide linker, a flexible linker, a rigid linker, a helical linker, or a non-helical linker.

[0293] 284. The multifunctional molecule of embodiment 282 or 283, wherein the linker is a peptide linker. 285. The multifunctional molecule of embodiment 284, wherein the peptide linker contains Gly and Ser.

[0294] 286. From SEQ ID NOs: 7248 - 7251 or 7252 - 7253 and 77 - 78 The multifunctional molecule of embodiment 285, comprising the selected amino acid sequence. 287. (i) A first antigen-binding domain that selectively binds to a TCRBV antigen, and (ii) An NK cell engager, such as an anti-NKp30, anti-NKp46, anti-NKG2D, or anti-CD16 antibody molecule A multifunctional molecule comprising the same.

[0295] 288. The multifunctional molecule of embodiment 287, wherein the NK cell engager comprises an anti-NKp30 antibody molecule. 289. The multifunctional molecule of embodiment 287, wherein the NK cell engager comprises an anti-NKp46 antibody molecule.

[0296] 290. (i) A T cell receptor variable beta (TCRBV), such as a first antigen-binding domain that binds to, for example, selectively binds to a TCRBV antigen, and (ii) A cell death receptor signal engager A multifunctional molecule comprising the same.

[0297] 291. (i) A T cell receptor variable beta (TCRBV), such as a first antigen-binding domain that binds to, for example, selectively binds to a TCRBV antigen, and (ii) A cytokine inhibitor molecule, such as a TGF-beta inhibitor A multifunctional molecule comprising the same.

[0298] 292. The multifunctional molecule according to any one of embodiments 1 to 291, wherein the multifunctional molecule binds monovalently to the TCRBV antigen. 293. The multifunctional molecule according to any one of embodiments 1 to 291, wherein the multifunctional molecule binds polyvalently, for example, divalently, trivalently, tetravalently, pentavalently, hexavalently, heptavalently, octavalently, nonavalently, or decavalently, to the TCRBV antigen.

[0299] 294. The multifunctional molecule according to any one of embodiments 2 to 261, wherein the multifunctional molecule binds to the TCRBV antigen on lymphocytes expressing the TCRBV antigen. A multifunctional molecule that binds monovalently to immune cells, for example, via an immune cell engager, of any of the preceding embodiments.

[0300] A multifunctional molecule of any one of embodiments 1 to 294, wherein the multifunctional molecule binds to immune cells polyvalently, for example, divalently, trivalently, tetravalently, pentavalently, hexavalently, heptavalently, octavalently, nonavalently, or decavalently, for example, via an immune cell engager.

[0301] A multifunctional molecule of any of the preceding embodiments, further comprising a heavy chain constant region that mediates antibody-dependent cell cytotoxicity (ADCC), for example, an Fc region. A multifunctional molecule of any of the preceding embodiments, further comprising a heavy chain constant region that mediates complement-dependent cytotoxicity (e.g., via C1q), for example, an Fc region.

[0302] A nucleic acid molecule encoding a multifunctional molecule of any one of embodiments 1 to 298. A vector, for example, an expression vector, comprising the nucleic acid molecule of embodiment 299. A host cell comprising the nucleic acid molecule of embodiment 299 or the vector of embodiment 300.

[0303] A method of making, for example, producing, a multifunctional molecule or an antibody molecule of any one of embodiments 1 to 298, the method comprising culturing the host cell of embodiment 301 under suitable conditions, for example, conditions suitable for gene expression and / or homoor heterodimerization.

[0304] A pharmaceutical composition comprising a multifunctional molecule of any one of embodiments 1 to 298 and a pharmaceutically acceptable carrier, excipient, or stabilizer. A method of treating TCR bias, the method comprising administering to a subject in need thereof a multifunctional molecule of any one of embodiments 1 to 298, wherein the multifunctional molecule is administered in an amount effective to treat TCR bias.

[0305] 305. A method of treating an autoimmune disease (e.g., an autoimmune disease associated with TCR bias), comprising administering to a subject in need thereof a multifunctional molecule according to any one of Embodiments 1 to 298, wherein the multifunctional molecule is administered in an amount effective to treat the autoimmune disease.

[0306] 306. The method according to any one of Embodiments 304 or 305, further comprising the step of identifying, evaluating, or selecting a subject in need of treatment, wherein the step of identifying, evaluating, or selecting comprises determining whether the subject has TCR bias or an autoimmune disease (e.g., an autoimmune disease associated with TCR bias) (e.g., directly determining it or indirectly determining it, e.g., obtaining information regarding it).

[0307] 307. In response to determining that the subject has TCR bias or an autoimmune disease (e.g., an autoimmune disease associated with TCR bias), optionally, selecting a subject for treatment using a multifunctional molecule comprising an antigen-binding domain that binds to a TCRBV antigen (e.g., a TCRBV antigen corresponding to a biased TCRBV clone type), and administering a multifunctional molecule comprising an antigen-binding domain that binds to a TCRBV antigen (e.g., a TCRBV antigen corresponding to a biased TCRBV clone type). The method according to Embodiment 306, further comprising.

[0308] 308. A method of treating TCR bias, comprising: in response to determining that the subject has TCR bias, administering to a subject in need thereof a multifunctional molecule according to any one of claims 1 to 298, wherein the multifunctional molecule is administered in an amount effective to treat the TCR bias.

[0309] 309. A method of treating an autoimmune disease (e.g., an autoimmune disease associated with TCR bias), comprising: In response to determining that a subject has an autoimmune disease (e.g., an autoimmune disease associated with TCR bias), the method includes the step of administering to the subject in need thereof a multifunctional molecule according to any one of claims 1 to 298, wherein the multifunctional molecule is administered in an amount effective to treat the autoimmune disease (e.g., an autoimmune disease associated with TCR bias).

[0310] 310. The method according to any one of embodiments 304 to 309, wherein the subject has a TCR bias (e.g., a biased TCRBV clone type) and / or an autoimmune disease associated with said bias.

[0311] 311. A method of identifying a subject in need of treatment for cancer using a multifunctional molecule according to any one of embodiments 1 to 298, the method comprising determining whether the subject has a TCR bias (e.g., a biased TCRBV clone type) and / or an autoimmune disease associated with said bias (e.g., directly determining it or indirectly determining it, e.g., obtaining information regarding it), and in response to determining that the subject has a TCR bias (e.g., a biased TCRBV clone type) and / or an autoimmune disease associated with said bias, identifying the subject as a candidate for treatment using a multifunctional molecule comprising an antigen-binding domain that binds to a TCRBV antigen, and optionally not identifying the subject as a candidate for treatment using a multifunctional molecule comprising an antigen-binding domain that does not bind to the TCRBV antigen (e.g., that binds to a different TCRBV antigen).

[0312] 312. The method of embodiment 311, further comprising the step of treating (e.g., administering to) the subject with a multifunctional molecule comprising an antigen-binding domain that binds to a TCRBV antigen in response to identifying the subject as a candidate for treatment using a multifunctional molecule comprising an antigen-binding domain that binds to a TCRBV antigen.

[0313] A method for evaluating a subject in need of treatment of a TCR bias (e.g., a biased TCRBV clone type) and / or an autoimmune disease associated with said bias, the method comprising determining whether the subject has a TCR bias (e.g., directly determining it or indirectly determining it, e.g., obtaining information regarding it).

[0314] The method of embodiment 313, further comprising treating (e.g., administering to) the subject with a multifunctional molecule comprising an antigen-binding domain that binds to a TCRBV antigen in response to the evaluation.

[0315] The method of any one of embodiments 304 to 314, wherein the TCR bias is associated with an autoimmune disease. 316. The method of embodiment 315, wherein the autoimmune disease is selected from Churg-Strauss syndrome, sarcoidosis, systemic lupus erythematosus (SLE), type 1 diabetes, autoimmune hepatitis (e.g., type 1 or type 2), primary sclerosing cholangitis, primary biliary cirrhosis, multiple sclerosis, Guillain-Barré syndrome and AMAN (axonal and nerve neuropathy), chronic inflammatory demyelinating polyneuropathy (CIDP), transverse myelitis, Troxalant syndrome (THS), Devic's disease (neuromyelitis optica), paraneoplastic cerebellar degeneration (PCD), Lambert-Eaton syndrome, psoriasis, scleroderma, CREST (calcinosis, Raynaud's phenomenon, esophageal motility disorder, sclerodactyly, and telangiectasia) syndrome, dermatitis herpetiformis, dermatomyositis, bullous pemphigoid, cicatricial pemphigoid / benign mucous membrane pemphigoid, pemphigus, rheumatoid arthritis (RA), psoriatic arthritis, relapsing polychondritis, chronic recurrent multifocal osteomyelitis (CRMO), vasculitis, Kawasaki disease, granulomatosis with polyangiitis (GPA), Behçet's disease (vasculitis), Takayasu arteritis, polyarteritis nodosa, microscopic polyangiitis (MPA), leukocytoclastic vasculitis, Cogan syndrome, uveitis, peripheral uveitis (pars planitis), scleritis, autoimmune inner ear disease (AIED), Crohn's disease, ulcerative colitis (UC), Dressler syndrome, rheumatic fever, Evans syndrome, paroxysmal nocturnal hemoglobinuria (PNH), hemolytic anemia, thrombotic thrombocytopenic purpura (TTP), polymyositis, juvenile dermatomyositis (JDM) and juvenile polymyositis (JPM) including juvenile myositis (JM), Sjögren's syndrome, ocular cicatricial pemphigoid, or Hashimoto's thyroiditis.

[0316] 317. The method of any one of embodiments 304 to 316, further comprising the step of administering a second therapeutic treatment. 318. The method of embodiment 317, wherein the second therapeutic treatment comprises a therapeutic agent (e.g., a chemotherapeutic agent, a biologic agent, hormone therapy), radiation, or surgery.

[0317] A method of treating an autoimmune disease in a subject in need of treatment for an autoimmune disease (e.g., an autoimmune disease associated with TCR bias), the method comprising administering to the subject an effective amount, e.g., a therapeutically effective amount, of an antibody molecule that binds (e.g., specifically binds) to the T cell receptor beta variable region (TCRβV) (an "anti-TCRβV antibody molecule"), thereby treating the disorder.

[0318] A method of depleting a T cell population in a subject having an autoimmune disease (e.g., an autoimmune disease associated with TCR bias), the method comprising contacting the T cell population with an effective amount of an antibody molecule that binds (e.g., specifically binds) to the T cell receptor beta variable region (TCRβV) (an "anti-TCRβV antibody molecule").

[0319] The method of claim 320, wherein the contacting step occurs in vivo or in vitro. The anti-TCRβV antibody molecule is (i) not the antibody molecule disclosed in U.S. Patent No. 5,861,155; (ii) binds to TCRβV12 with an affinity and / or binding specificity less than (e.g., about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or less than about 2, 5, or 10-fold less than) the affinity and / or binding specificity of the 16G8 mouse antibody or its humanized version as described in U.S. Patent No. 5,861,155; (iii) binds to TCRβV12 with an affinity and / or binding specificity greater than (e.g., about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or greater than about 2, 5, 10-fold greater than) the affinity and / or binding specificity of the 16G8 mouse antibody or its humanized version as described in U.S. Patent No. 5,861,155; (iii) An affinity and / or binding specificity greater than that of the TM23 mouse antibody or its humanized version (e.g., about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more than about 2, 5, 10 - fold) for TCRβ V5 - 5 * 01 or TCRβ V5 - 1 * 01 and binds thereto; or (iv) An affinity and / or binding specificity greater than that of the TM23 mouse antibody or its humanized version (e.g., about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more than about 2, 5, 10 - fold) for TCRβ V5 - 5 * 01 or TCRβ V5 - 1 * 01 and binds thereto, the method according to any one of claims 319 to 321.

[0320] 323. The method according to any one of claims 319 to 322, wherein the anti - TCRβV antibody molecule comprises an Fc region, for example, an Fc region having effector functions, for example, an Fc region having antibody - dependent cell - mediated cytotoxicity (ADCC), antibody - dependent cell phagocytosis (ADCP) and / or complement - dependent cytotoxicity (CDC).

[0321] 324. The method according to claim 323, wherein the anti - TCRβV antibody molecule comprises an Fc region with enhanced effector functions as compared to, for example, the wild - type Fc region. 325. The method according to any one of claims 319 to 324, wherein the anti - TCRβV antibody molecule comprises a human IgG1 region or a human IgG4 region.

[0322] 326. The method according to any one of claims 319 or 321 to 325, wherein the autoimmune disease is Churg-Strauss syndrome, sarcoidosis, systemic lupus erythematosus (SLE), type 1 diabetes, autoimmune hepatitis (e.g., type 1 or type 2), primary sclerosing cholangitis, primary biliary cirrhosis, multiple sclerosis, Guillain-Barré syndrome and AMAN (axonal and nerve neuropathy), chronic inflammatory demyelinating polyneuropathy (CIDP), transverse myelitis, Troxalhanth syndrome (THS), Devic's disease (neuromyelitis optica), paraneoplastic cerebellar degeneration (PCD), Lambert-Eaton syndrome, psoriasis, scleroderma, CREST (calcinosis, Raynaud's phenomenon, esophageal motility disorder, sclerodactyly, and telangiectasia) syndrome, dermatitis herpetiformis, dermatomyositis, bullous pemphigoid, cicatricial pemphigoid / benign mucous membrane pemphigoid, pemphigus, rheumatoid arthritis (RA), psoriatic arthritis, relapsing polychondritis, chronic recurrent multifocal osteomyelitis (CRMO), vasculitis, Kawasaki disease, granulomatosis with polyangiitis (GPA), Behçet's disease (vasculitis), Takayasu arteritis, polyarteritis nodosa, microscopic polyangiitis (MPA), leukocytoclastic vasculitis, Cogan syndrome, uveitis, peripheral uveitis (pars planitis), scleritis, autoimmune inner ear disease (AIED), Crohn's disease, ulcerative colitis (UC), Dressler syndrome, rheumatic fever, Evans syndrome, paroxysmal nocturnal hemoglobinuria (PNH), hemolytic anemia, thrombotic thrombocytopenic purpura (TTP), polymyositis, juvenile dermatomyositis (JDM) and juvenile polymyositis (JPM) including juvenile myositis (JM), Sjögren's syndrome, ocular cicatricial pemphigoid, or Hashimoto's thyroiditis.

[0323] The method according to any one of claims 319 to 326, wherein the anti-TCRβV antibody molecule comprises one or more (e.g., all three) of the LC CDR1, LC CDR2, and LC CDR3 provided in Table 1A, 2A, 10A, 11A, 12A or 13A; and / or one or more (e.g., all three) of the HC CDR1, HC CDR2, and HC CDR3 provided in Table 1A, 2A, 10A, 11A, 12A or 13A, or a sequence having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto, and an antigen-binding domain.

[0324] The method according to any one of claims 319 to 327, wherein the anti-TCRβV antibody molecule comprises a variable heavy chain (VH) and / or a variable light chain (VL) provided in Table 1A, 2A, 10A, 11A, 12A or 13A, or a sequence having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto.

[0325] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

[0326] Other features and advantages of the present invention will be apparent from the following detailed description and claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0327]

Fig. 1A

Fig. 1B

Fig. 2A

Fig. 2B-1

Fig. 2B-2

Fig. 3

Fig. 4

Fig. 5

Mode for Carrying Out the Invention

[0328] (i) A T cell receptor variable beta (TCRBV), for example, an antigen-binding domain that binds to, for example, selectively binds to a TCRBV antigen, and (ii) (a) an immune cell engager selected from a T cell engager, an NK cell engager (for example, a molecule that binds to NKp30, NKp46, NKG2D, or CD16), a B cell engager, a dendritic cell engager, or a macrophage cell engager; (b) a cytokine molecule or a cytokine inhibitor molecule; and (c) one, two, or all of a cell death receptor signal engager, a multifunctional molecule (sometimes referred to herein as a "bispecific molecule") containing a plurality (for example, two or more) of functionalities (or binding specificities) is disclosed herein. In some embodiments, the antigen-binding domain comprises a sequence or a portion of a sequence found in Table 13 or Table 14. In some embodiments, the immune cell engager comprises an NK cell engager comprising a sequence or a portion of a sequence found in Tables 7-10. In some embodiments, the antigen-binding domain comprises a sequence or a portion of a sequence found in Table 13 or Table 14, and the immune cell engager comprises an NK cell engager comprising a sequence or a portion of a sequence found in Tables 7-10.

[0329] In one embodiment, the bispecific or multifunctional molecule is a bispecific (or bifunctional) molecule, a trispecific (or trifunctional) molecule, or a tetraspecific (or tetrafunctional) molecule.

[0330] In some embodiments, the multifunctional molecule comprises an antigen-binding domain that binds to a TCRB on the surface of a lymphocyte, for example, a T cell. The TCRBV antigen. In some embodiments, the TCRBV antigen corresponds to a biased TCRBV clone type. For example, a TCR comprising the TCRBV antigen may be overexpressed in the TCR repertoire or lymphocyte (for example, T cell) pool of a subject (for example, a subject having an autoimmune disease associated with TCR bias), or expressed at a higher level than in other subjects (for example, non-autoimmune disease subjects).

[0331] Without being bound by theory, the multispecific or multifunctional molecules disclosed herein are expected to localize (e.g., crosslink) and / or activate immune effector cells (e.g., immune cells selected from T cells, NK cells, B cells, dendritic cells or macrophages) in the presence of cells (e.g., lymphocytes, e.g., T cells) expressing a TCRBV antigen (e.g., a TCRBV antigen corresponding to a biased TCRBV clonotype) on the cell surface. Increasing the proximity and / or activity of immune cells in the presence of cells (e.g., lymphocytes, e.g., T cells) expressing a TCRBV antigen (e.g., a TCRBV antigen corresponding to a biased TCRBV clonotype) using the multispecific or multifunctional molecules described herein is expected to enhance the immune response against the target cells, thereby providing a more effective therapy (e.g., by reducing the level of biased TCRs and / or T cells expressing a biased TCR). In another embodiment, targeting cells (e.g., lymphocytes, e.g., T cells) expressing a TCRBV antigen (e.g., a TCRBV antigen corresponding to a biased TCRBV clonotype) using a multifunctional molecule that also includes a cell death-inducing moiety (e.g., a cell death receptor signal engager) is thought to promote the death of the target cells (e.g., by reducing the level of biased TCRs and / or T cells expressing a biased TCR).

[0332] Without being bound by theory, in some embodiments, by utilizing multispecific or multifunctional molecules that are specific for a particular TCRBV antigen (e.g., a TCRBV antigen corresponding to a biased TCRBV clonotype) but do not have specificity for other or all types of T cell receptors, it is expected that the harmful effects of increasing the proximity or activity of immune cells to T cells generally or promoting cell death in T cells generally can be reduced. Thus, the use of the multispecific or multifunctional molecules disclosed herein can increase the proximity or activity of immune cells to cells containing a TCRBV antigen corresponding to a biased TCRBV clonotype without necessarily increasing the proximity or activity of immune cells to T cells generally, or can promote cell death in cells containing a TCRBV antigen corresponding to a biased TCRBV clonotype without necessarily increasing cell death in T cells generally.

[0333] Accordingly, in particular, provided herein are multispecific or multifunctional molecules (e.g., multispecific or multifunctional antibody molecules) comprising the foregoing moieties, nucleic acids encoding the same, methods of producing the foregoing molecules, and methods of using the foregoing molecules to treat a disease or disorder, e.g., an autoimmune disease or TCR bias.

[0334] Definitions In some embodiments, the multifunctional molecule comprises an immune cell engager. An "immune cell engager" refers to one or more binding specificities that bind and / or activate an immune cell, e.g., a cell involved in an immune response. In embodiments, the immune cell is selected from T cells, NK cells, B cells, dendritic cells, and / or macrophage cells. An immune cell engager is an antibody molecule, a receptor molecule (e.g., a full-length receptor, a receptor fragment, or a fusion thereof (e.g., a receptor-Fc fusion)), or a ligand molecule that binds to an immune cell antigen (e.g., a T cell, NK cell antigen, B cell antigen, dendritic cell antigen, and / or macrophage cell antigen) (e.g., a full-length ligand, a ligand fragment, or a fusion thereof (e.g., It may be a ligand-Fc fusion. In embodiments, the immune cell engager specifically binds to the target immune cell and, for example, preferentially binds to the target immune cell. For example, if the immune cell engager is an antibody molecule, it binds to an immune cell antigen (e.g., a T cell antigen, an NK cell antigen, a B cell antigen, a dendritic cell antigen, and / or a macrophage cell antigen) with a dissociation constant of less than about 10 nM.

[0335] In some embodiments, the multifunctional molecule comprises a cytokine molecule. As used herein, the term "cytokine molecule" refers to the full length, fragment or variant of a cytokine; further, a cytokine comprising a receptor domain, e.g., a cytokine receptor dimerization domain; or an agonist of a cytokine receptor that induces activation of at least one of the naturally occurring cytokines, e.g., an antibody molecule (e.g., an agonist antibody) against a cytokine receptor. In some embodiments, the cytokine molecule is selected from interleukin-2 (IL-2), interleukin-7 (IL-7), interleukin-12 (IL-12), interleukin-15 (IL-15), interleukin-18 (IL-18), interleukin-21 (IL-21), or interferon γ, or fragments or variants thereof, or any combination of the foregoing cytokines. The cytokine molecule can be monomeric or dimeric. In embodiments, the cytokine molecule can further comprise a cytokine receptor dimerization domain. In other embodiments, the cytokine molecule is an agonist of a cytokine receptor, e.g., an antibody molecule (e.g., an agonist antibody) against a cytokine receptor selected from IL-15Ra or IL-21R.

[0336] As used herein, for example, the term "molecule" as used in antibody molecules, cytokine molecules, receptor molecules includes full-length naturally occurring molecules, as well as variants, such as functional variants (e.g., cleaved, fragmented, mutated (e.g., substantially similar sequences) or derivatized forms thereof), provided that at least one function and / or activity of the unmodified (e.g., naturally occurring) molecule remains.

[0337] As used herein, the term "autoimmune" disease, disorder, or condition refers to a disease in which the body's immune system attacks its own cells or tissues. Autoimmune diseases can result in the production of autoantibodies that are inappropriately produced and / or overproduced against self-antigens or autoantigens. Examples of autoimmune diseases include, but are not limited to, cardiovascular diseases, rheumatic diseases, glandular diseases, gastrointestinal diseases, skin diseases, liver diseases, neurological diseases, muscle diseases, kidney diseases, reproductive diseases, connective tissue diseases, and systemic diseases. In some embodiments, autoimmune diseases are mediated by T cells, B cells, innate immune cells (e.g., macrophages, eosinophils, or natural killer cells), or the complement-mediated pathway.

[0338] Certain terms are defined below. As used herein, the articles "a" and "an" refer to one or more of the grammatical objects of the article, e.g., at least one. The use of the term "a" or "an" may mean "one" when used in conjunction with the term "comprising" herein, but is also consistent with the meaning of "one or more," "at least one," and "one or more than one."

[0339] As used herein, "about" and "substantially" generally mean the degree of acceptable error for a measured quantity given the nature or precision of the measurement. Exemplary degrees of error are within 20 percent (%) of a given range of values, typically within 10%, and more typically within 5%.

[0340] As used herein, an "antibody molecule" refers to a molecule that contains at least one immunoglobulin variable domain. It refers to a protein, e.g., an immunoglobulin chain or a fragment thereof, that includes a nucleotide sequence. Antibody molecules include antibodies (e.g., full-length antibodies) and antibody fragments. In certain embodiments, antibody molecules include antigen-binding or functional fragments of full-length antibodies or full-length immunoglobulin chains. For example, full-length antibodies are immunoglobulin (Ig) molecules (e.g., IgG antibodies) that occur naturally or are formed by recombinant processes of normal immunoglobulin gene fragments. In embodiments, antibody molecules refer to immunologically active antigen-binding portions of immunoglobulin molecules, e.g., antibody fragments. Antibody fragments, e.g., functional fragments, are portions of antibodies, e.g., Fab, Fab', F(ab')2, F(ab)2, variable fragments (Fv), domain antibodies (dAbs), or single-chain variable fragments (scFv). Functional antibody fragments bind to the same antigen recognized by intact (e.g., full-length) antibodies. The term "antibody fragment" or "functional fragment" also includes isolated fragments consisting of the variable regions, such as an "Fv" fragment consisting of the variable regions of the heavy and light chains, or a recombinant single chain polypeptide molecule in which the variable regions of the light and heavy chains are linked by a peptide linker ("scFv protein"). In some embodiments, an antibody fragment does not include a portion of an antibody that does not have antigen binding activity, such as an Fc fragment or a single amino acid residue. Exemplary antibody molecules include full length antibodies and antibody fragments, such as dAb (domain antibodies), single chain, Fab, Fab', and F(ab')2 fragments, and single chain variable fragments (scFv).

[0341] As used herein, "immunoglobulin variable domain sequence" refers to an amino acid sequence capable of forming the structure of an immunoglobulin variable domain. For example, the sequence may include all or part of the amino acid sequence of a naturally occurring variable domain. For example, the sequence may or may not include one, two or more N-terminal or C-terminal amino acids, or may include other modifications compatible with forming a protein structure.

[0342] In embodiments, the antibody molecule is monospecific and includes, for example, binding specificity for a single epitope. In some embodiments, the antibody molecule is multispecific; for example, it includes multiple immunoglobulin variable domain sequences, where the first immunoglobulin variable domain sequence has binding specificity for a first epitope and the second immunoglobulin variable domain sequence has binding specificity for a second epitope. In some embodiments, the antibody molecule is a bispecific antibody molecule. As used herein, a "bispecific antibody molecule" refers to an antibody molecule having specificity for more than one (e.g., two, three, four, or more) epitopes and / or antigens.

[0343] As used herein, an "antigen" (Ag) refers to a molecule capable of eliciting an immune response, for example, one that is associated with activation of certain immune cells and / or antibody production. Virtually any macromolecule, including most proteins or peptides, can be an antigen. Antigens can also be derived from genomic recombinants or DNA. For example, any DNA that includes a nucleotide sequence or partial nucleotide sequence encoding a protein capable of inducing an immune response encodes an "antigen." In embodiments, an antigen need not be encoded only by the full-length nucleotide sequence of a gene, nor need an antigen be encoded at all by a gene. In embodiments, an antigen can be synthesized or can be derived from a biological sample having other biological components, such as a tissue sample, blood sample, cell, or fluid. As used herein, a "TCRBV antigen" includes any TCR variable beta chain or portion thereof that can elicit an immune response or can be targeted by an antigen-binding domain. In some embodiments, a biased TCR clonotype can be characterized by one or more TCRBV antigens presented, for example, on the surface of most, if not all, of the cells that include the clonotype.

[0344] The "antigen-binding site" or "binding site" of an antibody molecule refers to the part of the antibody molecule involved in antigen binding, for example, part of an immunoglobulin (Ig) molecule. In embodiments, the antigen-binding site is the heavy It is formed by the amino acid residues of the variable regions (V) of the heavy chain (H) and the light chain (L). Three highly divergent stretches within the variable regions of the heavy and light chains, called hypervariable regions, are positioned between more conserved adjacent regions called "framework regions" (FR). The FR are amino acid sequences that are naturally found between and adjacent to the hypervariable regions in immunoglobulins. In embodiments, in an antibody molecule, the three hypervariable regions of the light chain and the three hypervariable regions of the heavy chain are arranged relative to each other in three-dimensional space to form an antigen-binding surface that is complementary to the three-dimensional surface of the binding antigen. Each of the three hypervariable regions of the heavy and light chains is called a "complementary determining region" or "CDR". The framework regions and CDRs are defined and described, for example, in Kabat, E.A. et al. (1991) Sequences of Proteins of Immunological Interest, 5th ed., U.S. Department of Health and Human Services, NIH Publication No. 91-3242, and Chothia, C. et al. (1987) J. Mol. Biol. 196: 901-917. Each variable chain (e.g., variable heavy chain and variable light chain) typically consists of three CDRs and four FRs, arranged in amino-terminal to carboxy-terminal order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.

[0345] As used herein, the term "immune cell" refers to any of a variety of cells that function in the immune system, e.g., defend against infection and foreign agents. In embodiments, the term includes white blood cells, e.g., neutrophils, eosinophils, basophils, lymphocytes, and monocytes. Innate white blood cells include phagocytes (e.g., macrophages, neutrophils, and dendritic cells), mast cells, eosinophils, basophils, and natural killer cells. Innate white blood cells identify and eliminate pathogens by attacking larger pathogens through contact or by engulfing and killing microorganisms and are mediators of the activation of the adaptive immune response. Cells of the adaptive immune system are a special type of white blood cell called lymphocytes. B cells and T cells are important types of lymphocytes and are derived from hematopoietic stem cells in the bone marrow. B cells are involved in the humoral immune response and T cells are involved in the cellular immune response. The term "immune cell" includes immune effector cells.

[0346] As used herein, the term "immune effector cell" refers to a cell that is involved in promoting an immune response, e.g., an immune effector response. Examples of immune effector cells include, but are not limited to, T cells, e.g., alpha / beta T cells and gamma / delta T cells, B cells, natural killer (NK) cells, natural killer T cells, and mast cells.

[0347] The term "effector function" or "effector response" refers to the specialized function of a cell. The effector function of a T cell can be, for example, cytolytic activity or helper activity including the secretion of cytokines.

[0348] The compositions and methods of the present invention include polypeptides and nucleic acids having a particular sequence, or a sequence that is substantially identical or similar thereto, e.g., a sequence that is at least 80%, 85%, 90%, 95% identical, or more identical, to a particular sequence. In the context of amino acid sequences, the term "substantially identical" as used herein refers to a first amino acid sequence that contains a sufficient number or a minimal number of amino acid residues such that: i) it is identical to a second amino acid sequence, or ii) it is a conservative substitution of aligned amino acid residues in the second amino acid sequence, such that the first and second amino acid sequences can have a common structural domain and / or a common functional activity. For example, an amino acid sequence containing a common structural domain has at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to a reference sequence, e.g., a sequence provided herein.

[0349] In the context of nucleotide sequences, the term "substantially identical" as used herein refers to a first nucleic acid sequence that contains a sufficient number or a minimal number of nucleotides such that the first and second nucleotide sequences are identical to aligned nucleotides in the second nucleic acid sequence so as to encode a polypeptide having a common functional activity, or to encode a common polypeptide structural domain or a common polypeptide functional activity. For example, a nucleotide sequence having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to a reference sequence, e.g., a sequence provided herein.

[0350] The term "variant" refers to a polypeptide having an amino acid sequence that is substantially identical to a reference amino acid sequence, or that is encoded by a substantially identical nucleotide sequence. In some embodiments, the variant is a functional variant.

[0351] The term "functional variant" refers to a polypeptide having an amino acid sequence that is substantially identical to a reference amino acid sequence, or a polypeptide that is encoded by a substantially identical nucleotide sequence and that may have one or more activations of the reference amino acid sequence.

[0352] The calculation of sequence homology or sequence identity (these terms are used interchangeably herein) is performed as follows. To determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., for optimal alignment, gaps may be introduced into one or both of the first and second amino acid or nucleic acid sequences, and non-homologous sequences may be disregarded for comparison purposes). In a preferred embodiment, the length of the reference sequence aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%, 60%, still more preferably at least 70%, 80%, 90%, 100% of the length of the reference sequence. Next, the amino acid residues or nucleotides at the corresponding amino acid positions or nucleotide positions are compared. If the position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, the molecules are identical at that position (as used herein, amino acid or nucleic acid "identity" is equal to amino acid or nucleic acid "homology").

[0353] The percent identity between two sequences is a function of the number of identical positions shared by the sequences, and takes into account the number of gaps that need to be introduced for optimal alignment of the two sequences and the length of each gap.

[0354] The comparison of sequences and determination of the percent identity between two sequences can be accomplished using a mathematical algorithm. In a preferred embodiment, the percent identity between two amino acid sequences is determined using the Needleman and Wunsch ((1970) J. Mol. Biol. 48:444-453) algorithm incorporated into the GAP program of the GCG software package (available at http: / / www.gcg.com) using either the Blossum62 matrix or PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. In yet another preferred embodiment, the percent identity between two nucleotide sequences is determined using the GAP program of the GCG software package (available at http: / / www.gcg.com) with the NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. A particularly preferred set of parameters (and those to be used unless otherwise specified) is the Blossum62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a frame shift gap penalty of 5. is the Blossum62 score matrix having a frame shift gap penalty of 5.

[0355] The percent identity between two amino acid sequences or nucleotide sequences can be determined using the algorithm of E. Meyers and W. Miller ((1989) CABIOS, 4:11-17) incorporated into the ALIGN program (version 2.0) using the PAM120 residue weight table, a gap length penalty of 12, and a gap penalty of 4.

[0356] The nucleic acid and protein sequences described herein can be used, for example, as "query sequences" for performing searches against public databases to identify other family members or related sequences. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul et al. (1990) J. Mol. Biol. 215:403-10. BLAST nucleotide searches can be performed using the NBLAST program, score = 100, wordlength = 12, to obtain nucleotide sequences homologous to the nucleic acid molecules of the invention. BLAST protein searches can be performed using the XBLAST program, score = 50, wordlength = 3, to obtain amino acid sequences homologous to the protein molecules of the invention. To obtain gapped alignments for comparison purposes, gapped BLAST can be utilized as described in Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402. When utilizing the BLAST and gapped BLAST programs, the default parameters of each program (e.g., XBLAST and NBLAST) can be used. See http: / / www.ncbi.nlm.nih.gov.

[0357] It is understood that the molecules of the invention may have additional conservative or non-essential amino acid substitutions that do not substantially affect their function. The term "amino acid" is intended to include both natural and synthetic molecules that contain both amino functionality and acid functionality and all molecules that can be included in a polymer of naturally occurring amino acids. Exemplary amino acids include naturally occurring amino acids; analogs, derivatives, and homologs thereof; amino acid analogs having variant side chains; and all stereoisomers of any of the foregoing. As used herein, the term "amino acid" includes both D or L optical isomers and peptidomimetics.

[0358] "Conservative amino acid substitution" means that an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains are defined in the art. These families include amino acids having basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[0359] The terms "polypeptide", "peptide" and "protein" (in the case of a single chain) are used interchangeably herein and refer to a polymer of amino acids of any length. The polymer can be linear or branched, can include modified amino acids, and can be interrupted by non-amino acids. These terms also include amino acid polymers that have been modified; e.g., any other manipulation such as disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or conjugation with a labeling component. A polypeptide can be isolated from a natural source and can be produced by recombinant techniques from eukaryotic or prokaryotic hosts or can be the product of synthetic procedures.

[0360] The terms "nucleic acid", "nucleic acid sequence", "nucleotide sequence", or "polynucleotide sequence", and "polynucleotide" are used interchangeably. They refer to polymeric forms of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. A polynucleotide can be either single-stranded or double-stranded, and if single-stranded, can be either the coding strand or the non-coding (antisense) strand. A polynucleotide can include modified nucleotides such as methylated nucleotides and nucleotide analogs. The sequence of nucleotides can be interrupted by non-nucleotide components. A polynucleotide can be further modified after polymerization, for example, by conjugation with a labeling component. A nucleic acid can be a recombinant polynucleotide, or a polynucleotide of genomic, cDNA, semi-synthetic, or synthetic origin that is linked to another polynucleotide in a non-natural or unnatural sequence.

[0361] The term "isolated", as used herein, refers to a material that has been removed from its original environment or natural environment (e.g., the natural environment if it occurs naturally). For example, a naturally occurring polynucleotide or polypeptide present in a living animal is not isolated, but the same polynucleotide or polypeptide separated by human intervention from some or all of the coexisting materials of the natural system is isolated. Such a polynucleotide can be part of a vector, and / or such a polynucleotide or polypeptide can be part of a composition, and such a vector or composition can still be isolated in that it is not part of the environment in which it is found in nature.

[0362] The various aspects of the invention are described in further detail below. Additional definitions are set forth throughout the specification. Antibody molecule In one embodiment, the antibody molecule binds to a TCRBV antigen, such as a TCRBV antigen corresponding to a (e.g., biased TCRBV clonotype). In some embodiments, the TCRBV antigen is, for example, a mammalian, e.g., human, TCRBV antigen. In some embodiments, the antibody molecule binds to a TCRBV antigen on a lymphocyte, such as a T cell, such as a mammalian, e.g., human, lymphocyte, such as a T cell. For example, the antibody molecule specifically binds to a TCRBV antigen expressed as a portion of a TCR containing TCRBV, e.g., on the surface of a lymphocyte, e.g., a T cell.

[0363] In one embodiment, the antibody molecule is a monospecific antibody molecule and binds to a single epitope. For example, a monospecific antibody molecule has a plurality of immunoglobulin variable domain sequences each of which binds to the same epitope.

[0364] In one embodiment, the antibody molecule is a multispecific or multifunctional antibody molecule. For example, it includes a plurality of immunoglobulin variable domain sequences, wherein a first one of the plurality of immunoglobulin variable domain sequences has binding specificity for a first epitope and a second one of the plurality of immunoglobulin variable domain sequences has binding specificity for a second epitope. In one embodiment, the first and second epitopes are on the same antigen, such as the same protein (or subunit of a multimeric protein). In one embodiment, the first and second epitopes overlap. In one embodiment, the first and second epitopes do not overlap. In one embodiment, the first and second epitopes are on different antigens, such as different proteins (or different subunits of a multimeric protein). In one embodiment, the multispecific antibody molecule includes a third, fourth, or fifth immunoglobulin variable domain. In one embodiment, the multispecific antibody molecule is a bispecific antibody molecule, a trispecific antibody molecule, or a tetra - specific antibody molecule. molecule, or a tetra - specific antibody molecule.

[0365] In one embodiment, the multispecific antibody molecule is a bispecific antibody molecule. The bispecific antibody has specificity for only two antigens. The bispecific antibody molecule is characterized by a first immunoglobulin variable domain sequence having binding specificity for a first epitope and a second immunoglobulin variable domain sequence having binding specificity for a second epitope. In one embodiment, the first and second epitopes are on the same antigen, for example, the same protein (or subunit of a multimeric protein). In one embodiment, the first and second epitopes overlap. In one embodiment, the first and second epitopes do not overlap. In one embodiment, the first and second epitopes are on different antigens, for example, different proteins (or different subunits of a multimeric protein). In one embodiment, the bispecific antibody molecule comprises a heavy chain variable domain sequence and a light chain variable domain sequence having binding specificity for a first epitope and a heavy chain variable domain sequence and a light chain variable domain sequence having binding specificity for a second epitope. In one embodiment, the bispecific antibody molecule comprises a half antibody having binding specificity for a first epitope and a half antibody having binding specificity for a second epitope. In one embodiment, the bispecific antibody molecule comprises a half antibody, or a fragment thereof, having binding specificity for a first epitope and a half antibody, or a fragment thereof, having binding specificity for a second epitope. In one embodiment, the bispecific antibody molecule comprises a scFv or Fab, or a fragment thereof, having binding specificity for a first epitope and a scFv or Fab, or a fragment thereof, having binding specificity for a second epitope.

[0366] In one embodiment, antibody molecules include antigen-binding fragments of antibodies (e.g., Fab, F(ab’)2, and Fv), in addition to diabodies and single-chain molecules. For example, an antibody molecule can include a heavy (H) chain variable domain sequence (abbreviated herein as VH) and a light (L) chain variable domain sequence (abbreviated herein as VL). In one embodiment, an antibody molecule comprises or consists of a heavy chain and a light chain (referred to herein as a half-antibody). In another example, an antibody molecule includes two heavy (H) chain variable domain sequences and two light (L) chain variable domain sequences, thereby forming two antigen-binding sites, e.g., Fab, Fab’, F(ab’)2, Fc, Fd, Fd’, Fv, single-chain antibody (e.g., scFv), single variable domain antibody, diabody (Dab) (bivalent and bispecific), and chimeric (e.g., humanized) antibodies, which may be produced by modification of a full-length antibody or synthesized de novo using recombinant DNA technology. These functional antibody fragments retain their ability to selectively bind to their respective antigen or receptor. Antibodies and antibody fragments can be from any class of antibodies including, but not limited to, IgG, IgA, IgM, IgD, and IgE, and from any subclass of antibodies (e.g., IgG1, IgG2, IgG3, and IgG4). Preparations of antibody molecules can be monoclonal or polyclonal. Antibody molecules can also be human, humanized, CDR-grafted, or in vitro-generated antibodies. An antibody can have a heavy chain constant region selected, for example, from IgG1, IgG2, IgG3, or IgG4. An antibody can also have a light chain selected, for example, from kappa or lambda. The term “immunoglobulin” (Ig) is used interchangeably herein with the term “antibody”.

[0367] Examples of antigen-binding fragments of antibody molecules include: (i) a Fab fragment, which is a monovalent fragment consisting of VL, VH, CL, and CH1 domains; (ii) an F(ab’)2 fragment, which is a divalent fragment containing two Fab fragments linked by a disulfide bridge in the hinge region; (iii) an Fd fragment, which consists of VH and CH1 domains; (iv) an Fv fragment, which consists of VL and VH domains of a single arm of an antibody; (v) a diabody (dAb) fragment, which consists of a VH domain; (vi) a camel or camelized variable domain; (vii) a single-chain Fv ( scFv), for example, see Bird et al., (1988) Science 242:423-426; and Huston et al., (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883), (viii) a single-domain antibody. These antibody fragments can be obtained using conventional techniques known to those skilled in the art, and the fragments are screened for utility in the same manner as intact antibodies.

[0368] Antibody molecules include intact molecules as well as functional fragments thereof. The constant region of an antibody molecule can be modified, for example, mutated, to modify the properties of the antibody (e.g., to increase or decrease one or more of Fc receptor binding, antibody glycosylation, the number of cysteine residues, effector cell function, or complement function).

[0369] The antibody molecule can also be a single-domain antibody. Single-domain antibodies can include antibodies in which the complementary determining regions are part of a single-domain polypeptide. Examples include, but are not limited to, heavy-chain antibodies, antibodies that are naturally lacking in light chains, single-domain antibodies derived from conventional four-chain antibodies, engineered antibodies, and single-domain scaffolds other than those derived from antibodies. The single-domain antibody can be any in the art or any future single-domain antibody. The single-domain antibody can be derived from any species including, but not limited to, mouse, human, camel, llama, fish, shark, goat, rabbit, and cow. According to another aspect of the invention, the single-domain antibody is a naturally occurring single-domain antibody known as a heavy-chain antibody lacking a light chain. Such single-domain antibodies are disclosed, for example, in WO9404678. For reasons of clarity, this variable domain derived from a heavy-chain antibody that is naturally lacking in a light chain is known herein as VHH or nanobody to distinguish it from the conventional VH of a four-chain immunoglobulin. Such VHH molecules can be derived from antibodies produced in Camelidae species such as camel, llama, dromedary, alpaca, and guanaco. Other species than Camelidae may produce heavy-chain antibodies that are naturally lacking in light chains, and such VHHs are within the scope of the invention.

[0370] The VH and VL regions can be further divided into hypervariable regions called "complementary determining regions" (CDRs) flanked by more conserved regions called "framework regions" (FR or FW).

[0371] The framework regions and the CDR ranges are precisely defined in a number of ways (see Kabat, E. A. et al., (1991) Sequences of Proteins of Immunological Interest, 5th ed., U.S. Department of Health and Human Services, NIH Publication No. 91-3242; Chothia, C. et al., (1987) J. Mol. Biol. 196:901-917; and the definition of AbM used by Oxford Molecular's AbM antibody modeling software. Generally, for example, see "Protein Sequence and Structure Analysis of Antibody Variable Domains", Antibody Engineering Lab Manual (Duebel, S. and Kontermann, R., eds., Springer-Verlag, Heidelberg)).

[0372] The terms "complementary determining region" and "CDR", as used herein, refer to the amino acid sequences within the antibody variable regions that confer antigen specificity and binding affinity. Generally, there are three CDRs (HCDR1, HCDR2, HCDR3) in each heavy chain variable region and three CDRs (LCDR1, LCDR2, LCDR3) in each light chain variable region.

[0373] The precise amino acid sequence boundaries of a given CDR are those of Kabat et al., (1991), "Sequences of Proteins of Immunological Interest", 5th ed., Public Health Service, National It can be determined using any of a number of known schemes, including those described in the Institutes of Health, Bethesda, MD (the "Kabat" numbering scheme), Al-Lazikani et al., (1997) JMB 273, pages 927-948 (the "Chothia" numbering scheme). As used herein, a CDR defined according to the "Chothia" numbering scheme may also be referred to as a "hypervariable loop".

[0374] For example, under Kabat, the CDR amino acid residues in the heavy chain variable domain (VH) are numbered 31-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3), and the CDR amino acid residues in the light chain variable domain (VL) are numbered 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3). Under Chothia, the CDR amino acids in VH are numbered 26-32 (HCDR1), 52-56 (HCDR2), and 95-102 (HCDR3), and the amino acid residues in VL are numbered 26-32 (LCDR1), 50-52 (LCDR2), and 91-96 (LCDR3).

[0375] Each VH and VL typically contains three CDRs and four FRs arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0376] The antibody molecule can be a polyclonal or monoclonal antibody. The term "monoclonal antibody" or "monoclonal antibody composition" as used herein refers to a preparation of antibody molecules of a single molecular composition. A monoclonal antibody composition exhibits a single binding specificity and affinity for a particular epitope. Monoclonal antibodies can be produced by hybridoma technology or by methods that do not use hybridoma technology (e.g., recombinant methods).

[0377] Antibodies can be produced recombinantly, for example, by phage display or combinatorial methods. Phage display and combinatorial methods for generating antibodies are known in the art (e.g., Ladner et al., U.S. Patent No. 5,223,409; Kang et al., International Publication WO92 / 18619; Dower et al., International Publication WO91 / 17271; Winter et al., International Publication WO92 / 20791; Markland et al., International Publication WO92 / 15679; Breitling et al., International Publication WO93 / 01288; McCafferty et al., International Publication WO92 / 01047; Garrard et al., International Publication WO92 / 09690; Ladner et al., International Publication WO90 / 02809; Fuchs et al., (1991) Bio / Technology 9:1370-1372; Hay et al., (1992) Hum Antibod Hybridomas 3:81-85; Huse et al., (1989) Science 246:1275-1281; Griffiths et al., (1993) EMBO J 12:725-734; Hawkins et al., (1992) J Mol Biol 226:889-896; Clackson et al., (1991) Nature 352:624-628; Gram et al., (1992) PNAS 89:3576-3580; Garrad et al., (1991) Bio / Technology 9:1373-1377; Hoogenboom et al., (1991) Nuc Acid Res 19:4133-4137; and Barbas et al., (1991) PNAS 88:7978-7982 (the entire contents of all of these are hereby incorporated by reference herein). as described).

[0378] In one embodiment, the antibody is a fully human antibody (e.g., an antibody made in a mouse genetically engineered to produce an antibody from human immunoglobulin sequences), or a non-human antibody, e.g., a rodent (mouse or rat), goat, primate (e.g., monkey), camel antibody. Preferably, the non-human antibody is a rodent (mouse or rat antibody). Methods for producing rodent antibodies are known in the art.

[0379] Human monoclonal antibodies can be generated using transgenic mice that have human immunoglobulin genes rather than mouse systems. Splenocytes from these transgenic mice immunized with the antigen of interest are used to produce hybridomas that secrete human mAbs having specific affinity for epitopes from human proteins (see, e.g., Wood et al., International Application WO91 / 00906; Kucherlapati et al., PCT International Publication WO91 / 10741; Lonberg et al., International Application WO92 / 03918; Kay et al., International Application 92 / 03917; Lonberg, N. et al., 1994 Nature 368:856-859; Green, L.L. et al., 1994 Nature Genet. 7:13-21; Morrison, S.L. et al., 1994 Proc. Natl. Acad. Sci. USA 81:6851-6855; Bruggeman et al., 1993 Year Immunol 7:33-40; Tuaillon et al., 1993 PNAS 90:3720-3724; Bruggeman et al., 1991 Eur J Immunol 21:1323-1326).

[0380] The antibody molecule can be one in which the variable region, or a portion thereof, e.g., a CDR, is produced in a non-human organism, e.g., a rat or mouse. Chimeric, CDR-grafted, and humanized antibodies are within the scope of the invention. Antibody molecules produced in a non-human organism, e.g., a rat or mouse, and then modified, e.g., in the variable framework or constant region, to reduce antigenicity in humans are within the scope of the invention.

[0381] The "effective human" protein is a protein that does not substantially induce a neutralizing antibody response, such as a human anti-mouse antibody (HAMA) response. HAMA can be a problem in a number of situations, for example, in the treatment of chronic or relapsing disease states where antibody molecules are repeatedly administered. The HAMA response can potentially render repeated antibody administration ineffective due to increased antibody clearance from the serum (see, for example, Saleh et al., Cancer Immunol. Immunother., 32:180-190 (1990)) and also due to potential allergic reactions (see, for example, LoBuglio et al., Hybridoma, 5:5117-5123 (1986)).

[0382] Chimeric antibodies can be produced by recombinant DNA techniques known in the art (Robinson et al., International Publication PCT / US86 / 02269; Akira et al., European Patent Application No. 184,187; Taniguchi, M., European Patent Application No. 171,496; Morrison et al., European Patent Application No. 173,494; Neuberger et al., International Application WO86 / 01533; Cabilly et al., U.S. Patent No. 4,816,567; Cabilly et al., European Patent Application No. 125,023; Better et al., (1988 Science 240:1041-1043); Liu et al., (1987) PNAS 84:3439-3443; Liu et al., 1987, J. Immunol. 139:3521-3526; Sun et al., (1987) PNAS 84:214-218; Nishimura et al., 1987, Canc. Res. 47:999-1005; Wood et al., (1985) Nature 314:446 -449; and Shaw et al., 1988, J. Natl Cancer Inst. 80:1553-1559).

[0383] Humanized or CDR-grafted antibodies have at least one or two (of the heavy and / or light immunoglobulin chains), and generally all three, recipient CDRs replaced with donor CDRs. The antibody may be replaced with at least a portion of the non-human CDRs, or only a portion of the CDRs may be replaced with non-human CDRs. It is only necessary to replace the number of CDRs required for binding to the antigen. Preferably, the donor is a rodent antibody, such as a rat or mouse antibody, and the recipient is a human framework or human consensus framework. Typically, the immunoglobulin providing the CDRs is called the "donor", and the immunoglobulin providing the framework is called the "acceptor". In one embodiment, the donor immunoglobulin is non-human (e.g., rodent). The acceptor framework is a naturally occurring (e.g., human) framework or consensus framework, or a sequence that is about 85% or higher, preferably 90%, 95%, 99% or more identical thereto.

[0384] As used herein, the term "consensus sequence" refers to a sequence formed from the amino acids (or nucleotides) that occur most frequently in a family of related sequences (see, e.g., Winnaker, From Genes to Clones (Verlagsgesellschaft, Weinheim, Germany 1987)). In a family of proteins, each position in the consensus sequence is occupied by the amino acid that occurs most frequently at that position in the family. If two amino acids occur equally frequently, either can be included in the consensus sequence. "Consensus framework" refers to the framework region in a consensus immunoglobulin sequence.

[0385] Antibody molecules can be humanized by methods known in the art (see, e.g., Morrison, S. L., 1985, Science 229:1202-1207, Oi et al., 1986, BioTechniques 4:214, and Queen et al., U.S. Patent Nos. 5,585,089, 5,693,761 and 5,693,762, the entire contents of all of which are hereby incorporated by reference).

[0386] Humanized or CDR-grafted antibody molecules can be produced by CDR-grafting or CDR replacement, in which one, two, or all of the CDRs of an immunoglobulin chain can be replaced. See, e.g., U.S. Patent No. 5,225,539; Jones et al., 1986 Nature 321:552-525; Verhoeyan et al., 1988 Science 239:1534; Beidler et al., 1988 J. Immunol. 141:4053-4060; Winter, U.S. Patent No. 5,225,539 (the entire contents of all of which are hereby expressly incorporated by reference). Winter describes CDR-grafting methods that can be used to prepare the humanized antibodies of the present invention (British Patent Application No. 2188638A, filed Mar. 26, 1987; Winter, U.S. Patent No. 5,225,539), the contents of which are hereby incorporated by reference.

[0387] Humanized antibody molecules with specific amino acid substitutions, deletions or additions are also within the scope of the present invention. The criteria for selecting amino acids from the donor are described in U.S. Patent No. 5,585,089, e.g., columns 12-16 of U.S. Patent No. 5,585,089 (the contents of which are hereby incorporated by reference). Other techniques for humanizing antibodies are described in Padlan et al., EP519596A1, published Dec. 23, 1992.

[0388] The antibody molecule can be a single-chain antibody. The single-chain antibody (scFV) may be engineered (e.g., Colcher, D. et al., (1999) Ann N Y Acad Sci 880:263-80; and Reiter, Y., (1996) Clin Cancer Res 2:245-52). The single-chain antibody can dimerize or multimerize to generate a multivalent antibody having specificity for different epitopes of the same target protein.

[0389] In yet other embodiments, the antibody molecule has a heavy chain constant region selected from, for example, the heavy chain constant regions of IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE, particularly, for example, the (e.g., human) heavy chain constant regions of IgG1, IgG2, IgG3, and IgG4. In another embodiment, the antibody molecule has a light chain constant region selected from, for example, the (e.g., human) light chain constant regions of kappa or lambda. The constant region can be altered, e.g., mutated, to modify the properties of the antibody (e.g., to increase or decrease one or more of Fc receptor binding, antibody glycosylation, number of cysteine residues, effector cell function, and / or complement function). In one embodiment, the antibody has effector function and can fix complement. In other embodiments, the antibody does not recruit effector cells and does not fix complement. In another embodiment, the antibody has a reduced ability or no ability to bind to Fc receptors. For example, it is an isotype or subtype, fragment or other mutant that does not support binding to Fc receptors, e.g., it has a mutagenized Fc receptor-binding region or lacks it.

[0390] Methods for altering the constant region of an antibody are known in the art. Antibodies having altered functions, such as altered effector ligands, e.g., FcRs on cells, or altered affinities for the C1 component of complement, can be produced by replacing at least one amino acid residue in the constant portion of the antibody with a different residue (see, e.g., EP388,151A1, U.S. Patent No. 5,624,821, and U.S. Patent No. 5,648,260, all of which are hereby incorporated by reference in their entirety). Similar types of alterations that reduce or eliminate these functions when applied to mouse, or other species, immunoglobulins can be described.

[0391] Antibody molecules can be derivatized or conjugated to another functional molecule, e.g., another peptide or protein. As used herein, a “derivatized” antibody molecule is a modified antibody molecule. Methods of derivatization include, but are not limited to, the addition of a fluorescent moiety, a radioactive nucleotide, a toxin, an enzyme, or an affinity ligand, e.g., biotin. Thus, the antibody molecules of the invention are intended to include derivatives and other modified forms of the antibodies described herein, including immunoadhesion molecules. For example, an antibody molecule can be functionally linked (by chemical coupling, genetic fusion, non-covalent association, or otherwise) to one or more other molecular entities, e.g., another antibody (e.g., a bispecific antibody or diabody), a detectable agent, a cytotoxic agent, a pharmaceutical agent, and / or a protein or peptide that can mediate association with another molecule of an antibody or antibody portion (e.g., a streptavidin core region or polyhistidine tag).

[0392] One type of derivatized antibody molecule is produced by cross-linking two or more antibodies (of the same or different types, e.g., to create a bispecific antibody). Suitable cross-linkers include appropriate spacers (e.g., m-maleimidobenzoyl-N-hydroxysuccinimide ester) or homobifunctional (e.g., suberyl Heterobifunctional ones having two separately reactive groups separated by disuccinimidyl esters are mentioned. Such linkers are available from Pierce Chemical Company, Rockford, Ill.

[0393] Multispecific or multifunctional antibody molecules Exemplary structures of the multispecific and multifunctional molecules as defined herein are described throughout. Exemplary structures are further described in Weidle U et al., (2013) The Intriguing Options of Multispecific Antibody Formats for Treatment of Cancer. Cancer Genomics & Proteomics 10: 1 - 18 (2013); and Spiess C et al., (2015) Alternative molecular formats and therapeutic applications for bispecific antibodies. Molecular Immunology 67: 95 - 106 (the entire contents of each of these are hereby incorporated by reference herein).

[0394] In embodiments, the multispecific antibody molecule can include more than one antigen-binding site where different sites are specific for different antigens. In embodiments, the multispecific antibody molecule can bind to more than one (e.g., two or more) epitopes on the same antigen. In embodiments, the multispecific antibody molecule includes an antigen-binding site specific for a target cell (e.g., a lymphocyte (e.g., a T cell) containing a TCRBV antigen corresponding to a biased TCRBV clonotype) and a different antigen-binding site specific for an immune effector cell. In one embodiment, the multispecific antibody molecule is a bispecific antibody molecule. Bispecific antibody molecules can be classified into five different structural groups: (i) bispecific immunoglobulin G (BsIgG), (ii) IgG with an additional antigen-binding moiety, (iii) bispecific antibody fragments, (iv) bispecific fusion proteins, and (v) bispecific antibody conjugates.

[0395] BsIgG is a monovalent format for each antigen. Exemplary BsIgG formats include, but are not limited to, crossMab, DAF (two-in-one), DAF (four-in-one), DutaMab, DT-IgG, knobs-in-holes common LC, knobs-in-holes assembly, charge pair, Fab arm exchange, SEED body, triomab, LUZ-Y, Fcab, κλ body, orthogonal Fab. Spiess et al., Mol. Immunol. See Spiess et al., Mol. Immunol. 67(2015):95-106. Exemplary BsIgGs include catumaxomab (Fresenius Biotech, Trion Pharma, Neopharm) containing anti-CD3 and anti-EpCAM arms, and ertumaxomab (Neovii Biotech, Fresenius Biotech) targeting CD3 and HER2. In some embodiments, the BsIgG comprises heavy chains engineered for heterodimerization. For example, the heavy chains can be engineered for heterodimerization using the "knobs-into-holes" strategy, the SEED platform, common heavy chains (e.g., in a kappa lambda body), and the use of a heterodimeric Fc region. See Spiess et al., Mol. Immunol. 67(2015):95-106. Strategies that have been used to avoid homodimeric heavy chain pairing in BsIgGs include knobs-in-hole, duobody, azymetric, charge pair, HA-TF, SEED body, and differential protein A affinity. See the same reference. BsIgG can be produced by separate expression of the component antibodies in different host cells and subsequent purification / assembly into BsIgG. BsIgG can also be produced by expression of the component antibodies in a single host cell. BsIgG can be purified, for example, using affinity chromatography using protein A and sequential pH elution.

[0396] IgG with an additional antigen-binding part is another format of bispecific antibody molecules. For example, a monospecific IgG can be engineered to have bispecificity by adding an additional antigen-binding unit to the monospecific IgG at, for example, the N or C terminus of either the heavy or light chain. Exemplary additional antigen-binding units include single-domain antibodies (e.g., variable heavy or variable light chains), engineered protein scaffolds, and paired antibody variable domains (e.g., single-chain variable fragments or variable fragments). See the same reference. Examples of the added IgG formats include dual variable domain IgG (DVD-Ig), IgG(H)-scFv, scFv-(H)IgG, IgG(L)-scFv, scFv-(L)IgG, IgG(L,H)-Fv, IgG(H)-V, V(H)-IgG, IgG(L)-V, V(L)-IgG, KIH IgG-scFab, 2scFv-IgG, IgG-2scFv, scFv4-Ig, zybody, and DVI-IgG (4-in-1). See Spiess et al., Mol. Immunol. 67(2015):95-106. An example of IgG-scFv is MM-141 (Merrimack Pharmaceuticals) that binds to IGF-1R and HER3. Examples of DVD-Ig include ABT-981 (AbbVie) that binds to IL-1α and IL-1β, and ABT-122 (AbbVie) that binds to TNF and IL-17A.

[0397] Bispecific antibody fragments (BsAbs) are formats of bispecific antibody molecules that lack some or all of the antibody constant domains. For example, some BsAbs lack the Fc region. In embodiments, the bispecific antibody fragment comprises a heavy chain and a light chain region connected by a peptide linker that permits efficient expression of the BsAb in a single host cell. Exemplary bispecific antibody fragments include, but are not limited to, nanobodies, nanobody-HAS, BiTE, diabodies, DART, TandAb, scDiabody, scDiabody-CH3, diabody-CH3, tribodies, miniantibodies, minibodies, TriBi minibodies, scFv-CH3 KIH, Fab-scFv, scFv-CH-CL-scFv, F(ab’)2, F(ab’)2-scFv2, scFv-KIH, Fab-scFv-Fc, tetravalent HCAb, scDiabody-Fc, diabody-Fc, tandem scFv-Fc, and intrabodies. See the same reference. For example, the BiTE format comprises a tandem scFv, and the component scFvs bind to CD3 on T cells and to TCRBV antigens on lymphocytes, such as T cells.

[0398] Examples of bispecific fusion proteins include, for example, antibody fragments conjugated to other proteins to add additional specificity and / or functionality. An example of a bispecific fusion protein is immTAC, which comprises an anti-CD3 scFv conjugated to an affinity matured T cell receptor that recognizes an HLA-presented peptide. In embodiments, the dock-and-lock (DNL) method can be used to generate bispecific antibody molecules with higher valency. Also, fusion to an albumin-binding protein or human serum albumin can extend the serum half-life of the antibody fragment. See the same reference.

[0399] In embodiments, chemical conjugation, for example, chemical conjugation of antibodies and / or antibody fragments can be used to create BsAb molecules. See the same reference. Exemplary bispecific antibody conjugates include the CovX body format, in which a low molecular weight drug is site-specifically conjugated to each Fab arm or a single reactive lysine in an antibody or its fragment. In embodiments, conjugation improves the serum half-life of the low molecular weight drug. An exemplary CovX body is CVX-241 (NCT01004822), which comprises an antibody conjugated to two short peptides that inhibit either VEGF or Ang2. See the same reference.

[0400] Antibody molecules can be produced, for example, by recombinant expression of at least one or more components in a host system. Exemplary host systems include eukaryotic cells (e.g., mammalian cells, such as CHO cells, or insect cells, such as SF9 or S2 cells) and prokaryotic cells (e.g., E. coli). Bispecific antibody molecules can be produced by separate expression of the components in different host cells and subsequent purification / assembly. Alternatively, antibody molecules can be produced by expression of the components in a single host cell. Purification of bispecific antibody molecules can be performed by various methods, such as affinity chromatography, using, for example, Protein A and sequential pH elution. In other embodiments, affinity tags, such as histidine-containing tags, myc tags, or streptavidin tags, can be used for purification.

[0401] CDR grafted scaffold In an embodiment, the antibody molecule is a CDR-grafted scaffold domain. In an embodiment, the scaffold domain is based on a fibronectin domain, for example, a fibronectin type III domain. The overall fold of the fibronectin type III (Fn3) domain is closely related to that of the minimal functional antibody fragment, the variable domain of the antibody heavy chain. There are three loops at the ends of Fn3, and the positions of the BC, DE, and FG loops approximately correspond to those of CDR1, 2, and 3 of the VH domain of the antibody. Fn3 does not have a disulfide bond and thus, unlike antibodies and their fragments, is stable under reducing conditions (see, for example, WO98 / 56915, WO01 / 64942, WO00 / 34784). The Fn3 domain can be modified or altered (e.g., using the CDRs or hypervariable loops described herein) to select, for example, a domain that binds to an antigen / marker / cell described herein.

[0402] In embodiments, the scaffold domain, e.g., the folded domain, is based on a "minibody" scaffold created by deleting three beta strands from the heavy chain variable domain of an antibody, e.g., a monoclonal antibody (see, e.g., Tramontano et al., 1994, J Mol. Recognit. 7:9, and Martin et al., 1994, EMBO J. 13:5303-5309). The "minibody" can be used to present two hypervariable loops. In embodiments, the scaffold domain is a V-like domain (see, e.g., Coia et al., WO99 / 45110), or a domain derived from tendamistatin, a 74-residue six-stranded beta sheet sandwich held together by two disulfide bonds (see, e.g., McConnell and Hoess, 1995, J Mol. Biol. 250:460). For example, the loops of tendamistatin can be modified or altered (e.g., using CDRs or hypervariable loops) to select a domain that binds, e.g., to a marker / antigen / cell described herein. Another exemplary scaffold domain is a beta-sandwich structure derived from the extracellular domain of CTLA-4 (see, e.g., WO00 / 60070).

[0403] Other exemplary scaffold domains include, but are not limited to, T cell receptors, MHC proteins, extracellular domains (e.g., fibronectin type III repeats, EGF repeats), protease inhibitors (e.g., Kunitz domains, echistatin, and BPTI, etc.), TPR repeats, the trifoil structure, zinc finger domains, DNA-binding proteins, particularly monomeric DNA-binding proteins, RNA-binding proteins, enzymes, e.g., proteases (particularly inactivated proteases), RNases, chaperones, e.g., thioredoxin, and heat shock proteins, and intracellular signaling domains (e.g., SH2 and SH3 domains). See, for example, U.S. Patent Application Publication No. 20040009530 and U.S. Patent No. 7,501,121, which are incorporated herein by reference.

[0404] In embodiments, the scaffold domain is evaluated and selected, for example, by one or more of the following criteria: (1) amino acid sequence, (2) sequences of several homologous domains, (3) three-dimensional structure, and / or (4) stability data over a range of pH, temperature, salt, organic solvent, oxidant concentration. In embodiments, the scaffold domain is a small, stable protein domain, e.g., a protein of less than 100, 70, 50, 40, or 30 amino acids. The domain may contain one or more disulfide bonds or may chelate a metal, e.g., zinc.

[0405] Antibody-based fusions A variety of formats can be generated that contain additional binding entities attached to the N or C terminus of the antibody. These fusions having single-chain or disulfide-stabilized Fv or Fab result in the generation of tetravalent molecules with bivalent binding specificity for each antigen. Combinations of scFv and scFab with IgG allow the production of molecules that can recognize three or more different antigens.

[0406] Antibody-Fab fusions An antibody-Fab fusion is a bispecific antibody that includes a conventional antibody against a first target and a Fab against a second target fused to the C-terminus of the antibody heavy chain. Generally, the antibody and the Fab have a common light chain. The antibody fusion can be produced by (1) manipulating the DNA sequence of the target fusion and (2) transfecting the target DNA into a suitable host cell to express the fusion protein. As described by Coloma, J et al., (1997) Nature Biotech 15:159, the antibody-scFv fusion may be linked by a (Gly)-Ser linker between the C-terminus of the CH3 domain and the N-terminus of the scFv.

[0407] antibody-scFv fusion An antibody-scFv fusion is a bispecific antibody that includes a conventional antibody and an scFv of unique specificity fused to the C-terminus of the antibody heavy chain. The scFv can be fused to the C-terminus directly or through the heavy chain of the scFv via a linker peptide. The antibody fusion can be produced by (1) manipulating the DNA sequence of the target fusion and (2) transfecting the target DNA into a suitable host cell to express the fusion protein. As described by Coloma, J et al., (1997) Nature Biotech 15:159, the antibody-scFv fusion may be linked by a (Gly)-Ser linker between the C-terminus of the CH3 domain and the N-terminus of the scFv.

[0408] Variable domain immunoglobulin DVD The related format is a dual variable domain immunoglobulin (DVD) composed of VH and VL domains of a second specificity arranged at the N-terminus of the V domain by a shorter linker sequence.

[0409] Other exemplary bispecific antibody formats include, for example, the following: US Patent Application Publication No. 20160114057A1, US Patent Application Publication No. 20130243775A1, US Patent Application Publication No. 20140051833, US Patent Application Publication No. 20130022601, US Patent Application Publication No. 20150017187A1, US Patent Application Publication No. 20120201746A1, US Patent Application Publication No. 20150133638A1, US Patent Application Publication No. 20130266568A1, US Patent Application Publication No. 20160145340A1, WO2015127158A1, US Patent Application Publication No. 20150203591A1, US Patent Application Publication No. 20140322221A1, US Patent Application Publication No. 20130303396A1, US Patent Application Publication No. 20110293613, US Patent Application Publication Examples include those described in US Patent Application Publication No. 20130017200A1, US Patent Application Publication No. 20160102135A1, WO2015197598A2, WO2015197582A1, US Patent No. 9359437, US Patent Application Publication No. 20150018529, WO2016115274A1, WO2016087416A1, US Patent Application Publication No. 20080069820A1, US Patent No. 9145588B, US Patent No. 7919257, and US Patent Application Publication No. 20150232560A1. Exemplary multispecific molecules utilizing the full-length antibody-Fab / scFab format include the following: those described in US Patent No. 9382323B2, US Patent Application Publication No. 20140072581A1, US Patent Application Publication No. 20140308285A1, US Patent Application Publication No. 20130165638A1, US Patent Application Publication No. 20130267686A1, US Patent Application Publication No. 20140377269A1, US Patent No. 7741446B2, and WO1995009917A1. Exemplary multispecific molecules utilizing the domain exchange format include the following: those described in US Patent Application Publication No. 20150315296A1, WO2016087650A1, US Patent Application Publication No. 20160075785A1, WO2016016299A1, US Patent Application Publication No. 20160130347A1, US Patent Application Publication No. 20150166670, US Patent No. 8703132B2, US Patent Application Publication No. 20100316645, US Patent No. 8227577B2, and US Patent Application Publication No. 20130078249.

[0410] Fc-containing entity (minibody) Fc-containing entities, also known as minibodies, can be generated by fusing an scFv to the C-terminus of the constant heavy chain domain 3 (CH3-scFv) and / or the hinge region of an antibody with a different specificity (scFv-hinge-Fc). Trivalent entities having a disulfide-stabilized variable domain fused to the C-terminus of the CH3 domain of IgG (without a peptide linker) can also be produced.

[0411] Fc-containing multispecific molecule In some embodiments, the multispecific molecules disclosed herein include an immunoglobulin constant region (e.g., an Fc region). Exemplary Fc regions can be selected from the heavy chain constant regions of IgG1, IgG2, IgG3, or IgG4, more particularly, the heavy chain constant regions of human IgG1, IgG2, IgG3, or IgG4.

[0412] In some embodiments, the immunoglobulin chain constant region (e.g., an Fc region) is modified, e.g., mutated, to increase or decrease one or more of Fc receptor binding, antibody glycosylation, number of cysteine residues, effector cell function, or complement function.

[0413] In other embodiments, the boundary between the first and second immunoglobulin chain constant regions (e.g., the first and second Fc regions) is modified, e.g., mutated, to increase or decrease dimerization as compared to, for example, an unmanipulated boundary, e.g., a naturally occurring boundary. For example, dimerization of the immunoglobulin chain constant region (e.g., an Fc region) can be enhanced by providing one or more of paired holes and protrusions (“knob-in-hole”), electrostatic interactions, or strand exchange at the Fc boundary of the first and second Fc regions, thereby forming a higher ratio of heteromultimer:homomultimer as compared to, for example, an unmanipulated boundary.

[0414] In some embodiments, the multispecific molecule includes paired amino acid substitutions at one or more positions selected from 347, 349, 350, 351, 366, 368, 370, 392, 394, 395, 397, 398, 399, 405, 407, or 409 of, for example, the Fc region of human IgG1. For example, the immunoglobulin chain constant region (e.g., an Fc region) can include paired amino acid substitutions selected from T366S, L368A, or Y407V (e.g., corresponding to a hole or a hole), and T366W (e.g., corresponding to a protrusion or a knob).

[0415] In other embodiments, the multifunctional molecule comprises a half-life extender, such as human serum albumin or an antibody molecule against human serum albumin. Heterodimerizing antibody molecule and method of making the same Various methods for producing multispecific antibodies have been disclosed to address the problem of incorrect heavy chain pairing. Exemplary methods are described below. Exemplary multispecific antibody formats and methods of making said multispecific antibodies are also disclosed, for example, in Speiss et al., Molecular Immunology 67 (2015) pages 95 - 106, and Klein et al., mAb 4:6, pages 653 - 663, November / December 2012 (the respective contents of which are hereby incorporated by reference herein).

[0416] Heterodimerizing bispecific antibodies are based on the native IgG structure, with two binding arms recognizing different antigens. IgG-derived formats that allow defined monovalent (and simultaneous) antigen binding are generated by forced heavy chain heterodimerization combined with techniques that minimize light chain mispairing (such as the common light chain). Forced heavy chain heterodimerization can be obtained, for example, using the knob-in-hole or the strand exchange engineering domain (SEED).

[0417] Knob-in-hole The knob-in-hole described in U.S. Patent No. 5,731,116, U.S. Patent No. 7,476,724 and Ridgway, J et al., (1996) Prot. Engineering 9(7):617 - 621 generally involves (1) mutating the CH3 domain of one or both antibodies to promote heterodimerization, and (2) combining the mutated antibodies under conditions that promote heterodimerization. The "knob" or "protrusion" is typically created by replacing a small amino acid in the parental antibody with a larger amino acid (e.g., T366Y or T366W), and the "hole" or "pore" is created by replacing a larger residue in the parental antibody with a smaller amino acid (e.g., Y407T, T366S, L368A and / or Y407V).

[0418] For bispecific antibodies containing an Fc domain, the introduction of specific mutations into the constant region of the heavy chain can be utilized to promote the correct heterodimerization of the Fc portion. Some such techniques are reviewed in Klein et al. (mAb (2012) 4:6, pages 1-11) (the content of which is hereby incorporated by reference in its entirety herein). These techniques include the "knob-into-hole" (KiH) approach involving the introduction of a bulky residue into one of the CH3 domains of one of the antibody heavy chains. This bulky residue fits into a complementary "hole" in the other CH3 domain of the paired heavy chain, thereby promoting the correct pairing of the heavy chains (see, for example, U.S. Patent No. 7,642,228).

[0419] Exemplary KiH mutations include S354C, T366W in the "knob" heavy chain and Y349C, T366S, L368A, Y407V in the "hole" heavy chain. Other exemplary KiH mutations are provided in Table 1, along with stabilizing Fc cysteine mutations as needed.

[0420]

Table 1

[0421] Other Fc mutations have been provided by Igawa and Tsunoda, who identified three negatively charged residues in the CH3 domain of one chain that pair with three positively charged residues in the CH3 domain of the other chain. These specific charged residue pairs are E356-K439, E357-K370, D399-K409 and their reverse. By introducing at least two of K370E, K409D, K439E in chain B in addition to the following three mutations in chain A: E356K, E357K and D399K, either alone or in combination with newly identified disulfide bridges, they were able to act favorably on very efficient heterodimerization while simultaneously suppressing homodimerization (Martens T et al., "A novel one-armed antic-Met antibody inhibits glioblastoma growth in vivo.", Clin Cancer Res 2006;12:6144-52; PMID:17062691). Xencor defined 41 variant pairs based on a combination of structural calculations and sequence information, which were then screened for maximum heterodimerization to define the combination of S364H, F405A (HA) on chain A and Y349T, T394F (TF) on chain B (Moore GL et al., "A novel bispecific antibody format enables simultaneous bivalent and monovalent co-engagement of distinct target antigens.", MAbs 2011;3:546-57; PMID:22123055).

[0422] Other exemplary Fc mutations for promoting heterodimerization of bispecific antibodies include the following references (the contents of each of which are hereby incorporated by reference herein): WO2016071377A1, US Patent Application Publication No. 20140079689A1, US Patent Application Publication No. 20160194389A1, US Patent Application Publication No. 20160257763, WO2016071376A2, WO2015107026A1, WO2015107025A1, WO2015107015A1, US Patent Application Publication No. 20150353636A1, US Patent Application Publication No. 20140199294A1, US Patent No. 7750128B2, US Patent Application Publication No. 20160229915A1, US Patent Application Publication No. 20150344570A1, US Patent No. 8003774A1, US Patent Application Publication No. 20150337049A1, US Patent Application Publication No. 20150175707A1, US Patent Application Publication No. 20140242075A1, US Patent Application Publication No. 201 No. 30195849A1, US Patent Application Publication No. 20120149876A1, US Patent Application Publication No. 20140200331A1, US Patent No. 9309311B2, US Patent No. 8586713, US Patent Application Publication No. 20140037621A1, US Patent Application Publication No. 20130178605A1, US Patent Application Publication No. 20140363426A1, US Patent Application Publication No. 20140051835A1, and US Patent Application Publication No. 20110054151A1.

[0423] Stabilizing cysteine mutations have also been used in combination with KiH and other Fc heterodimerization promoting variants (see, for example, US Patent No. 7183076). Other exemplary cysteine modifications include, for example, those disclosed in US Patent Application Publication No. 20140348839A1, US Patent No. 7855275B2, and US Patent No. 9000130B2.

[0424] Chain Exchange Operation Domain (SEED) There is a heterodimeric Fc platform known that supports the design of bispecific and asymmetric fusion proteins by engineering a swap operation domain (SEED) C(H)3 heterodimer. These derivatives of the human IgG and IgA C(H)3 domains create complementary human SEED C(H)3 heterodimers composed of alternating segments of the human IgA and IgG C(H)3 sequences. The resulting pairs of SEED C(H)3 domains preferentially associate to form heterodimers when expressed in mammalian cells. The SEED body (Sb) fusion protein may be genetically linked to one or more fusion partners and consists of [IgG1 hinge]-C(H)2-[SEED C(H)3] (e.g., see Davis JH et al., "SEEDbodies: fusion proteins based on strand exchange engineered domain (SEED) CH3 heterodimers in an Fc analogue platform for asymmetric binders or immunofusions and bispecific antibodies.", Protein Eng Des Sel 2010;23:195-202; PMID:20299542 and U.S. Patent No. 8,871,912, the contents of each of which are hereby incorporated by reference herein).

[0425] DuoBody The "DuoBody" technology for producing bispecific antibodies with correct heavy chain pairing is known. The DuoBody technology involves three basic steps for generating stable bispecific human IgG1 antibodies in a post-production exchange reaction. In the first step, two IgG1s each containing a single matched mutation in the third constant (CH3) domain are produced separately using standard mammalian recombinant cell lines. Thereafter, these IgG1 antibodies are purified according to standard methods for recovery and purification. After production and purification (post-production), the two antibodies are recombined under controlled laboratory conditions to yield a bispecific antibody product with very high yields (typically >95%) (see, for example, Labrijn et al., PNAS 2013;110(13):5145-5150 and Labrijn et al., Nature Protocols 2014;9(10):2450-63, the contents of each of which are hereby incorporated by reference).

[0426] Electrostatic interaction Methods for making multispecific antibodies using CH3 amino acid changes with charged amino acids such that homodimer formation is electrostatically unfavorable are disclosed. EP1870459 and WO2009089004 describe other strategies that favor heterodimer formation upon co-expression of different antibody domains in host cells. In these methods, the heavy chain constant domain 3 (CH3), building the CH3-CH3 interface in both CH3 domains One or more residues are replaced with charged amino acids such that homodimer formation is electrostatically unfavorable and heterodimerization is electrostatically favorable. Additional methods for making multispecific molecules using electrostatic interactions are described in references including US Patent Application Publication No. 20100015133, US Patent No. 8592562B2, US Patent No. 9200060B2, US Patent Application Publication No. 20140154254A1, and US Patent No. 9358286A1, the contents of each of which are hereby incorporated by reference.

[0427] Common light chain Light chain mispairing needs to be avoided in order to generate a homogeneous preparation of bispecific IgG. One way to achieve this is through the use of the common light chain principle, i.e., by combining two binders that share one light chain but still have separate specificities. An exemplary method to enhance the formation of the desired bispecific antibody from a mixture of monomers is by providing a common variable light chain for interacting with each of the heteromeric variable heavy chain regions of the bispecific antibody. Compositions of bispecific antibodies having a common light chain and methods for their production are disclosed, for example, in U.S. Patent No. 7,183,076 B2, U.S. Patent Application Publication No. 2011 / 0177073 A1, EP 2847231 A1, WO 2016 / 079081 A1, and EP 3055329 A1 (the contents of each of which are hereby incorporated by reference herein).

[0428] CrossMab Another option for reducing light chain mispairing is CrossMab technology, which avoids non-specific L chain mispairing by exchanging the CH1 and CL domains in the Fab of one half of a bispecific antibody. Such crossover variants retain binding specificity and affinity, but the two arms are made different so that L chain mispairing is prevented. (As reviewed by Klein et al., supra) CrossMab technology involves domain swapping between the heavy and light chains to promote the formation of correct pairings. Briefly, a two-step modification process is applied to construct a bispecific IgG-like CrossMab antibody that can bind to two antigens by using two separate light chain-heavy chain pairs. First, the dimerization interface is engineered into the C-terminus of each heavy chain using a heterodimerization approach, such as the knob-into-hole (KiH) technology, to ensure that only heterodimers of two separate heavy chains from one antibody (e.g., antibody A) and a second antibody (e.g., antibody B) are efficiently formed. Next, the constant heavy chain 1 (CH1) and constant light chain (CL) domains of one antibody (antibody A) are exchanged while keeping the variable heavy chain (VH) and variable light chain (VL) domains consistent. The exchange of the CH1 and CL domains ensures that only the desired bispecific CrossMab is efficiently formed because the modified antibody (antibody A) light chain dimerizes efficiently only with the modified antibody (antibody A) heavy chain and the unmodified antibody (antibody B) light chain dimerizes efficiently only with the unmodified antibody (antibody B) heavy chain (see, for example, Cain, C., SciBX 4(28); doi:10.1038 / scibx.2011.783, the contents of which are hereby incorporated by reference).

[0429] Common heavy chain Exemplary methods for enhancing the formation of a desired bispecific antibody from a mixture of monomers involve providing a common variable heavy chain for interacting with each of the heteromeric variable light chain regions of the bispecific antibody. Compositions of bispecific antibodies having a common heavy chain and methods for their production are disclosed, for example, in U.S. Patent Application Publication No. 20120184716, U.S. Patent Application Publication No. 20130317200, and U.S. Patent Application Publication No. 20160264685A1, the contents of each of which are hereby incorporated by reference herein.

[0430] Amino acid modifications Alternative compositions of multispecific antibodies having correct light chain pairing and methods for their production include various amino acid modifications. For example, Zymeworks describes one or more amino acid modifications in the CH1 and / or CL domains, one or more amino acid modifications in the VH and / or VL domains, or combinations thereof, which are part of the interface between the light and heavy chains and create preferential pairing of each heavy chain with a desired light chain, such that when two heavy chains and two light chains of a heterodimer pair are co-expressed in a cell, the heavy chain of the first heterodimer preferentially pairs with one of the light chains over the others (see, for example, WO2015181805). Other exemplary methods are described in WO2016026943 (Argen-X), U.S. Patent Application Publication No. 20150211001, U.S. Patent Application Publication No. 20140072581A1, U.S. Patent Application Publication No. 20160039947A1, and U.S. Patent Application Publication No. 20150368352.

[0431] Lambda / kappa format Multispecific molecules (e.g., multispecific antibody molecules) comprising lambda light chain polypeptides and kappa light chain polypeptides can be used to enable heterodimerization. Methods for generating bispecific antibody molecules comprising lambda light chain polypeptides and kappa light chain polypeptides are disclosed in PCT / US17 / 53053, filed September 22, 2017, which is hereby incorporated by reference in its entirety herein.

[0432] In an embodiment, the multispecific molecule includes a multispecific antibody molecule, for example, an antibody molecule including two binding specificities, for example, a bispecific antibody molecule. The multispecific antibody molecule includes a lambda light chain polypeptide 1 (LLCP1) specific for a first epitope, a heavy chain polypeptide 1 (HCP1) specific for the first epitope, a kappa light chain polypeptide 2 (KLCP2) specific for a second epitope, and a heavy chain polypeptide 2 (HCP2) specific for the second epitope. It includes.

[0433] "Lambda light chain polypeptide 1 (LLCP1)", as the term is used herein, refers to a polypeptide that, when combined with its cognate heavy chain variable region, mediates specific binding to its epitope and can complex with HCP1, including a light chain (LC) sequence sufficient for this. In one embodiment, it includes all or a fragment of the CH1 region. In one embodiment, LLCP1 includes LC-CDR1, LC-CDR2, LC-CDR3, FR1, FR2, FR3, FR4, and CH1, or sequences therefrom sufficient to mediate specific binding to its epitope and complex with HCP1. LLCP1, together with its HCP1, provides specificity for the first epitope (KLCP2, together with its HCP2, provides specificity for the second epitope). As described elsewhere herein, LLCP1 has a higher affinity for HCP1 than for HCP2.

[0434] "Kappa light chain polypeptide 2 (KLCP2)", as the term is used herein, refers to a polypeptide that, when combined with its cognate heavy chain variable region, contains a light chain (LC) sequence sufficient to mediate specific binding to its epitope and to complex with HCP2. In one embodiment, it includes all or a fragment of the CH1 region. In one embodiment, KLCP2 includes LC-CDR1, LC-CDR2, LC-CDR3, FR1, FR2, FR3, FR4, and CH1, or sequences therefrom sufficient to mediate specific binding to its epitope and to complex with HCP2. KLCP2, together with its HCP2, provides specificity for a second epitope (LLCP1, together with its HCP1, provides specificity for a first epitope).

[0435] "Heavy chain polypeptide 1 (HCP1)", as the term is used herein, refers to a polypeptide that, when combined with its cognate LLCP1, contains a heavy chain (HC) sequence sufficient to mediate specific binding to its epitope and to complex with HCP1, e.g., an HC variable region sequence. In one embodiment, it includes all or a fragment of the CH1 region. In one embodiment, it includes all or a fragment of the CH2 and / or CH3 regions. In one embodiment, HCP1 includes HC-CDR1, HC-CDR2, HC-CDR3, FR1, FR2, FR3, FR4, CH1, CH2, and CH3, or (i) sequences therefrom sufficient to mediate specific binding to its epitope and to complex with LLCP1, (ii) to preferentially complex with LLCP1 as contrasted with KLCP2 as described herein, and (iii) to preferentially complex with HCP2 as contrasted with another molecule of HCP1 as described herein. HCP1, together with its LLCP1, provides specificity for a first epitope (KLCP2, together with its HCP2, provides specificity for a second epitope).

[0436] "Heavy chain polypeptide 2 (HCP2)", as the term is used herein, refers to a polypeptide comprising a heavy chain (HC) sequence, e.g., an HC variable region sequence, that is sufficient to mediate specific binding to its epitope and complex with HCP1 when combined with cognate LLCP1. In one embodiment, it comprises all or a fragment of the CH1 region. In one embodiment, it comprises all or a fragment of the CH2 and / or CH3 regions. In one embodiment, HCP1 comprises HC-CDR1, HC-CDR2, HC-CDR3, FR1, FR2, FR3, FR4, CH1, CH2, and CH3, or sequences therefrom sufficient to (i) mediate specific binding to its epitope, complex with KLCP2, (ii) preferentially complex with KLCP2 as contrasted with LLCP1 as described herein, and (iii) preferentially complex with HCP1 as contrasted with another molecule of HCP2 as described herein. HCP2, together with its KLCP2, provides specificity for a second epitope (LLCP1, together with its HCP1, provides specificity for a first epitope).

[0437] In some embodiments of the multispecific antibody molecules disclosed herein, LLCP1 has a higher affinity for HCP1 than for HCP2, and / or KLCP2 has a higher affinity for HCP2 than for HCP1.

[0438] In embodiments, the affinity of LLCP1 for HCP1 is sufficiently higher than its affinity for HCP2 such that, under preselected conditions, e.g., in an aqueous buffer at pH 7, e.g., in physiological saline at pH 7, or under physiological conditions, at least 75, 80, 90, 95, 98, 99, 99.5, or 99.9% of the multispecific antibody molecules have LLCP1 complexed or associated with HCP1.

[0439] In some embodiments of the multispecific antibody molecules disclosed herein, HCP1 has a higher affinity for HCP2 than for a second molecule of HCP1 and / or HCP2 has a higher affinity for HCP1 than for a second molecule of HCP2.

[0440] In embodiments, the affinity of HCP1 for HCP2 is sufficiently higher than its affinity for a second molecule of HCP1 such that, under preselected conditions, for example, in an aqueous buffer at pH 7, for example, in physiological saline at pH 7, or under physiological conditions, at least 75%, 80, 90, 95, 98, 99, 99.5 or 99.9% of the multispecific antibody molecules have HCP1 complexed with or bound to HCP2.

[0441] In another aspect, methods of making or producing multispecific antibody molecules are disclosed herein. The method comprises under conditions where (i)-(iv) associate, (i) providing a first heavy chain polypeptide (e.g., a heavy chain polypeptide comprising one, two, three or all of a first heavy chain variable region (first VH), first CH1, first heavy chain constant region (e.g., first CH2, first CH3, or both)), (ii) providing a second heavy chain polypeptide (e.g., a heavy chain polypeptide comprising one, two, three or all of a second heavy chain variable region (second VH), second CH1, second heavy chain constant region (e.g., second CH2, second CH3, or both)), (iii) providing a lambda chain polypeptide (e.g., a lambda light chain variable region (VLλ), lambda light chain constant chain (VLλ), or both) that preferentially associates with the first heavy chain polypeptide (e.g., first VH), and (iv) providing a kappa chain polypeptide (e.g., a lambda light chain variable region (VLκ), lambda light chain constant chain (VLκ), or both) that preferentially associates with the second heavy chain polypeptide (e.g., second VH) are included.

[0442] In embodiments, the first and second heavy chain polypeptides form an Fc boundary that enhances heterodimerization. In embodiments, (i)-(iv) (e.g., nucleic acids encoding (i)-(iv)) are introduced into a single cell, e.g., a single mammalian cell, e.g., a CHO cell. In embodiments, (i)-(iv) are expressed in the cell.

[0443] In embodiments, (i)-(iv) (e.g., nucleic acids encoding (i)-(iv)) are introduced into different cells, e.g., different mammalian cells, e.g., two or more CHO cells. In embodiments, (i)-(iv) are expressed in the cell.

[0444] In one embodiment, the method further comprises purifying the cell-expressed antibody molecule, e.g., using lambda and / or kappa specific purification, e.g., affinity chromatography.

[0445] In embodiments, the method further comprises evaluating the cell-expressed multispecific antibody molecule. For example, the purified cell-expressed multispecific antibody molecule can be analyzed by techniques known in the art, including mass spectrometry. In one embodiment, the purified cell-expressed antibody molecule is digested with a cleavage, e.g., papain, to yield Fab portions and evaluated using mass spectrometry.

[0446] In embodiments, the method produces correctly paired kappa / lambda multispecific, e.g., bispecific, antibody molecules in high yields, e.g., at least 75%, 80, 90, 95, 98, 99, 99.5 or 99.9%.

[0447] In other embodiments, the multispecific, e.g., bispecific, antibody molecule is (i) A first heavy chain polypeptide (HCP1) (for example, a heavy chain polypeptide comprising one, two, three, or all of a first heavy chain variable region (first VH), a first CH1, a first heavy chain constant region (for example, a first CH2, a first CH3, or both)), which binds to, for example, a first epitope, HCP1, (ii) A second heavy chain polypeptide (HCP2) (for example, a heavy chain polypeptide comprising one, two, three, or all of a second heavy chain variable region (second VH), a second CH1, a second heavy chain constant region (for example, a second CH2, a second CH3, or both)), which binds to, for example, a second epitope, HCP2, (iii) A lambda light chain polypeptide (LLCP1) (for example, a lambda light chain variable region (VLl), a lambda light chain constant chain (VLl), or both) that preferentially associates with a first heavy chain polypeptide (for example, a first VH), which binds to, for example, a first epitope, LLCP1, and (iv) A kappa light chain polypeptide (KLCP2) (for example, a lambda light chain variable region (VLk), a lambda light chain constant chain (VLk), or both) that preferentially associates with a second heavy chain polypeptide (for example, a second VH), which binds to, for example, a second epitope, KLCP2 comprising.

[0448] In embodiments, the first and second heavy chain polypeptides form an Fc boundary that enhances heterodimerization. In embodiments, the multispecific antibody molecule comprises a first binding specificity comprising a hybrid VLl-CLl heterodimerized to a first heavy chain variable region connected to an Fc constant, CH2-CH3 domain (having a knob modification), and a second binding specificity comprising a hybrid VLk-CLk heterodimerized to a second heavy chain variable region connected to an Fc constant, CH2-CH3 domain (having a hole modification).

[0449] TCR beta V antigen-binding domain The diversity of the immune system enables defense against a vast number of pathogens. Since the size of the germline genome is limited, diversity is achieved not only by the process of V(D)J recombination but also by deletions at the nucleotide junctions (junctions between V-D segments and D-J segments) and the addition of nucleotides that are pseudorandom and not templated. The TCR beta gene undergoes genetic rearrangement to generate diversity.

[0450] The TCR V beta repertoire varies between individuals and populations due to, for example, seven frequently occurring inactivating polymorphisms in functional gene segments and large insertion / deletion-related polymorphisms encompassing two V beta gene segments. The present disclosure provides, for example, antibody molecules and fragments thereof that bind, such as specifically bind, to, for example, human TCR beta V chains (TCRβV), such as TCRβV gene families (also called groups), such as TCRβV subfamilies (also called subgroups), as described herein. TCR beta V families and subfamilies are known in the art, for example, as described in Yassai et al. (2009) Immunogenetics 61(7):493-502; Wei S. and Concannon P. (1994) Human Immunology 41(3):201-206. The antibodies described herein can be recombinant antibodies, such as recombinant non-mouse antibodies, such as recombinant human or humanized antibodies.

[0451] In one aspect, the present disclosure provides anti-TCRβV antibody molecules that bind to human TCRβV, such as TCRβV families, such as gene families or variants thereof. In some embodiments, the TCRBV gene family includes one or more subfamilies, for example, as described herein, for example, as described in FIG. 3, Table 8A or 8B. In some embodiments, the TCRβV gene family includes the TCRβ V6 subfamily, the TCRβ V10 subfamily, the TCRβ V12 subfamily, the TCRβ V5 subfamily, the TCRβ V7 subfamily, the TCRβ V11 subfamily, the TCRβ V14 subfamily, the TCRβ V16 subfamily, the TCRβ V18 subfamily, the TCRβ V9 subfamily, the TCRβ V13 subfamily, the TCRβ V4 subfamily, the TCRβ V3 subfamily, the TCRβ V2 subfamily, the TCRβ V15 subfamily, the TCRβ V30 subfamily, the TCRβ V19 subfamily, the TCRβ V27 subfamily, the TCRβ V28 subfamily, the TCRβ V24 subfamily, the TCRβ V20 subfamily, the TCRβ V25 subfamily, the TCRβ V29 subfamily, the TCRβ V1 subfamily, the TCRβ V17 subfamily, the TCRβ V21 subfamily, the TCRβ V23 subfamily, or the TCRβ V26 subfamily.

[0452] In some embodiments, the TCRβ V6 subfamily is also known as TCRβ V13.1. In some embodiments, the TCRβ V6 subfamily is TCRβ V6-4 * 01, TCRβ V6-4 * 02, TCRβ V6-9 * 01, TCRβ V6-8 * 01, TCRβ V6-5 * 01, TCRβ V6-6 * 02, TCRβ V6-6 * 01, TCRβ V6-2 * 01, TCRβ V6-3 *01 or TCRβ V6-1 * 01, or variants thereof. In some embodiments, TCRβ V6 is TCRβ V6-4 * 01, or variants thereof. In some embodiments, TCRβ V6 is TCRβ V6-4 * 02, or variants thereof. In some embodiments, TCRβ V6 is TCRβ V6-9 * 01, or variants thereof. In some embodiments, TCRβ V6 is TCRβ V6-8 * 01, or variants thereof. In some embodiments, TCRβ V6 is TCRβ V6-5 * 01, or variants thereof. In some embodiments, TCRβ V6 is TCRβ V6-6 * 02, or variants thereof. In some embodiments, TCRβ V6 is TCRβ V6-6 * 01, or variants thereof. In some embodiments, TCRβ V6 is TCRβ V6-2 * 01, or variants thereof. In some embodiments, TCRβ V6 is TCRβ V6-3 * 01, or variants thereof. In some embodiments, TCRβ V6 is TCRβ V6-1 * 01, or variants thereof.

[0453] In some embodiments, TCRβ V6 is TCRβ V6-5 * 01, or variants thereof. In some embodiments, TCRβ V6, e.g., TCRβ V6-5 * 01 is recognized, e.g., bound, by SEQ ID NO: 1 and / or SEQ ID NO: 2. In some embodiments, TCRβ V6, e.g., TCRβ V6-5 * 01 is recognized, e.g., bound, by SEQ ID NO: 9 and / or SEQ ID NO: 10. In some embodiments, TCRβ V6 is recognized, e.g., bound, by SEQ ID NO: 9 and / or SEQ ID NO: 11.

[0454] In some embodiments, the TCRβ V10 subfamily is also known as TCRβ V12. In some embodiments, the TCRβ V10 subfamily is TCRβ V10-1 * 01, TCRβ V10-1 * 02, TCRβ V10-3 * 01 or TCRβ V10-2 * 01, or variants thereof.

[0455] In some embodiments, the TCRβ V12 subfamily is also known as TCRβ V8.1. In some embodiments, the TCRβ V12 subfamily is TCRβ V12-4 * 01, TCRβ V12-3 * 01, or TCRβ V12-5 * 01, or variants thereof. In some embodiments, TCRβ V12 is recognized, e.g., bound, by SEQ ID NO: 15 and / or SEQ ID NO: 16. In some embodiments, TCRβ V12 is recognized, e.g., bound, by any one of SEQ ID NOs: 23-25 and / or any one of SEQ ID NOs: 26-30.

[0456] In some embodiments, the TCRβ V5 subfamily is TCRβ V5-5 * 01, TCRβ V5-6 * 01, TCRβ V5-4 * 01, TCRβ V5-8 * 01, TCRβ V5-1 * 01, or selected from variants thereof.

[0457] In some embodiments, the TCRβ V7 subfamily is TCRβ V7-7 * 01, TCRβ V7-6 * 01, TCRβ V7-8 * 02, TCRβ V7-4 * 01, T CRβ V7-2 * 02, TCRβ V7-2 *03. TCRβ V7-2 * 01. TCRβ V7-3 * 01. TCRβ V7-9 * 03. or TCRβ V7-9 * 01. or includes variants thereof.

[0458] In some embodiments, the TCRβ V11 subfamily is TCRβ V11-1 * 01. TCRβ V11-2 * 01 or TCRβ V11-3 * 01. or includes variants thereof.

[0459] In some embodiments, the TCRβ V14 subfamily is TCRβ V14 * 01. or includes its variant. In some embodiments, the TCRβ V16 subfamily is TCRβ V16 * 01. or includes its variant.

[0460] In some embodiments, the TCRβ V18 subfamily is TCRβ V18 * 01. or includes its variant. In some embodiments, the TCRβ V9 subfamily is TCRβ V9 * 01 or TCRβ V9 * 02. or includes its variant.

[0461] In some embodiments, the TCRβ V13 subfamily is TCRβ V13 * 01. or includes its variant. In some embodiments, the TCRβ V4 subfamily is TCRβ V4-2 * 01. TCRβ V4-3 * 01. or TCRβ V4-1 * 01. or includes variants thereof.

[0462] In some embodiments, the TCRβ V3 subfamily is TCRβ V3-1 *01, or a variant thereof. In some embodiments, the TCRβ V2 subfamily is TCRβ V2 * 01, or a variant thereof.

[0463] In some embodiments, the TCRβ V15 subfamily is TCRβ V15 * 01, or a variant thereof. In some embodiments, the TCRβ V30 subfamily is TCRβ V30 * 01, or TCRβ V30 * 02, or a variant thereof.

[0464] In some embodiments, the TCRβ V19 subfamily is TCRβ V19 * 01, or TCRβ V19 * 02, or a variant thereof. In some embodiments, the TCRβ V27 subfamily is TCRβ V27 * 01, or a variant thereof.

[0465] In some embodiments, the TCRβ V28 subfamily is TCRβ V28 * 01, or a variant thereof. In some embodiments, the TCRβ V24 subfamily is TCRβ V24-1 * 01, or a variant thereof.

[0466] In some embodiments, the TCRβ V20 subfamily is TCRβ V20-1 * 01, or TCRβ V20-1 * 02, or a variant thereof. In some embodiments, the TCRβ V25 subfamily is TCRβ V25-1 * 01, or a variant thereof.

[0467] In some embodiments, the TCRβ V29 subfamily is TCRβ V29-1 *0 1, or a variant thereof.

[0468] [Table 2]

[0469] [Table 3]

[0470] Anti-TCRβV antibody Despite having low sequence similarity (e.g., low sequence identity among different antibody molecules that recognize different TCRβV subfamilies), a novel class of antibodies is disclosed herein that recognize structurally conserved regions, such as domains, on the TCRβV protein and have similar functions (e.g., similar cytokine profiles), i.e., the discovery of anti-TCRβV antibody molecules disclosed herein. Thus, the anti-TCRβV antibody molecules disclosed herein share structure-function relationships.

[0471] In some embodiments, the anti-TCRβV antibody molecules disclosed herein do not recognize, e.g., do not bind to, the interface of the TCRβV:TCR alpha complex. In some embodiments, the anti-TCRβV antibody molecules disclosed herein do not recognize, e.g., do not bind to, the constant region of the TCRβV protein. An exemplary antibody that binds to the constant region of the TCRBV region is JOVI.1 described by Viney et al. (Hybridoma. 1992 Dec;11(6):701-13).

[0472] In some embodiments, the anti-TCRβV antibody molecules disclosed herein do not recognize, e.g., do not bind to, one or more (e.g., all) of the complementarity determining regions (e.g., CDR1, CDR2, and / or CDR3) of the TCRβV protein.

[0473] In some embodiments, the anti-TCRβV antibody molecules disclosed herein bind to (e.g., specifically bind to) the TCRβV region. In some embodiments, the binding of the anti-TCRβV antibody molecules disclosed herein results in a cytokine profile that is different from the cytokine profile of a T cell engager (a "non-TCRβV-binding T cell engager") that binds to a receptor or molecule other than the TCRβV region. In some embodiments, the non-TCRβV-binding T cell engager comprises an antibody that binds to a CD3 molecule (e.g., a CD3 epsilon (CD3e) molecule), or a TCR alpha (TCRα) molecule. In some embodiments, the non-TCRβV-binding T cell engager is an OKT3 antibody or an SP34-2 antibody.

[0474] In one aspect, the disclosure provides anti-TCRβV antibody molecules that bind to one or more of the human TCRβVs, e.g., TCRβV gene families, e.g., TCRβV subfamilies, as described herein, e.g., in FIG. 3, Table 8A, or Table 8B. In some embodiments, the anti-TCRβV antibody molecule is a TCRβ V6 subfamily, TCRβ V10 subfamily, TCRβ V12 subfamily, TCRβ V5 subfamily, TCRβ V7 subfamily, TCRβ V11 subfamily, TCRβ V14 subfamily, TCRβ V16 subfamily, TCRβ V18 subfamily, TCRβ V9 subfamily, TCRβ V13 subfamily, TCRβ V4 subfamily, TCRβ V3 subfamily, TCRβ V2 subfamily, T Binds to one or more TCRβV subfamilies selected from the CRβ V15 subfamily, TCRβ V30 subfamily, TCRβ V19 subfamily, TCRβ V27 subfamily, TCRβ V28 subfamily, TCRβ V24 subfamily, TCRβ V20 subfamily, TCRβ V25 subfamily, TCRβ V29 subfamily, TCRβ V1 subfamily, TCRβ V17 subfamily, TCRβ V21 subfamily, TCRβ V23 subfamily, or TCRβ V26 subfamily, or variants thereof.

[0475] In some embodiments, the anti-TCRβV antibody molecule binds to the TCRβ V6 subfamily comprising TCRβ V6-4*01, TCRβ V6-4*02, TCRβ V6-9*01, TCRβ V6-8*01, TCRβ V6-5*01, TCRβ V6-6*02, TCRβ V6-6*01, TCRβ V6-2*01, TCRβ V6-3*01 or TCRβ V6-1*01, or variants thereof. In some embodiments, the TCRβ V6 subfamily comprises TCRβ V6-5*01, or variants thereof. In some embodiments, TCRβ V6 comprises TCRβ V6-4*01, or variants thereof. In some embodiments, TCRβ V6 comprises TCRβ V6-4*02, or variants thereof. In some embodiments, TCRβ V6 comprises TCRβ V6-9*01, or variants thereof. In some embodiments, TCRβ V6 comprises TCRβ V6-8*01, or variants thereof. In some embodiments, TCRβ V6 includes TCRβ V6-5*01, or a variant thereof. In some embodiments, TCRβ V6 includes TCRβ V6-6*02, or a variant thereof. In some embodiments, TCRβ V6 includes TCRβ V6-6*01, or a variant thereof. In some embodiments, TCRβ V6 includes TCRβ V6-2*01, or a variant thereof. In some embodiments, TCRβ V6 includes TCRβ V6-3*01, or a variant thereof. In some embodiments, TCRβ V6 includes TCRβ V6-1*01, or a variant thereof.

[0476] In some embodiments, the anti-TCRβV antibody molecule binds to the TCRβ V10 subfamily that includes TCRβ V10-1*01, TCRβ V10-1*02, TCRβ V10-3*01 or TCRβ V10-2*01, or a variant thereof.

[0477] In some embodiments, the anti-TCRβV antibody molecule binds to the TCRβ V12 subfamily that includes TCRβ V12-4*01, TCRβ V12-3*01 or TCRβ V12-5*01, or a variant thereof.

[0478] In some embodiments, the anti-TCRβV antibody molecule binds to the TCRβ V5 subfamily that includes TCRβ V5-5*01, TCRβ V5-6*01, TCRβ V5-4*01, TCRβ V5-8*01, TCRβ V5-1*01, or a variant thereof.

[0479] In some embodiments, the anti-TCRβV antibody molecule does not bind to TCRβ V12, or binds to TCRβ V12 with an affinity and / or binding specificity that is lower (e.g., about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or about 2, 5, or 10 times lower) than the affinity and / or binding specificity of the 16G8 mouse antibody or its humanized version described in U.S. Patent No. 5,861,155.

[0480] In some embodiments, the anti-TCRβV antibody molecule has a higher affinity and / or binding specificity (e.g., about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or about 2, 5, or 10 times higher) than the affinity and / or binding specificity of the 16G8 mouse antibody or its humanized version described in U.S. Patent No. 5,861,155. and binds to TCRβ V12.

[0481] In some embodiments, the anti-TCRβV antibody molecule has a higher affinity and / or binding specificity (e.g., about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or about 2, 5, or 10 times higher) than the affinity and / or binding specificity of the 16G8 mouse antibody or its humanized version described in U.S. Patent No. 5,861,155 and binds to a TCRβV region other than TCRβ V12 (e.g., a TCRβV region described herein, e.g., the TCRβ V6 subfamily (e.g., TCRβ V6-5*01)).

[0482] In some embodiments, the anti-TCRβV antibody molecule does not bind to either TCRβ V5-5*01 or TCRβ V5-1*01, or has a lower affinity and / or binding specificity (e.g., about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or about 2, 5, or 10 times lower) than the affinity and / or binding specificity of mouse antibody C or its humanized version described in U.S. Patent No. 5,861,155 and binds to TCRβ V5-5*01 or TCRβ V5-1*01.

[0483] In some embodiments, the anti-TCRβV antibody molecule has a higher affinity and / or binding specificity (e.g., about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or about 2, 5, or 10 times higher) than the affinity and / or binding specificity of mouse antibody C or its humanized version described in U.S. Patent No. 5,861,155 and binds to TCRβ V5-5*01 or TCRβ V5-1*01.

[0484] In some embodiments, the anti-TCRβV antibody molecule binds to a TCRβV region other than TCRβ V5-5*01 or TCRβ V5-1*01 (e.g., a TCRβV region described herein, e.g., the TCRβ V6 subfamily (e.g., TCRβ V6-5*01)) with an affinity and / or binding specificity that is higher (e.g., about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or about 2, 5, or 10 times higher) than the affinity and / or binding specificity of the murine antibody C or its humanized version described in U.S. Patent No. 5,861,155.

[0485] Anti-TCRβ V6 antibody Thus, in one aspect, the present disclosure provides an anti-TCRβV antibody molecule that binds to the TCRβ V6 subfamily, including human TCRβ V6, e.g., TCRβ V6-4*01, TCRβ V6-4*02, TCRβ V6-9*01, TCRβ V6-8*01, TCRβ V6-5*01, TCRβ V6-6*02, TCRβ V6-6*01, TCRβ V6-2*01, TCRβ V6-3*01 or TCRβ V6-1*01. In some embodiments, the TCRβ V6 subfamily includes TCRβ V6-5*01 or a variant thereof. In some embodiments, TCRβ V6 includes TCRβ V6-4*01, or a variant thereof. In some embodiments, TCRβ V6 is TCRβ comprises V6-4*02, or a variant thereof. In some embodiments, TCRβ V6 comprises TCRβ V6-9*01, or a variant thereof. In some embodiments, TCRβ V6 comprises TCRβ V6-8*01, or a variant thereof. In some embodiments, TCRβ V6 comprises TCRβ V6-5*01, or a variant thereof. In some embodiments, TCRβ V6 comprises TCRβ V6-6*02, or a variant thereof. In some embodiments, TCRβ V6 comprises TCRβ V6-6*01, or a variant thereof. In some embodiments, TCRβ V6 comprises TCRβ V6-2*01, or a variant thereof. In some embodiments, TCRβ V6 comprises TCRβ V6-3*01, or a variant thereof. In some embodiments, TCRβ V6 comprises TCRβ V6-1*01, or a variant thereof.

[0486] In some embodiments, TCRβ V6-5*01 is encoded by the nucleic acid sequence of SEQ ID NO: 43, or a sequence having 85%, 90%, 95%, 99% or higher identity thereto.

[0487] SEQ ID NO: 43 ATGAGCATCGGCCTCCTGTGCTGTGCAGCCTTGTCTCTCCTGTGGGCAGGTCCAGTGAATGCTGGTGTCACTCAGACCCCAAAATTCCAGGTCCTGAAGACAGGACAGAGCATGACACTGCAGTGTGCCCAGGATATGAACCATGAATACATGTCCTGGTATCGACAAGACCCAGGCATGGGGCTGAGGCTGATTCATTACTCAGTTGGTGCTGGTATCACTGACCAAGGAGAAGTCCCCAATGGCTACAATGTCTCCAGATCAACCACAGAGGATTTCCCGCTCAGGCTGCTGTCGGCTGCTCCCTCCCAGACATCTGTGTACTTCTGTGCCAGCAGTTACTC In some embodiments, TCRβ V6-5*01 comprises the amino acid sequence of SEQ ID NO: 44, or an amino acid sequence having 85%, 90%, 95%, 99% or higher identity thereto.

[0488] SEQ ID NO: 44 MSIGLLCCAALSLLWAGPVNAGVTQTPKFQVLKTGQSMTLQCAQDMNHEYMSWYRQDPGMGLRLIHYSVGAGITDQGEVPNGYNVSRSTTEDFPLRLLSAAPSQTSVYFCASSY In some embodiments, an anti-TCRβV antibody molecule, e.g., an anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule, is a non-mouse antibody molecule, e.g., a human or humanized antibody molecule. In some embodiments, an anti-TCRβV antibody molecule, e.g., an anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule, is a human antibody molecule. In some embodiments, an anti-TCRβV antibody molecule, e.g., an anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule, is a humanized antibody molecule.

[0489] In some embodiments, an anti-TCRβV antibody molecule, e.g., an anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule, is isolated or recombinant. In some embodiments, an anti-TCRβV antibody molecule, e.g., an anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule, comprises at least one antigen-binding region, e.g., a variable region or an antigen-binding fragment thereof, from an antibody selected from any one of the antibodies described herein, e.g., any one of A-H.1 to A-H.68, e.g., A-H.1, A-H.2 or A-H.68, or described in Table 1A, or encoded by a nucleotide sequence in Table 1A, or a sequence substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical) to any of the foregoing sequences.

[0490] In some embodiments, an anti-TCRβV antibody molecule, such as an anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule, comprises at least one, two, three, or four variable regions from an antibody selected from any one of the antibodies described herein, such as any one of A-H.1 to A-H.68, such as A-H.1, A-H.2, or A-H.68, or described in Table 1A, or a nucleotide sequence in Table 1A, or an antibody encoded by a sequence that is substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical) to any of the foregoing sequences.

[0491] In some embodiments, an anti-TCRβV antibody molecule, such as an anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule, comprises at least one or two heavy chain variable regions from an antibody molecule selected from any one of the antibodies described herein, such as any one of A-H.1 to A-H.68, such as A-H.1, A-H.2, or A-H.68, or described in Table 1A, or a nucleotide sequence in Table 1A, or an antibody encoded by a sequence that is substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical) to any of the foregoing sequences.

[0492] In some embodiments, the anti-TCRβV antibody molecule comprises a heavy chain variable region (VH) having the consensus sequence of SEQ ID NO: 231 or 3290. In some embodiments, an anti-TCRβV antibody molecule, such as an anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule, comprises at least one or two light chain variable regions from an antibody selected from any one of the antibodies described herein, such as any one of A-H.1 to A-H.68, such as A-H.1, A-H.2 or A-H.68, or as described in Table 1A, or a nucleotide sequence in Table 1A, or an antibody encoded by a sequence that is substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical) to any of the foregoing sequences.

[0493] In some embodiments, the anti-TCRβV antibody molecule comprises a light chain variable region (VL) having the consensus sequence of SEQ ID NO: 230 or 3289. In some embodiments, an anti-TCRβV antibody molecule, such as an anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule, comprises a heavy chain constant region of IgG4, such as human IgG4. In yet another embodiment, an anti-TCRβV antibody molecule, such as an anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule, comprises a heavy chain constant region of IgG1, such as human IgG1. In one embodiment, the heavy chain constant region comprises the amino acid sequence shown in Table 3A, or a sequence that is substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical) thereto.

[0494] In some embodiments, an anti-TCRβV antibody molecule, such as an anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule, comprises a kappa light chain constant region, such as human kappa light chain constant region. In one embodiment, the light chain constant region comprises the amino acid sequence shown in Table 3A, or a sequence that is substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical) thereto.

[0495] In some embodiments, an anti-TCRβV antibody molecule, such as an anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule, comprises at least one, two, or three complementarity determining regions (CDRs) from the heavy chain variable region (VH) of an antibody selected from any one of the antibodies described herein, such as any one of A-H.1 to A-H.68, such as A-H.1, A-H.2 or A-H.68, or described in Table 1A, or a nucleotide sequence in Table 1A, or a sequence that is substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical) to any of the foregoing sequences.

[0496] In some embodiments, an anti-TCRβV antibody molecule, such as an anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule, comprises at least one, two, or three CDRs (or all of the CDRs together) from the heavy chain variable region comprising the amino acid sequence shown in Table 1A or encoded by the nucleotide sequence shown in Table 1A. In one embodiment, one or more of the CDRs (or all of the CDRs together) have one, two, three, four, five, six or more changes, such as amino acid substitutions or deletions, compared to the amino acid sequence shown in Table 1A or encoded by the nucleotide sequence shown in Table 1A. shown or have one, two, three, four, five, six or more changes, such as amino acid substitutions or deletions, compared to the amino acid sequence shown in Table 1A or encoded by the nucleotide sequence shown in Table 1A.

[0497] In some embodiments, an anti-TCRβV antibody molecule, such as an anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule, comprises at least one, two, or three complementarity determining regions (CDRs) from the light chain variable region of an antibody selected from any one of the antibodies described herein, such as any one of A-H.1 to A-H.68, such as A-H.1, A-H.2 or A-H.68, or described in Table 1A, or a nucleotide sequence in Table 1A, or a sequence that is substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical) to any of the foregoing sequences.

[0498] In some embodiments, an anti-TCRβV antibody molecule, such as an anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule, comprises at least one, two, or three CDRs (or all of the CDRs together) from a light chain variable region comprising the amino acid sequence shown in Table 1A or encoded by the nucleotide sequence shown in Table 1A. In one embodiment, one or more of the CDRs (or all of the CDRs together) have one, two, three, four, five, six, or more changes, such as amino acid substitutions or deletions, compared to the amino acid sequence shown in Table 1A or encoded by the nucleotide sequence shown in Table 1A.

[0499] In some embodiments, an anti-TCRβV antibody molecule, such as an anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule, comprises at least one, two, three, four, five, or six CDRs (or all of the CDRs together) from the heavy and light chain variable regions comprising the amino acid sequence shown in Table 1A or encoded by the nucleotide sequence shown in Table 1A. In one embodiment, one or more of the CDRs (or all of the CDRs together) have one, two, three, four, five, six, or more changes, such as amino acid substitutions or deletions, compared to the amino acid sequence shown in Table 1A or encoded by the nucleotide sequence shown in Table 1A.

[0500] In some embodiments, an anti-TCRβV antibody molecule, such as an anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule, comprises an antibody described herein, such as an antibody selected from any one of A-H.1 to A-H.68, such as A-H.1, A-H.2 or A-H.68, or an antibody described in Table 1A or all six CDRs from an antibody encoded by the nucleotide sequence in Table 1A, or CDRs that are closely related, such as CDRs that are identical or have at most 2, 3 or 4 amino acid changes (e.g., substitutions, deletions or insertions, such as conservative substitutions). In some embodiments, an anti-TCRβV antibody molecule, such as an anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule, may comprise any CDR described herein.

[0501] In some embodiments, an anti-TCRβV antibody molecule, such as an anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule, comprises an antibody described herein, such as an antibody selected from any one of A-H.1 to A-H.68, such as A-H.1, A-H.2 or A-H.68, or at least 1, 2, or 3 CDRs according to Kabat et al. from the heavy chain variable region of the antibody described in Table 1A (e.g., at least 1, 2, or 3 CDRs according to the definition of Kabat shown in Table 1A), or sequences that are substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical) to any of the aforementioned sequences, or compared to 1, 2, or 3 CDRs according to Kabat et al. shown in Table 1A, sequences having at least 1 amino acid change but at most 2, 3 or 4 changes (e.g., substitutions, deletions or insertions, such as conservative substitutions). Included are sequences having at least 1 amino acid change but at most 2, 3 or 4 changes (e.g., substitutions, deletions or insertions, such as conservative substitutions).

[0502] In some embodiments, an anti-TCRβV antibody molecule, e.g., an anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule, is an antibody selected from any one of the antibodies described herein, e.g., any one of A-H.1 to A-H.68, e.g., A-H.1, A-H.2 or A-H.68, or at least one, two, or three CDRs (e.g., at least one, two, or three CDRs according to the Kabat definition shown in Table 1A) from the light chain variable region of the antibodies described in Table 1A, or is substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical) to any of the foregoing sequences, or has at least one amino acid change compared to one, two, or three CDRs by Kabat et al. shown in Table 1A, but includes a sequence having two, three, or four or fewer changes (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions).

[0503] In some embodiments, an anti-TCRβV antibody molecule, e.g., an anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule, is an antibody selected from any one of the antibodies described herein, e.g., any one of A-H.1 to A-H.68, e.g., A-H.1, A-H.2 or A-H.68, or at least one, two, three, four, five, or six CDRs (e.g., at least one, two, three, four, five, or six CDRs according to the Kabat definition shown in Table 1A) from the heavy and light chain variable regions of the antibodies described in or encoded by the nucleotide sequences in Table 1A, or is substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical) to any of the foregoing sequences, or has at least one amino acid change compared to one, two, three, four, five, or six CDRs by Kabat et al. shown in Table 1A, but includes a sequence having two, three, or four or fewer changes (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions).

[0504] In some embodiments, an anti-TCRβV antibody molecule, such as an anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule, is an antibody selected from any one of the antibodies described herein, such as any one of A-H.1 to A-H.68, such as A-H.1, A-H.2 or A-H.68, or as described in Table 1A, or all six CDRs according to Kabat et al. from the heavy and light chain variable regions of the antibody encoded by the nucleotide sequence in Table 1A (e.g., all six CDRs according to the definition of Kabat shown in Table 1A), or is substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical) to any of the foregoing sequences, or has at least one amino acid change compared to all six CDRs according to Kabat et al. shown in Table 1A, but has two, three or four or fewer changes (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions). In one embodiment, an anti-TCRβV antibody molecule, such as an anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule, may comprise any CDR described herein.

[0505] In some embodiments, an anti-TCRβV antibody molecule, such as an anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule, has at least one, two, or three hypervariable loops with the same canonical structure as the corresponding hypervariable loops of the antibodies described herein, such as any one of A-H.1 to A-H.68, such as A-H.1, A-H.2 or A-H.68, e.g., the same canonical structure as at least loop 1 and / or loop 2 of the heavy and / or light chain variable domains of the antibodies described herein. For example, see Chothia et al., (1992) J. Mol. Biol. 227:799-817; Tomlinson et al., (1992) J. Mol. Biol. 227:776-798 for descriptions of the canonical structures of hypervariable loops. These structures can be determined by examination of the tables described in these references.

[0506] In some embodiments, an anti-TCRβV antibody molecule, e.g., an anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule, is selected from any one of the antibodies described herein, e.g., any one of A-H.1 to A-H.68, e.g., A-H.1, A-H.2 or A-H.68, or at least one, two, or three CDRs (e.g., at least one, two, or three CDRs according to the definition of Chothia shown in Table 1A) from the heavy chain variable region of the antibodies described in Table 1A, or is substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical) to any of the foregoing sequences, or is at least one amino acid change compared to one, two, or three CDRs by Chothia et al. shown in Table 1A, but has two, three, or four or fewer changes (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions).

[0507] In some embodiments, an anti-TCRβV antibody molecule, e.g., an anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule, is an antibody selected from any one of the antibodies described herein, e.g., any one of A-H.1 to A-H.68, e.g., A-H.1, A-H.2 or A-H.68, or at least one, two, or three CDRs (e.g., at least one, two, or three CDRs according to the definition of Chothia shown in Table 1A) from the light chain variable region of the antibodies described in Table 1A, or is substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical) to any of the foregoing sequences, or is at least one amino acid change compared to one, two, or three CDRs by Chothia et al. shown in Table 1A, but has two, three, or four or fewer changes (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions).

[0508] In some embodiments, an anti-TCRβV antibody molecule, such as an anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule, is an antibody selected from any one of the antibodies described herein, such as any one of A-H.1 to A-H.68, e.g., A-H.1, A-H.2 or A-H.68, or as described in Table 1A, or at least 1, 2, 3, 4, 5, or 6 CDRs (e.g., at least 1, 2, 3, 4, 5, or 6 CDRs according to the definition of Chothia) from the heavy and light chain variable regions of an antibody encoded by the nucleotide sequence in Table 1A, or is substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical) to any of the foregoing sequences, or has at least one amino acid change compared to 1, 2, 3, 4, 5, or 6 CDRs by Chothia as shown in Table 1A, but has 2, 3 or 4 or fewer changes (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions).

[0509] In some embodiments, an anti-TCRβV antibody molecule, such as an anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule, is an antibody selected from any one of the antibodies described herein, such as any one of A-H.1 to A-H.68, e.g., A-H.1, A-H.2 or A-H.68, or as described in Table 1A, or all 6 CDRs by Chothia from the heavy and light chain variable regions of an antibody encoded by the nucleotide sequence in Table 1A (e.g., 6 according to the definition of Chothia shown in Table 1A) All CDRs), or are substantially identical to any of the foregoing sequences (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical), or compared to all six CDRs by Chothia et al. shown in Table 1A, are at least one amino acid change, but contain sequences having two, three or four or fewer changes (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions). In one embodiment, an anti-TCRβV antibody molecule, e.g., an anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule, may contain any CDR described herein.

[0510] In some embodiments, an anti-TCRβV antibody molecule, e.g., an anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule, contains CDRs or a combination of hypervariable loops defined according to Kabat et al., Chothia et al., or described in Table 1A.

[0511] In some embodiments, an anti-TCRβV antibody molecule, e.g., an anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule, may contain any combination of CDRs or hypervariable loops according to the definitions of Kabat and Chothia.

[0512] In some embodiments, the combined CDRs shown in Table 1A are CDRs that include Kabat CDRs and Chothia CDRs. In some embodiments, an anti-TCRβV antibody molecule, e.g., an anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule, contains a combination of CDRs or hypervariable loops identified as combined CDRs in Table 1A. In some embodiments, an anti-TCRβV antibody molecule, e.g., an anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule, may contain any combination of CDRs or hypervariable loops by the "combined" CDRs described in Table 1A.

[0513] In one embodiment, for example, in one embodiment including a variable region, a CDR (e.g., a combinatorial CDR, a Chothia CDR, or a Kabat CDR), or other sequences mentioned herein, for example, in Table 1A, the antibody molecule is a monospecific antibody molecule, a bispecific antibody molecule, a bivalent antibody molecule, a dual paratopic antibody molecule, or an antibody molecule comprising an antigen-binding fragment of an antibody, e.g., a half-antibody or an antigen-binding fragment of a half-antibody. In certain embodiments, the antibody molecule comprises a multispecific molecule, e.g., a bispecific molecule, as described herein.

[0514] In one embodiment, an anti-TCRβV antibody molecule, e.g., an anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule, (i) one, two, or all of the light chain complementarity-determining region 1 (LC CDR1), light chain complementarity-determining region 2 (LC CDR2), and light chain complementarity-determining region 3 (LC CDR3) of SEQ ID NO: 2, SEQ ID NO: 10, or SEQ ID NO: 11, and / or (ii) one, two, or all of the heavy chain complementarity-determining region 1 (HC CDR1), heavy chain complementarity-determining region 2 (HC CDR2), and heavy chain complementarity-determining region 3 (HC CDR3) of SEQ ID NO: 1 or SEQ ID NO: 9 is included.

[0515] In some embodiments, an anti-TCRβV antibody molecule, e.g., an anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule, comprises LC CDR1, LC CDR2, and LC CDR3 of SEQ ID NO: 2, and HC CDR1, HC...

Claims

1. (i) A first antigen-binding domain that binds to, for example, selectively binds to a T cell receptor variable beta (TCRBV), such as a TCRBV antigen, and (ii) (a) An immunocyte engager selected from an NK cell engager, a T cell engager, a B cell engager, a dendritic cell engager, or a macrophage cell engager; (b) A cytokine molecule or a cytokine inhibitor molecule; and (c) A cell death receptor signal engager One, two, or all of A multifunctional molecule comprising.

2. (i) A first antigen-binding domain that binds to, for example, selectively binds to a T cell receptor variable beta (TCRBV), such as a TCRBV antigen, and (ii) An NK cell engager, such as an anti-NKp30, anti-NKp46, anti-NKG2D, or anti-CD16 antibody molecule A multifunctional molecule comprising.

3. The multifunctional molecule according to claim 2, wherein the NK cell engager comprises an anti-NKp30 antibody molecule.

4. The multifunctional molecule according to claim 2, wherein the NK cell engager comprises an anti-NKp46 antibody molecule.

5. (i) A first antigen-binding domain that binds to, for example, selectively binds to a T cell receptor variable beta (TCRBV), such as a TCRBV antigen, and (ii) A cell death receptor signal engager A multifunctional molecule comprising.

6. (i) A first antigen-binding domain that binds to, for example, selectively binds to a T cell receptor variable beta (TCRBV), such as a TCRBV antigen, and (ii) A cytokine inhibitor molecule A multifunctional molecule comprising.

7. A nucleic acid molecule encoding the multifunctional molecule according to any one of claims 1 to 6.

8. A vector, such as an expression vector, comprising the nucleic acid molecule according to claim 7.

9. A host cell comprising the nucleic acid molecule according to claim 7 or the vector according to claim 8.

10. A method for producing, for example, generating the multifunctional molecule or antibody molecule according to any one of claims 1 to 6, the method comprising culturing the host cell according to claim 9 under appropriate conditions, such as conditions suitable for gene expression and / or homoor heterodimerization.

11. A pharmaceutical composition comprising the multifunctional molecule according to any one of claims 1 to 6 and a pharmaceutically acceptable carrier, excipient, or stabilizer.

12. A method of treating TCR bias, comprising administering to a subject in need thereof a multifunctional molecule according to any one of claims 1 to 6, wherein the multifunctional molecule is administered in an amount effective to treat TCR bias. A method. **Claim 13** A method of treating an autoimmune disease (e.g., an autoimmune disease associated with TCR bias), comprising administering to a subject in need thereof a multifunctional molecule according to any one of claims 1 to 6, wherein the multifunctional molecule is administered in an amount effective to treat the autoimmune disease. **Claim 14** A method of treating TCR bias, comprising, in response to determining that the subject has TCR bias, administering to a subject in need thereof a multifunctional molecule according to any one of claims 1 to 6 wherein the multifunctional molecule is administered in an amount effective to treat TCR bias. A method. **Claim 15** A method of treating an autoimmune disease (e.g., an autoimmune disease associated with TCR bias), comprising, in response to determining that the subject has an autoimmune disease (e.g., an autoimmune disease associated with TCR bias), administering to a subject in need thereof a multifunctional molecule according to any one of claims 1 to 6 wherein the multifunctional molecule is administered in an amount effective to treat the autoimmune disease (e.g., an autoimmune disease associated with TCR bias). A method. **Claim 16** A method of identifying a subject in need of treatment for cancer using a multifunctional molecule according to any one of claims 1 to 6, comprising determining whether the subject has TCR bias (e.g., a biased TCRBV clone type) and / or an autoimmune disease associated with said bias (e.g., directly or indirectly determining it, e.g., obtaining information about it), and in response to determining that the subject has TCR bias (e.g., a biased TCRBV clone type) and / or an autoimmune disease associated with said bias, identifying the subject as a candidate for treatment using a multifunctional molecule comprising an antigen-binding domain that binds to a TCRBV antigen, and optionally not identifying the subject as a candidate for treatment using a multifunctional molecule comprising an antigen-binding domain that does not bind to the TCRBV antigen (e.g., binds to a different TCRBV antigen). A method. **Claim 17** A method for evaluating a subject in need of treatment for a TCR bias (e.g., a biased TCRBV clone type) and / or an autoimmune disease associated with said bias, the method comprising determining whether the subject has a TCR bias (e.g., directly determining it or indirectly determining it, e.g., obtaining information regarding it).

18. A method of treating an autoimmune disease in a subject in need of treatment for an autoimmune disease (e.g., an autoimmune disease associated with TCR bias), the method comprising administering to the subject an effective amount, e.g., a therapeutically effective amount, of an antibody molecule that binds (e.g., specifically binds) to the T cell receptor beta variable region (TCRβV) (“anti-TCRβV antibody molecule”), thereby treating the disorder.

19. A method of depleting a T cell population in a subject having an autoimmune disease (e.g., an autoimmune disease associated with TCR bias), the method comprising contacting the T cell population with an effective amount of an antibody molecule that binds (e.g., specifically binds) to the T cell receptor beta variable region (TCRβV) (“anti-TCRβV antibody molecule”).

20. The method according to claim 19, wherein the contacting step occurs in vivo or in vitro. described method.

21. The anti-TCRβV antibody molecule is (i) not the antibody molecule disclosed in U.S. Patent No. 5,861,155; (ii) binds to TCRβV12 with an affinity and / or binding specificity less than (e.g., about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or less than about 2, 5, or one tenth of) the affinity and / or binding specificity of the 16G8 mouse antibody or its humanized version, as described in U.S. Patent No. 5,861,155; (iii) binds to TCRβV12 with an affinity and / or binding specificity greater than (e.g., about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or greater than about 2, 5, 10 times) the affinity and / or binding specificity of the 16G8 mouse antibody or its humanized version, as described in U.S. Patent No. 5,861,155; (iii) having an affinity and / or binding specificity greater than (e.g., about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or greater than about 2, 5, 10-fold) the affinity and / or binding specificity of the TM23 mouse antibody or its humanized version, and binding to TCRβ V5-5 ＊ 01 or TCRβ V5-1 ＊ 01; or (iv) As described in U.S. Patent No. 5,861,155, with an affinity and / or binding specificity greater than that of the TM23 mouse antibody or its humanized version (e.g., about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more than about 2, 5, 10-fold), the method according to any one of claims 18 to 20, which binds to TCRβ V5-5 ＊ 01 or TCRβ V5-1 ＊ 01.

22. The method according to any one of claims 18 to 21, wherein the anti-TCRβV antibody molecule comprises an Fc region, for example, an Fc region having effector functions, for example, an Fc region having antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell phagocytosis (ADCP) and / or complement-dependent cytotoxicity (CDC).

23. The method according to claim 22, wherein the anti-TCRβV antibody molecule comprises an Fc region having enhanced effector function as compared to, for example, a wild-type Fc region.

24. The method according to any one of claims 18 to 23, wherein the anti-TCRβV antibody molecule comprises a human IgG1 region or a human IgG4 region.

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