Antibody molecule binding to NKP30 and uses thereof

Abstract

The present invention provides a composition comprising a preparation having an antigen-binding domain that binds to NKp30, a method for treating a pathological condition or disease in a subject, and a method for proliferating and / or activating NK cells using the same.

Description

Technology Field

[0001] Cross-reference

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 589,149 filed on October 10, 2023, which is incorporated herein by reference in its entirety. Background Technology

[0003] Natural killer (NK) cells recognize and destroy tumor and virus-infected cells in an antibody-independent manner. The regulation of NK cells is mediated by activating and inhibiting receptors on the surface of NK cells. One family of activating receptors is the natural cytotoxic receptor (NCR), which includes NKp30, NKp44, and NKp46.

[0004] Given the importance of immune checkpoint pathways in regulating immune responses, there is a need to develop novel agents that activate the immune system by modulating the activity of immunosuppressive proteins such as PD-1. Such agents can be used, for example, in cancer immunotherapy and in the treatment of other pathological conditions such as chronic infections.

[0005] In one embodiment, particularly, a composition comprising a formulation comprising an antigen-binding domain that binds to NKp30 is provided herein, wherein the antigen-binding domain comprises a heavy chain variable region (VH) comprising:

[0006] (i) heavy chain complementarity determining region 3 (VHCDR3) comprising the sequence of GDWHYFDY (SEQ No. 504); and

[0007] (ii) heavy chain complementarity determining region 1 (VHCDR1) containing the sequence of GFSIIRVGYHWN (SEQ No. 502), heavy chain complementarity determining region 2 (VHCDR2) containing the sequence of YIHRSGSTSYNPSLKS (SEQ No. 503), or a combination thereof.

[0008] In some embodiments, the antigen binding domain comprises VH comprising VHCDR1 having the sequence of GFSIIRVGYHWN (SEQ No. 502), VHCDR2 having the sequence of YIHRSGSTSYNPSLKS (SEQ No. 503), and VHCDR3 having the sequence of GDWHYFDY (SEQ No. 504).

[0009] In some embodiments, the antigen binding domain comprises a light chain complementarity determining region 1 (VLCDR1) comprising the sequence of SGEKLSDKYVH (SEQ No. 26), a light chain complementarity determining region 2 (VLCDR2) comprising the sequence of ENDRRPS (SEQ No. 28), a light chain complementarity determining region 3 (VLCDR3) comprising the sequence of QFWDSTNSAV (SEQ No. 86), or any combination thereof.

[0010] In some embodiments, the antigen binding domain comprises a VL comprising a VLCDR1 containing the sequence of SGEKLSDKYVH (SEQ No. 26), a VLCDR2 containing the sequence of ENDRRPS (SEQ No. 28), and a VLCDR3 containing the sequence of QFWDSTNSAV (SEQ No. 86).

[0011] In some embodiments, the antigen binding domain comprises a VH having at least 75% sequence identity with the sequence of SEQ ID NO. 505.

[0012] In some embodiments, the antigen binding domain comprises a VH containing the sequence of SEQ ID NO. 505.

[0013] In some embodiments, the antigen binding domain comprises a VL having a sequence having at least 75% sequence identity with the sequence of SEQ ID NO. 13.

[0014] In some embodiments, the antigen binding domain comprises a VL containing the sequence of SEQ ID NO. 13.

[0015] In some embodiments, the antigen binding domain comprises a VH having at least 75% sequence identity with the sequence of SEQ ID NO. 505 and a VL having at least 75% sequence identity with the sequence of SEQ ID NO. 13.

[0016] In some embodiments, the antigen binding domain comprises VH containing the sequence of SEQ ID NO. 505 and VL containing the sequence of SEQ ID NO. 13.

[0017] In some embodiments, the antigen-binding domain comprises a whole antibody or its antigen-binding fragment, a divalent antibody, a bispecific antibody, or a biparatope antibody.

[0018] In some embodiments, the antigen-binding fragment is Fab, F(ab')2, Fv, short-chain Fv(scFv), diabody, single-domain antibody, or camelid antibody.

[0019] In some embodiments, the antigen binding domain is Fab or scFv.

[0020] In some embodiments, the antigen binding domain comprises one or more heavy chain constant regions selected from the group consisting of IgG1 heavy chain constant region or a functional fragment thereof, IgG2 heavy chain constant region or a functional fragment thereof, IgG3 heavy chain constant region or a functional fragment thereof, IgGA1 heavy chain constant region or a functional fragment thereof, IgGA2 heavy chain constant region or a functional fragment thereof, IgG4 heavy chain constant region or a functional fragment thereof, IgJ heavy chain constant region or a functional fragment thereof, IgM heavy chain constant region or a functional fragment thereof, IgD heavy chain constant region or a functional fragment thereof, and IgE heavy chain constant region or a functional fragment thereof.

[0021] In some embodiments, one or more heavy chain invariant regions comprise a sequence having at least 75% sequence identity with any one of the heavy chain invariant region sequences listed in Tables 3, 7, 8, 16, 17, 39, 40, 42C, 43A, 43B, and 46–51.

[0022] In some embodiments, one or more heavy chain invariant regions comprise a sequence having at least 75% sequence identity with the sequence of SEQ ID NO. 73, the sequence of SEQ ID NO. 76, the sequence of SEQ ID NO. 74, the sequence of SEQ ID NO. 75, the sequence of SEQ ID NO. 76, the sequence of SEQ ID NO. 40, the sequence of SEQ ID NO. 41, the sequence of SEQ ID NO. 42, the sequence of SEQ ID NO. 3645, the sequence of SEQ ID NO. 3646, the sequence of SEQ ID NO. 3649, the sequence of SEQ ID NO. 3451, the sequence of SEQ ID NO. 506, the sequence of SEQ ID NO. 507, the sequence of SEQ ID NO. 3647, the sequence of SEQ ID NO. 3648, the sequence of SEQ ID NO. 508 or 8686, the sequence of SEQ ID NO. 513, the sequence of SEQ ID NO. 514, the sequence of SEQ ID NO. 515, the sequence of SEQ ID NO. 516, the sequence of SEQ ID NO. 3447, or the sequence of SEQ ID NO. 511.

[0023] In some embodiments, the antigen binding domain comprises a kappa light chain constant region or a functional fragment thereof, a lambda light chain constant region or a functional fragment thereof, or a combination thereof.

[0024] In some embodiments, the kappa light chain invariant region comprises a sequence having at least 75% sequence identity with any one of the light chain invariant region sequences listed in Tables 3, 7, 8, 16, 17, 39, 40, 42C, 43A, 43B, and 46 to 51.

[0025] In some embodiments, the kappa light chain invariant region comprises a sequence having at least 75% sequence identity with the sequence of SEQ ID NO. 39 or the sequence of SEQ ID NO. 3644.

[0026] In some embodiments, the formulation is a polypeptide molecule.

[0027] In some embodiments, the formulation is a multifunctional molecule.

[0028] In some embodiments, the formulation is a multispecific molecule.

[0029] In some embodiments, the formulation comprises at least one cytokine molecule or a functional fragment or variant thereof or a functional fragment or variant thereof, an antigen-binding domain that binds to a T cell receptor beta chain constant domain (TRBC), a second antigen-binding domain that binds to NKp30, an immune cell engager, a tumor-targeting moiety, or any combination thereof.

[0030] In some embodiments, the formulation comprises at least two non-adjacent polypeptide chains, including a first polypeptide chain and a second polypeptide chain;

[0031] Here, the first polypeptide chain comprises the following configuration from the N-terminus to the C-terminus:

[0032] A-[First member of the dimerization module]-C,

[0033] The second polypeptide chain comprises the following structure from the N-terminus to the C-terminus:

[0034] B-[Second member of the dimerization module]-D;

[0035] The first member of the dimerization module comprises a first immunoglobulin constant domain; and the second member of the dimerization module comprises a second immunoglobulin constant domain;

[0036] At least one of A, B, C, and D is an antigen-binding domain; and

[0037] The first polypeptide chain and the second polypeptide chain are

[0038] Dimerization can be achieved through the interaction between the first member of the dimerization module and the second member of the dimerization module.

[0039] In some embodiments, (i) A is an antigen-binding domain, and B, C, and D are independently (a) an absence, (b) at least one cytokine molecule or a functional fragment or variant thereof, (c) an antigen-binding domain that binds to a T cell receptor beta-chain constant domain (TRBC), (d) a second antigen-binding domain that binds to NKp30, (e) an immune cell binder, or (f) a tumor-targeting moiety;

[0040] (ii) B is an antigen-binding domain, and A, C, and D are independently (a) absence, (b) at least one cytokine molecule or a functional fragment or variant thereof, (c) an antigen-binding domain that binds to a T cell receptor beta-chain constant domain (TRBC), (d) a second antigen-binding domain that binds to NKp30, (e) an immune cell binder, or (f) a tumor-targeting moiety;

[0041] (iii) C is an antigen-binding domain, and A, B, and D are independently (a) absence, (b) at least one cytokine molecule or a functional fragment or variant thereof, (c) an antigen-binding domain that binds to a T cell receptor beta-chain constant domain (TRBC), (d) a second antigen-binding domain that binds to NKp30, (e) an immune cell binder, or (f) a tumor-targeting moiety;

[0042] (iv) D is an antigen-binding domain, and A, B, and C are independently (a) absence, (b) at least one cytokine molecule or a functional fragment or variant thereof, (c) an antigen-binding domain that binds to a T cell receptor beta chain constant domain (TRBC), (d) a second antigen-binding domain that binds to NKp30, (e) an immune cell binder, or (f) a tumor-targeting moiety.

[0043] In some embodiments, the formulation comprises at least one cytokine molecule or a functional fragment or variant thereof.

[0044] In some embodiments, at least one cytokine molecule or a functional fragment or variant thereof comprises IL-2 or a functional fragment or variant thereof, IL-7 or a functional fragment or variant thereof, IL-12 or a functional fragment or variant thereof, IL-15 or a functional fragment or variant thereof, IL-18 or a functional fragment or variant thereof, IL-21 or a functional fragment or variant thereof, interferon gamma or a functional fragment or variant thereof, or any combination thereof.

[0045] In some embodiments, at least one cytokine molecule or a functional fragment or variant thereof comprises a sequence having at least 75% sequence identity with any one sequence selected from the group consisting of cytokine sequences listed in Tables 40, 42C, 43A, 43B, and 46–51.

[0046] In some embodiments, at least one cytokine molecule or a functional fragment or variant thereof comprises any one sequence selected from the group consisting of cytokine sequences listed in Tables 40, 42C, 43A, 43B, and 46–51.

[0047] In some embodiments, at least one cytokine molecule or a functional fragment or variant thereof comprises IL-2 or a functional fragment or variant thereof.

[0048] In some embodiments, IL-2 or a functional fragment or variant thereof comprises a sequence having at least 75% sequence identity with the sequence of SEQ ID NO. 2191, the sequence of SEQ ID NO. 2270, or the sequence of SEQ ID NO. 2280.

[0049] In some embodiments, IL-2 or a functional fragment or variant thereof comprises the sequence of SEQ ID NO. 2191, the sequence of SEQ ID NO. 2270, or the sequence of SEQ ID NO. 2280.

[0050] In some embodiments, at least one cytokine molecule or a functional fragment or variant thereof comprises IL-15 or a functional fragment or variant thereof.

[0051] In some embodiments, IL-15 or a functional fragment or variant thereof comprises a sequence having at least 75% sequence identity with the sequence of SEQ ID NO. 2170, SEQ ID NO. 3670, or SEQ ID NO. 3673.

[0052] In some embodiments, IL-15 or a functional fragment or variant thereof comprises the sequence of SEQ ID NO. 2170, the sequence of SEQ ID NO. 3670, or the sequence of SEQ ID NO. 3673.

[0053] In some embodiments, at least one cytokine molecule or its functional fragment or variant further comprises an IL15R-alpha dimerization domain covalently linked to IL-15 or its functional fragment or variant.

[0054] In some embodiments, the IL15R alpha dimerization domain comprises the IL-15 receptor alpha sushi domain or a functional fragment or variant thereof.

[0055] In some embodiments, the IL15R alpha dimerization domain comprises a sequence having at least 75% sequence identity with the sequence of SEQ ID NO. 3472.

[0056] In some embodiments, the IL15R alpha dimerization domain comprises the sequence of SEQ ID NO. 3472.

[0057] In some embodiments, IL-15 or a functional fragment or variant thereof is sharedly linked to the IL15R-alpha dimerization domain via a linker.

[0058] In some embodiments, IL-15 or a functional fragment or variant thereof is covalently linked to the IL15R-alpha dimerization domain through a linker containing the sequence of SEQ ID NO. 3473.

[0059] In some embodiments, at least one cytokine molecule or a functional fragment or variant thereof comprises a sequence having at least 75% sequence identity with the sequence of SEQ ID NO. 3474, SEQ ID NO. 3671, or SEQ ID NO. 8689.

[0060] In some embodiments, at least one cytokine molecule or a functional fragment or variant thereof comprises the sequence of SEQ ID NO. 3474, the sequence of SEQ ID NO. 3671, or the sequence of SEQ ID NO. 8689.

[0061] In some embodiments, at least one cytokine molecule or a functional fragment or variant thereof comprises IL-7 or a functional fragment or variant thereof.

[0062] In some embodiments, IL-7 or a functional fragment or variant thereof comprises a sequence having at least 75% sequence identity with the sequence of SEQ ID NO. 3540.

[0063] In some embodiments, IL-7 or a functional fragment or variant thereof comprises the sequence of SEQ ID NO. 3540.

[0064] In some embodiments, at least one cytokine molecule or a functional fragment or variant thereof comprises IL-12 or a functional fragment or variant thereof.

[0065] In some embodiments, IL-12 or a functional fragment or variant thereof comprises a sequence having at least 75% sequence identity with the sequence of SEQ ID NO. 3542, the sequence of SEQ ID NO. 3543, or the sequence of SEQ ID NO. 3545.

[0066] In some embodiments, IL-12 or a functional fragment or variant thereof comprises the sequence of SEQ ID NO. 3542, the sequence of SEQ ID NO. 3543, or the sequence of SEQ ID NO. 3545.

[0067] In some embodiments, at least one cytokine molecule or a functional fragment or variant thereof comprises IL-18 or a functional fragment or variant thereof.

[0068] In some embodiments, IL-18 or a functional fragment or variant thereof comprises a sequence having at least 75% sequence identity with the sequence of SEQ ID NO. 2192.

[0069] In some embodiments, IL-18 or a functional fragment or variant thereof comprises the sequence of SEQ ID NO. 2192.

[0070] In some embodiments, at least one cytokine molecule or a functional fragment or variant thereof comprises IL-21 or a functional fragment or variant thereof.

[0071] In some embodiments, IL-21 or a functional fragment or variant thereof comprises a sequence having at least 75% sequence identity with the sequence of SEQ ID NO. 3540.

[0072] In some embodiments, IL-21 or a functional fragment or variant thereof comprises the sequence of SEQ ID NO. 3540.

[0073] In some embodiments, the formulation includes an antigen-binding domain that binds to TRBC.

[0074] In some embodiments, the antigen-binding domain that binds to TRBC binds to TRBC1 or TRBC2.

[0075] In some embodiments, the antigen-binding domain that binds to TRBC binds to TRBC1, wherein the antigen-binding domain that binds to TRBC comprises a VH comprising the following:

[0076] (i) A VHCDR1 comprising a sequence having at least 75% sequence identity with any one sequence selected from the group of VHCDR1 sequences listed in Tables 2A, 2B, 3A, 3B, 5A, 5B, 7, 8, 16, 39, and 40;

[0077] (ii) A VHCDR2 comprising a sequence having at least 75% sequence identity with any one sequence selected from the group consisting of VHCDR2 sequences listed in Tables 2A, 2B, 3A, 3B, 5A, 5B, 7, 8, 16, 39, and 40;

[0078] (iii) a VHCDR3 comprising a sequence having at least 75% sequence identity with any one sequence selected from the group consisting of VHCDR3 sequences listed in Tables 2A, 2B, 3A, 3B, 5A, 5B, 7, 8, 16, 39, and 40; or

[0079] (iv) Any combination of these.

[0080] In some embodiments, the antigen-binding domain that binds to TRBC binds to TRBC1, wherein the antigen-binding domain that binds to TRBC comprises a VH comprising the following:

[0081] (i) A VHCDR1 comprising any one sequence selected from the group consisting of VHCDR1 sequences listed in Tables 2A, 2B, 3A, 3B, 5A, 5B, 7, 8, 16, 39, and 40;

[0082] (ii) A VHCDR2 comprising any one sequence selected from the group consisting of VHCDR2 sequences listed in Tables 2A, 2B, 3A, 3B, 5A, 5B, 7, 8, 16, 39, and 40;

[0083] (iii) a VHCDR3 comprising any one sequence selected from the group consisting of VHCDR3 sequences listed in Tables 2A, 2B, 3A, 3B, 5A, 5B, 7, 8, 16, 39, and 40; or

[0084] (iv) Any combination of these.

[0085] In some embodiments, the antigen-binding domain that binds to TRBC binds to TRBC1, wherein the antigen-binding domain that binds to TRBC comprises a VH having at least 75% sequence identity with any one sequence selected from the group of VH sequences listed in Tables 7, 8, 16, 39, and 40.

[0086] In some embodiments, the antigen binding domain that binds to TRBC binds to TRBC1, wherein the antigen binding domain that binds to TRBC comprises a VH comprising any one sequence selected from the group consisting of VH sequences listed in Tables 7, 8, 16, 39, and 40.

[0087] In some embodiments, the antigen-binding domain that binds to TRBC binds to TRBC1, wherein the antigen-binding domain that binds to TRBC comprises a VL comprising the following:

[0088] (i) A VLCDR1 comprising a sequence having at least 75% sequence identity with any one sequence selected from the group of VLCDR1 sequences listed in Tables 4, 6, 7, 8, 16, 39, and 40;

[0089] (ii) A VLCDR2 comprising a sequence having at least 75% sequence identity with any one sequence selected from the group of VLCDR2 sequences listed in Tables 4, 6, 7, 8, 16, 39, and 40;

[0090] (iii) a VLCDR3 comprising a sequence having at least 75% sequence identity with any one sequence selected from the group consisting of VLCDR3 sequences listed in Tables 4, 6, 7, 8, 16, 39, and 40; or

[0091] (iv) Any combination of these.

[0092] In some embodiments, the antigen-binding domain that binds to TRBC binds to TRBC1, wherein the antigen-binding domain that binds to TRBC comprises a VL comprising the following:

[0093] (i) A VLCDR1 comprising any one sequence selected from the group consisting of VLCDR1 sequences listed in Tables 4, 6, 7, 8, 16, 39, and 40;

[0094] (ii) A VLCDR2 comprising any one sequence selected from the group consisting of VLCDR2 sequences listed in Tables 4, 6, 7, 8, 16, 39, and 40;

[0095] (iii) a VLCDR3 comprising any one sequence selected from the group consisting of VLCDR3 sequences listed in Tables 4, 6, 7, 8, 16, 39, and 40; or

[0096] (iv) Any combination of these.

[0097] In some embodiments, the antigen-binding domain that binds to TRBC binds to TRBC1, wherein the antigen-binding domain that binds to TRBC comprises a VL having at least 75% sequence identity with any one sequence selected from the group of VL sequences listed in Tables 7, 8, 16, 39, and 40.

[0098] In some embodiments, the antigen-binding domain that binds to TRBC binds to TRBC1, wherein the antigen-binding domain that binds to TRBC comprises a VL comprising any one sequence selected from the group consisting of VL sequences listed in Tables 7, 8, 16, 39, and 40.

[0099] In some embodiments, the antigen-binding domain that binds to TRBC binds to TRBC2, wherein the antigen-binding domain that binds to TRBC comprises a VH comprising the following:

[0100] (i) A VHCDR1 comprising a sequence having at least 75% sequence identity with any one sequence selected from the group of VHCDR1 sequences listed in Tables 9A, 9B, 11, 12, 13, 14, 15, 17, 39, and 40;

[0101] (ii) A VHCDR2 comprising a sequence having at least 75% sequence identity with any one sequence selected from the group of VHCDR2 sequences listed in Tables 9A, 9B, 12, 13, 14, 15, 17, 39, and 40;

[0102] (iii) a VHCDR3 comprising a sequence having at least 75% sequence identity with any one sequence selected from the group consisting of VHCDR3 sequences listed in Tables 9A, 9B, 12, 13, 14, 15, 17, 39, and 40; or

[0103] (iv) Any combination of these.

[0104] In some embodiments, the antigen-binding domain that binds to TRBC binds to TRBC2, wherein the antigen-binding domain that binds to TRBC comprises a VH comprising the following:

[0105] (i) A VHCDR1 comprising any one sequence selected from the group consisting of VHCDR1 sequences listed in Tables 9A, 9B, 12, 13, 14, 15, 17, 39, and 40;

[0106] (ii) A VHCDR2 comprising any one sequence selected from the group consisting of VHCDR2 sequences listed in Tables 9A, 9B, 12, 13, 14, 15, 17, 39, and 40;

[0107] (iii) a VHCDR3 comprising any one sequence selected from the group consisting of VHCDR3 sequences listed in Tables 9A, 9B, 12, 13, 14, 15, 17, 39, and 40; or

[0108] (iv) Any combination of these.

[0109] In some embodiments, the antigen-binding domain that binds to TRBC binds to TRBC2, wherein the antigen-binding domain that binds to TRBC comprises a VH having at least 75% sequence identity with any one sequence selected from the group consisting of VH sequences listed in Tables 11, 15, 17, 39, and 40.

[0110] In some embodiments, the antigen-binding domain that binds to TRBC binds to TRBC2, wherein the antigen-binding domain that binds to TRBC comprises a VH comprising any one sequence selected from the group consisting of VH sequences listed in Tables 11, 15, 17, 39, and 40.

[0111] In some embodiments, the antigen-binding domain that binds to TRBC binds to TRBC2, wherein the antigen-binding domain that binds to TRBC comprises a VL comprising the following:

[0112] (i) A VLCDR1 comprising a sequence having at least 75% sequence identity with any one sequence selected from the group of VLCDR1 sequences listed in Tables 10, 12, 13, 14, 15, 17, 39, and 40;

[0113] (ii) A VLCDR2 comprising a sequence having at least 75% sequence identity with any one sequence selected from the group of VLCDR2 sequences listed in Tables 10, 12, 13, 14, 15, 17, 39, and 40;

[0114] (iii) a VLCDR3 comprising a sequence having at least 75% sequence identity with any one sequence selected from the group consisting of VLCDR3 sequences listed in Tables 10, 12, 13, 14, 15, 17, 39, and 40; or

[0115] (iv) Any combination of these.

[0116] In some embodiments, the antigen-binding domain that binds to TRBC binds to TRBC2, wherein the antigen-binding domain that binds to TRBC comprises a VL comprising the following:

[0117] (i) A VLCDR1 comprising any one sequence selected from the group consisting of VLCDR1 sequences listed in Tables 10, 12, 13, 14, 15, 17, 39, and 40;

[0118] (ii) A VLCDR2 comprising any one sequence selected from the group consisting of VLCDR2 sequences listed in Tables 10, 12, 13, 14, 15, 17, 39, and 40;

[0119] (iii) a VLCDR3 comprising any one sequence selected from the group consisting of VLCDR3 sequences listed in Tables 10, 12, 13, 14, 15, 17, 39, and 40; or

[0120] (iv) Any combination of these.

[0121] In some embodiments, the antigen-binding domain that binds to TRBC binds to TRBC2, wherein the antigen-binding domain that binds to TRBC comprises a VL having at least 75% sequence identity with any one sequence selected from the group of VL sequences listed in Tables 11, 15, 17, 39, and 40.

[0122] In some embodiments, the antigen-binding domain that binds to TRBC binds to TRBC2, wherein the antigen-binding domain that binds to TRBC comprises a VL comprising any one sequence selected from the group consisting of VL sequences listed in Tables 11, 15, 17, 39, and 40.

[0123] In some embodiments, the antigen-binding domain that binds to TRBC binds to TRBC2, wherein the antigen-binding domain that binds to TRBC comprises a sequence having at least 75% sequence identity with any one sequence selected from the group consisting of scFv sequences listed in Tables 12, 15, 17, 39, and 40.

[0124] In some embodiments, the antigen-binding domain that binds to TRBC binds to TRBC2, wherein the antigen-binding domain that binds to TRBC comprises a VL comprising any one sequence selected from the group consisting of scFV sequences listed in Tables 12, 15, 17, 39, and 40.

[0125] In some embodiments, the formulation includes a second antigen-binding domain that binds to NKp30.

[0126] In some embodiments, the antigen binding domain and the second antigen binding domain that bind to NKp30 are identical.

[0127] In some embodiments, the antigen binding domain and the second antigen binding domain that bind to NKp30 are different.

[0128] In some embodiments, the second antigen-binding domain that binds to NKp30 comprises a heavy chain variable region (VH) comprising:

[0129] (i) heavy chain complementarity determining region 3 (VHCDR3) comprising the sequence of GDWHYFDY (SEQ No. 504); and

[0130] (ii) heavy chain complementarity determining region 1 (VHCDR1) containing the sequence of GFSIIRVGYHWN (SEQ No. 502), heavy chain complementarity determining region 2 (VHCDR2) containing the sequence of YIHRSGSTSYNPSLKS (SEQ No. 503), or a combination thereof.

[0131] In some embodiments, the second antigen-binding domain that binds to NKp30 comprises VH comprising VHCDR1 having the sequence of GFSIIRVGYHWN (SEQ No. 502), VHCDR2 having the sequence of YIHRSGSTSYNPSLKS (SEQ No. 503), and VHCDR3 having the sequence of GDWHYFDY (SEQ No. 504).

[0132] In some embodiments, the second antigen-binding domain that binds to NKp30 comprises a light chain complementarity determining region 1 (VLCDR1) comprising the sequence of SGEKLSDKYVH (SEQ No. 26), a light chain complementarity determining region 2 (VLCDR2) comprising the sequence of ENDRRPS (SEQ No. 28), a light chain complementarity determining region 3 (VLCDR3) comprising the sequence of QFWDSTNSAV (SEQ No. 86), or any combination thereof.

[0133] In some embodiments, the second antigen-binding domain that binds to NKp30 comprises VL comprising VLCDR1 having the sequence of SGEKLSDKYVH (SEQ No. 26), VLCDR2 having the sequence of ENDRRPS (SEQ No. 28), and VLCDR3 having the sequence of QFWDSTNSAV (SEQ No. 86).

[0134] In some embodiments, the second antigen-binding domain that binds to NKp30 comprises a VH having at least 75% sequence identity with the sequence of SEQ ID NO. 505.

[0135] In some embodiments, the second antigen-binding domain that binds to NKp30 comprises a VH containing the sequence of SEQ ID NO. 505.

[0136] In some embodiments, the second antigen-binding domain that binds to NKp30 comprises a VL having a sequence having at least 75% sequence identity with the sequence of SEQ ID NO. 13.

[0137] In some embodiments, the second antigen-binding domain that binds to NKp30 comprises a VL containing the sequence of SEQ ID NO. 13.

[0138] In some embodiments, the second antigen-binding domain that binds to NKp30 comprises a VH having at least 75% sequence identity with the sequence of SEQ ID NO. 505 and a VL having at least 75% sequence identity with the sequence of SEQ ID NO. 13.

[0139] In some embodiments, the second antigen-binding domain that binds to NKp30 comprises VH containing the sequence of SEQ ID NO. 505 and VL containing the sequence of SEQ ID NO. 13.

[0140] In some embodiments, the second antigen-binding domain that binds to NKp30 comprises a heavy chain variable region (VH) comprising:

[0141] (i) A VHCDR1 comprising a sequence having at least 75% sequence identity with any one sequence selected from the group of VHCDR1 sequences listed in Tables 20A, 20B, 21A, 21B, 23A, 23B, 25, 26, 16, and 17;

[0142] (ii) A VHCDR2 comprising a sequence having at least 75% sequence identity with any one sequence selected from the group consisting of VHCDR2 sequences listed in Tables 20A, 20B, 21A, 21B, 23A, 23B, 25, 26, 16, and 17;

[0143] (iii) a VHCDR3 comprising a sequence having at least 75% sequence identity with any one sequence selected from the group consisting of VHCDR3 sequences listed in Tables 20A, 20B, 21A, 21B, 23A, 23B, 25, 26, 16, and 17; or

[0144] (iv) Any combination of these.

[0145] In some embodiments, the second antigen-binding domain that binds to NKp30 comprises a VH comprising the following:

[0146] (i) A VHCDR1 comprising any one sequence selected from the group consisting of VHCDR1 sequences listed in Tables 20A, 20B, 21A, 21B, 23A, 23B, 25, 26, 16, and 17;

[0147] (ii) A VHCDR2 comprising any one sequence selected from the group consisting of VHCDR2 sequences listed in Tables 20A, 20B, 21A, 21B, 23A, 23B, 25, 26, 16, and 17;

[0148] (iii) a VHCDR3 comprising any one sequence selected from the group consisting of VHCDR3 sequences listed in Tables 20A, 20B, 21A, 21B, 23A, 23B, 25, 26, 16, and 17; or

[0149] (iv) Any combination of these.

[0150] In some embodiments, the second antigen-binding domain that binds to NKp30 comprises a VH having at least 75% sequence identity with any one sequence selected from the group of VH sequences listed in Tables 25, 26, 16, and 17.

[0151] In some embodiments, the second antigen-binding domain that binds to NKp30 comprises a VH comprising any one sequence selected from the group consisting of VH sequences listed in Tables 25, 26, 16, and 17.

[0152] In some embodiments, the second antigen-binding domain that binds to NKp30 comprises a VL comprising the following:

[0153] (i) A VLCDR1 comprising a sequence having at least 75% sequence identity with any one sequence selected from the group of VLCDR1 sequences listed in Tables 22, 24, 25, 26, 16, and 17;

[0154] (ii) A VLCDR2 comprising a sequence having at least 75% sequence identity with any one sequence selected from the group of VLCDR2 sequences listed in Tables 22, 24, 25, 26, 16, and 17;

[0155] (iii) a VLCDR3 comprising a sequence having at least 75% sequence identity with any one sequence selected from the group consisting of VLCDR3 sequences listed in Tables 22, 24, 25, 26, 16, and 17; or

[0156] (iv) Any combination of these.

[0157] In some embodiments, the second antigen-binding domain that binds to NKp30 comprises a VL comprising the following:

[0158] (i) A VLCDR1 comprising any one sequence selected from the group consisting of VLCDR1 sequences listed in Tables 22, 24, 25, 26, 16, and 17;

[0159] (ii) A VLCDR2 comprising any one sequence selected from the group consisting of VLCDR2 sequences listed in Tables 22, 24, 25, 26, 16, and 17;

[0160] (iii) a VLCDR3 comprising any one sequence selected from the group consisting of VLCDR3 sequences listed in Tables 22, 24, 25, 26, 16, and 17; or

[0161] (iv) Any combination of these.

[0162] In some embodiments, the second antigen-binding domain that binds to NKp30 comprises a VL having at least 75% sequence identity with any one sequence selected from the group consisting of VH sequences listed in Tables 25, 26, 16, and 17.

[0163] In some embodiments, the second antigen-binding domain that binds to NKp30 comprises a VL comprising any one sequence selected from the group consisting of VL sequences listed in Tables 25, 26, 16, and 17.

[0164] In some embodiments, the formulation includes immune cell binders.

[0165] In some embodiments, the immune cell binder is selected from the group consisting of T cell binders, NK cell binders, B cell binders, dendritic cell binders, and macrophage cell binders.

[0166] In some embodiments, the formulation includes a T cell binder.

[0167] In some embodiments, the T cell binder includes an antigen-binding domain that binds to the T cell receptor beta chain region (TCRβV).

[0168] In some embodiments, the antigen-binding domain that binds to the T cell receptor beta chain region (TCRβV) binds to the TCRβV of any one T cell receptor beta chain (TCRβ) subfamily member selected from the group consisting of the TCRβ subfamily members listed in Tables 29 and 42A.

[0169] In some embodiments, the antigen-binding domain that binds to TCRβV comprises a heavy chain variable region (VH) comprising the following:

[0170] (i) A VHCDR1 comprising a sequence having at least 75% sequence identity with any one sequence selected from the group consisting of VHCDR1 sequences listed in Tables 30, 32, 42B, 42C, 43A, 43B, and 43–51;

[0171] (ii) A VHCDR2 comprising a sequence having at least 75% sequence identity with any one sequence selected from the group consisting of VHCDR2 sequences listed in Tables 30, 32, 42B, 42C, 43A, 43B, and 43–51;

[0172] (iii) a VHCDR3 comprising a sequence having at least 75% sequence identity with any one sequence selected from the group consisting of VHCDR3 sequences listed in Tables 30, 32, 42B, 42C, 43A, 43B, and 43–51; or

[0173] (iv) Any combination of these.

[0174] In some embodiments, the antigen-binding domain that binds to TCRβV comprises a VH comprising the following:

[0175] (i) A VHCDR1 comprising any one sequence selected from the group consisting of VHCDR1 sequences listed in Tables 30, 32, 42B, 42C, 43A, 43B, and 43–51;

[0176] (ii) A VHCDR2 comprising any one sequence selected from the group consisting of VHCDR2 sequences listed in Tables 30, 32, 42B, 42C, 43A, 43B, and 43–51;

[0177] (iii) a VHCDR3 comprising any one sequence selected from the group consisting of VHCDR3 sequences listed in Tables 30, 32, 42B, 42C, 43A, 43B, and 43–51; or

[0178] (iv) Any combination of these.

[0179] In some embodiments, the antigen-binding domain that binds to TCRβV comprises a VH having at least 75% sequence identity with any one sequence selected from the group consisting of VH sequences listed in Tables 30, 32, 42B, 42C, 43A, 43B, and 43–51.

[0180] In some embodiments, the antigen-binding domain that binds to TCRβV comprises a VH comprising any one sequence selected from the group consisting of VH sequences listed in Tables 30, 32, 42B, 42C, 43A, 43B, and 43 to 51.

[0181] In some embodiments, the antigen-binding domain that binds to TCRβV comprises a VL containing the following:

[0182] (i) A VLCDR1 comprising a sequence having at least 75% sequence identity with any one sequence selected from the group consisting of VLCDR1 sequences listed in Tables 30, 32, 42B, 42C, 43A, 43B, and 43–51;

[0183] (ii) A VLCDR2 comprising a sequence having at least 75% sequence identity with any one sequence selected from the group consisting of VLCDR2 sequences listed in Tables 30, 32, 42B, 42C, 43A, 43B, and 43–51;

[0184] (iii) a VLCDR3 comprising a sequence having at least 75% sequence identity with any one sequence selected from the group consisting of VLCDR3 sequences listed in Tables 30, 32, 42B, 42C, 43A, 43B, and 43–51; or

[0185] (iv) Any combination of these.

[0186] In some embodiments, the antigen-binding domain that binds to TCRβV comprises a VL containing the following:

[0187] (i) A VLCDR1 comprising any one sequence selected from the group consisting of VLCDR1 sequences listed in Tables 30, 32, 42B, 42C, 43A, 43B, and 43–51;

[0188] (ii) A VLCDR2 comprising any one sequence selected from the group consisting of VLCDR2 sequences listed in Tables 30, 32, 42B, 42C, 43A, 43B, and 43–51;

[0189] (iii) a VLCDR3 comprising any one sequence selected from the group consisting of VLCDR3 sequences listed in Tables 30, 32, 42B, 42C, 43A, 43B, and 43–51; or

[0190] (iv) Any combination of these.

[0191] In some embodiments, the antigen-binding domain that binds to TCRβV comprises a VL having at least 75% sequence identity with any one sequence selected from the group consisting of VL sequences listed in Tables 30, 32, 42B, 42C, 43A, 43B, and 43–51.

[0192] In some embodiments, the antigen-binding domain that binds to TCRβV comprises a VL comprising any one sequence selected from the group consisting of VL sequences listed in Tables 30, 32, 42B, 42C, 43A, 43B, and 43 to 51.

[0193] In some embodiments, the formulation includes an NK cell binder.

[0194] In some embodiments, the NK cell binder does not bind to NKp30.

[0195] In some embodiments, the NK cell binder comprises a sequence having at least 75% sequence identity with any one sequence selected from the group consisting of the sequences listed in Table 27.

[0196] In some embodiments, the NK cell binder comprises any one sequence selected from the group consisting of the sequences listed in Table 27.

[0197] In some embodiments, the formulation includes a tumor-targeting moiety.

[0198] In some embodiments, the antigen-binding domain that binds to TRBC, the second antigen-binding domain that binds to NKp30, the immune cell binder, the tumor-targeting moiety, or any combination thereof is a whole antibody or its antigen-binding fragment, a divalent antibody, a bispecific antibody, or a biparatope antibody.

[0199] In some embodiments, the antigen-binding domain that binds to TRBC, the second antigen-binding domain that binds to NKp30, the immune cell binder, the tumor-targeting moiety, or any combination thereof is Fab, F(ab')2, Fv, short-chain Fv(scFv), Diabadi, a single-domain antibody, or a camelid antibody.

[0200] In some embodiments, the antigen-binding domain that binds to TRBC, the second antigen-binding domain that binds to NKp30, the immune cell binder, the tumor-targeting moiety, or any combination thereof is Fab or scFv.

[0201] In some embodiments, an antigen-binding domain that binds to TRBC, a second antigen-binding domain that binds to NKp30, an immune cell binder, a tumor-targeting moiety, or any combination thereof comprises one or more heavy-chain constant regions selected from the group consisting of an IgG1 heavy-chain constant region or a functional fragment thereof, an IgG2 heavy-chain constant region or a functional fragment thereof, an IgG3 heavy-chain constant region or a functional fragment thereof, an IgGA1 heavy-chain constant region or a functional fragment thereof, an IgGA2 heavy-chain constant region or a functional fragment thereof, an IgG4 heavy-chain constant region or a functional fragment thereof, an IgJ heavy-chain constant region or a functional fragment thereof, an IgM heavy-chain constant region or a functional fragment thereof, an IgD heavy-chain constant region or a functional fragment thereof, and an IgE heavy-chain constant region or a functional fragment thereof.

[0202] In some embodiments, one or more heavy chain invariant regions comprise a sequence having at least 75% sequence identity with any one of the heavy chain invariant region sequences listed in Tables 3, 7, 8, 16, 17, 39, 40, 42C, 43A, 43B, and 46–51.

[0203] In some embodiments, one or more heavy chain invariant regions comprise a sequence having at least 75% sequence identity with the sequence of SEQ ID NO. 73, the sequence of SEQ ID NO. 76, the sequence of SEQ ID NO. 74, the sequence of SEQ ID NO. 75, the sequence of SEQ ID NO. 76, the sequence of SEQ ID NO. 40, the sequence of SEQ ID NO. 41, the sequence of SEQ ID NO. 42, the sequence of SEQ ID NO. 3645, the sequence of SEQ ID NO. 3646, the sequence of SEQ ID NO. 3649, the sequence of SEQ ID NO. 3451, the sequence of SEQ ID NO. 506, the sequence of SEQ ID NO. 507, the sequence of SEQ ID NO. 3647, the sequence of SEQ ID NO. 3648, the sequence of SEQ ID NO. 508 or 8686, the sequence of SEQ ID NO. 513, the sequence of SEQ ID NO. 514, the sequence of SEQ ID NO. 515, the sequence of SEQ ID NO. 516, the sequence of SEQ ID NO. 3447, or the sequence of SEQ ID NO. 511.

[0204] In some embodiments, an antigen-binding domain that binds to TRBC, a second antigen-binding domain that binds to NKp30, an immune cell binder, a tumor-targeting moiety, or any combination thereof comprises a kappa light chain constant region or a functional fragment thereof, a lambda light chain constant region or a functional fragment thereof, or a combination thereof.

[0205] In some embodiments, the kappa light chain invariant region comprises a sequence having at least 75% sequence identity with any one of the light chain invariant region sequences listed in Tables 3, 7, 8, 16, 17, 39, 40, 42C, 43A, 43B, and 46 to 51.

[0206] In some embodiments, the kappa light chain invariant region comprises a sequence having at least 75% sequence identity with the sequence of SEQ ID NO. 39 or the sequence of SEQ ID NO. 3644.

[0207] In some embodiments, a first member of the dimerization module includes a first Fc region, and a second member of the dimerization module includes a second Fc region.

[0208] In some embodiments, the first Fc region is selected from the group consisting of IgG1 Fc region or its functional fragment, IgG2 Fc region or its functional fragment, IgG3 Fc region or its functional fragment, IgGA1 Fc region or its functional fragment, IgGA2 Fc region or its functional fragment, IgG4 Fc region or its functional fragment, IgJ Fc region or its functional fragment, IgM Fc region or its functional fragment, IgD Fc region or its functional fragment, and IgE Fc region or its functional fragment.

[0209] In some embodiments, the second Fc region is selected from the group consisting of IgG1 Fc region or its functional fragment, IgG2 Fc region or its functional fragment, IgG3 Fc region or its functional fragment, IgGA1 Fc region or its functional fragment, IgGA2 Fc region or its functional fragment, IgG4 Fc region or its functional fragment, IgJ Fc region or its functional fragment, IgM Fc region or its functional fragment, IgD Fc region or its functional fragment, and IgE Fc region or its functional fragment.

[0210] In some embodiments, the first Fc region, the second Fc region, or a combination thereof comprises an Fc interface having one or more of a paired co-protrusion, electrostatic interaction, or strand-exchange, and the dimerization of the first Fc region and the second Fc region is enhanced as indicated by a greater ratio of heteropolymer to homopolymer form compared to the dimerization of the Fc region having a non-operated interface.

[0211] In some implementations,

[0212] (i) The first Fc region includes the following:

[0213] (a) Ala at location 297 of the heavy chain invariant region according to EU numbering,

[0214] (b) Cys at location 349 of the heavy chain invariant region according to EU numbering,

[0215] (c) Ser at location 366 of the heavy chain invariant region according to EU numbering,

[0216] (d) Ala at position 368 of the heavy chain invariant region according to EU numbering; and

[0217] (e) Val at position 407 of the heavy chain invariant region according to EU numbering; and

[0218] (ii) The second Fc region includes the following:

[0219] (a) Ala at location 297 of the heavy chain invariant region according to EU numbering,

[0220] (b) Cys at position 354 of the heavy chain invariant region according to EU numbering, and

[0221] (c) Trp at location 366 of the heavy chain invariant region according to EU numbering.

[0222] In some implementations,

[0223] (i) The first Fc region includes the following:

[0224] (a) Ala at location 297 of the heavy chain invariant region according to EU numbering,

[0225] (b) Cys at position 354 of the heavy chain invariant region according to EU numbering, and

[0226] (c) Trp at position 366 of the heavy chain invariant region according to EU numbering; and

[0227] (ii) The second Fc region includes the following:

[0228] (a) Ala at location 297 of the heavy chain invariant region according to EU numbering,

[0229] (b) Cys at location 349 of the heavy chain invariant region according to EU numbering,

[0230] (c) Ser at location 366 of the heavy chain invariant region according to EU numbering,

[0231] (d) Ala at position 368 of the heavy chain invariant region according to EU numbering; and

[0232] (e) Val. at location 407 of the heavy chain invariant region according to EU numbering.

[0233] In some embodiments, the first Fc region includes a mutation that reduces Fc receptor binding to the formulation compared to the formulation without the mutation.

[0234] In some embodiments, the first Fc region comprises an Asn297Ala (N297A) mutation or a Leu234Ala / Leu235Ala (LALA) mutation according to EU numbering, or a combination thereof.

[0235] In some embodiments, the second Fc region includes a mutation that reduces Fc receptor binding to the formulation compared to the formulation without the mutation.

[0236] In some embodiments, the second Fc region comprises an Asn297Ala (N297A) mutation or a Leu234Ala / Leu235Ala (LALA) mutation according to EU numbering, or a combination thereof.

[0237] In some embodiments, the first Fc region comprises a sequence having at least 75% sequence identity with any one sequence selected from the group consisting of the sequence of SEQ ID NO. 73, the sequence of SEQ ID NO. 76, the sequence of SEQ ID NO. 74, the sequence of SEQ ID NO. 75, the sequence of SEQ ID NO. 40, the sequence of SEQ ID NO. 41, the sequence of SEQ ID NO. 42, the sequence of SEQ ID NO. 3645, the sequence of SEQ ID NO. 3646, the sequence of SEQ ID NO. 3649, the sequence of SEQ ID NO. 8687, the sequence of SEQ ID NO. 506, the sequence of SEQ ID NO. 507, the sequence of SEQ ID NO. 3647, the sequence of SEQ ID NO. 3648, the sequence of SEQ ID NO. 508 or 8686, the sequence of SEQ ID NO. 513, the sequence of SEQ ID NO. 514, the sequence of SEQ ID NO. 515, the sequence of SEQ ID NO. 516, the sequence of SEQ ID NO. 3447, and the sequence of SEQ ID NO. 511.

[0238] In some embodiments, the second Fc region comprises a sequence having at least 75% sequence identity with any one sequence selected from the group consisting of the sequence of SEQ ID NO. 73, the sequence of SEQ ID NO. 76, the sequence of SEQ ID NO. 74, the sequence of SEQ ID NO. 75, the sequence of SEQ ID NO. 40, the sequence of SEQ ID NO. 41, the sequence of SEQ ID NO. 42, the sequence of SEQ ID NO. 3645, the sequence of SEQ ID NO. 3646, the sequence of SEQ ID NO. 3649, the sequence of SEQ ID NO. 8687, the sequence of SEQ ID NO. 506, the sequence of SEQ ID NO. 507, the sequence of SEQ ID NO. 3647, the sequence of SEQ ID NO. 3648, the sequence of SEQ ID NO. 508 or 8686, the sequence of SEQ ID NO. 513, the sequence of SEQ ID NO. 514, the sequence of SEQ ID NO. 515, the sequence of SEQ ID NO. 516, the sequence of SEQ ID NO. 3447, and the sequence of SEQ ID NO. 511.

[0239] In some embodiments, the first Fc region comprises any one sequence selected from the group consisting of the sequence of SEQ ID NO. 73, the sequence of SEQ ID NO. 76, the sequence of SEQ ID NO. 74, the sequence of SEQ ID NO. 75, the sequence of SEQ ID NO. 40, the sequence of SEQ ID NO. 41, the sequence of SEQ ID NO. 42, the sequence of SEQ ID NO. 3645, the sequence of SEQ ID NO. 3646, the sequence of SEQ ID NO. 3649, the sequence of SEQ ID NO. 8687, the sequence of SEQ ID NO. 506, the sequence of SEQ ID NO. 507, the sequence of SEQ ID NO. 3647, the sequence of SEQ ID NO. 3648, the sequence of SEQ ID NO. 508 or 8686, the sequence of SEQ ID NO. 513, the sequence of SEQ ID NO. 514, the sequence of SEQ ID NO. 515, the sequence of SEQ ID NO. 516, the sequence of SEQ ID NO. 3447, and the sequence of SEQ ID NO. 511.

[0240] In some embodiments, the second Fc region comprises any one sequence selected from the group consisting of the sequence of SEQ ID NO. 73, the sequence of SEQ ID NO. 76, the sequence of SEQ ID NO. 74, the sequence of SEQ ID NO. 75, the sequence of SEQ ID NO. 40, the sequence of SEQ ID NO. 41, the sequence of SEQ ID NO. 42, the sequence of SEQ ID NO. 3645, the sequence of SEQ ID NO. 3646, the sequence of SEQ ID NO. 3649, the sequence of SEQ ID NO. 8687, the sequence of SEQ ID NO. 506, the sequence of SEQ ID NO. 507, the sequence of SEQ ID NO. 3647, the sequence of SEQ ID NO. 3648, the sequence of SEQ ID NO. 508 or 8686, the sequence of SEQ ID NO. 513, the sequence of SEQ ID NO. 514, the sequence of SEQ ID NO. 515, the sequence of SEQ ID NO. 516, the sequence of SEQ ID NO. 3447, and the sequence of SEQ ID NO. 511.

[0241] In some embodiments, the first Fc region comprises a sequence having at least 75% sequence identity with any one sequence selected from the group consisting of the sequence of SEQ ID NO. 3648, the sequence of SEQ ID NO. 508 or 8686, the sequence of SEQ ID NO. 506, the sequence of SEQ ID NO. 507, the sequence of SEQ ID NO. 3477, the sequence of SEQ ID NO. 511, the sequence of SEQ ID NO. 515, and the sequence of SEQ ID NO. 516.

[0242] In some embodiments, the second Fc region comprises a sequence having at least 75% sequence identity with the sequence of SEQ ID NO. 3648, the sequence of SEQ ID NO. 508 or 8686, the sequence of SEQ ID NO. 506, the sequence of SEQ ID NO. 507, the sequence of SEQ ID NO. 3477, the sequence of SEQ ID NO. 511, the sequence of SEQ ID NO. 515, or the sequence of SEQ ID NO. 516.

[0243] In some embodiments, the first Fc region comprises any one sequence selected from the group consisting of sequences of SEQ ID NO. 3648, SEQ ID NO. 508, SEQ ID NO. 506, SEQ ID NO. 507, SEQ ID NO. 3477, SEQ ID NO. 511, SEQ ID NO. 515, or SEQ ID NO. 516.

[0244] In some embodiments, the second Fc region comprises any one sequence selected from the group consisting of the sequences of SEQ ID NO. 3648, SEQ ID NO. 508, SEQ ID NO. 506, SEQ ID NO. 507, SEQ ID NO. 3477, SEQ ID NO. 511, SEQ ID NO. 515, or SEQ ID NO. 516.

[0245] In some implementations,

[0246] (i) The first Fc region comprises the sequence of SEQ ID NO. 506 or the sequence of SEQ ID NO. 507, and the second Fc region comprises the sequence of SEQ ID NO. 3648 or the sequence of SEQ ID NO. 508 or 8686;

[0247] (ii) The first Fc region includes the sequence of SEQ ID NO. 3447 or the sequence of SEQ ID NO. 511, and the second Fc region includes the sequence of SEQ ID NO. 515 or the sequence of SEQ ID NO. 516.

[0248] In some implementations,

[0249] (i) The first Fc region comprises the sequence of SEQ ID NO. 3648 or the sequence of SEQ ID NO. 508 or 8686, and the second Fc region comprises the sequence of SEQ ID NO. 506 or the sequence of SEQ ID NO. 507;

[0250] (ii) The first Fc region includes the sequence of SEQ ID NO. 515 or the sequence of SEQ ID NO. 516, and the second Fc region includes the sequence of SEQ ID NO. 3447 or the sequence of SEQ ID NO. 511.

[0251] In some embodiments, the formulation comprises a first polypeptide, a second polypeptide, and at least one cytokine molecule or a functional fragment or variant thereof, wherein the first polypeptide and the second polypeptide are not adjacent;

[0252] (i) The first polypeptide is

[0253] (a) Connected to an antigen-binding domain including VH and VL, or

[0254] (b) comprising a first member of a dimerization module connected to a first portion of an antigen-binding domain containing VH, wherein the formulation further comprises a third polypeptide comprising a second portion of an antigen-binding domain containing VL, and the third polypeptide is not adjacent to the first polypeptide and the second polypeptide; and

[0255] (ii) The second polypeptide comprises a second member of the dimerized molecule, and

[0256] Here, at least one cytokine molecule or a functional fragment or variant thereof is covalently linked to the second polypeptide or the first polypeptide.

[0257] In some embodiments, the formulation comprises a first polypeptide, a second polypeptide, and at least one cytokine molecule or a functional fragment or variant thereof, wherein the first polypeptide and the second polypeptide are not adjacent;

[0258] (i) the first polypeptide comprises a first member of a dimerization module connected to a first portion of an antigen-binding domain comprising VH, wherein the formulation further comprises a third polypeptide comprising a second portion of an antigen-binding domain comprising VL, and the third polypeptide is not adjacent to the first polypeptide and the second polypeptide; and

[0259] (ii) The second polypeptide comprises a second member of the dimerized molecule, and

[0260] Here, at least one cytokine molecule or a functional fragment or variant thereof is covalently linked to the second polypeptide.

[0261] In some embodiments, at least one cytokine molecule or a functional fragment or variant thereof is covalently linked to the N-terminus of the second polypeptide, the C-terminus of the second polypeptide, or a combination thereof.

[0262] In some embodiments, at least one cytokine molecule or a functional fragment or variant thereof is within a single continuous polypeptide chain of the second polypeptide.

[0263] In some embodiments, the first polypeptide comprises the following:

[0264] (i) an antigen-binding domain comprising VH and VL, wherein the antigen-binding domain further comprises a heavy chain constant domain 1 (CH1) connected to VH; or

[0265] (ii) A first portion of a first antigen-binding domain comprising VH, wherein the first portion of the antigen-binding domain further comprises CH1 connected to VH.

[0266] In some embodiments, CH1 is connected to the C-terminus of VH.

[0267] In some embodiments, the formulation includes the following:

[0268] (i) a first polypeptide comprising an antigen-binding domain comprising VH and VL, wherein the antigen-binding domain further comprises a light chain constant domain (CL) connected to VL; or

[0269] (ii) a first polypeptide comprising a first portion of an antigen-binding domain; and a third polypeptide comprising a second portion of an antigen-binding domain, wherein the second portion of the antigen-binding domain further comprises a CL connected to VL.

[0270] In some embodiments, CL is connected to the C-terminal of VL.

[0271] In some embodiments, a first member of the dimerization module is connected to the C-terminus of an antigen-binding domain comprising VH and VL, or to the C-terminus of a first portion of a first antigen-binding domain comprising VH.

[0272] In some embodiments, the second polypeptide comprises the following:

[0273] (i) an antigen-binding domain that binds to TRBC, a second antigen-binding domain that binds to NKp30, an immune cell binder, or a tumor-targeting moiety comprising VH and VL, wherein the antigen-binding domain that binds to TRBC, a second antigen-binding domain that binds to NKp30, an immune cell binder, or a tumor-targeting moiety further comprising a heavy chain constant domain 1 (CH1) connected to VH; or

[0274] (ii) a tumor targeting moiety comprising a first portion of an antigen-binding domain that binds to TRBC, a second antigen-binding domain that binds to NKp30, an immune cell binder, or VH, wherein the tumor targeting moiety further comprises a first portion of an antigen-binding domain that binds to TRBC, a second antigen-binding domain that binds to NKp30, an immune cell binder, or a tumor targeting moiety further comprising a CH1 connected to VH.

[0275] In some embodiments, CH1 is connected to the C-terminus of VH.

[0276] In some embodiments, the formulation includes the following:

[0277] (i) a second polypeptide comprising an antigen-binding domain that binds to TRBC, a second antigen-binding domain that binds to NKp30, an immune cell binder, or a tumor-targeting moiety comprising VH and VL, wherein the antigen-binding domain that binds to TRPC, the second antigen-binding domain that binds to NKp30, the immune cell binder, or the tumor-targeting moiety further comprises a light chain constant domain (CL) connected to VL; or

[0278] (ii) a second polypeptide comprising a first portion of an antigen-binding domain that binds to TRBC, a second antigen-binding domain that binds to NKp30, an immune cell binder, or a tumor-targeting moiety,

[0279] The formulation further comprises a fourth polypeptide comprising a second portion of an antigen-binding domain that binds to TRBC, a second antigen-binding domain that binds to NKp30, an immune cell binder, or a tumor-targeting moiety, wherein the fourth polypeptide is not adjacent to the first polypeptide, the second polypeptide, and the third polypeptide, and

[0280] A second polypeptide comprising a second portion of an antigen-binding domain that binds to TRBC, a second antigen-binding domain that binds to NKp30, an immune cell binder, or a tumor-targeting moiety further comprising a CL connected to VL.

[0281] In some embodiments, CL is connected to the C-terminal of VL.

[0282] In some embodiments, a second member of the dimerization module is connected to the C-terminus of a tumor targeting moiety comprising an antigen-binding domain that binds to TRBC, a second antigen-binding domain that binds to NKp30, an immune cell binder, or VH and VL, or is connected to the C-terminus of a tumor targeting moiety comprising a first portion of an antigen-binding domain that binds to TRBC, a second antigen-binding domain that binds to NKp30, an immune cell binder, or VH.

[0283] In some embodiments, the formulation further comprises the following:

[0284] (i) a linker between a first member of the dimerization module and an antigen-binding domain comprising VH and VL or a first portion of an antigen-binding domain comprising VH;

[0285] (ii) a linker between a second member of the dimerization module and an antigen-binding domain that binds to TRBC, a second antigen-binding domain that binds to NKp30, an immune cell binder, or a tumor-targeting moiety comprising VH and VL, or a first portion of the antigen-binding domain that binds to TRBC, a second antigen-binding domain that binds to NKp30, an immune cell binder, or a tumor-targeting moiety comprising VH;

[0286] (iii) a linker between the VH and VL of the antigen-binding domain;

[0287] (iv) Linker between CH1 and VH of the antigen-binding domain;

[0288] (v) Linker between CL and VL of the antigen-binding domain;

[0289] (vi) an antigen-binding domain that binds to TRBC, a second antigen-binding domain that binds to NKp30, an immune cell binder, or a linker between VH and VL of a tumor-targeting moiety;

[0290] (vii) an antigen-binding domain that binds to TRBC, a second antigen-binding domain that binds to NKp30, an immune cell binder, or a linker between CH1 and VH of a tumor-targeting moiety;

[0291] (viii) an antigen-binding domain that binds to TRBC, a second antigen-binding domain that binds to NKp30, an immune cell binder, or a linker between CL and VL of a tumor-targeting moiety;

[0292] (ix) a linker between at least one cytokine molecule or a functional fragment or variant thereof and a first polypeptide, a linker between at least one cytokine molecule or a functional fragment or variant thereof and a second polypeptide, or any combination thereof; or

[0293] (x) Any combination of these.

[0294] In some embodiments, the linker is selected from the group consisting of a cleavable linker, a non-cleavable linker, a peptide linker, a flexible linker, a rigid linker, a helical linker, and a non-helical linker.

[0295] In some embodiments, the linker is a peptide linker comprising the sequence of SEQ ID NO. 3473, SEQ ID NO. 3308, or SEQ ID NO. 8291.

[0296] In some embodiments, at least one cytokine molecule or a functional fragment or variant thereof is connected to the N-terminus of the first polypeptide, the C-terminus of the first polypeptide, the N-terminus of the second polypeptide, the C-terminus of the second polypeptide, the N-terminus of the third polypeptide, and the C-terminus of the third polypeptide, or any combination thereof.

[0297] In some embodiments, at least one cytokine molecule or a functional fragment or variant thereof is in a single continuous polypeptide chain of a first polypeptide, a second polypeptide, or a third cytokine polypeptide to which at least one cytokine molecule or a functional fragment or variant thereof is connected.

[0298] In some embodiments, the formulation includes the following:

[0299] (i) a first polypeptide comprising a first portion of a dimerization module connected to the C-terminus of a first portion of an antigen-binding domain;

[0300] (ii) a second polypeptide comprising a second portion of a dimerization module;

[0301] (iii) a third polypeptide comprising a second portion of an antigen-binding domain; and

[0302] (iv) At least one cytokine molecule or a functional fragment or variant thereof covalently linked to the N-terminus of the second polypeptide.

[0303] In some embodiments, the formulation includes the following:

[0304] (i) A first polypeptide chain comprising the sequence of SEQ ID NO. 505 and the sequence of SEQ ID NO. 3649 or SEQ ID NO. 3451;

[0305] (ii) a second polypeptide chain comprising the sequence of SEQ ID NO. 3472, the sequence of SEQ ID NO. 2170, and the sequence of SEQ ID NO. 3648 or SEQ ID NO. 508 or 8686; and

[0306] (iii) A third polypeptide comprising the sequence of SEQ ID NO. 13 and the sequence of SEQ ID NO. 3644.

[0307] In some embodiments, the formulation includes the following:

[0308] (i) A first polypeptide chain comprising the sequence of SEQ ID NO. 505 operably connected to the sequence of SEQ ID NO. 3649 or SEQ ID NO. 3451 in the direction from the N-terminus to the C-terminus;

[0309] (ii) a second polypeptide chain comprising the sequence of SEQ ID NO. 3472 operably linked to the sequence of SEQ ID NO. 2170 operably linked to the sequence of SEQ ID NO. 3648 or SEQ ID NO. 508 or 8686 in the direction from the N-terminus to the C-terminus; and

[0310] (iii) A third polypeptide comprising the sequence of SEQ ID NO. 13 operably connected to the sequence of SEQ ID NO. 3644 from the N-terminus to the C-terminus.

[0311] In some embodiments, the second polypeptide chain comprises, in the direction from the N-terminus to the C-terminus, the sequence of SEQ ID NO. 3472, which is operably connected to the sequence of SEQ ID NO. 2170, which is operably connected to the sequence of SEQ ID NO. 3473, which is operably connected to the sequence of SEQ ID NO. 3308, which is operably connected to the sequence of SEQ ID NO. 3648, SEQ ID NO. 508, or SEQ ID NO. 8686.

[0312] In some embodiments, the formulation includes the following:

[0313] (i) A first polypeptide chain comprising the sequence of SEQ ID NO. 505 and the sequence of SEQ ID NO. 3649 or SEQ ID NO. 3451;

[0314] (ii) a second polypeptide chain comprising the sequence of SEQ ID NO. 3474 and the sequence of SEQ ID NO. 3648 or SEQ ID NO. 508 or 8686; and

[0315] (iii) A third polypeptide comprising the sequence of SEQ ID NO. 13 and the sequence of SEQ ID NO. 3644.

[0316] In some embodiments, the formulation includes the following:

[0317] (i) A first polypeptide chain comprising the sequence of SEQ ID NO. 505 operably connected to the sequence of SEQ ID NO. 3649 or SEQ ID NO. 3451 in the direction from the N-terminus to the C-terminus;

[0318] (ii) a second polypeptide chain comprising the sequence of SEQ ID NO. 3474 operably connected to the sequence of SEQ ID NO. 3648 or SEQ ID NO. 508 or 8686 in the direction from the N-terminus to the C-terminus; and

[0319] (iii) A third polypeptide comprising the sequence of SEQ ID NO. 13 operably connected to the sequence of SEQ ID NO. 3644 from the N-terminus to the C-terminus.

[0320] In some embodiments, the second polypeptide comprises the sequence of SEQ ID NO. 3474, which is operably linked to the sequence of SEQ ID NO. 3308, which is operably linked to the sequence of SEQ ID NO. 3648, SEQ ID NO. 508, or SEQ ID NO. 8686, in the direction from the N-terminus to the C-terminus.

[0321] In some embodiments, the formulation includes the following:

[0322] (i) A first polypeptide chain comprising a sequence having at least 75% sequence identity with the sequence of SEQ ID NO. 500;

[0323] (ii) a second polypeptide chain comprising a sequence having at least 75% sequence identity with the sequence of SEQ ID NO. 3420; and

[0324] (iii) A third polypeptide comprising a sequence having at least 75% sequence identity with the sequence of SEQ ID NO. 501.

[0325] In some embodiments, the formulation includes the following:

[0326] (i) A first polypeptide chain comprising a sequence having at least 75% sequence identity with the sequence of SEQ ID NO. 4702;

[0327] (ii) a second polypeptide chain comprising a sequence having at least 75% sequence identity with the sequence of SEQ ID NO. 4703; and

[0328] (iii) A third polypeptide comprising a sequence having at least 75% sequence identity with the sequence of SEQ ID NO. 501.

[0329] In some embodiments, the formulation includes the following:

[0330] (i) A first polypeptide chain comprising the sequence of SEQ ID NO. 500;

[0331] (ii) a second polypeptide chain comprising the sequence of SEQ ID NO. 3420; and

[0332] (iii) A third polypeptide comprising the sequence of SEQ ID NO. 501.

[0333] In some embodiments, the formulation includes the following:

[0334] (i) A first polypeptide chain comprising the sequence of SEQ ID NO. 4702;

[0335] (ii) a second polypeptide chain comprising the sequence of SEQ ID NO. 3420; and

[0336] (iii) A third polypeptide comprising the sequence of SEQ ID NO. 4703. In some embodiments, the formulation comprises the following:

[0337] (i) A first polypeptide chain comprising the sequence of SEQ ID NO. 505 and the sequence of SEQ ID NO. 3649 or SEQ ID NO. 3451;

[0338] (ii) a second polypeptide chain comprising the sequence of SEQ ID NO. 3472, the sequence of SEQ ID NO. 3670, and the sequence of SEQ ID NO. 3648 or SEQ ID NO. 508 or 8686; and

[0339] (iii) A third polypeptide comprising the sequence of SEQ ID NO. 13 and the sequence of SEQ ID NO. 3644.

[0340] In some embodiments, the formulation includes the following:

[0341] (i) A first polypeptide chain comprising the sequence of SEQ ID NO. 505 operably connected to the sequence of SEQ ID NO. 3649 or SEQ ID NO. 3451 in the direction from the N-terminus to the C-terminus;

[0342] (ii) a second polypeptide chain comprising the sequence of SEQ ID NO. 3472 operably linked to the sequence of SEQ ID NO. 3670 operably linked to the sequence of SEQ ID NO. 3648 or SEQ ID NO. 508 or 8686 in the direction from the N-terminus to the C-terminus; and

[0343] (iii) A third polypeptide comprising the sequence of SEQ ID NO. 13 operably connected to the sequence of SEQ ID NO. 3644 from the N-terminus to the C-terminus.

[0344] In some embodiments, the second polypeptide comprises, in the direction from the N-terminus to the C-terminus, the sequence of SEQ ID NO. 3472, which is operably linked to the sequence of SEQ ID NO. 3308, which is operably linked to the sequence of SEQ ID NO. 3670, which is operably linked to the sequence of SEQ ID NO. 3473.

[0345] In some embodiments, the formulation includes the following:

[0346] (i) A first polypeptide chain comprising the sequence of SEQ ID NO. 505 and the sequence of SEQ ID NO. 3649 or SEQ ID NO. 3451;

[0347] (ii) a second polypeptide chain comprising the sequence of SEQ ID NO. 3671 and the sequence of SEQ ID NO. 3648 or SEQ ID NO. 508 or 8686; and

[0348] (iii) A third polypeptide comprising the sequence of SEQ ID NO. 13 and the sequence of SEQ ID NO. 3644.

[0349] In some embodiments, the formulation includes the following:

[0350] (i) A first polypeptide chain comprising the sequence of SEQ ID NO. 505 operably connected to the sequence of SEQ ID NO. 3649 or SEQ ID NO. 3451 in the direction from the N-terminus to the C-terminus;

[0351] (ii) a second polypeptide chain comprising the sequence of SEQ ID NO. 3671 operably connected to the sequence of SEQ ID NO. 3648 or SEQ ID NO. 508 or 8686 in the direction from the N-terminus to the C-terminus; and

[0352] (iii) A third polypeptide comprising the sequence of SEQ ID NO. 13 and the sequence of SEQ ID NO. 3644 in the direction from the N-terminus to the C-terminus.

[0353] In some embodiments, the second polypeptide comprises the sequence of SEQ ID NO. 3671 operably linked to the sequence of SEQ ID NO. 3308 operably linked to the sequence of SEQ ID NO. 3648 or SEQ ID NO. 508 or 8686 in the direction from the N-terminus to the C-terminus.

[0354] In some embodiments, the formulation includes the following:

[0355] (i) A first polypeptide chain comprising a sequence having at least 75% sequence identity with the sequence of SEQ ID NO. 500;

[0356] (ii) a second polypeptide chain comprising a sequence having at least 75% sequence identity with the sequence of SEQ ID NO. 3669; and

[0357] (iii) A third polypeptide comprising a sequence having at least 75% sequence identity with the sequence of SEQ ID NO. 501.

[0358] In some embodiments, the formulation includes the following:

[0359] (i) A first polypeptide chain comprising a sequence having at least 75% sequence identity with the sequence of SEQ ID NO. 4702;

[0360] (ii) a second polypeptide chain comprising a sequence having at least 75% sequence identity with the sequence of SEQ ID NO. 4708; and

[0361] (iii) A third polypeptide comprising a sequence having at least 75% sequence identity with the sequence of SEQ ID NO. 501.

[0362] In some embodiments, the formulation includes the following:

[0363] (i) A first polypeptide chain comprising the sequence of SEQ ID NO. 500;

[0364] (ii) a second polypeptide chain comprising the sequence of SEQ ID NO. 3669; and

[0365] (iii) A third polypeptide comprising the sequence of SEQ ID NO. 501.

[0366] In some embodiments, the formulation includes the following:

[0367] (i) A first polypeptide chain comprising the sequence of SEQ ID NO. 4702;

[0368] (ii) a second polypeptide chain comprising the sequence of SEQ ID NO. 4708; and

[0369] (iii) A third polypeptide comprising the sequence of SEQ ID NO. 501.

[0370] In some embodiments, the formulation includes the following:

[0371] (i) A first polypeptide chain comprising the sequence of SEQ ID NO. 505 and the sequence of SEQ ID NO. 3649 or SEQ ID NO. 3451;

[0372] (ii) a second polypeptide chain comprising the sequence of 2270 and the sequence of SEQ ID NO. 3648 or SEQ ID NO. 508 or 8686; and

[0373] (iii) A third polypeptide comprising the sequence of SEQ ID NO. 13 and the sequence of SEQ ID NO. 3644.

[0374] In some embodiments, the formulation includes the following:

[0375] (i) A first polypeptide chain comprising the sequence of SEQ ID NO. 505 operably connected to the sequence of SEQ ID NO. 3649 or SEQ ID NO. 3451 in the direction from the N-terminus to the C-terminus;

[0376] (ii) a second polypeptide chain comprising the sequence of 2270 operably connected to the sequence of SEQ ID NO. 3648 or SEQ ID NO. 508 or 8686 in the direction from the N-terminus to the C-terminus; and

[0377] (iii) A third polypeptide comprising the sequence of SEQ ID NO. 13 and the sequence of SEQ ID NO. 3644.

[0378] In some embodiments, the second polypeptide comprises the sequence of SEQ ID NO. 2270, which is operably linked to the sequence of SEQ ID NO. 3308, which is operably linked to the sequence of SEQ ID NO. 3648, SEQ ID NO. 508, or SEQ ID NO. 8686, in the direction from the N-terminus to the C-terminus.

[0379] In some embodiments, the formulation includes the following:

[0380] (i) A first polypeptide chain comprising a sequence having at least 75% sequence identity with the sequence of SEQ ID NO. 500;

[0381] (ii) a second polypeptide chain comprising a sequence having at least 75% sequence identity with the sequence of SEQ ID NO. 3340; and

[0382] (iii) A third polypeptide comprising a sequence having at least 75% sequence identity with the sequence of SEQ ID NO. 501.

[0383] In some embodiments, the formulation includes the following:

[0384] (i) A first polypeptide chain comprising a sequence having at least 75% sequence identity with the sequence of SEQ ID NO. 4702;

[0385] (ii) a second polypeptide chain comprising a sequence having at least 75% sequence identity with the sequence of SEQ ID NO. 4706; and

[0386] (iii) A third polypeptide comprising a sequence having at least 75% sequence identity with the sequence of SEQ ID NO. 501.

[0387] In some embodiments, the formulation includes the following:

[0388] (i) A first polypeptide chain comprising the sequence of SEQ ID NO. 500;

[0389] (ii) a second polypeptide chain comprising the sequence of SEQ ID NO. 3340; and

[0390] (iii) A third polypeptide comprising the sequence of SEQ ID NO. 501.

[0391] In some embodiments, the formulation includes the following:

[0392] (i) A first polypeptide chain comprising the sequence of SEQ ID NO. 4702;

[0393] (ii) a second polypeptide chain comprising the sequence of SEQ ID NO. 4706; and

[0394] (iii) A third polypeptide comprising the sequence of SEQ ID NO. 501.

[0395] In some embodiments, the formulation comprises a first polypeptide and a second polypeptide, wherein the first polypeptide and the second polypeptide are not adjacent,

[0396] (i) The first polypeptide is

[0397] (a) connected to an antigen-binding domain comprising VH of the antigen-binding domain and VL of the antigen-binding domain,

[0398] (b) comprising a first member of a dimerization module connected to a first portion of an antigen-binding domain comprising VH of the antigen-binding domain, wherein the formulation further comprises a third polypeptide comprising a second portion of an antigen-binding domain comprising VL of the antigen-binding domain, and the third polypeptide is not adjacent to the first polypeptide and the second polypeptide; and

[0399] (ii) The second polypeptide is

[0400] (a) Connected to an antigen-binding domain that binds to a T cell receptor beta-chain constant domain (TRBC) including VH and VL, or

[0401] (b) a second member of a dimerization module connected to a first portion of an antigen-binding domain that binds to TRBC, comprising VH of an antigen-binding domain that binds to TRBC, wherein the formulation further comprises a fourth polypeptide comprising a second portion of an antigen-binding domain that binds to TRBC, comprising VL of an antigen-binding domain, and the fourth polypeptide is not adjacent to the first polypeptide, the second polypeptide, and the third polypeptide.

[0402] In some embodiments, the formulation comprises a first polypeptide and a second polypeptide, wherein the first polypeptide and the second polypeptide are not adjacent,

[0403] (i) the first polypeptide comprises a first member of a dimerization module connected to a first portion of an antigen-binding domain comprising VH of the antigen-binding domain, wherein the formulation further comprises a third polypeptide comprising a second portion of an antigen-binding domain comprising VL of the antigen-binding domain, and the third polypeptide is not adjacent to the first polypeptide and the second polypeptide; and

[0404] (ii) The second polypeptide comprises a second member of a dimerization module connected to an antigen-binding domain that binds to TRBC.

[0405] In some embodiments, the formulation includes the following:

[0406] (i) a first polypeptide chain comprising the sequence of SEQ ID NO. 505, the sequence of SEQ ID NO. 13, and the sequence of SEQ ID NO. 3447 or SEQ ID NO. 511;

[0407] (ii) a third polypeptide comprising the sequence of SEQ ID NO. 8308 and the sequence of SEQ ID NO. 3644; and

[0408] (iii) a second polypeptide chain comprising the sequence of SEQ ID NO. 517 and the sequence of SEQ ID NO. 513 or SEQ ID NO. 514; or a second polypeptide chain comprising the sequence of SEQ ID NO. 512 and the sequence of SEQ ID NO. 513 or SEQ ID NO. 514.

[0409] In some embodiments, the formulation includes the following:

[0410] (i) A first polypeptide chain comprising the sequence of SEQ ID NO. 505, operably connected to the sequence of SEQ ID NO. 13, operably connected to the sequence of SEQ ID NO. 3447 or SEQ ID NO. 511, in the direction from the N-terminus to the C-terminus;

[0411] (ii) a third polypeptide comprising the sequence of SEQ ID NO. 8308 operably linked to the sequence of SEQ ID NO. 3644 from the N-terminus to the C-terminus; and

[0412] (iii) a second polypeptide chain comprising the sequence of SEQ ID NO. 517 operably connected to the sequence of SEQ ID NO. 513 or SEQ ID NO. 514 in the direction from the N-terminus to the C-terminus; or a second polypeptide chain comprising the sequence of SEQ ID NO. 512 operably connected to the sequence of SEQ ID NO. 513 or SEQ ID NO. 514 in the direction from the N-terminus to the C-terminus.

[0413] In some embodiments, the first polypeptide comprises the sequence of SEQ ID NO. 505, which is operably linked to the sequence of SEQ ID NO. 8291, which is operably linked to the sequence of SEQ ID NO. 13, in the direction from the N-terminus to the C-terminus.

[0414] In some embodiments, the formulation includes the following:

[0415] (i) A first polypeptide chain comprising a sequence having at least 75% sequence identity with the sequence of SEQ ID NO. 509 or the sequence of SEQ ID NO. 4704;

[0416] (ii) a third polypeptide comprising a sequence having at least 75% sequence identity with the sequence of SEQ ID NO. 7380; and

[0417] (iii) A second polypeptide chain comprising a sequence having at least 75% sequence identity with the sequence of SEQ ID NO. 7382, the sequence of SEQ ID NO. 4677, the sequence of SEQ ID NO. 7379, or the sequence of 4675.

[0418] In some embodiments, the formulation includes the following:

[0419] (i) A first polypeptide chain comprising the sequence of SEQ ID NO. 509 or the sequence of SEQ ID NO. 4704;

[0420] (ii) a third polypeptide comprising the sequence of SEQ ID NO. 7380; and

[0421] (iii) A second polypeptide chain comprising the sequence of SEQ ID NO. 7382, the sequence of SEQ ID NO. 4677, the sequence of SEQ ID NO. 7379, or the sequence of 4675.

[0422] In some embodiments, the formulation comprises a first polypeptide chain comprising the sequence of SEQ ID NO. 505, the sequence of SEQ ID NO. 13, and the sequence of SEQ ID NO. 3447 or SEQ ID NO. 511.

[0423] In some embodiments, the formulation comprises a first polypeptide chain comprising the sequence of SEQ ID NO. 505, which is operably linked to the sequence of SEQ ID NO. 13, which is operably linked to the sequence of SEQ ID NO. 3447 or SEQ ID NO. 511, in the direction from the N-terminus to the C-terminus.

[0424] In some embodiments, the first polypeptide comprises the sequence of SEQ ID NO. 505, which is operably linked to the sequence of SEQ ID NO. 8291, which is operably linked to the sequence of SEQ ID NO. 13, in the direction from the N-terminus to the C-terminus.

[0425] In some embodiments, the formulation comprises a first polypeptide chain comprising a sequence having at least 75% sequence identity with the sequence of SEQ ID NO. 509.

[0426] In some embodiments, the formulation comprises a first polypeptide chain comprising the sequence of SEQ ID NO. 509.

[0427] In some embodiments, the formulation includes the following:

[0428] (i) a third polypeptide comprising the sequence of SEQ ID NO. 7419 and the sequence of SEQ ID NO. 3644; and

[0429] (ii) a second polypeptide chain comprising the sequence of SEQ ID NO. 7420 and the sequence of SEQ ID NO. 513 or SEQ ID NO. 514; or a second polypeptide chain comprising the sequence of SEQ ID NO. 7423 and the sequence of SEQ ID NO. 513 or SEQ ID NO. 514.

[0430] In some embodiments, the formulation includes the following:

[0431] (i) a third polypeptide comprising the sequence of SEQ ID NO. 7419 operably linked to the sequence of SEQ ID NO. 3644 from the N-terminus to the C-terminus; and

[0432] (ii) a second polypeptide chain comprising the sequence of SEQ ID NO. 7420 operably connected to the sequence of SEQ ID NO. 513 or SEQ ID NO. 514 in the direction from the N-terminus to the C-terminus; or a second polypeptide chain comprising the sequence of SEQ ID NO. 7423 operably connected to the sequence of SEQ ID NO. 513 or SEQ ID NO. 514.

[0433] In some embodiments, the formulation includes the following:

[0434] (i) a third polypeptide comprising a sequence having at least 75% sequence identity with the sequence of SEQ ID NO. 7439; and

[0435] (ii) A second polypeptide chain comprising a sequence having at least 75% sequence identity with the sequence of SEQ ID NO. 4680, the sequence of SEQ ID NO. 7438, the sequence of SEQ ID NO. 4681, or the sequence of SEQ ID NO. 7440.

[0436] In some embodiments, the formulation includes the following:

[0437] (i) a third polypeptide comprising the sequence of SEQ ID NO. 7439; and

[0438] (ii) A second polypeptide chain comprising the sequence of SEQ ID NO. 4680, the sequence of SEQ ID NO. 7438, the sequence of SEQ ID NO. 4681, or the sequence of 7440.

[0439] In some embodiments, the formulation includes the following:

[0440] (i) a third polypeptide comprising the sequence of SEQ ID NO. 8012 and the sequence of SEQ ID NO. 3644; and

[0441] (ii) A second polypeptide chain comprising the sequence of SEQ ID NO. 8011 and the sequence of SEQ ID NO. 513 or SEQ ID NO. 514.

[0442] In some embodiments, the formulation includes the following:

[0443] (i) a third polypeptide comprising the sequence of SEQ ID NO. 8012 operably linked to the sequence of SEQ ID NO. 3644 from the N-terminus to the C-terminus; and

[0444] (ii) A second polypeptide chain comprising the sequence of SEQ ID NO. 8011 operably connected to the sequence of SEQ ID NO. 513 or SEQ ID NO. 514 in the direction from the N-terminus to the C-terminus.

[0445] In some embodiments, the formulation includes the following:

[0446] (i) a third polypeptide comprising a sequence having at least 75% sequence identity with the sequence of SEQ ID NO. 8002; and

[0447] (ii) A second polypeptide chain comprising a sequence having at least 75% sequence identity with the sequence of SEQ ID NO. 8001 or 8697.

[0448] In some embodiments, the formulation includes the following:

[0449] (i) a third polypeptide comprising a sequence having at least 75% sequence identity with the sequence of SEQ ID NO. 8002; and

[0450] (ii) A second polypeptide chain comprising a sequence having at least 75% sequence identity with the sequence of SEQ ID NO. 4682.

[0451] In some embodiments, the formulation includes the following:

[0452] (i) a third polypeptide comprising the sequence of SEQ ID NO. 8002; and

[0453] (ii) A second polypeptide chain comprising the sequence of SEQ ID NO. 8001 or 8697.

[0454] In some embodiments, the formulation includes the following:

[0455] (i) a third polypeptide comprising the sequence of SEQ ID NO. 8002; and

[0456] (ii) A second polypeptide chain comprising the sequence of SEQ ID NO. 4682.

[0457] In some embodiments, the formulation includes the following:

[0458] (i) a third polypeptide comprising the sequence of SEQ ID NO. 8014 and the sequence of SEQ ID NO. 3644; and

[0459] (ii) a second polypeptide chain comprising the sequence of SEQ ID NO. 8013 and the sequence of SEQ ID NO. 513 or SEQ ID NO. 514, a second polypeptide chain comprising the sequence of SEQ ID NO. 8299 and the sequence of SEQ ID NO. 513 or SEQ ID NO. 514, or a second polypeptide chain comprising the sequence of SEQ ID NO. 8303 and the sequence of SEQ ID NO. 513 or SEQ ID NO. 514.

[0460] In some embodiments, the formulation includes the following:

[0461] (i) a third polypeptide comprising the sequence of SEQ ID NO. 8014 operably linked to the sequence of SEQ ID NO. 3644 from the N-terminus to the C-terminus; and

[0462] (ii) a second polypeptide chain comprising the sequence of SEQ ID NO. 8013 operably linked to the sequence of SEQ ID NO. 513 or SEQ ID NO. 514 in the direction from the N-terminus to the C-terminus, a second polypeptide chain comprising the sequence of SEQ ID NO. 8299 operably linked to the sequence of SEQ ID NO. 513 or SEQ ID NO. 514, or a second polypeptide chain comprising the sequence of SEQ ID NO. 8303 operably linked to the sequence of SEQ ID NO. 513 or SEQ ID NO. 514.

[0463] In some embodiments, the formulation includes the following:

[0464] (i) a third polypeptide comprising a sequence having at least 75% sequence identity with the sequence of SEQ ID NO. 8005; and

[0465] (ii) A second polypeptide chain comprising a sequence having at least 75% sequence identity with the sequence of SEQ ID NO. 8004, the sequence of SEQ ID NO. 4685, the sequence of SEQ ID NO. 8007, the sequence of SEQ ID NO. 4686, the sequence of SEQ ID NO. 4687, or the sequence of SEQ ID NO. 8009.

[0466] In some embodiments, the formulation includes the following:

[0467] (i) a third polypeptide comprising the sequence of SEQ ID NO. 8005; and

[0468] (ii) A second polypeptide chain comprising the sequence of SEQ ID NO. 8004, the sequence of SEQ ID NO. 4685, the sequence of SEQ ID NO. 8007, the sequence of SEQ ID NO. 4686, the sequence of SEQ ID NO. 4687, or the sequence of SEQ ID NO. 8009.

[0469] In some embodiments, the formulation further comprises at least one cytokine molecule or a functional fragment or variant thereof, wherein the at least one cytokine molecule or a functional fragment or variant thereof is connected to a first member of a dimerization module, a second member of a dimerization module, an antigen-binding domain, an antigen-binding domain that binds to TRBC, or any combination thereof.

[0470] In some embodiments, at least one cytokine molecule or a functional fragment or variant thereof comprises IL-2 or a functional fragment or variant thereof, IL-7 or a functional fragment or variant thereof, IL-12 or a functional fragment or variant thereof, IL-15 or a functional fragment or variant thereof, IL-18 or a functional fragment or variant thereof, IL-21 or a functional fragment or variant thereof, interferon gamma or a functional fragment or variant thereof, or any combination thereof.

[0471] In some embodiments, at least one cytokine molecule or a functional fragment or variant thereof comprises a sequence having at least 75% sequence identity with any one sequence selected from the group consisting of cytokine sequences listed in Tables 40, 42C, 43A, 43B, and 46–51.

[0472] In some embodiments, at least one cytokine molecule or a functional fragment or variant thereof comprises any one sequence selected from the group consisting of cytokine sequences listed in Tables 40, 42C, 43A, 43B, and 46–51.

[0473] In some embodiments, at least one cytokine molecule or a functional fragment or variant thereof comprises IL-2 or a functional fragment or variant thereof.

[0474] In some embodiments, IL-2 or a functional fragment or variant thereof comprises a sequence having at least 75% sequence identity with the sequence of SEQ ID NO. 2191, the sequence of SEQ ID NO. 2270, or the sequence of SEQ ID NO. 2280.

[0475] In some embodiments, IL-2 or a functional fragment or variant thereof comprises the sequence of SEQ ID NO. 2191, the sequence of SEQ ID NO. 2270, or the sequence of SEQ ID NO. 2280.

[0476] In some embodiments, at least one cytokine molecule or a functional fragment or variant thereof comprises IL-15 or a functional fragment or variant thereof.

[0477] In some embodiments, IL-15 or a functional fragment or variant thereof comprises a sequence having at least 75% sequence identity with the sequence of SEQ ID NO. 2170, SEQ ID NO. 3670, or SEQ ID NO. 3673.

[0478] In some embodiments, IL-15 or a functional fragment or variant thereof comprises the sequence of SEQ ID NO. 2170, the sequence of SEQ ID NO. 3670, or the sequence of SEQ ID NO. 3673.

[0479] In some embodiments, at least one cytokine molecule or its functional fragment or variant further comprises an IL15R-alpha dimerization domain covalently linked to IL-15 or its functional fragment or variant.

[0480] In some embodiments, the IL15R alpha dimerization domain comprises the IL-15 receptor alpha sushi domain or a functional fragment or variant thereof.

[0481] In some embodiments, the IL15R alpha dimerization domain comprises a sequence having at least 75% sequence identity with the sequence of SEQ ID NO. 3472.

[0482] In some embodiments, the IL15R alpha dimerization domain comprises the sequence of SEQ ID NO. 3472.

[0483] In some embodiments, IL-15 or a functional fragment or variant thereof is sharedly linked to the IL15R-alpha dimerization domain via a linker.

[0484] In some embodiments, IL-15 or a functional fragment or variant thereof is covalently linked to the IL15R-alpha dimerization domain through a linker containing the sequence of SEQ ID NO. 3473.

[0485] In some embodiments, at least one cytokine molecule or a functional fragment or variant thereof comprises a sequence having at least 75% sequence identity with the sequence of SEQ ID NO. 3474, SEQ ID NO. 3671, or SEQ ID NO. 8689.

[0486] In some embodiments, at least one cytokine molecule or a functional fragment or variant thereof comprises the sequence of SEQ ID NO. 3474, the sequence of SEQ ID NO. 3671, or the sequence of SEQ ID NO. 8689.

[0487] In some embodiments, at least one cytokine molecule or a functional fragment or variant thereof comprises IL-7 or a functional fragment or variant thereof.

[0488] In some embodiments, IL-7 or a functional fragment or variant thereof comprises a sequence having at least 75% sequence identity with the sequence of SEQ ID NO. 3540.

[0489] In some embodiments, IL-7 or a functional fragment or variant thereof comprises the sequence of SEQ ID NO. 3540.

[0490] In some embodiments, at least one cytokine molecule or a functional fragment or variant thereof comprises IL-12 or a functional fragment or variant thereof.

[0491] In some embodiments, IL-12 or a functional fragment or variant thereof comprises a sequence having at least 75% sequence identity with the sequence of SEQ ID NO. 3542, the sequence of SEQ ID NO. 3543, or the sequence of SEQ ID NO. 3545.

[0492] In some embodiments, IL-12 or a functional fragment or variant thereof comprises the sequence of SEQ ID NO. 3542, the sequence of SEQ ID NO. 3543, or the sequence of SEQ ID NO. 3545.

[0493] In some embodiments, at least one cytokine molecule or a functional fragment or variant thereof comprises IL-18 or a functional fragment or variant thereof.

[0494] In some embodiments, IL-18 or a functional fragment or variant thereof comprises a sequence having at least 75% sequence identity with the sequence of SEQ ID NO. 2192.

[0495] In some embodiments, IL-18 or a functional fragment or variant thereof comprises the sequence of SEQ ID NO. 2192.

[0496] In some embodiments, at least one cytokine molecule or a functional fragment or variant thereof comprises IL-21 or a functional fragment or variant thereof.

[0497] In some embodiments, IL-21 or a functional fragment or variant thereof comprises a sequence having at least 75% sequence identity with the sequence of SEQ ID NO. 3540.

[0498] In some embodiments, IL-21 or a functional fragment or variant thereof comprises the sequence of SEQ ID NO. 3540.

[0499] In another embodiment, a recombinant polynucleotide molecule comprising a polynucleotide sequence encoding a formulation of a composition as provided herein is provided herein.

[0500] In some embodiments, the recombinant polynucleotide molecule is an isolated recombinant polynucleotide molecule.

[0501] In another embodiment, a vector comprising a recombinant polynucleotide molecule as provided herein is provided herein.

[0502] In another embodiment, a cell comprising a recombinant polynucleotide molecule as provided herein or a vector as provided herein is provided herein.

[0503] In another embodiment, a method for preparing a formulation of any one of the compositions provided herein is provided, said method comprising the step of culturing cells provided herein under conditions suitable for the expression of the formulation.

[0504] In another embodiment, a pharmaceutical composition comprising a composition as provided herein, a recombinant polynucleotide molecule as provided herein, a vector as provided herein, or a cell as provided herein, and a pharmaceutically acceptable carrier, excipient, or diluent is provided herein.

[0505] In another aspect, a method for treating a condition or disease in a subject requiring treatment is provided herein, said method comprises the step of treating the condition or disease in the subject by administering to the subject a therapeutically effective amount of a composition as provided herein, a recombinant polynucleotide molecule as provided herein, a vector as provided herein, a cell as provided herein, a pharmaceutical composition as provided herein, or any combination thereof.

[0506] In some embodiments, the pathological condition or disease is cancer.

[0507] In some embodiments, the cancer is leukemia or lymphoma.

[0508] In some embodiments, the cancer is selected from the group consisting of acquired immunodeficiency syndrome (AIDS)-associated lymphoma, angioimmunoblastic T-cell lymphoma, adult T-cell leukemia / lymphoma, Burkitt lymphoma, central nervous system (CNS) lymphoma, diffuse large B-cell lymphoma (DLBCL), lymphoblastic lymphoma, mantle cell lymphoma (MCL), peripheral T-cell lymphoma (PTCL), transformed follicular and transformed mucosa-associated lymphoid tissue (MALT) lymphoma, cutaneous T-cell lymphoma (mycosis fungoides and Sézary syndrome), follicular lymphoma, lymphoplasmocyte lymphoma / Waldenstrom macroglobulinemia, marginal zone B-cell lymphoma, gastric mucosa-associated lymphoid tissue (MALT) lymphoma, chronic lymphocytic leukemia / small cell lymphocytic lymphoma (CLL / SLL), extranodal T- / NK-cell lymphoma, and anaplastic large cell lymphoma.

[0509] In some embodiments, peripheral T-cell lymphoma (PTCL) is hepatosplenic T-cell lymphoma (HSGDTCL), subcutaneous panniculitis-like T-cell lymphoma, or enteropathy-associated T-cell lymphoma.

[0510] In some embodiments, cutaneous T-cell lymphoma is mycosis fungoides and Sèzary syndrome.

[0511] In some embodiments, the extranodal T- / NK-cell lymphoma is of the nasal type.

[0512] In some embodiments, the anaplastic giant cell lymphoma is primary cutaneous anaplastic giant cell lymphoma or systemic anaplastic giant cell lymphoma.

[0513] In some embodiments, cancer is a solid tumor.

[0514] In some embodiments, the solid tumor is selected from the group consisting of pancreatic cancer, breast cancer, colorectal cancer, colon cancer, lung cancer, skin cancer, ovarian cancer, liver cancer, esophageal cancer, endometrial cancer, stomach cancer, head and neck cancer, kidney cancer, and prostate cancer.

[0515] In some embodiments, the pancreatic cancer is a pancreatic adenocarcinoma or a pancreatic ductal adenocarcinoma.

[0516] In some embodiments, the lung cancer is small cell lung cancer (SCLC) or non-small cell lung cancer (NSCLC).

[0517] In another embodiment, a method for proliferating and / or activating NK cells in an NK cell population is provided herein, the method comprising the step of proliferating and / or activating NK cells in an NK cell population by contacting an NK cell population with an effective amount of a composition as provided herein, a recombinant polynucleotide molecule as provided herein, a vector as provided herein, a cell as provided herein, a pharmaceutical composition as provided herein, or any combination thereof.

[0518] In some embodiments, the step of proliferating and / or activating NK cells is the step of proliferating and / or activating in vivo or in vitro. Brief explanation of the drawing

[0519] Novel features of the present invention are specifically set forth in the appended claims. The features and advantages of the present invention will be better understood by referring to the following detailed description and the accompanying drawings, which describe exemplary embodiments in which the principles of the present invention are used: Figures 1a~1d Figure [] is a schematic diagram of an exemplary format and composition of a formulation as provided herein. In some embodiments, the formulation comprises an antigen-binding domain that binds to NKp30 (“NKp30”) and an antigen-binding domain that binds to TRBC1 (“TRBC1”). In some embodiments, the antigen-binding domain that binds to NKp30 comprises scFv. In some embodiments, the antigen-binding domain that binds to NKp30 comprises Fab. In some embodiments, the antigen-binding domain that binds to TRBC1 comprises scFv: In some embodiments, the antigen-binding domain that binds to TRBC1 comprises Fab: Fig. 1a Figure 1 illustrates a formulation comprising anti-TRBC1 Fab, anti-NKp30 scFv, and an Fc dimer. The Fc dimer comprises two Fc chains. The C-terminus of the heavy chain of anti-TRBC1 Fab is fused to the N-terminus of one Fc chain. Anti-NKp30 scFv is fused to the N-terminus of the other Fc chain. Fig. 1b Figure 1 illustrates a formulation comprising anti-TRBC1 Fab, anti-NKp30 Fab, and an Fc dimer. The Fc dimer comprises two Fc chains. The C-terminus of the heavy chain of anti-TRBC1 Fab is fused to the N-terminus of one Fc chain. The C-terminus of the heavy chain of anti-NKp30 Fab is fused to the N-terminus of the other Fc chain. Fig. 1c Figure 1 illustrates a formulation comprising anti-TRBC1 scFv, anti-NKp30 Fab, and an Fc dimer. The Fc dimer comprises two Fc chains. Anti-TRBC1 scFv is fused to the N-terminus of the other Fc chain. The C-terminus of the heavy chain of anti-NKp30 Fab is fused to the N-terminus of one Fc chain. Fig. 1dThe figure illustrates a formulation comprising anti-NKp30 scFv, anti-TRBC1 scFv, and an Fc dimer. The Fc dimer comprises two Fc chains. Anti-TRBC1 scFv is fused to the N-terminus of one Fc chain. Anti-NKp30 scFv is fused to the N-terminus of the other Fc chain. In some embodiments, an antigen-binding domain that binds to NKp30 is connected to the C-terminus of a first member of the dimerization module or to the C-terminus of a second member of the dimerization module. In some embodiments, the formulation comprises two or more antigen-binding domains that bind to NKp30, and the two or more antigen-binding domains that bind to NKp30 are independently connected to the N-terminus of a first member of the dimerization module, the N-terminus of a second member of the dimerization module, the C-terminus of a first member of the dimerization module, the C-terminus of a second member of the dimerization module, or any combination thereof. In some embodiments, the antigen-binding domain that binds to TRBC1 is connected to the C-terminus of a first member of the dimerization module or the C-terminus of a second member of the dimerization module. In some embodiments, the formulation comprises two or more antigen-binding domains that bind to TRBC1, and the two or more antigen-binding domains that bind to TRBC1 are independently connected to the N-terminus of a first member of the dimerization module, the N-terminus of a second member of the dimerization module, the C-terminus of a first member of the dimerization module, the C-terminus of a second member of the dimerization module, or any combination thereof. Figures 2a~2dFigure [] is a schematic diagram of an exemplary format and composition of a formulation as provided herein. In some embodiments, the formulation comprises an antigen-binding domain that binds to NKp30 (“NKp30”) and an antigen-binding domain that binds to TRBC2 (“TRBC2”). In some embodiments, the antigen-binding domain that binds to NKp30 comprises scFv. In some embodiments, the antigen-binding domain that binds to NKp30 comprises Fab. In some embodiments, the antigen-binding domain that binds to TRBC2 comprises scFv: In some embodiments, the antigen-binding domain that binds to TRBC2 comprises Fab: Fig. 2a Figure 1 illustrates a formulation comprising anti-TRBC2 Fab, anti-NKp30 scFv, and an Fc dimer. The Fc dimer comprises two Fc chains. The C-terminus of the heavy chain of anti-TRBC2 Fab is fused to the N-terminus of one Fc chain. Anti-NKp30 scFv is fused to the N-terminus of the other Fc chain. Fig. 2b Figure 1 illustrates a formulation comprising anti-TRBC2 Fab, anti-NKp30 Fab, and an Fc dimer. The Fc dimer comprises two Fc chains. The C-terminus of the heavy chain of anti-TRBC2 Fab is fused to the N-terminus of one Fc chain. The C-terminus of the heavy chain of anti-NKp30 Fab is fused to the N-terminus of the other Fc chain. Fig. 2c Figure 1 illustrates a formulation comprising anti-TRBC2 scFv, anti-NKp30 Fab, and an Fc dimer. The Fc dimer comprises two Fc chains. Anti-TRBC2 scFv is fused to the N-terminus of the other Fc chain. The C-terminus of the heavy chain of anti-NKp30 Fab is fused to the N-terminus of one Fc chain. Fig. 2dThe figure illustrates a formulation comprising anti-NKp30 scFv, anti-TRBC2 scFv, and an Fc dimer. The Fc dimer comprises two Fc chains. Anti-TRBC2 scFv is fused to the N-terminus of one Fc chain. Anti-NKp30 scFv is fused to the N-terminus of the other Fc chain. In some embodiments, an antigen-binding domain that binds to NKp30 is connected to the C-terminus of a first member of the dimerization module or to the C-terminus of a second member of the dimerization module. In some embodiments, the formulation comprises two or more antigen-binding domains that bind to NKp30, and the two or more antigen-binding domains that bind to NKp30 are independently connected to the N-terminus of a first member of the dimerization module, the N-terminus of a second member of the dimerization module, the C-terminus of a first member of the dimerization module, the C-terminus of a second member of the dimerization module, or any combination thereof. In some embodiments, the antigen-binding domain that binds to TRBC2 is connected to the C-terminus of a first member of the dimerization module or the C-terminus of a second member of the dimerization module. In some embodiments, the formulation comprises two or more antigen-binding domains that bind to TRBC2, and the two or more antigen-binding domains that bind to TRBC2 are independently connected to the N-terminus of a first member of the dimerization module, the N-terminus of a second member of the dimerization module, the C-terminus of a first member of the dimerization module, the C-terminus of a second member of the dimerization module, or any combination thereof. Figures 3a~3b ...is a schematic diagram of an exemplary format and composition of a formulation as provided herein. In some embodiments, the formulation comprises an antigen-binding domain that binds to NKp30 (“Nkp30”) and at least one cytokine molecule or a functional fragment or variant thereof (“cytokine”). In some embodiments, the antigen-binding domain that binds to NKp30 comprises scFv. In some embodiments, the antigen-binding domain that binds to NKp30 comprises Fab. Fig. 3aFigure 1 illustrates a formulation comprising anti-NKp30 scFv, a cytokine molecule, and an Fc dimer. The Fc dimer comprises two Fc chains. Anti-NKp30 scFv is fused to the N-terminus of one Fc chain. The cytokine molecule is fused to the N-terminus of the other Fc chain. Fig. 3b The figure illustrates a formulation comprising anti-NKp30 Fab, a cytokine molecule, and an Fc dimer. The Fc dimer comprises two Fc chains. The C-terminus of the heavy chain of anti-NKp30 Fab is fused to the N-terminus of one Fc chain. The cytokine molecule is fused to the N-terminus of the other Fc chain. In some embodiments, an antigen-binding domain that binds to NKp30 is connected to the C-terminus of a first member of the dimerization module or to the C-terminus of a second member of the dimerization module. In some embodiments, the formulation comprises two or more antigen-binding domains that bind to NKp30, and the two or more antigen-binding domains that bind to NKp30 are independently connected to the N-terminus of a first member of the dimerization module, the N-terminus of a second member of the dimerization module, the C-terminus of a first member of the dimerization module, the C-terminus of a second member of the dimerization module, or any combination thereof. In some embodiments, at least one cytokine molecule or a functional fragment or variant thereof is connected to the C-terminus of a first member of the dimerization module or the C-terminus of a second member of the dimerization module. In some embodiments, the formulation comprises two or more cytokine molecules or functional fragments or variants thereof, and the two or more cytokine molecules or functional fragments or variants thereof are independently connected to the N-terminus of a first member of a dimerization module, the N-terminus of a second member of a dimerization module, the C-terminus of a first member of a dimerization module, the C-terminus of a second member of a dimerization module, or any combination thereof. In some embodiments, the formulation comprises an antigen-binding domain that binds to NKp30, an antigen-binding domain that binds to TRBC1, and at least one cytokine molecule or a functional fragment or variant thereof. In some embodiments, the antigen-binding domain that binds to NKp30, the antigen-binding domain that binds to TRBC1, and at least one cytokine molecule or a functional fragment or variant thereof are independently connected to the N-terminus of a first member of a dimerization module, the N-terminus of a second member of a dimerization module, the C-terminus of a first member of a dimerization module, the C-terminus of a second member of a dimerization module, or any combination thereof. In some embodiments, the formulation comprises an antigen-binding domain that binds to NKp30, an antigen-binding domain that binds to TRBC2, and at least one cytokine molecule or a functional fragment or variant thereof. In some embodiments, an antigen-binding domain that binds to NKp30, an antigen-binding domain that binds to TRBC2, and at least one cytokine molecule or a functional fragment or variant thereof are independently connected to the N-terminus of a first member of a dimerization module, the N-terminus of a second member of a dimerization module, the C-terminus of a first member of a dimerization module, the C-terminus of a second member of a dimerization module, or any combination thereof. In some embodiments, the formulation comprises an antigen-binding domain that binds to NKp30, an antigen-binding domain that binds to TRBC1 and / or an antigen-binding domain that binds to TRBC2, and at least one cytokine molecule or a functional fragment or variant thereof.In some embodiments, an antigen-binding domain that binds to NKp30, an antigen-binding domain that binds to TRBC1 and / or an antigen-binding domain that binds to TRBC2, and at least one cytokine molecule or a functional fragment or variant thereof is independently connected to the N-terminus of the first member of the dimerization module, the N-terminus of the second member of the dimerization module, the C-terminus of the first member of the dimerization module, the C-terminus of the second member of the dimerization module, or any combination thereof. Specific details for implementing the invention

[0520] definition

[0521] In some embodiments, the multifunctional molecule comprises an immune cell engager. “Immune cell engager” refers to one or more binding specificities that bind to and / or activate immune cells, e.g., cells involved in an immune response. In embodiments, the immune cells are selected from T cells, NK cells, B cells, dendritic cells, and / or macrophage cells. The immune cell engager may be an antibody molecule, a receptor molecule (e.g., full-length receptor, receptor fragment, or fusion thereof (e.g., receptor-Fc fusion)), or a ligand molecule (e.g., full-length ligand, ligand fragment, or fusion thereof (e.g., ligand-Fc fusion)) that binds to immune cell antigens (e.g., T cell, NK cell antigen, B cell antigen, dendritic cell antigen, and / or macrophage cell antigen). In embodiments, the immune cell engager specifically binds to target immune cells, e.g., preferentially to target immune cells. For example, if the immune cell binder is an antibody molecule, it binds to immune cell antigens (e.g., T cell antigen, NK cell antigen, B cell antigen, dendritic cell antigen, and / or macrophage cell antigen) with a dissociation constant of less than about 10 nM.

[0522] In some embodiments, the polyfunctional molecule comprises a cytokine molecule. As used herein, “cytokine molecule” refers to a full length, fragment, or variant of a cytokine; a cytokine further comprising a receptor domain, e.g., a cytokine receptor dimerization domain; or an agonist of a cytokine receptor, e.g., an antibody molecule against a cytokine receptor (e.g., an agonist antibody) (inducing the activity of at least one naturally occurring cytokine). In some embodiments, the cytokine molecule is selected from interleukin-2 (IL-2), interleukin-7 (IL-7), interleukin-12 (IL-12), interleukin-15 (IL-15), interleukin-18 (IL-18), interleukin-21 (IL-21), or interferon gamma, or a fragment or variant thereof, or a combination of any one of the aforementioned cytokines. The cytokine molecule may be a monomer or a dimer. In an embodiment, the cytokine molecule may further comprise a cytokine receptor dimerization domain. In another embodiment, the cytokine molecule is an antibody molecule (e.g., agonist antibody) against a cytokine receptor selected from an agonist of the cytokine receptor, e.g., IL-15Ra or IL-21R.

[0523] As used herein, the term “molecule” as used for, for example, antibody molecules, cytokine molecules, and receptor molecules includes not only whole naturally occurring molecules but also variants, for example, functional variants (e.g., truncated forms, fragment forms, mutated forms (e.g., substantially similar sequences), or derivatized forms, provided that at least one function and / or activity of the unmodified (e.g., naturally occurring) molecule remains.

[0524] In some embodiments, the pluripotent molecule comprises a stromal modifying moiety. As used herein, “stromal modifying moiety” refers to a preparation capable of altering, for example, degrading a component of the substrate, for example, a protein (e.g., an enzyme). In embodiments, the component of the substrate is selected from, for example, an ECM component, for example, a glycosaminoglycan, for example, hyaluronan (also known as hyaluronic acid or HA), chondroitin sulfate, chondroitin, dermatan sulfate, heparin sulfate, heparin, entactin, tenacin, aggrecan, and keratin sulfate; or an extracellular protein, for example, collagen, laminin, elastin, fibrinogen, fibronectin, and vitronectin.

[0525] Specific terms are defined below.

[0526] As used herein, the singular articles (“a” and “an”) refer to one or more, two or more, e.g., at least one grammatical object of the article. When used herein with the term “including,” the use of the singular expression (“a” or “an”) may mean “one,” but is also consistent with the meanings of “one or more,” “at least one,” and “one or more.”

[0527] As used herein, “approximately” and “roughly” generally refer to an acceptable degree of error for a measured quantity, taking into account the nature or precision of the measurement. An exemplary degree of error is within 20 percent (%) of the range of a given value, typically 10 percent, and more typically 5 percent.

[0528] As used herein, “antibody molecule” refers to a protein comprising at least one immunoglobulin variable domain sequence, e.g., an immunoglobulin chain or a fragment thereof. The antibody molecule comprises an antibody (e.g., a full-length antibody) and an antibody fragment. In one embodiment, the antibody molecule comprises an antigen-binding fragment or a functional fragment of a full-length antibody or a full-length immunoglobulin chain. For example, a full-length antibody is an immunoglobulin (Ig) molecule (e.g., an IgG antibody) that occurs naturally or is formed by a normal immunoglobulin gene fragment recombination process. In an embodiment, the antibody molecule refers to an immunologically active antigen-binding portion of an immunoglobulin molecule, e.g., an antibody fragment. The antibody fragment, e.g., a functional fragment, is a part of an antibody, e.g., Fab, Fab′, F(ab′)2, F(ab)2, a variable fragment (Fv), a domain antibody (dAb), or a short-chain variable fragment (scFv). A functional antibody fragment binds to the same antigen recognized by an intact (e.g., full-length) antibody. The term “antibody fragment” or “functional fragment” also includes an isolated fragment consisting of variable regions, such as a “Fv” fragment consisting of variable regions of a heavy chain and a light chain, or a recombinant short-chain polypeptide molecule (“scFv protein”) in which the variable regions of a light chain and a heavy chain are joined by a peptide linker. In some embodiments, the antibody fragment does not include a portion of the antibody that is not antigen-binding active, such as an Fc fragment or a single amino acid residue. An exemplary antibody molecule includes a full-length antibody and antibody fragments, such as a dAb (domain antibody), a short-chain, Fab, Fab', and F(ab')2 fragment, and a short-chain variable fragment (scFv).

[0529] As used herein, “immunoglobulin variable domain sequence” refers to an amino acid sequence capable of forming the structure of an immunoglobulin variable domain. For example, the sequence may include all or part of the amino acid sequence of a naturally occurring variable domain. For example, the sequence may include or not include one, two, or more N-terminal or C-terminal amino acids, or may include other modifications compatible with the formation of the protein structure.

[0530] In an embodiment, the antibody molecule is monospecific and includes, for example, binding specificity to a single epitope. In some embodiments, the antibody molecule is multispecific and includes, for example, a plurality of immunoglobulin variable domain sequences, wherein a first immunoglobulin variable domain sequence has binding specificity to a first epitope and a second immunoglobulin variable domain sequence has binding specificity to a second epitope. In some embodiments, the antibody molecule is a bispecific antibody molecule. As used herein, “bispecific antibody molecule” refers to an antibody molecule having specificity to two or more (e.g., two, three, four or more) epitopes and / or antigens.

[0531] As used herein, “antigen” (Ag) refers to a molecule capable of inducing an immune response, for example, involved in the activation of specific immune cells and / or antibody production. Any macromolecule, including almost any protein or peptide, may be an antigen. Antigens may also be derived from genomic recombination or DNA. For example, any DNA containing a nucleotide sequence or partial nucleotide sequence encoding a protein capable of inducing an immune response encodes an “antigen.” In embodiments, the antigen does not need to be encoded solely by the full nucleotide sequence of a gene, nor does the antigen need to be encoded by a gene at all. In embodiments, the antigen may be synthesized or derived from a biological sample, for example, a tissue sample, a tumor sample, a cell, or a fluid containing other biological components. As used herein, “tumor antigen” or, interchangeably, “cancer antigen” comprises any molecule present in or associated with cancer, for example, cancer cells or the tumor microenvironment capable of inducing an immune response. As used herein, “immune cell antigen” comprises any molecule present on or associated with immune cells capable of inducing an immune response.

[0532] The “antigen binding site” or “binding portion” of an antibody molecule refers to the portion of an antibody molecule, for example, 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 chain (H) and light chain (L) chains. Three highly divergent stretches within the variable regions of the heavy and light chains, referred to as hypervariable regions, are positioned between more conserved lateral stretches referred to as “framework regions” (FR). FR is an amino acid sequence naturally found in immunoglobulins between and adjacent to the hypervariable regions. In an embodiment, in the antibody molecule, the three hypervariable regions of the light chain and the three hypervariable regions of the heavy chain are positioned relative to each other in three-dimensional space to form an antigen binding surface complementary to the three-dimensional surface of the bound antigen. The three hypervariable regions of each of the heavy and light chains are referred to as “complementary determining regions” or “CDRs.” Framework regions and CDRs have been defined and described, for example, in the literature by Kabat, EA et al. [(1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242] and in the literature by Chothia, C. et al. [(1987) J. Mol. Biol. 196:901-917], or by the ImMunoGeneTics (IMGT) numbering system. Each variable chain (e.g., variable heavy chain and variable light chain) generally consists of three CDRs and four FRs arranged in the following amino acid sequence from amino terminus to carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.

[0533] As used herein, the terms “NKp30” (also known as Natural Cytotoxicity Inducing Receptor 3), CD337 (Differentiation Cluster 337), NCR3, 1C7, LY117, MALS, Natural Cytotoxicity Inducing Receptor 3, NCR3, nkp30, or NKP30, when used interchangeably herein, refer to a Type I transmembrane receptor belonging to the immunoglobulin (Ig) superfamily expressed in quiescent and active natural killer (NK) cells. In some embodiments, NKp30 may induce a cytotoxic response to tumor cells. The term “NKp30” includes isoforms, mammalian (e.g., human) NKp30, human species homologs, and analogs comprising at least one common epitope with NKp30. Human NKp30 includes isoforms comprising, but not limited to: NKp30A, NKp30B, or NKp30C. Additionally, the term “NKp30” includes any one of a recombinant or naturally occurring form of NKp30 or a variant or homolog thereof that has or retains NKp30 activity (e.g., at least 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% activity). In some embodiments, the variant or homolog has at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity over the entire sequence or a portion of the sequence (e.g., a 50, 100, 150, or 200 consecutive amino acid portion) compared to naturally occurring NKp30. In some embodiments, NKp30 is substantially identical to the protein identified by UniProt reference number O14931 or a variant or homolog having substantial identity therewith.

[0534] As used herein, the terms “T cell receptor beta variable chain,” “T cell receptor beta chain variable region,” “TCRβV,” “TCRβV,” “TCRβV,” “TCRβv,” “TCRβv,” “TCRβv,” “TCRβv,” “T cell receptor variable beta chain,” “TCRβV,” “TCR Vβ,” “TCRV β,” “TCRβV,” “TCRv β,” “TCR vβ,” “TCRBV,” “TCRVB,” “TRBV,” or “TCR beta V” are used interchangeably herein and refer to an extracellular region of a T cell receptor beta chain containing an antigen recognition domain of a T cell receptor. The term TCRβV includes isoforms, mammalian (e.g., human) TCRβV, human species homologs, and analogs comprising at least one common epitope with TCRβV. Human TCRβV includes a gene group comprising subfamilies, including but not limited to the following: 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, or TCRβ V29 subfamily. In some embodiments, the TCRβ V6 subgroup 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. In some embodiments, TCRβV includes TCRβ V6-5*01.TCRβ V6-5*01 is also known as TRBV65; TCRβV 6S5; TCRβV 13S1; or TCRβV 13.1. The amino acid sequence of TCRβ V6-5*01, e.g. human TCRβ V6-5*01, is known in the art, as provided, e.g. by IMGT ID L36092.

[0535] As used herein, “cancer” may include all types of tumorigenic processes and / or cancerous growths. In embodiments, cancer includes primary tumors as well as metastatic tissues or cells, tissues, or organs that have transformed into malignancy. In embodiments, cancer includes all histopathologies and stages of cancer (e.g., invasive / severe stages). In embodiments, cancer includes recurrent and / or resistant cancer. The terms “cancer” and “tumor” may be used interchangeably. For example, both terms include solid and liquid tumors. As used herein, the terms “cancer” or “tumor” include precancerous as well as malignant cancers and tumors.

[0536] As used herein, “immune cell” refers to any of the various cells that function in the immune system, for example, to protect against agents of infection and foreign substances. In embodiments, this term includes leukocytes, e.g., neutrophils, eosinophils, basophils, lymphocytes, and monocytes. Innate leukocytes include phagocytes (e.g., macrophages, neutrophils, and dendritic cells), mast cells, eosinophils, basophils, and natural killer cells. Innate leukocytes identify and eliminate pathogens by attacking larger pathogens through contact or by phagocytosing and killing microorganisms, and are mediators in the activation 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 and originate from hematopoietic stem cells in the bone marrow. B cells are involved in humoral immune responses, while T cells are involved in cell-mediated immune responses. The term “immune cell” includes immune effector cells.

[0537] “Immune effector cells” refers to cells involved in an immune response, for example, in the promotion of an immune effector response, as the term is used herein. Examples of immune effector cells include, but are not limited to, T cells, e.g., alpha / beta T cells and gamma / delta T cells, B cells, natural killer (NK) cells, natural killer T (NK T) cells, and mast cells.

[0538] The terms “effector function” or “effector response” refer to specific functions of a cell. For example, the effector function of a T cell may be cytolytic activity, including the secretion of cytokines, or helper activity.

[0539] The compositions and methods of the present invention comprise the specified sequence, or a sequence substantially identical or similar thereto, for example, a sequence of polypeptides and nucleic acids having at least 80%, 85%, 90%, or 95% or more sequence identity with the specified sequence. In the context of amino acid sequences, the term “substantially identical” is used herein to refer to a first amino acid containing a sufficient or minimum number of amino acid residues that are i) identical to or ii) conservative substitutions thereof with an amino acid residue aligned in a second amino acid sequence so that the first and second amino acid sequences may have a common structural domain and / or common functional activity. For example, an amino acid sequence containing a common structural domain having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with a reference sequence, for example, a sequence provided herein.

[0540] In the context of nucleotide sequences, the term “substantially identical” is used herein to refer to a first nucleic acid sequence containing a sufficient or minimum number of nucleotides identical to those in the second nucleic acid sequence aligned to encode a polypeptide having common functional activity, or to a reference sequence, e.g., a nucleotide sequence having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with respect to a reference sequence, e.g., a sequence provided herein.

[0541] 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, the variant is a functional variant.

[0542] The term “functional variant” refers to a polypeptide that has a substantially identical amino acid sequence to a reference amino acid sequence, is encoded by a substantially identical nucleotide sequence, and may have one or more activities of the reference amino acid sequence.

[0543] The term “scFv” refers to a fusion protein comprising at least one antibody fragment comprising a variable region of a light chain and at least one antibody fragment comprising a variable region of a heavy chain, wherein the light chain and heavy chain variable regions are adjacently connected via a short flexible polypeptide linker and may be expressed as a short-chain polypeptide, and the scFv retains the specificity of the intact antibody from which the scFv is derived. Unless otherwise specified, as used herein, the scFv is, for example, V in any order for the N-terminal and C-terminal ends of the polypeptide. L and V H It can have a variable region, and scFv is V L -Linker-V H Includes or V H -Linker-V L It may include.

[0544] The terms “Complementary Determining Region” and “CDR” are used interchangeably herein and refer to the sequence of amino acids within an antibody variable region that confers antigen specificity and binding affinity. For example, generally, each heavy chain variable region has three CDRs (HCDR1, HCDR2, and HCDR3), and each light chain variable region has three CDRs (LCDR1, LCDR2, and LCDR3). The precise amino acid sequence boundaries of a given CDR can be determined using any one of several well-known methods, including the following: Kabat et al. (1991), “Sequences of Proteins of Immunological Interest,” 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (“Kabat” numbering scheme)], the literature by Al-Lazikani et al. [(1997) JMB 273,927-948 (“Chothia” numbering scheme)], the ImMunoGeneTics (IMGT) numbering scheme, or any combination thereof. Under the Kabat numbering scheme, in some embodiments, a heavy chain variable domain (V H The CDR amino acid residues within are numbered 31–35 (HCDR1), 50–65 (HCDR2), and 95–102 (HCDR3); and the light chain variable domain (V L The CDR amino acid residues within are numbered 24–34 (LCDR1), 50–56 (LCDR2), and 89–97 (LCDR3). Under the Chothia numbering scheme, in some embodiments, V H The CDR amino acids within are numbered 26–32 (HCDR1), 52–56 (HCDR2), and 95–102 (HCDR3); V LThe CDR amino acid residues within are numbered 26–32 (LCDR1), 50–52 (LCDR2), and 91–96 (LCDR3). In the combined Kabat and Chothia numbering scheme, in some embodiments, the CDR corresponds to an amino acid residue that is part of the Kabat CDR, the Chothia CDR, or both. For example, in some embodiments, the CDR is V H , for example, mammals V H , for example, human V H Amino acid residues 26–35 (HCDR1), 50–65 (HCDR2), and 95–102 (HCDR3) at; and V L , for example, mammals V L , for example, human V L It corresponds to amino acid residues 24–34 (LCDR1), 50–56 (LCDR2), and 89–97 (LCDR3) in.

[0545] The “humanized” form of a non-human (e.g., rodent) antibody is a chimeric immunoglobulin, immunoglobulin chain, or fragment thereof (e.g., Fv, Fab, Fab', F(ab')2, or other antigen-binding subsequences of the antibody) containing a minimal sequence derived from a non-human immunoglobulin. In most cases, the humanized antibody and its antibody fragment are human immunoglobulins (recipient antibodies or antibody fragments) in which residues from the recipient’s complementarity determining region (CDR) are replaced with residues from the CDR of a non-human species (donor antibody), such as mouse, rat, or rabbit, to possess the desired specificity, affinity, and ability. In some cases, residues from the human immunoglobulin’s Fv framework region (FR) are replaced with corresponding non-human residues. Additionally, the humanized antibody / antibody fragment may contain residues that are not found in the recipient antibody, nor in the introduced CDR or framework sequence. These modifications can further improve and optimize the performance of the antibody or antibody fragment. Generally, the humanized antibody or antibody fragment thereof will contain at least one substantially variable domain, typically both, wherein all or substantially all of the CDR region corresponds to the CDR region of a non-human immunoglobulin and all or substantial portion of the FR region is the FR region of a human immunoglobulin sequence. The humanized antibody or antibody fragment may also contain at least a portion of the immunoglobulin constant region (Fc), generally at least a portion of the human immunoglobulin. For further details, refer to Jones et al. [Nature, 321: 522-525, 1986]; Reichmann et al. [Nature, 332: 323-329, 1988]; and Presta [Curr. Op. Struct. Biol., 2: 593-596, 1992].

[0546] “Fully human” refers to immunoglobulins, such as antibodies or antibody fragments, where the entire molecule is of human origin or consists of an amino acid sequence identical to the human form of the antibody or immunoglobulin.

[0547] The term “pecifically bind” refers to a situation where an antibody or ligand recognizes and binds to a cognate binding partner protein present in a sample (e.g., a stimulating and / or co-stimulating molecule present on a T cell), but the antibody or ligand does not substantially recognize or bind to any other molecule within the sample.

[0548] As used herein, “immune cell” refers to any of the various cells that function in the immune system, for example, to protect against agents of infection and foreign substances. In embodiments, this term includes leukocytes, e.g., neutrophils, eosinophils, basophils, lymphocytes, and monocytes. Innate leukocytes include phagocytes (e.g., macrophages, neutrophils, and dendritic cells), mast cells, eosinophils, basophils, and natural killer cells. Innate leukocytes identify and eliminate pathogens by attacking larger pathogens through contact or by phagocytosing and killing microorganisms, and are mediators in the activation 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 and originate from hematopoietic stem cells in the bone marrow. B cells are involved in humoral immune responses, while T cells are involved in cell-mediated immune responses. The term “immune cell” includes immune effector cells.

[0549] As used herein, the term “immune effector cell” refers to a cell involved in promoting an immune response, e.g., an immune effector response. Examples of immune effector cells include, but are not limited to, T cells (e.g., alpha / beta T cells, gamma / delta T cells, CD4+ T cells, CD8+ T cells), B cells, natural killer (NK) cells, natural killer T (NK T) cells, monocytes, macrophages, neutrophils, basophils, dendritic cells, and mast cells.

[0550] The terms “effector function” or “effector response” refer to specific functions of a cell. For example, the effector function of a T cell may be cytolytic activity involving the secretion of cytokines (e.g., CD8+ T cells) or helper activity (e.g., CD4+ T cells).

[0551] The term “antigen-presenting cell” or “APC” refers to immune system cells, such as accessory cells (e.g., B cells, dendritic cells, etc.), that display foreign antigens complexed with major histocompatibility complexes (MHCs) on their surfaces. T cells can recognize these complexes using T cell receptors (TCRs). APCs process the antigens and present them to T cells.

[0552] The term “substantially purified cell” or “substantially purified cell population” refers to a cell or cell population that is essentially free of other cell types. A substantially purified cell also refers to a cell isolated from other cell types that are normally associated in a natural state. In some cases, a substantially purified cell population refers to a homogeneous cell population. In other cases, the term simply refers to cells isolated from cells that are naturally associated with them in a natural state. In some embodiments, the cells are cultured in a test tube. In other embodiments, the cells are not cultured in a test tube.

[0553] “Derived” indicates the relationship between the first molecule and the second molecule, as the term is used herein. This generally refers to the structural similarity between the first molecule and the second molecule and does not imply or imply that the method or source for the first molecule derived from the second molecule is limited. For example, in the case of an intracellular signaling domain derived from the CD3zeta molecule, the intracellular signaling domain possesses sufficient CD3zeta structure to have the necessary function, namely the ability to generate a signal under appropriate conditions. This does not imply or imply that the specific method for producing the intracellular signaling domain is limited, for example, that to provide the intracellular signaling domain, one must necessarily start with the CD3zeta sequence and delete unwanted sequences or introduce mutations to reach the intracellular signaling domain.

[0554] The term “encoding” refers to the unique properties of a specific sequence of nucleotides within a polynucleotide, such as a gene, cDNA, or mRNA, to function as a template for the synthesis of other polymers and macromolecules in biological processes having a defined nucleotide sequence (e.g., rRNA, tRNA, and mRNA) or a defined amino acid sequence and the biological properties derived therefrom. Thus, a gene, cDNA, or RNA is said to encode a protein when the transcription and translation of the mRNA corresponding to that gene produces a protein in a cell or other biological system. Both the coding strand, whose nucleotide sequence is identical to the mRNA sequence and is generally provided in sequence lists, and the non-coding strand used as a template for the transcription of the gene or cDNA may be referred to as encoding the protein or other product of that gene or cDNA.

[0555] The calculation of homology or sequence identity between sequences (terms used interchangeably herein) is performed as follows.

[0556] 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., for optimal alignment, a gap may be introduced in one or both of the first and second amino acid or nucleic acid sequences, and non-homologous sequences may be ignored for comparison purposes). In a preferred embodiment, the length of the reference sequence aligned for comparison 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. Then, amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are compared. If a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, the molecules are identical at that position (as used herein, amino acid or nucleic acid “identity” is equivalent to amino acid or nucleic acid “homology”).

[0557] The percentage of identity between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps and the length of each gap that needs to be introduced for optimal alignment of the two sequences.

[0558] The comparison of sequences and the determination of the percentage of identity between two sequences can be achieved using a mathematical algorithm. In a preferred embodiment, the percentage of identity between two amino acid sequences is Needleman and Wunsch (1970) J. Mol. Biol.It is determined using the algorithm (48:444-453), which is incorporated into the GAP program within the GCG software package (available at gcg.com) using the Blossum 62 matrix or PAM250 matrix, and gap weights of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6. In another preferred embodiment, the percentage of identity between two nucleotide sequences is determined using the GAP program within the GCG software package (available at gcg.com), using the NWSgapdna.CMP matrix and gap weights of 40, 50, 60, 70, or 80, and length weights of 1, 2, 3, 4, 5, or 6. A particularly desirable parameter set (and a parameter set to be used unless otherwise specified) is a Blossum 62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5.

[0559] The percentage of identity between two amino acid or nucleotide sequences, using the PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4, as incorporated by E. Meyers and W. Miller (1989) in the ALIGN program (version 2.0). CABIOS It can be determined using the algorithm of , 4:11-17).

[0560] The nucleic acid and protein sequences described herein may be used as “query sequences” to perform a search on public databases to identify, for example, other family members or related sequences. Such a search is based on the literature of Altschul et al. [(1990) J. Mol.This can be performed using the NBLAST and XBLAST programs (version 2.0) from Biol. 215:403-10. BLAST nucleotide search can be performed with the NBLAST program, score = 100, word length = 12 to obtain nucleotide sequences homologous to the nucleic acid molecule of the present invention. BLAST protein search can be performed with the XBLAST program, score = 50, word length = 3 to obtain amino acid sequences homologous to the protein molecule of the present invention. To obtain gap alignment for comparison, gap BLAST is performed according to Altschul et al.'s literature [(1997) Nucleic Acids Res. It may be used as described in [25:3389-3402]. When using BLAST and Gapped BLAST programs, the default parameters of each program (e.g., XBLAST and NBLAST) may be used. See ncbi.nlm.nih.gov.

[0561] It will be understood that the molecules of the present invention may have additional conservative or non-essential amino acid substitutions that do not substantially affect their function.

[0562] The term “amino acid” is intended to include both natural and synthetic molecules that contain both amino acid functional groups and acid functional groups and may be included in polymers of naturally occurring amino acids. Exemplary amino acids include naturally occurring amino acids; analogs, derivatives, and components thereof; amino acid analogs having variant side chains; and all stereoisomers of any one of the foregoing. As used herein, the term “amino acid” includes both D- or L-optical isomers and peptide mimics.

[0563] “Conservative amino acid substitution” is the replacement of an amino acid residue with an amino acid residue having a similar side chain. A series of amino acid residues having similar side chains is defined in the art. This series includes amino acids having a basic side chain (e.g., lysine, arginine, histidine), an acidic side chain (e.g., aspartic acid, glutamic acid), an uncharged polar side chain (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), a nonpolar side chain (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), a beta-branched side chain (e.g., threonine, valine, isoleucine), and an aromatic side chain (e.g., tyrosine, phenylalanine, tryptophan, histidine). Such conservative modifications include amino acid substitution, addition, and deletion. Modifications may be introduced into antibodies or antibody fragments by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative substitution is the replacement of an amino acid residue with an amino acid residue having a similar side chain. The series of amino acid residues having similar side chains is defined in the relevant art. These series include amino acids having basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Accordingly, one or more amino acid residues in the CAR may be replaced with other amino acid residues of the same side chain series, and the modified CAR may be tested using the functional assays described herein.

[0564] The terms “polypeptide,” “peptide,” and “protein” (if short-chain) are used interchangeably herein to refer to a polymer of amino acids of any length. The polymer may be linear or branched, may contain modified amino acids, or may be interrupted by non-amino acids. The terms also include modified amino acid polymers; for example, any other operations such as disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or conjugation with labeling components. The polypeptide may be isolated from natural sources, produced by recombinant technology from eukaryotic or prokaryotic hosts, or be the product of a synthetic process.

[0565] The terms “nucleic acid,” “nucleic acid sequence,” “nucleotide sequence,” or “polynucleotide sequence,” and “polynucleotide” are used interchangeably. They refer to polymeric forms of nucleotides, deoxyribonucleotides, or ribonucleotides, or analogs thereof, of any length. Polynucleotides may be single-stranded or double-stranded, and if single-stranded, may be a coding strand or a non-coding (antisense) strand. Polynucleotides may include modified nucleotides, such as methylated nucleotides and nucleotide analogs. The sequence of nucleotides may be disrupted by non-nucleotide components. Polynucleotides may be further modified after polymerization, for example, by conjugation with labeling components. Nucleic acids may be recombinant polynucleotides, or polynucleotides of genomic, cDNA, semi-synthetic, or synthetic origin linked to other polynucleotides of non-natural or non-natural arrangements.

[0566] As used herein, the term “isolated” refers to material removed from its original or natural environment (e.g., the natural environment if it occurs naturally). For example, naturally occurring polynucleotides or polypeptides present in living animals are not isolated, but the same polynucleotides or polypeptides separated from some or all of the coexisting material in nature by human intervention are isolated. Such polynucleotides may be part of a vector and / or may be part of a composition, and such vector or composition may still be isolated in that it is not part of the environment in which it is found in nature.

[0567] The term “endogenous” refers to any substance produced from or within an organism, cell, tissue, or system.

[0568] The term “exogenous” refers to any substance introduced or generated outside of an organism, cell, tissue, or system.

[0569] The term “expression” refers to the transcription and / or translation of a specific nucleotide sequence induced by a promoter.

[0570] The term “transfer vector” refers to a composition of material comprising isolated nucleic acids that can be used to deliver the isolated nucleic acids into a cell. A number of vectors are known in the art, including but not limited to linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Accordingly, the term “transcription vector” includes autonomously replicating plasmids or viruses. The term should also be interpreted to further include non-plasmid and non-viral compounds that facilitate the delivery of nucleic acids into a cell, such as, for example, polylysine compounds, liposomes, etc. Examples of viral transfer vectors include, but are not limited to, adenovirus vectors, adeno-associated virus vectors, retrovirus vectors, lentivirus vectors, etc.

[0571] The term “expression vector” refers to a vector comprising a recombinant polynucleotide containing an expression control sequence operably linked to a nucleotide sequence to be expressed. The expression vector contains sufficient cis-acting elements for expression; other elements for expression may be supplied by a host cell or in an in vitro expression system. The expression vector comprises all that are known in the art, including cosmids, plasmids (e.g., in a naked state or contained within a liposome), and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that contain recombinant polynucleotides.

[0572] As used herein, the term “vector” refers to any vehicle that may be used to deliver and / or express a nucleic acid molecule. This may be a delivery vector or an expression vector as described herein.

[0573] The term “lentivirus” refers to a genus of retroviruses. Lentiviruses are unique among retroviruses that can infect non-dividing cells; and because they can deliver a significant amount of genetic information to the DNA of host cells, they are one of the most efficient used in gene delivery vector methods.

[0574] The term “lentiviral vector” includes vectors derived from at least a portion of the lentiviral genome, particularly including self-inactivating lentiviral vectors such as those provided in the following literature: Milone et al. [Mol. Ther. 17(8): 1453-1464 (2009)]. Other examples of lentiviral vectors available for clinical use include, but are not limited to, Oxford BioMedica’s LENTIVECTOR® gene delivery technology, Lentigen’s LENTIMAX™ vector system, etc. Non-clinical types of lentiviral vectors are also available and will be known to those skilled in the art.

[0575] The terms “operably linked” or “transcriptional regulation” refer to a functional coupling between a regulatory sequence and a heterogeneous nucleic acid sequence, resulting in the expression of the latter. For example, a first nucleic acid sequence is operably linked to a second nucleic acid sequence when the first nucleic acid sequence is positioned in a functional relationship with the second nucleic acid sequence. For example, if a promoter influences the transcription or expression of a coding sequence, the promoter is operably linked to the coding sequence. Operatably linked DNA sequences may be adjacent to each other and, for example, within the same read frame if necessary to link two protein coding regions.

[0576] The term “parenteral” administration for immunogenic compositions includes, for example, subcutaneous (sc), intravenous (iv), intramuscular (im), or intrasternal injection, intratumoral, or infusion techniques.

[0577] The term “promoter” refers to a DNA sequence that is recognized by the cell’s synthetic machinery or introduces the synthetic machinery necessary to initiate the specific transcription of a polynucleotide sequence.

[0578] The term “promoter / regulatory sequence” refers to a nucleic acid sequence required for the expression of a gene product operably linked to a promoter / regulatory sequence. In some cases, this sequence may be a core promoter sequence, and in other cases, this sequence may include enhancer sequences and other regulatory elements required for the expression of the gene product. The promoter / regulatory sequence may, for example, express the gene product in a tissue-specific manner.

[0579] The term “constitutive promoter” refers to a nucleotide sequence that, when operably linked with a polynucleotide encoding or specifying a gene product, causes the cell to produce the gene product under most or all physiological conditions.

[0580] The term “inducible promoter” refers to a nucleotide sequence that, when operably linked with a polynucleotide encoding or specifying a gene product, causes the cell to substantially produce a gene product only when a derivative corresponding to the promoter is present in the cell.

[0581] The term “tissue-specific promoter” refers to a nucleotide sequence that, when operably linked with a nucleotide encoded by a gene or specified by the promoter, causes the cell to substantially produce a gene product only when the cell is a cell of the tissue type corresponding to the promoter.

[0582] As used herein, “transient” refers to the expression of a non-integrated transplanted gene for a period of hours, days, or weeks, wherein the expression period is shorter than the expression period of the gene when integrated into the genome or contained within a plasmid replicon stable in a host cell.

[0583] The terms “transfection,” “transformation,” or “transduction” refer to the process in which exogenous nucleic acids are delivered or introduced into a host cell. “Transfected,” “transformed,” or “transduced” cells are cells that have been transfected, transformed, or introduced with exogenous nucleic acids. Cells include primary target cells and their progeny.

[0584] The term “Chimeric Antigen Receptor” or alternatively “CAR” is used interchangeably herein and refers to a recombinant polypeptide construct comprising a cytoplasmic signaling domain (also referred to herein as an “intracellular signaling domain”) comprising at least an extracellular antigen binding domain, a transmembrane domain, and a functional signaling domain derived from a stimulating molecule as defined below. In some embodiments, the domains of the CAR polypeptide construct are located within the same polypeptide chain, for example, a chimeric fusion protein. In some embodiments, the domains of the CAR polypeptide construct are not adjacent to each other, for example, within different polypeptide chains. In one embodiment, the stimulating molecule of the CAR is a zeta chain associated with a T cell receptor complex. In one embodiment, the cytoplasmic signaling domain comprises a primary signaling domain (e.g., the primary signaling domain of CD3-zeta). In one embodiment, the cytoplasmic signaling domain further comprises one or more functional signaling domains derived from at least one co-stimulating molecule as defined below. In one embodiment, the co-stimulator is selected from 4-1BB (i.e., CD137), CD27, ICOS, and / or CD28. In one embodiment, the CAR comprises a chimeric fusion protein comprising an extracellular antigen recognition domain, a transmembrane domain, and an intracellular signaling domain comprising a functional signaling domain derived from the stimulator. In one embodiment, the CAR comprises a chimeric fusion protein comprising an extracellular antigen recognition domain, a transmembrane domain, and an intracellular signaling domain comprising a functional signaling domain derived from the co-stimulator and a functional signaling domain derived from the stimulator.In one embodiment, the CAR comprises a chimeric fusion protein comprising an extracellular antigen recognition domain, a transmembrane domain, and an intracellular signaling domain comprising two functional signaling domains derived from one or more co-stimulatory molecule(s) and a functional signaling domain derived from a stimulatory molecule. In one embodiment, the CAR comprises a chimeric fusion protein comprising an extracellular antigen recognition domain, a transmembrane domain, and an intracellular signaling domain comprising at least two functional signaling domains derived from one or more co-stimulatory molecule(s) and a functional signaling domain derived from a stimulatory molecule. In one embodiment, the CAR comprises any leader sequence at the amino-terminus (N-ter) of the CAR fusion protein. In one embodiment, the CAR further comprises a leader sequence at the N-terminus of the extracellular antigen recognition domain, wherein the leader sequence is optionally cleaved from the antigen recognition domain (e.g., scFv) during cell processing and localization of the CAR to the cell membrane.

[0585] As used herein, the term “signaling domain” refers to a functional part of a protein that functions as an effector by transmitting information within a cell and regulating cell activity through a defined signaling pathway, thereby generating a second messenger or responding to such messenger.

[0586] As used herein, the term “intracellular signaling domain” refers to the intracellular portion of a molecule. The intracellular signaling domain may generate signals that promote immune effector functions in CAR-containing cells, e.g., CART cells or CAR-expressing NK cells. Examples of immune effector functions in CART cells or CAR-expressing NK cells include cytolytic activity and helper activity, including the secretion of cytokines. In embodiments, the intracellular signaling domain transduces an effector function signal and induces the cell to perform a specific function. The entire intracellular signaling domain may be used, but in many cases, it is not necessary to use the entire chain. To the extent that a truncated portion of the intracellular signaling domain is used, said truncated portion may be used in place of the whole chain, provided that it transduces an effector function signal. Accordingly, the term “intracellular signaling domain” is intended to include any truncated portion of the intracellular signaling domain sufficient to transduce an effector function signal. In some embodiments, the intracellular signaling domain comprises a primary intracellular signaling domain. An exemplary primary intracellular signaling domain comprises those derived from molecules responsible for primary stimulation or antigen-dependent simulation. In one embodiment, the intracellular signaling domain may comprise a co-stimulating intracellular domain. An exemplary co-stimulating intracellular signaling domain comprises those derived from molecules responsible for co-stimulating signals or antigen-independent stimulation. For example, in the case of CAR-expressing immune effector cells, e.g., CART cells or CAR-expressing NK cells, the primary intracellular signaling domain may comprise the cytoplasmic sequence of a T cell receptor, and the co-stimulating intracellular signaling domain may comprise the cytoplasmic sequence from a co-receptor or a co-stimulating molecule.The primary intracellular signaling domain may include a signaling motif known as an immune receptor tyrosine-based activation motif or ITAM. Examples of ITAMs containing a primary cytoplasmic signaling sequence include, but are not limited to, those derived from CD3 zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CDS, CD22, CD79a, CD79b, CD278 (“ICOS”), FcεRI, CD66d, DAP10, and DAP12.

[0587] The term “zeta” or alternatively “zeta chain”, “CD3-zeta” or “TCR-zeta” is defined as the protein provided as GenBank accession number BAG36664.1, or equivalent residues from non-human species, e.g., mouse, rodent, monkey, primate, etc., and “zeta stimulating domain” or alternatively “CD3-zeta stimulating domain” or “TCR-zeta stimulating domain” is defined as amino acid residues from the cytoplasmic domain of the zeta chain sufficient to functionally transmit the initial signal required for T cell activation. In one embodiment, the cytoplasmic domain of zeta comprises residues 52 to 164 of GenBank accession number BAG36664.1, or equivalent residues from non-human species, e.g., mouse, rodent, monkey, primate, etc., which are functional heterologs thereof.

[0588] The term “co-stimulator” refers to a homologous binding partner on a T cell that specifically binds to a co-stimulatory ligand and mediates a co-stimulatory response by the T cell, such as proliferation, to an unrestricted degree. A co-stimulator is a cell surface molecule other than an antigen receptor or its ligand that is necessary for an efficient immune response. Co-stimulating molecules include, but are not limited to: MHC class I molecules, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocyte-activating molecules (SLAM proteins), activated NK cell receptors, BTLA, Toll ligand receptors, OX40, CD2, CD7, CD27, CD28, CD30, CD40, CDS, ICAM-1, LFA-1 (CD11a / CD18), 4-1BB (CD137), B7-H3, CDS, ICAM-1, ICOS (CD278), GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8 alpha, CD8 beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, V L A1, CD49a, ITGA4, IA4, CD49D, ITGA6, V LA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, Ligands that specifically bind to GADS, SLP-76, PAG / Cbp, CD19a, and CD83.

[0589] “Co-stimulatory intracellular signaling domain” refers to the intracellular portion of a co-stimulatory molecule. The intracellular signaling domain may include the entire intracellular portion of the molecule from which the intracellular signaling domain is derived, the entire intrinsic intracellular signaling domain, or a functional fragment thereof.

[0590] As used herein, the term “signaling pathway” refers to the biochemical relationships between various signaling molecules that play a role in transmitting signals from one part of a cell to another.

[0591] As used herein, the term “cell surface receptor” includes molecules and molecular complexes capable of receiving signals and transmitting signals across a cell membrane.

[0592] The terms “antitumor effect” or “anticancer effect,” used interchangeably herein, refer to biological effects that may be manifested by various means, including but not limited to, for example, a reduction in tumor volume, a reduction in the number of tumor cells, a reduction in the number of metastases, an increase in life expectancy, a reduction in tumor cell proliferation, a reduction in tumor cell survival, or the alleviation of various physiological symptoms associated with cancerous pathology. The “antitumor effect” may first manifest through the ability of peptides, polynucleotides, cells, and antibodies to prevent the development of tumors.

[0593] As used interchangeably herein, the terms “cancer” or “tumor” may include all types of tumorigenic processes and / or cancerous growths. In embodiments, cancer includes primary tumors as well as metastatic tissues or cells, tissues, or organs that have transformed into malignancy. In embodiments, cancer includes all histopathologies and stages of cancer, e.g., invasive / severe stages. In embodiments, cancer includes recurrent and / or resistant cancer. For example, both terms include solid and liquid tumors. As used herein, the term “cancer” includes precancerous cancers and tumors as well as malignant cancers and tumors.

[0594] The term “autologous” refers to any substance originating from an entity that is subsequently reintroduced into the same entity.

[0595] The term “allogenic” refers to any substance derived from a different animal of the same species as the individual into which the substance is introduced. Two or more individuals are referred to as allogenic when genes at one or more loci are not identical. In some modalities, allogenic substances from individuals of the same species may not be genetically similar enough to interact antigenically.

[0596] The term “xenogenetic” refers to a graft derived from an animal of a different species.

[0597] As used herein, the term “apheresis” refers to an in vitro process known in the art of drawing blood from a donor or patient, separating selected specific component(s), and passing the remainder through a device that returns it to the donor or patient’s blood circulation, for example, by re-transfusion. Accordingly, in this context, “apheresis sample” refers to a sample obtained using apheresis.

[0598] The term “combination” refers to a combination therapy fixed in the form of a single dosing unit, or a combination administration in which a compound and a combination partner (e.g., another drug as described below, also referred to as a “therapeutic agent” or “co-preparation”) may be administered simultaneously and independently, or separately at a time interval, particularly at a time interval that allows the combination partners to exhibit cooperation (e.g., synergy). Single components may be packaged in a kit or packaged separately. One or both of the components (e.g., powder or liquid) may be reconstituted or diluted to a desired dosage before administration. As used herein, the terms “co-administration” or “combination” encompass the administration of a selected combination partner to a single subject (e.g., a patient) requiring it, and are intended to include a therapeutic mode in which the formulations do not need to be administered via the same route of administration or simultaneously. As used herein, the term “pharmaceutical combination” refers to a product formed by mixing or combining two or more active ingredients, and includes both fixed combinations and non-fixed combinations of active ingredients. The term “fixed combination” means that both active ingredients (e.g., a compound and a combination partner) are administered to a patient simultaneously in the form of a single entity or dosage. The term “non-fixed combination” means that both active ingredients (e.g., a compound and a combination partner) are administered to a patient simultaneously, incidentally, or sequentially without specific time limits as separate entities, wherein such administration provides the two compounds to the patient’s body at a therapeutically effective level. Sequential administration also applies to cocktail therapy, e.g., administering three or more active ingredients.

[0599] The terms “effective amount” or “therapeutic effective amount” are used interchangeably herein and refer to an amount of a compound, formulation, substance, or composition as described herein that is effective in achieving a specific biological result.

[0600] As used herein, the term “treat, treatment, and treating” refers to reducing or alleviating the progression, severity, and / or duration of a proliferative disorder by administering one or more therapies (e.g., one or more therapeutic agents such as CARs), or alleviating one or more symptoms of a proliferative disorder (preferably, one or more distinguishable symptoms). In certain embodiments, the term “treat” refers to alleviating at least one measurable physical parameter of a proliferative disorder, such as tumor growth (which may not be identifiable per patient). In other embodiments, the term “treat” refers to physically inhibiting the progression of a proliferative disorder, for example by stabilizing a distinguishable symptom, or physiologically inhibiting it, for example by stabilizing a physical parameter, or both. In other embodiments, the term “treat” refers to a reduction or stabilization of tumor size or the number of cancer cells.

[0601] As used herein, the term “therapeutic” means treatment. Therapeutic effects are achieved by the reduction, suppression, remission, or eradication of the disease state.

[0602] As used herein, the term “prophylaxis” refers to preventive or protective treatment for a disease or diseased condition.

[0603] The term “subject” is intended to include living organisms (e.g., mammals, humans) in which an immune response may be induced.

[0604] Various aspects of the present invention are described in more detail below. Additional definitions are provided throughout this specification.

[0605] antibody molecules

[0606] In one embodiment, the antibody molecule binds to a cancer antigen, for example, a tumor antigen or a stromal antigen. In some embodiments, the cancer antigen is, for example, a mammalian, for example, a human cancer antigen. In other embodiments, the antibody molecule binds to an immune cell antigen, for example, a mammalian, for example, a human immune cell antigen. For example, the antibody molecule has an epitope on the cancer antigen or immune cell antigen, for example It specifically binds to linear or stereoepipeds.

[0607] In one embodiment, the antibody molecule is a single-specific antibody molecule and binds to a single epitope. For example, a single-specific antibody molecule having a plurality of immunoglobulin variable domain sequences, each of which binds to the same epitope.

[0608] In one embodiment, the antibody molecule is a multispecific or multifunctional antibody molecule, for example, comprising a plurality of immunoglobulin variable domain sequences, wherein the plurality of first immunoglobulin variable domain sequences have binding specificity for a first epitope and the plurality of second immunoglobulin variable domain sequences have binding specificity for a second epitope. In one embodiment, the first and second epitopes are on the same antigen, for example, the same protein (or subunit of a multimeric protein). In one embodiment, the first and second epitopes overlap. In one embodiment, the first and second epitopes do not overlap. In one embodiment, the first and second epitopes are on different antigens, for example, different proteins (or different subunits of a multimeric protein). In one embodiment, the 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 quadruplicative antibody molecule.

[0609] In one embodiment, the multispecific antibody molecule is a bispecific antibody molecule. The bispecific antibody has specificity for two or fewer antigens. The bispecific antibody molecule is characterized by a first immunoglobulin variable domain sequence having binding specificity for a first epitope and a second immunoglobulin variable domain sequence having binding specificity for a second epitope. In one embodiment, the first and second epitopes are on the same antigen, for example, the same protein (or subunit of a multimeric protein). In one embodiment, the first and second epitopes overlap. In one embodiment, the first and second epitopes do not overlap. In one embodiment, the first and second epitopes are on different antigens, for example, different proteins (or different subunits of a multimeric protein). In one embodiment, the bispecific antibody molecule comprises a heavy chain variable domain sequence and a light chain variable domain sequence having binding specificity for a first epitope, and a heavy chain variable domain sequence and a light chain variable domain sequence having binding specificity for a second epitope. In one embodiment, the bispecific antibody molecule comprises a half antibody having binding specificity for a first epitope and a half antibody having binding specificity for a second epitope. In one embodiment, the bispecific antibody molecule comprises a half antibody or a fragment thereof having binding specificity for a first epitope, and a half antibody or a fragment thereof having binding specificity for a second epitope. In one embodiment, the bispecific antibody molecule comprises a scFv or Fab, or a fragment thereof having binding specificity for a first epitope, and a scFv or Fab, or a fragment thereof having binding specificity for a second epitope.

[0610] In one embodiment, the antibody molecule comprises a diabody and a short-chain molecule, as well as an antigen-binding fragment of the antibody (e.g., Fab, F(ab')2, and Fv). For example, the antibody molecule may comprise a heavy chain (H) variable domain sequence (abbreviated as VH herein) and a light chain (L) variable domain sequence (abbreviated as VL herein). In one embodiment, the antibody molecule comprises or consists of a heavy chain and a light chain (referred to herein as a half-antibody). In other examples, antibody molecules comprise two heavy chain (H) variable domain sequences and two light chain (L) variable domain sequences, thereby forming two antigen-binding sites, e.g., Fab, Fab', F(ab')2, Fc, Fd, Fd', Fv, short-chain antibodies (e.g., scFv), single-variable domain antibodies, diabodies (Dab) (bivalent and bispecific), and chimeric (e.g., humanized) antibodies, which may be produced using recombinant DNA technology by modification of whole antibodies or synthetic de novo antibodies. These functional antibody fragments possess the ability to selectively bind to their respective antigens or receptors. Antibodies and antibody fragments may be derived from any class of antibodies, including but not limited to IgG, IgA, IgM, IgD, and IgE, and any subclass of antibodies (e.g., IgG1, IgG2, IgG3, and IgG4). The preparation of antibody molecules may be monoclonal or polyclonal. The antibody molecule may also be a human, humanized, CDR-transplanted, or in vitro-generated antibody. The antibody may have a heavy chain constant region selected from, for example, IgG1, IgG2, IgG3, or IgG4. The antibody may also have a light chain selected from, for example, kappa or lambda. The term “immunoglobulin” (Ig) is used interchangeably with the term “antibody” herein.

[0611] Examples of antigen-binding fragments of antibody molecules include: (i) a Fab fragment, a monovalent fragment consisting of VL, VH, CL, and CH1 domains; (ii) an F(ab')2 fragment, a divalent fragment comprising two Fab fragments connected by a disulfide bridge at a hinge region; (iii) an Fd fragment consisting of VH and CH1 domains; (iv) an Fv fragment consisting of VL and VH domains of a single arm of the antibody; (v) a diabody (dAb) fragment consisting of a VH domain; (vi) a camelid or camelid variable domain; (vii) a single-chain Fv (scFv). For example, see the literature of Bird et al. [(1988)] Science 242:423-426]; and the literature of Huston et al.[(1988) Proc. Natl. Acad. Sci. USA 85:5879-5883); (viii) single-domain antibodies. These antibody fragments are obtained using conventional techniques known to those skilled in the art, and the fragments are screened for utility in the same way as the whole antibody.

[0612] Antibody molecules contain not only the intact molecule but also its functional fragments. The constant region of the antibody molecule can be mutated to modify the properties of the antibody (e.g., to increase or decrease one or more of Fc receptor binding, antibody glycosylation, the number of cysteine ​​residues, effector cell function, or complement function).

[0613] The antibody molecule may also be a single-domain antibody. A single-domain antibody may include an antibody in which the complementarity determining region is part of a single-domain polypeptide. Examples include, but are not limited to, heavy-chain antibodies, antibodies naturally lacking a light chain, single-domain antibodies derived from conventional 4-chain antibodies, engineered antibodies, and single-domain scaffolds other than those derived from antibodies. A single-domain antibody may be any of the single-domain antibodies in the art or any future single-domain antibody. A single-domain antibody may 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 known as a heavy-chain antibody lacking a light chain. Such a single-domain antibody is disclosed, for example, in WO 9404678. For clarity, such variable domains derived from heavy-chain antibodies that naturally lack light chains are known herein as VHH or nanobodies to distinguish them from the conventional VH of four-chain immunoglobulins. These VHH molecules Camelidae It can be derived from antibodies grown in species, for example, camels, llamas, dromedals, alpacas, and guanacos. Camelidae Other species can produce heavy-chain antibodies that naturally lack light chains; such VHHs are within the scope of the present invention.

[0614] The VH and VL regions can be subdivided into a supervariable region referred to as the “complementarity determination region” (CDR), which is interspersed with a more conserved region referred to as the “framework region” (FR or FW).

[0615] The framework domain and the degree of CDR have been accurately defined by multiple methods (Kabat, EA et al. [(1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242]; Chothia, C. et al. [(1987) J. Mol. Biol . 196:901-917]; and AbM definitions used by Oxford Molecular's AbM antibody modeling software). Generally, for example, Protein Sequence and Structure Analysis of Antibody Variable Domains. In: Antibody Engineering Lab Manual (Ed.: Duebel, S. and Kontermann, R., Springer-Verlag, Heidelberg).

[0616] The terms “complementary determining region” and “CDR” as used herein refer to amino acid sequences within antibody variable regions that confer antigen specificity and binding affinity. Generally, each heavy chain variable region (HCDR1, HCDR2, HCDR3) has three CDRs and each light chain variable region (LCDR1, LCDR2, LCDR3) has three CDRs.

[0617] The exact amino acid sequence boundaries of a given CDR are Kabat et al. [(1991), “Sequences of Proteins of Immunological Interest,” 5 th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (“Kabat” numbering system)], Al-Lazikani et al. [(1997) JMBIt may be determined using any one of a number of known methods, including those described by 273,927-948 (“Chothia” numbering scheme). As used herein, a CDR defined according to the “Chothia” numbering scheme is also referred to as a “supervariable loop.”

[0618] For example, under Kabat, the CDR amino acid residues in the heavy chain variable domain (VH) are numbered 31-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3); and the CDR amino acid residues in the light chain variable domain (VL) are numbered 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3). Under Chothia, the CDR amino acids in VH are numbered 26-32 (HCDR1), 52-56 (HCDR2), and 95-102 (HCDR3); and the amino acid residues in VL are numbered 26-32 (LCDR1), 50-52 (LCDR2), and 91-96 (LCDR3).

[0619] Each VH and VL typically contains three CDRs and four FRs arranged in the following order from the amino terminus to the carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0620] Antibody molecules can be polyclonal or monoclonal antibodies.

[0621] As used herein, the terms “monoclonal antibody” or “monoclonal antibody composition” refer to the preparation of antibody molecules of a single-molecule composition. Monoclonal antibody compositions exhibit single-binding specificity and affinity for specific epitopes. Monoclonal antibodies may be prepared by hybridoma technology or by a method that does not use hybridoma technology (e.g., a recombinant method).

[0622] Antibodies can be produced recombinantly, for example by phage display or by a combination method.

[0623] Phage display and combination methods for generating antibodies are known in the art (e.g., U.S. Patent Publication No. 5,223,409 of Ladner et al., the contents of which are incorporated herein by reference; International Publication No. WO 92 / 18619 of Kang et al.; International Publication No. WO 91 / 17271 of Dower et al.; International Publication No. WO 92 / 20791 of Winter et al.; International Publication No. WO 92 / 15679 of Markland et al.; International Publication No. WO 93 / 01288 of Breitling et al.; International Publication No. WO 92 / 01047 of McCafferty et al.; International Publication No. WO 92 / 09690 of Garrard et al.; International Publication No. WO 90 / 02809 of Ladner et al.; and the literature of Fuchs et al. [(1991)] Bio / Technology 9:1370-1372]; Hay et al.'s literature[(1992) Hum Antibod Hybridomas 3:81-85]; Huse et al.'s literature[(1989) Science 246:1275-1281]; Griffths et al.'s literature[(1993) EMBO J 12:725-734]; Hawkins et al.'s literature[(1992) J Mol Biol 226:889-896]; Clackson et al.'s literature[(1991) Nature 352:624-628]; Gram et al.'s literature[(1992) PNAS 89:3576-3580]; Garrad et al.'s literature[(1991) Bio / Technology 9:1373-1377]; the literature of Hoogenboom et al.[(1991) Nuc Acid Res 19:4133-4137]; and the literature of Barbas et al.[(1991) PNAS A gripping display and combination method as described in [88:7978-7982]).

[0624] In one embodiment, the antibody is a complete human antibody (e.g., an antibody produced in a mouse genetically engineered to produce an antibody from a human immunoglobulin sequence), or a non-human antibody, e.g., a rodent (mouse or rat), goat, primate (e.g., monkey), or camel antibody. Preferably, the non-human antibody is a rodent (mouse or rat antibody). Methods for producing rodent antibodies are known in the art.

[0625] Human monoclonal antibodies can be generated using genetically modified mice carrying human immunoglobulin genes rather than mouse systems. Using splenocytes from these genetically modified mice immunized with the antigen of interest, hybridomas are produced that secrete human mAbs having specific affinity for epitopes from human proteins (e.g., 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]; the literature of Morrison, SL et al.[1994 Proc. Natl. Acad. Sci. USA 81:6851-6855]; Bruggeman et al. [1993 Year Immunol 7:33-40]; Tuaillon et al. [1993 PNAS 90:3720-3724]; Bruggeman et al.'s literature[1991 Eur J Immunol [See 21:1323-1326]).

[0626] The antibody molecule may have a variable region or part thereof, for example, a CDR, generated in a non-human organism, for example, a rat or a mouse. Chimeras, CDR-transplants, and humanized antibodies are within the invention. Antibody molecules generated in a non-human organism, for example, a rat or a mouse, and then modified, for example, in a variable framework or an invariant region, to reduce antigenicity in humans are within the invention.

[0627] “Effectively human” proteins are proteins that do not substantially induce a neutralizing antibody response, for example, a human anti-rat antibody (HAMA) response. HAMA can be problematic in many situations, for example, when antibody molecules are repeatedly administered for the treatment of chronic or recurrent disease conditions. The HAMA response is due to increased antibody clearance from the serum (e.g., Saleh et al.'s literature [ Cancer Immunol. Immunother. [See , 32:180-190 (1990)]) and also due to potential allergic reactions (e.g., the literature of LoBuglio et al. [ Hybridoma [Refer to , 5:5117-5123 (1986)] Repeated antibody administration can potentially make it ineffective.

[0628] Chimeric antibodies can be produced by recombinant DNA technology known in the art (International patent publication PCT / US86 / 02269 by Lobinson et al.; European patent application 184,187 by Akira et al.; European patent application 171,496 by Taniguchi, M.; European patent application 173,494 by Morrison et al.; International application WO 86 / 01533 by Neuberger et al.; U.S. patent number 4,816,567 by Cabilly et al.; European patent application 125,023 by Cabilly et al.; literature by Better et al. [(1988 Science 240:1041-1043)]; Liu et al.'s literature[(1987) PNAS84:3439-3443]; Liu et al. [1987, J. Immunol. 139:3521-3526]; Sun et al.'s literature[(1987) PNAS 84:214-218]; the literature of Nishimura et al.[1987, Canc. Res. 47:999-1005]; Wood et al.'s literature[(1985) Nature 314:446-449]; and the literature of Shaw et al.[1988, J. Natl Cancer Inst. [Refer to 80:1553-1559]).

[0629] The humanized or CDR-transplanted antibody will have at least one or two, but generally three, recipient CDRs (of heavy and / or light chain immunoglobulin chains) replaced by the donor CDR. The antibody may be replaced by at least a portion of the non-human CDRs, or only a portion of the CDRs may be replaced by non-human CDRs. Only the number of CDRs required to bind to the antigen needs to be replaced. Preferably, the donor will be a rodent antibody, e.g., a rat or mouse antibody, and the recipient will be a human framework or a human consensus framework. Generally, the immunoglobulin providing the CDRs is referred to as the “donor,” and the immunoglobulin providing the framework is referred to as the “recipient.” In one embodiment, the donor immunoglobulin is non-human (e.g., rodent). The recipient framework is a naturally occurring (e.g., human) framework or a consensus framework, or a sequence that is at least 85%, preferably 90%, 95%, or 99% identical to it.

[0630] As used herein, the term “consensus sequence” refers to a sequence formed from the most frequently occurring amino acids (or nucleotides) in a series of related sequences (see, for example, Winnaker’s work [From Genes to Clones (Verlagsgesellschaft, Weinheim, Germany 1987)]). In a protein series, each position of the consensus sequence is occupied by the most frequently occurring amino acid at that position in the series. If two amino acids occur equally frequently, either one may be included in the consensus sequence. The “consensus framework” refers to the framework region of the consensus immunoglobulin sequence.

[0631] Antibody molecules can be humanized by methods known in the art ( for example, Morrison, SL, 1985, Science 229:1202-1207, Oi etc, 1986, BioTechniques 4:214, and Queen etc. , described in US 5,585,089, US 5,693,761 and US 5,693,762, the contents of all of which are incorporated herein by reference).

[0632] Humanized or CDR-transplanted antibody molecules may be produced by CDR-transplantation or CDR substitution, wherein one, two, or all CDRs of the immunoglobulin chain may be substituted. For example, U.S. Patent 5,225,539; Jones et al. [1986 Nature 321:552-525]; Verhoeyan et al. [1988 ScienceReferences are made to 239:1534; Beidler et al. [1988 J. Immunol. 141:4053-4060]; and Winter’s U.S. Patent 5,225,539, the contents of all of which are expressly incorporated herein by reference. Winter describes a CDR-transplant method that can be used to produce the humanized antibody of the present invention (UK patent application GB 2188638A filed March 26, 1987; Winter’s U.S. Patent 5,225,539), the contents of which are expressly incorporated herein by reference.

[0633] Additionally, humanized antibody molecules having specific amino acids substituted, deleted, or added are within the scope of the present invention. Criteria for selecting amino acids from a donor are described in U.S. Patent 5,585,089, e.g., columns 12-16 of U.S. Patent 5,585,089, the contents of which are incorporated herein by reference. Other techniques for humanizing antibodies are described in EP 519596 A1 by Padlan et al. (published December 23, 1992).

[0634] Antibody molecules can be short-chain antibodies. Short-chain antibodies (scFVs) can be engineered (e.g., Colcher, D. et al.

[1999] Ann NY Acad Sci 880:263-80]; and the literature of Reiter, Y. [(1996) Clin Cancer Res [See 2:245-52]). Single-chain antibodies can be dimerized or multimerized to produce multivalent antibodies that have specificity for different epitopes of the same target protein.

[0635] In another embodiment, 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; in particular, it has a heavy chain constant region selected from, for example, the (e.g., human) heavy chain constant regions of IgG1, IgG2, IgG3, and IgG4. In another embodiment, the antibody molecule has a light chain constant region selected from, for example, the (e.g., human) light chain constant regions of kappa or lambda. The constant regions may be altered, for example, mutated, to modify the properties of the antibody (e.g., to increase or decrease one or more of Fc receptor binding, antibody glycosylation, number of cysteine ​​residues, effector cell function, and / or complement function). In one embodiment, the antibody has an effector function; and can immobilize complement. In another embodiment, the antibody does not mobilize effector cells or immobilize complement. In another embodiment, the antibody has reduced or no ability to bind to the Fc receptor. For example, this is an isoform or subform, fragment, or other mutant that does not support binding to the Fc receptor, and has, for example, a mutated or deleted Fc receptor binding region.

[0636] Methods for modifying the constant region of an antibody are known in the art. Modified functions, for example, antibodies having altered affinities 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 No. 5,624,821 and U.S. Patent No. 5,648,260, the contents of all of which are incorporated herein by reference). Similar types of modifications may be described, which, when applied to rodents or other species, will reduce or eliminate these functions in the immunoglobulin.

[0637] Antibody molecules may be derivatized or linked to another functional molecule (e.g., another peptide or protein). As used herein, a “derivativeized” antibody molecule is a modified one. Derivatization methods include, but are not limited to, the addition of affinity ligands such as fluorescent moiety, radionucleotide, toxin, enzyme, or biotin. Accordingly, the antibody molecules of the present invention are intended to include derivatized and otherwise modified forms of the antibodies described herein, including immunoadhesive molecules. For example, the antibody molecule may be functionally linked to one or more other molecular entities (by chemical coupling, genetic fusion, non-covalent bonding, etc.), such as other antibodies (e.g., bispecific antibodies or diabodies), detectable agents, cytotoxic agents, pharmaceutical agents, and / or proteins or peptides capable of mediating binding to the antibody or other molecules of the antibody or antibody portion (e.g., streptavidin core region or polyhistidine tag).

[0638] One type of derivatized antibody molecule is produced by crosslinking two or more antibodies of the same or different types (e.g., to produce a bispecific antibody). Suitable crosslinkers include those that are heterofunctional (e.g., m-maleimidobenzoyl-N-hydroxysuccinimide ester) or homodifunctional (e.g., disuccinimidyl suberate) having two distinct reactive groups separated by a suitable spacer. Such linkers are available from Pierce Chemical Company (Rockford, II).

[0639] Multispecific or multifunctional antibody molecules

[0640] As used herein, a “multifunctional” or “multispecific” molecule refers to a molecule having two or more functional groups, e.g., two or more binding specificities, e.g., a polypeptide. In some embodiments, the functional groups may include one or more immune cell binders, one or more tumor binding molecules, one or more cytokine molecules, one or more substrate modifiers, and other moiety as described herein. In some embodiments, the multispecific molecule is a multispecific antibody molecule, e.g., a bispecific antibody molecule.

[0641] The exemplary structures of multispecific and multifunctional molecules defined herein are described in their entirety. Exemplary structures are further described below, the full contents of each of which are incorporated herein by reference: the work of 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].

[0642] In an embodiment, the multispecific antibody molecule may comprise two or more antigen-binding sites, wherein different sites are specific to different antigens. In an embodiment, the multispecific antibody molecule may bind to more than one (e.g., two or more) epitopes on the same antigen. In an embodiment, the multispecific antibody molecule comprises an antigen-binding site specific to target cells (e.g., cancer cells) and different antigen-binding sites specific to immune effector cells. In one embodiment, the multispecific antibody molecule is a bispecific antibody molecule. Bispecific antibody molecules may be classified 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.

[0643] BsIgG is a format that is monovalent to each antigen. Exemplary BsIgG formats include, but are not limited to, crossMab, DAF(2-in-1), DAF(4-in-1), DutaMab, DT-IgG, Knob-in-Hole common LC, Knob-in-Hole assembly, charge pair, Fab-arm exchange, SEEDbody, Triomab, LUZ-Y, Fcab, kl-body, and orthogonal Fab. Spiess See the literature by et al. [Mol. Immunol. 67(2015):95-106]. Exemplary BsIgGs include catumaxomab (Fresenius Biotech, Trion Pharma, Neopharm) containing anti-CD3 and anti-EpCAM subarms; and ertumaxomab (Neovii Biotech, Fresenius Biotech) targeting CD3 and HER2. In some embodiments, BsIgGs comprise a heavy chain engineered for heterodimerization. For example, the heavy chain may be engineered for heterodimerization using the “knov-into-hole” strategy, the SEED platform, a common heavy chain (e.g., within a kl-body), and the heterodimer Fc region. Spiess See the literature by et al. [Mol. Immunol. 67(2015):95-106] . Strategies used to avoid homomer heavy chain pairing in BsIgG include knobe-in-hole, duobodies, enzymes, charge pairs, HA-TF, SEED bodies, and differential protein A affinity. See above. BsIgG can be produced by the separate expression of component antibodies in different host cells and subsequent purification / assembly into BsIgG. BsIgG can also be produced by the expression of component antibodies in a single host cell. BsIgG can be purified using affinity chromatography, for example, with protein A and sequential pH elution.

[0644] IgG with an additional antigen-binding moiety attached is another format of a bispecific antibody molecule. For example, monospecific IgG can be engineered to possess bispecificity by attaching an additional antigen-binding unit to the monospecific IgG, for example, at the N-terminus or C-terminus of the heavy or light chain. Exemplary additional antigen-binding units include a single-domain antibody (e.g., variable heavy chain or variable light chain), an engineered protein scaffold, and a paired antibody variable domain (e.g., a single-chain variable fragment or variable fragment). See above. Examples of the attached IgG formats include dual variable domain IgG (DVD-Ig), IgG(H)-scFv, scFv-(H)IgG, IgG(L)-scFv, scFv-(L)IgG, IgG(L,H)-Fv, IgG(H)-V, V(H)-IgG, IgG(L)-V, V(L)-IgG, KIH IgG-scFab, 2scFv-IgG, IgG-2scFv, scFv4-Ig, zybody, and DVI-IgG(4-in-1). SpiessSee Mol. Immunol. 67(2015):95-106. An example of IgG-scFv is MM-141 (Merrimack Pharmaceuticals), which binds to IGF-1R and HER3. Examples of DVD-Ig include ABT-981 (AbbVie), which binds to IL-1α and IL-1β; and ABT-122 (AbbVie), which binds to TNF and IL-17A.

[0645] A bispecific antibody fragment (BsAb) is a format of a bispecific antibody molecule lacking some or all of the antibody constant domain. For example, some BsAbs lack the Fc region. In an embodiment, the bispecific antibody fragment comprises a heavy chain and a light chain region joined by a peptide linker that allows for efficient expression of the BsAb in a single host cell. Exemplary bispecific antibody fragments include, but are not limited to, nanobodies, nanobodies-HAS, BiTE, diabodies, DART, TandAb, sc-diabodies, sc-diabodies-CH3, diabodies-CH3, tribodies, miniantibodies, minibodies, TriBi minibodies, scFv-CH3 KIH, Fab-scFv, scFv-CH-CL-scFv, F(ab')2, F(ab')2-scFv2, scFv-KIH, Fab-scFv-Fc, tetravalent HCAb, sc-diabodies-Fc, diabodies-Fc, tandem scFv-Fc, and intrabodies. See above. For example, the BiTE format includes tandem scFv, wherein the component scFv binds to CD3 on T cells and surface antigens on cancer cells.

[0646] Bispecific fusion proteins comprise, for example, antibody fragments linked to other proteins to add additional specificity and / or functionality. An example of a bispecific fusion protein is an immTAC containing an anti-CD3 scFv linked to an affinity-mature T cell receptor that recognizes an HLA-presenting peptide. In an embodiment, a dock-and-lock (DNL) method may be used to generate bispecific antibody molecules having a higher valence. Additionally, fusion to an albumin-binding protein or human serum albumin may extend the serum half-life of the antibody fragment. See above.

[0647] In an embodiment, a BsAb molecule may be produced using chemical conjugation, e.g., chemical conjugation of an antibody and / or antibody fragment. See above. An exemplary bispecific antibody conjugate comprises a CovX-body format in which a low molecular weight drug is site-specifically conjugated to a single reactive lysine in each Fab arm or antibody or fragment thereof. In an embodiment, the conjugation improves the serum half-life of the low molecular weight drug. An exemplary CovX-body is CVX-241 (NCT01004822), which comprises an antibody conjugated to two short peptides that inhibit VEGF or Ang2. See above.

[0648] Antibody molecules may be produced in a host system, for example, by the recombinant expression of at least one component. Exemplary host systems include eukaryotic cells (e.g., mammalian cells, e.g., CHO cells, or insect cells, e.g., SF9 or S2 cells) and prokaryotic cells (e.g., E. coli). Bispecific antibody molecules may be produced by the separate expression of components in different host cells and subsequent purification / assembly. Alternatively, antibody molecules may be produced by the expression of components in a single host cell. Purification of bispecific antibody molecules may be performed by various methods, such as affinity chromatography, using, for example, protein A and sequential pH elution. In other embodiments, affinity tags, for example, histidine-containing tags, myc tags, or streptavidin tags may be used for purification.

[0649] CDR-Implanted Scaffold

[0650] In an embodiment, the antibody molecule is a CDR-transplanted scaffold domain. In an embodiment, the scaffold domain is based on a fibronectin domain, e.g., a fibronectin type III domain. The total folding of the fibronectin type III (Fn3) domain is closely related to the folding of the smallest functional antibody fragment, which is the variable domain of the antibody heavy chain. At the end of Fn3 are the following three loops; the positions of the BC, DE, and FG loops correspond approximately to the positions of CDR1, 2, and 3 of the antibody's VH domain. Fn3 does not possess disulfide bonds; therefore, unlike the antibody and its fragments, Fn3 is stable under reducing conditions (see, e.g., WO 98 / 56915; WO 01 / 64942; WO 00 / 34784). The Fn3 domain may be modified or varied (e.g., using the CDR or hypervariable loop described herein) to select a domain that binds to the antigen / marker / cell described herein.

[0651] In an embodiment, the scaffold domain, e.g., the folded domain, is based on a “minibody” scaffold generated by deleting three beta strands from the heavy chain variable domain of an antibody, e.g., a monoclonal antibody (see, e.g., Tramontano et al. [1994, J Mol. Recognit. 7:9]; and Martin et al. [1994, EMBO J. 13:5303-5309]). The “minibody” can be used to present two hypervariable loops. In an embodiment, the scaffold domain is a V-shaped domain (e.g., Coia et al. WO 99 / 45110) or a domain derived from tendamistatin, which is a 74-residue, 6-strand beta-sheet sandwich held together by two disulfide bonds (e.g., see McConnell and Hoess [1995, J Mol. Biol. 250:460]). For example, the loop of tenamistatin may be modified or varied (e.g., using a CDR or a supervariable loop) to select a domain that binds to the marker / antigen / cell described herein. Another exemplary scaffold domain is a beta-sandwich structure derived from the extracellular domain of CTLA-4 (e.g., see WO 00 / 60070).

[0652] 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, ECOTI, BPTI, etc.); TPR repeats; trifoil structures; zinc finger domains; DNA-binding proteins; particularly monomeric DNA-binding proteins; RNA-binding proteins; enzymes, e.g., proteases (particularly inactivated proteases), RNases; chaperones, e.g., thioredoxin; and heat shock proteins; and intracellular signaling domains (e.g., SH2 and SH3 domains). For example, refer to US 20040009530 and US 7,501,121 incorporated herein by reference.

[0653] In an embodiment, the scaffold domain is evaluated and selected, for example, by one or more of the following criteria: (1) amino acid sequence, (2) sequence of several homologous domains, (3) three-dimensional structure, and / or (4) stability data over a range of pH, temperature, salinity, organic solvent, and oxidant concentration. In an embodiment, the scaffold domain is a small and stable protein domain, for example, a protein of 100, 70, 50, 40, or fewer than 30 amino acids. The domain may contain one or more disulfide bonds or may chelate a metal, for example, zinc.

[0654] antibody-based fusion

[0655] Various formats containing additional binding entities attached to the N or C terminals of antibodies can be produced. These fusions with single-chain or disulfide-stabilized Fv or Fab result in the production of tetravalent molecules with divalent binding specificity for each antigen. The combination of IgG with scFv and scFab enables the production of molecules capable of recognizing three or more different antigens.

[0656] Antibody-Fab Fusion

[0657] An antibody-Fab fusion is a bispecific antibody comprising a traditional antibody for a first target and a Fab for a second target fused to the C-terminus of the antibody heavy chain. Generally, the antibody and the Fab will have a common light chain. Antibody fusions are (1) Manipulating the DNA sequence of the targeted fusion, and (2) To express a fusion protein, it can be produced by transfecting target DNA into an appropriate host cell. Antibody-scFv fusion is Coloma, J. etc. (1997) Nature Biotech As described in 15:159, it appears that the C-terminus of the CH3 domain and the N-terminus of the scFv can be connected by a (Gly)-Ser linker.

[0658] Antibody-scFv fusion

[0659] Antibody-scFv fusion It is a bispecific antibody comprising a traditional antibody of intrinsic specificity and a scFv fused to the C-terminus of the antibody heavy chain. The scFv can be fused to the C-terminus directly via the scFv heavy chain or via a linker peptide. Antibody fusion is (1) Manipulating the DNA sequence of the targeted fusion, and (2) To express a fusion protein, it can be produced by transfecting target DNA into an appropriate host cell. Antibody-scFv fusion is Coloma, J. etc. (1997) Nature Biotech As described in 15:159, it appears that the C-terminus of the CH3 domain and the N-terminus of the scFv can be connected by a (Gly)-Ser linker.

[0660] Variable Domain Immunoglobulin DVD

[0661] The relevant format is a dual variable domain immunoglobulin (DVD), which consists of VH and VL domains at a second specificity position on the N-terminus of the V domain by a shorter linker sequence.

[0662] Other exemplary multispecific antibody formats include those described below: e.g., US20160114057A1, US20130243775A1, US20140051833, US20130022601, US20150017187A1, US20120201746A1, US20150133638A1, US20130266568A1, US20160145340A1, WO2015127158A1, US20150203591A1, US20140322221A1, US20130303396A1, US20110293613, US20130017200A1, US20160102135A1, WO2015197598A2, WO2015197582A1, US9359437, US20150018529, WO2016115274A1, WO2016087416A1, US20080069820A1, US9145588B, US7919257, and US20150232560A1. Exemplary multispecific molecules using the whole antibody-Fab / scFab format include those described below: US9382323B2, US20140072581A1, US20140308285A1, US20130165638A1, US20130267686A1, US20140377269A1, US7741446B2, and WO1995009917A1. Exemplary multispecific molecules using a domain exchange format include those described below: US20150315296A1, WO2016087650A1, US20160075785A1, WO2016016299A1, US20160130347A1, US20150166670, US8703132B2, US20100316645, US8227577B2, US20130078249.

[0663] Fc-containing entity (mini-antibody)

[0664] Fc-containing entities, also known as mini-antibodies, can be produced by fusing scFv to the C-terminus of the constant heavy chain domain 3 (CH3-scFv) and / or to the hinge domain of an antibody with different specificity (scFv-hinge-Fc). A trivalent entity having a disulfide-stabilized variable domain (without a peptide linker) fused to the C-terminus of the CH3 domain of IgG may also be produced.

[0665] Fc-containing multispecific molecules

[0666] In some embodiments, the multispecific molecule disclosed herein comprises an immunoglobulin constant region (e.g., an Fc region). An exemplary Fc region may be selected from a heavy chain constant region of IgG1, IgG2, IgG3, or IgG4; more specifically, from a heavy chain constant region of human IgG1, IgG2, IgG3, or IgG4.

[0667] In some embodiments, the immunoglobulin chain constant region (e.g., Fc region) is modified, e.g., mutated 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.

[0668] In another embodiment, the interface of the first and second immunoglobulin chain constant regions (e.g., first and second Fc regions) is modified, for example, or mutated to increase or decrease dimerization compared to, for example, an unmanipulated interface, e.g., a naturally occurring interface. For example, dimerization of the immunoglobulin chain constant regions (e.g., Fc regions) may be enhanced by providing an Fc interface of the first and second Fc regions having one or more of paired protrusion-cavities (“knob-in-holes”), electrostatic interactions, or strand-exchanges, so as to form a greater ratio of heteropolymers to homopolymers compared to, for example, an unmanipulated interface.

[0669] In some embodiments, the multispecific molecule comprises, for example, a paired amino acid substitution at a position selected from one or more of 347, 349, 350, 351, 366, 368, 370, 392, 394, 395, 397, 398, 399, 405, 407, or 409 in the Fc region of human IgG1. The immunoglobulin chain constant region (e.g., the Fc region) may comprise a paired amino acid substitution selected from the following: T366S, L368A, or Y407V (e.g., corresponding to a cavity or hole), and T366W (e.g., corresponding to a protrusion or knob).

[0670] In another embodiment, the multifunctional molecule includes a half-life extender, for example, human serum albumin or an antibody molecule against human serum albumin.

[0671] Heteromerized antibody molecule and method of preparation

[0672] Various methods for producing multispecific antibodies to address the problem of inaccurate heavy chain pairing have been disclosed. Exemplary methods are described below. Exemplary multispecific antibody formats and methods for producing said multispecific antibodies are 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 full contents of each of these are incorporated herein by reference.

[0673] Heterodimeric bispecific antibodies are based on a natural IgG structure, where two binding arms recognize different antigens. IgG-derived formats that enable defined monovalent (and simultaneous) antigen binding are generated by forced heavy chain heterodimerization combined with techniques to minimize light chain pairing errors (e.g., common light chains). Forced heavy chain heterodimerization can be obtained, for example, using a knob-in-hole OR strand exchange operation domain (SEED).

[0674] Knob-in-Hole

[0675] US 5,731,116, US 7,476,724 and Ridgway, J. etc. (1996) Prot. Engineering The knob-in-hole described in 9(7): 617-621 is extensively involved in the following: (1) Mutating the CH3 domain of one or both antibodies to promote heterodimerization; and (2) combining the mutated antibodies under conditions that promote heterodimerization. “Knobs” or “protrusions” are generally created by substituting a small amino acid in the parent antibody with a larger amino acid (e.g., T366Y or T366W); and “holes” or “cavities” are created by substituting a larger residue in the parent antibody with a smaller amino acid (e.g., Y407T, T366S, L368A, and / or Y407V).

[0676] For bispecific antibodies containing an Fc domain, introducing specific mutations into the constant region of the heavy chain may be used to facilitate the precise heteromerization of the Fc portion. 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 a “knob-into-hole” (KiH) approach involving the introduction of a bulky residue into one of the CH3 domains of one of the antibody heavy chains. This bulky residue is inserted into a complementary “hole” in the other CH3 domain of the paired heavy chain to facilitate the precise pairing of the heavy chains (see, for example, US7642228).

[0677] Exemplary KiH mutations include S354C and T366W in the “knob” heavy chain and Y349C, T366S, L368A, and Y407V in the “hall” heavy chain. Other exemplary KiH mutations with additional selective stabilizing Fc cysteine ​​mutations are provided in Table 1.

[0678] Other Fc mutations are provided by Igawa and Tsunoda, who identified three negatively charged residues in the CH3 domain of one chain, which are paired with three positively charged residues in the CH3 domain of the other chain. These specific charged residue pairs are as follows: E356-K439, E357-K370, D399-K409, and vice versa. By introducing the following three mutations in chain A: E356K, E357K, and D399K, as well as at least two of K370E, K409D, and K439E in chain B, either alone or in combination with a newly identified disulfide bridge, it was possible to be favorable for highly efficient heterodimerization while inhibiting homodimerization (Martens T et al. A novel one-armed anti-Met antibody inhibits glioblastoma growth in vivo. Clin Cancer Res 2006; 12:6144-52; PMID:17062691). Xencor defined 41 variant pairs based on combining structural calculations and subsequently screened sequence information for maximum heteromerization, defining combinations of S364H, F405A (HA) on chain A and Y349T, T394F (TF) on chain B (Moore GL et al. A novel bispecific antibody format enables simultaneous bivalent and monovalent co-engagement of distinct target antigens. MAbs 2011; 3:546-57; PMID: 22123055).

[0679] Other exemplary Fc mutations for promoting the heteromerization 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, US20160229915A1, US20150344570A1, US8003774A1, US20150337049A1, US20150175707A1, US20140242075A1, US20130195849A1, US20120149876A1, US20140200331A1, US9309311B2, US8586713, US20140037621A1, US20130178605A1, US20140363426A1, US20140051835A1 and US20110054151A1.

[0680] Stabilizing cysteine ​​mutants have also been used in combination with KiH and other Fc heteromerization-promoting mutants, see, for example, US7183076. Other exemplary cysteine ​​modifications include those disclosed, for example, in US20140348839A1, US7855275B2, and US9000130B2.

[0681] Strand Exchange Engineered Domain (SEED)

[0682] A heterodimeric Fc platform is known that supports the design of bispecific and asymmetric fusion proteins by devising strand-exchange manipulation domain (SEED) C(H)3 heterodimers. These derivatives of human IgG and IgA C(H)3 domains generate complementary human SEED C(H)3 heterodimers consisting of alternating segments of human IgA and IgG C(H)3 sequences. The generated SEED C(H)3 domain pairs preferentially bind to form a heterodimer when expressed in mammalian cells. The SEEDbody(Sb) fusion protein consists of [IgG1 hinge]-C(H)2-[SEED C(H)3], which may be genetically linked to one or more fusion partners (e.g., see the works of Davis JH et al. incorporated herein by reference [SEEDbodies: fusion proteins based on strand exchange engineered domain (SEED) CH3 heterodimers in an Fc analogue platform for asymmetric binders or immunofusions and bispecific antibodies. Protein Eng Des Sel 2010; 23:195-202; PMID:20299542] and US8871912, respectively).

[0683] Duobody

[0684] “DuoBody” technology for producing bispecific antibodies with precise heavy chain pairing is known. DuoBody technology comprises three basic steps for generating stable bispecific human IgG1 antibodies in a post-production exchange reaction. In the first step, two IgG1 antibodies, each containing a single matching mutation in the third constant (CH3) domain, are produced separately using a standard mammalian recombinant cell line. These IgG1 antibodies are then purified according to standard processes for recovery and purification. After production and purification (post-production), the two antibodies are recombined under customized laboratory conditions to produce bispecific antibody products in very high yields (typically >95%) (see, e.g., Labrijn et al., PNAS 2013;110(13):5145-5150 and Labrijn et al., Nature Protocols 2014;9(10):2450-63, the contents of which are incorporated herein by reference).

[0685] electrostatic interaction

[0686] A method for preparing a multispecific antibody using a CH3 amino acid change having a charged amino acid such that homodimer formation is electrostatically undesirable is disclosed. EP1870459 and WO 2009089004 describe other strategies for favoring heterodimer formation upon co-expression of different antibody domains in host cells. In these methods, one or more residues constituting the heavy chain constant domain 3 (CH3), and the CH3-CH3 interface at both CH3s, are replaced with a charged amino acid such that homodimer formation is electrostatically undesirable and heterodimerization is electrostatically favorable. Additional methods for preparing a multispecific molecule 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.

[0687] common light chain

[0688] To produce a homogeneous formulation of bispecific IgG, it is necessary to avoid light chain pairing errors. One way to achieve this is to use the common light chain principle, that is, to combine two binders that share a single light chain but still possess distinct specificities. An exemplary method for enhancing the formation of a desired bispecific antibody from a mixture of monomers is by providing a common variable light chain to interact with each of the heteromeric variable heavy chain regions of the bispecific antibody. For example, compositions and methods for producing bispecific antibodies having a common light chain as disclosed in US7183076B2, US20110177073A1, EP2847231A1, WO2016079081A1, and EP3055329A1, the contents of which are incorporated herein by reference.

[0689] Common heavy chain

[0690] An exemplary method for enhancing the formation of a desired bispecific antibody from a mixture of monomers is by providing a common variable heavy chain to interact with each of the heteromeric variable light chain regions of the bispecific antibody. Compositions and methods for producing a bispecific antibody having a common heavy chain are disclosed, for example, in US20120184716, US20130317200, and US20160264685A1, the contents of which are incorporated herein by reference.

[0691] CrossMab

[0692] Another option for reducing light chain error-pairing is CrossMab technology, which avoids non-specific L-chain error-pairing by exchanging the CH1 and CL domains at half of the Fab of a bispecific antibody. These crossover variants maintain binding specificity and affinity but differ the two arms to prevent L-chain error-pairing. CrossMab technology (Kellen et al. The above ) involves domain exchange between the heavy chain and light chain to facilitate the formation of precise pairings. Briefly, to construct a bispecific IgG-like CrossMab antibody capable of binding to two antigens by using two distinct light-heavy chain pairs, a two-step modification process is applied. First, the dimerization interface is manipulated at the C-terminus of each heavy chain using a heteromerization approach, e.g., the Knob-Into-Hole (KiH) technique, to ensure that only two distinct heavy chain heteromers are efficiently formed from one antibody (e.g., Antibody A) and a second antibody (e.g., Antibody B). Next, the constant heavy chain 1 (CH1) and constant light chain (CL) domains of one antibody are exchanged (Antibody A) to consistently maintain the variable heavy chain (VH) and variable light chain (VL) domains. The exchange of the CH1 and CL domains ensured that the modified antibody (Antibody A) light chain dimerizes efficiently only with the modified antibody (Antibody A) heavy chain, whereas the unmodified antibody (Antibody B) light chain dimerizes efficiently only with the unmodified antibody (Antibody B) heavy chain; thus, only the desired bispecific CrossMab will be efficiently formed (see, for example, the work of Cain, C. incorporated herein by reference [SciBX 4(28); doi:10.1038 / scibx.2011.783]).

[0693] Amino acid modification

[0694] Alternative compositions and methods for producing multispecific antibodies having precise light chain pairings include various amino acid modifications. For example, Zymeworks describes a heterodimer having one or more amino acid modifications in the CH1 and / or CL domains and one or more amino acid modifications in the VH and / or VL domains, or a combination thereof, which are part of the interface between the light chain and the heavy chain and create a preferential pairing between each heavy chain and the desired light chain, so that when the two heavy chains and two light chains of the heterodimer pair are co-expressed in a cell, the heavy chain of the first heterodimer preferentially pairs with one of the light chains rather than one of the light chains (see, e.g., WO2015181805). Other exemplary methods are described in WO2016026943 (Argen-X), US20150211001, US20140072581A1, US20160039947A1, and US20150368352.

[0695] Lambda / Kappa format

[0696] A multispecific molecule (e.g., a multispecific antibody molecule) comprising a lambda light chain polypeptide and a kappa light chain polypeptide may be used to allow heteromerization. A method for producing a bispecific antibody molecule comprising a lambda light chain polypeptide and a kappa light chain polypeptide is disclosed in PCT / US17 / 53053 filed September 22, 2017, the entirety of which is incorporated herein by reference.

[0697] In an embodiment, the multispecific molecule comprises a multispecific antibody molecule, for example, an antibody molecule comprising two binding specificities, for example, a bispecific antibody molecule. The multispecific antibody molecule comprises the following:

[0698] Lambda light chain polypeptide 1 (LLCP1) specific to the first epitope;

[0699] Heavy chain polypeptide 1 (HCP1) specific to the first epitope;

[0700] Kappa light chain polypeptide 2 (KLCP2) specific to the second epitope; and

[0701] Heavy chain polypeptide 2 (HCP2) specific to the second epitope.

[0702] As used herein, the term “lambda light chain polypeptide 1 (LLCP1)” refers to a polypeptide comprising a light chain (LC) sequence sufficient to mediate specific binding to its epitope and form a complex with HCP1 when combined with a homologous heavy chain variable region. In one embodiment, it comprises all or a fragment of the CH1 region. In one embodiment, LLCP1 comprises a sequence sufficient to mediate specific binding to LC-CDR1, LC-CDR2, LC-CDR3, FR1, FR2, FR3, FR4, and CH1, or its epitope, and a complex with HCP1. LLCP1 provides specificity for a first epitope with its HCP1 (KLCP2 provides specificity for a second epitope with its HCP2). As described elsewhere in this invention, LLCP1 has a higher affinity for HCP1 than HCP2.

[0703] “Kappa light chain polypeptide 2 (KLCP2)” refers to a polypeptide comprising a light chain (LC) sequence sufficient to mediate specific binding to its epitope and form a complex with HCP2 when combined with a homologous heavy chain variable region, as the term is used herein. In one embodiment, it comprises all or a fragment of the CH1 region. In one embodiment, KLCP2 comprises LC-CDR1, LC-CDR2, LC-CDR3, FR1, FR2, FR3, FR4, and CH1, or a sequence sufficient therefrom, to mediate the specific binding of the epitope and the complex with HCP2. KLCP2 provides specificity for a second epitope with its HCP2 (whereas LLCP1 provides specificity for a first epitope with its HCP1).

[0704] As used herein, the term “heavy chain polypeptide 1 (HCP1)” refers to a polypeptide comprising a heavy chain (HC) sequence, e.g., a HC variable region sequence, sufficient to mediate specific binding to its epitopes when combined with cognate LLCP1 and to form a complex with HCP1. In one embodiment, it comprises all or a fragment of the CH1 region. In one embodiment, it comprises all or a fragment of the CH2 and / or CH3 region. In one embodiment, HCP1 comprises HC-CDR1, HC-CDR2, HC-CDR3, FR1, FR2, FR3, FR4, CH1, CH2, and CH3, or a sufficient sequence thereof, and (i) mediates the specific binding of an epitope and a complex with LLCP1, (ii) preferentially forms a complex with LLCP1 as described herein in contrast to KLCP2, and (iii) preferentially forms a complex with HCP2 as described herein in contrast to other molecules of HCP1. HCP1 provides specificity for a first epitope with its LLCP1 (KLCP2 provides specificity for a second epitope with its HCP2).

[0705] As used herein, the term “heavy chain polypeptide 2 (HCP2)” refers to a polypeptide comprising a sufficient heavy chain (HC) sequence, e.g., a sequence of the HC variable region, which, when combined with the homologous LLCP1, mediates specific binding to its epitopes and can form a complex with HCP1. In one embodiment, it comprises all or a fragment of the CH1 region. In one embodiment, it comprises all or a fragment of the CH2 and / or CH3 region. In one embodiment, HCP1 comprises HC-CDR1, HC-CDR2, HC-CDR3, FR1, FR2, FR3, FR4, CH1, CH2, and CH3, or a sufficient sequence thereof, and (i) mediates the specific binding of an epitope and a complex with KLCP2, (ii) preferentially forms a complex with KLCP2 as described herein in contrast to LLCP1, and (iii) preferentially forms a complex with HCP1 in contrast to other molecules of HCP2. HCP2 provides specificity for a second epitope with KLCP2 (LLCP1 provides specificity for a first epitope with HCP1).

[0706] In some embodiments of the multispecific antibody molecule disclosed herein:

[0707] LLCP1 has a higher affinity for HCP1 than HCP2; and / or

[0708] KLCP2 has a higher affinity for HCP2 than HCP1.

[0709] In an embodiment, the affinity of LLCP1 for HCP1 is sufficiently greater than the affinity for HCP2, so that under predetermined conditions, for example in an aqueous buffer of pH 7, for example in a saline solution of pH 7, or under physiological conditions, at least 75, 80, 90, 95, 98, 99, 99.5, or 99.9% of the molecules of the multispecific antibody form a complex with or interface with LLCP1 HCP1.

[0710] In some embodiments of the multispecific antibody molecule disclosed herein:

[0711] HCP1 has a greater affinity for HCP2 than the second molecule of HCP1; and / or

[0712] HCP2 has a greater affinity for HCP1 than the second molecule of HCP2.

[0713] In an embodiment, the affinity of HCP1 for HCP2 is sufficiently greater than the affinity of a second molecule of HCP1, so that under predetermined conditions, for example, at pH 7, in an aqueous buffer, for example, at pH 7, in saline, or under physiological conditions, at least 75%, 80%, 90%, 95%, 98%, 99%, 99.5%, or 99.9% of the molecules of the multispecific antibody form a complex with HCP1 or interface with HCP2.

[0714] In another aspect, a method for manufacturing or producing a multispecific antibody molecule is described herein. The method comprises the following:

[0715] (i) providing a first heavy chain polypeptide (e.g., a heavy chain polypeptide comprising one, two, three, or all of a first heavy chain variable region (first VH), a first CH1, and a first heavy chain constant region (e.g., a first CH2, a first CH3, or both));

[0716] (ii) providing a second heavy chain polypeptide (e.g., a heavy chain polypeptide comprising one, two, three, or all of a second heavy chain variable region (second VH), a second CH1, and a second heavy chain constant region (e.g., a second CH2, a second CH3, or both));

[0717] (iii) providing a lambda chain polypeptide (e.g., lambda light chain variable region (VLλ), lambda light chain constant chain (VLλ), or both) that preferentially binds to the first heavy chain polypeptide (e.g., first VH); and

[0718] (iv) a step of providing a kappa light chain polypeptide (e.g., kappa light chain variable region (VLκ), kappa light chain constant chain (VLκ), or both) that preferentially binds to a second heavy chain polypeptide (e.g., second VH) under conditions where (i) to (iv) are connected.

[0719] In an embodiment, the first and second heavy chain polypeptides form an Fc interface that enhances heteromerization.

[0720] In an embodiment, (i) to (iv) (e.g., nucleic acid encoding (i) to (iv)) is introduced into a single cell, e.g., a single mammalian cell, e.g., a CHO cell. In an embodiment, (i) to (iv) are expressed in the cell.

[0721] In an embodiment, (i) to (iv) (e.g., nucleic acid encoding (i) to (iv)) are introduced into different cells, e.g., different mammalian cells, e.g., two or more CHO cells. In an embodiment, (i) to (iv) are expressed in the cells.

[0722] In one embodiment, the method further comprises the step of purifying the cell-expressed antibody molecule using, for example, lambda- and / or-kappa-specific purification, e.g., affinity chromatography.

[0723] In an embodiment, the method further comprises the step of evaluating a cell-expressed multispecific antibody molecule. For example, the purified cell-expressed multispecific antibody molecule may be analyzed by techniques known in the art, including mass spectrometry. In one embodiment, the purified cell-expressed antibody molecule is cleaved, for example, by digestion with papain to obtain a Fab moiety, and evaluated using mass spectrometry.

[0724] In an embodiment, the method produces accurately paired kappa / lambda multiple-specific, e.g. bispecific, antibody molecules in high yield (e.g., in a yield of at least 75%, 80%, 90%, 95%, 98%, 99%, 99.5%, or 99.9%).

[0725] In another embodiment, a multispecific, e.g. bispecific antibody molecule comprises the following:

[0726] (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., a first CH2, a first CH3, or both), e.g., an HCP1 that binds to a first epitope;

[0727] (ii) a second heavy chain polypeptide (HCP2) (e.g., a heavy chain polypeptide comprising one, two, three, or all of a second heavy chain variable region (second VH), a second CH1, and a second heavy chain constant region (e.g., a second CH2, a second CH3, or both), e.g., an HCP2 that binds to a second epitope;

[0728] (iii) a lambda light chain polypeptide (LLCP1) that preferentially binds to the first heavy chain polypeptide (e.g., the first VH) (e.g., a lambda light chain variable region (VLλ), a lambda light chain constant chain (VLλ), or both), for example, an LLCP1 that binds to the first epitope; and

[0729] (iv) a kappa light chain polypeptide (e.g., kappa light chain variable region (VLκ), kappa light chain constant chain (VLκ), or both) that preferentially binds to the second heavy chain polypeptide (e.g., second VH), e.g., KLCP2 that binds to the second epitope.

[0730] In an embodiment, the first and second heavy chain polypeptides form an Fc interface that enhances heteromerization. In an embodiment, the multispecific antibody molecule has a first binding specificity comprising a heteromerized hybrid VLλ-CLλ, CH2-CH3 domain (having a knob modification) in a first heavy chain variable region connected to an Fc constant, and a second binding specificity comprising a heteromerized hybrid VLκ-CLκ, CH2-CH3 domain (having a hole modification) in a second heavy chain variable region connected to an Fc constant.

[0731] TRBC1 and TRBC2 antigen binding domains

[0732] The present disclosure provides, in particular, an antibody molecule, for example, a multispecific (e.g., bispecific, trispecific, quadrspecific) or multifunctional molecule, comprising one or more antigen-binding domains that bind to a tumor antigen on a lymphoma cell (e.g., a T cell). In some embodiments, the tumor antigen comprises a T cell receptor comprising TRBC1 or TRBC2. In some embodiments, the antigen-binding domain preferentially binds to a T cell receptor comprising TRBC1 (e.g., compared to a T cell receptor comprising TRBC2). In some embodiments, the antigen-binding domain preferentially binds to a T cell receptor comprising TRBC2 (e.g., compared to a T cell receptor comprising TRBC1). In some embodiments, the multifunctional molecule comprises one or more antigen-binding domains that selectively target lymphocytes expressing TRBC1 or TRBC2. In some embodiments, the antigen binding domain selectively targets lymphocytes expressing a T cell receptor including TRBC1 or a T cell receptor including TRBC2.

[0733] T cell receptors (TCRs) are receptors found on the surface of lymphocytes, specifically on T lymphocytes (T cells). TCRs play a role in recognizing antigen fragments presented by major histocompatibility complex (MHC) molecules on other immune cells (e.g., B cells) by transmitting signals via associated CD3 and activating T cells. In humans, almost all TCRs are heterodimers containing alpha and beta chains. Both the alpha and beta chains of the TCR contain variable and constant regions. The variable regions of the alpha and beta chains are encoded by distinct DNA elements (elements V, D, and J for the beta chain; elements V and J for the alpha chain). Recombination of these elements results in significant changes to the antigen-binding specificity of the TCR. The constant region of the TCR beta chain is selected from two different domains: beta constant domain 1 and beta constant domain 2. Although we do not wish to be bound by theory, it is believed that most TCRs containing beta chains contain either beta invariant domain 1 or beta invariant domain 2, but not both invariant domain 1 and invariant domain 2.

[0734] In some embodiments, the multifunctional or multispecific molecule or antibody molecule of the present application comprises an antigen-binding domain that binds to a neutral antigen on lymphoma cells (e.g., T cells), e.g., a T cell receptor comprising TRBC1, a T cell receptor comprising TRBC1 and TRBC2, or TRBC2. In some embodiments, the multifunctional or multispecific molecule or antibody molecule of the present application selectively targets lymphocytes expressing a T cell receptor comprising TRBC1, a T cell receptor comprising TRBC1 and TRBC2, or a T cell receptor comprising TRBC2. While it is most common for lymphocytes or lymphoma cells presenting a T cell receptor comprising TRBC1 or TRBC2 to be T cells, cancer causes significant disruption in non-disease expression patterns. Therefore, in some embodiments, the lymphoma cells or lymphocytes may not be T cells. In some embodiments, the lymphoma cells or lymphocytes are B cells. In some embodiments, the lymphoma cells or lymphocytes are natural killer cells.

[0735] In some embodiments, the antigen-binding domain (e.g., first antigen-binding domain) comprises any CDR amino acid sequence, framework region (FWR) amino acid sequence, or variable region amino acid sequence of an anti-TRBC1 antibody known in the art. In some embodiments, the CDR amino acid sequence, framework region (FWR) amino acid sequence, or variable region amino acid sequence is selected from JOVI.1.

[0736] TRBC1 antigen binding domain

[0737] In some embodiments, the antigen-binding domain that binds to TRBC1 comprises one or more CDRs disclosed in Table 2A or Table 2B, Table 3A or Table 3B, or Table 4 (e.g., VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2, and / or VLCDR3), or a sequence having at least 85%, 90%, 95%, or 99% identity with respect to this. In some embodiments, the antigen-binding domain that binds to TRBC1 comprises one or more framework regions disclosed in Table 2A or Table 2B, Table 3A or Table 3B, or Table 4 (e.g., VHFWR1, VHFWR2, VHFWR3, VHFWR4, VLFWR1, VLFWR2, VLFWR3, and / or VLFWR4), or a sequence having at least 85%, 90%, 95%, or 99% identity with respect to this. In some embodiments, the antigen-binding domain that binds to TRBC1 comprises VH and / or VL disclosed in Table 7, or a sequence having at least 85%, 90%, 95%, or 99% identity therewith. In some embodiments, the antigen-binding domain that binds to TRBC1 comprises the amino acid sequence disclosed in Table 8, or a sequence having at least 85%, 90%, 95%, or 99% identity therewith.

[0738] In some embodiments, the antigen-binding domain that binds to TRBC1 comprises one or more CDRs disclosed in Table 5A and / or 5B (e.g., VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2, and / or VLCDR3), or a sequence having at least 85%, 90%, 95%, or 99% identity with these. In some embodiments, the antigen-binding domain that binds to TRBC1 comprises one or more framework regions disclosed in Table 5A and / or 5B (e.g., VHFWR1, VHFWR2, VHFWR3, VHFWR4, VLFWR1, VLFWR2, VLFWR3, and / or VLFWR4), or a sequence having at least 85%, 90%, 95%, or 99% identity with these. In some embodiments, the antigen-binding domain that binds to TRBC1 comprises VH and / or VL disclosed in Table 7, or a sequence having at least 85%, 90%, 95%, or 99% identity with it.

[0739] In some embodiments, the antigen binding domain that binds to TRBC1 comprises VH, comprising heavy chain complementarity determining region 1 (VHCDR1), VHCDR2, and VHCDR3, and VL, comprising light chain complementarity determining region 1 (VLCDR1), VLCDR2, and VLCDR3.

[0740] In some embodiments, VHCDR1, VHCDR2, and VHCDR3 are Sequence number Each includes the amino acid sequences of 8643, 7355, and 202 (or sequences having at least 85%, 90%, 95%, or 99% identity with these). In some embodiments, VHCDR1, VHCDR2, and VHCDR3 are Sequence number Each includes the amino acid sequences of 8643, 201, and 202 (or sequences having at least 85%, 90%, 95%, or 99% identity with these). In some embodiments, VHCDR1, VHCDR2, and VHCDR3 are Sequence numberEach includes the amino acid sequences of 7354, 201, and 202 (or sequences having at least 85%, 90%, 95%, or 99% identity with these). In some embodiments, VHCDR1, VHCDR2, and VHCDR3 are Sequence number Each includes the amino acid sequences of 7354, 7355, and 202 (or sequences having at least 85%, 90%, 95%, or 99% identity with these).

[0741] In some embodiments, VLCDR1, VLCDR2, and VLCDR3 are Sequence number Each includes the amino acid sequence of 223, 224, and 225 or 8680 (or a sequence having at least 85%, 90%, 95%, or 99% identity with this). In some embodiments, VLCDR1, VLCDR2, and VLCDR3 are Sequence number Each includes an amino acid sequence of 7367, 224, and 225 or 8680 (or a sequence having at least 85%, 90%, 95%, or 99% identity with such a sequence). In some embodiments, VLCDR1, VLCDR2, and VLCDR3 are Sequence number Each includes the amino acid sequences of 223, 7368, and 225 or 8680 (or sequences having at least 85%, 90%, 95%, or 99% identity with these). In some embodiments, VLCDR1, VLCDR2, and VLCDR3 are Sequence number Each includes the amino acid sequences of 223, 224, and 7369 (or sequences having at least 85%, 90%, 95%, or 99% identity with these). In some embodiments, VLCDR1, VLCDR2, and VLCDR3 are Sequence number Each includes the amino acid sequences of 7367, 7368, and 7369 (or sequences having at least 85%, 90%, 95%, or 99% identity with these).

[0742] In some embodiments, VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2, and VLCDR3 are Sequence numberEach includes the amino acid sequences of 8643, 7355, 202, 223, 224, and 225 or 8680 (or sequences having at least 85%, 90%, 95%, or 99% identity with these). In some embodiments, VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2, and VLCDR3 are Sequence number Each comprises the amino acid sequences of 8643, 201, 202, 223, 224, and 225 or 8680 (or sequences having at least 85%, 90%, 95%, or 99% identity with these). In some embodiments, VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2, and VLCDR3 comprise the following amino acid sequences: Sequence number 8643, 7355, 202, 7367, 224, and 225 or 8680, each (or a sequence having at least 85%, 90%, 95%, or 99% identity with this); Sequence number 8643, 7355, 202, 223, 7368, and 225 or 8680, respectively (or sequences having at least 85%, 90%, 95%, or 99% identity with these); Sequence number 8643, 7355, 202, 223, 224, and 7369, respectively (or sequences having at least 85%, 90%, 95%, or 99% identity with these); Sequence number 8643, 7355, 202, 7367, 7368, and 7369, respectively (or sequences having at least 85%, 90%, 95%, or 99% identity with these); Sequence number 8643, 201, 202, 7367, 224, and 225 or 8680, each (or a sequence having at least 85%, 90%, 95%, or 99% identity with this); Sequence number 8643, 201, 202, 223, 7368, and 225 or 8680, respectively (or sequences having at least 85%, 90%, 95%, or 99% identity with these); Sequence number8643, 201, 202, 223, 224, and 7369, respectively (or sequences having at least 85%, 90%, 95%, or 99% identity with these); Sequence number 8643, 201, 202, 7367, 7368, and 7369, respectively (or sequences having at least 85%, 90%, 95%, or 99% identity with these); Sequence number 7354, 201, 202, 223, 224, and 225 or 8680, each (or a sequence having at least 85%, 90%, 95%, or 99% identity with this); Sequence number 7354, 201, 202, 7367, 224, and 225 or 8680, each (or a sequence having at least 85%, 90%, 95%, or 99% identity with this); Sequence number 7354, 201, 202, 223, 7368, and 225 or 8680, respectively (or sequences having at least 85%, 90%, 95%, or 99% identity with these); Sequence number 7354, 201, 202, 223, 224, and 7369, each (or sequences having at least 85%, 90%, 95%, or 99% identity with these); Sequence number 7354, 201, 202, 7367, 7368, and 7369, respectively (or sequences having at least 85%, 90%, 95%, or 99% identity with these); Sequence number 7354, 7355, 202, 223, 224, and 225 or 8680, each (or a sequence having at least 85%, 90%, 95%, or 99% identity with this); Sequence number 7354, 7355, 202, 7367, 224, and 225 or 8680, each (or a sequence having at least 85%, 90%, 95%, or 99% identity with this); Sequence number 7354, 7355, 202, 223, 7368, and 225 or 8680, each (or a sequence having at least 85%, 90%, 95%, or 99% identity with this); Sequence number7354, 7355, 202, 223, 224, and 7369, each (or a sequence having at least 85%, 90%, 95%, or 99% identity with this); or Sequence number 7354, 7355, 202, 7367, 7368, and 7369, respectively (or sequences having at least 85%, 90%, 95%, or 99% identity with these).

[0743] In some embodiments, VH is Sequence number A sequence of amino acids selected from the group consisting of 7351, 253, 250-252, 254, 7343, 7344, 7350, and 7352 (or a sequence having at least 85%, 90%, 95%, or 99% identity with this), and / or VL is Sequence number It comprises an amino acid sequence selected from the group consisting of 258, 255–257, 259, 260, and 7357–7360 (or a sequence having at least 85%, 90%, 95%, or 99% identity with this). In some embodiments, VH and VL are Sequence number Each includes the amino acid sequences of 7351 and 258 (or sequences having at least 85%, 90%, 95%, or 99% identity with these). In some embodiments, VH and VL are Sequence number Each includes the amino acid sequences of 253 and 258 (or sequences having at least 85%, 90%, 95%, or 99% identity with these).

[0744] In some embodiments, an antigen-binding domain (e.g., a first antigen-binding domain) that binds to a tumor antigen on a lymphoma cell (e.g., a T cell receptor including TRBC1, TRBC1, TRBC2, or TRBC2 comprises any CDR amino acid sequence, framework region (FWR) amino acid sequence, or variable region amino acid sequence of Table 1, Table 2A or Table 2B, Table 3A or Table 3B, Table 4, Table 7, and Table 8. In some embodiments, an antigen-binding domain (e.g., a first antigen-binding domain) that binds to a tumor antigen on a lymphoma cell (e.g., a T cell receptor including TRBC1, TRBC1, TRBC2, or TRBC2 comprises heavy chain and / or light chain amino acid sequences of Table 8. In some embodiments, an antigen-binding domain (e.g., a first antigen-binding domain) that selectively targets a T cell receptor containing TRBC1, a T cell receptor containing TRBC1, a T cell receptor containing TRBC2, or a lymphocyte expressing TRBC2 comprises any CDR amino acid sequence, framework region (FWR) amino acid sequence, or variable region amino acid sequence disclosed in Table 1, Table 2A or Table 2B, Table 3A or Table 3B, Table 4, Table 7, and Table 8. In some embodiments, an antigen-binding domain (e.g., a first antigen-binding domain) that selectively targets a T cell receptor containing TRBC1, a T cell receptor containing TRBC1, a T cell receptor containing TRBC2, or a lymphocyte expressing TRBC2 comprises the heavy chain and / or light chain amino acid sequence of Table 8. An antigen-binding domain that binds to a tumor antigen containing TRBC1 or selectively targets a lymphocyte expressing TRBC1 may be said to target TRBC1 (i.e., a TRBC1-targeting antigen-binding domain).An antigen-binding domain that binds to tumor antigens containing TRBC2 or selectively targets lymphocytes expressing TRBC2 can be said to target TRBC2 (i.e., called a TRBC2-targeting antigen-binding domain).

[0745] In some embodiments, the antigen-binding domain targeting TRBC1 is Sequence number The heavy chain complementarity determining region 1 (VHCDR1) amino acid sequence of 8643 (or a sequence with 1, 2, 3, or 4 or fewer mutations, e.g., substitutions, additions, or deletions), Sequence number The VHCDR2 amino acid sequence of 201 (or a sequence having 1, 2, 3, or 4 or fewer mutations, e.g., substitutions, additions, or deletions), and / or Sequence number It comprises a VH comprising a 202-amino acid sequence of VHCDR3 (or a sequence having 1, 2, 3, or 4 or fewer mutations, e.g., substitutions, additions, or deletions). In some embodiments, the TRBC1 antigen-binding domain is Sequence number 8643 The VHCDR1 amino acid sequence, Sequence number The VHCDR2 amino acid sequence of 201, and / or Sequence number It includes VH containing the 202 VHCDR3 amino acid sequence.

[0746] In some embodiments, the antigen-binding domain targeting TRBC1 is Sequence number The amino acid sequence of the light chain complementarity determining region 1 (VLCDR1) of 223 (or a sequence with 1, 2, 3, or 4 or fewer mutations, e.g., substitutions, additions, or deletions), Sequence number 224 VLCDR2 amino acid sequences (or sequences with 1, 2, 3, or 4 or fewer mutations, e.g., substitutions, additions, or deletions), and / or Sequence numberVL comprises a VLCDR3 amino acid sequence of 225 or 8680 (or a sequence having 1, 2, 3, or 4 or fewer mutations, e.g., substitutions, additions, or deletions). In some embodiments, the antigen-binding domain targeting TRBC1 comprises Sequence number 223 VLCDR1 amino acid sequence, Sequence number 224 VLCDR2 amino acid sequences, and / or Sequence number It includes VL containing a VLCDR3 amino acid sequence of 225 or 8680.

[0747] In some embodiments, the antigen-binding domain targeting TRBC1 is Sequence number Amino acid sequence of heavy chain framework region 1 (VHFWR1) of 203, Sequence number 204 VHFWR2 amino acid sequence, Sequence number The VHFWR3 amino acid sequence of 8679, and / or Sequence number It contains VH containing the 206 VHFWR4 amino acid sequence.

[0748] In some embodiments, the antigen-binding domain targeting TRBC1 is Sequence number amino acid sequence of light chain framework region 1 (VLFWR1) of 226, Sequence number 227 VLFWR2 amino acid sequence, Sequence number The 228th VLFWR3 amino acid sequence, and / or Sequence number It contains VL containing the 229 VLFWR4 amino acid sequence.

[0749] In some embodiments, the antigen-binding domain targeting TRBC1 is Sequence number The VHFWR1 amino acid sequence of 203 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations relative to this sequence, e.g., substitutions, additions, or deletions), Sequence number The VHFWR2 amino acid sequence of 204 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations relative to this sequence, e.g., substitutions, additions, or deletions), Sequence number 8679The VHFWR3 amino acid sequence (or a sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 or fewer mutations, e.g., substitutions, additions, or deletions), and / or Sequence number It contains VH containing the 206 VHFWR4 amino acid sequence.

[0750] In some embodiments, the antigen-binding domain targeting TRBC1 is Sequence number The VLFWR1 amino acid sequence of 226 (or a sequence with 1, 2, or 3 or fewer mutations, e.g., substitutions, additions, or deletions), Sequence number The VLFWR2 amino acid sequence of 227 (or a sequence having one or fewer mutations, e.g., substitutions, additions, or deletions), Sequence number The VLFWR3 amino acid sequence of 228 (or a sequence having one or fewer mutations, e.g., substitutions, additions, or deletions), and / or Sequence number It contains VL containing the 229 VLFWR4 amino acid sequence.

[0751] In some embodiments, the antigen-binding domain targeting TRBC1 is Sequence number Amino acid sequence of heavy chain framework region 1 (VHFWR1) of 207, Sequence number 208 VHFWR2 amino acid sequence, Sequence number The 209th VHFWR3 amino acid sequence, and / or Sequence number It contains VH containing the 210 VHFWR4 amino acid sequence.

[0752] In some embodiments, the antigen-binding domain targeting TRBC1 is Sequence number The VHFWR1 amino acid sequence of 207 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations relative to this sequence, e.g., substitutions, additions, or deletions), Sequence number The VHFWR2 amino acid sequence of 208 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations relative to this sequence, e.g., substitutions, additions, or deletions), Sequence number The VHFWR3 amino acid sequence of 209 (or a sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 or fewer mutations, e.g., substitutions, additions, or deletions), and / or Sequence number It contains VH containing the 210 VHFWR4 amino acid sequence.

[0753] In some embodiments, the antigen-binding domain targeting TRBC1 is Sequence number 207 amino acid sequence of heavy chain framework region 1 (VHFWR1), Sequence number 208 The VHFWR2 amino acid sequence, Sequence number The 213-amino acid sequence of VHFWR3, and / or Sequence number 210 It contains VH containing the VHFWR4 amino acid sequence.

[0754] In some embodiments, the antigen-binding domain targeting TRBC1 is Sequence number 207 The VHFWR1 amino acid sequence (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations relative to this sequence, e.g., substitutions, additions, or deletions), Sequence number 208 The VHFWR2 amino acid sequence (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations relative to this sequence, e.g., substitutions, additions, or deletions), Sequence number The VHFWR3 amino acid sequence of 213 (or a sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 or fewer mutations, e.g., substitutions, additions, or deletions), and / or Sequence number 210 It contains VH containing the VHFWR4 amino acid sequence.

[0755] In some embodiments, the antigen-binding domain targeting TRBC1 is Sequence number 7345 amino acid sequence of heavy chain framework region 1 (VHFWR1), Sequence number 208 The VHFWR2 amino acid sequence, Sequence number The VHFWR3 amino acid sequence of 217, and / or Sequence number 210 It contains VH containing the VHFWR4 amino acid sequence.

[0756] In some embodiments, the antigen-binding domain targeting TRBC1 is Sequence number 7345 The VHFWR1 amino acid sequence (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations relative to this sequence, e.g., substitutions, additions, or deletions), Sequence number 208 The VHFWR2 amino acid sequence (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations relative to this sequence, e.g., substitutions, additions, or deletions), Sequence number The VHFWR3 amino acid sequence of 217 (or a sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 or fewer mutations, e.g., substitutions, additions, or deletions), and / or Sequence number 210 It contains VH containing the VHFWR4 amino acid sequence.

[0757] In some embodiments, the antigen-binding domain targeting TRBC1 is Sequence number 207 amino acid sequence of heavy chain framework region 1 (VHFWR1), Sequence number 212 The VHFWR2 amino acid sequence, Sequence number The 221st VHFWR3 amino acid sequence, and / or Sequence number 210 It contains VH containing the VHFWR4 amino acid sequence.

[0758] In some embodiments, the antigen-binding domain targeting TRBC1 is Sequence number 207 The VHFWR1 amino acid sequence (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations relative to this sequence, e.g., substitutions, additions, or deletions), Sequence number 212The VHFWR2 amino acid sequence (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations relative to this sequence, e.g., substitutions, additions, or deletions), Sequence number The VHFWR3 amino acid sequence of 221 (or a sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 or fewer mutations, e.g., substitutions, additions, or deletions), and / or Sequence number 210 It contains VH containing the VHFWR4 amino acid sequence.

[0759] In some embodiments, the antigen-binding domain targeting TRBC1 is Sequence number amino acid sequence of light chain framework region 1 (VLFWR1) of 230, Sequence number VLFWR2 amino acid sequence of 231, Sequence number The 232-amino acid sequence of VLFWR3, and / or Sequence number It contains VL containing the VLFWR4 amino acid sequence of 8685.

[0760] In some embodiments, the antigen-binding domain targeting TRBC1 is Sequence number The VLFWR1 amino acid sequence of 230 (or a sequence with 1, 2, or 3 or fewer mutations, e.g., substitutions, additions, or deletions), Sequence number The VLFWR2 amino acid sequence of 231 (or a sequence having one or fewer mutations, e.g., substitutions, additions, or deletions), Sequence number The 232-amino acid sequence of VLFWR3 (or a sequence having one or fewer mutations, e.g., substitutions, additions, or deletions), and / or Sequence number It contains VL containing the VLFWR4 amino acid sequence of 8685.

[0761] In some embodiments, the antigen-binding domain targeting TRBC1 is Sequence number amino acid sequence of light chain framework region 1 (VLFWR1) of 234, Sequence number VLFWR2 amino acid sequence of 235, Sequence numberThe 236-amino acid sequence of VLFWR3, and / or Sequence number It contains VL containing the VLFWR4 amino acid sequence of 237.

[0762] In some embodiments, the antigen-binding domain targeting TRBC1 is Sequence number The VLFWR1 amino acid sequence of 234 (or a sequence with 1, 2, or 3 or fewer mutations, e.g., substitutions, additions, or deletions), Sequence number The VLFWR2 amino acid sequence of 235 (or a sequence with one or fewer mutations, e.g., substitutions, additions, or deletions), Sequence number The 236-amino acid sequence of VLFWR3 (or a sequence having one or fewer mutations, e.g., substitutions, additions, or deletions), and / or Sequence number It contains VL containing the VLFWR4 amino acid sequence of 237.

[0763] In some embodiments, the antigen-binding domain targeting TRBC1 is Sequence number Amino acid sequence of light chain framework region 1 (VLFWR1) of 238, Sequence number VLFWR2 amino acid sequence of 239, Sequence number 240 VLFWR3 amino acid sequences, and / or Sequence number It contains VL containing the VLFWR4 amino acid sequence of 241.

[0764] In some embodiments, the antigen-binding domain targeting TRBC1 is Sequence number The VLFWR1 amino acid sequence of 238 (or a sequence with 1, 2, or 3 or fewer mutations, e.g., substitutions, additions, or deletions), Sequence number The VLFWR2 amino acid sequence of 239 (or a sequence having one or fewer mutations, e.g., substitutions, additions, or deletions), Sequence number A 240-amino acid sequence of VLFWR3 (or a sequence with one or fewer mutations, e.g., substitutions, additions, or deletions), and / or Sequence numberIt contains VL containing the VLFWR4 amino acid sequence of 241.

[0765] In some embodiments, the antigen-binding domain targeting TRBC1 is Sequence number Amino acid sequence of light chain framework region 1 (VLFWR1) of 242, Sequence number VLFWR2 amino acid sequence of 243, Sequence number 240 VLFWR3 amino acid sequences, and / or Sequence number It contains VL containing the 245 VLFWR4 amino acid sequence.

[0766] In some embodiments, the antigen-binding domain targeting TRBC1 is Sequence number The VLFWR1 amino acid sequence of 242 (or a sequence with 1, 2, or 3 or fewer mutations, e.g., substitutions, additions, or deletions), Sequence number The VLFWR2 amino acid sequence of 243 (or a sequence with one or fewer mutations, e.g., substitutions, additions, or deletions), Sequence number 240 The VLFWR3 amino acid sequence (or a sequence having one or fewer mutations, e.g., a substitution, addition, or deletion), and / or Sequence number It contains VL containing the 245 VLFWR4 amino acid sequence.

[0767] In some embodiments, the antigen-binding domain targeting TRBC1 is Sequence number amino acid sequence of light chain framework region 1 (VLFWR1) of 246, Sequence number 247 VLFWR2 amino acid sequence, Sequence number 240 The VLFWR3 amino acid sequence of, and / or Sequence number It contains VL containing the 249 VLFWR4 amino acid sequence.

[0768] In some embodiments, the antigen-binding domain targeting TRBC1 is Sequence numberThe VLFWR1 amino acid sequence of 246 (or a sequence with 1, 2, or 3 or fewer mutations, e.g., substitutions, additions, or deletions), Sequence number The VLFWR2 amino acid sequence of 247 (or a sequence having one or fewer mutations, e.g., substitutions, additions, or deletions), Sequence number 240 The VLFWR3 amino acid sequence (or a sequence having one or fewer mutations, e.g., a substitution, addition, or deletion), and / or Sequence number It contains VL containing the 249 VLFWR4 amino acid sequence.

[0769] In some embodiments, the antigen-binding domain targeting TRBC1 is Sequence number A sequence of 250 amino acids (or Sequence number It comprises a VH comprising 250 and an amino acid sequence having at least about 77%, 80%, 85%, 90%, 95%, or 99% sequence identity. In some embodiments, the antigen-binding domain targeting TRBC1 is Sequence number The amino acid sequence of 255 (or Sequence number It includes a VL comprising an amino acid sequence having at least about 93%, 95%, or 99% sequence identity with 255. In some embodiments, the antigen-binding domain targeting TRBC1 is Sequence number VH comprising a sequence of 250 amino acids. In some embodiments, the antigen-binding domain targeting TRBC1 includes Sequence number It contains VL containing a 255 amino acid sequence.

[0770] In some embodiments, the antigen-binding domain targeting TRBC1 is Sequence number VH containing a 250-amino acid sequence and Sequence number It contains VL containing a 255 amino acid sequence.

[0771] In some embodiments, the antigen-binding domain targeting TRBC1 is Sequence number The amino acid sequence of 251 (or Sequence numberIt includes a VH comprising an amino acid sequence having at least about 77%, 80%, 85%, 90%, 95%, or 99% sequence identity with 251. In some embodiments, the antigen-binding domain targeting TRBC1 is Sequence number It contains VH containing an amino acid sequence of 251.

[0772] In some embodiments, the antigen-binding domain targeting TRBC1 is Sequence number 252 amino acid sequences (or Sequence number VH comprising 252 and an amino acid sequence having at least about 77%, 80%, 85%, 90%, 95%, or 99% sequence identity. In some embodiments, the antigen-binding domain targeting TRBC1 is Sequence number It contains VH containing a 252 amino acid sequence.

[0773] In some embodiments, the antigen-binding domain targeting TRBC1 is Sequence number The amino acid sequence of 253 (or Sequence number VH comprising 253 and an amino acid sequence having at least about 77%, 80%, 85%, 90%, 95%, or 99% sequence identity. In some embodiments, the antigen-binding domain targeting TRBC1 is Sequence number It contains VH containing a 253 amino acid sequence.

[0774] In some embodiments, the antigen-binding domain targeting TRBC1 is Sequence number The 254-amino acid sequence (or Sequence number VH comprising 254 and an amino acid sequence having at least about 77%, 80%, 85%, 90%, 95%, or 99% sequence identity. In some embodiments, the antigen-binding domain targeting TRBC1 is Sequence number It contains VH containing a sequence of 254 amino acids.

[0775] In some embodiments, the antigen-binding domain targeting TRBC1 is Sequence number256 amino acid sequences (or Sequence number It includes a VL comprising an amino acid sequence having at least about 93%, 95%, or 99% sequence identity with 256. In some embodiments, the antigen-binding domain targeting TRBC1 comprises Sequence number It contains VL containing a 256-amino acid sequence.

[0776] In some embodiments, the antigen-binding domain targeting TRBC1 is Sequence number The amino acid sequence of 257 (or Sequence number It includes a VL comprising an amino acid sequence having at least about 93%, 95%, or 99% sequence identity with 257. In some embodiments, the antigen-binding domain targeting TRBC1 comprises Sequence number It contains VL containing an amino acid sequence of 257.

[0777] In some embodiments, the antigen-binding domain targeting TRBC1 is Sequence number The amino acid sequence of 258 (or Sequence number It includes a VL comprising an amino acid sequence having at least about 93%, 95%, or 99% sequence identity with 258. In some embodiments, the antigen-binding domain targeting TRBC1 comprises Sequence number It contains VL containing a sequence of 258 amino acids.

[0778] In some embodiments, the antigen-binding domain targeting TRBC1 is Sequence number The amino acid sequence of 259 (or Sequence number It includes a VL comprising an amino acid sequence having at least about 93%, 95%, or 99% sequence identity with 259. In some embodiments, the antigen-binding domain targeting TRBC1 comprises Sequence number It contains VL containing the amino acid sequence of 259.

[0779] In some embodiments, the antigen-binding domain targeting TRBC1 is Sequence number 260 amino acid sequences (or Sequence numberIt includes a VL comprising an amino acid sequence having at least about 93%, 95%, or 99% sequence identity with 260. In some embodiments, the antigen-binding domain targeting TRBC1 is Sequence number It contains VL containing a sequence of 260 amino acids.

[0780] In some embodiments, the antigen-binding domain targeting TRBC1 is Sequence number The amino acid sequence of 6154 (or Sequence number It comprises a heavy chain comprising an amino acid sequence having at least about 93%, 95%, or 99% sequence identity with 6154. In some embodiments, the antigen-binding domain targeting TRBC1 comprises Sequence number It includes a heavy chain containing the amino acid sequence of 6154.

[0781] In some embodiments, the antigen-binding domain targeting TRBC1 is Sequence number The amino acid sequence of 4671 (or Sequence number It comprises a heavy chain comprising an amino acid sequence having at least about 93%, 95%, or 99% sequence identity with 4671. In some embodiments, the antigen-binding domain targeting TRBC1 comprises Sequence number It includes a heavy chain containing the amino acid sequence of 4671.

[0782] In some embodiments, the antigen-binding domain targeting TRBC1 is Sequence number The amino acid sequence of 6155 (or Sequence number It comprises a heavy chain comprising an amino acid sequence having at least about 93%, 95%, or 99% sequence identity with 6155. In some embodiments, the antigen-binding domain targeting TRBC1 comprises Sequence number It includes a heavy chain containing the amino acid sequence of 6155.

[0783] In some embodiments, the antigen-binding domain targeting TRBC1 is Sequence number The amino acid sequence of 4672 (or Sequence numberIt comprises a heavy chain comprising an amino acid sequence having at least about 93%, 95%, or 99% sequence identity with 4672. In some embodiments, the antigen-binding domain targeting TRBC1 comprises Sequence number It includes a heavy chain containing a sequence of 4672 amino acids.

[0784] In some embodiments, the antigen-binding domain targeting TRBC1 is Sequence number The amino acid sequence of 6156 (or Sequence number It comprises a light chain comprising an amino acid sequence having at least about 93%, 95%, or 99% sequence identity with 6156. In some embodiments, the antigen-binding domain targeting TRBC1 comprises Sequence number It contains a light chain containing the amino acid sequence of 6156.

[0785] In some embodiments, the antigen-binding domain targeting TRBC1 is Sequence number The amino acid sequence of 6167 (or Sequence number It comprises a heavy chain comprising an amino acid sequence having at least about 93%, 95%, or 99% sequence identity with 6167. In some embodiments, the antigen-binding domain targeting TRBC1 comprises Sequence number It includes a heavy chain containing the amino acid sequence of 6167.

[0786] In some embodiments, the antigen-binding domain targeting TRBC1 is Sequence number The amino acid sequence of 4673 (or Sequence number It comprises a heavy chain comprising an amino acid sequence having at least about 93%, 95%, or 99% sequence identity with 4673. In some embodiments, the antigen-binding domain targeting TRBC1 comprises Sequence number It includes a heavy chain containing the amino acid sequence of 4673.

[0787] In some embodiments, the antigen-binding domain targeting TRBC1 is Sequence number The amino acid sequence of 6168 (or Sequence numberIt comprises a heavy chain comprising an amino acid sequence having at least about 93%, 95%, or 99% sequence identity with 6168. In some embodiments, the antigen-binding domain targeting TRBC1 comprises Sequence number It includes a heavy chain containing the amino acid sequence of 6168.

[0788] In some embodiments, the antigen-binding domain targeting TRBC1 is Sequence number The amino acid sequence of 4674 (or Sequence number It comprises a heavy chain comprising an amino acid sequence having at least about 93%, 95%, or 99% sequence identity with 4674. In some embodiments, the antigen-binding domain targeting TRBC1 comprises Sequence number It includes a heavy chain containing a sequence of 4674 amino acids.

[0789] In some embodiments, the antigen-binding domain targeting TRBC1 is Sequence number The amino acid sequence of 6169 (or Sequence number It comprises a light chain comprising an amino acid sequence having at least about 93%, 95%, or 99% sequence identity with 6169. In some embodiments, the antigen-binding domain targeting TRBC1 comprises Sequence number It includes a light chain containing the amino acid sequence of 6169.

[0790] In some embodiments, the antigen-binding domain targeting TRBC1 is Sequence number The amino acid sequence of 6154 (or Sequence number A heavy chain comprising an amino acid sequence having at least about 93%, 95%, or 99% sequence identity with 6154 and Sequence number The amino acid sequence of 6156 (or Sequence number It comprises a light chain comprising an amino acid sequence having at least about 93%, 95%, or 99% sequence identity with 6156. In some embodiments, the antigen-binding domain targeting TRBC1 comprises Sequence number A heavy chain containing the amino acid sequence of 6154 and Sequence numberIt contains a light chain containing the amino acid sequence of 6156.

[0791] In some embodiments, the antigen-binding domain targeting TRBC1 is Sequence number The amino acid sequence of 6155 (or Sequence number A heavy chain comprising an amino acid sequence having at least about 93%, 95%, or 99% sequence identity with 6155 and Sequence number The amino acid sequence of 6156 (or Sequence number It comprises a light chain comprising an amino acid sequence having at least about 93%, 95%, or 99% sequence identity with 6156. In some embodiments, the antigen-binding domain targeting TRBC1 comprises Sequence number A heavy chain containing the amino acid sequence of 6155 and Sequence number It contains a light chain containing the amino acid sequence of 6156.

[0792] In some embodiments, the antigen-binding domain targeting TRBC1 is Sequence number The amino acid sequence of 6167 (or Sequence number A heavy chain comprising an amino acid sequence having at least about 93%, 95%, or 99% sequence identity with 6167 and Sequence number The amino acid sequence of 6169 (or Sequence number It comprises a light chain comprising an amino acid sequence having at least about 93%, 95%, or 99% sequence identity with 6169. In some embodiments, the antigen-binding domain targeting TRBC1 comprises Sequence number A heavy chain containing the amino acid sequence of 6167 and Sequence number It includes a light chain containing the amino acid sequence of 6169.

[0793] In some embodiments, the antigen-binding domain targeting TRBC1 is Sequence number The amino acid sequence of 6168 (or Sequence number A heavy chain comprising an amino acid sequence having at least about 93%, 95%, or 99% sequence identity with 6168 and Sequence number The amino acid sequence of 6169 (or Sequence numberIt comprises a light chain comprising an amino acid sequence having at least about 93%, 95%, or 99% sequence identity with 6169. In some embodiments, the antigen-binding domain targeting TRBC1 comprises Sequence number A heavy chain containing the amino acid sequence of 6168 and Sequence number It includes a light chain containing the amino acid sequence of 6169.

[0794] TRBC2 antigen binding domain

[0795] In some embodiments, the antigen-binding domain that binds to TRBC2 comprises one or more CDRs disclosed in Table 9A, Table 9B, or Table 10 (e.g., VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2, and / or VLCDR3), or a sequence having at least 85%, 90%, 95%, or 99% identity with respect to this. In some embodiments, the antigen-binding domain that binds to TRBC2 comprises one or more framework regions disclosed in Table 9A, Table 9B, or Table 10 (e.g., VHFWR1, VHFWR2, VHFWR3, VHFWR4, VLFWR1, VLFWR2, VLFWR3, and / or VLFWR4), or a sequence having at least 85%, 90%, 95%, or 99% identity with respect to this. In some embodiments, the antigen-binding domain that binds to TRBC2 comprises VH and / or VL disclosed in Table 11, or a sequence having at least 85%, 90%, 95%, or 99% identity with it. In some embodiments, the antigen-binding domain that binds to TRBC2 comprises the amino acid sequence disclosed in Table 12, or a sequence having at least 85%, 90%, 95%, or 99% identity with it.

[0796] In some embodiments, the antigen binding domain that binds to TRBC2 comprises VH, comprising heavy chain complementarity determining region 1 (VHCDR1), VHCDR2, and VHCDR3, and VL, comprising light chain complementarity determining region 1 (VLCDR1), VLCDR2, and VLCDR3.

[0797] In some embodiments, VHCDR1, VHCDR2, and VHCDR3 are GX1X2MH (where X1 in the sequence is Y or F, and X2 is P, H, V, Y, K, or A) and Sequence number Each includes the amino acid sequences of 201 and 7442 (or sequences having at least 85%, 90%, 95%, or 99% identity with these). In some embodiments, VHCDR1, VHCDR2, and VHCDR3 are Sequence number Each includes the amino acid sequences of 7422, 201, and 7403 (or sequences having at least 85%, 90%, 95%, or 99% identity with these). In some embodiments, VHCDR1, VHCDR2, and VHCDR3 are Sequence number Each includes the amino acid sequences of 7401, 201, and 7403 (or sequences having at least 85%, 90%, 95%, or 99% identity with these). In some embodiments, VHCDR1, VHCDR2, and VHCDR3 are Sequence number Each includes the amino acid sequences of 7394, 201, and 307 (or sequences having at least 85%, 90%, 95%, or 99% identity with these). In some embodiments, VHCDR1, VHCDR2, and VHCDR3 are Sequence number Each includes the amino acid sequences of 8643, 201, and 8695 (or sequences having at least 85%, 90%, 95%, or 99% identity with these). In some embodiments, VHCDR1, VHCDR2, and VHCDR3 are Sequence number Each includes the amino acid sequences of 8643, 201, and 7400 (or sequences having at least 85%, 90%, 95%, or 99% identity with these). In some embodiments, VHCDR1, VHCDR2, and VHCDR3 are Sequence numberEach includes the amino acid sequences of 7405, 201, and 7403 (or sequences having at least 85%, 90%, 95%, or 99% identity with these). In some embodiments, VHCDR1, VHCDR2, and VHCDR3 are Sequence number Each includes the amino acid sequences of 7407, 201, and 7403 (or sequences having at least 85%, 90%, 95%, or 99% identity with these). In some embodiments, VHCDR1, VHCDR2, and VHCDR3 are Sequence number Each includes the amino acid sequences of 7427, 201, and 7403 (or sequences having at least 85%, 90%, 95%, or 99% identity with these). In some embodiments, VHCDR1, VHCDR2, and VHCDR3 are Sequence number Each includes the amino acid sequences of 7430, 201, and 7403 (or sequences having at least 85%, 90%, 95%, or 99% identity with these).

[0798] In some embodiments, VLCDR1, VLCDR2, and VLCDR3 are Sequence number Each includes an amino acid sequence of 7443, 224, and 225 or 8680 (or a sequence having at least 85%, 90%, 95%, or 99% identity with this). In some embodiments, VLCDR1, VLCDR2, and VLCDR3 are Sequence number Each includes an amino acid sequence of 7410, 224, and 225 or 8680 (or a sequence having at least 85%, 90%, 95%, or 99% identity with such a sequence). In some embodiments, VLCDR1, VLCDR2, and VLCDR3 are Sequence number Each includes the amino acid sequences of 7409, 224, and 225 or 8680 (or sequences having at least 85%, 90%, 95%, or 99% identity with these).

[0799] In some embodiments, VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2, and VLCDR3 are GX1X2MH (where X1 in the sequence is Y or F, and X2 is P, H, V, Y, K, or A) and Sequence number Each includes the amino acid sequences of 201, 7442, 7443, 224, and 225 or 8680 (or sequences having at least 85%, 90%, 95%, or 99% identity with these). In some embodiments, VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2, and VLCDR3 are Sequence number Each includes the amino acid sequences of 7422, 201, 7403, 7410, 224, and 225 or 8680 (or sequences having at least 85%, 90%, 95%, or 99% identity with these). In some embodiments, VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2, and VLCDR3 are Sequence number Each comprises the amino acid sequences of 7401, 201, 7403, 7410, 224, and 225 or 8680 (or sequences having at least 85%, 90%, 95%, or 99% identity with these). In some embodiments, VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2, and VLCDR3 each comprise the following amino acid sequences: Sequence number 7394, 201, 307, 7410, 224, and 225 or 8680 (or sequences having at least 85%, 90%, 95%, or 99% identity with these); Sequence number 8643, 201, 8695, 7410, 224, and 225 or 8680 (or a sequence having at least 85%, 90%, 95%, or 99% identity with this); Sequence number 8643, 201, 7400, 7410, 224, and 225 or 8680 (or a sequence having at least 85%, 90%, 95%, or 99% identity with this); Sequence number7405, 201, 7403, 7410, 224, and 225 or 8680 (or a sequence having at least 85%, 90%, 95%, or 99% identity with this); Sequence number 7407, 201, 7403, 7410, 224, and 225 or 8680 (or a sequence having at least 85%, 90%, 95%, or 99% identity with this); Sequence number 7427, 201, 7403, 7410, 224, and 225 or 8680 (or a sequence having at least 85%, 90%, 95%, or 99% identity with this); Sequence number 7430, 201, 7403, 7410, 224, and 225 or 8680 (or a sequence having at least 85%, 90%, 95%, or 99% identity with this); Sequence number 7422, 201, 7403, 7409, 224, and 225 or 8680 (or a sequence having at least 85%, 90%, 95%, or 99% identity with this); Sequence number 7401, 201, 7403, 7409, 224, and 225 or 8680 (or a sequence having at least 85%, 90%, 95%, or 99% identity with this); Sequence number 7394, 201, 307, 7409, 224, and 225 or 8680 (or sequences having at least 85%, 90%, 95%, or 99% identity with these); Sequence number 8643, 201, 8695, 7409, 224, and 225 or 8680 (or a sequence having at least 85%, 90%, 95%, or 99% identity with this); Sequence number 8643, 201, 7400, 7409, 224, and 225 or 8680 (or a sequence having at least 85%, 90%, 95%, or 99% identity with this); Sequence number 7405, 201, 7403, 7409, 224, and 225 or 8680 (or a sequence having at least 85%, 90%, 95%, or 99% identity with this); Sequence number7407, 201, 7403, 7409, 224, and 225 or 8680 (or a sequence having at least 85%, 90%, 95%, or 99% identity with this); Sequence number 7427, 201, 7403, 7409, 224, and 225 or 8680 (or a sequence having at least 85%, 90%, 95%, or 99% identity with this); or Sequence number 7430, 201, 7403, 7409, 224, and 225 or 8680 (or a sequence having at least 85%, 90%, 95%, or 99% identity with this).

[0800] In some embodiments, VH is Sequence number A sequence of amino acids selected from the group consisting of 7420, 7423, 7411, 7412, 7413, 7414, 7415, 7416, 7417, 7425, 7428, and 7431 (or a sequence having at least 85%, 90%, 95%, or 99% identity with this), and / or VL is Sequence number It comprises an amino acid sequence selected from the group consisting of 7419 and 7418 (or a sequence having at least 85%, 90%, 95%, or 99% identity with respect to this). In some embodiments, VH and VL are Sequence number Each includes the amino acid sequences of 7420 and 7419 (or sequences having at least 85%, 90%, 95%, or 99% identity with these). In some embodiments, VH and VL are Sequence number Each includes the amino acid sequences of 7423 and 7419 (or sequences having at least 85%, 90%, 95%, or 99% identity with these). In some embodiments, VH and VL are Sequence number Each includes the amino acid sequences of 7411 and 7419 (or sequences having at least 85%, 90%, 95%, or 99% identity with these). In some embodiments, VH and VL are Sequence numberEach includes the amino acid sequences of 7412 and 7419 (or sequences having at least 85%, 90%, 95%, or 99% identity with these). In some embodiments, VH and VL are Sequence number Each includes the amino acid sequences of 7413 and 7419 (or sequences having at least 85%, 90%, 95%, or 99% identity with these). In some embodiments, VH and VL are Sequence number Each includes the amino acid sequences of 7414 and 7419 (or sequences having at least 85%, 90%, 95%, or 99% identity with these). In some embodiments, VH and VL are Sequence number Each includes the amino acid sequences of 7415 and 7419 (or sequences having at least 85%, 90%, 95%, or 99% identity with these). In some embodiments, VH and VL are Sequence number Each includes the amino acid sequences of 7416 and 7419 (or sequences having at least 85%, 90%, 95%, or 99% identity with these). In some embodiments, VH and VL are Sequence number Each includes the amino acid sequences of 7417 and 7419 (or sequences having at least 85%, 90%, 95%, or 99% identity with these). In some embodiments, VH and VL are Sequence number Each includes the amino acid sequences of 7425 and 7419 (or sequences having at least 85%, 90%, 95%, or 99% identity with these). In some embodiments, VH and VL are Sequence number Each includes the amino acid sequences of 7428 and 7419 (or sequences having at least 85%, 90%, 95%, or 99% identity with these). In some embodiments, VH and VL are Sequence number Each includes the amino acid sequences of 7431 and 7419 (or sequences having at least 85%, 90%, 95%, or 99% identity with these). In some embodiments, VH and VL are Sequence numberEach includes the amino acid sequences of 7420 and 7418 (or sequences having at least 85%, 90%, 95%, or 99% identity with these). In some embodiments, VH and VL are Sequence number Each includes the amino acid sequences of 7423 and 7418 (or sequences having at least 85%, 90%, 95%, or 99% identity with these). In some embodiments, VH and VL are Sequence number Each includes the amino acid sequences of 7411 and 7418 (or sequences having at least 85%, 90%, 95%, or 99% identity with these). In some embodiments, VH and VL are Sequence number Each includes the amino acid sequences of 7412 and 7418 (or sequences having at least 85%, 90%, 95%, or 99% identity with these). In some embodiments, VH and VL are Sequence number Each includes the amino acid sequences of 7413 and 7418 (or sequences having at least 85%, 90%, 95%, or 99% identity with these). In some embodiments, VH and VL are Sequence number Each includes the amino acid sequences of 7414 and 7418 (or sequences having at least 85%, 90%, 95%, or 99% identity with these). In some embodiments, VH and VL are Sequence number Each includes the amino acid sequences of 7415 and 7418 (or sequences having at least 85%, 90%, 95%, or 99% identity with these). In some embodiments, VH and VL are Sequence number Each includes the amino acid sequences of 7416 and 7418 (or sequences having at least 85%, 90%, 95%, or 99% identity with these). In some embodiments, VH and VL are Sequence number Each includes the amino acid sequences of 7417 and 7418 (or sequences having at least 85%, 90%, 95%, or 99% identity with these). In some embodiments, VH and VL are Sequence numberEach includes the amino acid sequences of 7425 and 7418 (or sequences having at least 85%, 90%, 95%, or 99% identity with these). In some embodiments, VH and VL are Sequence number Each includes the amino acid sequences of 7428 and 7418 (or sequences having at least 85%, 90%, 95%, or 99% identity with these). In some embodiments, VH and VL are Sequence number Each includes the amino acid sequences of 7431 and 7418 (or sequences having at least 85%, 90%, 95%, or 99% identity with these).

[0801] In some embodiments, the antigen-binding domain that binds to TRBC2 is Sequence number It includes an amino acid sequence selected from the group consisting of 7433, 7434, 7435, 7436, and 7437 (or a sequence having at least 85%, 90%, 95%, or 99% identity with this).

[0802] In some embodiments, the bispecific antibody is considered herein to comprise a first antigen-binding domain that binds to TRBC2. In some embodiments, an exemplary TRBC2 binding domain comprises the heavy and light chains of a TRBC2 binding antibody (e.g., TRBC2 binder-1 or TRBC2 binder-2 or TRCBC2 binder-3 or TRCBC2 binder-4). In some embodiments, an exemplary TRBC2 binding domain Sequence number A heavy chain variable domain selected from 8011, 8013, 8020, or 8303, or Sequence number A sequence that is at least 90% identical to 8011, 8013, 8020, or 8303; and Sequence number A light chain variable domain selected from 8012, 8014, 8014, or 8014, or Sequence number It includes sequences that are at least 90% identical to 1, 3, 31, or 33, and any combination thereof.

[0803] In some embodiments, an exemplary TRBC2 binding domain comprises the heavy and light chains of a TRBC2 binding antibody, e.g., TRBC2 binder-1. In some embodiments, an exemplary TRBC2 binding domain Sequence number 8011 It includes at least 90% identical heavy chain amino acid sequences, for example, a heavy chain variable domain (VH). In some embodiments, an exemplary TRBC2 binding domain is Sequence number It includes a heavy chain variable domain that is at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to 8011. In some embodiments, the TRBC2 binding domain is Sequence number It includes a heavy chain containing the amino acid sequence of 8011.

[0804] In some implementations, an exemplary TRBC2 combined domain is Sequence number 8012 It includes an amino acid sequence within the light chain that is at least 95% identical to, for example, a light chain variable domain (VL). In some embodiments, the light chain variable domain is Sequence number It is at least 96%, at least 97%, at least 98%, or at least 99% identical to 8012. In some implementations, the TRBC2 combined domain is Sequence number It comprises a light chain comprising the amino acid sequence of 8012. In some embodiments, the bispecific antibody comprises a first antigen-binding domain that binds to TRBC2, and the first antigen-binding domain comprises: a TRBC2 binder; or a TRBC2 binder having one or more amino acid sequences described in Table 13 or Table 14; or a heavy chain VH domain of a TRBC2 binder having either one of the domains described in Table 13 or Table 14, or having one or more domains having one or more amino acid modifications compared to the amino acid sequence of the domain described in Table 13 or Table 14 ( Sequence number 8011) and light chain VL domain ( Sequence number Includes 8012).

[0805] In some embodiments, an exemplary TRBC2 binding domain comprises the heavy chain and light chain of a TRBC2 binding antibody, e.g., TRBC2 binder-2. In some embodiments, an exemplary TRBC2 binding domain Sequence number 8013 and includes a heavy chain that is at least 95% identical. In some embodiments, an exemplary TRBC2 binding domain is Sequence number It includes a heavy chain that is at least 96%, at least 97%, at least 98%, or at least 99% identical to 8013. In some embodiments, an exemplary TRBC2 binding domain is Sequence number 8014 It includes a light chain that is at least 95% identical to and. In some embodiments, an exemplary TRBC2 binding domain is Sequence number It comprises a light chain that is at least 96%, at least 97%, at least 98%, or at least 99% identical to 8014. In some embodiments, the bispecific antibody comprises a first antigen-binding domain that binds to TRBC2, and the first antigen-binding domain comprises a TRBC2 binder; or a heavy chain VH of a TRBC2 binder having one or more domains having one or more amino acid modifications compared to the amino acid sequence of a domain described in Table 13 ( Sequence number 8013) and light chain VL( Sequence number Includes 8014).

[0806] In some implementations, an exemplary TRBC2 combined domain is Sequence number 8020 It includes at least 90% identical heavy chain amino acid sequences, for example, a heavy chain variable domain (VH). In some embodiments, an exemplary TRBC2 binding domain is Sequence number It includes at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical heavy chain variable domains with 8020.

[0807] In some implementations, an exemplary TRBC2 combined domain is Sequence number 8014It includes an amino acid sequence within the light chain that is at least 90% identical to, for example, a light chain variable domain (VL). In some embodiments, an exemplary TRBC2 binding domain is Sequence number It comprises a light chain variable domain that is at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to 8014. In some embodiments, the bispecific antibody comprises a first antigen-binding domain that binds to TRBC2, and the first antigen-binding domain comprises a TRBC2 binder; or a heavy chain VH of a TRBC2 binder having one or more domains having one or more amino acid modifications compared to the amino acid sequence of a domain described in Table 13 or Table 14 ( Sequence number 8020 ) and light chain VL( Sequence number 8014 Includes ).

[0808] In some implementations, an exemplary TRBC2 combined domain is Sequence number 8303 It includes at least 90% identical heavy chain amino acid sequences, for example, a heavy chain variable domain (VH). In some embodiments, an exemplary TRBC2 binding domain is Sequence number 8303 It includes at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical heavy chain variable domains.

[0809] In some implementations, an exemplary TRBC2 combined domain is Sequence number 8014It includes an amino acid sequence within the light chain that is at least 90% identical to, for example, a light chain variable domain (VL). In some embodiments, the bispecific antibody comprises a first antigen-binding domain that binds to TRBC2, and the first antigen-binding domain comprises a TRBC2 binder; or a heavy chain VH of a TRBC2 binder having one or more domains having one or more amino acid modifications compared to the amino acid sequence of a domain described in Table 13 ( Sequence number 8013 ) and light chain VL( Sequence number 8024 Includes ).

[0810] For example, the TRBC2-linked heavy chain domain is Sequence number 8011 Amino acid sequence QVQLVQSGAEVKKPGSSVKVSCKASPRGFYGYHMHWVRQAPGQGLEWMGFINPYTNDIQYNERFRGRVTITSDESTTTAYMELSSLRSEDTAVYYCAMGNGKWGDGAYRFFDLWGQGTLVTVSS (Sequence No. 8011) It may include, and the TRBC2-binding light chain domain is Sequence number 8012 The amino acid sequence of DVVMTQSPLSLPVTLGQPASISCRSSENLVHSNGRTYLQWYQQRPGQSPRLLIY RVSNRFP GVPDRFSGSGSGTDFTLKISRVEAEDVGVYFCSQSSLEPYTFGGGTKVEIK (Sequence No. 8012) It may include.

[0811] For example, the TRBC2-linked heavy chain domain is Sequence number 8013 The order of

[0812] QVQLVQSGAEVKKPGSSVKVSCKASPRGFYGYHMHWVRQAPGQGLEWMGFINPYNNHIQYNERFRGRVTITSDESTTTAYMELSSLRSEDTAVYYCALGNGKWGDGAYRFFDFWGQGTLVTVSS (Sequence No. 8013) It may include, and the TRBC2-binding light chain domain is Sequence number 8014 The order of

[0813] DVVMTQSPLSLPVTLGQPASISCRSSKNLVHSNGRTYLQWYQQRPGQSPRLLIY RVSNRFP GVPDRFSGSGSGTDFTLKISRVEAEDVGVYFCSQSTREPYTFGGGTKVEIK (Sequence No. 8014) It may include.

[0814] The TRBC2-linked heavy chain domain is Sequence number 8020 sequence

[0815] QVQLVQSGAEVKKPGSSVKVSCKASPRGFYGYHMHWVRQAPGQGLEWMG FINPYNNHIQYNERFRG RVTITSDESTTTAYMELSSLRSEDTAVYYCALGEGKWGDGAYRFFDFWGQGTLVTVSS (Sequence No. 8020) It may include, and the TRBC2-binding light chain domain is Sequence number 8014 Sequence of DVVMTQSPLSLPVTLGQPASISCRSSKNLVHSNGRTYLQWYQQRPGQSPRLLIY RVSNRFP GVPDRFSGSGSGTDFTLKISRVEAEDVGVYFCSQSTREPYTFGGGTKVEIK (Sequence No. 8014) It may include.

[0816] The TRBC2-linked heavy chain domain is Sequence number 8303 sequence

[0817] QVQLVQSGAEVKKPGSSVKVSCKASPRGFYGYHMHWVRQAPGQGLEW MGFINPYNNHIQYNERFRG RVTITSDESTTTAYMELSSLRSEDTAVYYCALGAGKWGDGAYRFFDFWGQGTLVTVSS (Sequence No. 8303) It may include, and the TRBC2-binding light chain domain is Sequence number 8014 Sequence of DVVMTQSPLSLPVTLGQPASISCRSSKNLVHSNGRTYLQWYQQRPGQSPRLLIY RVSNRFP GVPDRFSGSGSGTDFTLKISRVEAEDVGVYFCSQSTREPYTFGGGTKVEIK (Sequence No. 8014) It may include.

[0818] In some embodiments, the TRBC2 antigen binding domain is Sequence number 8024 Compared to the VH domain of an antibody as described in, the heavy chain variable domain within the VH domain may contain mutations, and the mutations may be selected from T28K mutations, Y32F mutations, and A100N mutations. In some embodiments, the TRBC2 antigen-binding domain is Sequence number 8024 Compared to the VH domain of an antibody as described in, the heavy chain variable domain within the VH domain comprises one or more mutations, and the mutations are selected from T28K mutations, Y32F mutations, and A100N mutations. In some embodiments, the VH domain may comprise mutations, and the mutations Sequence number 8024 It may be at position V2, or Y27, or G31, or R98, or Y102, or N103, or A107. In some embodiments, the VH domain of the TRBC2 antigen-binding domain is Sequence number 8024 Compared to the VH domain of an antibody such as that described in, it may contain V2K or V2R mutations. In some embodiments, the VH domain of the TRBC2 antigen-binding domain is Sequence number 8024 Compared to the VH domain of an antibody such as that described in, it may include Y27F, Y27M, Y27N, or Y27W mutations. In some embodiments, the VH domain of the TRBC2 antigen-binding domain is Sequence number 8024 Compared to the VH domain of an antibody such as that described in, it may include G31K, G31R, or G31S mutations. In some embodiments, the VH domain of the TRBC2 antigen-binding domain is Sequence number 8024 Compared to the VH domain of an antibody such as that described in, it may contain an R98K mutation. In some embodiments, the VH domain of the TRBC2 antigen-binding domain is Sequence number 8024Compared to the VH domain of the antibody as described in, it may include Y102F or Y102L mutations. In some embodiments, the VH domain of the TRBC2 antigen-binding domain is Sequence number 8024 Compared to the VH domain of the antibody described in [the text], it may include N193A, N193E, N103F, N103H, N103L, N103M, N103Q, N103S, N103W, or N103Y mutations.

[0819] In some embodiments, the TRBC2 antigen-binding domain may include a mutation in the light chain variable domain, and the mutation Sequence number 8025 It may be at position N35 or R55 for the VL domain of the antibody as described in. In some embodiments, the VL domain of the TRBC2 antigen-binding domain is Sequence number It may include N35M, N35F, N35Y, N35K, or N35R mutations at position 35 for the VL domain of the antibody as described in 8025.

[0820] Sequence number 8024 - Reference antibody VH domain:

[0821] QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYVMHWVRQAPGQGLEWMGFINPYNDDIQSNERFRGRVTMTRDT SISTAYMELSRLRSDDTAVYYCARGAGYNFDGAYRFFDFWGQGTMVTVSS (Sequence number 8024 ).

[0822] Sequence number 8025 - Reference antibody VL domain:

[0823] DIVMTQSPLSLPVTPGEPASISCRSSQRLVHSNGNTYLHWYLQKPGQSPRLLIYRVSNRFPGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC SQSTHVPYT FGQGTKLEIK (Sequence number 8025 ).

[0824] In some embodiments, the bispecific antibody comprises a second antigen-binding domain that binds to NKp30. In some embodiments, an exemplary NKp30 binding domain comprises the heavy chain and light chain of NKp30 binder 1. In some embodiments, an exemplary NKp30 binding domain is Sequence number 8006 or 8698 It includes a heavy chain having the amino acid sequence of. In some embodiments, an exemplary NKp30 binding domain is Sequence number 8006 or 8698 It includes a heavy chain having an amino acid sequence that is at least 90% identical to the amino acid sequence of. In some embodiments, an exemplary NKp30 binding domain is Sequence number 8006 or 8698 It includes a heavy chain having an amino acid sequence that is at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence of.

[0825] In some embodiments, an exemplary NKp30 binding domain comprises a heavy chain and a light chain of NKp30 binder 1. In some embodiments, an exemplary NKp30 binding domain is Sequence number It includes a heavy chain having an amino acid sequence of 4,684 or 8,690. In some embodiments, an exemplary NKp30 binding domain is Sequence number 4684 or 8690 It includes a heavy chain having an amino acid sequence that is at least 90% identical to the amino acid sequence of. In some embodiments, an exemplary NKp30 binding domain is Sequence number It includes a heavy chain having an amino acid sequence that is at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence of 4684 or 8690.

[0826] In some implementations, an exemplary NKp30 binding domain is Sequence number 8003 It includes the light chain of. In some embodiments, the exemplary NKp30 binding domain includes the light chain of SEQ ID NO. 4683. .In some implementations, an exemplary NKp30 binding domain is Sequence number 8003 It includes a light chain having an amino acid sequence that is at least 90% identical to the amino acid sequence of. In some embodiments, an exemplary NKp30 binding domain is Sequence number 4683 It includes a light chain having an amino acid sequence that is at least 90% identical to the amino acid sequence of. In some embodiments, an exemplary NKp30 binding domain is Sequence number 8003 It includes a light chain having an amino acid sequence that is at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence of. In some embodiments, an exemplary NKp30 binding domain is Sequence number 4683 It includes a light chain having an amino acid sequence that is at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence of . In some embodiments, a bispecific antibody comprising a second antigen-binding domain that binds to NKp30 comprises a heavy chain and a light chain of NKp30 binder 1 having the amino acid sequence described below.

[0827] An exemplary NKp30-linked heavy chain domain is Sequence number 8030 The sequence of: EIQLLESGGGLVQPGGSLRLSCAVSGFSITTTGYHWNWVRQAPGKGLEWVGYIYSSGSTSYNPSLKSRFTISRDTSKNTFYLQMNSLRAEDTAVYYCARGDWHYFDYWGQGTMVTVSS (Sequence No. 8030) It may include.

[0828] An exemplary NKp30-binding light chain domain is Sequence No. 13 The sequence of: DSVTTQSPLSLPVTLGQPASISCSGEKLSDKYVHWYQQRPGQSPRMLIYENDRRPSGVPDRFSGSNSGNDATLKISRVEAEDVGVYFCQFWDSTNSAVFGGGTKVEIK (Sequence No. 13) It may include.

[0829] In some embodiments, an exemplary bispecific antibody comprises: (i) Sequence number The heavy chain sequence of 8011 or Sequence number 8011 and at least 90% identical sequence; and Sequence number 8012 The light chain sequence of or Sequence number 8012 A first antigen-binding domain comprising a TRBC2 binding domain having at least 95% identical sequence; and (ii) Sequence number 8030 and 13 A second antigen-binding domain comprising an NKp30 binding domain having heavy chain and light chain sequences, respectively.

[0830] In some embodiments, an exemplary bispecific antibody comprises: (i) Sequence number The heavy chain sequence of 8013 or Sequence number 8013 and at least 95% identical sequence; and Sequence number The light chain sequence of 8014 or Sequence number 8014 A first antigen-binding domain comprising a TRBC2 binding domain having at least 95% identical sequence; and (ii) Sequence number 8030 and 13 A second antigen-binding domain comprising an NKp30 binding domain having heavy chain and light chain sequences, respectively.

[0831] In some embodiments, an exemplary bispecific antibody comprises: (i) Sequence number 8011 heavy chain sequence of or Sequence number 8011 and at least 90% identical sequence; and Sequence number 8012 light chain sequence or Sequence number 8012 A first antigen-binding domain comprising a TRBC2 binding domain having at least 95% identical sequence; and (ii) Sequence number 8030 and at least 90% identical and Sequence No. 13 A second antigen-binding domain comprising an NKp30 binding domain having at least 90% identical heavy chain and light chain sequences, respectively.

[0832] In some embodiments, an exemplary bispecific antibody comprises: (i) Sequence number 8013 heavy chain sequence of or Sequence number 8013 and at least 95% identical sequence; and Sequence number 8014 The light chain sequence of or Sequence number 8014 A first antigen-binding domain comprising a TRBC2 binding domain having at least 95% identical sequence; and (ii) Sequence number 8030 and at least 95% identical and Sequence No. 13 A second antigen-binding domain comprising an NKp30 binding domain having at least 90% identical heavy chain and light chain sequences, respectively.

[0833] In some embodiments, the bispecific antibody comprises: (i) a first antigen-binding domain comprising a TRBC2 binding domain comprising a short-chain variable fragment (scFv), wherein the scFv comprises a heavy chain and a light chain connected by a short linker; and (ii) a second antigen-binding domain comprising an NKp30 binding domain comprising a short-chain variable fragment (scFv), wherein the scFv comprises a heavy chain and a light chain connected by a short linker.

[0834] In some embodiments, the heavy chain of the TRBC2 binding domain comprises HC CDR1, CDR2, and CDR3. In some embodiments, the light chain of the TRBC2 binding domain comprises LC CDR1, CDR2, and CDR3. In one embodiment, the heavy chain of the TRBC2 binding domain comprises PRGFYGYHMH (SEQ No. 8272) {or PRGFYGY (according to the Chothia numbering system Sequence number 8041 )}, or PRGFYGYAMH( Sequence number 8701 It includes HC CDR1 having an amino acid sequence including ). In one embodiment, the heavy chain of the TRBC2 binding domain is the amino acid sequence RSGFHGYAMH( Sequence number 8207 ) or PRGFHGYHMH( Sequence number 8211It includes HC CDR1 having ). In some embodiments, the heavy chain of the TRBC2 binding domain is the amino acid sequence SRSGFHGYAMH( Sequence number 8215 It includes HC CDR1 containing ). In some embodiments, the heavy chain of the TRBC2 binding domain is the amino acid sequence PRGFYGYHMH( Sequence number 8272 It includes HC CDR1 containing ). In some embodiments, the heavy chain of the TRBC2 binding domain is the amino acid sequence RSSQNLVHSNGRTYLH( Sequence number 8226 It includes HC CDR1 containing ). In one embodiment, the heavy chain of the TRBC2 binding domain is the amino acid sequence MGFINPYTNDIQYNERFRG( Sequence number 8042 It includes HC CDR2 having ). In one embodiment, the heavy chain of the TRBC2 binding domain is the amino acid sequence GNGKWGDGAYRFFDL( Sequence number 8043 It includes HC CDR3 having ). In one embodiment, the heavy chain of the TRBC2 binding domain is the amino acid sequence FINPYNNHIQYNERFRG( Sequence number 8044 ) or FINPYNDDIQYNQKFQG( Sequence number 8208 ) or YINPYNRDIKYNQKFQG ( Sequence number 8212 It includes HC CDR2 containing ). In some embodiments, HC CDR2 is the amino acid sequence FINPYNHAIKYNQKFQG( Sequence number 8213 ) has. In some embodiments, HC CDR2 has the amino acid sequence YINPYTGDIKYNERFRG( Sequence number 8217 ) has. In some embodiments, HC CDR2 has the amino acid sequence FINPYNDDIQYNERFRG( Sequence number 8221 ) has. In some embodiments, HC CDR2 has the amino acid sequence TINPYNAEIKYNQKFQG( Sequence number 8222 ) has. In some embodiments, HC CDR2 has the amino acid sequence TINPYNRDIQYNQKFQG( Sequence number 8225 ) has. In some embodiments, HC CDR2 has the amino acid sequence FINPYNRDIKYNERFRG( Sequence number 8228 ) has. In some embodiments, HC CDR2 has the amino acid sequence AINPYTNDIKYNERFRG( Sequence number 8229 ) has. In some embodiments, HC CDR2 has the amino acid sequence AINPYTNHIQYNERFRG( Sequence number 8230 ) has. In some embodiments, HC CDR2 has the amino acid sequence AINPYTRAIKYNERFRG( Sequence number 8231 ) has. In some embodiments, HC CDR2 has the amino acid sequence TINPYNGDIQYNERFRG( Sequence number 8232 ) has. In some embodiments, HC CDR2 has the amino acid sequence AINPYNTDIKYNERFRG( ...

Claims

Claim 1 A composition comprising a formulation having an antigen-binding domain that binds to NKp30, wherein the antigen-binding domain comprises a heavy chain variable region (VH) comprising: (i) a heavy chain complementarity determining region 3 (VHCDR3) comprising the sequence of GDWHYFDY (SEQ No. 504); and (ii) a heavy chain complementarity determining region 1 (VHCDR1) comprising the sequence of GFSIIRVGYHWN (SEQ No. 502), a heavy chain complementarity determining region 2 (VHCDR2) comprising the sequence of YIHRSGSTSYNPSLKS (SEQ No. 503), or a combination thereof. Claim 2 A composition according to claim 1, wherein the antigen binding domain comprises VH including VHCDR1 having the sequence GFSIIRVGYHWN (SEQ No. 502), VHCDR2 having the sequence YIHRSGSTSYNPSLKS (SEQ No. 503), and VHCDR3 having the sequence GDWHYFDY (SEQ No. 504). Claim 3 A composition according to claim 1 or 2, wherein the antigen binding domain comprises a light chain complementarity determining region 1 (VLCDR1) comprising the sequence of SGEKLSDKYVH (SEQ No. 26), a light chain complementarity determining region 2 (VLCDR2) comprising the sequence of ENDRRPS (SEQ No. 28), a light chain complementarity determining region 3 (VLCDR3) comprising the sequence of QFWDSTNSAV (SEQ No. 86), or any combination thereof, a light chain variable region (VL). Claim 4 A composition according to any one of claims 1 to 3, wherein the antigen binding domain comprises VL comprising VLCDR1 having the sequence of SGEKLSDKYVH (SEQ No. 26), VLCDR2 having the sequence of ENDRRPS (SEQ No. 28), and VLCDR3 having the sequence of QFWDSTNSAV (SEQ No. 86). Claim 5 A composition according to any one of claims 1 to 4, wherein the antigen-binding domain comprises a VH having at least 75% sequence identity with the sequence of SEQ ID NO.

505. Claim 6 A composition according to any one of claims 1 to 5, wherein the antigen binding domain comprises a VH comprising the sequence of SEQ ID NO.

505. Claim 7 A composition according to any one of claims 1 to 6, wherein the antigen-binding domain comprises a VL having at least 75% sequence identity with the sequence of SEQ ID NO.

13. Claim 8 A composition according to any one of claims 1 to 7, wherein the antigen binding domain comprises a VL comprising the sequence of SEQ ID NO.

13. Claim 9 A composition according to any one of claims 1 to 8, wherein the antigen-binding domain comprises a VH having at least 75% sequence identity with the sequence of SEQ ID NO. 505 and a VL having at least 75% sequence identity with the sequence of SEQ ID NO.

13. Claim 10 A composition according to any one of claims 1 to 9, wherein the antigen binding domain comprises VH comprising the sequence of SEQ ID NO. 505 and VL comprising the sequence of SEQ ID NO.

13. Claim 11 A composition according to any one of claims 1 to 10, wherein the antigen-binding domain is a whole antibody or an antigen-binding fragment thereof, a divalent antibody, a bispecific antibody, or a biparatope antibody. Claim 12 A composition according to either claim 1 or claim 11, wherein the antigen-binding domain is Fab, F(ab')2, Fv, short-chain Fv(scFv), diabadi, a single-domain antibody, or a camelid antibody. Claim 13 A composition according to any one of claims 1 to 12, wherein the antigen binding domain is Fab or scFv. Claim 14 A composition according to any one of claims 1 to 13, wherein the antigen binding domain comprises one or more heavy chain constant regions selected from the group consisting of IgG1 heavy chain constant region or a functional fragment thereof, IgG2 heavy chain constant region or a functional fragment thereof, IgG3 heavy chain constant region or a functional fragment thereof, IgGA1 heavy chain constant region or a functional fragment thereof, IgGA2 heavy chain constant region or a functional fragment thereof, IgG4 heavy chain constant region or a functional fragment thereof, IgJ heavy chain constant region or a functional fragment thereof, IgM heavy chain constant region or a functional fragment thereof, IgD heavy chain constant region or a functional fragment thereof, and IgE heavy chain constant region or a functional fragment thereof. Claim 15 A composition according to claim 14, wherein one or more heavy chain invariant regions comprises a sequence having at least 75% sequence identity with any one of the heavy chain invariant region sequences listed in Tables 3, 7, 8, 16, 17, 39, 40, 42C, 43A, 43B, and 46 to 51. Claim 16 In claim 14 or 15, the one or more heavy chain invariant regions are sequences having at least 75% sequence identity with the sequence of SEQ ID NO. 73, the sequence of SEQ ID NO. 76, the sequence of SEQ ID NO. 74, the sequence of SEQ ID NO. 75, the sequence of SEQ ID NO. 76, the sequence of SEQ ID NO. 40, the sequence of SEQ ID NO. 41, the sequence of SEQ ID NO. 42, the sequence of SEQ ID NO. 3645, the sequence of SEQ ID NO. 3646, the sequence of SEQ ID NO. 3649, the sequence of SEQ ID NO. 3451, the sequence of SEQ ID NO. 506, the sequence of SEQ ID NO. 507, the sequence of SEQ ID NO. 3647, the sequence of SEQ ID NO. 3648, the sequence of SEQ ID NO. 508 or 8686, the sequence of SEQ ID NO. 513, the sequence of SEQ ID NO. 514, the sequence of SEQ ID NO. 515, the sequence of SEQ ID NO. 516, the sequence of SEQ ID NO. 3447, or the sequence of SEQ ID NO.

511. A composition including. Claim 17 A composition according to any one of claims 1 to 16, wherein the antigen-binding domain comprises a kappa light chain constant region or a functional fragment thereof, a lambda light chain constant region or a functional fragment thereof, or a combination thereof. Claim 18 A composition according to claim 17, wherein the kappa light chain invariant region comprises a sequence having at least 75% sequence identity with any one of the light chain invariant region sequences listed in Tables 3, 7, 8, 16, 17, 39, 40, 42C, 43A, 43B, and 46 to 51. Claim 19 A composition according to claim 17 or 18, wherein the kappa light chain invariant region comprises a sequence having at least 75% sequence identity with the sequence of SEQ ID NO. 39 or the sequence of SEQ ID NO. 3644. Claim 20 A composition according to any one of claims 1 to 19, wherein the preparation is a polypeptide molecule. Claim 21 A composition according to any one of claims 1 to 20, wherein the formulation is a multifunctional molecule. Claim 22 A composition according to any one of claims 1 to 21, wherein the formulation is a multispecific molecule. Claim 23 A composition according to either claim 21 or 22, wherein the formulation comprises at least one cytokine molecule or a functional fragment or variant thereof or a functional fragment or variant thereof, an antigen-binding domain that binds to a T cell receptor beta chain constant domain (TRBC), a second antigen-binding domain that binds to NKp30, an immune cell engager, a tumor-targeting moiety, or any combination thereof. Claim 24 A composition according to any one of claims 1 to 23, wherein the formulation comprises at least two discontinuous polypeptide chains including a first polypeptide chain and a second polypeptide chain; wherein the first polypeptide chain comprises the following configuration in the direction from the N-terminus to the C-terminus: A-[first member of the dimerization module]-C, and the second polypeptide chain comprises the following configuration in the direction from the N-terminus to the C-terminus: B-[second member of the dimerization module]-D; wherein the first member of the dimerization module comprises a first immunoglobulin constant domain; and the second member of the dimerization module comprises a second immunoglobulin constant domain; and at least one of A, B, C, and D is an antigen-binding domain; and wherein the first polypeptide chain and the second polypeptide chain are dimerized through an interaction between the first member of the dimerization module and the second member of the dimerization module. Claim 25 In claim 24, (i) A is an antigen-binding domain, and B, C, and D are independently (a) absent, (b) at least one cytokine molecule or a functional fragment or variant thereof, (c) an antigen-binding domain that binds to a T cell receptor beta-chain constant domain (TRBC), (d) a second antigen-binding domain that binds to NKp30, (e) an immune cell binder, or (f) a tumor-targeting moiety; (ii) B is an antigen-binding domain, and A, C, and D are independently (a) absent, (b) at least one cytokine molecule or a functional fragment or variant thereof, (c) an antigen-binding domain that binds to a T cell receptor beta-chain constant domain (TRBC), (d) a second antigen-binding domain that binds to NKp30, (e) an immune cell binder, or (f) a tumor-targeting moiety; and (iii) C is an antigen-binding domain, and A, B, and D are independently (a) absent, (b) at least one cytokine molecule or A composition comprising: (c) an antigen-binding domain that binds to a T cell receptor beta-chain constant domain (TRBC), (d) a second antigen-binding domain that binds to NKp30, (e) an immune cell binder, or (f) a tumor-targeting moiety; and (iv) D is an antigen-binding domain, and A, B, and C are independently (a) absent, (b) at least one cytokine molecule or a functional fragment or variant thereof, (c) an antigen-binding domain that binds to a T cell receptor beta-chain constant domain (TRBC), (d) a second antigen-binding domain that binds to NKp30, (e) an immune cell binder, or (f) a tumor-targeting moiety. Claim 26 A composition according to any one of claims 21 to 25, wherein the formulation comprises at least one cytokine molecule or a functional fragment or variant thereof. Claim 27 A composition according to any one of claims 23 to 26, wherein the at least one cytokine molecule or its functional fragment or variant comprises IL-2 or its functional fragment or variant, IL-7 or its functional fragment or variant, IL-12 or its functional fragment or variant, IL-15 or its functional fragment or variant, IL-18 or its functional fragment or variant, IL-21 or its functional fragment or variant, interferon gamma or its functional fragment or variant, or any combination thereof. Claim 28 A composition according to any one of claims 23 to 27, wherein the at least one cytokine molecule or its functional fragment or variant comprises a sequence having at least 75% sequence identity with any one sequence selected from the group consisting of cytokine sequences listed in Tables 40, 42C, 43A, 43B, and 46 to 51. Claim 29 A composition according to any one of claims 23 to 28, wherein the at least one cytokine molecule or a functional fragment or variant thereof comprises any one sequence selected from the group consisting of cytokine sequences listed in Tables 40, 42C, 43A, 43B, and 46 to 51. Claim 30 A composition according to any one of claims 23 to 29, wherein at least one cytokine molecule or a functional fragment or variant thereof comprises IL-2 or a functional fragment or variant thereof. Claim 31 A composition according to any one of claims 23 to 30, wherein the IL-2 or its functional fragment or variant comprises a sequence having at least 75% sequence identity with the sequence of SEQ ID NO. 2191, the sequence of SEQ ID NO. 2270, or the sequence of SEQ ID NO. 2280. Claim 32 A composition according to any one of claims 23 to 31, wherein the IL-2 or its functional fragment or variant comprises the sequence of SEQ ID NO. 2191, the sequence of SEQ ID NO. 2270, or the sequence of SEQ ID NO. 2280. Claim 33 A composition according to any one of claims 23 to 29, wherein at least one cytokine molecule or a functional fragment or variant thereof comprises IL-15 or a functional fragment or variant thereof. Claim 34 A composition according to any one of claims 23 to 29 and 33, wherein the IL-15 or its functional fragment or variant comprises a sequence having at least 75% sequence identity with the sequence of SEQ ID NO. 2170, SEQ ID NO. 3670, or SEQ ID NO. 3673. Claim 35 A composition according to any one of claims 23 to 29, 33, and 34, wherein the IL-15 or its functional fragment or variant comprises the sequence of SEQ ID NO. 2170, the sequence of SEQ ID NO. 3670, or the sequence of SEQ ID NO. 3673. Claim 36 A composition according to any one of claims 23 to 29 and 33 to 35, wherein the at least one cytokine molecule or its functional fragment or variant further comprises an IL15R-alpha dimerization domain covalently linked to the IL-15 or its functional fragment or variant. Claim 37 A composition according to claim 36, wherein the IL15R-alpha dimerization domain comprises an IL-15 receptor alpha-sushi domain or a functional fragment or variant thereof. Claim 38 A composition according to claim 36 or 37, wherein the IL15R alpha dimerization domain comprises a sequence having at least 75% sequence identity with the sequence of SEQ ID NO. 3472. Claim 39 A composition according to any one of claims 36 to 38, wherein the IL15R alpha dimerization domain comprises the sequence of SEQ ID NO. 3472. Claim 40 A composition according to any one of claims 36 to 39, wherein the IL-15 or its functional fragment or variant is sharedly linked to the IL15R-alpha dimerization domain through a linker. Claim 41 A composition according to any one of claims 36 to 40, wherein the IL-15 or its functional fragment or variant is covalently linked to an IL15R-alpha dimerization domain through a linker comprising the sequence of SEQ ID NO. 3473. Claim 42 A composition according to any one of claims 23 to 29 and 33 to 41, wherein the at least one cytokine molecule or a functional fragment or variant thereof comprises a sequence having at least 75% sequence identity with the sequence of SEQ ID NO. 3474, SEQ ID NO. 3671, or SEQ ID NO. 8689. Claim 43 A composition according to any one of claims 23 to 29 and 33 to 42, wherein at least one cytokine molecule or a functional fragment or variant thereof comprises the sequence of SEQ ID NO. 3474, the sequence of SEQ ID NO. 3671, or the sequence of SEQ ID NO. 8689. Claim 44 A composition according to any one of claims 23 to 29, wherein at least one cytokine molecule or a functional fragment or variant thereof comprises IL-7 or a functional fragment or variant thereof. Claim 45 A composition according to any one of claims 23 to 29 and 44, wherein the IL-7 or its functional fragment or variant comprises a sequence having at least 75% sequence identity with the sequence of SEQ ID NO. 3540. Claim 46 A composition according to any one of claims 23 to 29, 44, and 45, wherein the IL-7 or a functional fragment or variant thereof comprises the sequence of SEQ ID NO. 3540. Claim 47 A composition according to any one of claims 23 to 29, wherein at least one cytokine molecule or a functional fragment or variant thereof comprises IL-12 or a functional fragment or variant thereof. Claim 48 A composition according to any one of claims 23 to 29 and 47, wherein the IL-12 or its functional fragment or variant comprises a sequence having at least 75% sequence identity with the sequence of SEQ ID NO. 3542, the sequence of SEQ ID NO. 3543, or the sequence of SEQ ID NO. 3545. Claim 49 A composition according to any one of claims 23 to 29, 47, and 48, wherein the IL-12 or its functional fragment or variant comprises the sequence of SEQ ID NO. 3542, the sequence of SEQ ID NO. 3543, or the sequence of SEQ ID NO. 3545. Claim 50 A composition according to any one of claims 23 to 29, wherein at least one cytokine molecule or a functional fragment or variant thereof comprises IL-18 or a functional fragment or variant thereof. Claim 51 A composition according to any one of claims 23 to 29 and 50, wherein the IL-18 or its functional fragment or variant comprises a sequence having at least 75% sequence identity with the sequence of SEQ ID NO. 2192. Claim 52 A composition according to any one of claims 23 to 29, 50, and 51, wherein the IL-18 or a functional fragment or variant thereof comprises the sequence of SEQ ID NO. 2192. Claim 53 A composition according to any one of claims 23 to 29, wherein at least one cytokine molecule or a functional fragment or variant thereof comprises IL-21 or a functional fragment or variant thereof. Claim 54 A composition according to any one of claims 23 to 29 and 53, wherein the IL-21 or a functional fragment or variant thereof comprises a sequence having at least 75% sequence identity with the sequence of SEQ ID NO. 3540. Claim 55 A composition according to any one of claims 23 to 29, 53, and 54, wherein the IL-21 or a functional fragment or variant thereof comprises the sequence of SEQ ID NO. 3540. Claim 56 A composition according to any one of claims 21 to 55, wherein the formulation comprises an antigen-binding domain that binds to TRBC. Claim 57 A composition according to any one of claims 23 to 56, wherein the antigen-binding domain binding to TRBC binds to TRBC1 or TRBC2. Claim 58 In claim 57, the antigen-binding domain binding to TRBC binds to TRBC1, and the antigen-binding domain binding to TRBC comprises a VH comprising the following: (i) a VHCDR1 comprising a sequence having at least 75% sequence identity with any one sequence selected from the group consisting of VHCDR1 sequences listed in Tables 2A, 2B, 3A, 3B, 5A, 5B, 7, 8, 16, 39, and 40; (ii) a VHCDR2 comprising a sequence having at least 75% sequence identity with any one sequence selected from the group consisting of VHCDR2 sequences listed in Tables 2A, 2B, 3A, 3B, 5A, 5B, 7, 8, 16, 39, and 40; (iii) Tables 2A, 2B, 3A, 3B, 5A, 5B, 7, 8, 16, 39, and a VHCDR3 comprising a sequence having at least 75% sequence identity with any one sequence selected from the group consisting of the VHCDR3 sequences listed in 40; or (iv) any combination thereof. Claim 59 A composition according to claim 57 or 58, wherein the antigen-binding domain binding to TRBC binds to TRBC1, and the antigen-binding domain binding to TRBC comprises a VH comprising the following: (i) a VHCDR1 comprising any one sequence selected from the group consisting of VHCDR1 sequences listed in Tables 2A, 2B, 3A, 3B, 5A, 5B, 7, 8, 16, 39, and 40; (ii) a VHCDR2 comprising any one sequence selected from the group consisting of VHCDR2 sequences listed in Tables 2A, 2B, 3A, 3B, 5A, 5B, 7, 8, 16, 39, and 40; (iii) any one selected from the group consisting of VHCDR3 sequences listed in Tables 2A, 2B, 3A, 3B, 5A, 5B, 7, 8, 16, 39, and 40 VHCDR3 containing the sequence; or (iv) any combination thereof. Claim 60 A composition according to any one of claims 57 to 59, wherein the antigen-binding domain that binds to TRBC binds to TRBC1, and the antigen-binding domain that binds to TRBC comprises a VH having at least 75% sequence identity with any one sequence selected from the group consisting of VH sequences listed in Tables 7, 8, 16, 39, and 40. Claim 61 A composition according to any one of claims 57 to 60, wherein the antigen-binding domain that binds to TRBC binds to TRBC1, and the antigen-binding domain that binds to TRBC comprises a VH comprising any one sequence selected from the group consisting of VH sequences listed in Tables 7, 8, 16, 39, and 40. Claim 62 A composition according to any one of claims 57 to 60, wherein the antigen-binding domain binding to TRBC binds to TRBC1, and the antigen-binding domain binding to TRBC comprises a VL comprising the following: (i) a VLCDR1 comprising a sequence having at least 75% sequence identity with any one sequence selected from the group consisting of VLCDR1 sequences listed in Tables 4, 6, 7, 8, 16, 39, and 40; (ii) a VLCDR2 comprising a sequence having at least 75% sequence identity with any one sequence selected from the group consisting of VLCDR2 sequences listed in Tables 4, 6, 7, 8, 16, 39, and 40; (iii) a VLCDR3 comprising a sequence having at least 75% sequence identity with any one sequence selected from the group consisting of VLCDR3 sequences listed in Tables 4, 6, 7, 8, 16, 39, and 40; or (iv) any combination of these. Claim 63 A composition according to any one of claims 57 to 62, wherein the antigen-binding domain binding to TRBC binds to TRBC1, and the antigen-binding domain binding to TRBC comprises a VL comprising: (i) a VLCDR1 comprising any one sequence selected from the group consisting of VLCDR1 sequences listed in Tables 4, 6, 7, 8, 16, 39, and 40; (ii) a VLCDR2 comprising any one sequence selected from the group consisting of VLCDR2 sequences listed in Tables 4, 6, 7, 8, 16, 39, and 40; (iii) a VLCDR3 comprising any one sequence selected from the group consisting of VLCDR3 sequences listed in Tables 4, 6, 7, 8, 16, 39, and 40; or (iv) any combination thereof. Claim 64 A composition according to any one of claims 57 to 59, wherein the antigen-binding domain that binds to TRBC binds to TRBC1, and the antigen-binding domain that binds to TRBC comprises a VL having at least 75% sequence identity with any one sequence selected from the group consisting of VL sequences listed in Tables 7, 8, 16, 39, and 40. Claim 65 A composition according to any one of claims 57 to 60, wherein the antigen-binding domain that binds to TRBC binds to TRBC1, and the antigen-binding domain that binds to TRBC comprises a VL comprising any one sequence selected from the group consisting of VL sequences listed in Tables 7, 8, 16, 39, and 40. Claim 66 In claim 57, the antigen-binding domain binding to TRBC binds to TRBC2, and the antigen-binding domain binding to TRBC comprises a VH comprising the following: (i) a VHCDR1 comprising a sequence having at least 75% sequence identity with any one sequence selected from the group consisting of VHCDR1 sequences listed in Tables 9A, 9B, 11, 12, 13, 14, 15, 17, 39, and 40; (ii) a VHCDR2 comprising a sequence having at least 75% sequence identity with any one sequence selected from the group consisting of VHCDR2 sequences listed in Tables 9A, 9B, 12, 13, 14, 15, 17, 39, and 40; (iii) a VHCDR2 having at least 75% sequence identity with any one sequence selected from the group consisting of VHCDR2 sequences listed in Tables 9A, 9B, 12, 14, 13, 14, 15, 17, 39, and 40 VHCDR3 comprising a sequence having at least 75% sequence identity with any one sequence selected from the group of VHCDR3 sequences; or (iv) any combination thereof. Claim 67 A composition according to claim 57 or 66, wherein the antigen-binding domain binding to TRBC binds to TRBC2, and the antigen-binding domain binding to TRBC comprises a VH comprising: (i) a VHCDR1 comprising any one sequence selected from the group consisting of VHCDR1 sequences listed in Tables 9A, 9B, 12, 13, 14, 15, 17, 39, and 40; (ii) a VHCDR2 comprising any one sequence selected from the group consisting of VHCDR2 sequences listed in Tables 9A, 9B, 12, 13, 14, 15, 17, 39, and 40; (iii) a VHCDR3 comprising any one sequence selected from the group consisting of VHCDR3 sequences listed in Tables 9A, 9B, 12, 13, 14, 15, 17, 39, and 40; or (iv) any combination thereof. Claim 68 A composition according to any one of claims 57, 66, and 67, wherein the antigen-binding domain binding to TRBC binds to TRBC2, and the antigen-binding domain binding to TRBC comprises a VH having at least 75% sequence identity with any one sequence selected from the group consisting of VH sequences listed in Tables 11, 15, 17, 39, and 40. Claim 69 A composition according to any one of claims 57 and 66 to 68, wherein the antigen-binding domain that binds to TRBC binds to TRBC2, and the antigen-binding domain that binds to TRBC comprises a VH comprising any one sequence selected from the group consisting of VH sequences listed in Tables 11, 15, 17, 39, and 40. Claim 70 A composition according to any one of claims 57 and 66 to 69, wherein the antigen-binding domain binding to TRBC binds to TRBC2, and the antigen-binding domain binding to TRBC comprises a VL comprising the following: (i) a VLCDR1 comprising a sequence having at least 75% sequence identity with any one sequence selected from the group consisting of VLCDR1 sequences listed in Tables 10, 12, 13, 14, 15, 17, 39, and 40; (ii) a VLCDR2 comprising a sequence having at least 75% sequence identity with any one sequence selected from the group consisting of VLCDR2 sequences listed in Tables 10, 12, 13, 14, 15, 17, 39, and 40; (iii) from the group consisting of VLCDR3 sequences listed in Tables 10, 12, 13, 14, 15, 17, 39, and 40 VLCDR3 comprising a sequence having at least 75% sequence identity with any one selected sequence; or (iv) any combination thereof. Claim 71 A composition according to any one of claims 57 and 66 through 70, wherein the antigen-binding domain binding to TRBC binds to TRBC2, and the antigen-binding domain binding to TRBC comprises a VL comprising: (i) a VLCDR1 comprising any one sequence selected from the group consisting of VLCDR1 sequences listed in Tables 10, 12, 13, 14, 15, 17, 39, and 40; (ii) a VLCDR2 comprising any one sequence selected from the group consisting of VLCDR2 sequences listed in Tables 10, 12, 13, 14, 15, 17, 39, and 40; (iii) a VLCDR3 comprising any one sequence selected from the group consisting of VLCDR3 sequences listed in Tables 10, 12, 13, 14, 15, 17, 39, and 40; or (iv) any combination thereof. Claim 72 A composition according to any one of claims 57 and 66 to 71, wherein the antigen-binding domain that binds to TRBC binds to TRBC2, and the antigen-binding domain that binds to TRBC comprises a VL having at least 75% sequence identity with any one sequence selected from the group consisting of VL sequences listed in Tables 11, 15, 17, 39, and 40. Claim 73 A composition according to any one of claims 57 and 66 to 72, wherein the antigen-binding domain that binds to TRBC binds to TRBC2, and the antigen-binding domain that binds to TRBC comprises a VL comprising any one sequence selected from the group consisting of VL sequences listed in Tables 11, 15, 17, 39, and 40. Claim 74 A composition according to any one of claims 57 and 66 to 73, wherein the antigen-binding domain that binds to TRBC binds to TRBC2, and the antigen-binding domain that binds to TRBC comprises a sequence having at least 75% sequence identity with any one sequence selected from the group consisting of scFv sequences listed in Tables 12, 15, 17, 39, and 40. Claim 75 A composition comprising a VL according to any one of claims 57 and 66 to 74, wherein the antigen-binding domain that binds to TRBC binds to TRBC2, and the antigen-binding domain that binds to TRBC comprises any one sequence selected from the group consisting of scFv sequences listed in Tables 12, 15, 17, 39, and 40. Claim 76 A composition according to any one of claims 21 to 75, wherein the formulation comprises a second antigen-binding domain that binds to NKp30. Claim 77 A composition according to any one of claims 23 to 76, wherein the antigen-binding domain binding to NKp30 and the second antigen-binding domain are identical. Claim 78 A composition according to any one of claims 23 to 76, wherein the antigen-binding domain binding to NKp30 and the second antigen-binding domain are different. Claim 79 A composition according to any one of claims 23 to 77, wherein the second antigen-binding domain binding to NKp30 comprises a heavy chain variable region (VH) comprising: (i) a heavy chain complementarity determining region 3 (VHCDR3) comprising the sequence of GDWHYFDY (SEQ No. 504); and (ii) a heavy chain complementarity determining region 1 (VHCDR1) comprising the sequence of GFSIIRVGYHWN (SEQ No. 502), a heavy chain complementarity determining region 2 (VHCDR2) comprising the sequence of YIHRSGSTSYNPSLKS (SEQ No. 503), or a combination thereof. Claim 80 A composition according to any one of claims 23 to 77 and 79, wherein the second antigen-binding domain that binds to NKp30 comprises VH including VHCDR1 having the sequence GFSIIRVGYHWN (SEQ No. 502), VHCDR2 having the sequence YIHRSGSTSYNPSLKS (SEQ No. 503), and VHCDR3 having the sequence GDWHYFDY (SEQ No. 504). Claim 81 A composition according to any one of claims 23 to 77, 79, and 80, wherein the second antigen-binding domain that binds to NKp30 comprises a light chain complementarity determining region 1 (VLCDR1) comprising the sequence of SGEKLSDKYVH (SEQ No. 26), a light chain complementarity determining region 2 (VLCDR2) comprising the sequence of ENDRRPS (SEQ No. 28), a light chain complementarity determining region 3 (VLCDR3) comprising the sequence of QFWDSTNSAV (SEQ No. 86), or any combination thereof. Claim 82 A composition according to any one of claims 23 to 77 and 79 to 81, wherein the second antigen-binding domain that binds to NKp30 comprises VL comprising VLCDR1 having the sequence of SGEKLSDKYVH (SEQ No. 26), VLCDR2 having the sequence of ENDRRPS (SEQ No. 28), and VLCDR3 having the sequence of QFWDSTNSAV (SEQ No. 86). Claim 83 A composition according to any one of claims 23 to 77 and claims 79 to 82, wherein the second antigen-binding domain that binds to NKp30 comprises a VH having at least 75% sequence identity with the sequence of SEQ ID NO.

505. Claim 84 A composition according to any one of claims 23 to 77 and claims 79 to 83, wherein the second antigen-binding domain that binds to NKp30 comprises a VH comprising the sequence of SEQ ID NO.

505. Claim 85 A composition according to any one of claims 23 to 77 and claims 79 to 84, wherein the second antigen-binding domain that binds to NKp30 comprises a VL having a sequence having at least 75% sequence identity with the sequence of SEQ ID NO.

13. Claim 86 A composition according to any one of claims 23 to 77 and 79 to 85, wherein the second antigen-binding domain that binds to NKp30 comprises a VL having the sequence of SEQ ID NO.

13. Claim 87 A composition according to any one of claims 23 to 77 and 79 to 86, wherein the second antigen-binding domain that binds to NKp30 comprises a VH having at least 75% sequence identity with the sequence of SEQ ID NO. 505 and a VL having at least 75% sequence identity with the sequence of SEQ ID NO.

13. Claim 88 A composition according to any one of claims 23 to 77 and 79 to 87, wherein the second antigen-binding domain that binds to NKp30 comprises VH having the sequence of SEQ ID NO. 505 and VL having the sequence of SEQ ID NO.

13. Claim 89 A composition according to any one of claims 23 to 76 and 78, wherein the second antigen-binding domain binding to NKp30 comprises a heavy chain variable region (VH) comprising: (i) a VHCDR1 having at least 75% sequence identity with any one sequence selected from the group consisting of VHCDR1 sequences listed in Tables 20A, 20B, 21A, 21B, 23A, 23B, 25, 26, 16, and 17; (ii) a VHCDR2 having at least 75% sequence identity with any one sequence selected from the group consisting of VHCDR2 sequences listed in Tables 20A, 20B, 21A, 21B, 23A, 23B, 25, 26, 16, and 17; (iii) Tables 20A, 20B, 21A, 21B, A VHCDR3 comprising a sequence having at least 75% sequence identity with any one sequence selected from the group consisting of the VHCDR3 sequences listed in 23A, 23B, 25, 26, 16, and 17; or (iv) any combination thereof. Claim 90 A composition according to any one of claims 23 to 76, 78, and 89, wherein the second antigen-binding domain binding to NKp30 comprises a VH comprising the following: (i) a VHCDR1 comprising any one sequence selected from the group consisting of VHCDR1 sequences listed in Tables 20A, 20B, 21A, 21B, 23A, 23B, 25, 26, 16, and 17; (ii) a VHCDR2 comprising any one sequence selected from the group consisting of VHCDR2 sequences listed in Tables 20A, 20B, 21A, 21B, 23A, 23B, 25, 26, 16, and 17; (iii) Tables 20A, 20B, 21A, 21B, 23A, 23B, 25, 26, 16, and A VHCDR3 comprising any one sequence selected from the group consisting of the VHCDR3 sequences listed in 17; or (iv) any combination thereof. Claim 91 A composition according to any one of claims 23 to 76, 78, 89, and 90, wherein the second antigen-binding domain that binds to NKp30 comprises a VH having at least 75% sequence identity with any one sequence selected from the group consisting of VH sequences listed in Tables 25, 26, 16, and 17. Claim 92 A composition according to any one of claims 23 to 76, 78, and 89 to 91, wherein the second antigen-binding domain binding to NKp30 comprises a VH comprising any one sequence selected from the group consisting of VH sequences listed in Tables 25, 26, 16, and 17. Claim 93 A composition according to any one of claims 23 to 76, 78, and 89 to 92, wherein the second antigen-binding domain binding to NKp30 comprises a VL comprising the following: (i) a VLCDR1 comprising a sequence having at least 75% sequence identity with any one sequence selected from the group consisting of VLCDR1 sequences listed in Tables 22, 24, 25, 26, 16, and 17; (ii) a VLCDR2 comprising a sequence having at least 75% sequence identity with any one sequence selected from the group consisting of VLCDR2 sequences listed in Tables 22, 24, 25, 26, 16, and 17; (iii) a VLCDR3 comprising a sequence having at least 75% sequence identity with any one sequence selected from the group consisting of VLCDR3 sequences listed in Tables 22, 24, 25, 26, 16, and 17; or (iv) any combination of these. Claim 94 A composition according to any one of claims 23 to 76, 78, and 89 to 93, wherein the second antigen-binding domain binding to NKp30 comprises a VL comprising: (i) a VLCDR1 comprising any one sequence selected from the group consisting of VLCDR1 sequences listed in Tables 22, 24, 25, 26, 16, and 17; (ii) a VLCDR2 comprising any one sequence selected from the group consisting of VLCDR2 sequences listed in Tables 22, 24, 25, 26, 16, and 17; (iii) a VLCDR3 comprising any one sequence selected from the group consisting of VLCDR3 sequences listed in Tables 22, 24, 25, 26, 16, and 17; or (iv) any combination thereof. Claim 95 A composition according to any one of claims 23 to 76, 78, and 89 to 94, wherein the second antigen-binding domain that binds to NKp30 comprises a VL having at least 75% sequence identity with any one sequence selected from the group consisting of VL sequences listed in Tables 25, 26, 16, and 17. Claim 96 A composition according to any one of claims 23 to 76, 78, and 89 to 95, wherein the second antigen-binding domain that binds to NKp30 comprises a VL comprising any one sequence selected from the group consisting of VL sequences listed in Tables 25, 26, 16, and 17. Claim 97 A composition according to any one of claims 21 to 96, wherein the preparation comprises an immune cell binder. Claim 98 A composition according to any one of claims 23 to 97, wherein the immune cell binder is selected from the group consisting of T cell binders, NK cell binders, B cell binders, dendritic cell binders, and macrophage cell binders. Claim 99 A composition according to any one of claims 21 to 98, wherein the preparation comprises a T cell binder. Claim 100 A composition according to claim 98 or 99, wherein the T cell binder comprises an antigen-binding domain that binds to a T cell receptor beta chain region (TCRβV). Claim 101 A composition according to claim 100, wherein the antigen-binding domain binding to the T cell receptor beta chain region (TCRβV) binds to the TCRβV of any one T cell receptor beta chain (TCRβ) subfamily member selected from the group consisting of TCRβ subfamily members listed in Tables 29 and 42A. Claim 102 A composition according to claim 100 or 101, wherein the antigen-binding domain binding to TCRβV comprises a heavy chain variable region (VH) comprising: (i) a VHCDR1 having at least 75% sequence identity with any one sequence selected from the group consisting of VHCDR1 sequences listed in Tables 30, 32, 42B, 42C, 43A, 43B, and 43–51; (ii) a VHCDR2 having at least 75% sequence identity with any one sequence selected from the group consisting of VHCDR2 sequences listed in Tables 30, 32, 42B, 42C, 43A, 43B, and 43–51; (iii) any one sequence selected from the group consisting of VHCDR3 sequences listed in Tables 30, 32, 42B, 42C, 43A, 43B, and 43–51 and at VHCDR3 comprising a sequence having 75% sequence identity; or (iv) any combination thereof. Claim 103 A composition according to any one of claims 100 to 102, wherein the antigen-binding domain binding to TCRβV comprises a VH comprising the following: (i) a VHCDR1 comprising any one sequence selected from the group consisting of VHCDR1 sequences listed in Tables 30, 32, 42B, 42C, 43A, 43B, and 43–51; (ii) a VHCDR2 comprising any one sequence selected from the group consisting of VHCDR2 sequences listed in Tables 30, 32, 42B, 42C, 43A, 43B, and 43–51; (iii) a VHCDR3 comprising any one sequence selected from the group consisting of VHCDR3 sequences listed in Tables 30, 32, 42B, 42C, 43A, 43B, and 43–51; or (iv) any combination thereof. Claim 104 A composition according to any one of claims 100 to 103, wherein the antigen-binding domain binding to TCRβV comprises a VH having at least 75% sequence identity with any one sequence selected from the group consisting of VH sequences listed in Tables 30, 32, 42B, 42C, 43A, 43B, and 43 to 51. Claim 105 A composition according to any one of claims 100 to 104, wherein the antigen-binding domain binding to TCRβV comprises a VH comprising any one sequence selected from the group consisting of VH sequences listed in Tables 30, 32, 42B, 42C, 43A, 43B, and 43 to 51. Claim 106 A composition according to any one of claims 100 to 105, wherein the antigen-binding domain binding to TCRβV comprises a VL comprising the following: (i) a VLCDR1 having at least 75% sequence identity with any one sequence selected from the group consisting of VLCDR1 sequences listed in Tables 30, 32, 42B, 42C, 43A, 43B, and 43–51; (ii) a VLCDR2 having at least 75% sequence identity with any one sequence selected from the group consisting of VLCDR2 sequences listed in Tables 30, 32, 42B, 42C, 43A, 43B, and 43–51; (iii) any one sequence selected from the group consisting of VLCDR3 sequences listed in Tables 30, 32, 42B, 42C, 43A, 43B, and 43–51 and at VLCDR3 comprising a sequence having 75% sequence identity; or (iv) any combination thereof. Claim 107 A composition according to any one of claims 100 to 106, wherein the antigen-binding domain binding to TCRβV comprises a VL comprising: (i) a VLCDR1 comprising any one sequence selected from the group consisting of VLCDR1 sequences listed in Tables 30, 32, 42B, 42C, 43A, 43B, and 43–51; (ii) a VLCDR2 comprising any one sequence selected from the group consisting of VLCDR2 sequences listed in Tables 30, 32, 42B, 42C, 43A, 43B, and 43–51; (iii) a VLCDR3 comprising any one sequence selected from the group consisting of VLCDR3 sequences listed in Tables 30, 32, 42B, 42C, 43A, 43B, and 43–51; or (iv) any combination thereof. Claim 108 A composition according to any one of claims 100 to 107, wherein the antigen-binding domain that binds to TCRβV comprises a VL having at least 75% sequence identity with any one sequence selected from the group consisting of VL sequences listed in Tables 30, 32, 42B, 42C, 43A, 43B, and 43 to 51. Claim 109 A composition according to any one of claims 100 to 108, wherein the antigen-binding domain that binds to TCRβV comprises a VL comprising any one sequence selected from the group consisting of VL sequences listed in Tables 30, 32, 42B, 42C, 43A, 43B, and 43 to 51. Claim 110 A composition according to any one of claims 21 to 98, wherein the preparation comprises an NK cell binder. Claim 111 A composition according to claim 98 or 110, wherein the NK cell binder does not bind to NKp30. Claim 112 A composition according to any one of claims 98, 110, and 111, wherein the NK cell binder comprises a sequence having at least 75% sequence identity with any one sequence selected from the group consisting of sequences listed in Table 27. Claim 113 A composition according to any one of claims 98 and 110 to 112, wherein the NK cell binder comprises any one sequence selected from the group consisting of the sequences listed in Table 27. Claim 114 A composition according to any one of claims 21 to 113, wherein the formulation comprises a tumor-targeting moiety. Claim 115 A composition according to any one of claims 23 to 114, wherein the antigen-binding domain binding to TRBC, the second antigen-binding domain binding to NKp30, the immune cell binder, the tumor-targeting moiety, or any combination thereof is a whole antibody or an antigen-binding fragment thereof, a divalent antibody, a bispecific antibody, or a biparatope antibody. Claim 116 A composition according to any one of claims 23 to 115, wherein the antigen-binding domain binding to TRBC, the second antigen-binding domain binding to NKp30, the immune cell binder, the tumor-targeting moiety, or any combination thereof is Fab, F(ab')2, Fv, short-chain Fv(scFv), Diabadi, a single-domain antibody, or a camelid antibody. Claim 117 A composition according to any one of claims 23 to 116, wherein the antigen-binding domain binding to TRBC, the second antigen-binding domain binding to NKp30, the immune cell binder, the tumor-targeting moiety, or any combination thereof is Fab or scFv. Claim 118 A composition according to any one of claims 23 to 117, wherein the antigen-binding domain binding to TRBC, the second antigen-binding domain binding to NKp30, the immune cell binder, the tumor-targeting moiety, or any combination thereof comprises one or more heavy-chain constant regions selected from the group consisting of IgG1 heavy-chain constant region or a functional fragment thereof, IgG2 heavy-chain constant region or a functional fragment thereof, IgG3 heavy-chain constant region or a functional fragment thereof, IgGA1 heavy-chain constant region or a functional fragment thereof, IgGA2 heavy-chain constant region or a functional fragment thereof, IgG4 heavy-chain constant region or a functional fragment thereof, IgJ heavy-chain constant region or a functional fragment thereof, IgM heavy-chain constant region or a functional fragment thereof, IgD heavy-chain constant region or a functional fragment thereof, and IgE heavy-chain constant region or a functional fragment thereof. Claim 119 A composition according to claim 118, wherein one or more heavy chain invariant regions comprises a sequence having at least 75% sequence identity with any one of the heavy chain invariant region sequences listed in Tables 3, 7, 8, 16, 17, 39, 40, 42C, 43A, 43B, and 46 to 51. Claim 120 In claim 118 or 119, the one or more heavy chain invariant regions have at least 75% sequence identity with the sequence of SEQ ID NO. 73, the sequence of SEQ ID NO. 76, the sequence of SEQ ID NO. 74, the sequence of SEQ ID NO. 75, the sequence of SEQ ID NO. 76, the sequence of SEQ ID NO. 40, the sequence of SEQ ID NO. 41, the sequence of SEQ ID NO. 42, the sequence of SEQ ID NO. 3645, the sequence of SEQ ID NO. 3646, the sequence of SEQ ID NO. 3649, the sequence of SEQ ID NO. 3451, the sequence of SEQ ID NO. 506, the sequence of SEQ ID NO. 507, the sequence of SEQ ID NO. 3647, the sequence of SEQ ID NO. 3648, the sequence of SEQ ID NO. 508 or 8686, the sequence of SEQ ID NO. 513, the sequence of SEQ ID NO. 514, the sequence of SEQ ID NO. 515, the sequence of SEQ ID NO. 516, the sequence of SEQ ID NO. 3447, or the sequence of SEQ ID NO.

511. A composition comprising a sequence. Claim 121 A composition according to any one of claims 118 to 120, wherein the antigen-binding domain binding to TRBC, the second antigen-binding domain binding to NKp30, the immune cell binder, the tumor-targeting moiety, or any combination thereof comprises a kappa light chain constant region or a functional fragment thereof, a lambda light chain constant region or a functional fragment thereof, or a combination thereof. Claim 122 A composition according to claim 121, wherein the kappa light chain invariant region comprises a sequence having at least 75% sequence identity with any one of the light chain invariant region sequences listed in Tables 3, 7, 8, 16, 17, 39, 40, 42C, 43A, 43B, and 46 to 51. Claim 123 A composition according to claim 121 or 122, wherein the kappa light chain invariant region comprises a sequence having at least 75% sequence identity with the sequence of SEQ ID NO. 39 or the sequence of SEQ ID NO. 3644. Claim 124 A composition according to any one of claims 24 to 123, wherein the first member of the dimerization module comprises a first Fc region and the second member of the dimerization module comprises a second Fc region. Claim 125 A composition according to claim 124, wherein the first Fc region or the second Fc region comprises N297. Claim 126 A composition according to claim 124, wherein the first Fc region is selected from the group consisting of IgG1 Fc region or a functional fragment thereof, IgG2 Fc region or a functional fragment thereof, IgG3 Fc region or a functional fragment thereof, IgGA1 Fc region or a functional fragment thereof, IgGA2 Fc region or a functional fragment thereof, IgG4 Fc region or a functional fragment thereof, IgJ Fc region or a functional fragment thereof, IgM Fc region or a functional fragment thereof, IgD Fc region or a functional fragment thereof, and IgE Fc region or a functional fragment thereof. Claim 127 A composition according to claim 124 or 126, wherein the second Fc region is selected from the group consisting of IgG1 Fc region or a functional fragment thereof, IgG2 Fc region or a functional fragment thereof, IgG3 Fc region or a functional fragment thereof, IgGA1 Fc region or a functional fragment thereof, IgGA2 Fc region or a functional fragment thereof, IgG4 Fc region or a functional fragment thereof, IgJ Fc region or a functional fragment thereof, IgM Fc region or a functional fragment thereof, IgD Fc region or a functional fragment thereof, and IgE Fc region or a functional fragment thereof. Claim 128 A composition according to any one of claims 124 to 127, wherein the first Fc region, the second Fc region, or a combination thereof comprises an Fc interface having one or more of a paired cavity-protrusion, electrostatic interaction, or strand-exchange, and the dimerization of the first Fc region and the second Fc region is enhanced as indicated in a heteropolymer:homogeneous polymer form at a greater ratio compared to the dimerization of the Fc region having an unmanipulated interface. Claim 129 In claim 128, (i) the first Fc region comprises: (a) Ala at position 297 of the heavy chain invariant region according to EU numbering, (b) Cys at position 349 of the heavy chain invariant region according to EU numbering, (c) Ser at position 366 of the heavy chain invariant region according to EU numbering, (d) Ala at position 368 of the heavy chain invariant region according to EU numbering; and (e) Val at position 407 of the heavy chain invariant region according to EU numbering; and (ii) the second Fc region comprises: (a) Ala at position 297 of the heavy chain invariant region according to EU numbering, (b) Cys at position 354 of the heavy chain invariant region according to EU numbering, and (c) Trp at position 366 of the heavy chain invariant region according to EU numbering. Claim 130 In claim 128, (i) the first Fc region comprises: (a) Ala at position 297 of the heavy chain invariant region according to EU numbering, (b) Cys at position 354 of the heavy chain invariant region according to EU numbering, and (c) Trp at position 366 of the heavy chain invariant region according to EU numbering; and (ii) the second Fc region comprises: (a) Ala at position 297 of the heavy chain invariant region according to EU numbering, (b) Cys at position 349 of the heavy chain invariant region according to EU numbering, (c) Ser at position 366 of the heavy chain invariant region according to EU numbering, (d) Ala at position 368 of the heavy chain invariant region according to EU numbering; and (e) Val at position 407 of the heavy chain invariant region according to EU numbering. Claim 131 A composition according to any one of claims 124 to 130, wherein the first Fc region comprises a mutation that reduces Fc receptor binding to said preparation compared to a preparation without mutation. Claim 132 A composition according to any one of claims 124 to 131, wherein the first Fc region comprises an Asn297Ala (N297A) mutation or a Leu234Ala / Leu235Ala (LALA) mutation according to EU numbering, or a combination thereof. Claim 133 A composition according to any one of claims 124 to 132, wherein the second Fc region comprises a mutation that reduces Fc receptor binding to said preparation compared to said preparation without mutation. Claim 134 A composition according to any one of claims 124 to 133, wherein the second Fc region comprises an Asn297Ala (N297A) mutation or a Leu234Ala / Leu235Ala (LALA) mutation according to EU numbering, or a combination thereof. Claim 135 In any one of claims 124 to 134, the first Fc region is the sequence of SEQ ID NO. 73, the sequence of SEQ ID NO. 76, the sequence of SEQ ID NO. 74, the sequence of SEQ ID NO. 75, the sequence of SEQ ID NO. 40, the sequence of SEQ ID NO. 41, the sequence of SEQ ID NO. 42, the sequence of SEQ ID NO. 3645, the sequence of SEQ ID NO. 3646, the sequence of SEQ ID NO. 3649, the sequence of SEQ ID NO. 8687, the sequence of SEQ ID NO. 506, the sequence of SEQ ID NO. 507, the sequence of SEQ ID NO. 3647, the sequence of SEQ ID NO. 3648, the sequence of SEQ ID NO. 508 or 8686, the sequence of SEQ ID NO. 513, the sequence of SEQ ID NO. 514, the sequence of SEQ ID NO. 515, the sequence of SEQ ID NO. 516, the sequence of SEQ ID NO. 3447, the sequence of SEQ ID NO. 511, the sequence of SEQ ID NO. 4663, and the sequence of SEQ ID NO. 4664 A composition comprising a sequence having at least 75% sequence identity with any one sequence selected from the group consisting of the sequence of SEQ ID NO. 4665, the sequence of SEQ ID NO. 4666, the sequence of SEQ ID NO. 4667, the sequence of SEQ ID NO. 4668, the sequence of SEQ ID NO. 4669, and the sequence of SEQ ID NO. 4670. Claim 136 In any one of claims 124 to 135, the second Fc region is the sequence of SEQ ID NO. 73, the sequence of SEQ ID NO. 76, the sequence of SEQ ID NO. 74, the sequence of SEQ ID NO. 75, the sequence of SEQ ID NO. 40, the sequence of SEQ ID NO. 41, the sequence of SEQ ID NO. 42, the sequence of SEQ ID NO. 3645, the sequence of SEQ ID NO. 3646, the sequence of SEQ ID NO. 3649, the sequence of SEQ ID NO. 8687, the sequence of SEQ ID NO. 506, the sequence of SEQ ID NO. 507, the sequence of SEQ ID NO. 3647, the sequence of SEQ ID NO. 3648, the sequence of SEQ ID NO. 508 or 8686, the sequence of SEQ ID NO. 513, the sequence of SEQ ID NO. 514, the sequence of SEQ ID NO. 515, the sequence of SEQ ID NO. 516, the sequence of SEQ ID NO. 3447, the sequence of SEQ ID NO. 511, the sequence of SEQ ID NO. 4663, and the sequence of SEQ ID NO. 4664 A composition comprising a sequence having at least 75% sequence identity with any one sequence selected from the group consisting of the sequence of SEQ ID NO. 4665, the sequence of SEQ ID NO. 4666, the sequence of SEQ ID NO. 4667, the sequence of SEQ ID NO. 4668, the sequence of SEQ ID NO. 4669, and the sequence of SEQ ID NO. 4670. Claim 137 In any one of claims 124 to 136, the first Fc region is the sequence of SEQ ID NO. 73, the sequence of SEQ ID NO. 76, the sequence of SEQ ID NO. 74, the sequence of SEQ ID NO. 75, the sequence of SEQ ID NO. 40, the sequence of SEQ ID NO. 41, the sequence of SEQ ID NO. 42, the sequence of SEQ ID NO. 3645, the sequence of SEQ ID NO. 3646, the sequence of SEQ ID NO. 3649, the sequence of SEQ ID NO. 8687, the sequence of SEQ ID NO. 506, the sequence of SEQ ID NO. 507, the sequence of SEQ ID NO. 3647, the sequence of SEQ ID NO. 3648, the sequence of SEQ ID NO. 508 or 8686, the sequence of SEQ ID NO. 513, the sequence of SEQ ID NO. 514, the sequence of SEQ ID NO. 515, the sequence of SEQ ID NO. 516, the sequence of SEQ ID NO. 3447, the sequence of SEQ ID NO. 511, the sequence of SEQ ID NO. 4663, and the sequence of SEQ ID NO. 4664 A composition comprising any one sequence selected from the group consisting of the sequence of SEQ ID NO. 4665, the sequence of SEQ ID NO. 4666, the sequence of SEQ ID NO. 4667, the sequence of SEQ ID NO. 4668, the sequence of SEQ ID NO. 4669, and the sequence of SEQ ID NO. 4670. Claim 138 In any one of claims 124 to 137, the second Fc region is the sequence of SEQ ID NO. 73, the sequence of SEQ ID NO. 76, the sequence of SEQ ID NO. 74, the sequence of SEQ ID NO. 75, the sequence of SEQ ID NO. 40, the sequence of SEQ ID NO. 41, the sequence of SEQ ID NO. 42, the sequence of SEQ ID NO. 3645, the sequence of SEQ ID NO. 3646, the sequence of SEQ ID NO. 3649, the sequence of SEQ ID NO. 8687, the sequence of SEQ ID NO. 506, the sequence of SEQ ID NO. 507, the sequence of SEQ ID NO. 3647, the sequence of SEQ ID NO. 3648, the sequence of SEQ ID NO. 508 or 8686, the sequence of SEQ ID NO. 513, the sequence of SEQ ID NO. 514, the sequence of SEQ ID NO. 515, the sequence of SEQ ID NO. 516, the sequence of SEQ ID NO. 3447, the sequence of SEQ ID NO. 511, the sequence of SEQ ID NO. 4663, and the sequence of SEQ ID NO. 4664 A composition comprising any one sequence selected from the group consisting of the sequence of SEQ ID NO. 4665, the sequence of SEQ ID NO. 4666, the sequence of SEQ ID NO. 4667, the sequence of SEQ ID NO. 4668, the sequence of SEQ ID NO. 4669, and the sequence of SEQ ID NO. 4670. Claim 139 A composition according to any one of claims 124 to 136, wherein the first Fc region comprises a sequence having at least 75% sequence identity with any one sequence selected from the group consisting of the sequence of SEQ ID NO. 3648, the sequence of SEQ ID NO. 508 or 8686, the sequence of SEQ ID NO. 506, the sequence of SEQ ID NO. 507, the sequence of SEQ ID NO. 3477, the sequence of SEQ ID NO. 511, the sequence of SEQ ID NO. 515, and the sequence of SEQ ID NO.

516. Claim 140 A composition according to any one of claims 124 to 136 and 139, wherein the second Fc region comprises a sequence having at least 75% sequence identity with the sequence of SEQ ID NO. 3648, the sequence of SEQ ID NO. 508 or 8686, the sequence of SEQ ID NO. 506, the sequence of SEQ ID NO. 507, the sequence of SEQ ID NO. 3477, the sequence of SEQ ID NO. 511, the sequence of SEQ ID NO. 515, or the sequence of SEQ ID NO.

516. Claim 141 A composition according to any one of claims 124 to 136, 139, and 140, wherein the first Fc region comprises any one sequence selected from the group consisting of the sequences of SEQ ID NO. 3648, SEQ ID NO. 508, SEQ ID NO. 506, SEQ ID NO. 507, SEQ ID NO. 3477, SEQ ID NO. 511, SEQ ID NO. 515, or SEQ ID NO.

516. Claim 142 A composition according to any one of claims 124 to 136 and 139 to 141, wherein the second Fc region comprises any one sequence selected from the group consisting of the sequences of SEQ ID NO. 3648, SEQ ID NO. 508, SEQ ID NO. 506, SEQ ID NO. 507, SEQ ID NO. 3477, SEQ ID NO. 511, SEQ ID NO. 515, or SEQ ID NO.

516. Claim 143 A composition according to any one of claims 124 to 142, wherein (i) the first Fc region comprises the sequence of SEQ ID NO. 506 or the sequence of SEQ ID NO. 507, and the second Fc region comprises the sequence of SEQ ID NO. 3648 or the sequence of SEQ ID NO. 508 or 8686; or (ii) the first Fc region comprises the sequence of SEQ ID NO. 3447 or the sequence of SEQ ID NO. 511, and the second Fc region comprises the sequence of SEQ ID NO. 515 or the sequence of SEQ ID NO.

516. Claim 144 A composition according to any one of claims 124 to 142, wherein (i) the first Fc region comprises the sequence of SEQ ID NO. 3648 or the sequence of SEQ ID NO. 508 or 8686, and the second Fc region comprises the sequence of SEQ ID NO. 506 or the sequence of SEQ ID NO. 507; or (ii) the first Fc region comprises the sequence of SEQ ID NO. 515 or the sequence of SEQ ID NO. 516, and the second Fc region comprises the sequence of SEQ ID NO. 3447 or the sequence of SEQ ID NO.

511. Claim 145 A composition according to any one of claims 1 to 144, wherein the formulation comprises a first polypeptide, a second polypeptide, and at least one cytokine molecule or a functional fragment or variant thereof, wherein the first polypeptide and the second polypeptide are not adjacent; wherein: (i) the first polypeptide comprises (a) a first member of a dimerization module connected to an antigen-binding domain comprising VH and VL, or (b) a first member of a dimerization module connected to a first portion of an antigen-binding domain comprising VH, wherein the formulation further comprises a third polypeptide comprising a second portion of an antigen-binding domain comprising VL, and the third polypeptide is not adjacent to the first polypeptide and the second polypeptide; and (ii) the second polypeptide comprises a second member of a dimerization molecule, wherein the at least one cytokine molecule or a functional fragment or variant thereof is covalently connected to the second polypeptide or the first polypeptide. Claim 146 A composition according to any one of claims 1 to 145, wherein the formulation comprises a first polypeptide, a second polypeptide, and at least one cytokine molecule or a functional fragment or variant thereof, wherein the first polypeptide and the second polypeptide are not adjacent; wherein: (i) the first polypeptide comprises a first member of a dimerization module connected to a first portion of an antigen-binding domain comprising VH, and the formulation further comprises a third polypeptide comprising a second portion of an antigen-binding domain comprising VL, wherein the third polypeptide is not adjacent to the first polypeptide and the second polypeptide; and (ii) the second polypeptide comprises a second member of a dimerization molecule, wherein the at least one cytokine molecule or a functional fragment or variant thereof is covalently connected to the second polypeptide. Claim 147 A composition according to claim 146, wherein at least one cytokine molecule or a functional fragment or variant thereof is covalently connected to the N-terminus of the second polypeptide, the C-terminus of the second polypeptide, or a combination thereof. Claim 148 A composition according to claim 146 or 147, wherein at least one cytokine molecule or a functional fragment or variant thereof is present within a single continuous polypeptide chain of the second polypeptide. Claim 149 In any one of claims 145 to 148, the first polypeptide comprises the following composition: (i) an antigen-binding domain comprising VH and VL, wherein the antigen-binding domain further comprises a heavy chain constant domain 1 (CH1) connected to VH; or (ii) a first portion of the first antigen-binding domain comprising VH, wherein the first portion of the antigen-binding domain further comprises CH1 connected to VH. Claim 150 In claim 149, the composition wherein CH1 is connected to the C-terminus of VH. Claim 151 In any one of claims 145 to 150, the formulation comprises the following: a composition comprising: (i) a first polypeptide comprising an antigen-binding domain comprising VH and VL, wherein the antigen-binding domain further comprises a light chain constant domain (CL) connected to VL; or (ii) a first polypeptide comprising a first portion of the antigen-binding domain and a third polypeptide comprising a second portion of the antigen-binding domain, wherein the second portion of the antigen-binding domain further comprises a CL connected to VL. Claim 152 A composition according to claim 151, wherein the CL is connected to the C-terminal of the VL. Claim 153 A composition according to any one of claims 145 to 152, wherein the first member of the dimerization module is connected to the C-terminus of the antigen-binding domain comprising VH and VL, or to the C-terminus of the first portion of the first antigen-binding domain comprising VH. Claim 154 In any one of claims 145 to 153, the composition wherein the second polypeptide comprises: (i) an antigen-binding domain that binds to TRBC, a second antigen-binding domain that binds to NKp30, an immune cell binder, or a tumor-targeting moiety comprising VH and VL, further comprising a heavy chain constant domain 1 (CH1) connected to VH; or (ii) a tumor-targeting moiety comprising a first portion of an antigen-binding domain that binds to TRBC, a second antigen-binding domain that binds to NKp30, an immune cell binder, or VH, further comprising a first portion of an antigen-binding domain that binds to TRBC, a second antigen-binding domain that binds to NKp30, an immune cell binder, or a tumor-targeting moiety further comprising CH1 connected to VH. Claim 155 In claim 154, the composition wherein CH1 is connected to the C-terminus of VH. Claim 156 In any one of claims 145 to 155, the formulation comprises the following: a composition comprising: (i) a second polypeptide comprising an antigen-binding domain that binds to TRBC, a second antigen-binding domain that binds to NKp30, an immune cell binder, or a tumor-targeting moiety comprising VH and VL, wherein the antigen-binding domain that binds to TRBC, the second antigen-binding domain that binds to NKp30, the immune cell binder, or the tumor-targeting moiety further comprises a light chain constant domain (CL) connected to VL; or (ii) a second polypeptide comprising a first portion of an antigen-binding domain that binds to TRBC, a second antigen-binding domain that binds to NKp30, an immune cell binder, or a tumor-targeting moiety, wherein the preparation further comprises a fourth polypeptide comprising a second portion of the antigen-binding domain that binds to TRBC, the second antigen-binding domain that binds to NKp30, the immune cell binder, or the tumor-targeting moiety, wherein the fourth polypeptide is not adjacent to the first polypeptide, the second polypeptide, and the third polypeptide, and the second portion of the antigen-binding domain that binds to TRBC, the second antigen-binding domain that binds to NKp30, the immune cell binder, or the tumor-targeting moiety further comprises a CL connected to VL. Claim 157 In paragraph 156, the composition wherein the CL is connected to the C-terminus of the VL. Claim 158 A composition according to any one of claims 154 to 157, wherein the second member of the dimerization module is connected to the C-terminus of the tumor targeting moiety comprising the antigen binding domain that binds to TRBC, the second antigen binding domain that binds to NKp30, the immune cell binder, or VH and VL, or is connected to the C-terminus of the tumor targeting moiety comprising the first portion of the antigen binding domain that binds to TRBC, the second antigen binding domain that binds to NKp30, the immune cell binder, or VH. Claim 159 A composition according to any one of claims 145 to 158, wherein the formulation further comprises: (i) a linker between a first member of the dimerization module and the antigen-binding domain comprising VH and VL or a first portion of the antigen-binding domain comprising VH; (ii) a linker between a second member of the dimerization module and the antigen-binding domain binding to TRBC, the second antigen-binding domain binding to NKp30, the immune cell binder, or the tumor-targeting moiety comprising VH and VL, or the first portion of the antigen-binding domain binding to TRBC, the second antigen-binding domain binding to NKp30, the immune cell binder, or the tumor-targeting moiety comprising VH; (iii) a linker between VH and VL of the antigen-binding domain; (iv) a linker between CH1 and VH of the antigen-binding domain; (v) a linker between CL and VL of the antigen-binding domain; (vi) the antigen-binding domain binding to TRBC, (vii) the second antigen-binding domain binding to NKp30, the immune cell binder, or a linker between VH and VL of the tumor-targeting moiety; (viii) the antigen-binding domain binding to TRBC, the second antigen-binding domain binding to NKp30, the immune cell binder, or a linker between CH1 and VH of the tumor-targeting moiety; (viii) the antigen-binding domain binding to TRBC, the second antigen-binding domain binding to NKp30, the immune cell binder, or a linker between CL and VL of the tumor-targeting moiety; (ix) a linker between the at least one cytokine molecule or its functional fragment or variant and the first polypeptide, a linker between the at least one cytokine molecule or its functional fragment or variant and the second polypeptide, or any combination thereof; or (x) any combination thereof. Claim 160 A composition according to claim 159, wherein the linker is selected from the group consisting of a cutting linker, a non-cutting linker, a peptide linker, a flexible linker, a rigid linker, a helical linker, and a non-helical linker. Claim 161 A composition according to claim 160, wherein the linker is a peptide linker comprising the sequence of SEQ ID NO. 3473, SEQ ID NO. 3308, or SEQ ID NO. 8291. Claim 162 A composition according to any one of claims 145 to 161, wherein at least one cytokine molecule or a functional fragment or variant thereof is connected to the N-terminus of the first polypeptide, the C-terminus of the first polypeptide, the N-terminus of the second polypeptide, the C-terminus of the second polypeptide, the N-terminus of the third polypeptide, the C-terminus of the third polypeptide, or any combination thereof. Claim 163 A composition according to claim 162, wherein the at least one cytokine molecule or a functional fragment or variant thereof is located within a single continuous polypeptide chain of the first polypeptide, the second polypeptide, or the third cytokine polypeptide to which the at least one cytokine molecule or a functional fragment or variant thereof is connected. Claim 164 In any one of claims 145 to 163, the formulation comprises a composition including: (i) a first polypeptide comprising a first portion of a dimerization module connected to the C-terminus of a first portion of the antigen-binding domain; (ii) a second polypeptide comprising a second portion of the dimerization module; (iii) a third polypeptide comprising a second portion of the antigen-binding domain; and (iv) at least one cytokine molecule or a functional fragment or variant thereof covalently connected to the N-terminus of the second polypeptide. Claim 165 In any one of claims 24 to 164, the formulation comprises the following: a composition comprising: (i) a first polypeptide chain comprising the sequence of SEQ ID NO. 505 and the sequence of SEQ ID NO. 3649 or SEQ ID NO. 3451; (ii) a second polypeptide chain comprising the sequence of SEQ ID NO. 3472, the sequence of SEQ ID NO. 2170, and the sequence of SEQ ID NO. 3648 or SEQ ID NO. 508 or 8686; and (iii) a third polypeptide comprising the sequence of SEQ ID NO. 13 and the sequence of SEQ ID NO. 3644. Claim 166 In any one of claims 24 to 165, the formulation comprises: a composition comprising: (i) a first polypeptide chain comprising the sequence of SEQ ID NO. 505 operably linked to the sequence of SEQ ID NO. 3649 or SEQ ID NO. 3451 in the direction from the N-terminus to the C-terminus; (ii) a second polypeptide chain comprising the sequence of SEQ ID NO. 3472 operably linked to the sequence of SEQ ID NO. 2170 operably linked to the sequence of SEQ ID NO. 3648 or SEQ ID NO. 508 or 8686 in the direction from the N-terminus to the C-terminus; and (iii) a third polypeptide comprising the sequence of SEQ ID NO. 13 operably linked to the sequence of SEQ ID NO. 3644 in the direction from the N-terminus to the C-terminus. Claim 167 A composition according to claim 166, wherein the second polypeptide chain comprises, in the direction from the N-terminus to the C-terminus, the sequence of SEQ ID NO. 3472, which is operably connected to the sequence of SEQ ID NO. 2170, which is operably connected to the sequence of SEQ ID NO. 3473, which is operably connected to the sequence of SEQ ID NO. 3648, SEQ ID NO. 508, or SEQ ID NO. 8686. Claim 168 In any one of claims 24 to 167, the formulation comprises the following: (i) a first polypeptide chain comprising the sequence of SEQ ID NO. 505 and the sequence of SEQ ID NO. 3649 or SEQ ID NO. 3451; (ii) a second polypeptide chain comprising the sequence of SEQ ID NO. 3474 and the sequence of SEQ ID NO. 3648 or SEQ ID NO. 508 or 8686; and (iii) a third polypeptide comprising the sequence of SEQ ID NO. 13 and the sequence of SEQ ID NO. 3644. Claim 169 In any one of claims 24 to 168, the formulation comprises: a composition comprising: (i) a first polypeptide chain comprising the sequence of SEQ ID NO. 505 operably linked to the sequence of SEQ ID NO. 3649 or SEQ ID NO. 3451 in the direction from the N-terminus to the C-terminus; (ii) a second polypeptide chain comprising the sequence of SEQ ID NO. 3474 operably linked to the sequence of SEQ ID NO. 3648 or SEQ ID NO. 508 or 8686 in the direction from the N-terminus to the C-terminus; and (iii) a third polypeptide comprising the sequence of SEQ ID NO. 13 operably linked to the sequence of SEQ ID NO. 3644 in the direction from the N-terminus to the C-terminus. Claim 170 A composition according to claim 169, wherein the second polypeptide comprises the sequence of SEQ ID NO. 3474 operably linked to the sequence of SEQ ID NO. 3308 operably linked to the sequence of SEQ ID NO. 3648 or SEQ ID NO. 508 or 8686 in the direction from the N-terminus to the C-terminus. Claim 171 In any one of claims 24 to 170, the formulation comprises the following: (i) a first polypeptide chain comprising a sequence having at least 75% sequence identity with the sequence of SEQ ID NO. 500; (ii) a second polypeptide chain comprising a sequence having at least 75% sequence identity with the sequence of SEQ ID NO. 3420; and (iii) a third polypeptide comprising a sequence having at least 75% sequence identity with the sequence of SEQ ID NO.

501. Claim 172 In any one of claims 24 to 171, the formulation comprises the following: a composition comprising: (i) a first polypeptide chain comprising the sequence of SEQ ID NO. 500; (ii) a second polypeptide chain comprising the sequence of SEQ ID NO. 3420; and (iii) a third polypeptide comprising the sequence of SEQ ID NO.

501. Claim 173 In any one of claims 24 to 164, the formulation comprises the following: (i) a first polypeptide chain comprising the sequence of SEQ ID NO. 505 and the sequence of SEQ ID NO. 3649 or SEQ ID NO. 3451; (ii) a second polypeptide chain comprising the sequence of SEQ ID NO. 3472, the sequence of SEQ ID NO. 3670, and the sequence of SEQ ID NO. 3648 or SEQ ID NO. 508 or 8686; and (iii) a third polypeptide comprising the sequence of SEQ ID NO. 13 and the sequence of SEQ ID NO. 3644. Claim 174 In any one of claims 24 to 164 and 173, the formulation comprises: a composition comprising: (i) a first polypeptide chain comprising the sequence of SEQ ID NO. 505 operably linked to the sequence of SEQ ID NO. 3649 or SEQ ID NO. 3451 in the direction from the N-terminus to the C-terminus; (ii) a second polypeptide chain comprising the sequence of SEQ ID NO. 3472 operably linked to the sequence of SEQ ID NO. 3670 operably linked to the sequence of SEQ ID NO. 3648 or SEQ ID NO. 508 or 8686 in the direction from the N-terminus to the C-terminus; and (iii) a third polypeptide comprising the sequence of SEQ ID NO. 13 operably linked to the sequence of SEQ ID NO. 3644 in the direction from the N-terminus to the C-terminus. Claim 175 A composition according to claim 174, wherein the second polypeptide comprises, in the direction from the N-terminus to the C-terminus, the sequence of SEQ ID NO. 3472, which is operably linked to the sequence of SEQ ID NO. 3308, which is operably linked to the sequence of SEQ ID NO. 3670, which is operably linked to the sequence of SEQ ID NO. 3473, which is operably linked to the sequence of SEQ ID NO. 3648, SEQ ID NO. 508, or SEQ ID NO. 8686. Claim 176 In any one of claims 24 to 164 and 173 to 175, the formulation comprises the following: (i) a first polypeptide chain comprising the sequence of SEQ ID NO. 505 and the sequence of SEQ ID NO. 3649 or SEQ ID NO. 3451; (ii) a second polypeptide chain comprising the sequence of SEQ ID NO. 3671 and the sequence of SEQ ID NO. 3648 or SEQ ID NO. 508 or 8686; and (iii) a third polypeptide comprising the sequence of SEQ ID NO. 13 and the sequence of SEQ ID NO. 3644. Claim 177 In any one of claims 24 to 164 and 173 to 176, the formulation comprises the following: (i) a first polypeptide chain comprising the sequence of SEQ ID NO. 505 operably linked to the sequence of SEQ ID NO. 3649 or SEQ ID NO. 3451 in the direction from the N-terminus to the C-terminus; (ii) a second polypeptide chain comprising the sequence of SEQ ID NO. 3671 operably linked to the sequence of SEQ ID NO. 3648 or SEQ ID NO. 508 or 8686 in the direction from the N-terminus to the C-terminus; (iii) a third polypeptide comprising the sequence of SEQ ID NO. 13 and the sequence of SEQ ID NO. 3644 in the direction from the N-terminus to the C-terminus. Claim 178 A composition according to claim 177, wherein the second polypeptide comprises the sequence of SEQ ID NO. 3671 operably linked to the sequence of SEQ ID NO. 3308 operably linked to the sequence of SEQ ID NO. 3648 or SEQ ID NO. 508 or 8686 in the direction from the N-terminus to the C-terminus. Claim 179 In any one of claims 24 to 164 and claims 173 to 178, the formulation comprises the following: (i) a first polypeptide chain comprising a sequence having at least 75% sequence identity with the sequence of SEQ ID NO. 500; (ii) a second polypeptide chain comprising a sequence having at least 75% sequence identity with the sequence of SEQ ID NO. 3669; and (iii) a third polypeptide comprising a sequence having at least 75% sequence identity with the sequence of SEQ ID NO.

501. Claim 180 In any one of claims 24 to 164 and 173 to 179, the formulation comprises the following: (i) a first polypeptide chain comprising the sequence of SEQ ID NO. 500; (ii) a second polypeptide chain comprising the sequence of SEQ ID NO. 3669; and (iii) a third polypeptide comprising the sequence of SEQ ID NO.

501. Claim 181 In any one of claims 24 to 164, the formulation comprises: (i) a first polypeptide chain comprising the sequence of SEQ ID NO. 505 and the sequence of SEQ ID NO. 3649 or SEQ ID NO. 3451; (ii) a second polypeptide chain comprising the sequence of 2270 and the sequence of SEQ ID NO. 3648 or SEQ ID NO. 508 or 8686; and (iii) a third polypeptide comprising the sequence of SEQ ID NO. 13 and the sequence of SEQ ID NO. 3644. Claim 182 In any one of claims 24 to 164 and 181, the formulation comprises the following: a composition comprising: (i) a first polypeptide chain comprising the sequence of SEQ ID NO. 505 operably linked to the sequence of SEQ ID NO. 3649 or SEQ ID NO. 3451 in the direction from the N-terminus to the C-terminus; (ii) a second polypeptide chain comprising the sequence of 2270 operably linked to the sequence of SEQ ID NO. 3648 or SEQ ID NO. 508 or 8686 in the direction from the N-terminus to the C-terminus; and (iii) a third polypeptide comprising the sequence of SEQ ID NO. 13 and the sequence of SEQ ID NO. 3644. Claim 183 A composition according to claim 182, wherein the second polypeptide comprises the sequence of SEQ ID NO. 2270 operably linked to the sequence of SEQ ID NO. 3308 operably linked to the sequence of SEQ ID NO. 3648 or SEQ ID NO. 508 or 8686 in the direction from the N-terminus to the C-terminus. Claim 184 In any one of claims 24 to 164 and claims 181 to 183, the formulation comprises the following: a composition comprising: (i) a first polypeptide chain comprising a sequence having at least 75% sequence identity with the sequence of SEQ ID NO. 500; (ii) a second polypeptide chain comprising a sequence having at least 75% sequence identity with the sequence of SEQ ID NO. 3340; and (iii) a third polypeptide comprising a sequence having at least 75% sequence identity with the sequence of SEQ ID NO.

501. Claim 185 In any one of claims 24 to 164 and 181 to 184, the formulation comprises the following: a composition comprising: (i) a first polypeptide chain comprising the sequence of SEQ ID NO. 500; (ii) a second polypeptide chain comprising the sequence of SEQ ID NO. 3340; and (iii) a third polypeptide comprising the sequence of SEQ ID NO.

501. Claim 186 In any one of claims 1 to 145, the formulation comprises a first polypeptide and a second polypeptide, wherein the first polypeptide and the second polypeptide are not adjacent; (i) the first polypeptide comprises a first member of a dimerization module connected to an antigen binding domain comprising (a) VH of the antigen binding domain and VL of the antigen binding domain, or (b) a first member of a dimerization module connected to a first portion of the antigen binding domain comprising VH of the antigen binding domain, wherein the formulation further comprises a third polypeptide comprising a second portion of the antigen binding domain comprising VL of the antigen binding domain, and the third polypeptide is not adjacent to the first polypeptide and the second polypeptide; and (ii) the second polypeptide comprises (a) a second member of a dimerization module connected to an antigen-binding domain that binds to a T cell receptor beta chain constant domain (TRBC) including VH and VL, or (b) a second member of a dimerization module connected to a first portion of an antigen-binding domain that binds to a TRBC including VH of the antigen-binding domain that binds to a TRBC, wherein the formulation further comprises a fourth polypeptide comprising a second portion of an antigen-binding domain that binds to a TRBC including VL of the antigen-binding domain, and wherein the fourth polypeptide is not adjacent to the first polypeptide, the second polypeptide, and the third polypeptide. Claim 187 A composition according to any one of claims 1 to 145 and 186, wherein the formulation comprises a first polypeptide and a second polypeptide, wherein the first polypeptide and the second polypeptide are not adjacent; (i) the first polypeptide comprises a first member of a dimerization module connected to a first portion of an antigen-binding domain comprising VH of the antigen-binding domain, wherein the formulation further comprises a third polypeptide comprising a second portion of an antigen-binding domain comprising VL of the antigen-binding domain, wherein the third polypeptide is not adjacent to the first polypeptide and the second polypeptide; and (ii) the second polypeptide comprises a second member of a dimerization module connected to the antigen-binding domain that binds to TRBC. Claim 188 In any one of claims 1 to 145, 186, and 187, the formulation comprises the following: (i) a first polypeptide chain comprising the sequence of SEQ ID NO. 505, the sequence of SEQ ID NO. 13, and the sequence of SEQ ID NO. 3447 or SEQ ID NO. 511; (ii) a third polypeptide comprising the sequence of SEQ ID NO. 8308 and the sequence of SEQ ID NO. 3644; and (iii) a second polypeptide chain comprising the sequence of SEQ ID NO. 517 and the sequence of SEQ ID NO. 513 or SEQ ID NO. 514; or a second polypeptide chain comprising the sequence of SEQ ID NO. 512 and the sequence of SEQ ID NO. 513 or SEQ ID NO.

514. Claim 189 In any one of claims 1 to 145 and 186 to 188, the formulation comprises: a composition comprising: (i) a first polypeptide chain comprising the sequence of SEQ ID NO. 505 operably linked to the sequence of SEQ ID NO. 13 operably linked to the sequence of SEQ ID NO. 3447 or SEQ ID NO. 511 in the direction from the N-terminus to the C-terminus; (ii) a third polypeptide comprising the sequence of SEQ ID NO. 8308 operably linked to the sequence of SEQ ID NO. 3644 in the direction from the N-terminus to the C-terminus; and (iii) a second polypeptide chain comprising the sequence of SEQ ID NO. 517 operably linked to the sequence of SEQ ID NO. 513 or SEQ ID NO. 514 in the direction from the N-terminus to the C-terminus; or a second polypeptide chain comprising the sequence of SEQ ID NO. 512 operably linked to the sequence of SEQ ID NO. 513 or SEQ ID NO. 514 in the direction from the N-terminus to the C-terminus. Claim 190 A composition according to claim 189, wherein the first polypeptide comprises the sequence of SEQ ID NO. 505 operably linked to the sequence of SEQ ID NO. 8291 operably linked to the sequence of SEQ ID NO. 13 in the direction from the N-terminus to the C-terminus. Claim 191 In any one of claims 1 to 145 and 186 to 190, the formulation comprises: a composition comprising: (i) a first polypeptide chain comprising a sequence having at least 75% sequence identity with the sequence of SEQ ID NO. 509; (ii) a third polypeptide comprising a sequence having at least 75% sequence identity with the sequence of SEQ ID NO. 7380; and (iii) a second polypeptide chain comprising a sequence having at least or 75% sequence identity with the sequence of SEQ ID NO. 7382, the sequence of SEQ ID NO. 4677, the sequence of SEQ ID NO. 7379, or the sequence of SEQ ID NO. 4675. Claim 192 In any one of claims 1 to 145 and 186 to 191, the formulation comprises: a composition comprising: (i) a first polypeptide chain comprising the sequence of SEQ ID NO. 509 or the sequence of SEQ ID NO. 4704; (ii) a third polypeptide comprising the sequence of SEQ ID NO. 7380; and (iii) a second polypeptide chain comprising the sequence of SEQ ID NO. 7382, the sequence of SEQ ID NO. 4677, the sequence of SEQ ID NO. 7379, or the sequence of 4675. Claim 193 A composition according to any one of claims 1 to 145, wherein the formulation comprises a first polypeptide chain comprising the sequence of SEQ ID NO. 505, the sequence of SEQ ID NO. 13, and the sequence of SEQ ID NO. 3447 or SEQ ID NO.

511. Claim 194 A composition according to any one of claims 1 to 145 and 193, wherein the formulation comprises the sequence of SEQ ID NO. 505 operably linked to the sequence of SEQ ID NO. 13 operably linked to the sequence of SEQ ID NO. 3447 or SEQ ID NO. 511 in the direction from the N-terminus to the C-terminus. Claim 195 A composition according to claim 194, wherein the first polypeptide comprises the sequence of SEQ ID NO. 505 operably linked to the sequence of SEQ ID NO. 8291 operably linked to the sequence of SEQ ID NO. 13 in the direction from the N-terminus to the C-terminus. Claim 196 A composition according to any one of claims 1 to 145 and claims 193 to 195, wherein the formulation comprises a first polypeptide having a sequence having at least 75% sequence identity with the sequence of SEQ ID NO.

509. Claim 197 A composition according to any one of claims 1 to 145 and claims 193 to 196, wherein the formulation comprises a first polypeptide comprising the sequence of SEQ ID NO.

509. Claim 198 A composition according to any one of claims 1 to 145 and claims 193 to 197, wherein the formulation comprises a first polypeptide comprising the sequence of SEQ ID NO.

509. Claim 199 In any one of claims 193 to 198, the composition comprises: (i) a third polypeptide comprising the sequence of SEQ ID NO. 7419 and the sequence of SEQ ID NO. 3644; and (ii) a second polypeptide chain comprising the sequence of SEQ ID NO. 7420 and the sequence of SEQ ID NO. 513 or SEQ ID NO. 514; or a second polypeptide chain comprising the sequence of SEQ ID NO. 7423 and the sequence of SEQ ID NO. 513 or SEQ ID NO.

514. Claim 200 In any one of claims 193 to 199, the formulation comprises: a composition comprising: (i) a third polypeptide comprising the sequence of SEQ ID NO. 7419 operably linked to the sequence of SEQ ID NO. 3644 in the direction from the N-terminus to the C-terminus; and (ii) a second polypeptide chain comprising the sequence of SEQ ID NO. 7420 operably linked to the sequence of SEQ ID NO. 513 or SEQ ID NO. 514 in the direction from the N-terminus to the C-terminus; or a second polypeptide chain comprising the sequence of SEQ ID NO. 7423 operably linked to the sequence of SEQ ID NO. 513 or SEQ ID NO.

514. Claim 201 In any one of claims 193 to 200, the formulation comprises: a composition comprising: (i) a third polypeptide having at least 75% sequence identity with the sequence of SEQ ID NO. 7439; and (ii) a second polypeptide chain having at least 75% sequence identity with the sequence of SEQ ID NO. 4680, the sequence of SEQ ID NO. 7438, the sequence of SEQ ID NO. 4681, or the sequence of SEQ ID NO. 7440. Claim 202 In any one of claims 193 to 201, the formulation comprises: a composition comprising: (i) a third polypeptide comprising the sequence of SEQ ID NO. 7439; and (ii) a second polypeptide chain comprising the sequence of SEQ ID NO. 4680, the sequence of SEQ ID NO. 7438, the sequence of SEQ ID NO. 4681, or the sequence of 7440. Claim 203 In any one of claims 193 to 198, the formulation comprises: a composition comprising: (i) a third polypeptide comprising the sequence of SEQ ID NO. 8012 and the sequence of SEQ ID NO. 3644; and (ii) a second polypeptide chain comprising the sequence of SEQ ID NO. 8011 and the sequence of SEQ ID NO. 513 or SEQ ID NO.

514. Claim 204 In any one of claims 193 to 198 and 203, the formulation comprises: a composition comprising: (i) a third polypeptide comprising the sequence of SEQ ID NO. 8012 operably linked to the sequence of SEQ ID NO. 3644 in the direction from the N-terminus to the C-terminus; and (ii) a second polypeptide chain comprising the sequence of SEQ ID NO. 8011 operably linked to the sequence of SEQ ID NO. 513 or SEQ ID NO. 514 in the direction from the N-terminus to the C-terminus. Claim 205 In any one of claims 193 to 198, 203, and 204, the formulation comprises: a composition comprising: (i) a third polypeptide having a sequence having at least 75% sequence identity with the sequence of SEQ ID NO. 8002; and (ii) a second polypeptide chain having a sequence having at least 75% sequence identity with the sequence of SEQ ID NO. 8001 or 8697. Claim 206 In any one of claims 193 to 198 and 203 to 205, the formulation comprises: a composition comprising: (i) a third polypeptide comprising the sequence of SEQ ID NO. 8002; and (ii) a second polypeptide chain comprising the sequence of SEQ ID NO. 8001 or 8697. Claim 207 In any one of claims 193 to 198, the composition comprises: (i) a third polypeptide comprising the sequence of SEQ ID NO. 8014 and the sequence of SEQ ID NO. 3644; and (ii) a second polypeptide chain comprising the sequence of SEQ ID NO. 8013 and the sequence of SEQ ID NO. 513 or SEQ ID NO. 514, a second polypeptide chain comprising the sequence of SEQ ID NO. 8299 and the sequence of SEQ ID NO. 513 or SEQ ID NO. 514, or a second polypeptide chain comprising the sequence of SEQ ID NO. 8303 and the sequence of SEQ ID NO. 513 or SEQ ID NO.

514. Claim 208 In any one of claims 193 to 198 and 207, the formulation comprises: a composition comprising: (i) a third polypeptide comprising the sequence of SEQ ID NO. 8014 operably linked to the sequence of SEQ ID NO. 3644 in the direction from the N-terminus to the C-terminus; and (ii) a second polypeptide chain comprising the sequence of SEQ ID NO. 8013 operably linked to the sequence of SEQ ID NO. 513 or SEQ ID NO. 514 in the direction from the N-terminus to the C-terminus, a second polypeptide chain comprising the sequence of SEQ ID NO. 8299 operably linked to the sequence of SEQ ID NO. 513 or SEQ ID NO. 514, or a second polypeptide chain comprising the sequence of SEQ ID NO. 8303 operably linked to the sequence of SEQ ID NO. 513 or SEQ ID NO.

514. Claim 209 In any one of claims 193 to 198, 207, and 208, the formulation comprises: a composition comprising: (i) a third polypeptide having at least 75% sequence identity with the sequence of SEQ ID NO. 8005; and (ii) a second polypeptide chain having at least 75% sequence identity with the sequence of SEQ ID NO. 8004, the sequence of SEQ ID NO. 4685, the sequence of SEQ ID NO. 8007, the sequence of SEQ ID NO. 4686, the sequence of SEQ ID NO. 4687, or the sequence of SEQ ID NO. 8009. Claim 210 In any one of claims 193 to 198 and 207 to 209, the formulation comprises: a composition comprising: (i) a third polypeptide comprising the sequence of SEQ ID NO. 8005; and (ii) a second polypeptide chain comprising the sequence of SEQ ID NO. 8004, the sequence of SEQ ID NO. 4685, the sequence of SEQ ID NO. 8007, the sequence of SEQ ID NO. 4686, the sequence of SEQ ID NO. 4687, or the sequence of SEQ ID NO. 8009. Claim 211 A composition according to any one of claims 186 to 210, wherein the formulation further comprises at least one cytokine molecule or a functional fragment or variant thereof, wherein the at least one cytokine molecule or a functional fragment or variant thereof is connected to a first member of the dimerization module, a second member of the dimerization module, an antigen-binding domain, an antigen-binding domain that binds to TRBC, or any combination thereof. Claim 212 A composition according to claim 211, wherein at least one cytokine molecule or a functional fragment or variant thereof comprises IL-2 or a functional fragment or variant thereof, IL-7 or a functional fragment or variant thereof, IL-12 or a functional fragment or variant thereof, IL-15 or a functional fragment or variant thereof, IL-18 or a functional fragment or variant thereof, IL-21 or a functional fragment or variant thereof, interferon gamma or a functional fragment or variant thereof, or any combination thereof. Claim 213 A composition according to claim 211 or 212, wherein the at least one cytokine molecule or its functional fragment or variant comprises a sequence having at least 75% sequence identity with any one sequence selected from the group consisting of cytokine sequences listed in Tables 40, 42C, 43A, 43B, and 46–51. Claim 214 A composition according to any one of claims 211 to 213, wherein the at least one cytokine molecule or a functional fragment or variant thereof comprises any one sequence selected from the group consisting of cytokine sequences listed in Tables 40, 42C, 43A, 43B, and 46 to 51. Claim 215 A composition according to any one of claims 211 to 214, wherein at least one cytokine molecule or a functional fragment or variant thereof comprises IL-2 or a functional fragment or variant thereof. Claim 216 A composition according to any one of claims 212 to 215, wherein the IL-2 or its functional fragment or variant comprises a sequence having at least 75% sequence identity with the sequence of SEQ ID NO. 2191, the sequence of SEQ ID NO. 2270, or the sequence of SEQ ID NO. 2280. Claim 217 A composition according to any one of claims 212 to 216, wherein the IL-2 or its functional fragment or variant comprises the sequence of SEQ ID NO. 2191, the sequence of SEQ ID NO. 2270, or the sequence of SEQ ID NO. 2280. Claim 218 A composition according to any one of claims 211 to 214, wherein at least one cytokine molecule or a functional fragment or variant thereof comprises IL-15 or a functional fragment or variant thereof. Claim 219 A composition according to any one of claims 212 to 214 and 218, wherein the IL-15 or its functional fragment or variant comprises a sequence having at least 75% sequence identity with the sequence of SEQ ID NO. 2170, SEQ ID NO. 3670, or SEQ ID NO. 3673. Claim 220 A composition according to any one of claims 212 to 214, 218, and 219, wherein the IL-15 or its functional fragment or variant comprises the sequence of SEQ ID NO. 2170, the sequence of SEQ ID NO. 3670, or the sequence of SEQ ID NO. 3673. Claim 221 A composition according to any one of claims 212 to 214 and 218 to 220, wherein the at least one cytokine molecule or its functional fragment or variant further comprises an IL15R-alpha dimerization domain covalently linked to the IL-15 or its functional fragment or variant. Claim 222 A composition according to claim 221, wherein the IL15R-alpha dimerization domain comprises an IL-15 receptor alpha-sushi domain or a functional fragment or variant thereof. Claim 223 A composition according to claim 221 or 222, wherein the IL15R alpha dimerization domain comprises a sequence having at least 75% sequence identity with the sequence of SEQ ID NO. 3472. Claim 224 A composition according to any one of claims 221 to 223, wherein the IL15R alpha dimerization domain comprises the sequence of SEQ ID NO. 3472. Claim 225 A composition according to any one of claims 221 to 224, wherein the IL-15 or its functional fragment or variant is sharedly linked to the IL15R-alpha dimerization domain through a linker. Claim 226 A composition according to any one of claims 221 to 225, wherein the IL-15 or its functional fragment or variant is covalently linked to an IL15R-alpha dimerization domain through a linker comprising the sequence of SEQ ID NO. 3473. Claim 227 A composition according to any one of claims 212 to 214 and 218 to 226, wherein the at least one cytokine molecule or a functional fragment or variant thereof comprises a sequence having at least 75% sequence identity with the sequence of SEQ ID NO. 3474, SEQ ID NO. 3671, or SEQ ID NO. 8689. Claim 228 A composition according to any one of claims 212 to 214 and 218 to 227, wherein at least one cytokine molecule or a functional fragment or variant thereof comprises the sequence of SEQ ID NO. 3474, the sequence of SEQ ID NO. 3671, or the sequence of SEQ ID NO. 8689. Claim 229 A composition according to any one of claims 211 to 214, wherein at least one cytokine molecule or a functional fragment or variant thereof comprises IL-7 or a functional fragment or variant thereof. Claim 230 A composition according to any one of claims 212 to 214 and 229, wherein the IL-7 or its functional fragment or variant comprises a sequence having at least 75% sequence identity with the sequence of SEQ ID NO. 3540. Claim 231 A composition according to any one of claims 212 to 214, 229, and 230, wherein the IL-7 or its functional fragment or variant comprises the sequence of SEQ ID NO. 3540. Claim 232 A composition according to any one of claims 211 to 214, wherein at least one cytokine molecule or a functional fragment or variant thereof comprises IL-12 or a functional fragment or variant thereof. Claim 233 A composition according to any one of claims 212 to 214 and 232, wherein the IL-12 or its functional fragment or variant comprises a sequence having at least 75% sequence identity with the sequence of SEQ ID NO. 3542, the sequence of SEQ ID NO. 3543, or the sequence of SEQ ID NO. 3545. Claim 234 A composition according to any one of claims 212 to 214, 232, and 233, wherein the IL-12 or its functional fragment or variant comprises the sequence of SEQ ID NO. 3542, the sequence of SEQ ID NO. 3543, or the sequence of SEQ ID NO. 3545. Claim 235 A composition according to any one of claims 211 to 214, wherein at least one cytokine molecule or a functional fragment or variant thereof comprises IL-18 or a functional fragment or variant thereof. Claim 236 A composition according to any one of claims 212 to 214 and 235, wherein the IL-18 or its functional fragment or variant comprises a sequence having at least 75% sequence identity with the sequence of SEQ ID NO. 2192. Claim 237 A composition according to any one of claims 212 to 214, 235, and 236, wherein the IL-18 or a functional fragment or variant thereof comprises the sequence of SEQ ID NO. 2192. Claim 238 A composition according to any one of claims 211 to 214, wherein at least one cytokine molecule or a functional fragment or variant thereof comprises IL-21 or a functional fragment or variant thereof. Claim 239 A composition according to any one of claims 212 to 214 and 238, wherein the IL-21 or a functional fragment or variant thereof comprises a sequence having at least 75% sequence identity with the sequence of SEQ ID NO. 3540. Claim 240 A composition according to any one of claims 212 to 214, 238, and 239, wherein the IL-21 or a functional fragment or variant thereof comprises the sequence of SEQ ID NO. 3540. Claim 241 A recombinant polynucleotide molecule comprising a polynucleotide sequence encoding a formulation of a composition of any one of claims 1 to 240. Claim 242 In paragraph 241, the recombinant polynucleotide molecule is a recombinant polynucleotide molecule that is an isolated recombinant polynucleotide molecule. Claim 243 A vector comprising the recombinant polynucleotide molecule of claim 241 or 242. Claim 244 A cell containing the recombinant polynucleotide molecule of claim 241 or 242 or the vector of claim 243. Claim 245 A method for preparing a preparation of a composition of any one of claims 1 to 240, comprising the step of culturing the cells of claim 244 under conditions suitable for the expression of said preparation. Claim 246 A pharmaceutical composition comprising a composition of any one of claims 1 to 240, a recombinant polynucleotide molecule of claim 241 or 242, a vector of claim 243, or a cell of claim 244, and a pharmaceutically acceptable carrier, excipient, or diluent. Claim 247 A method for treating a condition or disease in a subject requiring treatment, comprising the step of administering to the subject a composition of any one of claims 1 to 240, a recombinant polynucleotide molecule of claim 241 or 242, a vector of claim 243, a cell of claim 244, or a therapeutically effective amount of a pharmaceutical composition of claim 246, or any combination thereof, to treat the condition or disease in the subject. Claim 248 In paragraph 247, the above pathological condition or disease is cancer, method. Claim 249 In paragraph 248, the above cancer is leukemia or lymphoma. Claim 250 A method according to claim 247 or 248, wherein the cancer is selected from the group consisting of acquired immunodeficiency syndrome (AIDS)-associated lymphoma, angioimmunoblastic T-cell lymphoma, adult T-cell leukemia / lymphoma, Burkitt lymphoma, central nervous system (CNS) lymphoma, diffuse large B-cell lymphoma (DLBCL), lymphoblastic lymphoma, mantle cell lymphoma (MCL), peripheral T-cell lymphoma (PTCL), transformed follicular and transformed mucosa-associated lymphoid tissue (MALT) lymphoma, cutaneous T-cell lymphoma (mycosis fungoides and Sézary syndrome), follicular lymphoma, lymphoplasmocytic lymphoma / Waldenstrom macroglobulinemia, marginal zone B-cell lymphoma, gastric mucosa-associated lymphoid tissue (MALT) lymphoma, chronic lymphocytic leukemia / small cell lymphocytic lymphoma (CLL / SLL), extranodal T- / NK-cell lymphoma, and anaplastic large cell lymphoma. Claim 251 In paragraph 250, the peripheral T-cell lymphoma (PTCL) is hepatosplenic T-cell lymphoma (HSGDTCL), subcutaneous panniculitis-like T-cell lymphoma, or enteropathy-associated T-cell lymphoma. Claim 252 In paragraph 250, the above cutaneous T-cell lymphoma is mycosis fungoides and Sézary syndrome, method. Claim 253 In paragraph 250, the above extranodal T- / NK-cell lymphoma is of the nasal type. Claim 254 In paragraph 250, the method wherein the anaplastic large cell lymphoma is primary cutaneous anaplastic large cell lymphoma or systemic anaplastic large cell lymphoma. Claim 255 In paragraph 248, the above cancer is a solid tumor, method. Claim 256 A method according to claim 255, wherein the solid tumor is selected from the group consisting of pancreatic cancer, breast cancer, colorectal cancer, colon cancer, lung cancer, skin cancer, ovarian cancer, liver cancer, esophageal cancer, endometrial cancer, stomach cancer, head and neck cancer, kidney cancer, and prostate cancer. Claim 257 In paragraph 256, the above pancreatic cancer is a pancreatic adenocarcinoma or a pancreatic ductal adenocarcinoma. Claim 258 In paragraph 256, the method wherein the lung cancer is small cell lung cancer (SCLC) or non-small cell lung cancer (NSCLC). Claim 259 In paragraph 247, the above pathological condition or disease is an autoimmune disease. Claim 260 In paragraph 259, the autoimmune disease is selected from the group consisting of: Amyotrophic Lateral Sclerosis (ALS), acute anterior uveitis (UAA), celiac disease (CD), ankylosing spondylitis (AS), childhood COVID-induced multisystem inflammatory syndrome (MIS-C), primary Sjögren's syndrome (PSS), Churg-Strauss syndrome, sarcoidosis, systemic lupus erythematosus (SLE), diabetes mellitus type 1, autoimmune hepatitis (e.g., type 1 or type 2), narcolepsy type 1, primary sclerosing cholangitis, primary biliary cirrhosis, multiple sclerosis, Guillain-Barre syndrome and AMAN (axonal and neuroneuropathy), chronic inflammatory demyelinating polyneuropathy (CIDP), transverse myelitis, Tolosa-Hunt syndrome (THS), Devic's disease (neuromyelitis optica), Paraneoplastic cerebellar hyperplasia (PCD), Lambert-Eaton syndrome, psoriasis, scleroderma, CREST syndrome (calcification, Raynaud's phenomenon, esophageal motility disorder, sclerosis, and telangiectasia), herpetic dermatitis, dermatomyositis, bullous pemphigus, pathological pemphigus / benign mucosal pemphigus, pemphigoid pregnancy, rheumatoid arthritis (RA), psoriatic arthritis, recurrent polychondritis, chronic recurrent multifocal osteomyelitis (CRMO), vasculitis, Kawasaki disease, artificial joint failure, granulomatosis with polyangiitis (GPA), Behcet's disease (vasculitis), Takayasu's arteritis, polyarteritis nodosum, polyangiitis microae (MPA), leukocytoclastic vasculitis, Cogan Syndrome, uveitis, peripheral uveitis (pars planitis), scleritis, autoimmune inner ear disease (AIED), Crohn's disease, ulcerative colitis (UC), Dressler syndrome, rheumatic fever, Evans syndrome, paroxysmal nocturnal hemoglobinuria (PNH), hemolytic anemia, thrombocytopenic purpura (TTP), polymyositis, juvenile myositis (JM) (including juvenile dermatomyositis (JDM) and juvenile polymyositis (JPM)), ocular pemphigus,and Hashimoto's thyroiditis. Claim 261 In paragraph 260, the above-mentioned autoimmune disease is multiple sclerosis, UAA, PsA, narcolepsy type I, ALS, Crohn's disease, celiac disease, diabetes mellitus type I, Sjögren's syndrome, ankylosing spondylitis, artificial joint failure, or COVID-induced multi-organ inflammatory syndrome, method. Claim 262 A method for proliferating and / or activating NK cells in an NK cell population, comprising the step of contacting an effective amount of a composition of any one of claims 1 to 240, a recombinant polynucleotide molecule of claim 241 or 242, a vector of claim 243, a cell of claim 244, or a pharmaceutical composition of claim 246, or any combination thereof, with said NK cell population to proliferate and / or activate the NK cells in said NK cell population. Claim 263 In paragraph 262, the step of proliferating and / or activating NK cells is in vivo or in vitro proliferation and / or activation, a method.

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