Multifunctional molecules that bind to T cells and their use for treating autoimmune disorders

Abstract

and (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 or a cytokine inhibitor molecule; and / or (iv) a death receptor signal enhancer. Further disclosed are nucleic acids encoding same, methods of producing said molecules, and methods of treating autoimmune diseases using said molecules.

Description

[Technical Field]

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

[0002] T cell-mediated antigen recognition depends on the interaction between the T cell receptor (TCR) and the antigen-receptor major histocompatibility complex (MHC). The heterodimeric TCR is composed of an α-chain and a β-chain combination (αβTCR), which is expressed by the majority of T cells, whereas the γδ-chain (γδTCR) is present in only approximately 1–5% of T cells. A highly diverse TCR repertoire is a fundamental characteristic 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 result from autoreactive TCR clones attacking the patient's own cells. Improved therapies for autoimmune diseases are needed. Summary of the Invention [Means for solving the problem]

[0004] The present disclosure particularly relates to novel multispecific or multifunctional molecules that include one, two, or all of: (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) a stromal-modifying moiety. The terms "multispecific" and "multifunctional" are used interchangeably herein.

[0005] Without wishing to be bound by theory, TCR bias may exist in autoimmune diseases. This bias may be associated with a dominant autoreactive TCR clone that causes the disease or is associated with symptoms. For example, rebalancing the TCR repertoire by removing or depleting T cells containing 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 cells (e.g., immune effector cells selected from NK cells, T cells, B cells, dendritic cells, or macrophages) in target cells (e.g., T cells containing a biased TCRBV clonotype or containing a TCRBV antigen corresponding to the biased TCRBV clonotype). Increasing the proximity and / or activity of immune cells using the multispecific molecules described herein is expected to enhance the immune response against target cells (e.g., TCRBV, e.g., T cells comprising a TCRBV antigen (e.g., a TCRBV antigen corresponding to a biased TCRBV clonotype) and thereby provide a more effective therapy (e.g., a more effective autoimmune disease therapy). Without being bound by theory, it is believed that a targeted local immune response against target cells (e.g., T cells comprising a biased TCRBV clonotype, e.g., T cells that do not comprise a biased TCRBV clonotype) reduces the systemic toxic effects of the multispecific molecules described herein. A targeted immune response against autoreactive T cell populations that targets non-autoreactive T cells to a lesser extent (e.g., does not target non-autoreactive T cells) is believed to have fewer adverse effects than systemic ablation of all T cells.

[0006] Thus, provided herein, inter alia, are multispecific molecules (e.g., multispecific or multifunctional antibody molecules) comprising the aforementioned portions, nucleic acids encoding the molecules, methods of producing the aforementioned molecules, and methods of treating autoimmune diseases using the aforementioned molecules. Also provided herein are anti-TCRβV antibody molecules, nucleic acids encoding the molecules, methods of producing the aforementioned molecules, and methods of treating autoimmune diseases using the anti-TCRβV antibody molecules.

[0007] Additionally provided are methods for depleting (e.g., in vivo depletion) of biased TCRBV clonotypes in the context of autoimmune disease, e.g., using multispecific molecules or anti-TCRβV antibody molecules. In some embodiments, the methods involve identifying a clonal bias in TCRBV usage in a patient, e.g., associated with an autoreactive subpopulation, and, in response to this analysis, administering a multifunctional molecule targeted to a TCRBV antigen corresponding to the biased TCRBV clonotype to reduce, e.g., eliminate, the clonal bias and promote, e.g., establish, normal TCRBV distribution.

[0008] Thus, in one aspect, the present disclosure provides a method for manufacturing a semiconductor device comprising: (i) a first antigen-binding domain that binds, e.g., selectively binds, 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) Death receptor (death receptor) signal engager with one, two, or all of the The present invention features a multifunctional molecule comprising:

[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 disclosure features a nucleic acid molecule that encodes a multifunctional molecule disclosed herein.

[0010] In another aspect, the disclosure features a vector, e.g., an expression vector, that includes a nucleic acid molecule disclosed herein. In another aspect, the disclosure features a host cell containing a nucleic acid molecule or vector disclosed herein.

[0011] In another aspect, the disclosure features a method of making, e.g., producing, a multifunctional molecule disclosed herein, the method including culturing a host cell disclosed herein under appropriate conditions, e.g., conditions suitable for gene expression and / or homo- or heterodimerization.

[0012] In another aspect, the disclosure features a pharmaceutical composition that includes a multifunctional molecule disclosed herein. In another aspect, the disclosure features a method of treating TCR bias, the method including 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 disclosure features a method of treating an autoimmune disease (e.g., an autoimmune disease associated with TCR bias), the method including 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 provides a method of identifying a subject in need of treatment for a TCR bias or an autoimmune disease (e.g., associated with a TCR bias) using a multifunctional molecule disclosed herein, comprising determining whether the subject has a TCR bias (e.g., a biased TCRBV clonotype) and / or an autoimmune disease associated with said bias (e.g., determining directly or indirectly, e.g., obtaining information regarding), In response to determining that a subject has a TCR bias (e.g., a biased TCRBV clonotype) and / or an autoimmune disease associated with said bias, the method features identifying the subject as a candidate for treatment using a multifunctional molecule comprising an antigen-binding domain that binds to a TCRBV antigen.

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

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

[0017] In another aspect, the 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) (an "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 an antibody molecule disclosed in US Pat. 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 about 2-, 5-, or 10-fold less) than the affinity and / or binding specificity of the 16G8 murine antibody or a humanized version thereof, as described in U.S. Patent No. 5,861,155.

[0020] In some embodiments, the anti-TCRβ antibody molecule binds to TCRβ V12 with an affinity and / or binding specificity that exceeds (e.g., about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or about 2-fold, 5-fold, or 10-fold greater than) that of the 16G8 murine antibody or a humanized version thereof described in U.S. Pat. No. 5,861,155.

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

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

[0023] In some embodiments, the anti-TCRβV antibody molecule comprises an Fc region, e.g., an Fc region with effector function, e.g., antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular 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, eg, compared to a 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, that includes a nucleic acid molecule disclosed herein.

[0026] In another aspect, the disclosure features a host cell containing 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 appropriate conditions, e.g., conditions suitable for gene expression and / or homo- or heterodimerization.

[0027] In another aspect, the disclosure features a pharmaceutical composition including an anti-TCRβV antibody molecule disclosed herein. Further features of any of the foregoing multifunctional molecules, nucleic acids, vectors, host cells, or methods include one or more of the following enumerated embodiments.

[0028] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein which equivalents are intended to be encompassed by the following recited embodiments.

[0029] Enumerated Embodiments 1. (i) a first antigen-binding domain that binds, e.g., selectively binds, 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) Death receptor signal engager with one, two, or all of the A multifunctional molecule comprising:

[0030] 2. The multifunctional molecule of embodiment 1, wherein the TCRBV antigen corresponds to a biased TCRBV clonotype, e.g., present in a subject, e.g., a patient, e.g., a subject or patient with an autoimmune disease.

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

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

[0033] 5. The antigen-binding domain is TCRβ V6 (e.g., TCRβ V6-5 * 5. The multifunctional molecule of any of embodiments 1 to 4, which specifically binds to .01). 6. The pluripotent cell molecule of embodiment 5, wherein the antigen-binding domain comprises at least one (e.g., one, two, three, or four) variable region or antigen-binding fragment thereof derived from antibody AH.1 or antibody AH.2, or as set forth in Table 1A, or encoded by a nucleotide sequence of 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.

[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 collectively all of the CDRs) derived from a heavy chain variable region comprising the amino acid sequence shown in Table 1A, or encoded by the nucleotide sequence shown in Table 1A (or the sequence having one, two, three, four, five, six or more alterations, e.g., amino acid substitutions or deletions, relative to the amino acid sequence shown in Table 1A, or 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 collectively all of the CDRs) derived 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 (or the amino acid sequence having one, two, three, four, five, six, or more alterations, e.g., amino acid substitutions or deletions, relative to the amino acid sequence shown in Table 1A, or the sequence encoded by the nucleotide sequence shown in Table 1A).

[0036] 9. The antigen-binding domain is (i) one, two or all of 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 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; 9. The multifunctional molecule of any of embodiments 5 to 8, comprising:

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

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

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

[0040] 13. The antigen-binding domain comprises: (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 9. The multifunctional molecule of any of embodiments 5 to 8, comprising:

[0041] 14. The antigen-binding domain comprises: (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; 9. The multifunctional molecule of any of embodiments 5 to 8, comprising:

[0042] 15. The antigen-binding domain comprises: (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 9. The multifunctional molecule of any of embodiments 5 to 8, comprising:

[0043] 16. The antigen-binding domain comprises: (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 9. The multifunctional molecule of any 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 9. The multifunctional molecule of any 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 9. The multifunctional molecule of any of embodiments 5 to 8, comprising:

[0046] 19. The multifunctional molecule of any of embodiments 5 to 18, wherein the antigen-binding domain comprises a light chain variable region (VL) comprising light chain framework region 1 (VLFWR1) of antibody AH.1 or antibody AH.2, e.g., of SEQ ID NO: 2, 10, or 11, e.g., as shown in Figure 1B.

[0047] 20. The multifunctional molecule of any of embodiments 5 to 19, wherein the antigen-binding domain comprises a light chain variable region (VL) comprising the light chain framework region 2 (VLFWR2) of antibody AH.1 or antibody AH.2, e.g., of SEQ ID NO: 2, 10, or 11, e.g., as shown in Figure 1B.

[0048] 21. The multifunctional molecule of any of embodiments 5 to 20, wherein the antigen-binding domain comprises a light chain variable region (VL) comprising light chain framework region 3 (VLFWR3) of antibody AH.1 or antibody AH.2, e.g., of SEQ ID NO: 2, 10, or 11, e.g., as shown in Figure 1B.

[0049] 22. The multifunctional molecule of any of embodiments 5 to 21, wherein the antigen-binding domain comprises a light chain variable region (VL) comprising light chain framework region 4 (VLFWR4) of antibody AH.1 or antibody AH.2, e.g., of SEQ ID NO: 2, 10, or 11, e.g., as shown in Figure 1B.

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

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

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

[0053] 26. The multifunctional molecule of any of embodiments 5 to 25, wherein the antigen-binding domain comprises a framework region comprising an alteration, substitution (e.g., a conservative substitution) at position 10 according to Kabat numbering, such as a light chain variable domain comprising framework region 1 (VLFWR1), and the alteration at position 10 is a substitution of phenylalanine, e.g., serine to phenylalanine.

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

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

[0056] 29. The multifunctional molecule of any 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, e.g., a substitution at position 10 according to Kabat numbering, for example, a serine to phenylalanine substitution; (b) framework region 2 (VLFWR2) comprising a histidine at position 36, e.g., a substitution at position 36 according to Kabat numbering, for example, a tyrosine to histidine substitution, and an alanine at position 46, e.g., a substitution at position 46 according to Kabat numbering, for example, an arginine to alanine substitution; and (c) framework region 3 (VLFWR3) comprising a phenylalanine at position 87, e.g., a substitution at position 87 according to Kabat numbering, for example, a tyrosine to phenylalanine substitution, e.g., as set forth in the amino acid sequence of SEQ ID NO: 10.

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

[0058] 31. The multifunctional molecule of any of embodiments 5 to 30, wherein the antigen-binding domain comprises a heavy chain variable region (VH) comprising heavy chain framework region 1 (VHFWR1) of antibody AH.1 or antibody AH.2, e.g., of SEQ ID NO: 1 or 9, e.g., as shown in Figure 1A.

[0059] 32. The multifunctional molecule of any of embodiments 5 to 31, wherein the antigen-binding domain comprises a heavy chain variable region (VH) comprising heavy chain framework region 2 (VHFWR2) of antibody AH.1 or antibody AH.2, e.g., of SEQ ID NO: 1 or 9, e.g., as shown in Figure 1A.

[0060] 33. The multifunctional molecule of any of embodiments 5 to 32, wherein the antigen-binding domain comprises a heavy chain variable region (VH) comprising heavy chain framework region 3 (VHFWR3) of antibody AH.1 or antibody AH.2, e.g., of SEQ ID NO: 1 or 9, e.g., as shown in Figure 1A.

[0061] 34. The multifunctional molecule of any of embodiments 5 to 33, wherein the antigen-binding domain comprises a heavy chain variable region (VH) comprising heavy chain framework region 4 (VHFWR4) of antibody AH.1 or antibody AH.2, e.g., of SEQ ID NO: 1 or 9, e.g., as shown in Figure 1A.

[0062] 35. The multifunctional molecule of any 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. The multifunctional molecule of any 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. The multifunctional molecule of any of embodiments 5 to 36, wherein the antigen-binding domain comprises a heavy chain variable domain comprising a framework region, e.g., framework region 3 (VHFWR3), comprising one or more (e.g., one or two) changes, e.g., 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 of threonine, e.g., glutamic acid, with threonine, and the change at position 94 is a substitution of glycine, e.g., arginine with glycine.

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

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

[0067] 40. The multifunctional molecule of any of embodiments 5 to 18, wherein the antigen-binding domain comprises heavy chain framework regions 1-4 of antibody AH.2, e.g., SEQ ID NO: 9; and light chain framework regions 1-4 of antibody AH.2, e.g., SEQ ID NO: 11, or those depicted in Figures 1A and 1B.

[0068] 41. The antigen-binding domain is a VH domain comprising the amino acid sequence of SEQ ID NO:9, an amino acid sequence 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 no more than 1, 2, 5, 10 or 15 amino acid residues; and / or a VL domain comprising the amino acid sequence of SEQ ID NO:10, an amino acid sequence 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. 19. The multifunctional molecule of any of embodiments 5 to 18, comprising:

[0069] 42. The antigen-binding domain is a VH domain comprising the amino acid sequence of SEQ ID NO:9, an amino acid sequence 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 no more than 1, 2, 5, 10 or 15 amino acid residues; and / or a VL domain comprising the amino acid sequence of SEQ ID NO:11, an amino acid sequence 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 no more than 1, 2, 5, 10 or 15 amino acid residues. 19. The multifunctional molecule of any of embodiments 5 to 18, comprising:

[0070] 43. The antigen-binding domain is selected from the group consisting of TCRβ V12 (e.g., TCRβ V12-3 * 5. The multifunctional molecule of any of embodiments 1 to 4, which specifically binds to .01). 44. The pluripotent stem cell molecule of embodiment 43, wherein the antigen-binding domain comprises at least one (e.g., one, two, three, or four) variable region, or antigen-binding fragment thereof, as set forth in Table 2A, or encoded by a nucleotide sequence of Table 2A, 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.

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

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

[0073] 47. The antigen-binding domain is (i) one, two, or all of 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: 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 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; 47. The multifunctional molecule of any 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 48. The multifunctional molecule of any of embodiments 43 to 47, comprising:

[0075] 49. The antigen-binding domain is (i) a light chain variable region (VL) comprising an LC CDR1 amino acid sequence of SEQ ID NO: 20, an LC CDR2 amino acid sequence of SEQ ID NO: 21, and an 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; 48. The multifunctional molecule of any 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 48. The multifunctional molecule of any 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 48. The multifunctional molecule of any 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 48. The multifunctional molecule of any 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 48. The multifunctional molecule of any of embodiments 43 to 47, comprising:

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

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

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

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

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

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

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

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

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

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

[0090] 64. The multifunctional molecule of any of embodiments 43 to 57, wherein the antigen-binding domain comprises a framework region, e.g., a light chain variable domain comprising framework region 1 (VLFWR1), comprising 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 an aspartic acid, e.g., alanine to asparagine, the change at position 2 is an asparagine, e.g., isoleucine to asparagine, and the change at position 4 is a leucine, e.g., methionine to leucine substitution.

[0091] 65. The multifunctional molecule of any of embodiments 43 to 57 or 64, wherein the antigen-binding domain comprises a framework region, e.g., a light chain variable domain comprising framework region 3 (VLFWR3), comprising 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 glycine, e.g., lysine to glycine substitution, the change at position 69 is an asparagine, e.g., tyrosine to asparagine substitution, and the change at position 71 is a tyrosine, e.g., phenylalanine to tyrosine substitution.

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

[0093] 67. The multifunctional molecule of any of embodiments 43 to 57, 64, or 65, 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 an alanine to aspartic acid substitution, and a substitution at position 2 according to Kabat numbering, for example an isoleucine to aparagine substitution, and (b) a framework region 3 (FR3) comprising a substitution at position 69 according to Kabat numbering, for example a threonine to aparagine substitution, and a substitution at position 71 according to Kabat numbering, for example a phenylalanine to tyrosine substitution, e.g., as set forth in the amino acid sequence of SEQ ID NO: 27.

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

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

[0096] 70. The multifunctional molecule of any of embodiments 43 to 69, wherein the antigen-binding domain comprises a heavy chain variable region (VH) comprising heavy chain framework region 1 (VHFWR1), e.g., of SEQ ID NO: 15 or 23-25, e.g., as shown in Figure 2A.

[0097] 71. The multifunctional molecule of any of embodiments 43 to 70, wherein the antigen-binding domain comprises a heavy chain variable region (VH) comprising heavy chain framework region 2 (VHFWR2), e.g., of SEQ ID NO: 15 or 23-25, e.g., as shown in Figure 2A.

[0098] 72. The multifunctional molecule of any of embodiments 43 to 71, wherein the antigen-binding domain comprises a heavy chain variable region (VH) comprising heavy chain framework region 3 (VHFWR3), e.g., of SEQ ID NO: 15 or 23-25, e.g., as shown in Figure 2A.

[0099] 73. The multifunctional molecule of any of embodiments 43 to 72, wherein the antigen-binding domain comprises a heavy chain variable region (VH) comprising heavy chain framework region 4 (VHFWR4), e.g., of SEQ ID NO: 15 or 23-25, e.g., as shown in Figure 2A.

[0100] 74. The multifunctional molecule of any 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: 23.

[0101] 75. The multifunctional molecule of any 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: 24.

[0102] 76. The multifunctional molecule of any 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. The multifunctional molecule of any 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 a VH domain comprising an amino acid sequence selected from the amino acid sequence of SEQ ID NO:23, SEQ ID NO:24, or SEQ ID NO:25, an amino acid sequence 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 no more than 1, 2, 5, 10, or 15 amino acid residues; and / or a VL domain comprising an amino acid sequence selected 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, 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 less amino acid residues. 74. The multifunctional molecule of any of embodiments 43 to 73, comprising:

[0105] 79. The antigen-binding domain is a VH domain comprising the amino acid sequence of SEQ ID NO:23, an amino acid sequence 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 no more than 1, 2, 5, 10, or 15 amino acid residues; and a VL domain comprising the amino acid sequence of SEQ ID NO:26, an amino acid sequence 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 no more than 1, 2, 5, 10, or 15 amino acid residues. 74. The multifunctional molecule of any of embodiments 43 to 73, comprising:

[0106] 80. The antigen-binding domain is a VH domain comprising the amino acid sequence of SEQ ID NO:23, an amino acid sequence 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 no more than 1, 2, 5, 10, or 15 amino acid residues; and a VL domain comprising the amino acid sequence of SEQ ID NO:27, an amino acid sequence 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 no more than 1, 2, 5, 10, or 15 amino acid residues. 74. The multifunctional molecule of any of embodiments 43 to 73, comprising:

[0107] 81. The antigen-binding domain is a VH domain comprising the amino acid sequence of SEQ ID NO:23, an amino acid sequence 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 no more than 1, 2, 5, 10, or 15 amino acid residues; and a VL domain comprising the amino acid sequence of SEQ ID NO:28, an amino acid sequence 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 no more than 1, 2, 5, 10, or 15 amino acid residues. 74. The multifunctional molecule of any of embodiments 43 to 73, comprising:

[0108] 82. The antigen-binding domain is a VH domain comprising the amino acid sequence of SEQ ID NO:23, an amino acid sequence 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 no more than 1, 2, 5, 10, or 15 amino acid residues; and a VL domain comprising the amino acid sequence of SEQ ID NO:29, an amino acid sequence 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 no more than 1, 2, 5, 10, or 15 amino acid residues. 74. The multifunctional molecule of any of embodiments 43 to 73, comprising:

[0109] 83. The antigen-binding domain is a VH domain comprising the amino acid sequence of SEQ ID NO:23, an amino acid sequence 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 no more than 1, 2, 5, 10, or 15 amino acid residues; and a VL domain comprising the amino acid sequence of SEQ ID NO:30, an amino acid sequence 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 no more than 1, 2, 5, 10, or 15 amino acid residues. 74. The multifunctional molecule of any of embodiments 43 to 73, comprising:

[0110] 84. The antigen-binding domain is a VH domain comprising the amino acid sequence of SEQ ID NO:24, an amino acid sequence 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 no more than 1, 2, 5, 10, or 15 amino acid residues; and a VL domain comprising the amino acid sequence of SEQ ID NO:26, an amino acid sequence 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 no more than 1, 2, 5, 10, or 15 amino acid residues. 74. The multifunctional molecule of any of embodiments 43 to 73, comprising:

[0111] 85. The antigen-binding domain is a VH domain comprising the amino acid sequence of SEQ ID NO:24, an amino acid sequence 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 no more than 1, 2, 5, 10, or 15 amino acid residues; and a VL domain comprising the amino acid sequence of SEQ ID NO:27, an amino acid sequence 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 no more than 1, 2, 5, 10, or 15 amino acid residues. 74. The multifunctional molecule of any of embodiments 43 to 73, comprising:

[0112] 86. The antigen-binding domain is a VH domain comprising the amino acid sequence of SEQ ID NO:24, an amino acid sequence 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 no more than 1, 2, 5, 10, or 15 amino acid residues; and a VL domain comprising the amino acid sequence of SEQ ID NO:28, an amino acid sequence 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 no more than 1, 2, 5, 10, or 15 amino acid residues. 74. The multifunctional molecule of any of embodiments 43 to 73, comprising:

[0113] 87. The antigen-binding domain is a VH domain comprising the amino acid sequence of SEQ ID NO:24, an amino acid sequence 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 no more than 1, 2, 5, 10, or 15 amino acid residues; and a VL domain comprising the amino acid sequence of SEQ ID NO:29, an amino acid sequence 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 no more than 1, 2, 5, 10, or 15 amino acid residues. 74. The multifunctional molecule of any of embodiments 43 to 73, comprising:

[0114] 88. The antigen-binding domain is a VH domain comprising the amino acid sequence of SEQ ID NO:24, an amino acid sequence 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 no more than 1, 2, 5, 10, or 15 amino acid residues; and a VL domain comprising the amino acid sequence of SEQ ID NO:30, an amino acid sequence 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 no more than 1, 2, 5, 10, or 15 amino acid residues. 74. The multifunctional molecule of any 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 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 no more than 1, 2, 5, 10, or 15 amino acid residues; and a VL domain comprising the amino acid sequence of SEQ ID NO:26, an amino acid sequence 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 no more than 1, 2, 5, 10, or 15 amino acid residues. 74. The multifunctional molecule of any of embodiments 43 to 73, comprising:

[0116] 90. The antigen-binding domain is a VH domain comprising the amino acid sequence of SEQ ID NO:25, an amino acid sequence 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 no more than 1, 2, 5, 10, or 15 amino acid residues; and a VL domain comprising the amino acid sequence of SEQ ID NO:27, an amino acid sequence 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 no more than 1, 2, 5, 10, or 15 amino acid residues. 74. The multifunctional molecule of any of embodiments 43 to 73, comprising:

[0117] 91. The antigen-binding domain is a VH domain comprising the amino acid sequence of SEQ ID NO:25, an amino acid sequence 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 no more than 1, 2, 5, 10, or 15 amino acid residues; and a VL domain comprising the amino acid sequence of SEQ ID NO:28, an amino acid sequence 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 no more than 1, 2, 5, 10, or 15 amino acid residues. 74. The multifunctional molecule of any of embodiments 43 to 73, comprising:

[0118] 92. The antigen-binding domain is a VH domain comprising the amino acid sequence of SEQ ID NO:25, an amino acid sequence 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 no more than 1, 2, 5, 10, or 15 amino acid residues; and a VL domain comprising the amino acid sequence of SEQ ID NO:29, an amino acid sequence 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 no more than 1, 2, 5, 10, or 15 amino acid residues. 74. The multifunctional molecule of any of embodiments 43 to 73, comprising:

[0119] 93. The antigen-binding domain is a VH domain comprising the amino acid sequence of SEQ ID NO:25, an amino acid sequence 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 no more than 1, 2, 5, 10, or 15 amino acid residues; and a VL domain comprising the amino acid sequence of SEQ ID NO:30, an amino acid sequence 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 no more than 1, 2, 5, 10, or 15 amino acid residues. 74. The multifunctional molecule of any 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 a K for binding between the first antigen-binding domain and a T cell receptor containing a TCRBV antigen. D is the K for binding between the first antigen-binding domain and a T cell receptor not containing the TCRBV antigen. D 94. The multifunctional molecule of any one of embodiments 1 to 93, wherein the multifunctional molecule has less than 40%, 30%, 20%, 10%, 1%, 0.1%, or 0.01% of the total mass of the multifunctional molecule.

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

[0122] 96. The multifunctional molecule of any of the preceding embodiments, wherein binding of the first antigen-binding domain to a TCRBV antigen, e.g., on a lymphocyte (e.g., a T cell), does not significantly activate the lymphocyte, e.g., a T cell (e.g., as measured by T cell proliferation, expression of a T cell activation marker (e.g., CD69 or CD25), and / or expression of cytokines (e.g., TNFα and IFNγ)).

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

[0124] 98. The multifunctional molecule of 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 immune cells, e.g., effector cells. 100. The multifunctional molecule of embodiment 98, wherein the immune cell engager binds to but does not activate immune cells, e.g., effector cells.

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

[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, e.g., the T cell engager is an anti-CD3 antibody molecule.

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

[0129] 104. The multifunctional molecule of embodiment 103, wherein the NK cell engager is selected from an antibody molecule, e.g., an antibody molecule or ligand that binds to (e.g., activates) 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; e.g., the NK cell engager is an antibody molecule or ligand that binds to (e.g., activates) NKp30.

[0130] 105. The multifunctional molecule of embodiment 103, wherein the NK cell engager is an antibody molecule, e.g., an antigen-binding domain. 106. The multifunctional molecule of any of embodiments 104 or 105, wherein the NK cell engager is capable of engaging NK cells.

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

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

[0133] 109. The 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 7-10, or sequences substantially identical thereto.

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

[0135] 112. The 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 TCRBV antigen. 113. The multifunctional molecule of any of embodiments 103 to 112, wherein the NK cells are NKp30-expressing NK cells and the multifunctional molecule activates the NK cells when a TCRBV antigen is also present.

[0136] 114. The multifunctional molecule of any of embodiments 103 to 113, wherein the NK cells are not NKp30-expressing NK cells and the multifunctional molecule does not activate NK cells if the TCRBV antigen is also present.

[0137] 115.NK cell engagers (i) a heavy chain variable region (VH) comprising a heavy chain complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 6000 (or a sequence having no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), a VHCDR2 amino acid sequence of SEQ ID NO: 6001 (or a sequence having no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), and / or a VHCDR3 amino acid sequence of SEQ ID NO: 6002 (or a sequence having no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions); and (ii) a light chain variable region (VL) comprising a light chain complementarity determining region 1 (VLCDR1) amino acid sequence of SEQ ID NO: 6063 (or a sequence having no more than one, two, three, or four mutations, e.g., substitutions, additions, or deletions), a VLCDR2 amino acid sequence of SEQ ID NO: 6064 (or a sequence having no more than one, two, three, or four mutations, e.g., substitutions, additions, or deletions), and / or a VLCDR3 amino acid sequence of SEQ ID NO: 6065 (or a sequence having no more than one, two, three, or four mutations, e.g., substitutions, additions, or deletions). 114. The multifunctional molecule of any of embodiments 103 to 113, comprising:

[0138] 116.NK cell engagers (i) a heavy chain variable region (VH) comprising the heavy chain complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 6000, the VHCDR2 amino acid sequence of SEQ ID NO: 6001, and / or the VHCDR3 amino acid sequence of SEQ ID NO: 6002; and (ii) a light chain variable region (VL) comprising the light chain complementarity-determining region 1 (VLCDR1) amino acid sequence of SEQ ID NO: 6063, the VLCDR2 amino acid sequence of SEQ ID NO: 6064, and / or the VLCDR3 amino acid sequence of SEQ ID NO: 6065 116. The multifunctional molecule of embodiment 115, comprising:

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

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

[0141] 119.NK cell engagers (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 to 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 to SEQ ID NO: 6135); 119. The multifunctional molecule of any one of embodiments 103 to 118, comprising:

[0142] 120. The multifunctional molecule of any of embodiments 103 to 119, 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 to SEQ ID NO: 6148 or 6149).

[0143] 121. The multifunctional molecule of any of embodiments 103 to 120, wherein 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 to SEQ ID NO: 6150).

[0144] 122. The multifunctional molecule of any 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 to 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 to SEQ ID NO: 6150).

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

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

[0147] 125. The multifunctional molecule of 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 to SEQ ID NO: 6123).

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

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

[0150] 128. The multifunctional molecule of embodiment 127, wherein the NK cell engager comprises 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 to SEQ ID NO: 6124).

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

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

[0153] 131. The multifunctional molecule of embodiment 130, wherein the NK cell engager comprises a VH comprising 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 to SEQ ID NO: 6125).

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

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

[0156] 134. The multifunctional molecule of embodiment 133, wherein the NK cell engager comprises 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).

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

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

[0159] 137. The multifunctional molecule of embodiment 136, wherein the NK cell engager comprises 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. The multifunctional molecule of any of embodiments 103 to 116, wherein the NK cell engager comprises a heavy chain variable region (VH) comprising the heavy chain framework region 1 (VHFWR1) amino acid sequence of SEQ ID NO: 6035 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations, e.g., substitutions, additions, or deletions therefrom), the VHFWR2 amino acid sequence of SEQ ID NO: 6036 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations, e.g., substitutions, additions, or deletions therefrom), the VHFWR3 amino acid sequence of SEQ ID NO: 6037 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations, e.g., substitutions, additions, or deletions therefrom), or the VHFWR4 amino acid sequence of SEQ ID NO: 6038 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations, e.g., substitutions, additions, or deletions therefrom).

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

[0162] 140. The multifunctional molecule of embodiment 139, wherein the NK cell engager comprises 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).

[0163] 141. The multifunctional molecule of any of embodiments 103 to 116 or 123 to 140, 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: 6077 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations, e.g., substitutions, additions, or deletions therefrom), the VLFWR2 amino acid sequence of SEQ ID NO: 6078 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations, e.g., substitutions, additions, or deletions therefrom), the VLFWR3 amino acid sequence of SEQ ID NO: 6079 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations, e.g., substitutions, additions, or deletions therefrom), or the VLFWR4 amino acid sequence of SEQ ID NO: 6080 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations, e.g., substitutions, additions, or deletions therefrom).

[0164] 142. The multifunctional molecule of embodiment 141, 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: 6077, the VLFWR2 amino acid sequence of SEQ ID NO: 6078, the VLFWR3 amino acid sequence of SEQ ID NO: 6079, or the VLFWR4 amino acid sequence of SEQ ID NO: 6080.

[0165] 143. The multifunctional molecule of embodiment 142, wherein the NK cell engager comprises a VL 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 to SEQ ID NO: 6137).

[0166] 144. The multifunctional molecule of any of embodiments 103 to 116 or 123 to 140, 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: 6081 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations, e.g., substitutions, additions, or deletions therefrom), the VLFWR2 amino acid sequence of SEQ ID NO: 6082 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations, e.g., substitutions, additions, or deletions therefrom), the VLFWR3 amino acid sequence of SEQ ID NO: 6083 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations, e.g., substitutions, additions, or deletions therefrom), or the VLFWR4 amino acid sequence of SEQ ID NO: 6084 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations, e.g., substitutions, additions, or deletions therefrom).

[0167] 145. The multifunctional molecule of embodiment 144, 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: 6081, the VLFWR2 amino acid sequence of SEQ ID NO: 6082, the VLFWR3 amino acid sequence of SEQ ID NO: 6083, or the VLFWR4 amino acid sequence of SEQ ID NO: 6084.

[0168] 146. The multifunctional molecule of embodiment 145, wherein the NK cell engager comprises a VL comprising the amino acid sequence of SEQ ID NO: 6138 (or an amino acid sequence having at least about 93%, 95%, or 99% sequence identity to SEQ ID NO: 6138).

[0169] 147. The multifunctional molecule of any of embodiments 103 to 116 or 123 to 140, 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: 6085 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations, e.g., substitutions, additions, or deletions therefrom), the VLFWR2 amino acid sequence of SEQ ID NO: 6086 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations, e.g., substitutions, additions, or deletions therefrom), the VLFWR3 amino acid sequence of SEQ ID NO: 6087 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations, e.g., substitutions, additions, or deletions therefrom), or the VLFWR4 amino acid sequence of SEQ ID NO: 6088 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations, e.g., substitutions, additions, or deletions therefrom).

[0170] 148. The multifunctional molecule of embodiment 147, 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: 6085, the VLFWR2 amino acid sequence of SEQ ID NO: 6086, the VLFWR3 amino acid sequence of SEQ ID NO: 6087, or the VLFWR4 amino acid sequence of SEQ ID NO: 6088.

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

[0172] 150. The multifunctional molecule of any of embodiments 103 to 116 or 123 to 140, 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: 6089 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations, e.g., substitutions, additions, or deletions therefrom), the VLFWR2 amino acid sequence of SEQ ID NO: 6090 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations, e.g., substitutions, additions, or deletions therefrom), the VLFWR3 amino acid sequence of SEQ ID NO: 6091 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations, e.g., substitutions, additions, or deletions therefrom), or the VLFWR4 amino acid sequence of SEQ ID NO: 6092 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations, e.g., substitutions, additions, or deletions therefrom).

[0173] 151. The multifunctional molecule of embodiment 150, 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: 6089, the VLFWR2 amino acid sequence of SEQ ID NO: 6090, the VLFWR3 amino acid sequence of SEQ ID NO: 6091, or the VLFWR4 amino acid sequence of SEQ ID NO: 6092.

[0174] 152. The multifunctional molecule of embodiment 151, wherein the NK cell engager comprises a VL 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 to SEQ ID NO: 6140).

[0175] 153. The multifunctional molecule of any of embodiments 103 to 116 or 123 to 140, 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: 6093 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations, e.g., substitutions, additions, or deletions therefrom), the VLFWR2 amino acid sequence of SEQ ID NO: 6094 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations, e.g., substitutions, additions, or deletions therefrom), the VLFWR3 amino acid sequence of SEQ ID NO: 6095 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations, e.g., substitutions, additions, or deletions therefrom), or the VLFWR4 amino acid sequence of SEQ ID NO: 6096 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations, e.g., substitutions, additions, or deletions therefrom).

[0176] 154. The multifunctional molecule of embodiment 153, wherein the NK cell engager comprises a light chain framework region 1 (VLFWR1) amino acid sequence of SEQ ID NO: 6093, a VLFWR2 amino acid sequence of SEQ ID NO: 6094, a VLFWR3 amino acid sequence of SEQ ID NO: 6095, or a VLFWR4 amino acid sequence 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.NK cell engagers (i) a heavy chain variable region (VH) comprising a heavy chain complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 6007 (or a sequence having no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), a VHCDR2 amino acid sequence of SEQ ID NO: 6008 (or a sequence having no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), and / or a VHCDR3 amino acid sequence of SEQ ID NO: 6009 (or a sequence having no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions); and (ii) a light chain variable region (VL) comprising a light chain complementarity determining region 1 (VLCDR1) amino acid sequence of SEQ ID NO: 6070 (or a sequence having no more than one, two, three, or four mutations, e.g., substitutions, additions, or deletions), a VLCDR2 amino acid sequence of SEQ ID NO: 6071 (or a sequence having no more than one, two, three, or four mutations, e.g., substitutions, additions, or deletions), and / or a VLCDR3 amino acid sequence of SEQ ID NO: 6072 (or a sequence having no more than one, two, three, or four mutations, e.g., substitutions, additions, or deletions). 115. The multifunctional molecule of any of embodiments 103 to 114, comprising:

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

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

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

[0182] 160.NK cell engagers, (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 to 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 to SEQ ID NO: 6136); 159. The multifunctional molecule of any one of embodiments 103 to 114 or 156 to 159, comprising:

[0183] 161. The multifunctional molecule of any of embodiments 103 to 114 or 156 to 160, 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).

[0184] 162. The multifunctional molecule of any of embodiments 103 to 114 or 156 to 161, wherein the NK cell engager comprises 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).

[0185] 163. The multifunctional molecule of any 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. The multifunctional molecule of any of embodiments 103 to 114, 156, or 157, wherein the NK cell engager comprises a heavy chain variable region (VH) comprising the heavy chain framework region 1 (VHFWR1) amino acid sequence of SEQ ID NO: 6039 (or a sequence having 1, 2, 3, 4, 5, or 6 or less mutations, e.g., substitutions, additions, or deletions therefrom), the VHFWR2 amino acid sequence of SEQ ID NO: 6040 (or a sequence having 1, 2, 3, 4, 5, or 6 or less mutations, e.g., substitutions, additions, or deletions therefrom), the VHFWR3 amino acid sequence of SEQ ID NO: 6041 (or a sequence having 1, 2, 3, 4, 5, or 6 or less mutations, e.g., substitutions, additions, or deletions therefrom), or the VHFWR4 amino acid sequence of SEQ ID NO: 6042 (or a sequence having 1, 2, 3, 4, 5, or 6 or less mutations, e.g., substitutions, additions, or deletions therefrom).

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

[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 to SEQ ID NO: 6129).

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

[0190] 168. The multifunctional molecule of embodiment 167, wherein the NK cell engager comprises a heavy chain variable region (VH) comprising the heavy chain framework region 1 (VHFWR1) amino acid sequence of SEQ ID NO: 6043, the VHFWR2 amino acid sequence of SEQ ID NO: 6044, the VHFWR3 amino acid sequence of SEQ ID NO: 6045, or the VHFWR4 amino acid sequence of SEQ ID NO: 6046.

[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 to SEQ ID NO: 6130).

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

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

[0194] 172. The multifunctional molecule of embodiment 171, wherein the NK cell engager comprises a VH comprising 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. The multifunctional molecule of any of embodiments 103 to 114, 156, or 157, wherein the NK cell engager comprises a heavy chain variable region (VH) comprising the heavy chain framework region 1 (VHFWR1) amino acid sequence of SEQ ID NO: 6051 (or a sequence having 1, 2, 3, 4, 5, or 6 or less mutations, e.g., substitutions, additions, or deletions therefrom), the VHFWR2 amino acid sequence of SEQ ID NO: 6052 (or a sequence having 1, 2, 3, 4, 5, or 6 or less mutations, e.g., substitutions, additions, or deletions therefrom), the VHFWR3 amino acid sequence of SEQ ID NO: 6053 (or a sequence having 1, 2, 3, 4, 5, or 6 or less mutations, e.g., substitutions, additions, or deletions therefrom), or the VHFWR4 amino acid sequence of SEQ ID NO: 6054 (or a sequence having 1, 2, 3, 4, 5, or 6 or less mutations, e.g., substitutions, additions, or deletions therefrom).

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

[0197] 175. The multifunctional molecule of embodiment 174, wherein the NK cell engager comprises 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 to SEQ ID NO: 6132).

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

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

[0200] 178. The 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. The multifunctional molecule of any of embodiments 103 to 114, 156, or 157, wherein the NK cell engager comprises a heavy chain variable region (VH) comprising the heavy chain framework region 1 (VHFWR1) amino acid sequence of SEQ ID NO: 6059 (or a sequence having 1, 2, 3, 4, 5, or 6 or less mutations, e.g., substitutions, additions, or deletions therefrom), the VHFWR2 amino acid sequence of SEQ ID NO: 6060 (or a sequence having 1, 2, 3, 4, 5, or 6 or less mutations, e.g., substitutions, additions, or deletions therefrom), the VHFWR3 amino acid sequence of SEQ ID NO: 6061 (or a sequence having 1, 2, 3, 4, 5, or 6 or less mutations, e.g., substitutions, additions, or deletions therefrom), or the VHFWR4 amino acid sequence of SEQ ID NO: 6062 (or a sequence having 1, 2, 3, 4, 5, or 6 or less mutations, e.g., substitutions, additions, or deletions therefrom).

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

[0203] 181. The multifunctional molecule of embodiment 180, wherein the NK cell engager comprises a VH comprising 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. The multifunctional molecule of any of embodiments 103 to 114, 156, 157, or 164 to 181, 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: 6097 (or a sequence having 1, 2, 3, 4, 5, or 6 or less mutations, e.g., substitutions, additions, or deletions therefrom), the VLFWR2 amino acid sequence of SEQ ID NO: 6098 (or a sequence having 1, 2, 3, 4, 5, or 6 or less mutations, e.g., substitutions, additions, or deletions therefrom), the VLFWR3 amino acid sequence of SEQ ID NO: 6099 (or a sequence having 1, 2, 3, 4, 5, or 6 or less mutations, e.g., substitutions, additions, or deletions therefrom), or the VLFWR4 amino acid sequence of SEQ ID NO: 6100 (or a sequence having 1, 2, 3, 4, 5, or 6 or less mutations, e.g., substitutions, additions, or deletions therefrom).

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

[0206] 184. The multifunctional molecule of embodiment 183, wherein the NK cell engager comprises a VL comprising the amino acid sequence of SEQ ID NO: 6142 (or an amino acid sequence having at least about 93%, 95%, or 99% sequence identity to SEQ ID NO: 6142).

[0207] 185. The multifunctional molecule of any of embodiments 103 to 114, 156, 157, or 164 to 181, 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: 6101 (or a sequence having 1, 2, 3, 4, 5, or 6 or less mutations, e.g., substitutions, additions, or deletions therefrom), the VLFWR2 amino acid sequence of SEQ ID NO: 6102 (or a sequence having 1, 2, 3, 4, 5, or 6 or less mutations, e.g., substitutions, additions, or deletions therefrom), the VLFWR3 amino acid sequence of SEQ ID NO: 6103 (or a sequence having 1, 2, 3, 4, 5, or 6 or less mutations, e.g., substitutions, additions, or deletions therefrom), or the VLFWR4 amino acid sequence of SEQ ID NO: 6104 (or a sequence having 1, 2, 3, 4, 5, or 6 or less mutations, e.g., substitutions, additions, or deletions therefrom).

[0208] 186. The multifunctional molecule of embodiment 185, 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: 6101, the VLFWR2 amino acid sequence of SEQ ID NO: 6102, the VLFWR3 amino acid sequence of SEQ ID NO: 6103, or the VLFWR4 amino acid sequence of SEQ ID NO: 6104.

[0209] 187. The multifunctional molecule of embodiment 186, wherein the NK cell engager comprises 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 to SEQ ID NO: 6143).

[0210] 188. The multifunctional molecule of any of embodiments 103 to 114, 156, 157, or 164 to 181, 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: 6105 (or a sequence having 1, 2, 3, 4, 5, or 6 or less mutations, e.g., substitutions, additions, or deletions therefrom), the VLFWR2 amino acid sequence of SEQ ID NO: 6106 (or a sequence having 1, 2, 3, 4, 5, or 6 or less mutations, e.g., substitutions, additions, or deletions therefrom), the VLFWR3 amino acid sequence of SEQ ID NO: 6107 (or a sequence having 1, 2, 3, 4, 5, or 6 or less mutations, e.g., substitutions, additions, or deletions therefrom), or the VLFWR4 amino acid sequence of SEQ ID NO: 6108 (or a sequence having 1, 2, 3, 4, 5, or 6 or less mutations, e.g., substitutions, additions, or deletions therefrom).

[0211] 189. The multifunctional molecule of embodiment 188, 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: 6105, the VLFWR2 amino acid sequence of SEQ ID NO: 6106, the VLFWR3 amino acid sequence of SEQ ID NO: 6107, or the VLFWR4 amino acid sequence of SEQ ID NO: 6108.

[0212] 190. The multifunctional molecule of embodiment 189, wherein the NK cell engager comprises a VL comprising 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. The multifunctional molecule of any of embodiments 103 to 114, 156, 157, or 164 to 181, 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: 6109 (or a sequence having 1, 2, 3, 4, 5, or 6 or less mutations, e.g., substitutions, additions, or deletions therefrom), the VLFWR2 amino acid sequence of SEQ ID NO: 6110 (or a sequence having 1, 2, 3, 4, 5, or 6 or less mutations, e.g., substitutions, additions, or deletions therefrom), the VLFWR3 amino acid sequence of SEQ ID NO: 6111 (or a sequence having 1, 2, 3, 4, 5, or 6 or less mutations, e.g., substitutions, additions, or deletions therefrom), or the VLFWR4 amino acid sequence of SEQ ID NO: 6112 (or a sequence having 1, 2, 3, 4, 5, or 6 or less mutations, e.g., substitutions, additions, or deletions therefrom).

[0214] 192. The multifunctional molecule of embodiment 191, 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: 6109, the VLFWR2 amino acid sequence of SEQ ID NO: 6110, the VLFWR3 amino acid sequence of SEQ ID NO: 6111, or the VLFWR4 amino acid sequence of SEQ ID NO: 6112.

[0215] 193. The multifunctional molecule of embodiment 192, wherein the NK cell engager comprises a VL comprising the amino acid sequence of SEQ ID NO: 6145 (or an amino acid sequence having at least about 93%, 95%, or 99% sequence identity to SEQ ID NO: 6145).

[0216] 194. The multifunctional molecule of any of embodiments 103 to 114, 156, 157, or 164 to 181, 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: 6113 (or a sequence having no more than 1, 2, 3, 4, 5, or 6 mutations, e.g., substitutions, additions, or deletions therefrom), the VLFWR2 amino acid sequence of SEQ ID NO: 6114 (or a sequence having no more than 1, 2, 3, 4, 5, or 6 mutations, e.g., substitutions, additions, or deletions therefrom), the VLFWR3 amino acid sequence of SEQ ID NO: 6115 (or a sequence having no more than 1, 2, 3, 4, 5, or 6 mutations, e.g., substitutions, additions, or deletions therefrom), or the VLFWR4 amino acid sequence of SEQ ID NO: 6116 (or a sequence having no more than 1, 2, 3, 4, 5, or 6 mutations, e.g., substitutions, additions, or deletions therefrom).

[0217] 195. The multifunctional molecule of embodiment 194, 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: 6113, the VLFWR2 amino acid sequence of SEQ ID NO: 6114, the VLFWR3 amino acid sequence of SEQ ID NO: 6115, or the VLFWR4 amino acid sequence of SEQ ID NO: 6116.

[0218] 196. The multifunctional molecule of embodiment 195, wherein the NK cell engager comprises a VL comprising the amino acid sequence of SEQ ID NO: 6146 (or an amino acid sequence having at least about 93%, 95%, or 99% sequence identity to SEQ ID NO: 6146).

[0219] 197. The multifunctional molecule of any of embodiments 103 to 114, 156, 157, or 164 to 181, 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 (or a sequence having 1, 2, 3, 4, 5, or 6 or less mutations, e.g., substitutions, additions, or deletions therefrom), the 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), the 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 the VLFWR4 amino acid sequence of SEQ ID NO: 6120 (or a sequence having 1, 2, 3, 4, 5, or 6 or less mutations, e.g., substitutions, additions, or deletions therefrom).

[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] 202. The multifunctional molecule of any of embodiments 200 or 201, 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 clonotype).

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

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

[0226] 205. The 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 to SEQ ID NO: 6182).

[0227] 206. The 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 to SEQ ID NO: 6183).

[0228] 207. The 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 to SEQ ID NO: 6181).

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

[0230] 210. The multifunctional molecule of any of embodiments 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 clonotype).

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

[0232] 212. The multifunctional molecule of any one of embodiments 208 to 211, wherein the multifunctional molecule does not activate NK cells if the NK cells are not NKG2D-expressing NK cells and a TCRBV antigen (e.g., a TCRBV antigen corresponding to a biased TCRBV clonotype) is also present.

[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. The multifunctional molecule of any of embodiments 208 to 214, wherein the NK cell engager comprises an 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 to SEQ ID NO: 6175).

[0236] 216. 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: 6179 (or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 6179).

[0237] 217. The multifunctional molecule of 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 to SEQ ID NO: 6180).

[0238] 218. The multifunctional molecule of any of embodiments 208 to 212, 216 or 217, wherein the NK cell engager comprises an 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 to SEQ ID NO: 6178).

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

[0240] 221. The multifunctional molecule of any of embodiments 219 or 220, 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 clonotype).

[0241] 222. The multifunctional molecule of any one of embodiments 219 to 221, wherein the multifunctional molecule activates the NK cell if the NK cell is a CD16-expressing NK cell and a TCRBV antigen (e.g., a TCRBV antigen corresponding to a biased TCRBV clonotype) is also present.

[0242] 223. The multifunctional molecule of any one of embodiments 219 to 222, wherein the multifunctional molecule does not activate NK cells if the NK cells are not CD16-expressing NK cells and a TCRBV antigen (e.g., a TCRBV antigen corresponding to a biased TCRBV clonotype) is also present.

[0243] 224. The multifunctional molecule of 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 to SEQ ID NO: 6185).

[0244] 225. The multifunctional molecule of 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 to SEQ ID NO: 6186).

[0245] 226. The multifunctional molecule of 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 to SEQ ID NO: 6184).

[0246] 227. The multifunctional molecule of embodiment 103, wherein the NK cell engager is a ligand, and optionally the ligand further comprises an immunoglobulin constant region, e.g., an Fc region. 228. The multifunctional molecule of embodiment 227, wherein the NK cell engager is a ligand for NKp44 or NKp46, for example, viral HA.

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

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

[0249] 232. The multifunctional molecule of embodiment 231, wherein the immune cell engager 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 a Toll-like receptor (e.g., TLR4, e.g., constitutively active TLR4) (caTLR4) or a TLR9 agonist); 41BB; a CD2 agonist; CD47; or a STING agonist, or a combination thereof.

[0250] 233. The multifunctional molecule of any one of embodiments 98 to 100, wherein the immune cell engager is a B cell engager, such as CD40L, OX40L, or CD70 ligand, or an antibody molecule that binds to OX40, CD40, or CD70.

[0251] 234. The multifunctional molecule of any one of embodiments 98 to 100, wherein the immune cell engager 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 a Toll-like receptor (TLR) (e.g., TLR4, e.g., constitutively active TLR4 (caTLR4) or a TLR9 agonist); CD47; or a STING agonist.

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

[0253] 236. The multifunctional molecule of embodiment 234 or 235, wherein the STING agonist comprises a cyclic dinucleotide, e.g., cyclic di-GMP (cdGMP), cyclic di-AMP (cdAMP), or a combination thereof, optionally with a 2',5' or 3',5' phosphate linkage, e.g., wherein the STING agonist is covalently coupled to the multifunctional molecule.

[0254] 237. The multifunctional molecule of any one of embodiments 1-97, wherein the multifunctional molecule comprises a cytokine molecule. 238. The multifunctional molecule of embodiment 237, wherein 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 gamma, or a fragment or variant thereof, or a combination of any of the foregoing cytokines.

[0255] 239. The multifunctional molecule of embodiment 237 or 238, wherein the cytokine molecule is a monomer or a dimer. 240. The multifunctional molecule of any one of embodiments 237 to 239, wherein the cytokine molecule further comprises a receptor dimerization domain, for example, an IL15Ralpha dimerization domain.

[0256] 241. The multifunctional molecule of embodiment 240, wherein the cytokine molecule (e.g., IL-15) and the receptor dimerization domain (e.g., IL15R alpha dimerization domain) are not covalently linked, e.g., are non-covalently associated.

[0257] 242. The multifunctional molecule of any of embodiments 1-97, wherein the multifunctional molecule comprises a cytokine inhibitor molecule. 243. The multifunctional molecule of embodiment 242, wherein the cytokine inhibitor molecule is a TGF-beta inhibitor.

[0258] 244. The multifunctional molecule of any of embodiments 242 or 243, wherein the TGF-beta inhibitor inhibits (e.g., reduces the activity of) (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] 245. The multifunctional molecule of any of embodiments 242 to 244, wherein the TGF-beta inhibitor comprises a portion of the TGF-beta receptor (e.g., the extracellular domain of the TGF-beta receptor) that is capable of inhibiting (e.g., reducing the activity of) TGF-beta, or a functional fragment or variant thereof.

[0260] 246. The multifunctional molecule of embodiment 245, wherein the TGF-beta inhibitor comprises a portion 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. The multifunctional molecule of 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. The multifunctional molecule of any of embodiments 1 to 97, wherein the multifunctional molecule comprises a death receptor signal engager selected from a TNF-related apoptosis-inducing ligand (TRAIL) molecule, a death receptor molecule, or an antigen-binding domain that specifically binds to a death receptor.

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

[0264] 250. The multifunctional molecule of any of embodiments 248 or 249, wherein the death receptor signal engager does not activate death receptor signaling on cells other than lymphocytes that contain TCRBV antigens.

[0265] 251. The multifunctional molecule of any of embodiments 248 to 250, wherein the death receptor signal engager comprises a TRAIL molecule, such as 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 of any of embodiments 251 or 252, wherein the TRAIL molecule comprises a truncated TRAIL polypeptide, eg, relative to a wild-type TRAIL polypeptide.

[0267] 254. The multifunctional molecule of embodiment 253, wherein the TRAIL molecule comprises a truncated TRAIL molecule comprising at least residues corresponding to amino acids 95-281 of human TRAIL, such as residues corresponding to amino acids 95-281 of human TRAIL.

[0268] 255. The multifunctional molecule of embodiment 254, wherein the TRAIL molecule comprises a truncated TRAIL polypeptide comprising amino acids 95 to 281 of human TRAIL, e.g., not including amino acids 1 to 94 of human TRAIL.

[0269] 256. The multifunctional molecule of embodiment 253, wherein the TRAIL molecule comprises a truncated TRAIL molecule comprising at least residues corresponding to amino acids 122-281 of human TRAIL, such as residues corresponding to amino acids 122-281 of human TRAIL.

[0270] 257. The multifunctional molecule of embodiment 256, wherein the TRAIL molecule comprises amino acids 122 to 281 of human TRAIL, such as a truncated TRAIL polypeptide that does not include amino acids 1 to 121 of human TRAIL.

[0271] 258. The multifunctional molecule of any of embodiments 251 to 257, wherein the death receptor signal engager comprises one, two, or three TRAIL molecules. 259. The multifunctional molecule of any of embodiments 248 to 250, wherein the death receptor signal engager comprises an antigen-binding domain that specifically binds to a death receptor, e.g., death receptor 4 (DR4) or death receptor 5 (DR5).

[0272] 260. The multifunctional molecule of embodiment 259, wherein the death receptor signal engager comprises one, two, or three antigen-binding domains that specifically bind to the death receptor. 261. The multifunctional molecule of any of embodiments 259 or 260, wherein the antigen-binding domain that specifically binds to a death receptor binds to DR5.

[0273] 262. The multifunctional molecule of any of embodiments 259 to 261, wherein the antigen-binding domain that specifically binds to a death receptor comprises tigatuzumab, drozitumab, or conatumumab.

[0274] 263. The multifunctional molecule of any of embodiments 248 to 262, wherein the 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] 264. The multifunctional molecule of any of embodiments 248 to 263, wherein the 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] 265. The multifunctional molecule of any of embodiments 248 to 263, wherein the 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] 266. The multifunctional molecule of any of embodiments 248 to 263, wherein the 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] 267. The multifunctional molecule of any of embodiments 248 to 263, wherein the 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. The multifunctional molecule of any of embodiments 248 to 263, wherein the 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. The multifunctional molecule of any of embodiments 248 to 263, wherein the 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. The multifunctional molecule of any of embodiments 248 to 263, wherein the 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. The multifunctional molecule of any of embodiments 248 to 263, wherein the 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. The multifunctional molecule of any of embodiments 248 to 263, wherein the 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. The multifunctional molecule of embodiment 102, wherein the T cell engager binds to TCRβ. 274. The multifunctional molecule of embodiment 273, wherein the T cell engager comprises an antigen-binding domain (e.g., an antibody molecule or fragment thereof) that binds to (e.g., and in some embodiments, activates) CD3.

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

[0286] 277. Multifunctional molecules are (i) immune cell engagers (e.g., T cell engagers, NK cell engagers, B cell engagers, dendritic cell engagers, or macrophage cell engagers) and cytokine molecules; (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) immune cell engagers (e.g., T cell engagers, NK cell engagers, B cell engagers, dendritic cell engagers, or macrophage cell engagers) and death receptor signal engagers; (iv) cytokine molecules and death receptor signal engagers; (v) cytokine inhibitor molecules and death receptor signal engagers; (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 death receptor signal engager, or (vii) immune cell engagers (e.g., T cell engagers, NK cell engagers, B cell engagers, dendritic cell engagers, or macrophage cell engagers), cytokine inhibitor molecules, and death receptor signal engagers 277. The multifunctional molecule of any one of embodiments 1 to 276, comprising:

[0287] 278. A multifunctional molecule comprising: A,B-[dimerization module]-C,-D where: (a) the dimerization module comprises an immunoglobulin constant domain, e.g., a heavy chain constant domain (e.g., a homodimeric or heterodimeric heavy chain constant region, e.g., an Fc region), or a constant domain of an immunoglobulin variable region (e.g., a Fab region); (b) A, B, C, and D are, independently, absent or selected from the group consisting of: (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 death receptor signal engager; or (v) a stromal-modifying moiety, with the proviso 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 278. The multifunctional molecule of any one of embodiments 1 to 277, wherein any of the remaining A, B, C, and D are absent or comprise one of an immune cell engager, a cytokine molecule, a cytokine inhibitor molecule, a death receptor signal engager, or a stromal-modifying moiety.

[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, e.g., a T cell engager, e.g., 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, e.g., an NK cell engager, e.g., 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 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, e.g., a T cell engager, e.g., 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, e.g., an NK cell engager, e.g., 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 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, e.g., a T cell engager, e.g., 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 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 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, e.g., a T cell engager, e.g., 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, e.g., a T cell engager, e.g., 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, e.g., a T cell engager, e.g., an anti-CD3 antibody molecule, and (b) a 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 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 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, 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; (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, 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; (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, e.g., an NK cell engager, e.g., an anti-NKp30, anti-NKp46, anti-NKG2D, or anti-CD16 antibody molecule, and (b) a 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, e.g., an NK cell engager, e.g., an anti-NKp30, anti-NKp46, anti-NKG2D, or anti-CD16 antibody molecule, and (b) a stroma-modifying 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, e.g., a T cell engager, e.g., 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, e.g., a T cell engager, e.g., 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, e.g., a T cell engager, e.g., an anti-CD3 antibody molecule, and (b) a 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 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 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, 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; (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, 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; (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, e.g., an NK cell engager, e.g., an anti-NKp30, anti-NKp46, anti-NKG2D, or anti-CD16 antibody molecule, and (b) a 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, e.g., a T cell engager, e.g., 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, e.g., a T cell engager, e.g., 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, e.g., a T cell engager, e.g., an anti-CD3 antibody molecule, and (b) a 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 death receptor signal engager; (40) The multifunctional molecule of embodiment 278, wherein 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 death receptor signal engager.

[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 regions) comprising one or more of paired holes and protrusions ("knobs-in-holes"), electrostatic interactions, or strand exchange.

[0290] 281. The multifunctional molecule of embodiment 280, wherein the one or more immunoglobulin chain constant regions (e.g., Fc regions) comprise an amino acid substitution at a position selected from one or more of 347, 349, 350, 351, 366, 368, 370, 392, 394, 395, 397, 398, 399, 405, 407, or 409 of the Fc region, e.g., of human IgG1, and optionally the one or more immunoglobulin chain constant regions (e.g., Fc regions) comprise an amino acid substitution 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 a combination thereof.

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

[0292] 283. The multifunctional molecule according to 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 comprises Gly and Ser.

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

[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 first antigen-binding domain that binds, e.g., selectively binds, a T cell receptor variable beta (TCRBV), e.g., a TCRBV antigen; and (ii) Death receptor signal activators A multifunctional molecule comprising:

[0297] 291. (i) a first antigen-binding domain that binds, e.g., selectively binds, a T cell receptor variable beta (TCRBV), e.g., a TCRBV antigen; and (ii) cytokine inhibitor molecules, e.g., TGF-beta inhibitors A multifunctional molecule comprising:

[0298] 292. The multifunctional molecule of any of embodiments 1 to 291, wherein the multifunctional molecule binds monovalently to the TCRBV antigen. 293. The multifunctional molecule of any one of embodiments 1 to 291, wherein the multifunctional molecule binds to the TCRBV antigen in a multivalent manner, e.g., bivalent, trivalent, tetravalent, pentavalent, hexavalent, heptavalent, octavalent, nonavalent, or decavalent manner.

[0299] 294. The multifunctional molecule of any of embodiments 2 to 261, wherein the multifunctional molecule binds to a TCRBV antigen on lymphocytes expressing the TCRBV antigen. 295. The multifunctional molecule of any of the preceding embodiments, wherein the multifunctional molecule monovalently binds to immune cells, e.g., via an immune cell engager.

[0300] 296. The multifunctional molecule of any one of embodiments 1 to 294, wherein the multifunctional molecule binds to immune cells multivalently, e.g., bivalently, trivalently, tetravalently, pentavalently, hexavalently, heptavalently, octavalently, nonavalently, or decavalently, e.g., via an immune cell engager.

[0301] 297. The multifunctional molecule of any of the preceding embodiments, further comprising a heavy chain constant region, e.g., an Fc region, that mediates antibody-dependent cellular cytotoxicity (ADCC). 298. The multifunctional molecule of any of the preceding embodiments, further comprising a heavy chain constant region, e.g., an Fc region, that mediates complement dependent cytotoxicity (e.g., via C1q).

[0302] 299. A nucleic acid molecule encoding the multifunctional molecule of any one of embodiments 1 to 298. 300. A vector, for example an expression vector, comprising the nucleic acid molecule of embodiment 299. 301. A host cell comprising the nucleic acid molecule of embodiment 299 or the vector of embodiment 300.

[0303] 302. A method for making, e.g., producing, a multifunctional molecule or antibody molecule of any one of embodiments 1 to 298, comprising culturing a host cell of embodiment 301 under suitable conditions, e.g., conditions suitable for gene expression and / or homo- or heterodimerization.

[0304] 303. A pharmaceutical composition comprising the multifunctional molecule of any one of embodiments 1 to 298 and a pharmaceutically acceptable carrier, excipient, or stabilizer. 304. A method for treating TCR bias, comprising administering to a subject in need thereof the multifunctional molecule of any one of embodiments 1 to 298, wherein the multifunctional molecule is administered in an amount effective to treat the TCR bias.

[0305] 305. A method for treating an autoimmune disease (e.g., an autoimmune disease associated with TCR bias), comprising administering to a subject in need thereof the multifunctional molecule of 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 of any of embodiments 304 or 305, further comprising identifying, assessing, or selecting a subject in need of treatment, wherein the identifying, assessing, or selecting comprises determining (e.g., directly or indirectly determining, e.g., obtaining information regarding) whether the subject has a TCR bias or an autoimmune disease (e.g., an autoimmune disease associated with a TCR bias).

[0307] 307. In response to determining that the subject has a TCR bias or an autoimmune disease (e.g., an autoimmune disease associated with a TCR bias), Optionally, selecting a subject for treatment with a multifunctional molecule comprising an antigen-binding domain that binds to a TCRBV antigen (e.g., a TCRBV antigen corresponding to a biased TCRBV clonotype); 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 clonotype); The method of embodiment 306, further comprising:

[0308] 308. A method for treating TCR bias, comprising: 298. A method comprising, in response to determining that a subject has a TCR bias, administering to a subject in need thereof a multifunctional molecule of 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 for treating an autoimmune disease (e.g., an autoimmune disease associated with TCR bias), comprising: 298. A method comprising, in response to determining that a subject has an autoimmune disease (e.g., an autoimmune disease associated with TCR bias), administering to a subject in need thereof a multifunctional molecule of 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 of any of embodiments 304 to 309, wherein the subject has a TCR bias (e.g., a biased TCRBV clonotype) and / or an autoimmune disease associated with said bias.

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

[0312] 312. The method of embodiment 311, further comprising, 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, treating the subject (e.g., administering to the subject) a multifunctional molecule comprising an antigen-binding domain that binds to a TCRBV antigen.

[0313] 313. A method of assessing a subject in need of treatment for TCR bias (e.g., biased TCRBV clonotype) and / or an autoimmune disease associated with said bias, comprising a step of determining whether the subject has a TCR bias (e.g., a step of directly determining or indirectly determining, e.g., a step of obtaining information regarding).

[0314] 314. The method of embodiment 313, further comprising, in response to the assessment, treating the subject (e.g., administering to the subject) a multifunctional molecule comprising an antigen-binding domain that binds to a TCRBV antigen.

[0315] 315. The method of any one of embodiments 304 to 314, wherein the TCR bias is associated with an autoimmune disease. 316. Autoimmune diseases include 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 neuronal neuropathy), chronic inflammatory demyelinating polyneuropathy (CIDP), transverse myelitis, Tolosa-Hunt syndrome (THS), Devic's disease (neuromyelitis optica), paraneoplastic cerebellar degeneration (PCD), Lambert-Eaton syndrome, psoriasis, scleroderma, CREST (calcinosis, Raynaud's phenomenon, esophageal dysmotility, digital sclerosis, and telangiectasia) syndrome, dermatitis herpetiformis, dermatomyositis, bullous pemphigoid, cicatricial pemphigoid / benign mucous membrane pemphigoid, pemphigoid, rheumatoid arthritis (RA), psoriatic arthritis, Arthritis, relapsing polychondritis, chronic relapsing polymyelitis (CRMO), vasculitis, Kawasaki disease, granulomatosis with polyangiitis (GPA), Behçet's disease (vasculitis), Takayasu's arteritis, polyarteritis nodosa, microscopic polyangiitis (MPA), leukocytoclastic vasculitis, Cogan's syndrome, uveitis, peripheral uveitis (pars planitis), scleritis, autoimmune inner ear disease (AIED), Crohn's disease, ulcerative colitis 316. The method of embodiment 315, wherein the anemia is selected from: uncontrolled myocardial infarction (UC), Dressler's syndrome, rheumatic fever, Evans' syndrome, paroxysmal nocturnal hemoglobinuria (PNH), hemolytic anemia, thrombotic thrombocytopenic purpura (TTP), polymyositis, juvenile myositis (JM), including juvenile dermatomyositis (JDM) and juvenile polymyositis (JPM), Sjogren's syndrome, ocular cicatricial pemphigoid, or Hashimoto's thyroiditis.

[0316] 317. The method of any of embodiments 304 to 316, further comprising 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 biological agent, a hormone therapy), radiation, or surgery.

[0317] 319. A method of treating an autoimmune disease (e.g., an autoimmune disease associated with TCR bias) in a subject in need thereof, comprising administering to the subject an effective amount, e.g., a therapeutically effective amount, of an antibody molecule that binds to (e.g., specifically binds to) a T-cell receptor beta variable region (TCRβV) (an "anti-TCRβV antibody molecule"), thereby treating the disorder.

[0318] 320. A method for depleting a T cell population in a subject having an autoimmune disease (e.g., an autoimmune disease associated with TCR bias), 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] 321. The method of claim 320, wherein the contacting step occurs in vivo or in vitro. 322.Anti-TCRβV antibody molecule, (i) is not an antibody molecule as disclosed in U.S. Patent No. 5,861,155; (ii) binds to TCRβV12 with an affinity and / or binding specificity that is less (e.g., about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or about 2-, 5-, or 10-fold less) than the affinity and / or binding specificity of the 16G8 murine antibody or a humanized version thereof, as described in U.S. Pat. No. 5,861,155; (iii) binds to TCRβV12 with an affinity and / or binding specificity that is greater than (e.g., about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or about 2-, 5-, 10-fold greater) than the affinity and / or binding specificity of the 16G8 murine antibody or a humanized version thereof, as described in U.S. Pat. No. 5,861,155; (iii) TCRβ V5-5 with an affinity and / or binding specificity that is greater than (e.g., about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or about 2-, 5-, 10-fold greater than) the affinity and / or binding specificity of the TM23 murine antibody or a humanized version thereof, as described in U.S. Pat. No. 5,861,155. * 01 or TCRβ V5-1 * Combine with 01; or (iv) TCRβ V5-5 with an affinity and / or binding specificity that is greater than (e.g., about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or about 2-, 5-, 10-fold greater than) the affinity and / or binding specificity of the TM23 murine antibody or a humanized version thereof, as described in U.S. Pat. No. 5,861,155. * 01 or TCRβ V5-1 * 322. The method of any one of claims 319 to 321, wherein the compound binds to .Ol.

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

[0321] 324. The method of claim 323, wherein the anti-TCRβV antibody molecule comprises an Fc region with enhanced effector function, e.g., compared to a wild-type Fc region. 325. The method of 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 of any one of claims 319 or 321 to 325, wherein the autoimmune disease is selected from the group consisting of 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 neuronal neuropathy), chronic inflammatory demyelinating polyneuropathy (CIDP), transverse myelitis, Tolosa-Hunt syndrome (THS), Devic's disease (neuromyelitis optica), paraneoplastic cerebellar degeneration (PCD), Lambert-Eaton syndrome, psoriasis, scleroderma, CREST (calcinosis, Raynaud's phenomenon, esophageal dysmotility, digital sclerosis, and telangiectasia) syndrome, dermatitis herpetiformis, dermatomyositis, bullous pemphigoid, cicatricial pemphigoid / benign mucous membrane pemphigoid. Acne, pemphigoid, rheumatoid arthritis (RA), psoriatic arthritis, relapsing polychondritis, chronic relapsing polymyelitis (CRMO), vasculitis, Kawasaki disease, granulomatosis with polyangiitis (GPA), Behçet's disease (vasculitis), Takayasu's arteritis, polyarteritis nodosa, microscopic polyangiitis (MPA), leukocytoclastic vasculitis, Cogan's syndrome, uveitis, peripheral uveitis (pars planitis), scleritis, autoimmune inner ear disease (AI) ED), Crohn's disease, ulcerative colitis (UC), Dressler's syndrome, rheumatic fever, Evans' syndrome, paroxysmal nocturnal hemoglobinuria (PNH), hemolytic anemia, thrombotic thrombocytopenic purpura (TTP), polymyositis, juvenile myositis (JM), including juvenile dermatomyositis (JDM) and juvenile polymyositis (JPM), Sjogren's syndrome, ocular cicatricial pemphigoid, or Hashimoto's thyroiditis.

[0323] 327. The method of any one of claims 319 to 326, wherein the anti-TCRβV antibody molecule comprises an antigen-binding domain comprising 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.

[0324] 328. The method of any one of claims 319 to 327, wherein the anti-TCRβV antibody molecule comprises a variable heavy chain (VH) and / or variable light chain (VL) provided in Table 1A, 2A, 10A, 11A, 12A, or 13A, or a sequence at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto.

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

[0326] Other features and advantages of the invention will be apparent from the following detailed description and claims. [Brief explanation of the drawings]

[0327] [Figure 1A]Figures 1A-1B show alignments of the mouse VH and VL framework 1, CDR1, framework 2, CDR2, framework 3, CDR3, and framework 4 regions of the antibody A source with their respective humanized sequences. Kabat CDRs are shown in bold, Chothia CDRs in italics, and combined CDRs in boxes. Backmutated framework positions are double underlined. Figure 1A shows the VH sequences of mouse antibody A (SEQ ID NO: 1) and humanized antibody AH (SEQ ID NO: 9). Figure 1B shows the VL sequences of mouse antibody A (SEQ ID NO: 2) and humanized antibody AH (SEQ ID NO: 10 and SEQ ID NO: 11). [Figure 1B] FIG. 1B shows the VL sequences of murine antibody A (SEQ ID NO: 2) and humanized antibody AH (SEQ ID NO: 10 and SEQ ID NO: 11). [Figure 2A] Figures 2A-2B show alignments of the mouse VH and VL framework 1, CDR1, framework 2, CDR2, framework 3, CDR3, and framework 4 regions of antibody B source with their respective humanized sequences. Kabat CDRs are shown in bold, Chothia CDRs in italics, and combined CDRs in boxes. Backmutated framework positions are double underlined. Figure 2A shows the VH sequences of mouse antibody B (SEQ ID NO: 15) and humanized VH sequences BH.1A-BH.1C (SEQ ID NOs: 23-25). Figure 2B shows the VL sequences of mouse antibody B (SEQ ID NO: 16) and humanized VL sequences BH.1D-BH.1H (SEQ ID NOs: 26-30). [Figure 2B-1] FIG. 2B shows the VL sequences of mouse antibody B (SEQ ID NO: 16) and humanized VL sequences BH.1D to BH.1H (SEQ ID NOs: 26 to 30). [Figure 2B-2] FIG. 2B shows the VL sequences of mouse antibody B (SEQ ID NO: 16) and humanized VL sequences BH.1D to BH.1H (SEQ ID NOs: 26 to 30). [Figure 3]FIG. 1 shows a phylogenetic tree of TCRBV gene families and subfamilies to which the corresponding antibodies were mapped. The subfamily identities are as follows: Subfamily A: TCRβ V6; Subfamily B: TCRβ V10; Subfamily C: TCRβ V12; Subfamily D: TCRβ V5; Subfamily E: TCRβ V7; Subfamily F: TCRβ V11; Subfamily G: TCRβ V14; Subfamily H: TCRβ V16; Subfamily I: TCRβ V18; Subfamily J: TCRβ V9; Subfamily K: TCRβ V13; Subfamily L: TCRβ V4; Subfamily M: TCRβ V3; Subfamily N: TCRβ V2; Subfamily O: TCRβ V15; Subfamily P: TCRβ V30; Subfamily Q: TCRβ V19; Subfamily R: TCRβ V27; Subfamily S: TCRβ V28; Subfamily T: TCRβ V24; Subfamily U: TCRβ V20; Subfamily V: TCRβ V25; and subfamily W: TCRβ V29 subfamily. Subfamily members are described in detail herein in the section entitled "TCR beta V (TCRβV)." [Figure 4] Figure 4 is a graph showing binding of NKp30 antibody to NK92 cells. Data was calculated as percent AF747 positive population. [Figure 5] Figure 5 is a graph showing activation of NK92 cells by NKp30 antibodies. Data was generated using a hamster anti-NKp30 mAb. DETAILED DESCRIPTION OF THE INVENTION

[0328] Disclosed herein are multifunctional molecules (sometimes referred to herein as "multispecific molecules") that comprise multiple (e.g., two or more) functionalities (or binding specificities), including: (i) an antigen-binding domain that binds to, e.g., selectively binds to, T-cell receptor variable beta (TCRBV), e.g., a TCRBV antigen; and (ii) one, two, or all of: (a) an immune cell engager selected from a T cell engager, an NK cell engager (e.g., 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) a death receptor signal engager. In some embodiments, the antigen-binding domain comprises a sequence or 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 portion of a sequence found in Tables 7-10. In some embodiments, the antigen binding domain comprises a sequence or 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 portion of a sequence found in Tables 7-10.

[0329] In one embodiment, the multispecific 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 TCRBV antigen on the surface of a lymphocyte, e.g., a T cell. In some embodiments, the TCRBV antigen corresponds to a biased TCRBV clonotype, e.g., a TCR comprising the TCRBV antigen may be overexpressed in the TCR repertoire or lymphocyte (e.g., T cell) pool of a subject (e.g., a subject with an autoimmune disease associated with a TCR bias) or may be expressed at a level higher than in other subjects (e.g., non-autoimmune disease subjects).

[0331] Without being bound by theory, the multispecific or multifunctional molecules disclosed herein are expected to localize (e.g., cross-link) and / or activate immune cells (e.g., immune effector 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) that express, for example, a TCRBV antigen (e.g., a TCRBV antigen corresponding to a biased TCRBV clonotype) on their cell surface. Using the multispecific or multifunctional molecules described herein to increase the proximity and / or activity of immune cells in the presence of cells (e.g., lymphocytes, e.g., T cells) that express a TCRBV antigen (e.g., a TCRBV antigen corresponding to a biased TCRBV clonotype) is expected to enhance the immune response against the target cells, thereby providing more effective therapy (e.g., by reducing the levels of biased TCRs and / or T cells expressing the biased TCR). In another embodiment, targeting a cell (e.g., a lymphocyte, e.g., a T cell) expressing a TCRBV antigen (e.g., a TCRBV antigen corresponding to a biased TCRBV clonotype) with a multifunctional molecule that also includes a cell death-inducing moiety (e.g., a death receptor signal engager) is believed to promote death of the target cell (e.g., by reducing levels of the biased TCR and / or T cells expressing the biased TCR).

[0332] Without being bound by theory, it is expected that in some embodiments, the adverse effects of increasing immune cell proximity or activity to T cells in general or promoting cell death in T cells in general may be mitigated 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 lack specificity for other or all types of T cell receptors. Thus, it is believed that the use of the multispecific or multifunctional molecules disclosed herein may increase immune cell proximity or activity to cells containing a TCRBV antigen corresponding to a biased TCRBV clonotype without necessarily increasing immune cell proximity or activity to T cells in general, or may promote cell death in cells containing a TCRBV antigen corresponding to a biased TCRBV clonotype without necessarily increasing cell death in T cells in general.

[0333] Thus, provided herein are, inter alia, multispecific or multifunctional molecules (e.g., multispecific or multifunctional antibody molecules) comprising the aforementioned portions, nucleic acids encoding same, methods of producing the aforementioned molecules, and methods of using the aforementioned molecules to treat a disease or disorder, e.g., an autoimmune disease or TCR bias.

[0334] definition In some embodiments, the multifunctional molecule comprises an immune cell engager. "Immune cell engager" refers to one or more binding specificities that bind to and / or activate immune cells, e.g., cells involved in an immune response. In embodiments, the immune cells are selected from T cells, NK cells, B cells, dendritic cells, and / or macrophage cells. The immune cell engager can be 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 (e.g., a full-length ligand, a ligand fragment, or a fusion thereof (e.g., a ligand-Fc fusion)) 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). In embodiments, the immune cell engager specifically binds to a target immune cell, e.g., preferentially binds to a 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, "cytokine molecule" refers to a full-length, fragment, or variant of a cytokine; a cytokine further comprising a receptor domain, e.g., a cytokine receptor dimerization domain; or an agonist of a cytokine receptor, e.g., an antibody molecule (e.g., an agonist antibody) against a cytokine receptor, which induces activation of at least one naturally occurring cytokine. 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 gamma, or a fragment or variant thereof, or a combination of any of the aforementioned cytokines. The cytokine molecule may be a monomer or a dimer. In embodiments, the cytokine molecule may further comprise a cytokine receptor dimerization domain. In other embodiments, the cytokine molecule is an agonist of a cytokine receptor, for example, an antibody molecule (eg, an agonist antibody) against a cytokine receptor selected from IL-15Ra or IL-21R.

[0336] As used herein, the term "molecule," as used in, e.g., antibody molecules, cytokine molecules, receptor molecules, includes full-length naturally occurring molecules as well as variants, e.g., functional variants (e.g., truncations, fragments, mutations (e.g., substantially similar sequences) or derivatized forms thereof), so long as 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 inappropriately and / or excessively produced autoantibodies against self- or autoantigens. Autoimmune diseases include, but are not limited to, cardiovascular, rheumatic, glandular, gastrointestinal, skin, liver, neurological, muscular, renal, reproductive, connective tissue, 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 complement-mediated pathways.

[0338] Certain terms are defined below. As used herein, the articles "a" and "an" refer to one or to more than one, e.g., at least one, of the grammatical object of the article. The use of the terms "a" or "an," when used with the term "comprising" herein, can mean "one," 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 "approximately" generally refer to an acceptable degree of error for the quantity measured given the nature or precision of the measurement. Exemplary degrees of error are within 20 percent (%), typically within 10%, and more typically within 5% of a given range of values.

[0340] As used herein, an "antibody molecule" refers to a protein, e.g., an immunoglobulin chain or fragment thereof, that contains at least one immunoglobulin variable domain sequence. Antibody molecules encompass antibodies (e.g., full-length antibodies) and antibody fragments. In certain embodiments, an antibody molecule comprises an antigen-binding or functional fragment of a full-length antibody or a full-length immunoglobulin chain. For example, a full-length antibody is an immunoglobulin (Ig) molecule (e.g., an IgG antibody) that occurs naturally or is formed by the recombinant process of normal immunoglobulin gene fragments. In embodiments, an antibody molecule refers to an immunologically active antigen-binding portion of an immunoglobulin molecule, e.g., an antibody fragment. An antibody fragment, e.g., a functional fragment, is a portion of an antibody, e.g., a Fab, Fab', F(ab')2, F(ab)2, variable fragment (Fv), domain antibody (dAb), or single-chain variable fragment (scFv). A functional antibody fragment binds to the same antigen recognized by an intact (e.g., full-length) antibody. 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, e.g., an Fc fragment or a single amino acid residue. Exemplary antibody molecules include full-length antibodies and antibody fragments, e.g., dAb (domain antibody), 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, e.g., comprises binding specificity for a single epitope. In some embodiments, the antibody molecule is multispecific, e.g., comprises multiple immunoglobulin variable domain sequences, wherein a first immunoglobulin variable domain sequence has binding specificity for a first epitope and a 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 that has specificity for more than one (e.g., two, three, four, or more) epitopes and / or antigens.

[0343] As used herein, "antigen" (Ag) refers to a molecule capable of eliciting an immune response, e.g., involving activation of certain immune cells and / or antibody production. Any macromolecule, including almost any protein or peptide, can be an antigen. Antigens can also be derived from genomic recombinants or DNA. For example, any DNA containing a nucleotide sequence or partial nucleotide sequence encoding a protein capable of eliciting an immune response encodes an "antigen." In embodiments, an antigen need not be encoded solely by the full-length nucleotide sequence of a gene, nor need it be encoded by a gene at all. In embodiments, antigens can be synthesized or derived from a biological sample with other biological components, e.g., a tissue sample, a blood sample, a cell, or a fluid. As used herein, "TCRBV antigen" includes any TCR variable beta chain or portion thereof that can elicit an immune response or be targeted by an antigen-binding domain. In some embodiments, a biased TCR clonotype can be characterized by one or more TCRBV antigens that are displayed by most, e.g., all, of the cells that comprise the clonotype, e.g., on their surface.

[0344] The "antigen-binding site" or "binding site" of an antibody molecule refers to the portion of an antibody molecule, e.g., an immunoglobulin (Ig) molecule, that participates in antigen binding. In embodiments, the antigen-binding site is formed by amino acid residues from the variable regions (V) of the heavy (H) and light (L) chains. Three highly divergent stretches within the variable regions of the heavy and light chains, called hypervariable regions, are located between more conserved adjacent regions called "framework regions" (FR). FRs are amino acid sequences 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 positioned relative to each other in three-dimensional space to form an antigen-binding surface complementary to the three-dimensional surface of a bound antigen. The three hypervariable regions of each of the heavy and light chains are referred to as "complementarity-determining regions" or "CDRs." 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., USDapartment 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 from amino terminus to carboxy terminus in the following amino acid order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.

[0345] As used herein, "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 leukocytes, e.g., neutrophils, eosinophils, basophils, lymphocytes, and monocytes. Innate leukocytes include phagocytes (e.g., macrophages, neutrophils, and dendritic cells), mast cells, eosinophils, basophils, and natural killer cells. Innate leukocytes 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 specialized type of leukocyte 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] The term "immune effector cell," as used herein, refers to a cell that is involved in an immune response, e.g., promoting 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 a specialized function of a cell. The effector function of a T cell can be, for example, cytolytic activity or helper activity, including secretion of cytokines.

[0348] The compositions and methods of the present invention encompass polypeptides and nucleic acids having a specified sequence or a sequence substantially identical or similar thereto, for example, a sequence at least 80%, 85%, 90%, 95% identical, or more identical to the specified sequence. In the context of amino acid sequences, the term "substantially identical" is used herein to refer to a first amino acid sequence containing a sufficient or minimal number of amino acid residues that are i) identical to a second amino acid sequence, or ii) are conservative substitutions for aligned amino acid residues in a second amino acid sequence, such that the first and second amino acid sequences may share 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, such as a sequence provided herein.

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

[0350] The term "variant" refers to a polypeptide having substantially the same amino acid sequence as a reference amino acid sequence or encoded by substantially the same 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 substantially identical to a reference amino acid sequence, or a polypeptide encoded by a substantially identical nucleotide sequence, which may have one or more activations of the reference amino acid sequence.

[0352] Calculations of homology or sequence identity between sequences (the terms are used interchangeably herein) are 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 can be introduced into one or both of the first and second amino acid or nucleic acid sequences, and non-homologous sequences can be ignored 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%, and even more preferably at least 70%, 80%, 90%, or 100% of the length of the reference sequence. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a 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 equivalent to amino acid or nucleic acid "homology").

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

[0354] Comparison of sequences and determination of percent identity between two sequences can be achieved using a mathematical algorithm. In a preferred embodiment, 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 a Blossum62 matrix or a PAM250 matrix, 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, percent identity between two nucleotide sequences is determined using the GAP program of the GCG software package (available at http: / / www.gcg.com) using a NWSgapdna.CMP matrix, 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 which should be used unless otherwise specified) is a Blossum62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5.

[0355] The percent identity between two amino acid 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 a 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 as "query sequences" to conduct searches against public databases, for example, 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, word length=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, word length=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 BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. See http: / / www.ncbi.nlm.nih.gov.

[0357] It is understood that the molecules of the present invention may have additional conservative or non-essential amino acid substitutions which do not substantially affect their function. The term "amino acid" is intended to encompass all molecules, whether natural or synthetic, that contain both amino and acid functionalities and can be included in naturally occurring amino acid polymers. Exemplary amino acids include naturally occurring amino acids; their analogs, derivatives and homologs; amino acid analogs with 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] A "conservative amino acid substitution" is one in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with 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" (when single-chain) are used interchangeably herein to refer to polymers of amino acids of any length. The polymers can be linear or branched, can contain modified amino acids, and can be interrupted by non-amino acids. These terms also encompass amino acid polymers that have been modified; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation, such as conjugation with a labeling component. Polypeptides can be isolated from natural sources, produced by recombinant techniques from eukaryotic or prokaryotic hosts, or can be the product of synthetic techniques.

[0360] The terms "nucleic acid," "nucleic acid sequence," "nucleotide sequence," or "polynucleotide sequence," and "polynucleotide" are used interchangeably. They refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or their analogs. A polynucleotide can be either single-stranded or double-stranded, and if single-stranded, can be the coding strand or the non-coding (antisense) strand. A polynucleotide can comprise 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 does not occur in nature or is linked to another polynucleotide in a non-naturally occurring sequence.

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

[0362] 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, e.g., a TCRBV antigen corresponding to a biased TCRBV clonotype. In some embodiments, the TCRBV antigen is, e.g., a mammalian, e.g., human, TCRBV antigen. In some embodiments, the antibody molecule binds to a TCRBV antigen on a lymphocyte, e.g., a T cell, e.g., a mammalian, e.g., human, lymphocyte, e.g., a T cell. For example, the antibody molecule specifically binds to a TCRBV antigen expressed, e.g., on the surface of a lymphocyte, e.g., a T cell, as part of a TCR that includes TCRBV.

[0363] In one embodiment, the antibody molecule is a monospecific antibody molecule, which binds to a single epitope, e.g., a monospecific antibody molecule has multiple immunoglobulin variable domain sequences that each bind to the same epitope.

[0364] In one embodiment, the antibody molecule is a multispecific or multifunctional antibody molecule, e.g., it comprises multiple immunoglobulin variable domain sequences, wherein a first of the multiple immunoglobulin variable domain sequences has binding specificity for a first epitope and a second of the multiple immunoglobulin variable domain sequences has binding specificity for a second epitope. In one embodiment, the first and second epitopes are on the same antigen, e.g., 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, e.g., different proteins (or different subunits of a multimeric protein). In one embodiment, the multispecific antibody molecule comprises 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 tetraspecific antibody molecule.

[0365] In one embodiment, a multispecific antibody molecule is a bispecific antibody molecule. A bispecific antibody has specificity for only two antigens. A bispecific antibody molecule is characterized by a first immunoglobulin variable domain sequence that has binding specificity for a first epitope and a second immunoglobulin variable domain sequence that has binding specificity for a second epitope. In one embodiment, the first and second epitopes are on the same antigen, e.g., 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, e.g., different proteins (or different subunits of a multimeric protein). In one embodiment, a bispecific antibody molecule comprises heavy and light chain variable domain sequences that have binding specificity for a first epitope and heavy and light chain variable domain sequences that have binding specificity for a second epitope. In one embodiment, a 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, a bispecific antibody molecule comprises a half antibody, or fragment thereof, having binding specificity for a first epitope and a half antibody, or fragment thereof, having binding specificity for a second epitope. In one embodiment, a bispecific antibody molecule comprises an scFv or Fab, or fragment thereof, having binding specificity for a first epitope and an scFv or Fab, or 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 antibodies (e.g., scFv), single variable domain antibodies, diabodies (Dab) (bivalent and bispecific), and chimeric (e.g., humanized) antibodies, which are produced by modification of full-length antibodies or can be derived using recombinant DNA technology. They may be synthesized de novo. These functional antibody fragments retain the ability to selectively bind to their respective antigens or receptors. Antibodies and antibody fragments can be from any class of antibody, including, but not limited to, IgG, IgA, IgM, IgD, and IgE, and from any subclass of antibody (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 can have heavy chain constant regions selected from, for example, IgG1, IgG2, IgG3, or IgG4. Antibodies can also have light chains selected from, for example, kappa or lambda. The term "immunoglobulin" (Ig) is used interchangeably with the term "antibody" herein.

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

[0368] Antibody molecules include intact molecules as well as functional fragments thereof. The constant regions of an antibody molecule 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, the number of cysteine ​​residues, effector cell function, or complement function).

[0369] The antibody molecule may also be a single-domain antibody. Single-domain antibodies can include antibodies whose complementary determining regions are portions of a single-domain polypeptide. Examples include, but are not limited to, heavy-chain antibodies, antibodies naturally lacking 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 may be any known in the art or any future single-domain antibody. The single-domain antibody may 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 present invention, the single-domain antibody is a naturally occurring single-domain antibody known as a heavy-chain antibody lacking light chains. Such single-domain antibodies are disclosed, for example, in WO9404678. For clarity, this variable domain derived from a heavy-chain antibody naturally lacking light chains is known herein as a VHH or nanobody to distinguish it from the conventional VH of four-chain immunoglobulins. Such VHH molecules can be derived from antibodies produced in Camelidae species, such as camel, llama, dromedary, alpaca and guanaco. Other non-Camelidae species may produce heavy chain antibodies that are naturally devoid of light chains, and such VHHs are within the scope of the present invention.

[0370] The VH and VL regions can be further divided into regions of hypervariability called "complementarity-determining regions" (CDRs), interspersed with regions that are more conserved called "framework regions" (FR or FW).

[0371] The extent of framework regions and CDRs has been precisely defined in a number of ways (see Kabat, E.A. et al., (1991) Sequences of Proteins of Immunological Interest, 5th ed., US Department of Health and Human Services, NIH Publication No. 91-3242; Chothia, C. et al., (1987) J. Mol. Biol. 196:901-917; and the AbM definitions used by Oxford Molecular's AbM antibody modeling software. See generally, e.g., "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 "complementarity determining region" and "CDR" as used herein refer to the amino acid sequences in an antibody variable region 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 can be determined using any of a number of known schemes, including those described in Kabat et al. (1991) "Sequences of Proteins of Immunological Interest," 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD ("Kabat" numbering scheme); Al-Lazikani et al. (1997) JMB 273, 927-948 ("Chothia" numbering scheme). As used herein, CDRs defined according to the "Chothia" numbering scheme are sometimes referred to as "hypervariable loops."

[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 the VH are numbered 26-32 (HCDR1), 52-56 (HCDR2), and 95-102 (HCDR3), and the amino acid residues in the VL are numbered 26-32 (LCDR1), 50-52 (LCDR2), and 91-96 (LCDR3).

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

[0376] The antibody molecule may be a polyclonal or monoclonal antibody. The terms "monoclonal antibody" or "monoclonal antibody composition," as used herein, refer to a preparation of antibody molecules of single molecular composition. A monoclonal antibody composition displays 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 recombinantly produced, 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. Pat. No. 5,223,409; Kang et al., International Publication No. WO 92 / 18619; Dower et al., International Publication No. WO 91 / 17271; Winter et al., International Publication No. WO 92 / 20791; Markland et al., International Publication No. WO 92 / 15679; Breitling et al., International Publication No. WO 93 / 01288; McCafferty et al., International Publication No. WO 92 / 01047; Garrard et al., International Publication No. WO 92 / 09690; Ladner et al., International Publication No. WO 90 / 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; Griffths 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 which are hereby incorporated by reference.

[0378] In one embodiment, the antibody is a fully human antibody (e.g., an antibody made in a mouse genetically engineered to produce antibodies from human immunoglobulin sequences), or a non-human antibody, such as a rodent (mouse or rat), goat, primate (e.g., monkey), or 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 carrying the human immunoglobulin genes rather than the mouse system. Spleen cells from these transgenic mice immunized with an antigen of interest are used to produce hybridomas secreting human mAbs with specific affinity for epitopes from human proteins (see, e.g., Wood et al., International Application WO 91 / 00906; Kucherlapati et al., PCT International Publication WO 91 / 10741; Lonberg et al., International Application WO 92 / 03918; Kay et al., International Application 92 / 03917; Lonberg, N. et al., 1994 Nature 368:856-859; Green, LL et al., 1994 Nature Genet. 7:13-21; Morrison, SL 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 may be one in which the variable region, or a portion thereof, such as the CDR, is generated in a non-human organism, such as a rat or a mouse. Chimeric, CDR-grafted, and humanized antibodies are within the scope of the present invention. Antibody molecules generated in a non-human organism, such as a rat or a mouse, and then modified, for example, in the variable framework or constant region to reduce antigenicity in humans are within the scope of the present invention.

[0381] An "effectively human" protein is one that does not substantially elicit a neutralizing antibody response, e.g., a human anti-mouse antibody (HAMA) response. HAMA can be problematic in many situations, for example, when antibody molecules are administered repeatedly, for example, in the treatment of chronic or recurring disease states. HAMA responses can render repeated antibody administration potentially ineffective due to increased antibody clearance from serum (see, e.g., Saleh et al., Cancer Immunol. Immunother., 32:180-190 (1990)) and also due to potential allergic reactions (see, e.g., 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 No. 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 WO 86 / 01533; Cabilly et al., U.S. Pat. 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, but generally all three, recipient CDRs (of the heavy and / or light immunoglobulin chains) replaced with donor CDRs. The antibody may have at least a portion of a non-human CDR replaced, 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 antigen binding. Preferably, the donor is a rodent antibody, e.g., a rat or mouse antibody, and the recipient is a human framework or human consensus framework. Typically, the immunoglobulin providing the CDRs is referred to as the "donor," and the immunoglobulin providing the framework is referred to as 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 more identical thereto, 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 the consensus immunoglobulin sequence.

[0385] Antibody molecules can be humanized by methods known in the art (see, e.g., Morrison, SL, 1985, Science 229:1202-1207; Oi et al., 1986, BioTechniques 4:214; and Queen et al., U.S. Pat. Nos. 5,585,089, 5,693,761, and 5,693,762, the 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-substitution, in which one, two, or all 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 contents of all of which are hereby expressly incorporated herein by reference). Winter describes a CDR-grafting method that can be used to prepare the humanized antibodies of the present invention (UK Patent Application No. 2188638A, filed March 26, 1987; Winter, U.S. Patent No. 5,225,539) (the contents of which are hereby expressly incorporated herein by reference).

[0387] Humanized antibody molecules in which specific amino acids are substituted, deleted, or added are also within the scope of the present invention. Criteria for selecting amino acids from donors 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., EP 519596 A1, published December 23, 1992.

[0388] The antibody molecule may be a single-chain antibody. Single-chain antibodies (scFV) may be engineered (see, e.g., Colcher, D. et al., (1999) Ann NY Acad Sci 880:263-80; and Reiter, Y., (1996) Clin Cancer Res 2:245-52). Single-chain antibodies can be dimerized or multimerized to generate multivalent antibodies with specificities 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, IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE heavy chain constant regions, particularly, for example, IgG1, IgG2, IgG3, and IgG4 (e.g., human) heavy chain constant regions. In another embodiment, the antibody molecule has a light chain constant region selected from, for example, kappa or lambda (e.g., human) light chain constant regions. 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, the number of cysteine ​​residues, effector cell function, and / or complement function). In one embodiment, the antibody has effector function and can fix complement. In another embodiment, the antibody does not recruit effector cells and does not fix complement. In another embodiment, the antibody has reduced or no ability to bind to Fc receptors. For example, it may be an isotype or subtype, fragment or other mutant that does not support binding to an Fc receptor, e.g., it has a mutagenized or deleted Fc receptor binding region.

[0390] Methods for modifying antibody constant regions are known in the art. Antibodies with altered functions, such as altered affinity for effector ligands, such as FcR on cells, or 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, for example, EP388,151A1, U.S. Pat. No. 5,624,821, and U.S. Pat. No. 5,648,260, the contents of all of which are hereby incorporated by reference). Similar types of modifications can be described that reduce or eliminate these functions when applied to murine or other species' immunoglobulins.

[0391] Antibody molecules can be derivatized or linked 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 radionucleotide, a toxin, an enzyme, or an affinity ligand, such as biotin. Thus, the antibody molecules of the present invention are intended to include derivatized and otherwise modified forms of the antibodies described herein, including immunoadhesion molecules. For example, an antibody molecule can be functionally linked (by chemical coupling, genetic fusion, noncovalent association, or otherwise) to one or more other molecular entities, such as 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 of the antibody or antibody portion with another molecule (e.g., a streptavidin core region or a polyhistidine tag).

[0392] One type of derivatized antibody molecule is produced by crosslinking two or more antibodies (of the same or different types, e.g., to create bispecific antibodies). Suitable crosslinkers include those that are heterobifunctional, with two independently reactive groups separated by an appropriate spacer (e.g., m-maleimidobenzoyl-N-hydroxysuccinimide ester) or homobifunctional (e.g., disuccinimidyl suberate). Such linkers are available from Pierce Chemical Company, Rockford, Ill.

[0393] Multispecific or multifunctional antibody molecules Exemplary structures of 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 which are hereby incorporated by reference).

[0394] In embodiments, a multispecific antibody molecule can contain more than one antigen-binding site, with different sites specific for different antigens. In embodiments, a multispecific antibody molecule can bind to more than one (e.g., two or more) epitopes on the same antigen. In embodiments, a multispecific antibody molecule contains an antigen-binding site specific for a target cell (e.g., a lymphocyte (e.g., 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, a 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 additional antigen-binding moieties appended, (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-hole assembly, charge pair, Fab arm exchange, SEED body, triomab, LUZ-Y, Fcab, κλ body, and orthogonal Fab. See Spiess et al., Mol. Immunol. 67(2015):95-106. Exemplary BsIgGs include catumaxomab (Fresenius Biotech, Trion Pharma, Neopharm), which contains an anti-CD3 arm and an anti-EpCAM arm, and ertumaxomab (Neovii Biotech, Fresenius Biotech), which targets CD3 and HER2. In some embodiments, the BsIgG contains a heavy chain engineered for heterodimerization. For example, heavy chains can be engineered for heterodimerization using the "knobs-into-holes" strategy, the SEED platform, a common heavy chain (e.g., in κλ bodies), and the use of heterodimeric Fc regions. See Spiess et al., Mol. Immunol. 67(2015):95-106. Strategies that have been used to avoid homodimeric heavy chain pairing in BsIgG include knobs-in-holes, duobodies, azymetrics, charge pairs, HA-TF, SEED bodies, and differential Protein A affinity. See ibid. BsIgG can be produced by separate expression of component antibodies in different host cells and subsequent purification / assembly into BsIgG. BsIgG can also be produced by expression of component antibodies in a single host cell. BsIgG can be purified using affinity chromatography, for example, using Protein A and sequential pH elution.

[0396] IgG with an additional antigen-binding moiety added is another format of bispecific antibody molecule. For example, a monospecific IgG can be engineered to have bispecificity by adding an additional antigen-binding unit to the monospecific IgG, for example, at 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 chains or variable light chains), engineered protein scaffolds, and paired antibody variable domains (e.g., single-chain variable fragments or variable fragments). See ibid. Examples of 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 an IgG-scFv is MM-141 (Merrimack Pharmaceuticals), which binds to IGF-1R and HER3. Examples of DVD-Igs include ABT-981 (AbbVie), which binds to IL-1α and IL-1β, and ABT-122 (AbbVie), which binds to TNF and IL-17A.

[0397] Bispecific antibody fragments (BsAbs) are a format of bispecific antibody molecule that lacks some or all of the antibody constant domains. For example, some BsAbs lack the Fc region. In embodiments, bispecific antibody fragments comprise heavy and light chain regions connected by a peptide linker that allows efficient expression of the BsAb in a single host cell. Exemplary bispecific antibody fragments include, but are not limited to, nanobodies, nanobody-HAS, BiTEs, diabodies, DARTs, TandAbs, scDiabodies, scDiabody-CH3s, diabody-CH3s, triplebodies, miniantibodies, minibodies, TriBiminibodies, scFv-CH3 KIHs, Fab-scFvs, scFv-CH-CL-scFvs, F(ab')2, F(ab')2-scFv2s, scFv-KIHs, Fab-scFv-Fc, tetravalent HCAbs, scDiabody-Fc, diabody-Fc, tandem scFv-Fc, and intrabodies. See ibid. For example, the BiTE format comprises tandem scFvs, where the component scFvs bind to CD3 on T cells and to the TCRBV antigen on lymphocytes, e.g., T cells.

[0398] Bispecific fusion proteins include, for example, antibody fragments linked to other proteins to add additional specificity and / or functionality. An example of a bispecific fusion protein is immTAC, which contains an anti-CD3 scFv linked 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. Fusion to albumin-binding proteins or human serum albumin can also extend the serum half-life of antibody fragments. See Id.

[0399] In embodiments, chemical conjugation, e.g., chemical conjugation of antibodies and / or antibody fragments, can be used to generate BsAb molecules. See ibid. An exemplary bispecific antibody conjugate includes 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, the 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 ibid.

[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 scaffolds In some embodiments, the antibody molecule is a CDR-grafted scaffold domain. In some embodiments, 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 smallest functional antibody fragment, the variable domain of an antibody heavy chain. There are three loops at the end of Fn3, and the positions of the BC, DE, and FG loops roughly correspond to those of CDR1, 2, and 3 of the VH domain of an antibody. Fn3 does not have disulfide bonds, and therefore, unlike antibodies and their fragments, Fn3 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 domains that bind to, for example, the antigens / markers / cells 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). A "minibody" can be used to display two hypervariable loops. In embodiments, the scaffold domain is a V-like domain (see, e.g., Coia et al., WO 99 / 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 domains that bind to, for example, the markers / antigens / cells 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, ecotin, BPTI, etc.), TPR repeats, trifoil structures, zinc finger domains, DNA-binding proteins, particularly monomeric DNA-binding proteins, RNA-binding proteins, enzymes, e.g., proteases (particularly inactivating proteases), RNases, chaperones, e.g., thioredoxin, and heat shock proteins, and intracellular signaling domains (e.g., SH2 and SH3 domains). See, e.g., U.S. Patent Application Publication No. 20040009530 and U.S. Patent No. 7,501,121, incorporated herein by reference.

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

[0405] Antibody-Based Fusions A variety of antibody formats can be generated containing additional binding entities attached to the N- or C-terminus of the antibody. These fusions with single-chain or disulfide-stabilized Fv or Fab result in the generation of tetravalent molecules with bivalent binding specificities for each antigen. The combination of scFv and scFab with IgG allows the production of molecules capable of recognizing three or more different antigens.

[0406] Antibody-Fab fusion An antibody-Fab fusion is a bispecific antibody that contains a traditional 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 Fab share a common light chain. Antibody fusions 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, antibody-scFv fusions 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 Antibody-scFv fusions are bispecific antibodies containing a traditional 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 of the scFv heavy chain either directly or via a linker peptide. Antibody fusions 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, antibody-scFv fusions 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 A related format is the dual variable domain immunoglobulin (DVD), which consists of a VH and VL domain of a second specificity positioned N-terminal to the V domain by a shorter linker sequence.

[0409] Other exemplary multispecific antibody formats include, for example, those described in U.S. Patent Application Publication No. 20160114057A1, U.S. Patent Application Publication No. 20130243775A1, U.S. Patent Application Publication No. 20140051833, U.S. Patent Application Publication No. 20130022601, U.S. Patent Application Publication No. 20150017187A1, U.S. Patent Application Publication No. 20120201746A1, U.S. Patent Application Publication No. 20150133638A1, U.S. Patent Application Publication No. 20130266568A1, U.S. Patent Application Publication No. 20160145340A1, WO2015127158A1, U.S. Patent Application Publication No. 20150203591 ... WO20150203591A1, U. 40322221A1, U.S. Patent Application Publication No. 20130303396A1, U.S. Patent Application Publication No. 20110293613, U.S. Patent Application Publication No. 20130017200A1, U.S. Patent Application Publication No. 20160102135A1, WO2015197598A2, WO2015197582A1, U.S. Patent No. 9,359,437, U.S. Patent Application Publication No. 20150018529, WO2016115274A1, WO2016087416A1, U.S. Patent Application Publication No. 20080069820A1, U.S. Patent No. 9,145,588B, U.S. Patent No. 7,919,257, and U.S. Patent Application Publication No. 20150232560A1. Exemplary multispecific molecules utilizing a full-length antibody-Fab / scFab format include those described in the following: U.S. Patent No. 9,382,323 B2, U.S. Patent Application Publication No. 20140072581 A1, U.S. Patent Application Publication No. 20140308285 A1, U.S. Patent Application Publication No. 20130165638 A1, U.S. Patent Application Publication No. 20130267686 A1, U.S. Patent Application Publication No. 20140377269 A1, U.S. Patent No. 7,741,446 B2, and WO1995009917 A1.Exemplary multispecific molecules utilizing the domain-swapping format include those described in the following: U.S. Patent Application Publication No. 20150315296A1, WO2016087650A1, U.S. Patent Application Publication No. 20160075785A1, WO2016016299A1, U.S. Patent Application Publication No. 20160130347A1, U.S. Patent Application Publication No. 20150166670, U.S. Patent No. 8703132B2, U.S. Patent Application Publication No. 20100316645, U.S. Patent No. 8227577B2, U.S. Patent Application Publication No. 20130078249.

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

[0411] Fc-containing multispecific molecules In some embodiments, the multispecific molecules disclosed herein comprise 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., Fc region) is altered, e.g., mutated, to increase or decrease one or more of Fc receptor binding, antibody glycosylation, the number of cysteine ​​residues, effector cell function, or complement function.

[0413] In other embodiments, the interface of the first and second immunoglobulin chain constant regions (e.g., first and second Fc regions) is altered, e.g., mutated, to increase or decrease dimerization, e.g., compared to an unengineered interface, e.g., a naturally occurring interface. For example, dimerization of immunoglobulin chain constant regions (e.g., Fc regions) can be enhanced by providing one or more of paired holes and protrusions ("knobs-in-holes"), electrostatic interactions, or strand exchange at the Fc interface of the first and second Fc regions, thereby forming a higher ratio of heteromultimers:homomultimers, e.g., compared to an unengineered interface.

[0414] In some embodiments, the multispecific molecule comprises paired amino acid substitutions, e.g., in the Fc region of human IgG1, 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. For example, the immunoglobulin chain constant region (e.g., Fc region) can comprise paired amino acid substitutions selected from T366S, L368A, or Y407V (e.g., corresponding to a pore or hole), and T366W (e.g., corresponding to a protrusion or knob).

[0415] In other embodiments, the multifunctional molecule comprises a half-life extender, for example, human serum albumin or an antibody molecule against human serum albumin. Heterodimerized antibody molecules and methods of production 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 for making said multispecific antibodies are also disclosed, for example, in Speiss et al., Molecular Immunology 67 (2015) 95-106, and Klein et al., mAb 4:6, 653-663, November / December 2012 (the contents of each of which are hereby incorporated by reference).

[0416] Heterodimerized bispecific antibodies are based on the natural IgG structure, with the two binding arms recognizing different antigens. IgG-derived formats that allow for defined monovalent (and simultaneous) antigen binding are generated by forced heavy chain heterodimerization combined with techniques that minimize light chain mispairing (e.g., common light chains). Forced heavy chain heterodimerization can be achieved, for example, using knobs-in-holes or strand-exchange engineered domains (SEEDs).

[0417] Knob-in-Hole Knobs-in-holes, as described in U.S. Patent Nos. 5,731,116, 7,476,724, and Ridgway, J. et al. (1996) Prot. Engineering 9(7):617-621, generally involve (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 parent antibody with a larger one (e.g., T366Y or T366W), while the "hole" or "pore" is created by replacing a larger residue in the parent antibody with a smaller one (e.g., Y407T, T366S, L368A, and / or Y407V).

[0418] For bispecific antibodies containing Fc domains, the introduction of specific mutations into the constant region of the heavy chain to promote correct heterodimerization of the Fc portion can be utilized. Several such techniques are reviewed in Klein et al. (mAb (2012) 4:6, pp. 1-11), the contents of which are hereby incorporated by reference in their entirety. These techniques include the "knob-into-hole" (KiH) approach, which involves 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 correct heavy chain pairing (see, e.g., U.S. Pat. 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, along with additional optional stabilizing Fc cysteine ​​mutations, are provided in Table 1.

[0420] [Table 1]

[0421] Other Fc mutations were provided by Igawa and Tsunoda, who identified three negatively charged residues in the CH3 domain of one chain that paired with three positively charged residues in the CH3 domain of the other chain. These specific pairs of charged residues were E356-K439, E357-K370, D399-K409, and vice versa. Introducing three mutations in chain A: E356K, E357K, and D399K, plus at least two of K370E, K409D, and K439E in chain B, alone or in combination with newly identified disulfide bridges, could favor highly 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 maximal heterodimerization, defining the combination 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 to promote heterodimerization of multispecific antibodies include those described in the following references, the contents of each of which are hereby incorporated by reference: WO2016071377A1, U.S. Patent Application Publication No. 20140079689A1, U.S. Patent Application Publication No. 20160194389A1, U.S. Patent Application Publication No. 20160257763 No. 2016071376A2, WO2015107026A1, WO2015107025A1, WO2015107015A1, U.S. Patent Application Publication No. 20150353636A1, U.S. Patent Application Publication No. 20140199294A1, U.S. Patent No. 7750128B2, U.S. Patent Application Publication No. 20160229915A1, U.S. Patent Application Publication No. 2015034 4570A1, U.S. Patent No. 8003774A1, U.S. Patent Application Publication No. 20150337049A1, U.S. Patent Application Publication No. 20150175707A1, U.S. Patent Application Publication No. 20140242075A1, U.S. Patent Application Publication No. 20130195849A1, U.S. Patent Application Publication No. 20120149876A1, U.S. Patent Application Publication No. 20140200331A1 No. 9,309,311 B2, U.S. Pat. No. 8,586,713, U.S. Patent Application Publication No. 20140037621 A1, U.S. Patent Application Publication No. 20130178605 A1, U.S. Patent Application Publication No. 20140363426 A1, U.S. Patent Application Publication No. 20140051835 A1, and U.S. Patent Application Publication No. 20110054151 A1.

[0423] Stabilizing cysteine ​​mutations have also been used in combination with KiH and other Fc heterodimerization-promoting variants (see, e.g., U.S. Patent No. 7,183,076). Other exemplary cysteine ​​modifications include those disclosed in, e.g., U.S. Patent Application Publication No. 20140348839A1, U.S. Patent No. 7,855,275B2, and U.S. Patent No. 9,000,130B2.

[0424] Strand exchange engineering domain (SEED) A heterodimeric Fc platform is known that supports the design of bispecific and asymmetric fusion proteins by engineering strand-exchange engineered domain (SEED) C(H)3 heterodimers. These derivatives of human IgG and IgA C(H)3 domains create complementary human SEED C(H)3 heterodimers composed of alternating segments of human IgA and IgG C(H)3 sequences. The resulting SEED C(H)3 domain pairs preferentially associate to form heterodimers when expressed in mammalian cells. SEED body (Sb) fusion proteins consist of [IgG1 hinge]-C(H)2-[SEED C(H)3] optionally genetically linked to one or more fusion partners (see, e.g., 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. Pat. No. 8,871,912, the contents of each of which are hereby incorporated by reference).

[0425] Duo Body "Duobody" technology for producing bispecific antibodies with correct heavy chain pairing is known. Duobody technology involves three basic steps to generate stable bispecific human IgG1 antibodies in a post-production exchange reaction. In the first step, two IgG1s, each containing a single matching mutation in the third constant (CH3) domain, are produced separately using a standard mammalian recombinant cell line. These IgG1 antibodies are then 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 produce a bispecific antibody product with very high yields (typically >95%) (see, e.g., 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 interactions A method for producing a multispecific antibody has been disclosed in which CH3 amino acid changes are made with charged amino acids so that homodimer formation is electrostatically unfavorable. EP1870459 and WO2009089004 describe other strategies for favoring heterodimer formation during co-expression of different antibody domains in a host cell. In these methods, one or more residues that make up the CH3-CH3 interface in the heavy chain constant domain 3 (CH3), both CH3 domains, are replaced with charged amino acids so that homodimer formation is electrostatically unfavorable and heterodimerization is electrostatically favorable. Additional methods of generating multispecific molecules using electrostatic interactions are described in references including U.S. Patent Application Publication No. 20100015133, U.S. Patent No. 8,592,562 B2, U.S. Patent No. 9,200,060 B2, U.S. Patent Application Publication No. 20140154254 A1, and U.S. Patent No. 9,358,286 A1, the contents of each of which are hereby incorporated by reference herein.

[0427] Common light chain Light chain mispairing must be avoided to produce a homogeneous preparation of bispecific IgG. One way to achieve this is through the use of the common light chain principle, i.e., combining two binders that share one light chain but still have separate specificities. An exemplary method for enhancing the formation of the desired bispecific antibody from a mixture of monomers is by providing a common variable light chain to interact with each of the heteromeric variable heavy chain regions of the bispecific antibody. Compositions of bispecific antibodies with a common light chain and methods for their production are disclosed, for example, in U.S. Pat. No. 7,183,076 B2, U.S. Patent Application Publication No. 20110177073 A1, EP2847231 A1, WO2016079081 A1, and EP3055329 A1 (the contents of each of which are hereby incorporated by reference).

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

[0429] Common heavy chain An exemplary method for enhancing the formation of a desired bispecific antibody from a mixture of monomers is by providing a common variable heavy chain for interaction 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.

[0430] Amino acid modifications Alternative compositions of multispecific antibodies with correct light chain pairing and their production methods include various amino acid modifications.For example, Zymeworks describes a heterodimer that has one or more amino acid modifications in CH1 and / or CL domain, one or more amino acid modifications in VH and / or VL domain, or a combination thereof, which are the interface between light chain and heavy chain, and create preferential pairing between each heavy chain and desired light chain, so that when the 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 other (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 and kappa light chain polypeptides can be used to enable heterodimerization. Methods for generating bispecific antibody molecules comprising lambda and kappa light chain polypeptides are disclosed in PCT / US17 / 53053, filed September 22, 2017, which is hereby incorporated by reference in its entirety.

[0432] In embodiments, multispecific molecules include multispecific antibody molecules, e.g., antibody molecules that comprise two binding specificities, e.g., bispecific antibody molecules. Multispecific antibody molecules include: Lambda light chain polypeptide 1 (LLCP1) specific for the first epitope; heavy chain polypeptide 1 (HCP1) specific for the first epitope; kappa light chain polypeptide 2 (KLCP2) specific for a second epitope, and Heavy chain polypeptide 2 (HCP2) specific for a second epitope Includes.

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

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

[0436] "Heavy chain polypeptide 2 (HCP2)," as that term is used herein, refers to a polypeptide that includes sufficient heavy chain (HC) sequence, e.g., HC variable region sequence, that, when combined with its cognate LLCP1, is capable of mediating specific binding to its epitope and complexing with HCP1. 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 sequences therefrom sufficient to (i) mediate specific binding of its epitope and complex with KLCP2, (ii) preferentially complex to KLCP2 as opposed to LLCP1, as described herein, and (iii) preferentially complex to HCP1 as opposed to another molecule of HCP2, as described herein. HCP2, together with its KLCP2, provides specificity for the second epitope (LLCP1, together with its HCP1, provides specificity for the 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 greater than its affinity for HCP2, such that under preselected conditions, e.g., in an aqueous buffer, e.g., at pH 7, e.g., in saline, e.g., at pH 7, or under physiological conditions, at least 75, 80, 90, 95, 98, 99, 99.5, or 99.9% of the multispecific antibody molecule has 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 greater than its affinity for a second molecule, such that under preselected conditions, e.g., in an aqueous buffer, e.g., at pH 7, e.g., in saline, e.g., 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 or associated with HCP2.

[0441] In another aspect, disclosed herein is a method for generating or producing a multispecific antibody molecule, the method comprising: (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), a first CH1, a first heavy chain constant region (e.g., a first CH2, a 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), a second CH1, a second heavy chain constant region (e.g., a second CH2, a second CH3, or both)); (iii) providing a lambda chain polypeptide (e.g., a lambda light chain variable region (VLλ), a lambda light chain constant chain (VLλ), or both) that preferentially associates with a first heavy chain polypeptide (e.g., a first VH); and (iv) providing a kappa chain polypeptide (e.g., a lambda light chain variable region (VLκ), a lambda light chain constant chain (VLκ), or both) that preferentially associates with a second heavy chain polypeptide (e.g., a second VH). Includes.

[0442] In embodiments, the first and second heavy chain polypeptides form an Fc interface 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 cells.

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

[0445] In embodiments, the method further comprises evaluating the cell-expressed multispecific antibody molecules. For example, the purified cell-expressed multispecific antibody molecules can be analyzed by techniques known in the art, including mass spectrometry. In one embodiment, the purified cell-expressed antibody molecules are cleaved, for example, digested with papain, to produce Fab portions, which are evaluated using mass spectrometry.

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

[0447] In other embodiments, the multispecific, e.g., bispecific, antibody molecule comprises: (i) a first heavy chain polypeptide (HCP1) (e.g., 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 (e.g., a first CH2, a first CH3, or both)), e.g., HCP1, which binds to a first epitope; (ii) a second heavy chain polypeptide (HCP2) (e.g., 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 (e.g., a second CH2, a second CH3, or both)), e.g., an HCP2 that binds to a second epitope; (iii) a lambda light chain polypeptide (LLCP1) (e.g., a lambda light chain variable region (VL1), a lambda light chain constant chain (VL1), or both) that preferentially associates with a first heavy chain polypeptide (e.g., a first VH), e.g., LLCP1 that binds a first epitope; and (iv) a kappa light chain polypeptide (KLCP2) (e.g., 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 (e.g., a second VH), e.g., KLCP2 that binds a second epitope. Includes.

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

[0449] TCR beta V antigen-binding domain Diversity in the immune system allows for defense against a vast array of pathogens. Because the size of the germline genome is limited, diversity is achieved not only through the process of V(D)J recombination, but also through deletion of nucleotide junctions (the junctions between the VD and DJ segments) and pseudorandom, non-templated addition of nucleotides. TCR beta genes undergo 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-associated polymorphisms encompassing two V beta gene segments. The present disclosure provides, inter alia, antibody molecules and fragments thereof that bind, e.g., specifically bind, to, e.g., human TCR beta V chains (TCRβV), e.g., TCRβV gene families (also referred to as groups), e.g., TCRβV subfamilies (also referred to as subgroups), e.g., as described herein. TCR beta V families and subfamilies are known in the art and are described, for example, 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, e.g., recombinant non-murine antibodies, e.g., recombinant human or humanized antibodies.

[0451] In some aspects, the present disclosure provides anti-TCRβ antibody molecules that bind to a human TCRβV, e.g., a TCRβV family, e.g., a gene family, or variants thereof. In some embodiments, the TCRβV gene family includes one or more subfamilies, e.g., as described herein, e.g., as described in Figure 3, Table 8A, or 8B. In some embodiments, the TCRβ V gene family is selected from the group consisting of 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, TCRβ 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 V1 subfamily, TCRβ V17 subfamily, TCRβ V21 subfamily, TCRβ V23 subfamily, or 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 also known as 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 a variant thereof. In some embodiments, the TCRβ V6 comprises TCRβ V6-4 * 01, or a variant thereof. In some embodiments, the TCRβ V6 comprises TCRβ V6-4 * 02, or a variant thereof. In some embodiments, the TCRβ V6 comprises TCRβ V6-9 * 01, or a variant thereof. In some embodiments, the TCRβ V6 comprises TCRβ V6-8 * 01, or a variant thereof. In some embodiments, the TCRβ V6 comprises TCRβ V6-5 * 01, or a variant thereof. In some embodiments, the TCRβ V6 comprises TCRβ V6-6 * 02, or a variant thereof. In some embodiments, the TCRβ V6 comprises TCRβ V6-6 * 01, or a variant thereof. In some embodiments, the TCRβ V6 comprises TCRβV6-2 * 01, or a variant thereof. In some embodiments, the TCRβ V6 comprises TCRβ V6-3 * 01, or a variant thereof. In some embodiments, the TCRβ V6 comprises TCRβ V6-1 * 01, or any variant thereof.

[0453] In some embodiments, TCRβ V6 is TCRβ V6-5 * 01, or a variant 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 also known as TCRβ V10-1 * 01. TCRβ V10-1 * 02. TCRβ V10-3 * 01 or TCRβ V10-2 * 01, or any variant thereof.

[0455] In some embodiments, the TCRβ V12 subfamily is also known as TCRβ V8.1. In some embodiments, the TCRβ V12 subfamily is also known as TCRβ V12-4 * 01. TCRβ V12-3 * 01, or TCRβ V12-5 * 01, or variants thereof. In some embodiments, TCR β V12 is recognized by, e.g., bound by, SEQ ID NO: 15 and / or SEQ ID NO: 16. In some embodiments, TCR β V12 is recognized by, 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 a variant 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. TCRβ 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 any variant thereof.

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

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

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

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

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

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

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

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

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

[0467] In some embodiments, the TCRβ V29 subfamily is TCRβ V29-1 * 01, or any variant thereof.

[0468] [Table 2]

[0469] [Table 3]

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

[0471] In some embodiments, the anti-TCRβV antibody molecules disclosed herein do not recognize, eg, 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 TCRβV region is JOVI.1, described in 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 (e.g., specifically bind) to the TCRβV region. In some embodiments, binding of the anti-TCRβV antibody molecules disclosed herein results in a cytokine profile that differs from the cytokine profile of a T cell engager that binds to a receptor or molecule other than the TCRβV region (a "non-TCRβV-binding T cell engager"). 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 present disclosure provides an anti-TCRβV antibody molecule that binds to one or more human TCRβVs, e.g., TCRβV gene families, e.g., TCRβV subfamilies, e.g., as described herein, e.g., in Figure 3, Table 8A, or Table 8B. In some embodiments, the anti-TCRβV antibody molecule is selected from the group consisting of 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, TCRβ 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 V1 subfamily, TCRβ V17 subfamily, TCRβ V21 subfamily, TCRβ It binds to one or more TCRβ V subfamilies selected from the V23 subfamily, or the TCRβ V26 subfamily, or variants thereof.

[0475] In some embodiments, the anti-TCRβV antibody molecule binds to a 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 a variant thereof. In some embodiments, the TCRβ V6 subfamily comprises TCRβ V6-5*01, or a variant thereof. In some embodiments, the TCRβ V6 comprises TCRβ V6-4*01, or a variant thereof. In some embodiments, the TCRβ V6 comprises TCRβ V6-4*02, or a variant thereof. In some embodiments, the 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.

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

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

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

[0479] In some embodiments, the anti-TCRβ 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-fold lower) than the affinity and / or binding specificity of the 16G8 murine antibody or a humanized version thereof described in U.S. Pat. No. 5,861,155.

[0480] In some embodiments, the anti-TCRβ antibody molecule binds to TCRβ V12 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 16G8 murine antibody or a humanized version thereof described in U.S. Pat. No. 5,861,155.

[0481] In some embodiments, the anti-TCRβ antibody molecule binds to a TCRβ V region other than TCRβ V12 (e.g., a TCRβ V region described herein, e.g., the TCRβ V6 subfamily (e.g., TCRβ V6-5*01)) with an affinity and / or binding specificity that is greater (e.g., about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or about 2-, 5-, or 10-fold greater) than the affinity and / or binding specificity of the 16G8 murine antibody or a humanized version thereof described in U.S. Pat. No. 5,861,155.

[0482] In some embodiments, the anti-TCRβ antibody molecule does not bind to TCRβ V5-5*01 or TCRβ V5-1*01, or binds to TCRβ V5-5*01 or TCRβ V5-1*01 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-fold lower) than the affinity and / or binding specificity of murine antibody C or a humanized version thereof described in U.S. Pat. No. 5,861,155.

[0483] In some embodiments, the anti-TCRβ antibody molecule binds to TCRβ V5-5*01 or TCRβ V5-1*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 murine antibody C or a humanized version thereof described in U.S. Pat. No. 5,861,155.

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

[0485] Anti-TCRβ V6 antibody Thus, in one aspect, the present disclosure provides anti-TCRβ V antibody molecules that bind to human TCRβ V6, e.g., the TCRβ V6 subfamily, including 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, the TCRβ V6 includes TCRβ V6-4*01 or a variant thereof. In some embodiments, the TCRβ V6 includes TCRβ 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 ATGAGCATCGGCCTCCTGTGCTGTGCAGCCTTGTCTCTCCTGGGCAGGTCCAGTGAATGCTGGTGTCACTCAGACCCCAAAATTCCAGGTCCTGAAGACAGGACAGAGCATGACACTGCAGTGTGCCCAGGATATGAACCATGAATACATGTCCTGGTATCGACAAGACC CAGGCATGGGGCTGAGGCTGATTCATTACTCAGTTGGTGCTGGTATCACTGACCAAGGAGAAGTCCCCAATGGCTACAATGTCTCCAGATCAACCACAGAGGATTTCCCGCTCAGGCTGCTGTCGGCTGCTCCCTCCCAGACATCTGTGTACTTCTGTGCCAGCAGTTACTC In some embodiments, TCR β V6-5*01 comprises the amino acid sequence of SEQ ID NO: 44, or an amino acid sequence having 85%, 90%, 95%, 99% or more identity thereto.

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

[0489] In some embodiments, the anti-TCRβ V antibody molecule, eg, anti-TCRβ V6 (eg, anti-TCRβ V6-5*01) antibody molecule, is isolated or recombinant. In some embodiments, the anti-TCRβ antibody molecule, e.g., 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 antigen-binding fragment thereof, from an antibody described herein, e.g., an antibody selected from any one of AH.1-AH.68, e.g., AH.1, AH.2, or AH.68, or an antibody 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, the anti-TCRβ antibody molecule, e.g., 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 described herein, e.g., an antibody selected from any one of AH.1-AH.68, e.g., AH.1, AH.2, or AH.68, or an antibody 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.

[0491] In some embodiments, the anti-TCRβ antibody molecule, e.g., 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 described herein, e.g., an antibody selected from any one of AH.1-AH.68, e.g., AH.1, AH.2, or AH.68, or an antibody molecule 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.

[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, the anti-TCRβ antibody molecule, e.g., 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 described herein, e.g., an antibody selected from any one of AH.1-AH.68, e.g., AH.1, AH.2, or AH.68, or an antibody 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.

[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, the anti-TCRβ antibody molecule, e.g., the anti-TCRβ V6 (e.g., the anti-TCRβ V6-5*01) antibody molecule, comprises a heavy chain constant region of an IgG4, e.g., a human IgG4. In yet another embodiment, the anti-TCRβ antibody molecule, e.g., the anti-TCRβ V6 (e.g., the anti-TCRβ V6-5*01) antibody molecule, comprises a heavy chain constant region of an IgG1, e.g., a human IgG1. In one embodiment, the heavy chain constant region comprises an amino acid sequence set forth in Table 3A, or a sequence substantially identical thereto (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical).

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

[0495] In some embodiments, the anti-TCRβ antibody molecule, e.g., 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 described herein, e.g., an antibody selected from any one of AH.1-AH.68, e.g., AH.1, AH.2, or AH.68, or an antibody 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.

[0496] In some embodiments, an anti-TCRβ antibody molecule, e.g., an anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule, comprises at least one, two, or three CDRs (or collectively all of the CDRs) from a 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 collectively all of the CDRs) have one, two, three, four, five, six, or more changes, e.g., 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, the anti-TCRβ antibody molecule, e.g., anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule, comprises at least one, two, or three complementarity determining regions (CDRs) from a light chain variable region of an antibody described herein, e.g., an antibody selected from any one of AH.1-AH.68, e.g., AH.1, AH.2, or AH.68, or an antibody 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.

[0498] In some embodiments, an anti-TCRβ antibody molecule, e.g., an anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule, comprises at least one, two, or three CDRs (or collectively all of the CDRs) 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 collectively all of the CDRs) have one, two, three, four, five, six, or more changes, e.g., 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-TCRβ antibody molecule, e.g., 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 collectively all of the CDRs) from heavy and light chain variable regions comprising the amino acid sequences shown in Table 1A or encoded by the nucleotide sequences shown in Table 1A. In one embodiment, one or more of the CDRs (or collectively all of the CDRs) have one, two, three, four, five, six, or more changes, e.g., 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, the anti-TCRβ antibody molecule, e.g., anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule, comprises all six CDRs from an antibody described herein, e.g., an antibody selected from any one of AH.1-AH.68, e.g., AH.1, AH.2, or AH.68, or an antibody described in Table 1A or encoded by the nucleotide sequence in Table 1A, or closely related CDRs, e.g., CDRs that are identical or have at least one amino acid change but no more than two, three, or four changes (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions). In some embodiments, the anti-TCRβ antibody molecule, e.g., anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule, may comprise any CDR described herein.

[0501] In some embodiments, an anti-TCR β antibody molecule, e.g., an anti-TCR β V6 (e.g., anti-TCR β V6-5*01) antibody molecule, comprises at least one, two, or three CDRs according to Kabat et al. (e.g., at least one, two, or three CDRs according to the Kabat definition as set out in Table 1A) from the heavy chain variable region of an antibody described herein, e.g., an antibody chosen from any one of AH.1-AH.68, e.g., AH.1, AH.2, or AH.68, or an antibody described 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, or has at least one amino acid change but no more than two, three, or four changes (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) compared to one, two, or three CDRs according to Kabat et al. as set out in Table 1A.

[0502] In some embodiments, an anti-TCR β antibody molecule, e.g., an anti-TCR β V6 (e.g., anti-TCR β V6-5*01) antibody molecule, comprises at least one, two, or three CDRs according to Kabat et al. (e.g., at least one, two, or three CDRs according to the Kabat definition as set out in Table 1A) from the light chain variable region of an antibody described herein, e.g., an antibody chosen from any one of AH.1-AH.68, e.g., AH.1, AH.2, or AH.68, or an antibody described 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, or has at least one amino acid change but no more than two, three, or four changes (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) compared to one, two, or three CDRs according to Kabat et al. as set out in Table 1A.

[0503] In some embodiments, an anti-TCRβ antibody molecule, e.g., an anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule, comprises at least one, two, three, four, five, or six CDRs according to Kabat et al. (e.g., at least one, two, three, four, five, or six CDRs according to the Kabat definition as set forth in Table 1A) from the heavy and light chain variable regions of an antibody described herein, e.g., an antibody chosen from any one of AH.1 through AH.68, e.g., AH.1, AH.2, or AH.68, or an antibody described in Table 1A or encoded by the nucleotide sequence in Table 1A. or one, two, three, four, five, or six CDRs), 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 foregoing sequences, or that have at least one amino acid change but no more than two, three, or four changes (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) compared to one, two, three, four, five, or six CDRs according to Kabat et al. set forth in Table 1A.

[0504] In some embodiments, an anti-TCR β antibody molecule, e.g., an anti-TCR β V6 (e.g., anti-TCR β V6-5*01) antibody molecule, comprises all six CDRs according to Kabat et al. (e.g., all six CDRs according to the Kabat definition as set out in Table 1A) from the heavy and light chain variable regions of an antibody described herein, e.g., an antibody chosen from any one of AH.1-AH.68, e.g., AH.1, AH.2, or AH.68, or an antibody described in Table 1A or encoded by the 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, or having at least one amino acid change but no more than two, three, or four changes (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) compared to all six CDRs according to Kabat et al. as set out in Table 1A. In one embodiment, the anti-TCRβ V antibody molecule, eg, anti-TCRβ V6 (eg, anti-TCRβ V6-5*01) antibody molecule, may comprise any of the CDRs described herein.

[0505] In some embodiments, an anti-TCRβ antibody molecule, e.g., an anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule, comprises at least one, two, or three hypervariable loops that have the same canonical structure as the corresponding hypervariable loops of an antibody described herein, e.g., an antibody selected from any one of AH.1-AH.68, e.g., AH.1, AH.2, or AH.68, e.g., the same canonical structure as at least loop 1 and / or loop 2 of the heavy and / or light chain variable domain of an antibody described herein. See, e.g., Chothia et al. (1992) J. Mol. Biol. 227:799-817; Tomlinson et al. (1992) J. Mol. Biol. 227:776-798 for a description of the canonical structures of hypervariable loops. These structures can be determined by inspection of the tables provided in these references.

[0506] In some embodiments, an anti-TCRβ V antibody molecule, such as anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule comprises at least one, two, or three CDRs according to Chothia et al. (e.g., at least one, two, or three CDRs according to the Chothia definition as set out in Table 1A) from the heavy chain variable region of an antibody described herein, e.g., selected from any one of AH.1 through AH.68, e.g., AH.1, AH.2, or AH.68, or an antibody described 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, or has at least one amino acid change, but no more than two, three, or four changes (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions), compared to one, two, or three CDRs according to Chothia et al. as set out in Table 1A.

[0507] In some embodiments, an anti-TCRβ V antibody molecule, such as anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule comprises at least one, two, or three CDRs according to Chothia et al. (e.g., at least one, two, or three CDRs according to the Chothia definition as set out in Table 1A) from the light chain variable region of an antibody described herein, e.g., an antibody chosen from any one of AH.1 through AH.68, e.g., AH.1, AH.2, or AH.68, or an antibody described 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, or has at least one amino acid change but no more than two, three, or four changes (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) compared to one, two, or three CDRs according to Chothia et al. as set out in Table 1A.

[0508] In some embodiments, an anti-TCRβ antibody molecule, e.g., an anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule, comprises at least one, two, three, four, five, or six CDRs according to Chothia et al. (e.g., at least one, two, three, four, five, or six CDRs according to the Chothia definition as set forth in Table 1A) from the heavy and light chain variable regions of an antibody described herein, e.g., an antibody chosen from any one of AH.1 through AH.68, e.g., AH.1, AH.2, or AH.68, or an antibody described in Table 1A or encoded by the nucleotide sequence in Table 1A. or 4, 5, or 6 CDRs), 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 foregoing sequences, or that have at least one amino acid change but no more than two, three, or four changes (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) compared to one, two, three, four, five, or six CDRs according to Chothia et al. shown in Table 1A.

[0509] In some embodiments, an anti-TCR β antibody molecule, e.g., an anti-TCR β V6 (e.g., anti-TCR β V6-5*01) antibody molecule, comprises all six CDRs according to Chothia et al. (e.g., all six CDRs according to the Chothia definition as set forth in Table 1A) from the heavy and light chain variable regions of an antibody described herein, e.g., an antibody chosen from any one of AH.1-AH.68, e.g., AH.1, AH.2, or AH.68, or an antibody described in Table 1A or encoded by the 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, or having at least one amino acid change but no more than two, three, or four changes (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) compared to all six CDRs according to Chothia et al. as set forth in Table 1A. In one embodiment, the anti-TCRβ V antibody molecule, eg, anti-TCRβ V6 (eg, anti-TCRβ V6-5*01) antibody molecule, may comprise any of the CDRs described herein.

[0510] In some embodiments, the anti-TCRβ antibody molecule, e.g., anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule, comprises a combination of CDRs or 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, eg, an anti-TCRβ V6 (eg, anti-TCRβ V6-5*01) antibody molecule, may contain any combination of CDRs or hypervariable loops according to the definition of Kabat and Chothia.

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

[0513] In one embodiment, e.g., comprising a variable region, CDR (e.g., a combined CDR, Chothia CDR, or Kabat CDR), or other sequence referred to herein, e.g., in Table 1A, the antibody molecule is a monospecific antibody molecule, a bispecific antibody molecule, a bivalent antibody molecule, a biparatopic 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, e.g., as described herein.

[0514] In one embodiment, the anti-TCRβ V antibody molecule, e.g., anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule is (i) one, two or all of 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 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; Includes.

[0515] In some embodiments, the anti-TCRβ V antibody molecule, e.g., the 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 CDR2, and HC CDR3 of SEQ ID NO:1.

[0516] 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: 10, and HC CDR1, HC CDR2, and HC CDR3 of SEQ ID NO: 9.

[0517] 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: 11, and HC CDR1, HC CDR2, and HC CDR3 of SEQ ID NO: 9.

[0518] In one embodiment, the anti-TCRβ V antibody molecule, e.g., anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule 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 Includes.

[0519] In some embodiments, the anti-TCRβ V antibody molecule, e.g., the anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule, 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; Includes.

[0520] In one embodiment, the anti-TCRβ V antibody molecule, e.g., anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule 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 Includes.

[0521] In some embodiments, the anti-TCRβ V antibody molecule, e.g., the anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule, 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 Includes.

[0522] In one embodiment, the anti-TCRβ V antibody molecule, e.g., anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule 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 Includes.

[0523] In some embodiments, the anti-TCRβ V antibody molecule, e.g., the anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule, 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 Includes.

[0524] In one embodiment, the light or heavy chain variable framework (e.g., a region encompassing at least FR1, FR2, FR3, and optionally FR4) of an anti-TCRβ antibody molecule, e.g., an anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule, contains (a) at least 80%, 85%, 87% of the amino acid residues from a human light or heavy chain variable framework, e.g., from a human mature antibody, a human germline sequence, or a human consensus sequence. (b) a light or heavy chain variable framework that comprises 90%, 92%, 93%, 95%, 97%, 98%, or 100% of the amino acid residues from a human light or heavy chain variable framework, e.g., 20% to 80%, 40% to 60%, 60% to 90%, or 70% to 95% of the light or heavy chain variable framework residues from a human mature antibody, a human germline sequence, or a human consensus sequence; (c) a non-human framework (e.g., a rodent framework); or (d) a non-human framework that has been modified, e.g., deimmunized or partially humanized, e.g., to remove antigenic or cytotoxic determinants. In one embodiment, the light or heavy chain variable framework region (particularly FR1, FR2 and / or FR3) comprises a light or heavy chain variable framework sequence that is at least 70, 75, 80, 85, 87, 88, 90, 92, 94, 95, 96, 97, 98, 99% identical or identical to the framework of a VL or VH segment of a human germline gene.

[0525] In some embodiments, an anti-TCR β antibody molecule, e.g., an anti-TCR β V6 (e.g., anti-TCR β V6-5*01) antibody molecule, comprises a heavy chain variable domain having at least one, two, three, four, five, six, seven, ten, fifteen, twenty or more changes, e.g., amino acid substitutions or deletions, from the amino acid sequence of any one of AH.1 to AH.68, e.g., AH.1, AH.2 or AH.68, e.g., from the amino acid sequence of the FR region in the entire variable region, e.g., as shown in FIG. 1A or SEQ ID NO:9.

[0526] Alternatively, or in combination with the heavy chain substitutions described herein, an anti-TCRβ antibody molecule, e.g., an anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule, comprises a light chain variable domain having at least one, two, three, four, five, six, seven, ten, fifteen, twenty or more amino acid changes, e.g., amino acid substitutions or deletions, from the amino acid sequence of any one of AH.1 to AH.68, e.g., AH.1, AH.2 or AH.68, e.g., from the amino acid sequence of the FR region in the entire variable region, e.g., as shown in Figure 1B, or SEQ ID NO:10 or SEQ ID NO:11.

[0527] In some embodiments, the anti-TCRβ antibody molecule, e.g., the anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule, comprises one, two, three, or four heavy chain framework regions shown in Figure 1A, or a sequence substantially identical thereto.

[0528] In some embodiments, the anti-TCRβ antibody molecule, e.g., the anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) a...

Claims

1. (i) a first antigen-binding domain that binds to a T cell receptor beta variable region (TCRβV); (ii) a second antigen-binding domain that binds to NKp30; and wherein the first antigen-binding domain comprises a single domain antibody, a camelid antibody, a single chain variable fragment (scFv), or a Fab, and the second antigen-binding domain activates NK cells expressing NKp30, and the second antigen-binding domain comprises (i) a heavy chain variable region (VH) comprising a heavy chain complementarity determining region 1 (HCCDR1) sequence, a heavy chain complementarity determining region 2 (HCDR2) sequence, and a heavy chain complementarity determining region 3 (HCCDR3) sequence, each comprising the sequences of SEQ ID NO: 6000, SEQ ID NO: 6001, and SEQ ID NO: 6002; and a light chain variable region (VL) comprising a light chain complementarity determining region 1 (LCCDR1) sequence, a light chain complementarity determining region 2 (LCCDR2) sequence, and a light chain complementarity determining region 3 (LCCDR3) sequence, each comprising the sequences of SEQ ID NO: 6063, SEQ ID NO: 6064, and SEQ ID NO: 7293; (ii) a VH comprising an HCCDR1 sequence, an HCCDR2 sequence, and an HCCDR3 sequence, each comprising the sequences of SEQ ID NO: 7313, SEQ ID NO: 6001, and SEQ ID NO: 6002; and a VL comprising an LCCDR1 sequence, an LCCDR2 sequence, and an LCCDR3 sequence, each comprising the sequences of SEQ ID NO: 6063, SEQ ID NO: 6064, and SEQ ID NO: 7293; (iii) VH comprising an HCCDR1 sequence, an HCCDR2 sequence, and an HCCDR3 sequence, each comprising the sequences of SEQ ID NO: 6007, SEQ ID NO: 6008, and SEQ ID NO: 6009; and VL comprising an LCCDR1 sequence, an LCCDR2 sequence, and an LCCDR3 sequence, each comprising the sequences of SEQ ID NO: 6070, SEQ ID NO: 6071, and SEQ ID NO: 6072; (iv) VH comprising an HCCDR1 sequence, an HCCDR2 sequence, and an HCCDR3 sequence comprising the sequences of SEQ ID NO: 7313, SEQ ID NO: 6008, and SEQ ID NO: 6009, respectively; and VL comprising an LCCDR1 sequence, an LCCDR2 sequence, and an LCCDR3 sequence comprising the sequences of SEQ ID NO: 6070, SEQ ID NO: 6071, and SEQ ID NO: 6072, respectively; (v) VH comprising the HCCDR1 sequence, the HCCDR2 sequence, and the HCCDR3 sequence, each comprising the sequences of SEQ ID NO: 7313, SEQ ID NO: 7385, and SEQ ID NO: 7315; and VL comprising the LCCDR1 sequence, the LCCDR2 sequence, and the LCCDR3 sequence, each comprising the sequences of SEQ ID NO: 6070, SEQ ID NO: 6064, and SEQ ID NO: 7321; (vi) VH comprising an HCCDR1 sequence, an HCCDR2 sequence, and an HCCDR3 sequence, each comprising the sequences of SEQ ID NO: 7313, SEQ ID NO: 7318, and SEQ ID NO: 6009; and VL comprising an LCCDR1 sequence, an LCCDR2 sequence, and an LCCDR3 sequence, each comprising the sequences of SEQ ID NO: 6070, SEQ ID NO: 6064, and SEQ ID NO: 7321; (vii) a VH comprising an HCCDR1 sequence, an HCCDR2 sequence, and an HCCDR3 sequence, each comprising the sequences of SEQ ID NO: 7313, SEQ ID NO: 6008, and SEQ ID NO: 6009; and a VL comprising an LCCDR1 sequence, an LCCDR2 sequence, and an LCCDR3 sequence, each comprising the sequences of SEQ ID NO: 6070, SEQ ID NO: 6071, and SEQ ID NO: 6072; or (viii) VH comprising an HCCDR1 sequence, an HCCDR2 sequence, and an HCCDR3 sequence comprising the sequences of SEQ ID NO: 7313, SEQ ID NO: 6001, and SEQ ID NO: 7315, respectively; and VL comprising an LCCDR1 sequence, an LCCDR2 sequence, and an LCCDR3 sequence comprising the sequences of SEQ ID NO: 7326, SEQ ID NO: 7327, and SEQ ID NO: 7329, respectively; A multispecific molecule comprising:

2. The multispecific molecule may comprise: A, B-[dimerization module]-C, -D where: (a) the dimerization module comprises an immunoglobulin constant domain; (b) At least one of A, B, C, and D comprises a first antigen-binding domain that binds to a TCRβV antigen, and any remaining A, B, C, and D comprises a second antigen-binding domain that binds to NKp30.

3. the second antigen-binding domain (i) VH comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 6121, and VL comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 7294; (ii) VH comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 6123, and VL comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 6137; (iii) VH comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 6124, and VL comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 6138; (iv) VH comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 6125, and VL comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 6139; (v) VH comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 6126, and VL comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 6140; (vi) VH comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 6127, and VL comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 6141; (vii) VH comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 6122, and VL comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 6136; (viii) VH comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 6129, and VL comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 6142; (ix) VH comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 6130, and VL comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 6143; (x) VH comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 6131, and VL comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 6144; (xi) VH comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 6132, and VL comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 6145; (xii) VH comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 6133, and VL comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 6146; (xiii) VH comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 6134, and VL comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 6147; (xiv) VH comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 7295, and VL comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 7296; (xv) VH comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 7297, and VL comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 7298; (xvi) VH comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 6122, and VL comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 6136; (xvii) VH comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 7298, and VL comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 7299; (xviii) VH comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 7300, and VL comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 7305; (xix) VH comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 7301, and VL comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 7306; (xx) VH comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 7302, and VL comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 7307; (xxi) a VH comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 7303, and a VL comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 7308; or (xxii) VH comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 7304, and VL comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 7309; 3. The multispecific molecule of claim 1 or 2, comprising:

4. the second antigen-binding domain (i) VH comprising the amino acid sequence set forth in SEQ ID NO: 6121, and VL comprising the amino acid sequence set forth in SEQ ID NO: 7294; (ii) VH comprising the amino acid sequence set forth in SEQ ID NO: 6123, and VL comprising the amino acid sequence set forth in SEQ ID NO: 6137; (iii) VH comprising the amino acid sequence set forth in SEQ ID NO: 6124, and VL comprising the amino acid sequence set forth in SEQ ID NO: 6138; (iv) VH comprising the amino acid sequence set forth in SEQ ID NO: 6125, and VL comprising the amino acid sequence set forth in SEQ ID NO: 6139; (v) VH comprising the amino acid sequence set forth in SEQ ID NO: 6126, and VL comprising the amino acid sequence set forth in SEQ ID NO: 6140; (vi) VH comprising the amino acid sequence set forth in SEQ ID NO: 6127, and VL comprising the amino acid sequence set forth in SEQ ID NO: 6141; (vii) VH comprising the amino acid sequence set forth in SEQ ID NO: 6122, and VL comprising the amino acid sequence set forth in SEQ ID NO: 6136; (viii) VH comprising the amino acid sequence set forth in SEQ ID NO: 6129, and VL comprising the amino acid sequence set forth in SEQ ID NO: 6142; (ix) VH comprising the amino acid sequence set forth in SEQ ID NO: 6130, and VL comprising the amino acid sequence set forth in SEQ ID NO: 6143; (x) VH comprising the amino acid sequence set forth in SEQ ID NO: 6131, and VL comprising the amino acid sequence set forth in SEQ ID NO: 6144; (xi) VH comprising the amino acid sequence set forth in SEQ ID NO: 6132, and VL comprising the amino acid sequence set forth in SEQ ID NO: 6145; (xii) VH comprising the amino acid sequence set forth in SEQ ID NO: 6133, and VL comprising the amino acid sequence set forth in SEQ ID NO: 6146; (xiii) VH comprising the amino acid sequence set forth in SEQ ID NO: 6134, and VL comprising the amino acid sequence set forth in SEQ ID NO: 6147; (xiv) VH comprising the amino acid sequence set forth in SEQ ID NO: 7295, and VL comprising the amino acid sequence set forth in SEQ ID NO: 7296; (xv) VH comprising the amino acid sequence set forth in SEQ ID NO: 7297, and VL comprising the amino acid sequence set forth in SEQ ID NO: 7298; (xvi) VH comprising the amino acid sequence set forth in SEQ ID NO: 6122, and VL comprising the amino acid sequence set forth in SEQ ID NO: 6136; (xvii) VH comprising the amino acid sequence set forth in SEQ ID NO: 7298, and VL comprising the amino acid sequence set forth in SEQ ID NO: 7299; (xviii) VH comprising the amino acid sequence set forth in SEQ ID NO: 7300, and VL comprising the amino acid sequence set forth in SEQ ID NO: 7305; (xix) VH comprising the amino acid sequence set forth in SEQ ID NO: 7301, and VL comprising the amino acid sequence set forth in SEQ ID NO: 7306; (xx) VH comprising the amino acid sequence set forth in SEQ ID NO: 7302, and VL comprising the amino acid sequence set forth in SEQ ID NO: 7307; (xxi) a VH comprising the amino acid sequence set forth in SEQ ID NO: 7303, and a VL comprising the amino acid sequence set forth in SEQ ID NO: 7308; or (xxii) VH comprising the amino acid sequence set forth in SEQ ID NO: 7304, and VL comprising the amino acid sequence set forth in SEQ ID NO: 7309; The multispecific molecule of any one of claims 1 to 3, comprising:

5. The multispecific molecule of any one of claims 1 to 4, wherein NK cells activated by binding of the second antigen-binding domain express increased levels of interferon gamma (IFNγ) compared to the level of IFNγ expressed following binding of an IgG antibody that does not comprise the second antigen-binding domain.

6. the first antigen-binding domain that binds to TCRβV is selected from the group consisting of TCRβV6 subfamily, TCRβV10 subfamily, TCRβV12 subfamily, TCRβV5 subfamily, TCRβV7 subfamily, TCRβV11 subfamily, TCRβV14 subfamily, TCRβV16 subfamily, TCRβV18 subfamily, TCRβV9 subfamily, TCRβV13 subfamily, TCRβV4 subfamily, TCRβV3 subfamily, TCRβV2 subfamily, and TCRβV15 subfamily; 6. The multispecific molecule of any one of claims 1 to 5, which binds to a TCRβV subfamily, including the TCRβV30 subfamily, the TCRβV19 subfamily, the TCRβV27 subfamily, the TCRβV28 subfamily, the TCRβV24 subfamily, the TCRβV20 subfamily, the TCRβV25 subfamily, the TCRβV29 subfamily, the TCRβV1 subfamily, the TCRβV17 subfamily, the TCRβV21 subfamily, the TCRβV23 subfamily, or the TCRβV26 subfamily.

7. The first antigen-binding domain that binds to TCRβV is selected from the group consisting of TCRβV6-4*01, TCRβV6-4*02, TCRβV6-9*01, TCRβV6-8*01, TCRβV6-5*01, TCRβV6-6*02, TCRβV6-6*01, TCRβV6-2*01, TCRβV6-3*01, TCRβV6-1*01, TCRβV12-4*01, TCRβV1 7. The multispecific molecule of any one of claims 1 to 6, which binds to TCRβV12-5*01, TCRβV5-5*01, TCRβV5-6*01, TCRβV5-4*01, TCRβV5-8*01, TCRβV5-1*01, TCRβV10-1*01, TCRβV10-1*02, TCRβV10-3*01, or TCRβV10-2*01.

8. A first antigen-binding domain that binds to TCRβV (i) a VH comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1131, and a VL comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1136; (ii) VH comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1132, and VL comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1137; (iii) VH comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1133, and VL comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1138; (iv) VH comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1134, and VL comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1139; (v) VH comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1135, and VL comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1140; (vi) VH comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1153, and VL comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1158; (vii) VH comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1154, and VL comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1159; (viii) VH comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1155, and VL comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1160; (ix) VH comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1156, and VL comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1161; (x) a VH comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1157, and a VL comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1162; (xi) VH comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1197, and VL comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1203; (xii) VH comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1198, and VL comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1204; (xiii) VH comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1199, and VL comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1205; (xiv) VH comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1200, and VL comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1206; or (xv) VH comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1201, and VL comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1207; The multispecific molecule of any one of claims 1 to 7, comprising:

9. A first antigen-binding domain that binds to TCRβV (i) a VH comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1220, and a VL comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1224; (ii) VH comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1221, and VL comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1225; (iii) VH comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1222, and VL comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1226; (iv) VH comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1223, and VL comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1227; (v) VH comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1241, and VL comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1244; (vi) VH comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1242, and VL comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1245; (vii) VH comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1243, and VL comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1246; (viii) VH comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1260, and VL comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1264; (ix) VH comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1261, and VL comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1265; (x) a VH comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1262, and a VL comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1266; (xi) VH comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1263, and VL comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1267; (xii) VH comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1283, and VL comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1279; (xiii) VH comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1284, and VL comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1280; (xiv) VH comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1285, and VL comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1281; or (xv) VH comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1286, and VL comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1282; The multispecific molecule of any one of claims 1 to 7, comprising:

10. A first antigen-binding domain that binds to TCRβV (i) a VH comprising the amino acid sequence set forth in SEQ ID NO: 1131, and a VL comprising the amino acid sequence set forth in SEQ ID NO: 1136; (ii) VH comprising the amino acid sequence set forth in SEQ ID NO: 1132, and VL comprising the amino acid sequence set forth in SEQ ID NO: 1137; (iii) VH comprising the amino acid sequence set forth in SEQ ID NO: 1133, and VL comprising the amino acid sequence set forth in SEQ ID NO: 1138; (iv) VH comprising the amino acid sequence set forth in SEQ ID NO: 1134, and VL comprising the amino acid sequence set forth in SEQ ID NO: 1139; (v) VH comprising the amino acid sequence set forth in SEQ ID NO: 1135, and VL comprising the amino acid sequence set forth in SEQ ID NO: 1140; (vi) VH comprising the amino acid sequence set forth in SEQ ID NO: 1153, and VL comprising the amino acid sequence set forth in SEQ ID NO: 1158; (vii) VH comprising the amino acid sequence set forth in SEQ ID NO: 1154, and VL comprising the amino acid sequence set forth in SEQ ID NO: 1159; (viii) VH comprising the amino acid sequence set forth in SEQ ID NO: 1155, and VL comprising the amino acid sequence set forth in SEQ ID NO: 1160; (ix) VH comprising the amino acid sequence set forth in SEQ ID NO: 1156, and VL comprising the amino acid sequence set forth in SEQ ID NO: 1161; (x) VH comprising the amino acid sequence set forth in SEQ ID NO: 1157, and VL comprising the amino acid sequence set forth in SEQ ID NO: 1162; (xi) VH comprising the amino acid sequence set forth in SEQ ID NO: 1197, and VL comprising the amino acid sequence set forth in SEQ ID NO: 1203; (xii) VH comprising the amino acid sequence set forth in SEQ ID NO: 1198, and VL comprising the amino acid sequence set forth in SEQ ID NO: 1204; (xiii) VH comprising the amino acid sequence set forth in SEQ ID NO: 1199, and VL comprising the amino acid sequence set forth in SEQ ID NO: 1205; (xiv) VH comprising the amino acid sequence set forth in SEQ ID NO: 1200, and VL comprising the amino acid sequence set forth in SEQ ID NO: 1206; or (xv) VH comprising the amino acid sequence set forth in SEQ ID NO: 1201, and VL comprising the amino acid sequence set forth in SEQ ID NO: 1207 The multispecific molecule of any one of claims 1 to 8, comprising:

11. A first antigen-binding domain that binds to TCRβV (i) a VH comprising the amino acid sequence set forth in SEQ ID NO: 1220, and a VL comprising the amino acid sequence set forth in SEQ ID NO: 1224; (ii) VH comprising the amino acid sequence set forth in SEQ ID NO: 1221, and VL comprising the amino acid sequence set forth in SEQ ID NO: 1225; (iii) VH comprising the amino acid sequence set forth in SEQ ID NO: 1222, and VL comprising the amino acid sequence set forth in SEQ ID NO: 1226; (iv) VH comprising the amino acid sequence set forth in SEQ ID NO: 1223, and VL comprising the amino acid sequence set forth in SEQ ID NO: 1227; (v) VH comprising the amino acid sequence set forth in SEQ ID NO: 1241, and VL comprising the amino acid sequence set forth in SEQ ID NO: 1244; (vi) VH comprising the amino acid sequence set forth in SEQ ID NO: 1242, and VL comprising the amino acid sequence set forth in SEQ ID NO: 1245; (vii) VH comprising the amino acid sequence set forth in SEQ ID NO: 1243, and VL comprising the amino acid sequence set forth in SEQ ID NO: 1246; (viii) VH comprising the amino acid sequence set forth in SEQ ID NO: 1260, and VL comprising the amino acid sequence set forth in SEQ ID NO: 1264; (ix) VH comprising the amino acid sequence set forth in SEQ ID NO: 1261, and VL comprising the amino acid sequence set forth in SEQ ID NO: 1265; (x) VH comprising the amino acid sequence set forth in SEQ ID NO: 1262, and VL comprising the amino acid sequence set forth in SEQ ID NO: 1266; (xi) VH comprising the amino acid sequence set forth in SEQ ID NO: 1263, and VL comprising the amino acid sequence set forth in SEQ ID NO: 1267; (xii) VH comprising the amino acid sequence set forth in SEQ ID NO: 1283, and VL comprising the amino acid sequence set forth in SEQ ID NO: 1279; (xiii) VH comprising the amino acid sequence set forth in SEQ ID NO: 1284, and VL comprising the amino acid sequence set forth in SEQ ID NO: 1280; (xiv) VH comprising the amino acid sequence set forth in SEQ ID NO: 1285, and VL comprising the amino acid sequence set forth in SEQ ID NO: 1281; or (xv) VH comprising the amino acid sequence set forth in SEQ ID NO: 1286, and VL comprising the amino acid sequence set forth in SEQ ID NO: 1282; The multispecific molecule of claim 10, comprising:

12. Multispecific molecules (i) a first heavy chain polypeptide that binds to TCRβV; (ii) a second heavy chain polypeptide that binds to NKp30; (iii) a first light chain polypeptide that binds to TCRβV; and (iv) a second light chain polypeptide that binds to NKp30 12. The multispecific molecule of claim 1, further comprising: a first heavy chain polypeptide and a second heavy chain polypeptide, wherein the first heavy chain polypeptide and the second heavy chain polypeptide form an Fc interface that enhances heterodimerization.

13. The multispecific molecule of claim 12 , wherein the first light chain polypeptide is a lambda light chain polypeptide and the second light chain polypeptide is a kappa light chain polypeptide.

14. The multispecific molecule of any one of claims 1 to 13, further comprising one or more cytokine molecules.

15. 15. The multispecific molecule of claim 14, wherein the cytokine molecule is selected from the group consisting of interleukin-2 (IL-2) or a functional fragment or variant thereof, interleukin-7 (IL-7) or a functional fragment or variant thereof, interleukin-12 (IL-12) or a functional fragment or variant thereof, interleukin-15 (IL-15) or a functional fragment or variant thereof, interleukin-18 (IL-18) or a functional fragment or variant thereof, interleukin-21 (IL-21) or a functional fragment or variant thereof, and interferon gamma or a functional fragment or variant thereof, and any combination thereof.

16. 16. The multispecific molecule of any one of claims 1 to 15, further comprising a first immunoglobulin variable region sequence and a second immunoglobulin variable region sequence, wherein the first immunoglobulin variable region sequence has binding specificity for TCRβV and the second immunoglobulin variable region sequence has binding specificity for NKp30.

17. The multispecific molecule of any one of claims 1 to 16, further comprising a linker between a first antigen-binding domain that binds to TCRβV and a second antigen-binding domain that binds to NKp30.

18. 18. The multispecific molecule of claim 17, wherein the linker is selected from the group consisting of a cleavable linker, a non-cleavable linker, a peptide linker, a flexible linker, a rigid linker, a helical linker, and a non-helical linker.

19. 20. A pharmaceutical composition comprising the multispecific molecule of any one of claims 1 to 18 for use in depleting a T cell population containing a biased TCRβV clonotype.

20. 20. The pharmaceutical composition of claim 19 for in vivo use.

21. A recombinant polynucleotide comprising a sequence encoding a multifunctional molecule described in any one of claims 1 to 18.

22. A method for producing a multifunctional molecule according to any one of claims 1 to 18, comprising culturing a host cell containing a recombinant polynucleotide according to claim 21 under conditions suitable for gene expression and / or homo- or heterodimerization.

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