Multifunctional molecule combined with T cell and application of multifunctional molecule to treatment of autoimmune diseases

Abstract

The invention provides a multifunctional molecule combined with T cells and application of the multifunctional molecule to treatment of autoimmune diseases. A multifunctional molecule comprising i) an antigen binding domain that binds to a TCR variable beta chain (TCRBV) antigen; and one, two or all of (ii) an immune cell adaptor (e.g., selected from an NK cell adaptor, a T cell adaptor, a B cell adaptor, a dendritic cell adaptor or a macrophage adaptor); (iii) a cytokine molecule or a cytokine inhibitor molecule; and / or (iv) a death receptor signal enhancer. In addition, nucleic acids encoding the multifunctional molecules, methods of producing the molecules, and methods of treating autoimmune diseases using the molecules are also disclosed.

Description

[0001] This application is a divisional application of Chinese patent application No. 202080030461.7, filed on February 21, 2020, entitled "Multifunctional molecule that binds to T cells and its use in treating autoimmune diseases" (the corresponding PCT application was filed on February 21, 2020, and has the application number PCT / US2020 / 019321). Related applications

[0002] This application claims priority to U.S. Provisional Application 62 / 808,713, filed February 21, 2019, and U.S. Provisional Application 62 / 957,045, filed January 3, 2020, the entire contents of each of which are incorporated herein by reference. Background Technology

[0003] T cell-mediated antigen recognition depends on the interaction between the T cell receptor (TCR) and the antigen-major histocompatibility complex (MHC). Heterodimeric TCRs consist of a combination of α and β chains expressed by most T cells (αβ TCR) or γδ chains present only in approximately 1–5% of T cells (γδ TCR). A highly diverse TCR repertoire is a fundamental property of an effective immune system. However, the immune repertoire can change dramatically with the onset and progression of diseases such as cancer, autoimmune diseases, inflammatory diseases, and infectious diseases.

[0004] Autoimmunity may arise from abnormal regulation of the immune system. This can manifest as autoreactive TCR clones attacking the patient's own cells. Improved therapies are needed for autoimmune diseases. Summary of the Invention

[0005] This disclosure relates in particular to novel multispecific or multifunctional molecules comprising (i) an antigen-binding domain that binds to a TCR variable β-chain (TCRBV) antigen on a T cell (e.g., a TCRRBV antigen corresponding to a biased TCR clonal type); and one, two, or all of the following: (ii) an immune cell adjuvant (e.g., selected from NK cell adjuvants, T cell adjuvants, B cell adjuvants, dendritic cell adjuvants, or macrophage adjuvants); (iii) a cytokine molecule; and / or (iv) a matrix modification portion. The terms “multispecific” and “multifunctional” are used interchangeably herein.

[0006] Not wishing to be limited by theory, TCR bias may exist in autoimmune diseases. This bias may be associated with the dominant autoreactive TCR clone causing the disease or with symptoms. Rebalancing the TCR repertoire (e.g., by eliminating or depleting T cells including autoreactive clones) can treat the associated autoimmune disease and / or alleviate its symptoms. Therefore, the multispecific or multifunctional molecules disclosed herein are expected to target (e.g., localize, bridge, and / or activate) target cells (e.g., T cells including biased TRBV clones or T cells including TRBV antigens corresponding to biased TRBV clones) with immune cells (e.g., immune effector cells selected from NK cells, T cells, B cells, dendritic cells, or macrophages). Using the multispecific molecules described herein to increase the proximity and / or activity of immune cells is expected to enhance the immune response to target cells (e.g., T cells containing TRBV, such as TRBV antigens (e.g., TRBV antigens corresponding to biased TRBV clones)), thereby providing more effective therapies (e.g., more effective treatments for autoimmune diseases). Not limited to theory, targeted, localized immune responses to target cells (e.g., including T cells with biased TCRBCV clonal patterns, but not T cells excluding TCRBCV clonal patterns) are thought to reduce the systemic toxicity of the multispecific molecules described herein. Targeted immune responses against autoreactive T cell populations that target non-autoreactive T cells to a lesser extent (e.g., not targeting non-autoreactive T cells) are thought to have fewer detrimental effects compared to systemic ablation of all T cells.

[0007] Therefore, this document provides, in particular, a multispecific molecule comprising the foregoing portions (e.g., a multispecific or multifunctional antibody molecule), a nucleic acid encoding said multispecific molecule, a method for generating said molecule, and a method for treating autoimmune diseases using said molecule. This document also provides an anti-TCRβV antibody molecule, a nucleic acid encoding said anti-TCRβV antibody molecule, a method for generating said molecule, and a method for treating autoimmune diseases using said anti-TCRβV antibody molecule.

[0008] Further methods are provided for, for example, in the case of autoimmune diseases, to deplete (e.g., in vivo depletion) biased TCRβV clonal types using multispecific molecules or anti-TCRβV antibody molecules. In some embodiments, the method includes identifying clonal bias in TCRβV use (e.g., associated with autoreactive subsets) in patients, and, in response to this analysis, administering a multifunctional molecule targeting a TCRβV antigen corresponding to the biased TCRβV clonal type to reduce, for example, eliminate said clonal bias and promote, for example, the establishment of normal TCRβV distribution.

[0009] Therefore, in one aspect, this disclosure is characterized by a multifunctional molecule comprising: (i) Binding to, for example, selectively binding to the first antigen-binding domain of a T-cell receptor variable β (TCRBV) antigen, such as the TCRBV antigen. as well as (ii) One, two, or all of the following: (a) Immune cell adjuvants selected from NK cell adjuvants, T cell adjuvants, B cell adjuvants, dendritic cell adjuvants or macrophage adjuvants; (b) Cytokine molecules or cytokine inhibitor molecules; and (c) Death receptor signaling adaptor.

[0010] In some implementations, the first antigen-binding domain includes an anti-TCRβV antibody molecule, for example, as described herein.

[0011] On the other hand, this disclosure is characterized by nucleic acid molecules that encode the multifunctional molecules disclosed herein.

[0012] On the other hand, this disclosure is characterized by a vector containing the nucleic acid molecules disclosed herein, such as an expression vector.

[0013] On the other hand, this disclosure is characterized by a host cell containing the nucleic acid molecules or vectors disclosed herein.

[0014] On the other hand, this disclosure is characterized by methods for preparing, for example, the multifunctional molecules disclosed herein, which include culturing the host cells disclosed herein under suitable conditions, for example, conditions suitable for gene expression and / or homo- or hetero-dimerization.

[0015] On the other hand, this disclosure is characterized by pharmaceutical compositions comprising the multifunctional molecules disclosed herein.

[0016] On the other hand, this disclosure is characterized by a method for treating TCR bias, the method comprising administering a multifunctional molecule disclosed herein to a subject in need, wherein the multifunctional molecule is administered in an amount effective in treating the TCR bias.

[0017] In another aspect, this disclosure is characterized by a method for treating autoimmune diseases (e.g., autoimmune diseases associated with TCR bias), the method comprising administering the multifunctional molecule disclosed herein to a subject in need, wherein the multifunctional molecule is administered in an amount effective in treating the autoimmune disease.

[0018] On the other hand, this disclosure is characterized by a method for identifying subjects requiring treatment for TCR-biased or (e.g., TCR-related) autoimmune diseases using the multifunctional molecules disclosed herein, the method comprising determining (e.g., directly or indirectly, for example, by obtaining information about) whether the subject has a TCR bias (e.g., a biased TCR BV clonal type) and / or an autoimmune disease associated with said bias, wherein: In response to determining that the subject has a TCR bias (e.g., a biased TCR clonal type) and / or an autoimmune disease associated with the bias, the subject is identified as a candidate for treatment with a multifunctional molecule containing an antigen-binding domain that binds to a TCR clonal antigen.

[0019] On the other hand, this disclosure is characterized by a method for evaluating subjects who require treatment for TCR bias (e.g., biased TCR BV clonal type) and / or autoimmune diseases associated with said bias, the method comprising determining (e.g., directly or indirectly, for example, by obtaining information about) whether the subject has TCR bias (e.g., biased TCR BV clonal type).

[0020] In another aspect, this article discloses a method for treating autoimmune diseases (e.g., autoimmune diseases associated with TCR bias) in subjects in need, the method comprising administering to the subject an effective amount, for example a therapeutically effective amount, of an antibody molecule (“anti-TCRβV antibody molecule”) that binds (e.g., specifically binds) to the variable region of T cell receptor β (TCRβV).

[0021] On the other hand, this disclosure provides a method for depleting a population of T cells in a subject suffering from an autoimmune condition (e.g., an autoimmune disease associated with TCR bias), the method comprising contacting the population of T cells with an effective amount of an antibody molecule (“anti-TCRβV antibody molecule”) that binds (e.g., specifically binds) to the variable region of the T cell receptor β (TCRβV).

[0022] In some implementations, the contact occurs inside the body or outside the body.

[0023] In some embodiments, the anti-TCRβV antibody molecule is not the antibody molecule disclosed in U.S. Patent 5,861,155.

[0024] In some embodiments, the anti-TCRβV antibody molecule binds to TCRβV12 with an affinity and / or binding specificity less than (e.g., less than about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or about 2, 5, or 10 times) that of the 16G8 mouse antibody or its humanized form as described in U.S. Patent 5,861,155.

[0025] In some embodiments, the anti-TCRβV antibody molecule binds to TCRβV12 with an affinity and / or binding specificity greater than (e.g., greater than about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or about 2, 5, or 10 times) that of the 16G8 mouse antibody or its humanized form as described in U.S. Patent 5,861,155.

[0026] In some embodiments, the anti-TCRβV antibody molecule binds to TCRβV5-5*01 or TCRβV5-1*01 with an affinity and / or binding specificity greater than (e.g., greater than about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or about 2, 5, or 10 times) that of the TM23 mouse antibody or its humanized form as described in U.S. Patent 5,861,155.

[0027] In some embodiments, the anti-TCRβV antibody molecule binds to TCRβV5-5*01 or TCRβV5-1*01 with an affinity and / or binding specificity greater than (e.g., greater than about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or about 2, 5, or 10 times) that of the TM23 mouse antibody or its humanized form as described in U.S. Patent 5,861,155.

[0028] In some embodiments, the anti-TCRβV antibody molecule includes an Fc region, for example, an Fc region having effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cytophagy (ADCP), and / or complement-dependent cytotoxicity (CDC).

[0029] In some embodiments, the anti-TCRβV antibody molecule includes, for example, an Fc region with enhanced effector function compared to the wild-type Fc region.

[0030] In some embodiments, the anti-TCRβV antibody molecule contains a human IgG1 region or a human IgG4 region.

[0031] On the other hand, this disclosure is characterized by a nucleic acid molecule encoding the anti-TCRβV antibody molecule disclosed herein.

[0032] On the other hand, this disclosure is characterized by a vector containing the nucleic acid molecules disclosed herein, such as an expression vector.

[0033] On the other hand, this disclosure is characterized by a host cell containing the nucleic acid molecules or vectors disclosed herein.

[0034] On the other hand, this disclosure is characterized by a method for preparing, for example, generating the anti-TCRβV antibody molecule disclosed herein, the method comprising culturing the host cells disclosed herein under suitable conditions, for example, conditions suitable for gene expression and / or homo- or hetero-dimerization.

[0035] On the other hand, this disclosure is characterized by pharmaceutical compositions comprising the anti-TCRβV antibody molecule disclosed herein.

[0036] Additional features of any of the aforementioned multifunctional molecules, nucleic acids, vectors, host cells, or methods include one or more of the embodiments listed below.

[0037] Those skilled in the art will recognize, or can determine, through routine experiments only, many equivalents of the particular embodiments of the invention described herein. These equivalents are intended to be covered by the embodiments listed below.

[0038] List of implementation plans 1. A multifunctional molecule comprising: (i) Binding to, for example, selectively binding to the first antigen-binding domain of a T-cell receptor variable β (TCRBV) antigen, such as the TCRBV antigen. as well as (ii) One, two, or all of the following: (a) Immune cell adjuvants selected from NK cell adjuvants, T cell adjuvants, B cell adjuvants, dendritic cell adjuvants or macrophage adjuvants; (b) Cytokine molecules or cytokine inhibitor molecules; and (c) Death receptor signaling adaptor.

[0039] 2. The multifunctional molecule of embodiment 1, wherein the TCR BV antigen corresponds to a biased TCR BV clonal type, which is present, for example, in a subject, such as a patient, for example, a subject or patient with an autoimmune disease.

[0040] 3. Any multifunctional molecule in the prior embodiment, wherein the multifunctional molecule: (i) Specific binding to TCRBV antigens, for example, epitopes that are the same as or similar to those recognized by the anti-TCRBV antibody molecules described herein; (ii) Shows the same or similar binding affinity or specificity as the anti-TCRBV antibody molecule described herein, or both; (iii) Inhibition, for example, competitive inhibition of the binding of the anti-TCRBV antibody molecule described herein; (iv) Epitopes that bind to the same or overlapping epitopes as the anti-TCRBV antibody molecules described herein; or (v) Competes with and / or binds to the same epitopes as the anti-TCRBV antibody molecules described herein.

[0041] 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 sequences substantially identical thereto.

[0042] 5. A multifunctional molecule of any one of embodiments 1-4, wherein the antigen-binding domain specifically binds to TCRβ V6 (e.g., TCRβ V6-5*01).

[0043] 6. The multifunctional 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 from antibody AH.1 or antibody AH.2 or encoded by nucleotide sequences as described in or 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.

[0044] 7. A multifunctional molecule according to embodiment 5 or 6, wherein the antigen-binding domain comprises at least one, two, or three (or all) CDRs from the heavy chain variable region, the heavy chain variable region comprising an amino acid sequence shown in Table 1A or an amino acid sequence encoded by a nucleotide sequence shown in Table 1A (or a 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 amino acid sequence encoded by a nucleotide sequence shown in Table 1A).

[0045] 8. A multifunctional molecule of any of embodiments 5-7, wherein the antigen-binding domain comprises at least one, two, or three (or all) CDRs from a light chain variable region, the light chain variable region comprising an amino acid sequence shown in Table 1A or an amino acid sequence encoded by a nucleotide sequence shown in Table 1A (or a 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 amino acid sequence encoded by a nucleotide sequence shown in Table 1A).

[0046] 9. A multifunctional molecule according to any one of embodiments 5-8, wherein the antigen-binding domain comprises: (i) One, two, or all of the light chain complementarity determination regions 1 (LCCDR1), 2 (LC CDR2), and 3 (LC CDR3) of SEQ ID NO: 2, SEQ ID NO: 10, or SEQ ID NO: 11, and / or (ii) One, two, or all of the heavy chain complementarity determination regions 1 (HC CDR1), 2 (HC CDR2), and 3 (HC CDR3) of SEQ ID NO: 1 or SEQ ID NO: 9.

[0047] 10. A multifunctional molecule of any one of embodiments 5-8, 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.

[0048] 11. A multifunctional molecule of any one of embodiments 5-8, 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.

[0049] 12. A multifunctional molecule of any one of embodiments 5-8, 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.

[0050] 13. A multifunctional molecule according to any one of embodiments 5-8, wherein 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 amino acid sequence of HC CDR1 of SEQ ID NO: 3, the amino acid sequence of HC CDR2 of SEQ ID NO: 4, or the amino acid sequence of HC CDR3 of SEQ ID NO: 5.

[0051] 14. A multifunctional molecule according to any one of embodiments 5-8, wherein 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) Heavy chain variable region (VH), wherein the heavy chain variable region comprises 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.

[0052] 15. A multifunctional molecule according to any one of embodiments 5-8, wherein 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.

[0053] 16. A multifunctional molecule according to any one of embodiments 5-8, wherein 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) Heavy chain variable region (VH), the heavy chain variable region 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.

[0054] 17. A multifunctional molecule according to any one of embodiments 5-8, wherein the antigen-binding domain comprises: (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.

[0055] 18. A multifunctional molecule according to any one of embodiments 5-8, wherein the antigen-binding domain comprises: (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) Heavy chain variable region (VH), wherein the heavy chain variable region comprises 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.

[0056] 19. A multifunctional molecule according to any one of embodiments 5-18, wherein the antigen-binding domain comprises a light chain variable region (VL), and the light chain variable region comprises antibody AH.1 or antibody AH.2 (e.g., as shown in Figure 18). Figure 1B As shown, for example, the light chain framework region 1 (VLFWR1) of SEQ ID NO: 2, 10 or 11.

[0057] 20. A multifunctional molecule according to any one of embodiments 5-19, wherein the antigen-binding domain comprises a light chain variable region (VL), and the light chain variable region comprises antibody AH.1 or antibody AH.2 (e.g., as shown in Figure 19). Figure 1B As shown, for example, the light chain framework region 2 (VLFWR2) of SEQ ID NO: 2, 10 or 11.

[0058] 21. A multifunctional molecule according to any one of embodiments 5-20, wherein the antigen-binding domain comprises a light chain variable region (VL), and the light chain variable region comprises antibody AH.1 or antibody AH.2 (e.g., as shown in Figure 20). Figure 1B As shown, for example, the light chain framework region 3 (VLFWR3) of SEQ ID NO: 2, 10 or 11.

[0059] 22. A multifunctional molecule according to any one of embodiments 5-21, wherein the antigen-binding domain comprises a light chain variable region (VL), and the light chain variable region comprises antibody AH.1 or antibody AH.2 (e.g., as shown in Figure 21). Figure 1B As shown, for example, the light chain framework region 4 (VLFWR4) of SEQ ID NO: 2, 10 or 11.

[0060] 23. A multifunctional molecule of any one of embodiments 5-22, wherein the antigen-binding domain comprises a light chain variable region (VL), the light chain variable region 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.

[0061] 24. A multifunctional molecule of any one of embodiments 5-22, wherein the antigen-binding domain comprises a light chain variable region (VL), the light chain variable region 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.

[0062] 25. A multifunctional molecule of any one of embodiments 5-22, wherein the antigen-binding domain comprises a light chain variable region (VL), the light chain variable region 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: 11.

[0063] 26. A multifunctional molecule of any of embodiments 5-25, wherein the antigen-binding domain comprises a light chain variable domain, the light chain variable domain comprising a framework region, for example, framework region 1 (VLFWR1), the framework region comprising a change, for example, a substitution at position 10 according to the Kabat number (e.g., a conservative substitution), wherein the change at position 10 is a change to phenylalanine, for example, a serine-to-phenylalanine substitution.

[0064] 27. A multifunctional molecule of any one of embodiments 5-26, wherein the antigen-binding domain comprises a light chain variable domain, the light chain variable domain comprising a framework region, for example, framework region 2 (VLFWR2), the framework region comprising one or more (e.g., one or two) alterations, for example, substitutions (e.g., conserved substitutions) at positions selected from 36 and 46 according to the Kabat number, wherein the alteration at position 36 is to histidine, for example, a tyrosine-to-histidine substitution, and the alteration at position 46 is to alanine, for example, an arginine-to-alanine substitution.

[0065] 28. A multifunctional molecule of any of embodiments 5-27, wherein the antigen-binding domain comprises a light chain variable domain, the light chain variable domain comprising a framework region, for example, framework region 3 (VLFWR3), the framework region comprising a change, for example, a substitution at position 87 according to the Kabat number (e.g., a conservative substitution), wherein the change at position 87 is a change to phenylalanine, for example, a tyrosine-to-phenylalanine substitution.

[0066] 29. A multifunctional molecule of any one of embodiments 5-28, wherein the antigen-binding domain comprises a light chain variable domain comprising (a) a framework region 1 (VLFWR1) comprising phenylalanine at position 10, for example, a substitution at position 10 according to the Kabat number, such as a serine-to-phenylalanine substitution; (b) a framework region 2 (VLFWR2) comprising histidine at position 36, for example, a substitution at position 36 according to the Kabat number, such as a tyrosine-to-histidine substitution, and alanine at position 46, for example, a substitution at position 46 according to the Kabat number, such as an arginine-to-alanine substitution; and (c) a framework region 3 (VLFWR3) comprising phenylalanine at position 87, for example, a substitution at position 87 according to the Kabat number, such as a tyrosine-to-phenylalanine substitution, such as the amino acid sequence shown in SEQ ID NO: 10.

[0067] 30. A multifunctional molecule of any one of embodiments 5-28, wherein the antigen-binding domain comprises a light chain variable domain comprising (a) a framework region 2 (FR2) comprising a histidine at position 36, for example, a substitution at position 36 according to a Kabat number, for example, a tyrosine-to-histidine substitution, and an alanine at position 46, for example, a substitution at position 46 according to a Kabat number, for example, an arginine-to-alanine substitution; and (b) a framework region 3 (FR3) comprising a phenylalanine at position 87, for example, a substitution at position 87 according to a Kabat number, for example, a tyrosine-to-phenylalanine substitution, for example, as shown in the amino acid sequence of SEQ ID NO: 11.

[0068] 31. A multifunctional molecule according to any one of embodiments 5-30, wherein the antigen-binding domain comprises a heavy chain variable region (VH), and the heavy chain variable region comprises antibody AH.1 or antibody AH.2 (e.g., as shown in Figure 30). Figure 1A As shown, for example, the heavy chain framework region 1 (VHFWR1) of SEQ ID NO: 1 or 9.

[0069] 32. A multifunctional molecule according to any one of embodiments 5-31, wherein the antigen-binding domain comprises a heavy chain variable region (VH), and the heavy chain variable region comprises antibody AH.1 or antibody AH.2 (e.g., as shown in Figure 31). Figure 1A As shown, for example, the heavy chain framework region 2 (VHFWR2) of SEQ ID NO: 1 or 9.

[0070] 33. A multifunctional molecule according to any one of embodiments 5-32, wherein the antigen-binding domain comprises a heavy chain variable region (VH), and the heavy chain variable region comprises antibody AH.1 or antibody AH.2 (e.g., as shown in Figure 32). Figure 1A As shown, for example, the heavy chain framework region 3 (VHFWR3) of SEQ ID NO: 1 or 9.

[0071] 34. A multifunctional molecule according to any one of embodiments 5-33, wherein the antigen-binding domain comprises a heavy chain variable region (VH), and the heavy chain variable region comprises antibody AH.1 or antibody AH.2 (e.g., as shown in Figure 33). Figure 1A As shown, for example, the heavy chain framework region 4 (VHFWR4) of SEQ ID NO: 1 or 9.

[0072] 35. A multifunctional molecule of any one of embodiments 5-34, wherein the antigen-binding domain comprises a heavy chain variable region (VH), the heavy chain variable region comprising 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.

[0073] 36. A multifunctional molecule of any one of embodiments 5-34, wherein the antigen-binding domain comprises a heavy chain variable region (VH), the heavy chain variable region comprising 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.

[0074] 37. A multifunctional molecule of any one of embodiments 5-36, wherein the antigen-binding domain comprises a heavy chain variable domain, the heavy chain variable domain comprising a framework region, for example, framework region 3 (VHFWR3), the framework region comprising one or more (e.g., one or two) alterations, for example, substitutions (e.g., conserved substitutions) at positions selected from 73 and 94 according to Kabat numbering, wherein the alteration at position 73 is a change to threonine, for example, a glutamic acid to threonine substitution, and the alteration at position 94 is a change to glycine, for example, an arginine to glycine substitution.

[0075] 38. A multifunctional molecule of any one of embodiments 5-37, wherein the antigen-binding domain comprises a heavy chain variable domain, the heavy chain variable domain comprising a framework region 3 (FR3), the framework region 3 comprising a threonine at position 73 (e.g., a substitution at position 73 according to the Kabat number, e.g., a glutamic acid to threonine substitution) and a glycine at position 94 (e.g., a substitution at position 94 according to the Kabat number, e.g., an arginine to glycine substitution), for example, as shown in the amino acid sequence of SEQ ID NO: 10.

[0076] 39. A multifunctional molecule according to any one of embodiments 5-18, wherein the antigen-binding domain comprises heavy chain framework regions 1-4 of antibody AH.1 (e.g., SEQ ID NO: 9); and antibody AH.1 (e.g., SEQ ID NO: 10 or as shown in the figure). Figure 1A and 1B Light chain framework areas 1-4 (as shown in the diagram).

[0077] 40. A multifunctional molecule according to any one of embodiments 5-18, wherein the antigen-binding domain comprises heavy chain framework regions 1-4 of antibody AH.2 (e.g., SEQ ID NO: 9); and antibody AH.2 (e.g., SEQ ID NO: 11 or as shown in the figure). Figure 1A and 1B Light chain framework areas 1-4 (as shown in the diagram).

[0078] 41. A multifunctional molecule according to any one of embodiments 5-18, wherein the antigen-binding domain comprises: VH domain, said VH domain comprising the amino acid sequence of SEQ ID NO: 9, an amino acid sequence that is at least about 85%, 90%, 95%, 99% or more identical to the amino acid sequence of SEQ ID NO: 9, or an amino acid sequence that differs from the amino acid sequence of SEQ ID NO: 9 by no more than 1, 2, 5, 10 or 15 amino acid residues; and / or VL domain, the VL domain comprising the amino acid sequence of SEQ ID NO: 10, an amino acid sequence that is at least about 85%, 90%, 95%, 99% or more identical to the amino acid sequence of SEQ ID NO: 10, or an amino acid sequence that differs from the amino acid sequence of SEQ ID NO: 10 by no more than 1, 2, 5, 10 or 15 amino acid residues.

[0079] 42. A multifunctional molecule according to any one of embodiments 5-18, wherein the antigen-binding domain comprises: VH domain, said VH domain comprising the amino acid sequence of SEQ ID NO: 9, an amino acid sequence that is at least about 85%, 90%, 95%, 99% or more identical to the amino acid sequence of SEQ ID NO: 9, or an amino acid sequence that differs from the amino acid sequence of SEQ ID NO: 9 by no more than 1, 2, 5, 10 or 15 amino acid residues; and / or VL domain, the VL domain comprising the amino acid sequence of SEQ ID NO: 11, an amino acid sequence that is at least about 85%, 90%, 95%, 99% or more identical to the amino acid sequence of SEQ ID NO: 11, or an amino acid sequence that differs from the amino acid sequence of SEQ ID NO: 11 by no more than 1, 2, 5, 10 or 15 amino acid residues.

[0080] 43. A multifunctional molecule of any one of embodiments 1-4, wherein the antigen-binding domain specifically binds to TCRβ V12 (e.g., TCRβ V12-3*01).

[0081] 44. The multifunctional 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 described in Table 2A or encoded by nucleotide sequences in 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 aforementioned sequences.

[0082] 45. A multifunctional molecule according to embodiment 43 or 44, wherein the antigen-binding domain comprises at least one, two, or three (or all) CDRs from the heavy chain variable region, the heavy chain variable region comprising an amino acid sequence shown in Table 2A or an amino acid sequence encoded by a nucleotide sequence shown in Table 2A (or a 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 the amino acid sequence encoded by a nucleotide sequence shown in Table 2A).

[0083] 46. ​​A multifunctional molecule of any of embodiments 43-45, wherein the antigen-binding domain comprises at least one, two, or three (or all) CDRs from a light chain variable region, the light chain variable region comprising an amino acid sequence shown in Table 2A or an amino acid sequence encoded by a nucleotide sequence shown in Table 2A (or a 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 the amino acid sequence encoded by a nucleotide sequence shown in Table 2A).

[0084] 47. A multifunctional molecule according to any one of embodiments 43-46, wherein the antigen-binding domain comprises: (i) One, two, or all of the light chain complementarity determination regions 1 (LC CDR1), 2 (LC CDR2), and 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 the heavy chain complementarity determination regions 1 (HC CDR1), 2 (HC CDR2), and 3 (HC CDR3) of SEQ ID NO: 15, SEQ ID NO: 23, SEQ ID NO: 24, or SEQ ID NO: 25.

[0085] 48. A multifunctional molecule according to any one of embodiments 43-47, wherein the antigen-binding domain comprises: (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.

[0086] 49. A multifunctional molecule according to any one of embodiments 43-47, wherein the antigen-binding domain comprises: (i) a light chain variable region (VL) comprising the LC CDR1 amino acid sequence of SEQ ID NO: 20, the LC CDR2 amino acid sequence of SEQ ID NO: 21, and the LC CDR3 amino acid sequence of SEQ ID NO: 2; and / or (ii) Heavy chain variable region (VH), the heavy chain variable region 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.

[0087] 50. A multifunctional molecule according to any one of embodiments 43-47, wherein the antigen-binding domain comprises: (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 amino acid sequence of HC CDR1 of SEQ ID NO: 57, the amino acid sequence of HC CDR2 of SEQ ID NO: 58, or the amino acid sequence of HC CDR3 of SEQ ID NO: 59.

[0088] 51. A multifunctional molecule according to any one of embodiments 43-47, wherein the antigen-binding domain comprises: (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) Heavy chain variable region (VH), wherein the heavy chain variable region comprises 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.

[0089] 52. A multifunctional molecule according to any one of embodiments 43-47, wherein the antigen-binding domain comprises: (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.

[0090] 53. A multifunctional molecule according to any one of embodiments 43-47, wherein the antigen-binding domain comprises: (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) Heavy chain variable region (VH), wherein the heavy chain variable region comprises 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.

[0091] 54. A multifunctional molecule according to any one of embodiments 43-53, wherein the antigen-binding domain comprises a light chain variable region (VL), the light chain variable region comprising (e.g., as...) Figure 2B As shown, for example, SEQ ID NO: 16 or 26-30) light chain framework region 1 (VLFWR1).

[0092] 55. A multifunctional molecule according to any one of embodiments 43-54, wherein the antigen-binding domain comprises a light chain variable region (VL), the light chain variable region comprising (e.g., as...) Figure 2B As shown, for example, SEQ ID NO: 16 or 26-30) light chain framework region 2 (VLFWR2).

[0093] 56. A multifunctional molecule according to any one of embodiments 43-55, wherein the antigen-binding domain comprises a light chain variable region (VL), the light chain variable region comprising (e.g., as...) Figure 2B As shown, for example, SEQ ID NO: 16 or 26-30) light chain framework region 3 (VLFWR3).

[0094] 57. A multifunctional molecule according to any one of embodiments 43-56, wherein the antigen-binding domain comprises a light chain variable region (VL), the light chain variable region comprising (e.g., as...) Figure 2B As shown, for example, SEQ ID NO: 16 or 26-30) light chain framework region 4 (VLFWR4).

[0095] 58. A multifunctional molecule of any one of embodiments 43-57, wherein the antigen-binding domain comprises a light chain variable region (VL), the light chain variable region 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.

[0096] 59. A multifunctional molecule of any one of embodiments 43-57, wherein the antigen-binding domain comprises a light chain variable region (VL), the light chain variable region 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.

[0097] 60. A multifunctional molecule of any one of embodiments 43-57, wherein the antigen-binding domain comprises a light chain variable region (VL), the light chain variable region 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.

[0098] 61. A multifunctional molecule of any one of embodiments 43-57, wherein the antigen-binding domain comprises a light chain variable region (VL), the light chain variable region 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.

[0099] 62. A multifunctional molecule of any one of embodiments 43-57, wherein the antigen-binding domain comprises a light chain variable region (VL), the light chain variable region 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.

[0100] 63. A multifunctional molecule of any one of embodiments 43-57, wherein the antigen-binding domain comprises a light chain variable region (VL), the light chain variable region 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.

[0101] 64. A multifunctional molecule of any of embodiments 43-57, wherein the antigen-binding domain comprises a light chain variable domain, the light chain variable domain comprising a framework region, for example, framework region 1 (VLFWR1), the framework region comprising one or more (e.g., one, two, or three) alterations, for example, substitutions (e.g., conserved substitutions) at positions selected from 1, 2, and 4 according to Kabat numbering, wherein the alteration at position 1 is to become aspartic, for example, alanine to aspartic, the alteration at position 2 is to become asparagine, for example, isoleucine to asparagine, and the alteration at position 4 is to become leucine, for example, methionine to leucine.

[0102] 65. A multifunctional molecule according to any one of embodiments 43-57 or 64, wherein the antigen-binding domain comprises a light chain variable domain, the light chain variable domain comprising a framework region, for example, framework region 3 (VLFWR3), the framework region comprising one or more (e.g., one, two or three) alterations, for example, substitutions (e.g., conserved substitutions) at positions selected from 66, 69 and 71 according to Kabat numbering, wherein the alteration at position 66 is a change to glycine, for example, a lysine-to-glycine substitution, the alteration at position 69 is a change to asparagine, for example, a tyrosine-to-asparagine substitution, and the alteration at position 71 is a change to tyrosine, for example, a phenylalanine-to-tyrosine substitution.

[0103] 66. A multifunctional molecule according to any one of embodiments 43-57, 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 a Kabat number, for example, an isoleucine to asparagine substitution; and a framework region 3 (FR3) comprising a substitution at position 69 according to a Kabat number, for example, a threonine to asparagine substitution, and a substitution at position 71 according to a Kabat number, for example, a phenylalanine to tyrosine substitution, for example, as shown in the amino acid sequence of SEQ ID NO: 26.

[0104] 67. A multifunctional molecule according to any one of embodiments 43-57, 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 the Kabat number (e.g., alanine to aspartic acid substitution) and a substitution at position 2 according to the Kabat number (e.g., isoleucine to asparagine substitution); and (b) a framework region 3 (FR3) comprising a substitution at position 69 according to the Kabat number (e.g., threonine to asparagine substitution) and a substitution at position 71 according to the Kabat number (e.g., phenylalanine to tyrosine substitution), for example, as shown in the amino acid sequence of SEQ ID NO: 27.

[0105] 68. A multifunctional molecule according to any one of embodiments 43-57, 64 or 65, wherein the antigen-binding domain comprises a light chain comprising (a) a framework region 1 (FR1) comprising a substitution at position 2 according to a Kabat number, such as a serine-to-asparagine substitution, and a substitution at position 4 according to a Kabat number, such as a methionine-to-leucine substitution; and (b) a framework region 3 (FR3) comprising a substitution at position 69 according to a Kabat number, such as a threonine-to-asparagine substitution, and a substitution at position 71 according to a Kabat number, such as a phenylalanine-to-tyrosine substitution, for example, the amino acid sequence as shown in SEQ ID NO:28.

[0106] 69. A multifunctional molecule according to any one of embodiments 43-57, 64 or 65, wherein the antigen-binding domain comprises a light chain comprising (a) a framework region 1 (FR1) comprising a substitution at position 2 according to a Kabat number, such as a serine-to-asparagine substitution; and (b) a framework region 3 (FR3) comprising a substitution at position 66 according to a Kabat number, such as a lysine-to-glycine substitution; a substitution at position 69 according to a Kabat number, such as a threonine-to-asparagine substitution; and a substitution at position 71 according to a Kabat number, such as an alanine-to-tyrosine substitution, for example, as shown in the amino acid sequence of SEQ ID NO: 29.

[0107] 70. A multifunctional molecule according to any one of embodiments 43-69, wherein the antigen-binding domain comprises a heavy chain variable region (VH), the heavy chain variable region comprising (e.g., as...) Figure 2A As shown, for example, SEQ ID NO: 15 or 23-25) heavy chain framework region 1 (VHFWR1).

[0108] 71. A multifunctional molecule according to any one of embodiments 43-70, wherein the antigen-binding domain comprises a heavy chain variable region (VH), the heavy chain variable region comprising (e.g., as...) Figure 2A As shown, for example, SEQ ID NO: 15 or 23-25) heavy chain framework region 2 (VHFWR2).

[0109] 72. A multifunctional molecule according to any one of embodiments 43-71, wherein the antigen-binding domain comprises a heavy chain variable region (VH), the heavy chain variable region comprising (e.g., as...) Figure 2A As shown, for example, SEQ ID NO: 15 or 23-25) heavy chain framework region 3 (VHFWR3).

[0110] 73. A multifunctional molecule according to any one of embodiments 43-72, wherein the antigen-binding domain comprises a heavy chain variable region (VH), the heavy chain variable region comprising (e.g., as...) Figure 2A As shown, for example, SEQ ID NO: 15 or 23-25) heavy chain framework region 4 (VHFWR4).

[0111] 74. A multifunctional molecule of any one of embodiments 43-73, wherein the antigen-binding domain comprises a heavy chain variable region (VH), the heavy chain variable region comprising 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.

[0112] 75. A multifunctional molecule of any one of embodiments 43-73, wherein the antigen-binding domain comprises a heavy chain variable region (VH), the heavy chain variable region comprising 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.

[0113] 76. A multifunctional molecule of any one of embodiments 43-73, wherein the antigen-binding domain comprises a heavy chain variable region (VH), the heavy chain variable region comprising 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.

[0114] 77. A multifunctional molecule of any one of embodiments 43-73, wherein the antigen-binding domain comprises a heavy chain and a light chain, the heavy chain comprising heavy chain framework regions 1-4 of SEQ ID NO: 23, 24 or 25; and the light chain comprising light chain framework regions 1-4 of SEQ ID NO: 26, 27, 28, 29 or 30.

[0115] 78. A multifunctional molecule according to any one of embodiments 43-73, wherein the antigen-binding domain comprises: VH domain, said VH domain comprising an amino acid sequence selected from the amino acid sequences of SEQ ID NO: 23, SEQ ID NO: 24 or SEQ ID NO: 25, an amino acid sequence that is at least about 85%, 90%, 95%, 99% or more identical to the amino acid sequence of SEQ ID NO: 23, SEQ ID NO: 24 or SEQ ID NO: 25, or an amino acid sequence that differs from the amino acid sequence of SEQ ID NO: 23, SEQ ID NO: 24 or SEQ ID NO: 25 by no more than 1, 2, 5, 10 or 15 amino acid residues; and / or The VL domain comprises an amino acid sequence selected from the amino acid sequences of SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29 or SEQ ID NO: 30, an amino acid sequence that is at least about 85%, 90%, 95%, 99% or more identical to the amino acid sequence of SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29 or SEQ ID NO: 30, or 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 no more than 1, 2, 5, 10 or 15 amino acid residues.

[0116] 79. A multifunctional molecule according to any one of embodiments 43-73, wherein the antigen-binding domain comprises: The VH domain comprises the amino acid sequence of SEQ ID NO: 23, an amino acid sequence that is at least about 85%, 90%, 95%, 99% or more identical to the amino acid sequence of SEQ ID NO: 23, or an amino acid sequence that differs from the amino acid sequence of SEQ ID NO: 23 by no more than 1, 2, 5, 10 or 15 amino acid residues; and VL domain, the VL domain comprising the amino acid sequence of SEQ ID NO: 26, an amino acid sequence that is at least about 85%, 90%, 95%, 99% or more identical to the amino acid sequence of SEQ ID NO: 26, or an amino acid sequence that differs from the amino acid sequence of SEQ ID NO: 26 by no more than 1, 2, 5, 10 or 15 amino acid residues.

[0117] 80. A multifunctional molecule according to any one of embodiments 43-73, wherein the antigen-binding domain comprises: The VH domain comprises the amino acid sequence of SEQ ID NO: 23, an amino acid sequence that is at least about 85%, 90%, 95%, 99% or more identical to the amino acid sequence of SEQ ID NO: 23, or an amino acid sequence that differs from the amino acid sequence of SEQ ID NO: 23 by no more than 1, 2, 5, 10 or 15 amino acid residues; and VL domain, the VL domain comprising the amino acid sequence of SEQ ID NO: 27, an amino acid sequence that is at least about 85%, 90%, 95%, 99% or more identical to the amino acid sequence of SEQ ID NO: 27, or an amino acid sequence that differs from the amino acid sequence of SEQ ID NO: 27 by no more than 1, 2, 5, 10 or 15 amino acid residues.

[0118] 81. A multifunctional molecule according to any one of embodiments 43-73, wherein the antigen-binding domain comprises: The VH domain comprises the amino acid sequence of SEQ ID NO: 23, an amino acid sequence that is at least about 85%, 90%, 95%, 99% or more identical to the amino acid sequence of SEQ ID NO: 23, or an amino acid sequence that differs from the amino acid sequence of SEQ ID NO: 23 by no more than 1, 2, 5, 10 or 15 amino acid residues; and VL domain, the VL domain comprising the amino acid sequence of SEQ ID NO: 28, an amino acid sequence that is at least about 85%, 90%, 95%, 99% or more identical to the amino acid sequence of SEQ ID NO: 28, or an amino acid sequence that differs from the amino acid sequence of SEQ ID NO: 28 by no more than 1, 2, 5, 10 or 15 amino acid residues.

[0119] 82. A multifunctional molecule according to any one of embodiments 43-73, wherein the antigen-binding domain comprises: The VH domain comprises the amino acid sequence of SEQ ID NO: 23, an amino acid sequence that is at least about 85%, 90%, 95%, 99% or more identical to the amino acid sequence of SEQ ID NO: 23, or an amino acid sequence that differs from the amino acid sequence of SEQ ID NO: 23 by no more than 1, 2, 5, 10 or 15 amino acid residues; and VL domain, the VL domain comprising the amino acid sequence of SEQ ID NO: 29, an amino acid sequence that is at least about 85%, 90%, 95%, 99% or more identical to the amino acid sequence of SEQ ID NO: 29, or an amino acid sequence that differs from the amino acid sequence of SEQ ID NO: 29 by no more than 1, 2, 5, 10 or 15 amino acid residues.

[0120] 83. A multifunctional molecule according to any one of embodiments 43-73, wherein the antigen-binding domain comprises: The VH domain comprises the amino acid sequence of SEQ ID NO: 23, an amino acid sequence that is at least about 85%, 90%, 95%, 99% or more identical to the amino acid sequence of SEQ ID NO: 23, or an amino acid sequence that differs from the amino acid sequence of SEQ ID NO: 23 by no more than 1, 2, 5, 10 or 15 amino acid residues; and VL domain, the VL domain comprising the amino acid sequence of SEQ ID NO: 30, an amino acid sequence that is at least about 85%, 90%, 95%, 99% or more identical to the amino acid sequence of SEQ ID NO: 30, or an amino acid sequence that differs from the amino acid sequence of SEQ ID NO: 30 by no more than 1, 2, 5, 10 or 15 amino acid residues.

[0121] 84. A multifunctional molecule according to any one of embodiments 43-73, wherein the antigen-binding domain comprises: The VH domain comprises the amino acid sequence of SEQ ID NO: 24, an amino acid sequence that is at least about 85%, 90%, 95%, 99% or more identical to the amino acid sequence of SEQ ID NO: 24, or an amino acid sequence that differs from the amino acid sequence of SEQ ID NO: 24 by no more than 1, 2, 5, 10 or 15 amino acid residues; and VL domain, the VL domain comprising the amino acid sequence of SEQ ID NO: 26, an amino acid sequence that is at least about 85%, 90%, 95%, 99% or more identical to the amino acid sequence of SEQ ID NO: 26, or an amino acid sequence that differs from the amino acid sequence of SEQ ID NO: 26 by no more than 1, 2, 5, 10 or 15 amino acid residues.

[0122] 85. A multifunctional molecule according to any one of embodiments 43-73, wherein the antigen-binding domain comprises: The VH domain comprises the amino acid sequence of SEQ ID NO: 24, an amino acid sequence that is at least about 85%, 90%, 95%, 99% or more identical to the amino acid sequence of SEQ ID NO: 24, or an amino acid sequence that differs from the amino acid sequence of SEQ ID NO: 24 by no more than 1, 2, 5, 10 or 15 amino acid residues; and VL domain, the VL domain comprising the amino acid sequence of SEQ ID NO: 27, an amino acid sequence that is at least about 85%, 90%, 95%, 99% or more identical to the amino acid sequence of SEQ ID NO: 27, or an amino acid sequence that differs from the amino acid sequence of SEQ ID NO: 27 by no more than 1, 2, 5, 10 or 15 amino acid residues.

[0123] 86. A multifunctional molecule according to any one of embodiments 43-73, wherein the antigen-binding domain comprises: The VH domain comprises the amino acid sequence of SEQ ID NO: 24, an amino acid sequence that is at least about 85%, 90%, 95%, 99% or more identical to the amino acid sequence of SEQ ID NO: 24, or an amino acid sequence that differs from the amino acid sequence of SEQ ID NO: 24 by no more than 1, 2, 5, 10 or 15 amino acid residues; and VL domain, the VL domain comprising the amino acid sequence of SEQ ID NO: 28, an amino acid sequence that is at least about 85%, 90%, 95%, 99% or more identical to the amino acid sequence of SEQ ID NO: 28, or an amino acid sequence that differs from the amino acid sequence of SEQ ID NO: 28 by no more than 1, 2, 5, 10 or 15 amino acid residues.

[0124] 87. A multifunctional molecule according to any one of embodiments 43-73, wherein the antigen-binding domain comprises: The VH domain comprises the amino acid sequence of SEQ ID NO: 24, an amino acid sequence that is at least about 85%, 90%, 95%, 99% or more identical to the amino acid sequence of SEQ ID NO: 24, or an amino acid sequence that differs from the amino acid sequence of SEQ ID NO: 24 by no more than 1, 2, 5, 10 or 15 amino acid residues; and VL domain, the VL domain comprising the amino acid sequence of SEQ ID NO: 29, an amino acid sequence that is at least about 85%, 90%, 95%, 99% or more identical to the amino acid sequence of SEQ ID NO: 29, or an amino acid sequence that differs from the amino acid sequence of SEQ ID NO: 29 by no more than 1, 2, 5, 10 or 15 amino acid residues.

[0125] 88. A multifunctional molecule according to any one of embodiments 43-73, wherein the antigen-binding domain comprises: The VH domain comprises the amino acid sequence of SEQ ID NO: 24, an amino acid sequence that is at least about 85%, 90%, 95%, 99% or more identical to the amino acid sequence of SEQ ID NO: 24, or an amino acid sequence that differs from the amino acid sequence of SEQ ID NO: 24 by no more than 1, 2, 5, 10 or 15 amino acid residues; and VL domain, the VL domain comprising the amino acid sequence of SEQ ID NO: 30, an amino acid sequence that is at least about 85%, 90%, 95%, 99% or more identical to the amino acid sequence of SEQ ID NO: 30, or an amino acid sequence that differs from the amino acid sequence of SEQ ID NO: 30 by no more than 1, 2, 5, 10 or 15 amino acid residues.

[0126] 89. A multifunctional molecule according to any one of embodiments 43-73, wherein the antigen-binding domain comprises: The VH domain comprises the amino acid sequence of SEQ ID NO: 25, an amino acid sequence that is at least about 85%, 90%, 95%, 99% or more identical to the amino acid sequence of SEQ ID NO: 25, or an amino acid sequence that differs from the amino acid sequence of SEQ ID NO: 25 by no more than 1, 2, 5, 10 or 15 amino acid residues; and VL domain, the VL domain comprising the amino acid sequence of SEQ ID NO: 26, an amino acid sequence that is at least about 85%, 90%, 95%, 99% or more identical to the amino acid sequence of SEQ ID NO: 26, or an amino acid sequence that differs from the amino acid sequence of SEQ ID NO: 26 by no more than 1, 2, 5, 10 or 15 amino acid residues.

[0127] 90. A multifunctional molecule according to any one of embodiments 43-73, wherein the antigen-binding domain comprises: The VH domain comprises the amino acid sequence of SEQ ID NO: 25, an amino acid sequence that is at least about 85%, 90%, 95%, 99% or more identical to the amino acid sequence of SEQ ID NO: 25, or an amino acid sequence that differs from the amino acid sequence of SEQ ID NO: 25 by no more than 1, 2, 5, 10 or 15 amino acid residues; and VL domain, the VL domain comprising the amino acid sequence of SEQ ID NO: 27, an amino acid sequence that is at least about 85%, 90%, 95%, 99% or more identical to the amino acid sequence of SEQ ID NO: 27, or an amino acid sequence that differs from the amino acid sequence of SEQ ID NO: 27 by no more than 1, 2, 5, 10 or 15 amino acid residues.

[0128] 91. A multifunctional molecule according to any one of embodiments 43-73, wherein the antigen-binding domain comprises: The VH domain comprises the amino acid sequence of SEQ ID NO: 25, an amino acid sequence that is at least about 85%, 90%, 95%, 99% or more identical to the amino acid sequence of SEQ ID NO: 25, or an amino acid sequence that differs from the amino acid sequence of SEQ ID NO: 25 by no more than 1, 2, 5, 10 or 15 amino acid residues; and VL domain, the VL domain comprising the amino acid sequence of SEQ ID NO: 28, an amino acid sequence that is at least about 85%, 90%, 95%, 99% or more identical to the amino acid sequence of SEQ ID NO: 28, or an amino acid sequence that differs from the amino acid sequence of SEQ ID NO: 28 by no more than 1, 2, 5, 10 or 15 amino acid residues.

[0129] 92. A multifunctional molecule according to any one of embodiments 43-73, wherein the antigen-binding domain comprises: The VH domain comprises the amino acid sequence of SEQ ID NO: 25, an amino acid sequence that is at least about 85%, 90%, 95%, 99% or more identical to the amino acid sequence of SEQ ID NO: 25, or an amino acid sequence that differs from the amino acid sequence of SEQ ID NO: 25 by no more than 1, 2, 5, 10 or 15 amino acid residues; and VL domain, the VL domain comprising the amino acid sequence of SEQ ID NO: 29, an amino acid sequence that is at least about 85%, 90%, 95%, 99% or more identical to the amino acid sequence of SEQ ID NO: 29, or an amino acid sequence that differs from the amino acid sequence of SEQ ID NO: 29 by no more than 1, 2, 5, 10 or 15 amino acid residues.

[0130] 93. A multifunctional molecule according to any one of embodiments 43-73, wherein the antigen-binding domain comprises: The VH domain comprises the amino acid sequence of SEQ ID NO: 25, an amino acid sequence that is at least about 85%, 90%, 95%, 99% or more identical to the amino acid sequence of SEQ ID NO: 25, or an amino acid sequence that differs from the amino acid sequence of SEQ ID NO: 25 by no more than 1, 2, 5, 10 or 15 amino acid residues; and VL domain, the VL domain comprising the amino acid sequence of SEQ ID NO: 30, an amino acid sequence that is at least about 85%, 90%, 95%, 99% or more identical to the amino acid sequence of SEQ ID NO: 30, or an amino acid sequence that differs from the amino acid sequence of SEQ ID NO: 30 by no more than 1, 2, 5, 10 or 15 amino acid residues.

[0131] 94. A multifunctional molecule according to any one of embodiments 1-93, wherein the first antigen-binding domain has a high affinity for a T-cell receptor containing a TCRBV antigen, optionally wherein the first antigen-binding domain binds to a K+ receptor containing a TCRBV antigen. D The K+ binding between the first antigen-binding domain and a T-cell receptor that does not contain the TCRBV antigen does not exceed the amount of K+ binding. D 40%, 30%, 20%, 10%, 1%, 0.1% or 0.01%.

[0132] 95. Any multifunctional molecule in the prior embodiment, wherein the binding of the first antigen-binding domain to the TCRBV antigen on, for example, lymphocytes (e.g., T cells) does not activate lymphocytes, such as T cells.

[0133] 96. Any multifunctional molecule in the preceding embodiments, wherein the binding of the first antigen-binding domain to the TCRBV antigen on, for example, lymphocytes (e.g., T cells) does not significantly activate lymphocytes such as T cells (e.g., as measured by T cell proliferation, expression of T cell activation markers (e.g., CD69 or CD25) and / or expression of cytokines (e.g., TNFα and IFNγ).

[0134] 97. Any multifunctional molecule in the preceding embodiments, wherein the multifunctional molecule preferentially binds to lymphocytes containing the TCRBV antigen compared to lymphocytes not containing the TCRBV antigen, 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.

[0135] 98. A multifunctional molecule according to any one of embodiments 1-97, wherein the multifunctional molecule comprises an immune cell adjuvant selected from NK cell adjuvants, T cell adjuvants, B cell adjuvants, dendritic cell adjuvants or macrophage adjuvants.

[0136] 99. The multifunctional molecule of embodiment 98, wherein the immune cell adaptor binds to and activates immune cells, such as effector cells.

[0137] 100. A multifunctional molecule according to embodiment 98, wherein the immune cell adaptor binds to but does not activate immune cells, such as effector cells.

[0138] 101. A multifunctional molecule according to any one of embodiments 98-100, wherein the immune cell adaptor is a T cell adaptor, for example, a T cell adaptor that mediates binding to and activation of T cells, or a T cell adaptor that mediates binding to T cells but does not mediate T cell activation.

[0139] 102. A multifunctional molecule according to embodiment 101, wherein the T cell adaptor binds to TCRα, TCRγ, TCRζ, ICOS, CD28, CD27, HVEM, LIGHT, CD40, 4-1BB, OX40, DR3, GITR, CD30, TIM1, SLAM, CD2, CD3, or CD226, for example, the T cell adaptor is an anti-CD3 antibody molecule.

[0140] 103. A multifunctional molecule according to any one of embodiments 98-100, wherein the immune cell adaptor is an NK cell adaptor, for example, an NK cell adaptor that mediates binding to and activation of NK cells, or an NK cell adaptor that mediates binding to NK cells but does not mediate NK cell activation.

[0141] 104. A multifunctional molecule of embodiment 103, wherein the NK cell adaptor is selected from antibody molecules, such as antigen-binding domains or ligands, said antibody molecules or ligands binding to (e.g., activating): 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, for example, said NK cell adaptor is an antibody molecule or ligand binding to (e.g., activating) NKp30.

[0142] 105. A multifunctional molecule according to embodiment 103, wherein the NK cell adaptor is an antibody molecule, for example, an antigen-binding domain.

[0143] 106. A multifunctional molecule according to embodiment 104 or 105, wherein the NK cell adaptor is capable of adapting to NK cells.

[0144] 107. A multifunctional molecule according to any one of embodiments 103-106, wherein the NK cell adaptor is an antibody molecule, for example, an antigen-binding domain, that binds to NKp30, NKp46, NKG2D, or CD16.

[0145] 108. Any multifunctional molecule in the preceding embodiments, wherein the multifunctional molecule: (i) Specifically binds to epitopes of NKp30, NKp46, NKG2D or CD16, for example, epitopes that are the same as or similar to epitopes recognized by the anti-NKp30, anti-NKp46, anti-NKG2D or anti-CD16 antibody molecules described herein; (ii) exhibiting the same or similar binding affinity or specificity, or both, as described herein with the anti-NKp30, anti-NKp46, anti-NKG2D, or anti-CD16 antibody molecules; (iii) Inhibit, for example, competitively inhibit the binding of the anti-NKp30, anti-NKp46, anti-NKG2D or anti-CD16 antibody molecules described herein; (iv) Epitopes that bind to the same or overlapping epitopes as the anti-NKp30, anti-NKp46, anti-NKG2D, or anti-CD16 antibody molecules described herein; or (v) Competes with and / or binds to the same epitopes as the anti-NKp30, anti-NKp46, anti-NKG2D or anti-CD16 antibody molecules described herein.

[0146] 109. A multifunctional molecule of any one of embodiments 103-108, 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 substantially the same sequence thereas.

[0147] 110. A multifunctional molecule according to any one of embodiments 103-109, wherein the NK cell adaptor is an antibody molecule that binds to NKp30, for example, an antigen-binding domain.

[0148] 111. A multifunctional molecule of any of embodiments 103-110, wherein the lysis of lymphocytes, such as lymphocytes containing TCRVB antigens corresponding to a biased TCRVB clone, is mediated by NKp30.

[0149] 112. A multifunctional molecule according to any one of embodiments 103-111, wherein the multifunctional molecule does not activate NK cells when incubated with NK cells in the absence of the TCRBV antigen.

[0150] 113. A multifunctional molecule according to any one of embodiments 103-112, wherein the multifunctional molecule activates the NK cell when the NK cell is an NK cell expressing NKp30 and when the TCRBV antigen is also present.

[0151] 114. A multifunctional molecule according to any one of embodiments 103-113, wherein the multifunctional molecule does not activate NK cells when the NK cells are not NK cells expressing NKp30 and the TCRBAV antigen is also present.

[0152] 115. A multifunctional molecule according to any one of embodiments 103-113, wherein the NK cell adaptor comprises: (i) a heavy chain variable region (VH) comprising the 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., substitution, addition, or deletion), the VHCDR2 amino acid sequence of SEQ ID NO: 6001 (or a sequence having no more than 1, 2, 3, or 4 mutations, e.g., substitution, addition, or deletion), and / or the VHCDR3 amino acid sequence of SEQ ID NO: 6002 (or a sequence having no more than 1, 2, 3, or 4 mutations, e.g., substitution, addition, or deletion), and (ii) Light chain variable region (VL), the light chain variable region comprising the amino acid sequence of light chain complementarity-determining region 1 (VLCDR1) of SEQ ID NO: 6063 (or a sequence having no more than 1, 2, 3 or 4 mutations, such as substitution, addition or deletion), the amino acid sequence of VLCDR2 of SEQ ID NO: 6064 (or a sequence having no more than 1, 2, 3 or 4 mutations, such as substitution, addition or deletion), and / or the amino acid sequence of VLCDR3 of SEQ ID NO: 6065 (or a sequence having no more than 1, 2, 3 or 4 mutations, such as substitution, addition or deletion).

[0153] 116. The multifunctional molecule of embodiment 115, wherein the NK cell adaptor comprises: (i) Heavy chain variable region (VH), said heavy chain variable region 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) Light chain variable region (VL), the light chain variable region comprising the amino acid sequence of light chain complementarity determination region 1 (VLCDR1) of SEQ ID NO: 6063, the amino acid sequence of VLCDR2 of SEQ ID NO: 6064 and / or the amino acid sequence of VLCDR3 of SEQ ID NO: 6065.

[0154] 117. A multifunctional molecule according to any one of embodiments 103-116, wherein the NK cell adaptor comprises: (1) Heavy chain variable region (VH), the heavy chain variable region comprising the heavy chain framework region 1 (VHFWR1) amino acid sequence of SEQ ID NO: 6003 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations therein, e.g., substitution, addition or deletion), the VHFWR2 amino acid sequence of SEQ ID NO: 6004 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations therein, e.g., substitution, addition or deletion), the VHFWR3 amino acid sequence of SEQ ID NO: 6005 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations therein, e.g., substitution, addition or deletion), or the VHFWR4 amino acid sequence of SEQ ID NO: 6006 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations therein, e.g., substitution, addition or deletion), and / or (2) Light chain variable region (VL), the light chain variable region comprising the light chain framework region 1 (VLFWR1) amino acid sequence of SEQ ID NO: 6066 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations, such as substitution, addition or deletion), the VLFWR2 amino acid sequence of SEQ ID NO: 6067 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations, such as substitution, addition or deletion), the VLFWR3 amino acid sequence of SEQ ID NO: 6068 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations, such as substitution, addition or deletion), or the VLFWR4 amino acid sequence of SEQ ID NO: 6069 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations, such as substitution, addition or deletion).

[0155] 118. The multifunctional molecule of embodiment 117, wherein the NK cell adaptor comprises: (1) Heavy chain variable region (VH), wherein the heavy chain variable region comprises 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) Light chain variable region (VL), wherein the light chain variable region comprises the amino acid sequence of light chain framework region 1 (VLFWR1) of SEQ ID NO: 6066, the amino acid sequence of VLFWR2 of SEQ ID NO: 6067, the amino acid sequence of VLFWR3 of SEQ ID NO: 6068 or the amino acid sequence of VLFWR4 of SEQ ID NO: 6069.

[0156] 119. A multifunctional molecule according to any one of embodiments 103-118, wherein the NK cell adaptor comprises: (i) VH, wherein the VH comprises the amino acid sequence of SEQ ID NO: 6121 (or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95% or 99% sequence identity with SEQ ID NO: 6121), and / or (ii) VL, wherein the VL comprises the amino acid sequence of SEQ ID NO: 6135 (or an amino acid sequence having at least about 93%, 95% or 99% sequence identity with SEQ ID NO: 6135).

[0157] 120. A multifunctional molecule according to any one of embodiments 103-119, wherein the NK cell adaptor comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 6148 or 6149 (or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95% or 99% sequence identity with SEQ ID NO: 6148 or 6149).

[0158] 121. A multifunctional molecule according to any one of embodiments 103-120, wherein the NK cell adaptor comprises a light chain comprising the amino acid sequence of SEQ ID NO: 6150 (or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95% or 99% sequence identity with SEQ ID NO: 6150).

[0159] 122. A multifunctional molecule according to any one of embodiments 103-121, wherein the NK cell adaptor comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 6148 or 6149 (or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with SEQ ID NO: 6148 or 6149), and a light chain comprising the amino acid sequence of SEQ ID NO: 6150 (or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with SEQ ID NO: 6150).

[0160] 123. A multifunctional molecule according to any one of embodiments 103-116, wherein the NK cell adaptor comprises a heavy chain variable region (VH), the heavy chain variable region comprising the heavy chain framework region 1 (VHFWR1) amino acid sequence of SEQ ID NO: 6014 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations therein, e.g., substitution, addition or deletion), the VHFWR2 amino acid sequence of SEQ ID NO: 6015 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations therein, e.g., substitution, addition or deletion), the VHFWR3 amino acid sequence of SEQ ID NO: 6016 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations therein, e.g., substitution, addition or deletion), or the VHFWR4 amino acid sequence of SEQ ID NO: 6017 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations therein, e.g., substitution, addition or deletion).

[0161] 124. The multifunctional molecule of embodiment 123, wherein the NK cell adaptor comprises a heavy chain variable region (VH), the heavy chain variable region 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.

[0162] 125. A multifunctional molecule of embodiment 124, wherein the NK cell adaptor comprises VH, the VH comprising the amino acid sequence of SEQ ID NO: 6123 (or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95% or 99% sequence identity with SEQ ID NO: 6123).

[0163] 126. A multifunctional molecule according to any one of embodiments 103-116, wherein the NK cell adaptor comprises a heavy chain variable region (VH), the heavy chain variable region comprising the heavy chain framework region 1 (VHFWR1) amino acid sequence of SEQ ID NO: 6018 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations therein, e.g., substitution, addition or deletion), the VHFWR2 amino acid sequence of SEQ ID NO: 6019 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations therein, e.g., substitution, addition or deletion), the VHFWR3 amino acid sequence of SEQ ID NO: 6020 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations therein, e.g., substitution, addition or deletion), or the VHFWR4 amino acid sequence of SEQ ID NO: 6021 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations therein, e.g., substitution, addition or deletion).

[0164] 127. The multifunctional molecule of embodiment 126, wherein the NK cell adaptor comprises a heavy chain variable region (VH), the heavy chain variable region 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.

[0165] 128. A multifunctional molecule of embodiment 127, wherein the NK cell adaptor comprises VH, the VH comprising the amino acid sequence of SEQ ID NO: 6124 (or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95% or 99% sequence identity with SEQ ID NO: 6124).

[0166] 129. A multifunctional molecule according to any one of embodiments 103-116, wherein the NK cell adaptor comprises a heavy chain variable region (VH), the heavy chain variable region comprising the heavy chain framework region 1 (VHFWR1) amino acid sequence of SEQ ID NO: 6022 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations therein, e.g., substitution, addition or deletion), the VHFWR2 amino acid sequence of SEQ ID NO: 6023 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations therein, e.g., substitution, addition or deletion), the VHFWR3 amino acid sequence of SEQ ID NO: 6024 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations therein, e.g., substitution, addition or deletion), or the VHFWR4 amino acid sequence of SEQ ID NO: 6025 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations therein, e.g., substitution, addition or deletion).

[0167] 130. The multifunctional molecule of embodiment 129, wherein the NK cell adaptor comprises a heavy chain variable region (VH), the heavy chain variable region 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.

[0168] 131. The multifunctional molecule of embodiment 130, wherein the NK cell adaptor comprises VH, the 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 with SEQ ID NO: 6125).

[0169] 132. A multifunctional molecule according to any one of embodiments 103-116, wherein the NK cell adaptor comprises a heavy chain variable region (VH), the heavy chain variable region comprising the heavy chain framework region 1 (VHFWR1) amino acid sequence of SEQ ID NO: 6026 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations therein, e.g., substitution, addition or deletion), the VHFWR2 amino acid sequence of SEQ ID NO: 6027 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations therein, e.g., substitution, addition or deletion), the VHFWR3 amino acid sequence of SEQ ID NO: 6028 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations therein, e.g., substitution, addition or deletion), or the VHFWR4 amino acid sequence of SEQ ID NO: 6029 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations therein, e.g., substitution, addition or deletion).

[0170] 133. The multifunctional molecule of embodiment 132, wherein the NK cell adaptor comprises a heavy chain variable region (VH), the heavy chain variable region comprising the amino acid sequence of heavy chain framework region 1 (VHFWR1) of SEQ ID NO: 6026, the amino acid sequence of VHFWR2 of SEQ ID NO: 6027, the amino acid sequence of VHFWR3 of SEQ ID NO: 6028, or the amino acid sequence of VHFWR4 of SEQ ID NO: 6029.

[0171] 134. The multifunctional molecule of embodiment 133, wherein the NK cell adaptor comprises VH, the 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 with SEQ ID NO: 6126).

[0172] 135. A multifunctional molecule according to any one of embodiments 103-116, wherein the NK cell adaptor comprises a heavy chain variable region (VH), the heavy chain variable region comprising the heavy chain framework region 1 (VHFWR1) amino acid sequence of SEQ ID NO: 6030 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations therein, e.g., substitution, addition or deletion), the VHFWR2 amino acid sequence of SEQ ID NO: 6032 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations therein, e.g., substitution, addition or deletion), the VHFWR3 amino acid sequence of SEQ ID NO: 6033 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations therein, e.g., substitution, addition or deletion), or the VHFWR4 amino acid sequence of SEQ ID NO: 6034 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations therein, e.g., substitution, addition or deletion).

[0173] 136. The multifunctional molecule of embodiment 135, wherein the NK cell adaptor comprises a heavy chain variable region (VH), the heavy chain variable region comprising the amino acid sequence of heavy chain framework region 1 (VHFWR1) of SEQ ID NO: 6030, the amino acid sequence of VHFWR2 of SEQ ID NO: 6032, the amino acid sequence of VHFWR3 of SEQ ID NO: 6033, or the amino acid sequence of VHFWR4 of SEQ ID NO: 6034.

[0174] 137. The multifunctional molecule of embodiment 136, wherein the NK cell adaptor comprises VH, the 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 with SEQ ID NO: 6127).

[0175] 138. A multifunctional molecule according to any one of embodiments 103-116, wherein the NK cell adaptor comprises a heavy chain variable region (VH), the heavy chain variable region comprising the heavy chain framework region 1 (VHFWR1) amino acid sequence of SEQ ID NO: 6035 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations therein, e.g., substitution, addition or deletion), the VHFWR2 amino acid sequence of SEQ ID NO: 6036 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations therein, e.g., substitution, addition or deletion), the VHFWR3 amino acid sequence of SEQ ID NO: 6037 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations therein, e.g., substitution, addition or deletion), or the VHFWR4 amino acid sequence of SEQ ID NO: 6038 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations therein, e.g., substitution, addition or deletion).

[0176] 139. The multifunctional molecule of embodiment 138, wherein the NK cell adaptor comprises a heavy chain variable region (VH), the heavy chain variable region comprising the amino acid sequence of heavy chain framework region 1 (VHFWR1) of SEQ ID NO: 6035, the amino acid sequence of VHFWR2 of SEQ ID NO: 6036, the amino acid sequence of VHFWR3 of SEQ ID NO: 6037, or the amino acid sequence of VHFWR4 of SEQ ID NO: 6038.

[0177] 140. The multifunctional molecule of embodiment 139, wherein the NK cell adaptor comprises VH, the 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 with SEQ ID NO: 6128).

[0178] 141. A multifunctional molecule according to any one of embodiments 103-116 or 123-140, wherein the NK cell adaptor comprises a light chain variable region (VL), the light chain variable region comprising the amino acid sequence of light chain framework region 1 (VLFWR1) of SEQ ID NO: 6077 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations therein, e.g., substitution, addition or deletion), the amino acid sequence of VLFWR2 of SEQ ID NO: 6078 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations therein, e.g., substitution, addition or deletion), the amino acid sequence of VLFWR3 of SEQ ID NO: 6079 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations therein, e.g., substitution, addition or deletion), or the amino acid sequence of VLFWR4 of SEQ ID NO: 6080 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations therein, e.g., substitution, addition or deletion).

[0179] 142. The multifunctional molecule of embodiment 141, wherein the NK cell adaptor comprises a light chain variable region (VL), the light chain variable region comprising the amino acid sequence of light chain framework region 1 (VLFWR1) of SEQ ID NO: 6077, the amino acid sequence of VLFWR2 of SEQ ID NO: 6078, the amino acid sequence of VLFWR3 of SEQ ID NO: 6079, or the amino acid sequence of VLFWR4 of SEQ ID NO: 6080.

[0180] 143. The multifunctional molecule of embodiment 142, wherein the NK cell adaptor comprises VL, the 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 with SEQ ID NO: 6137).

[0181] 144. A multifunctional molecule according to any one of embodiments 103-116 or 123-140, wherein the NK cell adaptor comprises a light chain variable region (VL), the light chain variable region comprising the amino acid sequence of light chain framework region 1 (VLFWR1) of SEQ ID NO: 6081 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations therein, such as substitution, addition or deletion), the amino acid sequence of VLFWR2 of SEQ ID NO: 6082 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations therein, such as substitution, addition or deletion), the amino acid sequence of VLFWR3 of SEQ ID NO: 6083 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations therein, such as substitution, addition or deletion), or the amino acid sequence of VLFWR4 of SEQ ID NO: 6084 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations therein, such as substitution, addition or deletion).

[0182] 145. The multifunctional molecule of embodiment 144, wherein the NK cell adaptor comprises a light chain variable region (VL), the light chain variable region comprising the amino acid sequence of light chain framework region 1 (VLFWR1) of SEQ ID NO: 6081, the amino acid sequence of VLFWR2 of SEQ ID NO: 6082, the amino acid sequence of VLFWR3 of SEQ ID NO: 6083, or the amino acid sequence of VLFWR4 of SEQ ID NO: 6084.

[0183] 146. The multifunctional molecule of embodiment 145, wherein the NK cell adaptor comprises VL, the 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 with SEQ ID NO: 6138).

[0184] 147. A multifunctional molecule according to any one of embodiments 103-116 or 123-140, wherein the NK cell adaptor comprises a light chain variable region (VL), the light chain variable region comprising the amino acid sequence of light chain framework region 1 (VLFWR1) of SEQ ID NO: 6085 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations therein, e.g., substitution, addition or deletion), the amino acid sequence of VLFWR2 of SEQ ID NO: 6086 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations therein, e.g., substitution, addition or deletion), the amino acid sequence of VLFWR3 of SEQ ID NO: 6087 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations therein, e.g., substitution, addition or deletion), or the amino acid sequence of VLFWR4 of SEQ ID NO: 6088 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations therein, e.g., substitution, addition or deletion).

[0185] 148. The multifunctional molecule of embodiment 147, wherein the NK cell adaptor comprises a light chain variable region (VL), the light chain variable region comprising the amino acid sequence of light chain framework region 1 (VLFWR1) of SEQ ID NO: 6085, the amino acid sequence of VLFWR2 of SEQ ID NO: 6086, the amino acid sequence of VLFWR3 of SEQ ID NO: 6087, or the amino acid sequence of VLFWR4 of SEQ ID NO: 6088.

[0186] 149. The multifunctional molecule of embodiment 148, wherein the NK cell adaptor comprises VL, the 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 with SEQ ID NO: 6139).

[0187] 150. A multifunctional molecule according to any one of embodiments 103-116 or 123-140, wherein the NK cell adaptor comprises a light chain variable region (VL), the light chain variable region comprising the amino acid sequence of light chain framework region 1 (VLFWR1) of SEQ ID NO: 6089 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations therein, such as substitution, addition or deletion), the amino acid sequence of VLFWR2 of SEQ ID NO: 6090 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations therein, such as substitution, addition or deletion), the amino acid sequence of VLFWR3 of SEQ ID NO: 6091 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations therein, such as substitution, addition or deletion), or the amino acid sequence of VLFWR4 of SEQ ID NO: 6092 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations therein, such as substitution, addition or deletion).

[0188] 151. A multifunctional molecule according to embodiment 150, wherein the NK cell adaptor comprises a light chain variable region (VL), the light chain variable region comprising the amino acid sequence of light chain framework region 1 (VLFWR1) of SEQ ID NO: 6089, the amino acid sequence of VLFWR2 of SEQ ID NO: 6090, the amino acid sequence of VLFWR3 of SEQ ID NO: 6091, or the amino acid sequence of VLFWR4 of SEQ ID NO: 6092.

[0189] 152. The multifunctional molecule of embodiment 151, wherein the NK cell adaptor comprises VL, the 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 with SEQ ID NO: 6140).

[0190] 153. A multifunctional molecule according to any one of embodiments 103-116 or 123-140, wherein the NK cell adaptor comprises a light chain variable region (VL), the light chain variable region comprising the amino acid sequence of light chain framework region 1 (VLFWR1) of SEQ ID NO: 6093 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations therein, e.g., substitution, addition or deletion), the amino acid sequence of VLFWR2 of SEQ ID NO: 6094 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations therein, e.g., substitution, addition or deletion), the amino acid sequence of VLFWR3 of SEQ ID NO: 6095 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations therein, e.g., substitution, addition or deletion), or the amino acid sequence of VLFWR4 of SEQ ID NO: 6096 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations therein, e.g., substitution, addition or deletion).

[0191] 154. The multifunctional molecule of embodiment 153, wherein the NK cell adaptor comprises a light chain variable region (VL), the light chain variable region comprising the amino acid sequence of light chain framework region 1 (VLFWR1) of SEQ ID NO: 6093, the amino acid sequence of VLFWR2 of SEQ ID NO: 6094, the amino acid sequence of VLFWR3 of SEQ ID NO: 6095, or the amino acid sequence of VLFWR4 of SEQ ID NO: 6096.

[0192] 155. A multifunctional molecule according to embodiment 154, wherein the NK cell adaptor comprises VL, the 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 with SEQ ID NO: 6141).

[0193] 156. A multifunctional molecule according to any one of embodiments 103-114, wherein the NK cell adaptor comprises: (i) a heavy chain variable region (VH) comprising the 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., substitution, addition, or deletion), the VHCDR2 amino acid sequence of SEQ ID NO: 6008 (or a sequence having no more than 1, 2, 3, or 4 mutations, e.g., substitution, addition, or deletion), and / or the VHCDR3 amino acid sequence of SEQ ID NO: 6009 (or a sequence having no more than 1, 2, 3, or 4 mutations, e.g., substitution, addition, or deletion), and (ii) Light chain variable region (VL), the light chain variable region comprising the amino acid sequence of light chain complementarity-determining region 1 (VLCDR1) of SEQ ID NO: 6070 (or a sequence having no more than 1, 2, 3 or 4 mutations, such as substitution, addition or deletion), the amino acid sequence of VLCDR2 of SEQ ID NO: 6071 (or a sequence having no more than 1, 2, 3 or 4 mutations, such as substitution, addition or deletion), and / or the amino acid sequence of VLCDR3 of SEQ ID NO: 6072 (or a sequence having no more than 1, 2, 3 or 4 mutations, such as substitution, addition or deletion).

[0194] 157. The multifunctional molecule of embodiment 156, wherein the NK cell adaptor comprises: (i) Heavy chain variable region (VH), said heavy chain variable region comprising the heavy chain complementarity-determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 6007, the VHCDR2 amino acid sequence of SEQ ID NO: 6008 and / or the VHCDR3 amino acid sequence of SEQ ID NO: 6009, and (ii) Light chain variable region (VL), the light chain variable region comprising the amino acid sequence of light chain complementarity determination region 1 (VLCDR1) of SEQ ID NO: 6070, the amino acid sequence of VLCDR2 of SEQ ID NO: 6071 and / or the amino acid sequence of VLCDR3 of SEQ ID NO: 6072.

[0195] 158. A multifunctional molecule according to any one of embodiments 103-114, 156, or 157, wherein the NK cell adaptor comprises: (1) Heavy chain variable region (VH), the heavy chain variable region 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 therein, e.g., substitution, addition or deletion), 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 therein, e.g., substitution, addition or deletion), 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 therein, e.g., substitution, addition or deletion), 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 therein, e.g., substitution, addition or deletion), and / or (2) Light chain variable region (VL), the light chain variable region comprising the light chain framework region 1 (VLFWR1) amino acid sequence of SEQ ID NO: 6073 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations, such as substitution, addition or deletion), the VLFWR2 amino acid sequence of SEQ ID NO: 6074 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations, such as substitution, addition or deletion), the VLFWR3 amino acid sequence of SEQ ID NO: 6075 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations, such as substitution, addition or deletion), or the VLFWR4 amino acid sequence of SEQ ID NO: 6076 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations, such as substitution, addition or deletion).

[0196] 159. The multifunctional molecule of embodiment 158, wherein the NK cell adaptor comprises: (1) Heavy chain variable region (VH), wherein the heavy chain variable region comprises 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) Light chain variable region (VL), wherein the light chain variable region comprises the amino acid sequence of light chain framework region 1 (VLFWR1) of SEQ ID NO: 6073, the amino acid sequence of VLFWR2 of SEQ ID NO: 6074, the amino acid sequence of VLFWR3 of SEQ ID NO: 6075 or the amino acid sequence of VLFWR4 of SEQ ID NO: 6076.

[0197] 160. A multifunctional molecule according to any one of embodiments 103-114 or 156-159, wherein the NK cell adaptor comprises: (i) VH, wherein the VH comprises the amino acid sequence of SEQ ID NO: 6122 (or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95% or 99% sequence identity with SEQ ID NO: 6122), and / or (ii) VL, wherein the VL comprises the amino acid sequence of SEQ ID NO: 6136 (or an amino acid sequence having at least about 93%, 95% or 99% sequence identity with SEQ ID NO: 6136).

[0198] 161. A multifunctional molecule according to any one of embodiments 103-114 or 156-160, wherein the NK cell adaptor 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 with SEQ ID NO: 6151 or 6152).

[0199] 162. A multifunctional molecule according to any one of embodiments 103-114 or 156-161, wherein the NK cell adaptor 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 with SEQ ID NO: 6153).

[0200] 163. A multifunctional molecule according to any one of embodiments 103-114 or 156-162, wherein the NK cell adaptor 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 with 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 with SEQ ID NO: 6153).

[0201] 164. A multifunctional molecule according to any one of embodiments 103-114, 156 or 157, wherein the NK cell adaptor comprises a heavy chain variable region (VH), the heavy chain variable region comprising the heavy chain framework region 1 (VHFWR1) amino acid sequence of SEQ ID NO: 6039 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations therein, e.g., substitution, addition or deletion), the VHFWR2 amino acid sequence of SEQ ID NO: 6040 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations therein, e.g., substitution, addition or deletion), the VHFWR3 amino acid sequence of SEQ ID NO: 6041 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations therein, e.g., substitution, addition or deletion), or the VHFWR4 amino acid sequence of SEQ ID NO: 6042 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations therein, e.g., substitution, addition or deletion).

[0202] 165. The multifunctional molecule of embodiment 164, wherein the NK cell adaptor comprises a heavy chain variable region (VH), the heavy chain variable region comprising the amino acid sequence of heavy chain framework region 1 (VHFWR1) of SEQ ID NO: 6039, the amino acid sequence of VHFWR2 of SEQ ID NO: 6040, the amino acid sequence of VHFWR3 of SEQ ID NO: 6041, or the amino acid sequence of VHFWR4 of SEQ ID NO: 6042.

[0203] 166. A multifunctional molecule according to embodiment 165, wherein the NK cell adaptor comprises VH, the VH comprising the amino acid sequence of SEQ ID NO: 6129 (or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95% or 99% sequence identity with SEQ ID NO: 6129).

[0204] 167. A multifunctional molecule according to any one of embodiments 103-114, 156 or 157, wherein the NK cell adaptor comprises a heavy chain variable region (VH), the heavy chain variable region comprising the heavy chain framework region 1 (VHFWR1) amino acid sequence of SEQ ID NO: 6043 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations therein, e.g., substitution, addition or deletion), the VHFWR2 amino acid sequence of SEQ ID NO: 6044 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations therein, e.g., substitution, addition or deletion), the VHFWR3 amino acid sequence of SEQ ID NO: 6045 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations therein, e.g., substitution, addition or deletion), or the VHFWR4 amino acid sequence of SEQ ID NO: 6046 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations therein, e.g., substitution, addition or deletion).

[0205] 168. The multifunctional molecule of embodiment 167, wherein the NK cell adaptor comprises a heavy chain variable region (VH), the heavy chain variable region 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.

[0206] 169. A multifunctional molecule according to embodiment 168, wherein the NK cell adaptor comprises VH, the VH comprising the amino acid sequence of SEQ ID NO: 6130 (or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95% or 99% sequence identity with SEQ ID NO: 6130).

[0207] 170. A multifunctional molecule according to any one of embodiments 103-114, 156, or 157, wherein the NK cell adaptor comprises a heavy chain variable region (VH), the heavy chain variable region comprising the heavy chain framework region 1 (VHFWR1) amino acid sequence of SEQ ID NO: 6047 (or a sequence having no more than 1, 2, 3, 4, 5, or 6 mutations therein, e.g., substitution, addition, or deletion), the VHFWR2 amino acid sequence of SEQ ID NO: 6048 (or a sequence having no more than 1, 2, 3, 4, 5, or 6 mutations therein, e.g., substitution, addition, or deletion), the VHFWR3 amino acid sequence of SEQ ID NO: 6049 (or a sequence having no more than 1, 2, 3, 4, 5, or 6 mutations therein, e.g., substitution, addition, or deletion), or the VHFWR4 amino acid sequence of SEQ ID NO: 6050 (or a sequence having no more than 1, 2, 3, 4, 5, or 6 mutations therein, e.g., substitution, addition, or deletion).

[0208] 171. A multifunctional molecule according to embodiment 170, wherein the NK cell adaptor comprises a heavy chain variable region (VH), the heavy chain variable region 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.

[0209] 172. A multifunctional molecule according to embodiment 171, wherein the NK cell adaptor comprises VH, the 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 with SEQ ID NO: 6131).

[0210] 173. A multifunctional molecule according to any one of embodiments 103-114, 156 or 157, wherein the NK cell adaptor comprises a heavy chain variable region (VH), the heavy chain variable region comprising the heavy chain framework region 1 (VHFWR1) amino acid sequence of SEQ ID NO: 6051 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations therein, e.g., substitution, addition or deletion), the VHFWR2 amino acid sequence of SEQ ID NO: 6052 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations therein, e.g., substitution, addition or deletion), the VHFWR3 amino acid sequence of SEQ ID NO: 6053 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations therein, e.g., substitution, addition or deletion), or the VHFWR4 amino acid sequence of SEQ ID NO: 6054 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations therein, e.g., substitution, addition or deletion).

[0211] 174. The multifunctional molecule of embodiment 173, wherein the NK cell adaptor comprises a heavy chain variable region (VH), the heavy chain variable region comprising the amino acid sequence of heavy chain framework region 1 (VHFWR1) of SEQ ID NO: 6051, the amino acid sequence of VHFWR2 of SEQ ID NO: 6052, the amino acid sequence of VHFWR3 of SEQ ID NO: 6053, or the amino acid sequence of VHFWR4 of SEQ ID NO: 6054.

[0212] 175. A multifunctional molecule according to embodiment 174, wherein the NK cell adaptor comprises VH, the VH comprising the amino acid sequence of SEQ ID NO: 6132 (or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95% or 99% sequence identity with SEQ ID NO: 6132).

[0213] 176. A multifunctional molecule according to any one of embodiments 103-114, 156 or 157, wherein the NK cell adaptor comprises a heavy chain variable region (VH), the heavy chain variable region comprising the heavy chain framework region 1 (VHFWR1) amino acid sequence of SEQ ID NO: 6055 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations therein, e.g., substitution, addition or deletion), the VHFWR2 amino acid sequence of SEQ ID NO: 6056 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations therein, e.g., substitution, addition or deletion), the VHFWR3 amino acid sequence of SEQ ID NO: 6057 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations therein, e.g., substitution, addition or deletion), or the VHFWR4 amino acid sequence of SEQ ID NO: 6058 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations therein, e.g., substitution, addition or deletion).

[0214] 177. The multifunctional molecule of embodiment 176, wherein the NK cell adaptor comprises a heavy chain variable region (VH), the heavy chain variable region comprising the amino acid sequence of heavy chain framework region 1 (VHFWR1) of SEQ ID NO: 6055, the amino acid sequence of VHFWR2 of SEQ ID NO: 6056, the amino acid sequence of VHFWR3 of SEQ ID NO: 6057, or the amino acid sequence of VHFWR4 of SEQ ID NO: 6058.

[0215] 178. A multifunctional molecule according to embodiment 177, wherein the NK cell adaptor comprises VH, the 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 with SEQ ID NO: 6133).

[0216] 179. A multifunctional molecule according to any one of embodiments 103-114, 156 or 157, wherein the NK cell adaptor comprises a heavy chain variable region (VH), the heavy chain variable region comprising the heavy chain framework region 1 (VHFWR1) amino acid sequence of SEQ ID NO: 6059 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations therein, e.g., substitution, addition or deletion), the VHFWR2 amino acid sequence of SEQ ID NO: 6060 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations therein, e.g., substitution, addition or deletion), the VHFWR3 amino acid sequence of SEQ ID NO: 6061 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations therein, e.g., substitution, addition or deletion), or the VHFWR4 amino acid sequence of SEQ ID NO: 6062 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations therein, e.g., substitution, addition or deletion).

[0217] 180. A multifunctional molecule according to embodiment 179, wherein the NK cell adaptor comprises a heavy chain variable region (VH), the heavy chain variable region 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.

[0218] 181. A multifunctional molecule of embodiment 180, wherein the NK cell adaptor comprises VH, the 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 with SEQ ID NO: 6134).

[0219] 182. A multifunctional molecule comprising any one of embodiments 103-114, 156, 157 or 164-181, wherein the NK cell adaptor comprises a light chain variable region (VL), the light chain variable region comprising the amino acid sequence of light chain framework region 1 (VLFWR1) of SEQ ID NO: 6097 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations therein, such as substitution, addition or deletion), the amino acid sequence of VLFWR2 of SEQ ID NO: 6098 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations therein, such as substitution, addition or deletion), the amino acid sequence of VLFWR3 of SEQ ID NO: 6099 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations therein, such as substitution, addition or deletion), or the amino acid sequence of VLFWR4 of SEQ ID NO: 6100 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations therein, such as substitution, addition or deletion).

[0220] 183. The multifunctional molecule of embodiment 182, wherein the NK cell adaptor comprises a light chain variable region (VL), the light chain variable region comprising the amino acid sequence of light chain framework region 1 (VLFWR1) of SEQ ID NO: 6097, the amino acid sequence of VLFWR2 of SEQ ID NO: 6098, the amino acid sequence of VLFWR3 of SEQ ID NO: 6099, or the amino acid sequence of VLFWR4 of SEQ ID NO: 6100.

[0221] 184. A multifunctional molecule according to embodiment 183, wherein the NK cell adaptor comprises VL, the 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 with SEQ ID NO: 6142).

[0222] 185. A multifunctional molecule comprising any one of embodiments 103-114, 156, 157 or 164-181, wherein the NK cell adaptor comprises a light chain variable region (VL), the light chain variable region comprising the amino acid sequence of light chain framework region 1 (VLFWR1) of SEQ ID NO: 6101 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations therein, such as substitution, addition or deletion), the amino acid sequence of VLFWR2 of SEQ ID NO: 6102 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations therein, such as substitution, addition or deletion), the amino acid sequence of VLFWR3 of SEQ ID NO: 6103 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations therein, such as substitution, addition or deletion), or the amino acid sequence of VLFWR4 of SEQ ID NO: 6104 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations therein, such as substitution, addition or deletion).

[0223] 186. A multifunctional molecule according to embodiment 185, wherein the NK cell adaptor comprises a light chain variable region (VL), the light chain variable region comprising the amino acid sequence of light chain framework region 1 (VLFWR1) of SEQ ID NO: 6101, the amino acid sequence of VLFWR2 of SEQ ID NO: 6102, the amino acid sequence of VLFWR3 of SEQ ID NO: 6103, or the amino acid sequence of VLFWR4 of SEQ ID NO: 6104.

[0224] 187. A multifunctional molecule according to embodiment 186, wherein the NK cell adaptor comprises VL, the VL comprising the amino acid sequence of SEQ ID NO: 6143 (or an amino acid sequence having at least about 93%, 95% or 99% sequence identity with SEQ ID NO: 6143).

[0225] 188. A multifunctional molecule comprising any one of embodiments 103-114, 156, 157 or 164-181, wherein the NK cell adaptor comprises a light chain variable region (VL), the light chain variable region comprising the amino acid sequence of light chain framework region 1 (VLFWR1) of SEQ ID NO: 6105 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations therein, such as substitution, addition or deletion), the amino acid sequence of VLFWR2 of SEQ ID NO: 6106 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations therein, such as substitution, addition or deletion), the amino acid sequence of VLFWR3 of SEQ ID NO: 6107 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations therein, such as substitution, addition or deletion), or the amino acid sequence of VLFWR4 of SEQ ID NO: 6108 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations therein, such as substitution, addition or deletion).

[0226] 189. A multifunctional molecule according to embodiment 188, wherein the NK cell adaptor comprises a light chain variable region (VL), the light chain variable region comprising the amino acid sequence of light chain framework region 1 (VLFWR1) of SEQ ID NO: 6105, the amino acid sequence of VLFWR2 of SEQ ID NO: 6106, the amino acid sequence of VLFWR3 of SEQ ID NO: 6107, or the amino acid sequence of VLFWR4 of SEQ ID NO: 6108.

[0227] 190. A multifunctional molecule according to embodiment 189, wherein the NK cell adaptor comprises VL, the 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 with SEQ ID NO: 6144).

[0228] 191. A multifunctional molecule comprising any one of embodiments 103-114, 156, 157 or 164-181, wherein the NK cell adaptor comprises a light chain variable region (VL), the light chain variable region comprising the amino acid sequence of light chain framework region 1 (VLFWR1) of SEQ ID NO: 6109 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations therein, such as substitution, addition or deletion), the amino acid sequence of VLFWR2 of SEQ ID NO: 6110 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations therein, such as substitution, addition or deletion), the amino acid sequence of VLFWR3 of SEQ ID NO: 6111 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations therein, such as substitution, addition or deletion), or the amino acid sequence of VLFWR4 of SEQ ID NO: 6112 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations therein, such as substitution, addition or deletion).

[0229] 192. A multifunctional molecule according to embodiment 191, wherein the NK cell adaptor comprises a light chain variable region (VL), the light chain variable region comprising the amino acid sequence of light chain framework region 1 (VLFWR1) of SEQ ID NO: 6109, the amino acid sequence of VLFWR2 of SEQ ID NO: 6110, the amino acid sequence of VLFWR3 of SEQ ID NO: 6111, or the amino acid sequence of VLFWR4 of SEQ ID NO: 6112.

[0230] 193. A multifunctional molecule according to embodiment 192, wherein the NK cell adaptor comprises VL, the 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 with SEQ ID NO: 6145).

[0231] 194. A multifunctional molecule comprising any one of embodiments 103-114, 156, 157 or 164-181, wherein the NK cell adaptor comprises a light chain variable region (VL), the light chain variable region comprising the amino acid sequence of light chain framework region 1 (VLFWR1) of SEQ ID NO: 6113 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations therein, such as substitution, addition or deletion), the amino acid sequence of VLFWR2 of SEQ ID NO: 6114 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations therein, such as substitution, addition or deletion), the amino acid sequence of VLFWR3 of SEQ ID NO: 6115 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations therein, such as substitution, addition or deletion), or the amino acid sequence of VLFWR4 of SEQ ID NO: 6116 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations therein, such as substitution, addition or deletion).

[0232] 195. A multifunctional molecule according to embodiment 194, wherein the NK cell adaptor comprises a light chain variable region (VL), the light chain variable region comprising the amino acid sequence of light chain framework region 1 (VLFWR1) of SEQ ID NO: 6113, the amino acid sequence of VLFWR2 of SEQ ID NO: 6114, the amino acid sequence of VLFWR3 of SEQ ID NO: 6115, or the amino acid sequence of VLFWR4 of SEQ ID NO: 6116.

[0233] 196. A multifunctional molecule according to embodiment 195, wherein the NK cell adaptor comprises VL, the 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 with SEQ ID NO: 6146).

[0234] 197. A multifunctional molecule comprising any one of embodiments 103-114, 156, 157 or 164-181, wherein the NK cell adaptor comprises a light chain variable region (VL), the light chain variable region comprising the amino acid sequence of light chain framework region 1 (VLFWR1) of SEQ ID NO: 6117 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations therein, such as substitution, addition or deletion), the amino acid sequence of VLFWR2 of SEQ ID NO: 6118 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations therein, such as substitution, addition or deletion), the amino acid sequence of VLFWR3 of SEQ ID NO: 6119 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations therein, such as substitution, addition or deletion), or the amino acid sequence of VLFWR4 of SEQ ID NO: 6120 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations therein, such as substitution, addition or deletion).

[0235] 198. A multifunctional molecule according to embodiment 197, wherein the NK cell adaptor comprises a light chain variable region (VL), the light chain variable region comprising the amino acid sequence of light chain framework region 1 (VLFWR1) of SEQ ID NO: 6117, the amino acid sequence of VLFWR2 of SEQ ID NO: 6118, the amino acid sequence of VLFWR3 of SEQ ID NO: 6119, or the amino acid sequence of VLFWR4 of SEQ ID NO: 6120.

[0236] 199. A multifunctional molecule according to embodiment 198, wherein the NK cell adaptor comprises VL, the 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 with SEQ ID NO: 6147).

[0237] 200. A multifunctional molecule according to any one of embodiments 103-106, wherein the NK cell adaptor is an antibody molecule that binds to NKp46, for example, an antigen-binding domain.

[0238] 201. A multifunctional molecule of implementation scheme 200, wherein the lysis of lymphoma cells is mediated by NKp46.

[0239] 202. A multifunctional molecule of any one of embodiments 200 or 201, wherein the multifunctional molecule does not activate NK cells when incubated with NK cells in the absence of a TCRBAV antigen (e.g., a TCRBAV antigen corresponding to a biased TCRBAV clone).

[0240] 203. A multifunctional molecule according to any one of embodiments 200-202, wherein the multifunctional molecule activates the NK cells when the NK cells are NK cells expressing NKp46 and when a TCRBAV antigen (e.g., a TCRBAV antigen corresponding to a biased TCRBAV clonal type) is also present.

[0241] 204. A multifunctional molecule according to any one of embodiments 200-203, wherein the multifunctional molecule does not activate NK cells when the NK cells are not NK cells expressing NKp46 and a TCRBAV antigen (e.g., a TCRBAV antigen corresponding to a biased TCRBAV clonal type) is also present.

[0242] 205. A multifunctional molecule according to any one of embodiments 200-204, wherein the NK cell adaptor comprises VH, the VH comprising the amino acid sequence of SEQ ID NO: 6182 (or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95% or 99% sequence identity with SEQ ID NO: 6182).

[0243] 206. A multifunctional molecule according to any one of embodiments 200-205, wherein the NK cell adaptor comprises VL, the VL comprising the amino acid sequence of SEQ ID NO: 6183 (or an amino acid sequence having at least about 93%, 95% or 99% sequence identity with SEQ ID NO: 6183).

[0244] 207. Multifunctional molecules of 200-205, wherein the NK cell adaptor comprises scFV, the scFV comprising the amino acid sequence of SEQ ID NO: 6181 (or an amino acid sequence having at least about 93%, 95% or 99% sequence identity with SEQ ID NO: 6181).

[0245] 208. A multifunctional molecule according to any one of embodiments 103-106, wherein the NK cell adaptor is an antibody molecule that binds to NKG2D, for example, an antigen-binding domain.

[0246] 209. A multifunctional molecule according to implementation scheme 208, wherein the lysis of lymphoma cells is mediated by NKG2D.

[0247] 210. A multifunctional molecule of any one of embodiments 208 or 209, wherein the multifunctional molecule does not activate NK cells when incubated with NK cells in the absence of a TCRVB antigen (e.g., a TCRVB antigen corresponding to a biased TCRVB clone).

[0248] 211. A multifunctional molecule according to any one of embodiments 208-210, wherein the multifunctional molecule activates the NK cell when the NK cell is an NK cell expressing NKG2D and a TCRBAV antigen (e.g., a TCRBAV antigen corresponding to a biased TCRBAV clonal type) is also present.

[0249] 212. A multifunctional molecule according to any one of embodiments 208-211, wherein the multifunctional molecule does not activate NK cells when the NK cells are not NK cells expressing NKG2D and a TCRBAV antigen (e.g., a TCRBAV antigen corresponding to a biased TCRBAV clonal type) is also present.

[0250] 213. A multifunctional molecule of any one of embodiments 208-212, wherein the NK cell adaptor comprises VH, the 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 with SEQ ID NO: 6176).

[0251] 214. A multifunctional molecule of any one of embodiments 208-213, wherein the NK cell adaptor comprises VL, the 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 with SEQ ID NO: 6177).

[0252] 215. A multifunctional molecule of any one of embodiments 208-214, wherein the NK cell adaptor comprises scFV, the scFV comprising the amino acid sequence of SEQ ID NO: 6175 (or an amino acid sequence having at least about 93%, 95% or 99% sequence identity with SEQ ID NO: 6175).

[0253] 216. A multifunctional molecule of any one of embodiments 208-212, wherein the NK cell adaptor comprises VH, the VH comprising the amino acid sequence of SEQ ID NO: 6179 (or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95% or 99% sequence identity with SEQ ID NO: 6179).

[0254] 217. A multifunctional molecule according to any one of embodiments 208-212 or 216, wherein the NK cell adaptor comprises VL, the VL comprising the amino acid sequence of SEQ ID NO: 6180 (or an amino acid sequence having at least about 93%, 95% or 99% sequence identity with SEQ ID NO: 6180).

[0255] 218. A multifunctional molecule according to any one of embodiments 208-212, 216 or 217, wherein the NK cell adaptor comprises scFV, the scFV comprising the amino acid sequence of SEQ ID NO: 6178 (or an amino acid sequence having at least about 93%, 95% or 99% sequence identity with SEQ ID NO: 6178).

[0256] 219. A multifunctional molecule according to any one of embodiments 103-106, wherein the NK cell adaptor is an antibody molecule that binds to CD16, for example, an antigen-binding domain.

[0257] 220. A multifunctional molecule according to implementation scheme 219, wherein the lysis of lymphoma cells is mediated by CD16.

[0258] 221. A multifunctional molecule of any one of embodiments 219 or 220, wherein the multifunctional molecule does not activate NK cells when incubated with NK cells in the absence of a TCRBAV antigen (e.g., a TCRBAV antigen corresponding to a biased TCRBAV clone).

[0259] 222. A multifunctional molecule according to any one of embodiments 219-221, wherein the multifunctional molecule activates the NK cells when the NK cells are CD16-expressing NK cells and a TCRBAV antigen (e.g., a TCRBAV antigen corresponding to a biased TCRBAV clonal type) is also present.

[0260] 223. A multifunctional molecule of any of embodiments 219-222, wherein the multifunctional molecule does not activate NK cells when the NK cells are not CD16-expressing NK cells and a TCRBAV antigen (e.g., a TCRBAV antigen corresponding to a biased TCRBAV clonal type) is also present.

[0261] 224. A multifunctional molecule of any one of embodiments 219-223, wherein the NK cell adaptor comprises VH, the VH comprising the amino acid sequence of SEQ ID NO: 6185 (or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95% or 99% sequence identity with SEQ ID NO: 6185).

[0262] 225. A multifunctional molecule of any one of embodiments 219-224, wherein the NK cell adaptor comprises VL, the VL comprising the amino acid sequence of SEQ ID NO: 6186 (or an amino acid sequence having at least about 93%, 95% or 99% sequence identity with SEQ ID NO: 6186).

[0263] 226. A multifunctional molecule of any one of embodiments 219-225, wherein the NK cell adaptor comprises scFV, the scFV comprising the amino acid sequence of SEQ ID NO: 6184 (or an amino acid sequence having at least about 93%, 95% or 99% sequence identity with SEQ ID NO: 6184).

[0264] 227. A multifunctional molecule according to embodiment 103, wherein the NK cell adaptor is a ligand, optionally, the ligand further comprising an immunoglobulin constant region, such as an Fc region.

[0265] 228. A multifunctional molecule according to embodiment 227, wherein the NK cell adaptor is a ligand of NKp44 or NKp46, for example, viral HA.

[0266] 229. A multifunctional molecule according to embodiment 227, wherein the NK cell adaptor is a ligand of DAP10, for example, a co-receptor of NKG2D.

[0267] 230. A multifunctional molecule according to embodiment 227, wherein the NK cell adaptor is a ligand for CD16, such as a CD16a / b ligand, for example, the CD16a / b ligand further comprising an antibody Fc region.

[0268] 231. A multifunctional molecule of any one of embodiments 98-100, wherein the immune cell adaptor mediates binding to one or more of B cells, macrophages and / or dendritic cells, or mediates activation of one or more of B cells, macrophages and / or dendritic cells, or both.

[0269] 232. A multifunctional molecule according to embodiment 231, wherein the immune cell adaptor comprises a B cell adaptor, a macrophage adaptor, and / or a dendritic cell adaptor, selected from one or more of the following: a CD40 ligand (CD40L) or a CD70 ligand; an antibody molecule that binds to CD40 or CD70; an antibody molecule against OX40; an OX40 ligand (OX40L); an agonist of a Toll-like receptor (e.g., TLR4, such as constitutively active TLR4 (caTLR4) or a TLR9 agonist); 41BB; a CD2 agonist; CD47; or a STING agonist, or a combination thereof.

[0270] 233. A multifunctional molecule of any one of embodiments 98-100, wherein the immune cell adaptor is a B cell adaptor, such as a CD40L, OX40L, or CD70 ligand, or an antibody molecule that binds to OX40, CD40, or CD70.

[0271] 234. A multifunctional molecule of any one of embodiments 98-100, wherein the immune cell adaptor is a macrophage adaptor, for example, 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, such as constitutively active TLR4 (caTLR4) or a TLR9 agonist); CD47; or a STING agonist.

[0272] 235. A multifunctional molecule of any one of embodiments 98-100, wherein the immune cell adaptor is a dendritic cell adaptor, such as a CD2 agonist, an OX40 antibody, an OX40L, a 41BB agonist, a Toll-like receptor agonist or a fragment thereof (e.g., TLR4, such as constitutively active TLR4 (caTLR4)), a CD47 agonist or a STING agonist.

[0273] 236. A multifunctional molecule according to embodiment 234 or 235, wherein the STING agonist comprises a cyclic dinucleotide, such as cyclic diGMP (cdGMP), cyclic diAMP (cdAMP), or a combination thereof, optionally having a 2', 5' or 3', 5' phosphate bond, for example, wherein the STING agonist is covalently coupled to the multifunctional molecule.

[0274] 237. A multifunctional molecule of any one of embodiments 1-97, wherein the multifunctional molecule comprises a cytokine molecule.

[0275] 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-γ, or fragments or variants thereof, or any combination of the above cytokines.

[0276] 239. A multifunctional molecule according to embodiment 237 or 238, wherein the cytokine molecule is a monomer or a dimer.

[0277] 240. A multifunctional molecule of any one of embodiments 237-239, wherein the cytokine molecule further comprises a receptor dimerization domain, for example, an IL15Rα dimerization domain.

[0278] 241. A multifunctional molecule of embodiment 240, wherein the cytokine molecule (e.g., IL-15) and the receptor dimerization domain (e.g., IL15Rα dimerization domain) are not covalently linked, for example, are non-covalently associated.

[0279] 242. A multifunctional molecule of any one of embodiments 1-97, wherein the multifunctional molecule comprises a cytokine inhibitor molecule.

[0280] 243. The multifunctional molecule of embodiment 242, wherein the cytokine inhibitor molecule is a TGF-β inhibitor.

[0281] 244. A multifunctional molecule according to embodiment 242 or 243, wherein the TGF-β inhibitor inhibits (e.g., reduces the activity of) the following: (i) TGF-β1; (ii) TGF-β2; (iii) TGF-β3; (iv) (i) and (ii); (v) (i) and (iii); (vi) (ii) and (iii); or (vii) (i), (ii) and (iii).

[0282] 245. A multifunctional molecule of any one of embodiments 242-244, wherein the TGF-β inhibitor comprises a portion of the TGF-β receptor capable of inhibiting TGF-β (e.g., reducing its activity) (e.g., the extracellular domain of the TGF-β receptor), or a functional fragment or variant thereof.

[0283] 246. A multifunctional molecule of embodiment 245, wherein the TGF-β inhibitor comprises (i) TGFBR1; (ii) TGFBR2; (iii) TGFBR3; (iv) (i) and (ii); (v) (i) and (iii); (vi) (ii) and (iii); or (vii) portions of (i), (ii) and (iii).

[0284] 247. A multifunctional molecule of any one of embodiments 242-246, wherein the TGF-β 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 with it.

[0285] 248. A multifunctional molecule of any one of embodiments 1-97, wherein the multifunctional molecule comprises a death receptor signaling adaptor selected from TNF-associated apoptosis-inducing ligand (TRAIL) molecules, death receptor molecules, or antigen-binding domains that specifically bind to death receptors.

[0286] 249. A multifunctional molecule of embodiment 248, wherein the death receptor signaling adaptor activates death receptor signaling in lymphocytes (e.g., T cells) containing the TCRBV antigen, for example, and induces apoptosis or cell death in the cells.

[0287] 250. A multifunctional molecule according to embodiment 248 or 249, wherein the death receptor signaling adaptor does not activate death receptor signaling on cells other than lymphocytes containing the TCRBAV antigen.

[0288] 251. A multifunctional molecule of any one of embodiments 248-250, wherein the death receptor signaling adaptor comprises a TRAIL molecule, such as one or more TRAIL peptides or fragments thereof.

[0289] 252. A multifunctional molecule according to embodiment 251, wherein the TRAIL molecule specifically binds to death receptor 4 (DR4) or death receptor 5 (DR5).

[0290] 253. A multifunctional molecule according to embodiment 251 or 252, wherein the TRAIL molecule comprises, for example, a TRAIL polypeptide truncated relative to the wild-type TRAIL polypeptide.

[0291] 254. A multifunctional molecule of embodiment 253, wherein the TRAIL molecule contains at least residues corresponding to amino acids 95-281 of human TRAIL, for example, a truncated TRAIL molecule containing residues corresponding to amino acids 95-281 of human TRAIL.

[0292] 255. The multifunctional molecule of embodiment 254, wherein the TRAIL molecule comprises a truncated TRAIL polypeptide, the truncated TRAIL polypeptide comprising, for example, amino acids 95-281 of human TRAIL, but not amino acids 1-94 of human TRAIL.

[0293] 256. A multifunctional molecule of embodiment 253, wherein the TRAIL molecule contains at least residues corresponding to amino acids 122-281 of human TRAIL, for example, a truncated TRAIL molecule containing residues corresponding to amino acids 122-281 of human TRAIL.

[0294] 257. The multifunctional molecule of embodiment 256, wherein the TRAIL molecule comprises a truncated TRAIL polypeptide, the truncated TRAIL polypeptide comprising, for example, amino acids 122-281 of human TRAIL, but not amino acids 1-121 of human TRAIL.

[0295] 258. A multifunctional molecule of any one of embodiments 251-257, wherein the death receptor signaling adaptor comprises one, two, or three TRAIL molecules.

[0296] 259. A multifunctional molecule of any one of embodiments 248-250, wherein the death receptor signaling adaptor comprises an antigen-binding domain that specifically binds to a death receptor (e.g., death receptor 4 (DR4) or death receptor 5 (DR5)).

[0297] 260. A multifunctional molecule according to embodiment 259, wherein the death receptor signaling adaptor comprises one, two, or three antigen-binding domains that specifically bind to a death receptor.

[0298] 261. A multifunctional molecule according to implementation scheme 259 or 260, wherein an antigen-binding domain that specifically binds to the death receptor binds to DR5.

[0299] 262. A multifunctional molecule of any of embodiments 259-261, wherein the antigen-binding domain that specifically binds to the death receptor includes tigatuzumab, drozitumab, or conatumumab.

[0300] 263. A multifunctional molecule of any one of embodiments 248-262, wherein the death receptor signaling adaptor 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 with it.

[0301] 264. A multifunctional molecule of any one of embodiments 248-263, wherein the death receptor signaling adaptor 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 with it.

[0302] 265. A multifunctional molecule of any one of embodiments 248-263, wherein the death receptor signaling adaptor 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 with it.

[0303] 266. A multifunctional molecule of any one of embodiments 248-263, wherein the death receptor signaling adaptor 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 with it.

[0304] 267. A multifunctional molecule of any one of embodiments 248-263, wherein the death receptor signaling adaptor 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 with it.

[0305] 268. A multifunctional molecule of any one of embodiments 248-263, wherein the death receptor signaling adaptor 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 with it.

[0306] 269. A multifunctional molecule of any one of embodiments 248-263, wherein the death receptor signaling adaptor 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 with it.

[0307] 270. A multifunctional molecule of any one of embodiments 248-263, wherein the death receptor signaling adaptor 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 with it.

[0308] 271. A multifunctional molecule of any one of embodiments 248-263, wherein the death receptor signaling adaptor 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 with it.

[0309] 272. A multifunctional molecule of any one of embodiments 248-263, wherein the death receptor signaling adaptor 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 with it.

[0310] 273. The multifunctional molecule of embodiment 102, wherein the T cell adaptor binds to TCRβ.

[0311] 274. A multifunctional molecule of embodiment 273, wherein the T-cell adaptor comprises an antigen-binding domain (e.g., an antibody molecule or a fragment thereof) that binds to (e.g., and is activated in some embodiments) CD3.

[0312] 275. A multifunctional molecule according to embodiment 273 or 274, wherein the T cell adaptor does not bind to lymphocytes containing the TCRBV antigen.

[0313] 276. A multifunctional molecule according to any one of embodiments 273-275, wherein the T cell adaptor does not activate lymphocytes containing TCRBV.

[0314] 277. A multifunctional molecule according to any one of embodiments 1-276, wherein the multifunctional molecule comprises: (i) Immune cell adaptors (e.g., T cell adaptors, NK cell adaptors, B cell adaptors, dendritic cell adaptors, or macrophage adaptors) and cytokine molecules, (ii) Immune cell adjuvants (e.g., T cell adjuvants, NK cell adjuvants, B cell adjuvants, dendritic cell adjuvants, or macrophage adjuvants) and cytokine inhibitor molecules, (iii) Immune cell adjuvants (e.g., T cell adjuvants, NK cell adjuvants, B cell adjuvants, dendritic cell adjuvants, or macrophage adjuvants) and death receptor signaling adjuvants, (iv) Cytokine molecules and death receptor signaling adjuvants, (v) Cytokine inhibitor molecules and death receptor signaling adjuvants, (vi) Immune cell adjuvants (e.g., T cell adjuvants, NK cell adjuvants, B cell adjuvants, dendritic cell adjuvants, or macrophage adjuvants), cytokine molecules, and death receptor signaling adjuvants, or (vii) Immune cell adjuvants (e.g., T cell adjuvants, NK cell adjuvants, B cell adjuvants, dendritic cell adjuvants, or macrophage adjuvants), cytokine inhibitor molecules, and death receptor signaling adjuvants.

[0315] 278. A multifunctional molecule according to any one of embodiments 1-277, wherein the multifunctional molecule comprises the following configurations: A, B-[dimerization module]-C, -D, where: (a) The dimerization module includes an immunoglobulin constant domain, such as a heavy chain constant domain (e.g., a homodimer or heterodimer heavy chain constant region, such as an Fc region), or a constant domain of an immunoglobulin variable region (e.g., a Fab region); and (b) A, B, C, and D are independently absent; (i) an antigen-binding domain that selectively binds to the TCRVB antigen; (ii) an immune cell adjuvant selected from T cell adjuvants, NK cell adjuvants, B cell adjuvants, dendritic cell adjuvants, or macrophage adjuvants; (iii) a cytokine molecule or a cytokine inhibitor molecule; (iv) a death receptor signaling adjuvant; or (v) a matrix-modified portion, provided that: At least one, two, or three of A, B, C, and D contain an antigen-binding domain that selectively binds to the TCRBV antigen, and Any of the remaining items in A, B, C, and D are either absent or contain one of the following: immune cell adaptor, cytokine molecule, cytokine inhibitor molecule, death receptor signaling adaptor, or matrix modification motif.

[0316] 279. The multifunctional molecule of implementation scheme 278, wherein: (1) A contains an antigen-binding domain that selectively binds to the TCRVB antigen, and B, C or D contains an immune cell adaptor, such as a T cell adaptor, such as an anti-CD3 antibody molecule. (2) A contains an antigen-binding domain that selectively binds to the TCRVB antigen, and B, C or D contains an immune cell adaptor, such as an NK cell adaptor, such as an anti-NKp30, anti-NKp46, anti-NKG2D or anti-CD16 antibody molecule. (3) A contains an antigen-binding domain that selectively binds to the TCRVB antigen, and B, C, or D contains cytokine molecules; (4) A contains an antigen-binding domain that selectively binds to the TCRVB antigen, and B, C, or D contains a cytokine inhibitor molecule; (5) A contains an antigen-binding domain that selectively binds to the TCRVB antigen, and B, C, or D contains a death receptor signaling adaptor; (6) A contains a first antigen-binding domain that selectively binds to the TCRVB antigen, B contains a second antigen-binding domain that selectively binds to the TCRVB antigen, and C or D contains an immune cell adaptor, such as a T cell adaptor, such as an anti-CD3 antibody molecule. (7) A contains a first antigen-binding domain that selectively binds to the TCRVB antigen, B contains a second antigen-binding domain that selectively binds to the TCRVB antigen, and C or D contains an immune cell adaptor, such as an NK cell adaptor, such as an anti-NKp30, anti-NKp46, anti-NKG2D or anti-CD16 antibody molecule. (8) A contains a first antigen-binding domain that selectively binds to the TCRVB antigen, B contains a second antigen-binding domain that selectively binds to the TCRVB antigen, and C or D contains cytokine molecules. (9) A contains a first antigen-binding domain that selectively binds to the TCRVB antigen, B contains a second antigen-binding domain that selectively binds to the TCRVB antigen, and C or D contains a cytokine inhibitor molecule. (10) A contains a first antigen-binding domain that selectively binds to the TCRBV antigen, B contains a second antigen-binding domain that selectively binds to the TCRBV antigen, and C or D contains a death receptor signaling adaptor. (11) A contains a first antigen-binding domain that selectively binds to the TCRVB antigen, C contains a second antigen-binding domain that selectively binds to the TCRVB antigen, and B or D contains an immune cell adaptor, such as a T cell adaptor, such as an anti-CD3 antibody molecule. (12) A contains a first antigen-binding domain that selectively binds to the TCRVB antigen, C contains a second antigen-binding domain that selectively binds to the TCRVB antigen, and B or D contains an immune cell adaptor, such as an NK cell adaptor, such as an anti-NKp30, anti-NKp46, anti-NKG2D or anti-CD16 antibody molecule. (13) A contains a first antigen-binding domain that selectively binds to the TCRVB antigen, C contains a second antigen-binding domain that selectively binds to the TCRVB antigen, and B or D contains cytokine molecules. (14) A contains a first antigen-binding domain that selectively binds to the TCRVB antigen, C contains a second antigen-binding domain that selectively binds to the TCRVB antigen, and B or D contains a cytokine inhibitor molecule. (15) A contains a first antigen-binding domain that selectively binds to the TCRVB antigen, C contains a second antigen-binding domain that selectively binds to the TCRVB antigen, and B or D contains a death receptor signaling adaptor. (16) A contains a first antigen-binding domain that selectively binds to the TCRVB antigen, and B, C or D contains (a) an immune cell adaptor, such as an NK cell adaptor, such as an anti-NKp30, anti-NKp46, anti-NKG2D or anti-CD16 antibody molecule, and (b) a cytokine molecule. (17) A contains a first antigen-binding domain that selectively binds to the TCRVB antigen, and B, C or D contains (a) an immune cell adaptor, such as an NK cell adaptor, such as an anti-NKp30, anti-NKp46, anti-NKG2D or anti-CD16 antibody molecule, and (b) a cytokine inhibitor molecule. (18) A contains a first antigen-binding domain that selectively binds to the TCRVB antigen, and B, C or D contains (a) an immune cell adaptor, such as an NK cell adaptor, such as an anti-NKp30, anti-NKp46, anti-NKG2D or anti-CD16 antibody molecule, and (b) a death receptor signaling adaptor. (19) A contains a first antigen-binding domain that selectively binds to the TCRVB antigen, and B, C or D contains (a) an immune cell adaptor, such as a T cell adaptor, such as an anti-CD3 antibody molecule, and (b) a cytokine molecule. (20) A contains a first antigen-binding domain that selectively binds to the TCRVB antigen, and B, C or D contains (a) an immune cell adaptor, such as a T cell adaptor, such as an anti-CD3 antibody molecule, and (b) a cytokine inhibitor molecule. (21) A contains a first antigen-binding domain that selectively binds to the TCRVB antigen, and B, C or D contains (a) an immune cell adaptor, such as a T cell adaptor, such as an anti-CD3 antibody molecule, and (b) a death receptor signaling adaptor. (22) A contains a first antigen-binding domain that selectively binds to the TCRVB antigen, and B, C, or D contains (a) a cytokine molecule and (b) a death receptor signaling adaptor; (23) A contains a first antigen-binding domain that selectively binds to the TCRVB antigen, and B, C, or D contains (a) a cytokine inhibitor molecule and (b) a death receptor signaling adaptor; (24) A contains a first antigen-binding domain that selectively binds to the TCRVB antigen, B contains a second antigen-binding domain that selectively binds to the TCRVB antigen, and C or D contains (a) an immune cell adaptor, such as an NK cell adaptor, such as an anti-NKp30, anti-NKp46, anti-NKG2D or anti-CD16 antibody molecule, and (b) a cytokine molecule. (25) A contains a first antigen-binding domain that selectively binds to the TCRVB antigen, B contains a second antigen-binding domain that selectively binds to the TCRVB antigen, and C or D contains (a) an immune cell adaptor, such as an NK cell adaptor, such as an anti-NKp30, anti-NKp46, anti-NKG2D or anti-CD16 antibody molecule, and (b) a cytokine inhibitor molecule. (26) A contains a first antigen-binding domain that selectively binds to a TCRVB antigen, B contains a second antigen-binding domain that selectively binds to a TCRVB antigen, and C or D contains (a) an immune cell adaptor, such as an NK cell adaptor, such as an anti-NKp30, anti-NKp46, anti-NKG2D or anti-CD16 antibody molecule, and (b) a death receptor signaling adaptor. (27) A contains a first antigen-binding domain that selectively binds to the TCRVB antigen, B contains a second antigen-binding domain that selectively binds to the TCRVB antigen, and C or D contains (a) an immune cell adaptor, such as an NK cell adaptor, such as an anti-NKp30, anti-NKp46, anti-NKG2D or anti-CD16 antibody molecule, and (b) a matrix modification portion. (28) A contains a first antigen-binding domain that selectively binds to the TCRVB antigen, B contains a second antigen-binding domain that selectively binds to the TCRVB antigen, and C or D contains (a) an immune cell adaptor, such as a T cell adaptor, such as an anti-CD3 antibody molecule, and (b) a cytokine molecule. (29) A contains a first antigen-binding domain that selectively binds to the TCRVB antigen, B contains a second antigen-binding domain that selectively binds to the TCRVB antigen, and C or D contains (a) an immune cell adaptor, such as a T cell adaptor, such as an anti-CD3 antibody molecule, and (b) a cytokine inhibitor molecule. (30) A contains a first antigen-binding domain that selectively binds to a TCRVB antigen, B contains a second antigen-binding domain that selectively binds to a TCRVB antigen, and C or D contains (a) an immune cell adaptor, such as a T cell adaptor, such as an anti-CD3 antibody molecule, and (b) a death receptor signaling adaptor. (31) A contains a first antigen-binding domain that selectively binds to the TCRVB antigen, B contains a second antigen-binding domain that selectively binds to the TCRVB antigen, and C or D contains (a) a cytokine molecule and (b) a death receptor signaling adaptor. (32) A contains a first antigen-binding domain that selectively binds to the TCRVB antigen, B contains a second antigen-binding domain that selectively binds to the TCRVB antigen, and C or D contains (a) a cytokine inhibitor molecule and (b) a death receptor signaling adaptor. (33) A contains a first antigen-binding domain that selectively binds to the TCRVB antigen, C contains a second antigen-binding domain that selectively binds to the TCRVB antigen, and B or D contains (a) an immune cell adaptor, such as an NK cell adaptor, such as an anti-NKp30, anti-NKp46, anti-NKG2D or anti-CD16 antibody molecule, and (b) a cytokine molecule. (34) A contains a first antigen-binding domain that selectively binds to the TCRVB antigen, C contains a second antigen-binding domain that selectively binds to the TCRVB antigen, and B or D contains (a) an immune cell adaptor, such as an NK cell adaptor, such as an anti-NKp30, anti-NKp46, anti-NKG2D or anti-CD16 antibody molecule, and (b) a cytokine inhibitor molecule. (35) A contains a first antigen-binding domain that selectively binds to the TCRVB antigen, C contains a second antigen-binding domain that selectively binds to the TCRVB antigen, and B or D contains (a) an immune cell adaptor, such as an NK cell adaptor, such as an anti-NKp30, anti-NKp46, anti-NKG2D or anti-CD16 antibody molecule, and (b) a death receptor signaling adaptor. (36) A contains a first antigen-binding domain that selectively binds to the TCRVB antigen, C contains a second antigen-binding domain that selectively binds to the TCRVB antigen, and B or D contains (a) an immune cell adaptor, such as a T cell adaptor, such as an anti-CD3 antibody molecule, and (b) a cytokine molecule. (37) A contains a first antigen-binding domain that selectively binds to the TCRVB antigen, C contains a second antigen-binding domain that selectively binds to the TCRVB antigen, and B or D contains (a) an immune cell adaptor, such as a T cell adaptor, such as an anti-CD3 antibody molecule, and (b) a cytokine inhibitor molecule. (38) A contains a first antigen-binding domain that selectively binds to a TCRVB antigen, C contains a second antigen-binding domain that selectively binds to a TCRVB antigen, and B or D contains (a) an immune cell adaptor, such as a T cell adaptor, such as an anti-CD3 antibody molecule, and (b) a death receptor signaling adaptor. (39) A contains a first antigen-binding domain that selectively binds to the TCRVB antigen, C contains a second antigen-binding domain that selectively binds to the TCRVB antigen, and B or D contains (a) a cytokine molecule and (b) a death receptor signaling adaptor. (40) A contains a first antigen-binding domain that selectively binds to the TCRBV antigen, C contains a second antigen-binding domain that selectively binds to the TCRBV antigen, and B or D contains (a) a cytokine inhibitor molecule and (b) a death receptor signaling adaptor; or 280. A multifunctional molecule according to embodiment 278 or 279, wherein the dimerizing module comprises one or more immunoglobulin chain constant regions (e.g., Fc regions), the immunoglobulin chain constant regions comprising one or more of the following: paired cavity-protrusions (“knob-in-a hole”), electrostatic interactions, or chain exchange.

[0317] 281. A multifunctional molecule of embodiment 280, wherein one or more immunoglobulin chain constant regions (e.g., Fc regions) comprise amino acid substitutions at one or more of positions selected from, for example, the Fc region of human IgG1, such as positions 347, 349, 350, 351, 366, 368, 370, 392, 394, 395, 397, 398, 399, 405, 407, or 409, optionally wherein the one or more immunoglobulin chain constant regions (e.g., Fc regions) comprise amino acid substitutions selected from: T366S, L368A, or Y407V (e.g., corresponding to a cavity or mortis), or T366W (e.g., corresponding to a protrusion or pestle) or combinations thereof.

[0318] 282. A multifunctional molecule of any of embodiments 1-281, further comprising a linker, for example, a linker between one or more of the following: an antigen-binding domain and an immune cell adaptor, an antigen-binding domain and a cytokine molecule, an antigen-binding domain and a matrix-modified portion, an immune cell adaptor and a cytokine molecule, an immune cell adaptor and a matrix-modified portion, a cytokine molecule and a matrix-modified portion, an antigen-binding domain and a dimerization module, an immune cell adaptor and a dimerization module, a cytokine molecule and a dimerization module, or a matrix-modified portion and a dimerization module.

[0319] 283. The multifunctional molecule of embodiment 282, wherein the adapter is selected from: cleavable adapters, non-cleavable adapters, peptide adapters, flexible adapters, rigid adapters, helical adapters, or non-helical adapters.

[0320] 284. A multifunctional molecule according to embodiment 282 or 283, wherein the linker is a peptide linker.

[0321] 285. A multifunctional molecule according to embodiment 284, wherein the peptide linker comprises Gly and Ser.

[0322] 286. A multifunctional molecule according to embodiment 285, wherein the peptide linker comprises an amino acid sequence selected from SEQ ID NO: 7248-7251 or 7252-7253 and 77-78.

[0323] 287. A multifunctional molecule comprising: (i) the first antigen-binding domain that selectively binds to the TCRBV antigen, and (ii) NK cell adaptors, such as anti-NKp30, anti-NKp46, anti-NKG2D or anti-CD16 antibody molecules.

[0324] 288. A multifunctional molecule according to embodiment 287, wherein the NK cell adaptor comprises an anti-NKp30 antibody molecule.

[0325] 289. A multifunctional molecule according to embodiment 287, wherein the NK cell adaptor comprises an anti-NKp46 antibody molecule.

[0326] 290. A multifunctional molecule comprising: (i) a first antigen-binding domain, which binds to, for example, selectively binds to, T-cell receptor variable β (TCRBV), such as the TCRBV antigen, and (ii) Death receptor signaling adaptors.

[0327] 291. A multifunctional molecule comprising: (i) a first antigen-binding domain, which binds to, for example, selectively binds to, T-cell receptor variable β (TCRBV), such as the TCRBV antigen, and (ii) Cytokine inhibitor molecules, such as TGF-β inhibitors.

[0328] 292. A multifunctional molecule of any one of embodiments 1-291, wherein the multifunctional molecule is monovalently bound to the TCRBV antigen.

[0329] 293. A multifunctional molecule of any of embodiments 1-291, wherein the multifunctional molecule binds to the TCRBV antigen in a multivalent manner (e.g., divalent, trivalent, tetravalent, pentavalent, hexavalent, heptavalent, octavalent, nonavalent, or decavalent).

[0330] 294. A multifunctional molecule of any of embodiments 2-261, wherein the multifunctional molecule binds to the TCRBV antigen on lymphocytes expressing the TCRBV antigen.

[0331] 295. Any multifunctional molecule in the preceding embodiments, wherein the multifunctional molecule is monovalently bound to immune cells, for example via an immune cell adaptor.

[0332] 296. A multifunctional molecule of any of embodiments 1-294, wherein the multifunctional molecule is bound to immune cells in a multivalent manner (e.g., divalent, trivalent, tetravalent, pentavalent, hexavalent, heptavalent, octavalent, nonavalent, or decavalent) via an immune cell adaptor.

[0333] 297. Any multifunctional molecule in the prior embodiment further includes a heavy chain constant region, such as an Fc region, that mediates antibody-dependent cytotoxicity (ADCC).

[0334] 298. Any multifunctional molecule in the prior embodiment further includes (e.g., via C1q) a heavy chain constant region, such as an Fc region, that mediates complement-dependent cytotoxicity.

[0335] 299. A nucleic acid molecule encoding a multifunctional molecule of any one of embodiments 1-298.

[0336] 300. A vector, for example, an expression vector, comprising a nucleic acid molecule according to embodiment 299.

[0337] 301. A host cell comprising a nucleic acid molecule according to embodiment 299 or a vector according to embodiment 300.

[0338] 302. A method for preparing, for example, a multifunctional molecule or antibody molecule of any of embodiments 1-298, comprising culturing the host cell of embodiment 301 under suitable conditions, for example, conditions suitable for gene expression and / or homodimerization or heterodimerization.

[0339] 303. A pharmaceutical composition comprising a multifunctional molecule of any one of embodiments 1-298 and a pharmaceutically acceptable carrier, excipient, or stabilizer.

[0340] 304. A method for treating TCR bias, comprising administering to a subject in need a multifunctional molecule of any one of embodiments 1-298, wherein the multifunctional molecule is administered in an amount effective in treating the TCR bias.

[0341] 305. A method of treating an autoimmune disease (e.g., an autoimmune disease associated with TCR bias), comprising administering to a subject in need a multifunctional molecule of any one of embodiments 1-298, wherein the multifunctional molecule is administered in an amount effective in treating the autoimmune disease.

[0342] 306. The method of implementation scheme 304 or 305 further includes identifying, evaluating or selecting subjects who need treatment, wherein identifying, evaluating or selecting includes determining (e.g., directly or indirectly, for example, by obtaining information about) whether the subject has a TCR bias or an autoimmune disease (e.g., an autoimmune disease associated with a TCR bias).

[0343] 307. The method of implementation scheme 306 further includes, 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, the subject is selected to be treated with a multifunctional molecule comprising an antigen-binding domain that binds to a TCRVB antigen (e.g., a TCRVB antigen corresponding to a biased TCRVB clone), and The multifunctional molecule is applied, which includes an antigen-binding domain that binds to a TCRVB antigen (e.g., a TCRVB antigen corresponding to a biased TCRVB clone).

[0344] 308. A method for treating TCR bias, comprising: In response to determining that a subject has a TCR bias, a multifunctional molecule of any one of embodiments 1-298 is administered to the subject in need, wherein the multifunctional molecule is administered in an amount that is effective in treating the TCR bias.

[0345] 309. A method for treating an autoimmune disease (e.g., an autoimmune disease associated with TCR bias), comprising: In response to determining that a subject has an autoimmune disease (e.g., an autoimmune disease associated with TCR bias), a multifunctional molecule of any one of embodiments 1-298 is administered to the subject in need, wherein the multifunctional molecule is administered in an amount that is effective in treating the autoimmune disease (e.g., an autoimmune disease associated with TCR bias).

[0346] 310. The method of any one of embodiments 304-309, wherein the subject has a TCR bias (e.g., a biased TCR BV clone) and / or an autoimmune disease associated with said bias.

[0347] 311. A method for identifying a subject in need of cancer treatment using a multifunctional molecule of any one of embodiments 1-298, comprising determining (e.g., directly or indirectly, e.g., obtaining information about) whether the subject has a TCR bias (e.g., a biased TCR BV clonal type) and / or an autoimmune disease associated with said bias, wherein: In response to determining that the subject has a TCR bias (e.g., a biased TCR clonal type) and / or an autoimmune disease associated with said bias, the subject is identified as a candidate for treatment with a multifunctional molecule containing an antigen-binding domain that binds to a TCR clonal antigen, and optionally, is identified as not a candidate for treatment with a multifunctional molecule containing an antigen-binding domain that does not bind to said TCR clonal antigen (e.g., binds to a different TCR clonal antigen).

[0348] 312. The method of implementing scheme 311 further includes: In response to identifying the subject as a candidate for treatment with a multifunctional molecule containing an antigen-binding domain that binds to a TCRVB antigen, the subject is treated with (e.g., administered to the subject) a multifunctional molecule containing an antigen-binding domain that binds to a TCRVB antigen.

[0349] 313. A method for evaluating a subject who requires treatment for TCR bias (e.g., biased TCR BV clonal type) and / or an autoimmune disease associated with said bias, comprising determining (e.g., directly or indirectly, for example, by obtaining information about) whether the subject has TCR bias.

[0350] 314. The method of embodiment 313 further comprises, in response to the evaluation, treating the subject with (e.g., administered to the subject) a multifunctional molecule comprising an antigen-binding domain that binds to the TCRVB antigen.

[0351] 315. The method of any one of embodiments 304-314, wherein the TCR bias is associated with an autoimmune disease.

[0352] 316. The method of implementation scheme 315, wherein the autoimmune disease is selected from Churg-Strauss syndrome, sarcoidosis, systemic lupus erythematosus (SLE), type 1 diabetes, autoimmune hepatitis (e.g., type 1 or type 2), primary sclerosing cholangitis, primary biliary cirrhosis, multiple sclerosis, Guillain-Barre syndrome and AMAN (axonal and neuronal neuropathy), chronic inflammatory demyelinating polyneuropathy (CIDP), transverse myelitis, Tolosa-Hunt syndrome (THS), Dweck's disease (neuromyelitis optica), paraneoplastic cerebellar degeneration (PCD), Lambert-Eaton syndrome, psoriasis, scleroderma, CREST (calcification, Raynaud's phenomenon, esophageal motility disorder, finger sclerosis and telangiectasia) syndrome, herpetic dermatitis, dermatomyositis, bullous pemphigoid, cicatricial pemphigoid / benign mucosal pemphigoid, Pregnancy-related pemphigoid, rheumatoid arthritis (RA), psoriatic arthritis, relapsing polychondritis, chronic relapsing multifocal osteomyelitis (CRMO), vasculitis, Kawasaki disease, granulomatous polyangiitis (GPA), Behçet's disease (vasculitis), aortitis, nodular polyangiitis, microscopic polyangiitis (MPA), leukocyte rupture vasculitis, Cogan syndrome, uveitis, peripheral uveitis (paris plana inflammation), scleritis, autoimmune inner ear disease (AIED), Crohn's disease, ulcerative colitis (UC), Dereisler syndrome, rheumatic fever, Evans syndrome, paroxysmal nocturnal hemoglobinuria (PNH), hemolytic anemia, 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.

[0353] 317. The method of any one of embodiments 304-316, further comprising the application of a second therapeutic treatment.

[0354] 318. The method of embodiment 317, wherein the second therapeutic treatment includes a therapeutic agent (e.g., a chemotherapeutic agent, a biological agent, hormone therapy), radiation, or surgery.

[0355] 319. A method of treating an autoimmune disease (e.g., an autoimmune disease associated with TCR bias) in a subject in need, comprising administering to the subject an effective amount, for example a therapeutically effective amount, of an antibody molecule (“anti-TCRβV antibody molecule”) that binds (e.g., specifically binds) to the variable region of T cell receptor β (TCRβV).

[0356] 320. A method for depleting a population of T cells in a subject suffering from an autoimmune disease (e.g., an autoimmune disease associated with TCR bias), comprising contacting the population of T cells with an effective amount of an antibody molecule (“anti-TCRβV antibody molecule”) that binds (e.g., specifically binds) to the variable region of the T cell receptor β (TCRβV).

[0357] 321. The method according to embodiment 320, wherein the contact occurs in vivo or in vitro.

[0358] 322. The method according to any one of embodiments 319-321, wherein the anti-TCRβV antibody molecule: (i) It is not an antibody molecule disclosed in U.S. Patent 5,861,155; (ii) Binding to TCRβ V12 with an affinity and / or binding specificity less than (e.g., less than about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or about 2, 5 or 10 times) that of the 16G8 mouse antibody or its humanized form as described in U.S. Patent 5,861,155; (iii) Bind to TCRβ V12 with an affinity and / or binding specificity greater than (e.g., greater than about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or about 2, 5, or 10 times) that of the 16G8 mouse antibody or its humanized form as described in U.S. Patent 5,861,155; (iii) Binding to TCRβ V5-5*01 or TCRβ V5-1*01 with an affinity and / or binding specificity greater than (e.g., greater than about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or about 2, 5, or 10 times) that of the TM23 mouse antibody or its humanized form as described in U.S. Patent 5,861,155; or (iv) Bind to TCRβ V5-5*01 or TCRβ V5-1*01 with an affinity and / or binding specificity greater than (e.g., greater than about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or about 2, 5, or 10 times) that of the TM23 mouse antibody or its humanized form as described in U.S. Patent 5,861,155.

[0359] 323. The method according to any one of embodiments 319-322, wherein the anti-TCRβV antibody molecule comprises an Fc region, for example, an Fc region having effector functions (e.g., antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cytophagy (ADCP), and / or complement-dependent cytotoxicity (CDC)).

[0360] 324. The method of embodiment 323, wherein the anti-TCRβV antibody molecule comprises, for example, an Fc region having enhanced effector function compared to a wild-type Fc region.

[0361] 325. The method according to any one of embodiments 319-324, wherein the anti-TCRβV antibody molecule comprises a human IgG1 region or a human IgG4 region.

[0362] 326. The method according to any one of embodiments 319 or 321-325, wherein the autoimmune disease is selected from Churg-Strauss syndrome, sarcoidosis, systemic lupus erythematosus (SLE), type 1 diabetes mellitus, autoimmune hepatitis (e.g., type 1 or type 2), primary sclerosing cholangitis, primary biliary cirrhosis, multiple sclerosis, Guillain-Barre syndrome, and AMAN (axonal and sclerosing hepatitis). Neuronal neuropathy), chronic inflammatory demyelinating polyneuropathy (CIDP), transverse myelitis, Tolosa-Hunt syndrome (THS), Dweck's disease (neuromyelitis optica), paraneoplastic cerebellar degeneration (PCD), Lambert-Eaton syndrome, psoriasis, scleroderma, CREST (calcification, Raynaud's phenomenon, esophageal motility disorder, finger sclerosis and telangiectasia) syndrome, herpetic dermatitis, dermatomyositis, bullous pemphigoid, cicatricial pemphigoid / benign mucosal pemphigoid, gestational pemphigoid, rheumatoid arthritis (RA), psoriatic arthritis, relapsing polychondritis, chronic relapsing multifocal osteomyelitis (CRMO), vasculitis, Kawasaki disease, granulomatous polyneuropathy Gastritis (GPA), Behcet's disease (vasculitis), Takayasu arteritis, nodular polyangiitis, microscopic polyangiitis (MPA), leukocyte rupture vasculitis, Cogan syndrome, uveitis, peripheral uveitis (paris plana inflammation), scleritis, autoimmune inner ear disease (AIED), Crohn's disease, ulcerative colitis (UC), Dereisler syndrome, rheumatic fever, Evans syndrome, paroxysmal nocturnal hemoglobinuria (PNH), hemolytic anemia, 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.

[0363] 327. The method according to any one of embodiments 319-326, wherein the anti-TCRβV antibody molecule comprises an antigen-binding domain comprising one or more (e.g., all three) of LC CDR1, LC CDR2, and LC CDR3 provided in Tables 1A, 2A, 10A, 11A, 12A, or 13A; and / or one or more (e.g., all three) of HCCDR1, HC CDR2, and HC CDR3 provided in Tables 1A, 2A, 10A, 11A, 12A, or 13A, or a sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with them.

[0364] 328. The method according to any one of embodiments 319-327, wherein the anti-TCRβV antibody molecule comprises a variable heavy chain (VH) and / or a variable light chain (VL) provided in Tables 1A, 2A, 10A, 11A, 12A or 13A, or a sequence having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identity with it.

[0365] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Suitable methods and materials are described below, but methods and materials similar to or equivalent to those described herein may be used in the practice or testing of this invention. All publications, patent applications, patents, and other references mentioned herein are incorporated herein by reference in their entirety. In the event of conflict, this specification, including the definitions, shall prevail. Furthermore, materials, methods, and examples are illustrative only and are not intended to be limiting.

[0366] Other features and advantages of the invention will become apparent from the following detailed description and claims. Attached Figure Description

[0367] Figures 1A-1B The alignment of mouse VH and VL framework regions 1, CDR 1, 2, 2, 3, 3, and 4 derived from antibody A with their respective humanized sequences is shown. Kabat CDRs are shown in bold, Chothia CDRs in italics, and combined CDRs are shown in boxes. The locations of inverted mutation frameworks are indicated by double underlines. Figure 1A The VH sequence of mouse antibody A (SEQ ID NO: 1) and the VH sequence of humanized antibody AH (SEQ ID NO: 9) are shown. Figure 1B The VL sequences of mouse antibody A (SEQ ID NO: 2) and humanized antibody AH (SEQ ID NO: 10 and SEQ ID NO: 11) are shown.

[0368] Figures 2A-2B The alignment of mouse VH and VL framework regions 1, CDR 1, 2, CDR 2, 3, CDR 3, and 4 derived from antibody B with their respective humanized sequences is shown. Kabat CDRs are shown in bold, Chothia CDRs in italics, and combined CDRs are shown in boxes. The locations of inverted mutation frameworks are indicated by double underlines. Figure 2A The VH sequence of mouse antibody B (SEQ ID NO: 15) and the humanized VH sequences BH.1A to BH.1C (SEQ ID NO: 23-25) are shown. Figure 2BThe VL sequence of mouse antibody B (SEQ ID NO: 16) and the humanized VL sequences BH.1D to BH.1H (SEQ ID NO: 26-30) are shown.

[0369] Figure 3 A phylogenetic tree of the TCRBV gene family and subfamilies was drawn, and the corresponding antibodies were mapped. The subfamilies are as follows: Subfamilies A: TCRβ V6; Subfamilies B: TCRβ V10; Subfamilies C: TCRβ V12; Subfamilies D: TCRβ V5; Subfamilies E: TCRβ V7; Subfamilies F: TCRβ V11; Subfamilies G: TCRβ V14; Subfamilies H: TCRβ V16; Subfamilies I: TCRβ V18; Subfamilies J: TCRβ V9; Subfamilies K: TCRβ V13; Subfamilies L: TCRβ V4; Subfamilies M: TCRβ V3; Subfamilies N: TCRβ V2; Subfamilies O: TCRβ V15; Subfamilies P: TCRβ V30; Subfamilies Q: TCRβ V19; Subfamilies R: TCRβ V27; Subfamilies S: TCRβ V28; Subfamilies T: TCRβ V24; Subfamilies U: TCRβ V20; Subfamilies V: TCRβ V20; V25; and subfamily W: TCRβ V29 subfamily. Subfamily members are described in detail in the section entitled “TCR βV (TCRβV)”.

[0370] Figure 4 This is a graph showing the binding of NKp30 antibody to NK92 cells. The data are calculated as the percentage of the AF747 positive population.

[0371] Figure 5 This diagram illustrates the activation of NK92 cells via NKp30 antibody. Data was generated using hamster anti-NKp30 mAb. Detailed Implementation

[0372] This document discloses multifunctional molecules (also referred to herein as “multispecific molecules”) that include multiple (e.g., two or more) functions (or binding specificities) comprising (i) an antigen-binding domain that binds (e.g., selectively binds) to a T-cell receptor variable β (TCRBV) (e.g., a TCRVB antigen); and (ii) one, two, or all of the following: (a) an immune cell adjuvant selected from T-cell adjuvants, NK-cell adjuvants (e.g., molecules that bind to NKp30, NKp46, NKG2D, or CD16), B-cell adjuvants, dendritic cell adjuvants, or macrophage adjuvants; (b) cytokine molecules or cytokine inhibitor molecules; and (c) death receptor signaling adjuvants. In some embodiments, the antigen-binding domain comprises a sequence or a portion of a sequence as seen in Tables 13 or 14. In some embodiments, the immune cell adjuvant comprises an NK-cell adjuvant that comprises a sequence or a portion of a sequence as seen in Tables 7-10. In some embodiments, the antigen-binding domain comprises a sequence or a portion of a sequence as seen in Table 13 or 14, and the immune cell adaptor comprises an NK cell adaptor comprising a sequence or a portion of a sequence as seen in Tables 7-10.

[0373] In the implementation scheme, the multispecific or multifunctional molecule is a bispecific (or bifunctional) molecule, a trispecific (or trifunctional) molecule, or a tetraspecific (or tetrafunctional) molecule.

[0374] In some embodiments, the multifunctional molecule includes an antigen-binding domain that binds to a TCRBV antigen on the surface of lymphocytes, such as T cells. In some embodiments, the TCRBV antigen corresponds to a biased TCRBV clonal type; for example, a TCR containing the TCRBV antigen may be overexpressed in the TCR pool or lymphocyte (e.g., T cell) pool of a subject (e.g., a subject with an autoimmune disease associated with TCR bias), or expressed at a level higher than that in other subjects (e.g., subjects without autoimmune diseases).

[0375] Not limited to theory, the multispecific or multifunctional molecules disclosed herein are expected to localize (e.g., bridge) and / or activate immune cells (e.g., immune effector cells selected from NK cells, T cells, B cells, dendritic cells, or macrophages) in the presence of cells (e.g., lymphocytes, e.g., T cells) expressing TCR BV antigens (e.g., TCR BV antigens corresponding to biased TCR BV clonal types). 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) expressing TCR BV antigens (e.g., TCR BV antigens corresponding to biased TCR BV clonal types) is expected to enhance the immune response to target cells, thereby providing a more effective therapy (e.g., by reducing the levels of biased TCR and / or T cells expressing biased TCR). In another embodiment, targeting cells (e.g., lymphocytes, such as T cells) that express TCRBV antigens (e.g., TRBV antigens corresponding to biased TCRBV clonal types) with a multifunctional molecule that also contains a cell death-inducing portion (e.g., a death receptor signaling adaptor) is thought to induce the death of the target cells (e.g., by reducing the level of biased TCR and / or T cells expressing biased TCR).

[0376] Not limited to theory, in some embodiments, it is contemplated that the detrimental effects of generally increasing the proximity and / or activity of immune cells toward T cells or generally promoting T cell cell death can be mitigated by utilizing multispecific or multifunctional molecules that are specific to a particular TRBV antigen (e.g., a TRBV antigen corresponding to a biased TRBV clonal type) but nonspecific to other or all types of T cell receptors. In this way, the use of the multispecific or multifunctional molecules disclosed herein is considered to increase the proximity or activity of immune cells toward cells containing TRBV antigens corresponding to a biased TRBV clonal type without necessarily generally increasing the proximity and / or activity of immune cells toward T cells, or to promote cell death of cells containing TRBV antigens corresponding to a biased TRBV clonal type without necessarily generally increasing T cell death.

[0377] Therefore, this article provides, in particular, a multispecific or multifunctional molecule comprising the foregoing portions (e.g., a multispecific or multifunctional antibody molecule), a nucleic acid encoding the molecule, a method for producing the foregoing molecule, and a method for using the foregoing molecule to treat diseases or conditions such as autoimmune diseases or TCR bias. definition

[0378] In some embodiments, the multifunctional molecule includes an immune cell adaptor. An “immune cell adaptor” 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 macrophages. The immune cell adaptor 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 immune cell antigens (e.g., T cell, NK cell antigens, B cell antigens, dendritic cell antigens, and / or macrophage antigens). In embodiments, the immune cell adaptor specifically binds to target immune cells, for example, preferentially binding to target immune cells. For example, when the immune cell adaptor is an antibody molecule, it binds to immune cell antigens (e.g., T cell antigens, NK cell antigens, B cell antigens, dendritic cell antigens, and / or macrophage antigens) with a dissociation constant of less than about 10 nM.

[0379] In some embodiments, the multifunctional molecule includes cytokine molecules. As used herein, a "cytokine molecule" refers to the full length, fragment, or variant of a cytokine; it also includes a receptor domain, such as a cytokine with a cytokine receptor dimerization domain; or an agonist of a cytokine receptor, such as an antibody molecule (e.g., an agonist antibody) targeting a cytokine receptor that activates at least one activity of a 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-γ, or fragments or variants thereof, or any combination of the above cytokines. The cytokine molecule can be a monomer or a dimer. In embodiments, the cytokine molecule may further include a cytokine receptor dimerization domain. In other embodiments, the cytokine molecule is an agonist of a cytokine receptor, such as an antibody molecule (e.g., an agonist antibody) targeting a cytokine receptor selected from IL-15Ra or IL-21R.

[0380] As used herein, the term “molecule” in, for example, antibody molecules, cytokine molecules, receptor molecules, includes full-length, naturally occurring molecules, as well as variants, such as functional variants (e.g., truncated, fragmented, mutated (e.g., substantially similar sequences) or derived forms), provided that at least one function and / or activity of the unmodified (e.g., naturally occurring) molecule is preserved.

[0381] As used herein, the term "autoimmune disease," condition, or illness refers to a disease in which the body's immune system attacks its own cells or tissues. Autoimmune diseases can lead to the production of autoantibodies that inappropriately and / or excessively target self-antigens or autoantigens. Autoimmune diseases include, but are not limited to, cardiovascular diseases, rheumatoid diseases, adenomas, gastrointestinal diseases, skin diseases, liver diseases, nervous system diseases, muscle diseases, kidney diseases, reproductive-related diseases, connective tissue diseases, and systemic diseases. In some embodiments, autoimmune diseases are mediated by T-cell, B-cell, innate immune cells (e.g., macrophages, eosinophils, or natural killer cells), or complement-mediated pathways.

[0382] Some terms are defined as follows.

[0383] As used herein, the articles “a” and “an” refer to one or more (e.g., at least one) grammatical objects. When used with the term “including,” the use of the terms “a” or “an” can mean “one,” but also aligns with the meanings of “one or more,” “at least one,” and “one or more.”

[0384] As used herein, “about” and “approximately” generally refer to the acceptable degree of error of a measured quantity given the nature or precision of the measurement. Exemplary error levels are within 20% of a given range, typically within 10%, and more often within 5%.

[0385] As used herein, an "antibody molecule" refers to a protein that includes at least one immunoglobulin variable domain sequence, such as an immunoglobulin chain or a fragment thereof. Antibody molecules include antibodies (e.g., full-length antibodies) and antibody fragments. In embodiments, antibody molecules include antigen-binding or functional fragments of full-length antibodies, or full-length immunoglobulin chains. For example, a full-length antibody is a naturally occurring immunoglobulin (Ig) molecule (e.g., an IgG antibody) formed through a normal immunoglobulin gene fragment recombination process. In embodiments, an antibody molecule refers to the immunogenic antigen-binding portion of an immunoglobulin molecule, such as an antibody fragment. Antibody fragments, such as functional fragments, are part of an antibody, such as Fab, Fab′, F(ab′)2, F(ab)2, variable fragments (Fv), domain antibodies (dAb), or single-chain variable fragments (scFv). Functional antibody fragments bind to the same antigen recognized by the intact (e.g., full-length) antibody. The terms "antibody fragment" or "functional fragment" also include isolated fragments composed of variable regions (such as an "Fv" fragment composed of variable regions of a heavy chain and a light chain), or recombinant single-chain polypeptide molecules in which the light and heavy chain variable regions are linked by peptide linkers ("scFv protein"). In some embodiments, the antibody fragment does not include antibody portions that lack antigen-binding activity, such as Fc fragments or single amino acid residues. Exemplary antibody molecules include full-length antibodies and antibody fragments, such as dAb (domain antibody), single-chain, Fab, Fab', and F(ab')2 fragments, and single-chain variable fragments (scFv).

[0386] As used herein, an "immunoglobulin variable domain sequence" refers to an amino acid sequence that can form a structure that forms 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- or C-terminal amino acids, or may include other modifications compatible with the formation of the protein structure.

[0387] In some embodiments, the antibody molecule is monospecific, for example, it contains binding specificity for a single epitope. In some embodiments, the antibody molecule is multispecific, for example, it contains 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 is specific to more than one (e.g., two, three, four or more) epitopes and / or antigens.

[0388] As used herein, an "antigen" (Ag) is a molecule that can elicit an immune response, such as involving the activation of certain immune cells and / or antibody production. Any macromolecule, including virtually all proteins or peptides, can be an antigen. Antigens can also be derived from genomic recombinants or DNA. For example, any DNA containing a nucleotide sequence or a portion thereof 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. In embodiments, an antigen can be synthesized or can be derived from a biological sample, such as a tissue sample, blood sample, cells, or a fluid containing other biological components. As used herein, a "TCRBV antigen" includes any variable β chain of a TCR or a portion thereof that can elicit an immune response or be targeted by an antigen-binding domain. In some embodiments, a biased TCR clonal type may be characterized by one or more TCRRBV antigens being presented, for example, on the surface of most (e.g., all) cells containing that clonal type.

[0389] The "antigen-binding site" or "binding portion" of an antibody molecule refers to a part of an antibody molecule, such as an immunoglobulin (Ig) molecule, that participates in antigen binding. In an embodiment, the antigen-binding site is formed by amino acid residues in the variable (V) regions of the heavy (H) chain and the light (L) chain. Three highly divergent sequence segments within the variable regions of the heavy and light chains are called hypervariable regions, located between more conserved flanking sequence segments (called "framework regions" (FRs)). FRs are amino acid sequences naturally present in immunoglobulins and adjacent to and between them. In an embodiment, in an antibody molecule, the three hypervariable regions of the light chain and the three hypervariable regions of the heavy chain are arranged relative to each other in three-dimensional space to form an antigen-binding surface that is complementary to the three-dimensional surface of the binding antigen. The three hypervariable regions in each heavy and light chain are called "complementarity-determining regions" or "CDRs". Framework regions and CDRs have been defined and described, for example, in Kabat, EA, et al., (1991) Sequences of Proteins of Immunological Interest, 5th ed., US Department of Health and Human Services, NIH Publication No. 91-3242 and Chothia, C. et al., (1987) J. Mol. Biol. 196:901-917. Each variable chain (e.g., variable heavy chain and variable light chain) typically consists of three CDRs and four FRs, arranged in the following amino acid sequence from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.

[0390] As used herein, “immune cell” refers to any of the various cells that function in the immune system to protect, for example, from infectious agents and foreign substances. In embodiments, the term includes leukocytes, such as 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 recognize and eliminate pathogens by attacking larger pathogens through contact or by phagocytosis and killing microorganisms, and are mediators of adaptive immune responses. Cells of the adaptive immune system are a special type of leukocyte called lymphocytes. B cells and T cells are important types of lymphocytes that originate from hematopoietic stem cells in the bone marrow. B cells participate in humoral immune responses, while T cells participate in cell-mediated immune responses. The term “immune cell” also includes immune effector cells.

[0391] As used herein, the term "immune effector cell" refers to a cell that participates in an immune response, such as promoting an immune effector response. Examples of immune effector cells include, but are not limited to, T cells (e.g., α / β T cells and γ / δ T cells), B cells, natural killer (NK) cells, natural killer T (NK T) cells, and mast cells.

[0392] The term "effective function" or "effective response" refers to a cell's proprietary function. For example, T cell effector functions may include cytolytic activity or helper activity, including cytokine secretion.

[0393] The compositions and methods of the present invention comprise polypeptides and nucleic acids having a specified sequence or a sequence substantially identical or similar thereto (e.g., a sequence that is 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" as used herein means a first amino acid containing a sufficient or minimum number of amino acid residues that are i) identical to, or ii) have conserved substitutions with, the aligned amino acid residues in the second amino acid sequence, such that the first and second amino acid sequences may have common domains and / or common functional activities. For example, the amino acid sequence contains a common domain having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with a reference sequence (e.g., the sequence provided herein).

[0394] In the context of nucleotide sequences, the term "substantially identical" as used herein means that the first nucleic acid sequence contains a sufficient or minimum number of nucleotides identical to the aligned nucleotides in the second nucleic acid sequence, such that the first and second nucleotide sequences encode polypeptides with common functional activities, or polypeptides encoding common structural polypeptide domains or common functional polypeptide activities. For example, a nucleotide sequence having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with a reference sequence (e.g., the sequence provided herein).

[0395] The term "variant" refers to a polypeptide having a substantially identical amino acid sequence to a reference amino acid sequence, or encoded by a substantially identical nucleotide sequence. In some embodiments, a variant is a functional variant.

[0396] The term "functional variant" refers to a polypeptide that has a substantially identical amino acid sequence to a reference amino acid sequence, or is encoded by a substantially identical nucleotide sequence, and is capable of having one or more activities of the reference amino acid sequence.

[0397] The following calculations are performed to determine the homology or sequence identity between sequences (these terms are used interchangeably in this paper).

[0398] To determine the percentage of identity between two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., vacancies may be introduced in one or both of the first and second amino acid or nucleic acid sequences for optimal alignment, and non-homologous sequences may 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%, 100% of the length of the reference sequence. The amino acid residues or nucleotides at the 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, "identity" of an amino acid or nucleic acid is equivalent to "homology" of an amino acid or nucleic acid).

[0399] Taking into account the number of gaps that need to be included for optimal alignment of two sequences and the length of each gap, the percentage of identity between two sequences is a function of the number of identical positions shared by the sequences.

[0400] Mathematical algorithms can be used to compare sequences and determine the percentage of identity between two sequences. In a preferred embodiment, Needleman and Wunsch (1970) are used. J. Mol. BiolThe algorithm (48:444-453) (integrated into the GAP program in the GCG software package (available from http: / / www.gcg.com)) uses a Blossum 62 matrix or a PAM250 matrix with vacancy weights of 16, 14, 12, 10, 8, 6, or 4 and length weights of 1, 2, 3, 4, 5, or 6 to determine the percentage of identity between two amino acid sequences. In another preferred embodiment, the GAP program in the GCG software package (available from http: / / www.gcg.com) uses an NWSgapdna.CMP matrix with vacancy weights of 40, 50, 60, 70, or 80 and length weights of 1, 2, 3, 4, 5, or 6 to determine the percentage of identity between two nucleotide sequences. A particularly preferred set of parameters (unless otherwise specified, this set of parameters should be used) is a Blossum 62 scoring matrix with a vacancy penalty of 12, a vacancy extension penalty of 4, and a frameshift vacancy penalty of 5.

[0401] E. Meyers and W. Miller (1989) can be used. CABIOS The algorithm (which has been incorporated into the ALIGN program (version 2.0) uses the PAM120 weighted residue table with a vacancy length penalty of 12 and a vacancy penalty of 4 to determine the percentage of identity between two amino acid or nucleotide sequences.

[0402] The nucleic acid and protein sequences described in this article can be used as "query sequences" to search public databases, thereby identifying, for example, other family members or related sequences. Altschul et al. (1990) can be used as examples. J. Mol. Biol The NBLAST and XBLAST programs (version 2.0) of .215:403-10 can be used for searching. 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 present 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 present invention. For obtaining vacancy alignments for comparative purposes, Altschul et al. (1997) can be used. Nucleic Acids Res. The vacancy BLAST described in 25:3389-3402. When using the BLAST and vacancy BLAST procedures, the default parameters of the individual procedures (such as XBLAST and NBLAST) can be used. See http: / / www.ncbi.nlm.nih.gov.

[0403] It should be understood that the molecules of the present invention may have additional conserved or non-essential amino acid substitutions, which do not substantially affect their function.

[0404] The term "amino acid" is intended to encompass all molecules, whether natural or synthetic, that include both amino and acid functional groups and can be included in polymers of naturally occurring amino acids. Exemplary amino acids include naturally occurring amino acids; analogs, derivatives, and congeners; amino acid analogs with variable side chains; and all stereoisomers of any of the foregoing. As used herein, the term "amino acid" includes D- or L-optical isomers and peptide-like compounds.

[0405] "Conservative amino acid substitution" is a substitution in which an amino acid residue is replaced by an amino acid residue with 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), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), amino acids with nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), amino acids with β-branched side chains (e.g., threonine, valine, isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[0406] The terms “polypeptide,” “peptide,” and “protein,” used interchangeably herein, refer to polymers of amino acids of any length. The polymer may be linear or branched, may contain modified amino acids, and may be interrupted by non-amino acid components. The term also covers polymers of modified amino acids, where modifications are, for example, disulfide bond formation, glycosylation, esterification, acetylation, phosphorylation, or any other manipulation, such as conjugation with a labeled component. Polypeptides may be isolated from natural sources, generated from eukaryotic or prokaryotic hosts via recombinant techniques, or may be products of synthetic methods.

[0407] 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, namely deoxyribonucleotides or ribonucleotides or their analogues. Polynucleotides can be single-stranded or double-stranded, and if single-stranded, they can be coding or non-coding (antisense) strands. Polynucleotides can contain modified nucleotides, such as methylated nucleotides and nucleotide analogues. The sequence of nucleotides can be interrupted by non-nucleotide components. Polynucleotides can be further modified after polymerization, such as by conjugation with labeled components. Nucleic acids can be recombinant polynucleotides or polynucleotides of genomic, cDNA, semi-synthetic, or synthetic origin that are not naturally occurring or linked to another polynucleotide in a non-natural manner.

[0408] As used herein, the term "isolated" refers to material taken from its source or natural environment (e.g., the natural environment, if it exists naturally). For example, a naturally occurring polynucleotide or polypeptide present in a living animal is not isolated, but the same polynucleotide or polypeptide isolated from some or all of the coexisting substances in a natural system through human intervention is isolated. Such a polynucleotide may be part of a carrier, and / or such a polynucleotide or polypeptide may be part of a composition, and is still isolated because such a carrier or composition is not part of the environment in which it is found in nature.

[0409] The various aspects of the invention are described in further detail below. Other definitions are set forth throughout the specification. antibody molecules

[0410] In one embodiment, the antibody molecule binds to a TCRBV antigen, for example, (e.g., a TCRBV antigen corresponding to a biased TCRBV clonal type). In some embodiments, the TCRBV antigen is, for example, a mammalian (e.g., human) TCRBV antigen. In some embodiments, the antibody molecule binds to a TCRBV antigen on lymphocytes (e.g., T cells, for example, mammalian (e.g., human) lymphocytes, for example, T cells). For example, the antibody molecule specifically binds to a TCRBV antigen expressed on the surface of a lymphocyte (e.g., T cell) (e.g., as part of a TCR containing TCRBV).

[0411] In one embodiment, the antibody molecule is a monospecific antibody molecule that binds to a single epitope. For example, a monospecific antibody molecule has multiple immunoglobulin variable domain sequences, each of which binds to the same epitope.

[0412] In one embodiment, the antibody molecule is a multispecific or multifunctional antibody molecule, for example, comprising a plurality of immunoglobulin variable domain sequences, wherein a first immunoglobulin variable domain sequence of the plurality has binding specificity for a first epitope, and a second immunoglobulin variable domain sequence of the plurality has binding specificity for a second epitope. In one embodiment, the first and second epitopes are on the same antigen, such as the same protein (or a subunit of a multimeric protein). In one embodiment, the first and second epitopes overlap. In one embodiment, the first and second epitopes do not overlap. In one embodiment, the first and second epitopes are on different antigens, such as different proteins (or different subunits of a multimeric protein). In one embodiment, the multispecific antibody molecule 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.

[0413] In one embodiment, the multispecific antibody molecule is a bispecific antibody molecule. The bispecific antibody is specific to no more than two antigens. The bispecific antibody molecule is characterized by a first immunoglobulin variable domain sequence having binding specificity to a first epitope and a second immunoglobulin variable domain sequence having binding specificity to a second epitope. In one embodiment, the first and second epitopes are on the same antigen, for example, the same protein (or a subunit of a multimeric protein). In one embodiment, the first and second epitopes overlap. In one embodiment, the first and second epitopes do not overlap. In one embodiment, the first and second epitopes are on different antigens, for example, different proteins (or different subunits of a multimeric protein). In one embodiment, the bispecific antibody molecule comprises a heavy chain variable domain sequence and a light chain variable domain sequence having binding specificity to the first epitope, and a heavy chain variable domain sequence and a light chain variable domain sequence having binding specificity to the second epitope. In one embodiment, the bispecific antibody molecule comprises a hapten having binding specificity to the first epitope and a hapten having binding specificity to the second epitope. In one embodiment, the bispecific antibody molecule comprises a hapten or a fragment thereof having binding specificity to a first epitope and a hapten or a fragment thereof having binding specificity to a second epitope. In another embodiment, the bispecific antibody molecule comprises an scFv or Fab or a fragment thereof having binding specificity to the first epitope and an scFv or Fab or a fragment thereof having binding specificity to the second epitope.

[0414] In one embodiment, the antibody molecule comprises a biantibody, a single-chain molecule, and an antigen-binding fragment of the antibody (e.g., Fab, F(ab')2, and Fv). For example, the antibody molecule may 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, the antibody molecule comprises or consists of a heavy chain and a light chain (referred herein as a hapten). In another example, the antibody molecule comprises two heavy (H) chain variable domain sequences and two light (L) chain variable domain sequences, thereby forming two antigen-binding sites, such as Fab, Fab', F(ab')2, Fc, Fd, Fd', Fv, single-chain antibodies (e.g., scFv), single-variable domain antibodies, biantibodies (Dab) (bivalent and bispecific), and chimeric (e.g., humanized) antibodies, which can be generated by modifying an intact antibody or synthesized de novo using recombinant DNA technology. These functional antibody fragments retain the ability to selectively bind to their respective antigens or receptors. Antibodies and antibody fragments can be derived from any type of antibody, including but not limited to IgG, IgA, IgM, IgD, and IgE, and from any subclass of antibodies (e.g., IgG1, IgG2, IgG3, and IgG4). Antibody molecules can be monoclonal or polyclonal. Antibody molecules can also be human, humanized, CDR-transplanted, or in vitro generated antibodies. Antibodies can have a heavy chain constant region selected from, for example, IgG1, IgG2, IgG3, or IgG4. Antibodies may also have a light chain selected from, for example, κ or λ. The term “immunoglobulin” (Ig) is used interchangeably with the term “antibody” herein.

[0415] Examples of antigen-binding fragments in antibody molecules include: (i) Fab fragments, a monovalent fragment consisting of VL, VH, CL, and CH1 domains; (ii) F(ab')2 fragments, a bivalent fragment comprising two Fab fragments linked by disulfide bonds in the hinge region; (iii) Fd fragments consisting of VH and CH1 domains; (iv) Fv fragments consisting of the VL and VH domains of an antibody single arm; (v) biantibody (dAb) fragments consisting of the VH domain; (vi) camel or camelized variable domains; and (vii) single-chain Fv (scFv), see, for example, Bird et al., (1988). Science 242:423-426; and Huston et al., (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883; (viii) Single-domain antibodies. These antibody fragments were obtained using conventional techniques known to those skilled in the art, and the utility of the fragments was screened in the same manner as for intact antibodies.

[0416] Antibody molecules consist of the complete molecule and its functional fragments. Constant regions of an antibody molecule can be altered, for example by mutating them, to modify antibody properties (e.g., increasing or decreasing one or more of the following: Fc receptor binding, antibody glycosylation, number of cysteine ​​residues, effector cell function, or complement function).

[0417] Antibody molecules can also be single-domain antibodies. Single-domain antibodies can include antibodies whose complementarity-determining regions are part of a single-domain polypeptide. Examples include, but are not limited to, heavy-chain antibodies, naturally occurring antibodies lacking light chains, single-domain antibodies derived from conventional four-chain antibodies, engineered antibodies, and single-domain scaffolds not derived from antibodies. Single-domain antibodies can be any single-domain antibody in the art, or any future single-domain antibody. Single-domain antibodies can be derived from any species, including but not limited to mice, humans, camels, llamas, fish, sharks, goats, rabbits, and cattle. According to another aspect of the invention, a single-domain antibody is a naturally occurring single-domain antibody, referred to as a heavy-chain antibody lacking a light chain. Such a single-domain antibody is disclosed, for example, in WO 9404678. For clarity, such a variable domain derived from a naturally occurring heavy-chain antibody lacking a light chain is referred to herein as a VHH or nanobody to distinguish it from the conventional VH of a four-chain immunoglobulin. Such VHH molecules can be derived from antibodies produced in camelid species such as camels, llamas, dromedary camels, alpacas, and guanacos. Besides camelids, other species may also produce naturally occurring heavy chain antibodies lacking light chains; such VHHs are within the scope of this invention.

[0418] The VH and VL regions can be further subdivided into highly variable regions, known as the "complementary determination region" (CDR), interspersed with more conservative regions, known as the "framework region" (FR or FW).

[0419] The scope of the framework region and CDR has been precisely defined using various methods (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 definition used in Oxford Molecular's AbM antibody modeling software. See also, for example, protein sequence and structure analysis of antibody variable domains ( Protein Sequence and Structure Analysis of Antibody Variable Domains). Published in Antibody Engineering Lab Manual (edited by Duebel, S. and Kontermann, R., Springer-Verlag, Heidelberg).

[0420] As used herein, the terms “complementarity-determining region” and “CDR” refer to the amino acid sequence within the antibody variable region that confers antigen specificity and binding affinity. Typically, 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.

[0421] The precise amino acid sequence boundaries of a given CDR can be determined using any of a number of known schemes, including Kabat et al. (1991), “Sequences of Proteins of Immunological Interest,” 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD (“Kabat” numbering scheme); and Al-Lazikani et al. (1997). JMB The scheme described in 273,927-948 (“Chothia” numbering scheme). As used herein, the CDR defined according to the “Chothia” numbering scheme is sometimes also referred to as a “high-variable ring”.

[0422] 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); while the CDR amino acid residues in the light chain variable domain (VL) are numbered 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3). Under Chothia, the CDR amino acids in VH are numbered 26-32 (HCDR1), 52-56 (HCDR2), and 95-102 (HCDR3); while the amino acid residues in VL are numbered 26-32 (LCDR1), 50-52 (LCDR2), and 91-96 (LCDR3).

[0423] Each VH and VL typically includes three CDRs and four FRs, which are arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0424] Antibody molecules can be polyclonal or monoclonal antibodies.

[0425] As used herein, the term "monoclonal antibody" or "monoclonal antibody composition" refers to a formulation of an antibody molecule consisting of a single molecule. Monoclonal antibody compositions exhibit single binding specificity and affinity for a specific epitope. Monoclonal antibodies can be prepared using hybridoma technology or methods that do not use hybridoma technology (e.g., recombinant methods).

[0426] Antibodies can be generated through recombination, for example, by phage display or by combination methods.

[0427] Methods for phage display and assembly to generate antibodies are known in the art (e.g., in Ladner et al., U.S. Patent No. 5,223,409; Kang et al., International Publication No. WO 92 / 18619; Dower et al., International Publication No. WO91 / 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 The entire contents of 88:7978-7982 are incorporated herein by reference.

[0428] In one embodiment, the antibody is a fully human antibody (e.g., an antibody prepared in mice that have been genetically engineered to produce antibodies from human immunoglobulin sequences) or a non-human antibody, such as antibodies from rodents (mice or rats), goats, primates (e.g., monkeys), or camels. Preferably, the non-human antibody is a rodent antibody (mouse or rat antibody). Methods for producing rodent antibodies are known in the art.

[0429] Human monoclonal antibodies can be generated using transgenic mice carrying human immunoglobulin genes instead of mouse systems. Spleen cells from these transgenic mice, immunized with antigens of interest, are used to generate hybridomas that secrete human mAbs with specific affinity for epitopes of human proteins (see, for example, Wood et al., International Application WO 91 / 00906; Kucherlapati et al., PCT 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. US 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).

[0430] Antibody molecules can be those in which a variable region or a portion thereof, such as a CDR, is generated in a non-human organism, such as a rat or mouse. Chimeric, CDR-transplanted, and humanized antibodies are within the scope of this invention. Antibody molecules generated in a non-human organism, such as a rat or mouse, and then modified, for example, in a variable or constant region to reduce antigenicity in humans are also within the scope of this invention.

[0431] "Effective human" proteins are those that do not substantially elicit a neutralizing antibody response, such as a human anti-mouse antibody (HAMA) response. In many cases, such as if antibody molecules are repeatedly administered, for example, to treat chronic or relapsing diseases, HAMA can become problematic due to increased antibody clearance from the serum (see, for example, Saleh et al.). Cancer Immunol. Immunother. , 32:180-190 (1990)), and also due to potential allergic reactions (see, for example, LoBuglio et al., Hybridoma, 5:5117-5123 (1986)), HAMA response may render repeated antibody administration ineffective.

[0432] Chimeric antibodies can be produced using recombinant DNA techniques known in the art (see Robinson et al., International Patent 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., US Patent No. 4,816,567; Cabilly et al., European Patent Application No. 125,023; Better et al., (1988) Science 240:1041-1043); Liu et al., (1987) PNAS 84:3439-3443; Liu et al., 1987, J. Immunol. 139:3521-3526; Sun et al., (1987) PNAS 84:214-218; Nishimura et al., 1987, Canc. Res. 47:999-1005; Wood et al., (1985) Nature 314:446-449; and Shaw et al., 1988, J. Natl Cancer Inst. 80:1553-1559).

[0433] The humanized or CDR-transplanted antibody will have at least one or two (of the immunoglobulin heavy chain and / or light chain), but typically all three receptor CDRs are replaced by donor CDRs. The antibody may be replaced by at least a portion of a non-human CDR, or only some CDRs may be replaced by non-human CDRs. Only the number of CDRs required for antigen binding needs to be replaced. Preferably, the donor will be a rodent antibody, such as a rat or mouse antibody, and the receptor will be a human or shared human framework. Typically, the immunoglobulin providing the CDR is referred to as the "donor," and the immunoglobulin providing the framework is referred to as the "receptor." In one embodiment, the donor immunoglobulin is non-human (e.g., rodent). The receptor framework is a naturally occurring (e.g., human) framework or shared framework, or a sequence having about 85% or higher, preferably 90%, 95%, 99%, or higher, identity with it.

[0434] As used herein, the term "shared sequence" refers to a sequence formed by the most frequently occurring amino acid (or nucleotide) in a related sequence family (see, for example, Winnaker, From Genes to Clones (Verlagsgesellschaft, Weinheim, Germany 1987)). In a protein family, each position in the shared sequence is occupied by the most frequently occurring amino acid at that position in that family. If two amino acids occur with equal frequency, either one can be included in the shared sequence. "Shared framework" refers to the framework region in a shared immunoglobulin sequence.

[0435] Antibody molecules can be humanized using methods known in the art (see, for example, Morrison, SL, 1985). Science 229:1202-1207; Oi et al., 1986, BioTechniques 4:214 and Queen et al., US 5,585,089, US 5,693,761 and US 5,693,762, the entire contents of which are incorporated herein by reference.

[0436] Humanized or CDR-transplanted antibody molecules can be generated through CDR transplantation or CDR replacement, in which one, two, or all of the CDRs of the immunoglobulin chain can be replaced. See, for example, U.S. Patent 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 US 5,225,539, the entire contents of which are expressly incorporated herein by reference. Winter describes a CDR transplantation method that can be used to prepare the humanized antibodies of the present invention (UK patent application GB 2188638A filed March 26, 1987; Winter US 5,225,539), the contents of which are expressly incorporated herein by reference.

[0437] Humanized antibody molecules are also within the scope of this invention, wherein specific amino acids have been substituted, deleted, or added. A standard description of the selection of amino acids from donors is found in US 5,585,089, for example, in columns 12-16 of US 5,585,089, the contents of which are incorporated herein by reference. Other techniques for humanizing antibodies are described in Padlan et al., EP 519596 A1, published December 23, 1992.

[0438] Antibody molecules can be single-chain antibodies. Single-chain antibodies (scFVs) can be engineered (e.g., see 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 polymerized to produce multivalent antibodies specific to different epitopes of the same target protein.

[0439] In other embodiments, the antibody molecule has a heavy chain constant region selected from, for example, the heavy chain constant regions of IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE; particularly selected from, for example, the heavy chain constant regions of IgG1, IgG2, IgG3, and IgG4 (e.g., human). In another embodiment, the antibody molecule has a light chain constant region selected from, for example, κ or λ (e.g., human). The constant region can be altered, for example, by mutation, to modify the properties of the antibody (e.g., increasing or decreasing one or more of the following: Fc receptor binding, antibody glycosylation, number of cysteine ​​residues, effector cell function, and / or complement function). In one embodiment, the antibody has: effector function; and can repair complement. In other embodiments, the antibody does not: recruit effector cells; or repair complement. In another embodiment, the antibody has a reduced ability to bind to the Fc receptor or no ability to bind to the Fc receptor. For example, it is an isotype or subtype, fragment or other mutant that does not support binding to the Fc receptor, for example, it has a mutagenic or missing Fc receptor binding region.

[0440] Methods for altering the constant region of an antibody are known in the art. Antibodies with altered functions (e.g., 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 region of the antibody with a different residue (see, for example, EP 388,151 A1, U.S. Patent Nos. 5,624,821 and 5,648,260, the entire contents of which are incorporated herein by reference). Similar types of alterations can be described that, if applied to mice or other species, would reduce or eliminate these functions of the immunoglobulin.

[0441] Antibody molecules can be derived or linked to another functional molecule (e.g., another peptide or protein). As used herein, a “derived” antibody molecule is an antibody molecule that has been modified. Derivatization methods include, but are not limited to, the addition of a fluorescent moiety, a radioactive nucleotide, a toxin, an enzyme, or an affinity ligand such as biotin. Therefore, the antibody molecules of the present invention are intended to include the derived forms of antibodies described herein and other modified forms, including immunoadhesion molecules. For example, antibody molecules can be functionally linked (by chemical coupling, genetic fusion, non-covalent association, or other means) to one or more other molecular entities, such as another antibody (e.g., a bispecific antibody or a biantibody), a detectable reagent, a cytotoxic agent, a pharmaceutical reagent, and / or a protein or peptide (e.g., a streptavidin core region or a multihistidine tag) that can mediate the binding of an antibody or antibody moiety to another molecule.

[0442] One type of derived antibody molecule is produced by crosslinking two or more antibodies (of the same or different types, for example, to generate bispecific antibodies). Suitable crosslinking agents include heterobifunctional crosslinking agents having two distinct reactive groups separated by appropriate spacer groups (e.g., m-maleimide benzoyl-N-hydroxysuccinimide ester), or homobifunctional crosslinking agents (e.g., disuccinimide octanoate). Such linkers are available from Pierce Chemical Company, Rockford, Ill.

[0443] Multispecific or multifunctional antibody molecules The exemplary structures of the multispecific and multifunctional molecules defined herein are described in full. Further exemplary structures are described in the following reference: 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 which are incorporated herein by reference.

[0444] In one embodiment, a multispecific antibody molecule may contain more than one antigen-binding site, wherein different sites are specific to different antigens. In another embodiment, a multispecific antibody molecule may bind to more than one (e.g., two or more) epitopes on the same antigen. In yet another embodiment, a multispecific antibody molecule contains an antigen-binding site specific to target cells (e.g., lymphocytes (e.g., T cells) containing TCRAV antigens corresponding to a biased TCRAV clonal type) and different antigen-binding sites specific to immune effector cells. In one embodiment, a multispecific antibody molecule is a bispecific antibody molecule. Bispecific antibody molecules can be divided into five different structural groups: (i) bispecific immunoglobulin G (BsIgG); (ii) IgG with an additional antigen-binding moiety attached; (iii) bispecific antibody fragments; (iv) bispecific fusion proteins; and (v) bispecific antibody conjugates.

[0445] BsIgG is a monovalent form for each antigen. Exemplary forms of BsIgG include, but are not limited to, crossMab, DAF (dual-antigen), DAF (quadrivalent-antigen), DutaMab, DT-IgG, common LC with a club-and-mortar structure, club-and-mortar assembly, charge pairs, Fab arm exchange, SEEDbody, triomab, LUZ-Y, Fcab, κλ antibody, and orthogonal Fab. See Spiess et al., Mol. Immunol. 67(2015):95-106. Exemplary BsIgGs include catumaxomab (Fresenius Biotech, Trion Pharma, Neopharm), which comprises an anti-CD3 arm and an anti-EpCAM arm; and ertumaxomab (Neovii Biotech, Fresenius Biotech), which targets CD3 and HER2. In some embodiments, BsIgG comprises a heavy chain engineered for heterodimerization. For example, heavy chains can be engineered for heterodimerization using a "mortar and pestle" strategy, a SEED platform, common heavy chains (e.g., in kλ antibodies), and the use of heterodimeric Fc regions. See Spiess et al., Mol. Immunol. 67(2015):95-106. Strategies to avoid heavy chain pairing in homodimers of BsIgG include mortar and pestle structures, duobody, azymetric, charge pairs, HA-TF, SEEDbody, and differential A protein affinity. See previous literature. BsIgG can be generated by expressing component antibodies separately in different host cells and then purifying / assembling them into BsIgG. BsIgG can also be generated by expressing component antibodies in a single host cell. BsIgG can be purified using affinity chromatography, for example, using A protein and sequential pH elution.

[0446] IgG with an additional antigen-binding motif is another form of bispecific antibody molecule. For example, monospecific IgG can be engineered to be bispecific by attaching an additional antigen-binding unit to the monospecific IgG (e.g., at the N-terminus or C-terminus of the heavy or light chain). Exemplary additional antigen-binding units include single-domain antibodies (e.g., variable heavy or light chains), engineered protein scaffolds, and paired antibody variable domains (e.g., single-chain variable fragments or variable fragments). See previous literature. Examples of attached IgG forms 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 (quadruple). See Spiess et al., Mol. Immunol. 67(2015):95-106. An example of IgG-scFv is MM-141 (Merrimack Pharmaceuticals), which binds IGF-1R and HER3. Examples of DVD-Ig include ABT-981 (AbbVie), which binds IL-1α and IL-1β; and ABT-122 (AbbVie), which binds TNF and IL-17A.

[0447] Bispecific antibody fragments (BsAbs) are forms of bispecific antibody molecules lacking some or all of the antibody constant domains. For example, some BsAbs lack the Fc region. In embodiments, bispecific antibody fragments include heavy and light chain regions linked by peptide linkers that allow the BsAb to be efficiently expressed in a single host cell. Exemplary bispecific antibody fragments include, but are not limited to, nanobodies, nanobodies-HAS, BiTEs, biantibodies, DART, TandAbs, sc biantibodies, sc biantibodies-CH3, biantibodies-CH3, triple bodies, microbodies, microbodies, TriBi microbodies, scFv-CH3 KIH, Fab-scFv, scFv-CH-CL-scFv, F(ab')2, F(ab')2-scFv2, scFv-KIH, Fab-scFv-Fc, tetravalent HCAbs, sc biantibodies-Fc, biantibodies-Fc, tandem scFv-Fc, and intracellular antibodies. See previous literature. For example, the BiTE form includes tandem scFv, in which the scFv component binds to CD3 on T cells and surface antigens on cancer cells.

[0448] Bispecific fusion proteins include antibody fragments linked to other proteins, for example, to increase additional specificity and / or function. An example of a bispecific fusion protein is immTAC, which contains an anti-CD3 scFv linked to a mature T-cell receptor with affinity for recognizing an HLA-presenting peptide. In embodiments, a dock-and-lock (DNL) approach can be used to generate bispecific antibody molecules with higher valence states. Furthermore, fusions with albumin-binding proteins or human serum albumin can prolong the serum half-life of the antibody fragment. See previous literature.

[0449] In embodiments, chemical conjugation, such as chemical conjugation of antibodies and / or antibody fragments, can be used to generate BsAb molecules. See previous literature. Exemplary bispecific antibody conjugates include CovX forms, in which a low molecular weight drug is specifically conjugated to a single reactive lysine at a site in each Fab arm or antibody or fragment thereof. In embodiments, the conjugation improves the serum half-life of the low molecular weight drug. An exemplary CovX form is CVX-241 (NCT01004822), which comprises antibodies conjugated to two short peptides that inhibit VEGF or Ang2. See previous literature.

[0450] Antibody molecules can be generated by recombinant expression of, for example, 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 generated by individual expression of the components in different host cells followed by purification / assembly. Alternatively, antibody molecules can be generated by expressing the components in a single host cell. Purification of bispecific antibody molecules can be performed using various methods, such as affinity chromatography, for example, using protein A and continuous pH elution. In other embodiments, affinity tags can be used for purification, such as histidine-containing tags, myc tags, or streptavidin tags.

[0451] CDR implantation scaffold In the implementation scheme, the antibody molecule is a scaffold domain for CDR transplantation. In the implementation scheme, the scaffold domain is based on a fibronectin domain, such as the fibronectin type III domain. The overall folding of the fibronectin type III (Fn3) domain is closely related to the folding of the smallest functional antibody fragment (the variable domain of the antibody heavy chain). Fn3 has three loops at the end; the positions of the BC, DE, and FG loops roughly correspond to the positions of CDR1, 2, and 3 of the antibody's VH domain. Fn3 lacks disulfide bonds; therefore, unlike the antibody and its fragments, Fn3 is stable under reducing conditions (see, for example, WO 98 / 56915; WO 01 / 64942; WO 00 / 34784). The Fn3 domain can be modified (e.g., using the CDRs or hypervariable loops described herein) or altered, for example, to select a domain that binds to the antigen / marker / cell described herein.

[0452] In implementations, the scaffold domain, such as the folded domain, is an antibody-based “microantibody” scaffold, for example, generated by deleting three β chains from the heavy chain variable domain of a monoclonal antibody (see, e.g., Tramontano et al., 1994, J Mol. Recognit. 7:9; and Martin et al., 1994, EMBO J. 13:5303-5309). The “microantibody” can be used to present two hypervariable loops. In implementations, the scaffold domain is a V-like domain (see, e.g., Coia et al., WO99 / 45110) or a domain derived from tendamistatin, which is a 74-residue six-chain β-sheet sandwich structure held together by two disulfide bonds (see, e.g., McConnell and Hoess, 1995, J Mol. Biol. 250:460). For example, the loop of tendamistatin can be modified or altered (e.g., using CDR or hypervariable loops) to select a domain that binds to the marker / antigen / cell described herein. Another exemplary scaffold domain is a β-sandwich structure derived from the extracellular domain of CTLA-4 (see, for example, WO 00 / 60070).

[0453] 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, coliforms, BPTI, etc.); TPR repeats; trifoil structures; zinc finger domains; DNA-binding proteins; particularly monomeric DNA-binding proteins; RNA-binding proteins; enzymes, such as proteases (particularly inactivated proteases), RNases; protein chaperone molecules, such as thioredoxins and heat shock proteins; and intracellular signaling domains (e.g., SH2 and SH3 domains). See, for example, US 20040009530 and US7,501,121, which are incorporated herein by reference.

[0454] In the implementation scheme, the scaffold domain is evaluated and selected, for example, by one or more of the following criteria: (1) amino acid sequence, (2) sequences of several homologous domains, (3) 3D structure, and / or (4) stability data over a range of pH, temperature, salinity, organic solvents, and oxidant concentrations. In the implementation scheme, the scaffold domain is a small, stable protein domain, for example, in proteins with fewer than 100, 70, 50, 40, or 30 amino acids. The domain may include one or more disulfide bonds or may chelate a metal, such as zinc.

[0455] Antibody-based fusion Various forms can be generated, including additional binding entities attached to the N or C terminus of the antibody. These fusions with Fv or Fab stabilized by single-chain or disulfide bonds result in tetravalent molecules with bivalent binding specificity for each antigen. The binding of scFv and scFab to IgG can produce molecules that recognize three or more different antigens.

[0456] Antibody-Fab fusion Antibody-Fab fusions are bispecific antibodies comprising a conventional antibody targeting a first target fused to the C-terminus of the antibody heavy chain, and a Fab targeting a second target. Typically, the antibody and Fab will share a common light chain. Antibody fusions can be generated in the following ways: （1） The DNA sequence of the target fusion compound was engineered, and （2） Target DNA is transfected into suitable host cells to express fusion proteins. For example, Coloma, J. et al. (1997). Nature Biotech As described in 15:159, it appears that the antibody-scFv fusion can be linked via a (Gly)-Ser linker between the C-terminus of the CH3 domain and the N-terminus of the scFv.

[0457] Antibody-scFv fusion Antibody-scFv fusionIt is a bispecific antibody, comprising a conventional antibody fused to the C-terminus of the antibody heavy chain and an scFv with unique specificity. The scFv can be fused to the C-terminus directly via the scFv heavy chain or via a linker peptide. Antibody fusions can be generated in the following ways: （1） The DNA sequence of the target fusion compound was engineered, and （2） Target DNA is transfected into suitable host cells to express fusion proteins. For example, Coloma, J. et al. (1997). Nature Biotech As described in 15:159, it appears that the antibody-scFv fusion can be linked via a (Gly)-Ser linker between the C-terminus of the CH3 domain and the N-terminus of the scFv.

[0458] Variable Domain Immunoglobulin DVD One related form is the dual variable domain immunoglobulin (DVD), which consists of the VH and VL domains at a second specific position at the N-terminus of the V domain via a shorter linker sequence.

[0459] Other exemplary forms of multispecific antibodies include, for example, those described in the following patents: US20160114057A1, US20130243775A1, US20140051833, US20130022601, US20150017187A1, US20120201746A1, US20150133638A1, US20130266568A1, US20160145340A1, WO2015127158A1, US20150203591A1, US2014 0322221A1, US20130303396A1, US20110293613, US20130017200A1, US20160102135A1, WO2015197598A2, WO2015197582A1, US9359437, US20150018529, WO2016115274A1, WO2016087416A1, US20080069820A1, US9145588B, US7919257 and US20150232560A1. Exemplary multispecific molecules in the form of complete antibody-Fab / scFab include those described in the following patents: US9382323B2, US20140072581A1, US20140308285A1, US20130165638A1, US20130267686A1, US20140377269A1, US7741446B2, and WO1995009917A1. Exemplary multispecific molecules employing domain exchange include those described in the following patents: US20150315296A1, WO2016087650A1, US20160075785A1, WO2016016299A1, US20160130347A1, US20150166670, US8703132B2, US20100316645, US8227577B2, and US20130078249.

[0460] Entities containing Fc (microantibodies) Fc-containing entities (also known as microantibodies) can be generated by fusing scFv with the C-terminus of a constant heavy chain domain 3 (CH3-scFv) and / or with a hinge region of an antibody with different specificity (scFv-hinge-Fc). Trivalent entities with a disulfide-stabilized variable domain (without peptide linkers) fused to the C-terminus of the CH3 domain of IgG can also be prepared.

[0461] Fc-containing multispecific molecules In some embodiments, the multispecific molecules disclosed herein include immunoglobulin constant regions (e.g., Fc regions). Exemplary Fc regions may 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.

[0462] In some implementations, the constant region of the immunoglobulin chain (e.g., the Fc region) is altered, for example by mutating it, to increase or decrease one or more of the following: Fc receptor binding, antibody glycosylation, number of cysteine ​​residues, effector cell function, or complement function.

[0463] In other embodiments, the interface between the first and second immunoglobulin chain constant regions (e.g., the first and second Fc regions) is altered (e.g., mutated) to increase or decrease dimerization, for example, relative to an unengineered interface (e.g., a naturally occurring interface). For instance, dimerization of the immunoglobulin chain constant regions (e.g., the Fc regions) can be enhanced by providing one or more of the following to the Fc interface between the first and second Fc regions: paired protrusion-cavity structures (“mortar and pestle structures”), electrostatic interactions, or chain exchange, such that, for example, the ratio of heteropolymers to homopolymers is greater relative to an unengineered interface.

[0464] In some embodiments, the multispecific molecule includes paired amino acid substitutions at one or more positions selected from, for example, 347, 349, 350, 351, 366, 368, 370, 392, 394, 395, 397, 398, 399, 405, 407, or 409 of the Fc region of human IgG1. For example, the immunoglobulin chain constant region (e.g., the Fc region) may include paired amino acid substitutions selected from: T366S, L368A, or Y407V (e.g., corresponding to a cavity or mortis) and T366W (e.g., corresponding to a protrusion or pestle).

[0465] In other embodiments, the multifunctional molecule includes a half-life extender, such as human serum albumin, or an antibody molecule targeting human serum albumin.

[0466] Heterodimerized antibody molecules and their preparation methods Various methods for generating multispecific antibodies to address the problem of incorrect heavy chain pairing have been disclosed. Exemplary methods are described below. Exemplary forms of multispecific antibodies and methods for preparing said multispecific antibodies are also disclosed, for example, in Speiss et al., Molecular Immunology 67 (2015) 95–106; and Klein et al., mAbs 4:6, 653–663; November / December 2012; the entire contents of each of these are incorporated herein by reference.

[0467] Heterodimerized bispecific antibodies are based on the natural IgG structure, where the two binding arms recognize different antigens. Forced heavy chain heterodimerization, combined with techniques that minimize light chain (e.g., common light chain) mismatch, produces IgG-derived forms capable of achieving defined monovalent (and simultaneous) antigen binding. Forced heavy chain heterodimerization can be achieved using, for example, a pestle-and-mortar structure or a chain exchange engineered domain (SEED).

[0468] mortar and pestle structure The pestle and mortar structure is described in US 5,731,116, US 7,476,724, and Ridgway, J. et al. (1996). Prot. Engineering As described in 9(7): 617-621, generally speaking, it involves: （1） (1) Mutating the CH3 domain of one or two antibodies to promote heterodimerization; and (2) combining the mutated antibodies under conditions that promote heterodimerization. A "pestle" or "protrusion" is typically generated by replacing a small amino acid in the parent antibody with a larger amino acid (e.g., T366Y or T366W); a "mortar" or "cavity" is generated by replacing a larger residue in the parent antibody with a smaller amino acid (e.g., Y407T, T366S, L368A, and / or Y407V).

[0469] For bispecific antibodies including an Fc domain, proper heterodimerization of the Fc moiety can be facilitated by introducing specific mutations into the constant region of the heavy chain. Several such techniques are reviewed in Klein et al., (mAbs (2012) 4:6, 1-11), the contents of which are incorporated herein by reference in their entirety. These techniques include the "mortar and pestle" (KiH) approach, which involves introducing a bulky residue into a CH3 domain of an antibody heavy chain. This bulky residue coordinates to a complementary "mortar" in another CH3 domain of the paired heavy chain, thereby facilitating proper pairing of the heavy chains (see, for example, US7642228).

[0470] Exemplary KiH mutations include S354C and T366W in the "grossroots" heavy chain and Y349C, T366S, L368A, and Y407V in the "mortar" heavy chain. Table 1 provides other exemplary KiH mutations, as well as additional optional stabilizing Fc cysteine ​​mutations.

[0471] Table 1. Exemplary Fc KiH mutations and optional cysteine ​​mutations

[0472] Igawa and Tsunoda provided other Fc mutations, identifying three negatively charged residues in the CH3 domain of one strand that pair with three positively charged residues in the CH3 domain of the other strand. These specific charged residue pairs are: E356-K439, E357-K370, D399-K409, and vice versa. Introducing at least two of the following three mutations—E356K, E357K, and D399K—in chain A, alone or in combination with a newly discovered disulfide bridge, and at least two of the following three mutations—K370E, K409D, and K439E—in chain B promotes highly efficient heterodimerization while inhibiting 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, and then screened for the maximum heterodimerization, defining the combination of S364H and F405A (HA) on chain A and Y349T and T394F (TF) on chain B (Moore GL et al., Anovel bispecific antibody format enables simultaneous bivalent and monovalent co-engagement of distinct target antigens. MAbs 2011; 3:546-57; PMID: 22123055).

[0473] Other exemplary Fc mutations that promote heterodimerization of multispecific antibodies include those described in the following references, the contents of which are incorporated herein by reference: WO2016071377A1, US20140079689A1, US20160194389A1, US20160257763, WO2016071376A2, WO2015107026A1, WO2015107025A1, WO2015107015A1, US20150353636A1, US20140199294A1, US7750128B2, US201602299 15A1, US20150344570A1, US8003774A1, US20150337049A1, US20150175707A1, US20140242075A1, US20130195849A1, US20120149876A1, US20140200331A1, US9309311B2, US8586713, US20140037621A1, US20130178605A1, US20140363426A1, US20140051835A1, and US20110054151A1.

[0474] Stabilizing cysteine ​​mutations have also been used in combination with KiH and other variants that promote Fc heterodimerization, see, for example, US7183076. Other exemplary cysteine ​​modifications include, for example, those disclosed in US20140348839A1, US7855275B2, and US9000130B2.

[0475] Chain-switched engineered architecture (SEED) The heterodimer Fc platform, which supports the design of bispecific and asymmetric fusion proteins, is known by designing strand-exchange engineered domain (SEED)C(H)3 heterodimers. These derivatives of human IgG and IgA C(H)3 domains can form complementary human SEED C(H)3 heterodimers, which consist of alternating segments of human IgA and IgG C(H)3 sequences. When expressed in mammalian cells, the resulting pair of SEED C(H)3 domains preferentially associate to form heterodimers. The SEEDbody (Sb) fusion protein consists of [IgG1 hinge]-C(H)2-[SEED C(H)3] and can be genetically linked to one or more fusion partners (e.g., see Davis JH et al., SEEDbodies: fusion proteins based on strandexchange engineered domain (SEED) CH3 heterodimers in an Fc analogue platform for asymmetric binders or immunofusions and bispecific antibodies. ProteinEng Des Sel 2010; 23:195-202; PMID: 20299542 and US8871912. The contents of each of these are incorporated herein by reference).

[0476] Duobody The “Duobody” technique for generating bispecific antibodies with the correct heavy chain pairing is known. The DuoBody technique involves three basic steps for generating stable bispecific human IgG1 antibodies in a post-production exchange reaction. In the first step, two IgG1s are produced separately using a standard mammalian recombinant cell line, each containing a single matched mutation in the third constant (CH3) domain. These IgG1 antibodies are then purified according to standard methods for recovery and purification. After production and (post-production) purification, the two antibodies are recombined under custom laboratory conditions to produce bispecific antibody products in very high yields (typically >95%) (see, for example, Labrijn et al., PNAS 2013;110(13):5145-5150 and Labrijn et al., Nature Protocols 2014;9(10):2450-63, the contents of which are incorporated herein by reference).

[0477] electrostatic interaction Methods for preparing multispecific antibodies using charged amino acids and CH3 amino acid variations are disclosed, making homodimer formation electrostatically unfavorable. EP1870459 and WO 2009089004 describe other strategies that favor heterodimer formation when co-expressing different antibody domains in host cells. In these methods, replacing one or more residues in the heavy chain constant domain 3 (CH3) and the CH3-CH3 interface of two CH3 domains with charged amino acids makes homodimer formation electrostatically unfavorable and heterodimerization electrostatically favorable. Other methods for preparing multispecific molecules using electrostatic interactions are described in the following references, the contents of which are incorporated herein by reference, including US20100015133, US8592562B2, US9200060B2, US20140154254A1, and US9358286A1.

[0478] Common light chain Homogeneous formulations that avoid light chain mismatches to generate bispecific IgG are required. One way to achieve this is by using the principle of shared light chains, which involves combining two conjugates that share a light chain but still have different specificities. An exemplary method to enhance the formation of the desired bispecific antibody from a mixture of monomers is to provide a shared variable light chain to interact with each heteropolymer variable heavy chain region of the bispecific antibody. Compositions and methods for generating bispecific antibodies with a shared light chain are disclosed, for example, in US7183076B2, US20110177073A1, EP2847231A1, WO2016079081A1, and EP3055329A1, the contents of which are incorporated herein by reference.

[0479] CrossMab Another option for reducing light chain mismatch is the CrossMab technique, which avoids nonspecific L-chain mismatch by exchanging the CH1 and CL domains in the Fab of half of the bispecific antibody. Such a cross-linked variant retains binding specificity and affinity but makes the two arms so different that L-chain mismatch is prevented. CrossMab techniques (as outlined above by Klein et al.) involve domain swapping between the heavy and light chains to facilitate the formation of correct pairings. In short, a two-step modification process is employed to construct bispecific IgG-like CrossMab antibodies capable of binding to two antigens using two different light-heavy chain pairs. First, a dimerization interface is engineered to the C-terminus of each heavy chain using a heterodimerization method (e.g., the KiH technique) to ensure that only heterodimers from the two different heavy chains of one antibody (e.g., antibody A) and the second antibody (e.g., antibody B) are effectively formed. Next, the constant heavy chain 1 domain (CH1) and constant light chain domain (CL) of one antibody (antibody A) are exchanged, while the variable heavy chain (VH) and variable light chain (VL) domains remain unchanged. The exchange of the CH1 and CL domains ensures that the modified antibody (antibody A) light chain can only effectively dimerize with the modified antibody (antibody A) heavy chain, and the unmodified antibody (antibody B) light chain can only effectively dimerize with the unmodified antibody (antibody B) heavy chain; thus only the desired bispecific CrossMab is effectively formed (see, for example, Cain, C. SciBX 4(28); doi: 10.1038 / scibx.2011.783, the contents of which are incorporated herein by reference).

[0480] Common heavy chain An exemplary method for enhancing the formation of a desired bispecific antibody from a mixture of monomers is to provide a common variable heavy chain that interacts with each heteropolymer variable light chain region of the bispecific antibody. Compositions and methods for generating bispecific antibodies having a common heavy chain are disclosed, for example, in US20120184716, US20130317200, and US20160264685A1, the contents of which are incorporated herein by reference.

[0481] Amino acid modification Alternative compositions and methods for generating multispecific antibodies with the correct light chain pairings include various amino acid modifications. For example, Zymeworks describes heterodimers having one or more amino acid modifications in the CH1 and / or CL domains, one or more amino acid modifications in the VH and / or VL domains, or combinations thereof, which are part of the interface between the light and heavy chains and establish preferential pairing between each heavy chain and the desired light chain, such that when the two heavy chains and two light chains of a heterodimer pair are co-expressed in cells, the heavy chain of the first heterodimer preferentially pairs with one light chain rather than with each other (e.g., see WO2015181805). Other exemplary methods are described in WO2016026943 (Argen-X), US20150211001, US20140072581A1, US20160039947A1, and US20150368352.

[0482] λ / κ form Multispecific molecules comprising λ light chain peptides and κ light chain peptides (e.g., multispecific antibody molecules) can be used to allow heterodimerization. A method for generating bispecific antibody molecules comprising λ light chain peptides and κ light chain peptides is disclosed in PCT / US 17 / 53053, filed September 22, 2017, which is incorporated herein by reference in its entirety.

[0483] In the embodiments, multispecific molecules include multispecific antibody molecules, such as antibody molecules having two binding specificities, for example, bispecific antibody molecules. Multispecific antibody molecules include: λ light chain polypeptide 1 (LLCP1) is specific to the first epitope. Heavy chain polypeptide 1 (HCP1) is specific to the first epitope. κ light chain polypeptide 2 (KLCP2) is specific to the second epitope; and Heavy chain polypeptide 2 (HCP2) is specific to the second epitope.

[0484] As used herein, the term "λ light chain polypeptide 1 (LLCP1)" refers to a polypeptide containing a sufficient light chain (LC) sequence such that, upon binding to the homologous heavy chain variable region, it can mediate specific binding to its epitope and complexation with HCP1. In one embodiment, LLCP1 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 sequences therefrom sufficient to mediate specific binding to its epitope and complexation with HCP1. LLCP1, together with its HCP1, provides specificity for the first epitope (while KLCP2, together with its HCP2, provides specificity for the second epitope). As described elsewhere herein, LLCP1 has a higher affinity for HCP1 than for HCP2.

[0485] As used herein, the term "κ light chain polypeptide 2 (KLCP2)" refers to a polypeptide containing a sufficient light chain (LC) sequence such that, upon binding to the homologous heavy chain variable region, it can mediate specific binding to its epitope and complexation with HCP2. In some embodiments, KLCP2 contains all or a fragment of the CH1 region. In one embodiment, KLCP2 contains LC-CDR1, LC-CDR2, LC-CDR3, FR1, FR2, FR3, FR4, and CH1, or sequences therefrom sufficient to mediate specific binding to its epitope and complexation with HCP2. KLCP2, together with its HCP2, provides specificity for the second epitope (while LLCP1 and its HCP1 provide specificity for the first epitope).

[0486] As used herein, the term "heavy chain polypeptide 1 (HCP1)" refers to a polypeptide containing sufficient heavy chain (HC) sequence (e.g., HC variable region sequence) such that, upon binding to homologous LLCP1, it can mediate specific binding to its epitope and complexation with HCP1. In some embodiments, HCP1 comprises all or a fragment of the CH1 region. In one embodiment, HCP1 comprises all or a fragment of the CH2 and / or CH3 regions. In one embodiment, HCP1 comprises HC-CDR1, HC-CDR2, HC-CDR3, FR1, FR2, FR3, FR4, CH1, CH2, and CH3, or sequences therefrom sufficient to achieve: (i) mediating specific binding to its epitope and complexation with LLCP1; (ii) preferentially complexing with LLCP1 rather than KLCP2 as described herein; and (iii) preferentially complexing with HCP2 rather than another HCP1 molecule as described herein. HCP1, together with its LLCP1, provides specificity for the first epitope (while KLCP2, together with its HCP2, provides specificity for the second epitope).

[0487] As used herein, the term "heavy chain polypeptide 2 (HCP2)" refers to a polypeptide containing sufficient heavy chain (HC) sequence (e.g., HC variable region sequence) such that, upon binding to homologous LLCP1, it can mediate specific binding to its epitope and complexation with HCP1. In some embodiments, HCP2 comprises all or a fragment of the CH1 region. In some embodiments, HCP2 comprises all or a fragment of the CH2 and / or CH3 regions. In one embodiment, HCP1 comprises HC-CDR1, HC-CDR2, HC-CDR3, FR1, FR2, FR3, FR4, CH1, CH2, and CH3, or sequences therefrom sufficient to achieve: (i) mediating specific binding to its epitope and complexation with KLCP2; (ii) preferentially complexing with KLCP2 rather than LLCP1 as described herein; and (iii) preferentially complexing with HCP1 rather than another HCP2 molecule as described herein. HCP2, together with KLCP2, provides specificity for the second epitope (while LLCP1, together with HCP1, provides specificity for the first epitope).

[0488] In some implementation schemes 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.

[0489] In the implementation scheme, LLCP1 has a sufficiently high affinity for HCP1 to be greater than its affinity for HCP2, such that under preselected conditions, such as in an aqueous buffer (e.g., at pH 7), 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 LLCP1 complexed with or bound to HCP1.

[0490] In some implementation schemes of the multispecific antibody molecules disclosed herein: HCP1 has a greater affinity for HCP2 than for the second molecule of HCP1; and / or HCP2 has a greater affinity for HCP1 than for the second molecule of HCP2.

[0491] In the implementation scheme, HCP1 has a sufficiently greater affinity for HCP2 than for a second HCP1 molecule, such that under preselected conditions, such as in an aqueous buffer (e.g., at pH 7), 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 with or bound to HCP2.

[0492] On the other hand, this article discloses a method for preparing or generating multispecific antibody molecules. This method includes the association conditions of (i)-(iv): (i) Provide 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, and a first heavy chain constant region (e.g., first CH2, first CH3, or both)). (ii) Provide 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, and a second heavy chain constant region (e.g., second CH2, second CH3, or both)). (iii) Providing a λ-chain polypeptide (e.g., a λ-light variable region (VLλ), a λ-light constant chain (VLλ), or both) that preferentially associates with the first heavy chain polypeptide (e.g., the first VH); and (iv) Provide a κ-chain polypeptide (e.g., κ light variable region (VLκ), κ light constant chain (VLκ) or both) that preferentially associates with the second heavy chain polypeptide (e.g., second VH).

[0493] In the implementation scheme, the first heavy chain and the second heavy chain polypeptide form an Fc interface that enhances heterodimerization.

[0494] In one implementation, (i)-(iv) (e.g., nucleic acids encoding (i)-(iv)) is introduced into a single cell, such as a single mammalian cell, such as a CHO cell. In another implementation, (i)-(iv) is expressed in the cell.

[0495] In the implementation, (i)-(iv) (e.g., nucleic acids encoding (i)-(iv)) is introduced into different cells, such as different mammalian cells, such as two or more CHO cells. In the implementation, (i)-(iv) is expressed in the cells.

[0496] In one embodiment, the method further includes, for example, using λ-specific and / or κ-specific purification, such as affinity chromatography, to purify the cell-expressed antibody molecules.

[0497] In one embodiment, the method further includes evaluating multispecific antibody molecules expressed by cells. For example, purified multispecific antibody molecules expressed by cells can be analyzed using techniques known in the art, including mass spectrometry. In one embodiment, purified antibody molecules expressed by cells are lysed, for example, digested with papain to produce a Fab moiety, and evaluated using mass spectrometry.

[0498] In the implementation scheme, the method produces correctly paired κ / λ multispecific (e.g., bispecific) antibody molecules in high yields, such as at least 75%, 80%, 90%, 95%, 98%, 99%, 99.5%, or 99.9%.

[0499] In other embodiments, multispecific (e.g., bispecific) antibody molecules include: (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, and a first heavy chain constant region (e.g., first CH2, first CH3 or both), for example, wherein HCP1 binds to a first epitope; (ii) Second heavy chain polypeptide (HCP2) (e.g., a heavy chain polypeptide containing one, two, three or all of the second heavy chain variable region (second VH), second CH1, and second heavy chain constant region (e.g., second CH2, second CH3 or both), for example, wherein HCP2 binds to a second epitope; (iii) A λ light chain polypeptide (LLCP1) (e.g., λ light variable region (VL1), λ light constant chain (VL1), or both) that preferentially associates with a first heavy chain polypeptide (e.g., a first VH), for example, wherein LLCP1 binds to a first epitope; and (iv) A κ light chain polypeptide (KLCP2) (e.g., κ light variable region (VLk), κ light constant chain (VLk) or both) that preferentially associates with a second heavy chain polypeptide (e.g., second VH), for example, wherein KLCP2 binds to a second epitope.

[0500] In one embodiment, the first heavy chain polypeptide and the second heavy chain polypeptide form an Fc interface that enhances heterodimerization. In another embodiment, the multispecific antibody molecule has a first binding specificity and a second binding specificity, the first binding specificity comprising a hybrid VL1-CL1 heterodimerized to a first heavy chain variable region (with mortar modification) connected to the constant CH2-CH3 domain of the Fc, and the second binding specificity comprising a hybrid VLk-CLk heterodimerized to a second heavy chain variable region (with mortar modification) connected to the constant CH2-CH3 domain of the Fc. TCR βV antigen-binding domain

[0501] The diversity of the immune system enables protection against a wide range of pathogens. Due to the limited size of the germline genome, diversity can be achieved not only through V(D)J recombination but also through nucleotide linkages (links between VD and DJ segments) deletions and the addition of pseudo-random non-template nucleotides. The TCRβ gene exhibits diversity through gene arrangement.

[0502] Because, for example, seven inactivation polymorphisms frequently occur in functional gene segments, as well as large insertion / deletion-related polymorphisms covering two Vβ gene segments, all components of TCR Vβ vary from person to person and from population to population.

[0503] This disclosure provides, in particular, antibody molecules and fragments thereof that bind (e.g., specifically bind) to the human TCR βV chain (TCRβV), such as the TCRβV gene family (also called groups), such as the TCRβV subfamily (also called subgroups), as described herein. The TCRβV family and subfamilies are known in the art, for example, as described by Yassai et al. (2009). Immunogenetics 61(7) pp:493-502; Wei S. and Concannon P. (1994) Human Immunology As described in 41(3) pp:201-206. The antibodies described herein may be recombinant antibodies, such as recombinant non-mouse antibodies, such as recombinant human or humanized antibodies.

[0504] In one aspect, this disclosure provides anti-TCRβV antibody molecules that bind to human TCRβV, such as the TCRβV family, such as a gene family or variants thereof. In some embodiments, the TCRβV gene family includes, for example, as described herein. Figure 3 One or more subfamilies as described in Table 8A or Table 8B. In some implementations, the TCRβV gene family includes: 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 V17 subfamily, TCRβ V21 subfamily, and TCRβ... V23 subfamily or TCRβ V26 subfamily.

[0505] In some embodiments, the TCRβ V6 subfamily is also referred to as TCRβ V13.1. In some embodiments, the TCRβV6 subfamily includes: TCRβ V6-4*01, TCRβ V6-4*02, TCRβ V6-9*01, TCRβ V6-8*01, TCRβ V6-5*01, TCRβ V6-6*02, TCRβ V6-6*01, TCRβ V6-2*01, TCRβ V6-3*01, or TCRβ V6-1*01 or variants thereof. In some embodiments, TCRβ V6 includes TCRβ V6-4*01 or variants thereof. In some embodiments, TCRβ V6 includes TCRβ V6-4*02 or variants thereof. In some embodiments, TCRβ V6 includes TCRβ V6-9*01 or variants thereof. In some embodiments, TCRβ V6 includes TCRβ V6-8*01 or variants thereof. In some embodiments, TCRβ V6 includes TCRβV6-5*01 or a variant thereof. In some embodiments, TCRβ V6 includes TCRβ V6-6*02 or a variant thereof. In some embodiments, TCRβ V6 includes TCRβ V6-6*01 or a variant thereof. In some embodiments, TCRβ V6 includes TCRβ V6-2*01 or a variant thereof. In some embodiments, TCRβ V6 includes TCRβ V6-3*01 or a variant thereof. In some embodiments, TCRβ V6 includes TCRβ V6-1*01 or a variant thereof.

[0506] In some embodiments, TCRβ V6 includes TCRβ V6-5*01 or a variant thereof. In some embodiments, TCRβ V6, such as TCRβ V6-5*01, is identified, for example, by SEQ ID NO: 1 and / or SEQ ID NO: 2. In some embodiments, TCRβ V6, such as TCRβ V6-5*01, is identified, for example, by SEQ ID NO: 9 and / or SEQ ID NO: 10. In some embodiments, TCRβ V6 is identified, for example, by SEQ ID NO: 9 and / or SEQ ID NO: 11.

[0507] In some implementations, the TCRβ V10 subfamily is also referred to as TCRβ V12. In some implementations, the TCRβ V10 subfamily includes: TCRβ V10-1*01, TCRβ V10-1*02, TCRβ V10-3*01 or TCRβ V10-2*01 or variants thereof.

[0508] In some embodiments, the TCRβ V12 subfamily is also referred to as TCRβ V8.1. In some embodiments, the TCRβ V12 subfamily includes: TCRβ V12-4*01, TCRβ V12-3*01, or TCRβ V12-5*01 or variants thereof. In some embodiments, TCRβ V12 is identified by SEQ ID NO: 15 and / or SEQ ID NO: 16, for example, in combination. In some embodiments, TCRβ V12 is identified by any one of SEQ ID NO: 23-25 ​​and / or any one of SEQ ID NO: 26-30, for example, in combination.

[0509] In some implementations, the TCRβ V5 subfamily is selected from: TCRβ V5-5*01, TCRβ V5-6*01, TCRβ V5-4*01, TCRβ V5-8*01, TCRβ V5-1*01 or variants thereof.

[0510] In some implementations, the TCRβ V7 subfamily includes 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 variants thereof.

[0511] In some implementations, the TCRβ V11 subfamily includes: TCRβ V11-1*01, TCRβ V11-2*01, or TCRβV11-3*01 or variants thereof.

[0512] In some implementations, the TCRβ V14 subfamily includes TCRβ V14*01 or variants thereof.

[0513] In some implementations, the TCRβ V16 subfamily includes TCRβ V16*01 or variants thereof.

[0514] In some implementations, the TCRβ V18 subfamily includes TCRβ V18*01 or variants thereof.

[0515] In some implementations, the TCRβ V9 subfamily includes TCRβ V9*01 or TCRβ V9*02 or variants thereof.

[0516] In some implementations, the TCRβ V13 subfamily includes TCRβ V13*01 or variants thereof.

[0517] In some implementations, the TCRβ V4 subfamily includes TCRβ V4-2*01, TCRβ V4-3*01, or TCRβ V4-1*01 or variants thereof.

[0518] In some implementations, the TCRβ V3 subfamily includes TCRβ V3-1*01 or variants thereof.

[0519] In some implementations, the TCRβ V2 subfamily includes TCRβ V2*01 or variants thereof.

[0520] In some implementations, the TCRβ V15 subfamily includes TCRβ V15*01 or variants thereof.

[0521] In some implementations, the TCRβ V30 subfamily includes TCRβ V30*01 or TCRβ V30*02 or variants thereof.

[0522] In some implementations, the TCRβ V19 subfamily includes TCRβ V19*01 or TCRβ V19*02 or variants thereof.

[0523] In some implementations, the TCRβ V27 subfamily includes TCRβ V27*01 or variants thereof.

[0524] In some implementations, the TCRβ V28 subfamily includes TCRβ V28*01 or variants thereof.

[0525] In some implementations, the TCRβ V24 subfamily includes TCRβ V24-1*01 or a variant thereof.

[0526] In some implementations, the TCRβ V20 subfamily includes TCRβ V20-1*01 or TCRβ V20-1*02 or variants thereof.

[0527] In some implementations, the TCRβ V25 subfamily includes TCRβ V25-1*01 or a variant thereof.

[0528] In some implementations, the TCRβ V29 subfamily includes TCRβ V29-1*01 or a variant thereof.

[0529] Table 8A: List of TCRβV subfamilies and subfamilie members

[0530] Table 8B: Other TCRβV subfamilies Anti-TCRβV antibody

[0531] This paper discloses the discovery of a novel class of antibodies, namely the anti-TCRβV antibody molecules disclosed herein, which, despite having low sequence similarity (e.g., low sequence identity between different antibody molecules recognizing different TCRβV subfamilies), still recognize structurally conserved regions (e.g., domains) on the TCRβV protein and have similar functions (e.g., similar cytokine profiles). Therefore, the anti-TCRβV antibody molecules disclosed herein exhibit a structure-function relationship.

[0532] In some embodiments, the anti-TCRβV antibody molecules disclosed herein do not recognize (e.g., do not bind to) the TCRβV:TCRα complex interface.

[0533] 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 Viney et al., (…). Hybridoma JOVI.1 as described in December 1992; 11(6):701-13).

[0534] 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) complementarity-determining regions (e.g., CDR1, CDR2, and / or CDR3) of the TCRβV protein.

[0535] In some embodiments, the anti-TCRβV antibody molecules disclosed herein bind (e.g., specifically bind) to the TCRβV region. In some embodiments, the binding of the anti-TCRβV antibody molecules disclosed herein results in a cytokine profile different from that of T cell adaptors that bind to receptors or molecules other than the TCRβV region (“non-TCRβV-binding T cell adaptors”). In some embodiments, non-TCRβV-binding T cell adaptors include antibodies that bind to CD3 molecules (e.g., CD3ε (CD3e) molecules) or TCR alpha (TCRα) molecules. In some embodiments, non-TCRβV-binding T cell adaptors are OKT3 antibodies or SP34-2 antibodies.

[0536] On the one hand, this disclosure provides information related to human TCRβV, such as one or more members of the TCRβV gene family, such as the TCRβV subfamily, for example, as described herein. Figure 3Those anti-TCRβV antibody molecules described in Table 8A or Table 8B that are bound. In some implementations, the anti-TCRβV antibody molecule binds to one or more of the following TCRβV subfamilies: TCRβV6, TCRβV10, TCRβV12, TCRβV5, TCRβV7, TCRβV11, TCRβV14, TCRβV16, TCRβV18, TCRβV9, TCRβV13, TCRβV4, TCRβV3, TCRβV2, TCRβV15, TCRβV30, TCRβV19, TCRβV27, TCRβV28, TCRβV24, TCRβV20, TCRβV25, TCRβV29, TCRβV1, TCRβV17, TCRβV28, TCRβV29, TCRβV29, TCRβV29, TCRβV20 ... The V21 subfamily, the TCRβ V23 subfamily, or the TCRβ V26 subfamily or their variants.

[0537] In some embodiments, the anti-TCRβV antibody molecule binds to the TCRβ V6 subfamily, which includes: TCRβ V6-4*01, TCRβ V6-4*02, TCRβ V6-9*01, TCRβ V6-8*01, TCRβ V6-5*01, TCRβ V6-6*02, TCRβ V6-6*01, TCRβ V6-2*01, TCRβ V6-3*01, or TCRβ V6-1*01 or variants thereof. In some embodiments, the TCRβ V6 subfamily includes TCRβ V6-5*01 or variants thereof. In some embodiments, TCRβ V6 includes TCRβ V6-4*01 or variants thereof. In some embodiments, TCRβ V6 includes TCRβ V6-4*02 or variants thereof. In some embodiments, TCRβ V6 includes TCRβ V6-9*01 or variants thereof. In some embodiments, TCRβ V6 includes TCRβV6-8*01 or a variant thereof. In some embodiments, TCRβ V6 includes TCRβ V6-5*01 or a variant thereof. In some embodiments, TCRβ V6 includes TCRβ V6-6*02 or a variant thereof. In some embodiments, TCRβ V6 includes TCRβ V6-6*01 or a variant thereof. In some embodiments, TCRβ V6 includes TCRβ V6-2*01 or a variant thereof. In some embodiments, TCRβ V6 includes TCRβ V6-3*01 or a variant thereof. In some embodiments, TCRβ V6 includes TCRβ V6-1*01 or a variant thereof.

[0538] In some embodiments, the anti-TCRβV antibody molecule binds to the TCRβ V10 subfamily, which includes: TCRβ V10-1*01, TCRβ V10-1*02, TCRβ V10-3*01, or TCRβ V10-2*01 or variants thereof.

[0539] In some embodiments, the anti-TCRβV antibody molecule binds to the TCRβ V12 subfamily, which includes: TCRβ V12-4*01, TCRβ V12-3*01, or TCRβ V12-5*01 or variants thereof.

[0540] In some embodiments, the anti-TCRβV antibody molecule binds to the TCRβ V5 subfamily, which includes: TCRβ V5-5*01, TCRβ V5-6*01, TCRβ V5-4*01, TCRβ V5-8*01, TCRβ V5-1*01 or variants thereof.

[0541] In some embodiments, the anti-TCRβV antibody molecule does not bind to TCRβ V12, or binds to TCRβ V12 with an affinity and / or binding specificity less than (e.g., less than about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or about 2, 5, or 10 times) that of the 16G8 mouse antibody or its humanized form as described in U.S. Patent 5,861,155.

[0542] In some embodiments, the anti-TCRβV antibody molecule binds to TCRβV12 with an affinity and / or binding specificity greater than (e.g., greater than about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or about 2, 5, or 10 times) that of the 16G8 mouse antibody or its humanized form as described in U.S. Patent 5,861,155.

[0543] In some embodiments, the anti-TCRβV antibody molecule binds to the TCRβV region other than TCRβV12 with an affinity and / or binding specificity greater than (e.g., greater than about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or about 2, 5, or 10 times) that of the 16G8 mouse antibody or its humanized form as described in U.S. Patent 5,861,155. (e.g., the TCRβV region as described herein, such as the TCRβV6 subfamily (e.g., TCRβV6-5*01)).

[0544] In some embodiments, the anti-TCRβV 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 less than (e.g., less than about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or about 2, 5, or 10 times) that of the mouse antibody C or its humanized form as described in U.S. Patent 5,861,155.

[0545] In some embodiments, the anti-TCRβV antibody molecule binds to TCRβ V5-5*01 or TCRβ V5-1*01 with an affinity and / or binding specificity greater than (e.g., greater than about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or about 2, 5, or 10 times) that of the mouse antibody C or its humanized form as described in U.S. Patent 5,861,155.

[0546] In some embodiments, the anti-TCRβV antibody molecule binds to a TCRβV region other than TCRβV5-5*01 or TCRβV5-1*01 with an affinity and / or binding specificity greater than (e.g., greater than about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or about 2, 5, or 10 times) the affinity and / or binding specificity of the mouse antibody C or its humanized form as described in U.S. Patent 5,861,155. (e.g., the TCRβV region as described herein, such as the TCRβV6 subfamily (e.g., TCRβV6-5*01)). Anti-TCRβ V6 antibody

[0547] Therefore, in one aspect, this disclosure provides anti-TCRβV antibody molecules that bind to human TCRβ V6 (e.g., the TCRβ V6 subfamily), said 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, TCRβ V6 includes TCRβ V6-4*01 or a variant thereof. In some embodiments, TCRβ V6 includes TCRβV6-4*02 or a variant thereof. In some embodiments, TCRβ V6 includes TCRβ V6-9*01 or a variant thereof. In some embodiments, TCRβ V6 includes TCRβ V6-8*01 or a variant thereof. In some embodiments, TCRβ V6 includes TCRβ V6-5*01 or a variant thereof. In some embodiments, TCRβ V6 includes TCRβ V6-6*02 or a variant thereof. In some embodiments, TCRβ V6 includes TCRβ V6-6*01 or a variant thereof. In some embodiments, TCRβ V6 includes TCRβ V6-2*01 or a variant thereof. In some embodiments, TCRβ V6 includes TCRβ V6-3*01 or a variant thereof. In some embodiments, TCRβ V6 includes TCRβ V6-1*01 or a variant thereof.

[0548] In some implementations, 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 with it.

[0549] SEQ ID NO: 43 ATGAGCATCGGCCTCCTGTGCTGTGCAGCCTTGTCTCTCCTGTGGGCAGGTCCAGTGAATGCTGGTGTCACTCAGACCCAAAATTCCAGGTCCTGAAGACAGGACAGAGCATGACACTGCAGTGTGCCCAGGATATGAACCATGAATACATGTCCTGGTATCGACAAGACC CAGGCATGGGGCTGAGGCTGATTCATTACTCAGTTGGTGCTGGTATCACTGACCAAGGAGAAGTCCCCAATGGCTACAATGTCTCCAGATCAACCACAGAGGATTTCCCGCTCAGGCTGCTGTCGGCTGCTCCCTCCCAGACATCTGTGTACTTCTGTGCCAGCAGTTACTC In some embodiments, TCRβ V6-5*01 contains the amino acid sequence of SEQ ID NO: 44, or an amino acid sequence having 85%, 90%, 95%, 99% or higher identity with it.

[0550] SEQ ID NO: 44 MSIGLCCAALSLLWAGPVNAGVTQTPKFQVLKTGQSMTLQCAQDMNHEYMSWYRQDPGMGLRLIHYSVGAGITDQGEVPNGYNVSRSTTEDFPLRLLSAAPSQTSVYFCASSY In some embodiments, the anti-TCRβV antibody molecule, such as the anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule, is a non-mouse antibody molecule, such as a human or humanized antibody molecule. In some embodiments, the anti-TCRβV antibody molecule, such as the 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, such as the anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule, is a humanized antibody molecule.

[0551] In some implementations, the anti-TCRβV antibody molecule, such as the anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule, is isolated or recombinant.

[0552] In some embodiments, an anti-TCRβV antibody molecule, such as an anti-TCRβV6 (e.g., anti-TCRβV6-5*01) antibody molecule, includes at least one antigen-binding region, such as a variable region or an antigen-binding fragment thereof, from an antibody described herein (e.g., selected from any one of AH.1 to AH.68 (e.g., AH.1, AH.2, or AH.68), or an antibody described in Table 1A, or an antibody 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) to any of the aforementioned sequences).

[0553] In some embodiments, the anti-TCRβV antibody molecule, such as the anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule, includes at least one, two, three, or four variable regions from the antibody described herein (e.g., an antibody selected from any one of AH.1 to AH.68 (e.g., AH.1, AH.2, or AH.68), or the antibody described in Table 1A, or an antibody 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) to any of the aforementioned sequences).

[0554] In some embodiments, the anti-TCRβV antibody molecule, such as the anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule, includes at least one or two heavy chain variable regions from the antibody described herein (e.g., an antibody selected from any one of AH.1 to AH.68 (e.g., AH.1, AH.2, or AH.68), or the antibody described in Table 1A, or an antibody 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) to any of the aforementioned sequences).

[0555] In some embodiments, the anti-TCRβV antibody molecule contains a heavy chain variable region (VH) having a common sequence of SEQ ID NO: 231 or 3290.

[0556] In some embodiments, the anti-TCRβV antibody molecule, such as the anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule, includes at least one or two light chain variable regions from the antibody described herein (e.g., an antibody selected from any one of AH.1 to AH.68 (e.g., AH.1, AH.2, or AH.68), or the antibody described in Table 1A, or an antibody 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) to any of the aforementioned sequences).

[0557] In some embodiments, the anti-TCRβV antibody molecule contains a light chain variable region (VL) having a common sequence of SEQ ID NO: 230 or 3289.

[0558] In some embodiments, the anti-TCRβV antibody molecule, such as the anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule, includes a heavy chain constant region of IgG4 (e.g., human IgG4). In yet another embodiment, the anti-TCRβV antibody molecule, such as the anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule, includes a heavy chain constant region of IgG1 (e.g., human IgG1). In one embodiment, the heavy chain constant region comprises an amino sequence listed in Table 3A or a sequence substantially identical thereto (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99%, or more).

[0559] In some embodiments, the anti-TCRβV antibody molecule, such as the anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule, includes a κ light chain constant region, such as the human κ light chain constant region. In one embodiment, the light chain constant region comprises an amino sequence listed in Table 3A or a sequence substantially identical thereto (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99%, or more).

[0560] In some embodiments, anti-TCRβV antibody molecules, such as anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecules, include at least one, two, or three complementarity-determining regions (CDRs) of the heavy chain variable region (VH) of the antibody described herein (e.g., selected from any one of AH.1 to AH.68 (e.g., AH.1, AH.2, or AH.68), or the antibody described in Table 1A, or the antibody encoded by the nucleotide sequence in Table 1A, or the sequence substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99%, or more) to any of the aforementioned sequences).

[0561] In some embodiments, the anti-TCRβV antibody molecule, such as the anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule, comprises at least one, two, or three CDRs (or all CDRs) from the heavy chain variable region containing the amino acid sequence shown in Table 1A or the amino acid sequence encoded by the nucleotide sequence shown in Table 1A. In one embodiment, one or more CDRs (or all CDRs) have one, two, three, four, five, six, or more alterations, such as amino acid substitutions or deletions, relative to the amino acid sequence shown in Table 1A or the amino acid sequence encoded by the nucleotide sequence shown in Table 1A.

[0562] In some embodiments, the anti-TCRβV antibody molecule, such as the anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule, includes at least one, two, or three complementarity-determining regions (CDRs) of the light chain variable region of the antibody described herein (e.g., selected from any one of AH.1 to AH.68 (e.g., AH.1, AH.2, or AH.68), or the antibody described in Table 1A, or the antibody encoded by the nucleotide sequence in Table 1A, or the sequence substantially identical to any of the aforementioned sequences (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99%, or more identical)).

[0563] In some embodiments, the anti-TCRβV antibody molecule, such as the anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule, comprises at least one, two, or three CDRs (or all CDRs) from a light chain variable region containing an amino acid sequence encoded by the amino acid sequence shown in Table 1A or the amino acid sequence encoded by the nucleotide sequence shown in Table 1A. In one embodiment, one or more CDRs (or all CDRs) have one, two, three, four, five, six, or more alterations, such as amino acid substitutions or deletions, relative to the amino acid sequence shown in Table 1A or the amino acid sequence encoded by the nucleotide sequence shown in Table 1A.

[0564] In some embodiments, anti-TCRβV antibody molecules, such as anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecules, include at least one, two, three, four, five, or six CDRs (or all CDRs) from heavy chain variable regions and light chain variable regions comprising an amino acid sequence encoded by an amino acid sequence shown in Table 1A or by a nucleotide sequence shown in Table 1A. In one embodiment, one or more CDRs (or all CDRs) have one, two, three, four, five, six, or more alterations, such as amino acid substitutions or deletions, relative to the amino acid sequence shown in Table 1A or the amino acid sequence encoded by a nucleotide sequence shown in Table 1A.

[0565] In some embodiments, an anti-TCRβV antibody molecule, such as an anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule, includes all six CDRs of the antibodies described herein (e.g., antibodies selected from any one of AH.1 to AH.68 (e.g., AH.1, AH.2, or AH.68), or antibodies described in Table 1A, or antibodies encoded by the nucleotide sequences in Table 1A), or closely related CDRs, such as identical CDRs or CDRs having at least one amino acid change but no more than two, three, or four changes (e.g., substitution, deletion, or insertion, such as conserved substitution). In some embodiments, an anti-TCRβV antibody molecule, such as an anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule, may include any CDR described herein.

[0566] In some embodiments, the anti-TCRβV antibody molecule, such as the anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule, comprises an antibody from the antibody described herein (e.g., selected from any one of AH.1 to AH.68 (e.g., AH.1, AH.2, or AH.68), or the antibody described in Table 1A, or a sequence substantially identical to any of the sequences described above (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical)) of the heavy chain variable region of Kabat et al. (e.g., at least one, two, or three CDRs as defined by Kabat as listed in Table 1A); or at least one, two, or three CDRs having at least one amino acid change, but no more than two, three, or four changes (e.g., substitution, deletion, or insertion, such as a conserved substitution), relative to one, two, or three CDRs according to Kabat et al. shown in Table 1A.

[0567] In some embodiments, the anti-TCRβV antibody molecule, such as the anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule, comprises an antibody from the antibodies described herein (e.g., selected from any one of AH.1 to AH.68 (e.g., AH.1, AH.2, or AH.68), or the antibodies described in Table 1A, or a sequence substantially identical to any of the sequences described above (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical)) of the light chain variable region of at least one, two, or three CDRs according to Kabat et al. (e.g., at least one, two, or three CDRs as defined by Kabat as listed in Table 1A); or at least one, two, or three CDRs having at least one amino acid change, but no more than two, three, or four changes (e.g., substitution, deletion, or insertion, such as a conserved substitution), relative to one, two, or three CDRs according to Kabat et al. shown in Table 1A.

[0568] In some implementations, anti-TCRβV antibody molecules, such as anti-TCRβV6 (e.g., anti-TCRβ... V6-5*01) antibody molecules include antibodies derived from the antibodies described herein (e.g., selected from any one of AH.1 to AH.68 (e.g., AH.1, AH.2, or AH.68), or antibodies described in Table 1A, or antibodies encoded by nucleotide sequences in Table 1A; or sequences substantially identical to any of the sequences described above (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical)) of heavy chain variable regions and light chain variable regions according to at least one, two, three, four, five, or six CDRs (e.g., at least one, two, three, four, five, or six CDRs as defined by Kabat as listed in Table 1A); or at least one, two, three, four, five, or six CDRs with at least one amino acid change, but not more than two, three, or four changes (e.g., substitution, deletion, or insertion, such as a conserved substitution) relative to one, two, three, four, five, or six CDRs according to Kabat et al. as shown in Table 1A.

[0569] In some embodiments, anti-TCRβV antibody molecules, such as anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecules, include antibodies from the antibodies described herein (e.g., selected from any one of AH.1 to AH.68 (e.g., AH.1, AH.2, or AH.68), or antibodies described in Table 1A, or antibodies encoded by nucleotide sequences in Table 1A; or sequences substantially identical to any of the aforementioned sequences (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical)) of all six CDRs of the heavy chain variable region and light chain variable region according to Kabat et al. (e.g., all six CDRs as defined by Kabat as listed in Table 1A); or all six CDRs having at least one amino acid change, but no more than two, three, or four changes (e.g., substitution, deletion, or insertion, such as a conserved substitution), relative to all six CDRs according to Kabat et al. shown in Table 1A. In one embodiment, an anti-TCRβV antibody molecule, such as an anti-TCRβV6 (e.g., anti-TCRβV6-5*01) antibody molecule, may include any CDR described herein.

[0570] In some embodiments, anti-TCRβV antibody molecules, such as anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecules, include at least one, two, or three hypervariable rings having the same canonical structure as the corresponding hypervariable rings of the antibodies described herein (e.g., antibodies selected from any one of AH.1 to AH.68, e.g., AH.1, AH.2, or AH.68), for example, at least ring 1 and / or ring 2 of the heavy chain and / or light chain variable domains of the antibodies described herein. Descriptions of the canonical structures of hypervariable rings can be found, for example, in Chothia et al., (1992) J. Mol. Biol. 227:799-817; Tomlinson et al., (1992) J. Mol. Biol. 227:776-798. These structures can be determined by consulting the tables described in these references.

[0571] In some embodiments, the anti-TCRβV antibody molecule, such as the anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule, comprises an antibody from the antibody described herein (e.g., selected from any one of AH.1 to AH.68 (e.g., AH.1, AH.2, or AH.68), or the antibody described in Table 1A, or a sequence substantially identical to any of the sequences described above (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical)) of the heavy chain variable region according to Chothia et al. (e.g., at least one, two, or three CDRs as defined by Chothia as listed in Table 1A); or at least one, two, or three CDRs having at least one amino acid change, but no more than two, three, or four changes (e.g., substitution, deletion, or insertion, such as a conserved substitution), relative to one, two, or three CDRs according to Chothia et al. shown in Table 1A.

[0572] In some embodiments, the anti-TCRβV antibody molecule, such as the anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule, comprises at least one, two, or three CDRs of the light chain variable region according to Chothia et al. (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99%, or more identical) of any of the sequences described herein (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99%, or more) of any of the sequences substantially identical to any of the sequences listed above (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99%, or more)); or at least one, two, or three CDRs having at least one amino acid change, but no more than two, three, or four changes (e.g., substitution, deletion, or insertion, such as a conserved substitution), relative to one, two, or three CDRs according to Chothia et al. shown in Table 1A.

[0573] In some implementations, anti-TCRβV antibody molecules, such as anti-TCRβV6 (e.g., anti-TCRβ... V6-5*01) antibody molecules include antibodies derived from antibodies described herein (e.g., selected from any one of AH.1 to AH.68 (e.g., AH.1, AH.2, or AH.68), or antibodies described in Table 1A, or antibodies encoded by nucleotide sequences in Table 1A; or sequences substantially identical to any of the aforementioned sequences (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical)) with heavy chain variable regions and light chain variable regions according to Chothia et al. (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical)); or at least one, two, three, four, five or six CDRs of heavy chain variable regions as defined by Chothia et al., relative to one, two, three, four, five or six CDRs of light chain variable regions as defined by Chothia et al., as shown in Table 1A; but not exceeding two, three or four changes (e.g., substitution, deletion or insertion, such as conserved substitution).

[0574] In some embodiments, anti-TCRβV antibody molecules, such as anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecules, include antibodies from the antibodies described herein (e.g., selected from any one of AH.1 to AH.68 (e.g., AH.1, AH.2, or AH.68), or antibodies described in Table 1A, or antibodies encoded by nucleotide sequences in Table 1A; or sequences substantially identical to any of the aforementioned sequences (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical)) of all six CDRs of the heavy chain variable region and light chain variable region as described by Chothia et al. (e.g., all six CDRs as defined by Chothia as listed in Table 1A); or all six CDRs having at least one amino acid change, but no more than two, three, or four changes (e.g., substitution, deletion, or insertion, such as a conserved substitution), relative to all six CDRs as shown in Table 1A according to Chothia et al. In one embodiment, an anti-TCRβV antibody molecule, such as an anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule, may include any CDR described herein.

[0575] In some implementations, anti-TCRβV antibody molecules, such as anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecules, include combinations of CDRs or hypervariable rings as defined by Kabat et al., Chothia et al., or as described in Table 1A.

[0576] In some implementations, anti-TCRβV antibody molecules, such as anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecules, may contain any combination of CDRs or hypervariable rings as defined by Kabat and Chothia.

[0577] In some implementations, the CDRs listed in Table 1A are CDRs that include both the Kabat CDR and the Chothia CDR.

[0578] In some embodiments, anti-TCRβV antibody molecules, such as anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecules, comprise combinations of CDRs or hypervariable rings, which are identified as CDRs of combinations in Table 1A. In some embodiments, anti-TCRβV antibody molecules, such as anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecules, may contain any combination of CDRs or hypervariable rings of “combinations” CDRs as described in Table 1A.

[0579] In one embodiment, such as in embodiments containing a variable region, a CDR (e.g., a combined CDR, a Chothia CDR, or a Kabat CDR), or other sequences mentioned herein, such as in Table 1A, the antibody molecule is a monospecific antibody molecule, a bispecific antibody molecule, a bivalent antibody molecule, a bicomplementary antibody molecule, or an antibody molecule containing an antigen-binding fragment of an antibody (e.g., a hapten or an antigen-binding fragment of a hapten). In some embodiments, the antibody molecule includes a multispecific molecule, such as a bispecific molecule, for example, as described herein.

[0580] In one embodiment, the anti-TCRβV antibody molecule, such as the anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule, comprises: (i) One, two, or all of the light chain complementarity determination regions 1 (LCCDR1), 2 (LC CDR2), and 3 (LC CDR3) of SEQ ID NO: 2, SEQ ID NO: 10, or SEQ ID NO: 11, and / or (ii) One, two, or all of the heavy chain complementarity determination region 1 (HC CDR1), heavy chain complementarity determination region 2 (HC CDR2), and heavy chain complementarity determination region 3 (HC CDR3) of SEQ ID NO: 1 or SEQ ID NO: 9.

[0581] In some embodiments, anti-TCRβV antibody molecules, such as anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecules, include LC CDR1, LC CDR2 and LC CDR3 of SEQ ID NO: 2, and HC CDR1, HCCDR2 and HC CDR3 of SEQ ID NO: 1.

[0582] In some embodiments, anti-TCRβV antibody molecules, such as anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecules, include 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.

[0583] In some embodiments, anti-TCRβV antibody molecules, such as anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecules, include 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.

[0584] In one embodiment, the anti-TCRβV antibody molecule, such as the anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule, 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 amino acid sequence of HC CDR1 of SEQ ID NO: 3, the amino acid sequence of HC CDR2 of SEQ ID NO: 4, or the amino acid sequence of HC CDR3 of SEQ ID NO: 5.

[0585] In some embodiments, the anti-TCRβV antibody molecule, such as the anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule, includes: (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) Heavy chain variable region (VH), wherein the heavy chain variable region comprises 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.

[0586] In one embodiment, the anti-TCRβV antibody molecule, such as the anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule, 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.

[0587] In some embodiments, the anti-TCRβV antibody molecule, such as the anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule, includes: (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) Heavy chain variable region (VH), the heavy chain variable region 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...

Claims

1. A multifunctional molecule comprising: (i) Binding to, for example, selectively binding to the first antigen-binding domain of a T-cell receptor variable β (TCRBV) antigen, such as the TCRBV antigen. as well as (ii) One, two, or all of the following: (a) Immune cell adjuvants selected from NK cell adjuvants, T cell adjuvants, B cell adjuvants, dendritic cell adjuvants or macrophage adjuvants; (b) Cytokine molecules or cytokine inhibitor molecules; and (c) Death receptor signaling adaptor.

2. A multifunctional molecule comprising: (i) Binding to, for example, selectively binding to the first antigen-binding domain of a T-cell receptor variable β (TCRBV) antigen, such as the TCRBV antigen, and (ii) NK cell adaptors, such as anti-NKp30, anti-NKp46, anti-NKG2D or anti-CD16 antibody molecules.

3. A multifunctional molecule comprising: (i) Binding to, for example, selectively binding to the first antigen-binding domain of a T-cell receptor variable β (TCRBV) antigen, such as the TCRBV antigen, and (ii) Death receptor signaling adaptors.

4. A multifunctional molecule comprising: (i) Binding to, for example, selectively binding to the first antigen-binding domain of a T-cell receptor variable β (TCRBV) antigen, such as the TCRBV antigen, and (ii) Cytokine inhibitor molecules.

5. A nucleic acid molecule encoding a multifunctional molecule according to any one of claims 1-4.

6. A vector, for example, an expression vector, comprising the nucleic acid molecule according to claim 5.

7. A host cell comprising the nucleic acid molecule according to claim 5 or the vector according to claim 6.

8. A method for preparing, for example, a multifunctional molecule or antibody molecule according to any one of claims 1-4, comprising culturing the host cells according to claim 7 under suitable conditions, for example, conditions suitable for gene expression and / or homodimerization.

9. A pharmaceutical composition comprising a multifunctional molecule according to any one of claims 1-4 and a pharmaceutically acceptable carrier, excipient, or stabilizer.

10. A method of treating TCR bias, comprising administering to a subject in need a multifunctional molecule according to any one of claims 1-4, wherein the multifunctional molecule is administered in an amount effective in treating the TCR bias.

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