Multifunctional molecules that bind to T cell-associated cancer cells and their uses

Abstract

Disclosed are multifunctional molecules, including i) an antigen binding domain that binds to a T cell receptor β chain constant domain 1 or a T cell receptor β chain constant domain 2; and one, two or all of the following: (ii) an immune cell connector (e.g., selected from NK cell connectors, T cell connectors, B cell connectors, dendritic cell connectors or macrophage connectors); (iii) a cytokine molecule or a cytokine inhibitor molecule; (iv) a death receptor signal enhancer; and / or (v) a matrix modification portion. In addition, disclosed are nucleic acids encoding the multifunctional molecules, methods for producing the above molecules, and methods for treating cancer using the above molecules.

Description

[0001] Related Applications

[0002] This application claims priority to U.S. Serial No. 62 / 808,646, filed on February 21, 2019, the contents of which are incorporated herein by reference in their entirety.

[0003] Sequence Listing

[0004] This application contains a sequence listing submitted electronically in ASCII format, and the entire contents of which are incorporated herein by reference. The ASCII copy was created on February 19, 2020, is named E2070-7022WO_SL.txt, and is 810,998 bytes in size. Background Art

[0005] Lymphoma is a cancer caused by lymphocytes. T-cell lymphoma (TCL) is a lymphoma derived from T cells; these account for about 7% of all non-Hodgkin lymphomas in the United States. Common subtypes of TCL include: peripheral T-cell lymphoma, not otherwise specified (PTCLNOS), anaplastic large cell lymphoma (ALCL), angioimmunoblastic T-cell lymphoma (AITL) and cutaneous T-cell lymphoma (CTCL). Each type of TCL has its own pathology and symptoms. In view of the continued need for improved treatment of lymphomas (e.g., TCL), new compositions and treatments targeting lymphomas (e.g., TCL) are highly desirable. Summary of the invention

[0006] The present disclosure relates in particular to novel multispecific or multifunctional molecules comprising (i) an antigen binding domain that binds to a tumor antigen on a lymphoma cell (e.g., a T cell receptor comprising a T cell receptor β chain constant domain 1 (TRBC1) or a T cell receptor comprising a T cell receptor β chain constant domain 2 (TRBC2)); and one, two, or all of the following: (ii) an immune cell engager (e.g., selected from an NK cell engager, a T cell engager, a B cell engager, a dendritic cell engager, or a macrophage engager); (iii) a cytokine molecule; and / or (iv) a matrix modifying portion. The terms "multispecific" or "multifunctional" are used interchangeably herein.

[0007] Without wishing to be bound by theory, it is contemplated that the multispecific or multifunctional molecules disclosed herein target (e.g., localize, bridge and / or activate) immune cells (e.g., immune effector cells selected from NK cells, T cells, B cells, dendritic cells, or macrophages) to target cells (e.g., cancer cells (e.g., lymphoma cells)) expressing T cell receptors including TRBC1 or TRBC2, and / or alter the tumor stroma, e.g., alter the tumor microenvironment near the cancer site. It is contemplated that increasing the proximity and / or activity of immune cells using the multispecific molecules described herein can enhance the immune response to target cells (e.g., cancer cells, e.g., lymphoma cells), thereby providing more effective therapies (e.g., more effective cancer therapies). Without being bound by theory, targeted, localized immune responses to target cells (e.g., cancer cells) are believed to have the effect of reducing the systemic toxicity of the multispecific molecules described herein. Furthermore, where the target cancer cells are T cells (e.g., T cells expressing a T cell receptor comprising TRBC1 or TRBC2), a targeted immune response against the cancerous T cell population that targets non-cancerous T cells to a lesser extent (e.g., does not target non-cancerous T cells) is believed to have less deleterious effects than systemic elimination of all T cells.

[0008] Without wishing to be bound by theory, the clonally derived T-cell lymphoma is positive for either TRBC1 or TRBC2, but not both. In the case of a TRBC1+ T-cell malignancy, the anti-TRBC1 molecules disclosed herein (e.g., multifunctional molecules that bind to TRBC1 and NKp30) can deplete TRBC1+ cells while retaining TRBC2+ non-malignant T cells. Similarly, in the case of a TRBC2+ T-cell malignancy, the anti-TRBC2 molecules disclosed herein (e.g., multifunctional molecules that bind to TRBC2 and NKp30) can deplete TRBC2+ cells while retaining TRBC1+ non-malignant T cells.

[0009] Without wishing to be bound by theory, in some embodiments, the multifunctional molecules disclosed herein (e.g., anti-TRBC1 / NKp30 antibodies) activate NK cells only in the presence of cells expressing TRBC1. Without wishing to be bound by theory, in some embodiments, the multifunctional molecules disclosed herein (e.g., anti-TRBC2 / NKp30 antibodies) activate NK cells only in the presence of cells expressing TRBC2.

[0010] Thus, specifically provided herein are multispecific molecules (eg, multispecific or multifunctional antibody molecules) comprising the above-described portions, nucleic acids encoding the multispecific molecules, methods of producing the above-described molecules, and methods of using the above-described molecules to treat cancer.

[0011] In one aspect, provided herein is a multifunctional molecule comprising (i) a first antigen binding domain that binds to T cell receptor β chain constant domain 1 (TRBC1) or T cell receptor β chain constant domain 2 (TRBC2), and (ii) a second antigen binding domain that binds to NKp30.

[0012] In some embodiments, the first antigen binding domain binds to TRBC1. In some embodiments, the first antigen binding domain comprises one or more CDRs, framework regions, variable regions, or antigen binding domains disclosed in any one of Tables 2-6 and 19, or a sequence having at least 85%, 90%, 95%, or 99% identity thereto. In some embodiments, the first antigen binding domain comprises a VH comprising a heavy chain complementary determining region 1 (VHCDR1), a VHCDR2, and a VHCDR3; and a VL comprising a light chain complementary determining region 1 (VLCDR1), a VLCDR2, and a VLCDR3, wherein the VHCDR1, VHCDR2, and VHCDR3 comprise the amino acid sequences of: SEQ ID NOs: 7346, 7355, and 202, respectively; SEQ ID NOs: 7346, 201, and 202, respectively; SEQ ID NOs: 7354, 201, and 202, respectively; or SEQ ID NOs: 7354, 7355, and 202, respectively. In some embodiments, the VLCDR1, VLCDR2 and VLCDR3 comprise the amino acid sequences of: SEQ ID NOs: 223, 224 and 225, respectively; SEQ ID NOs: 7367, 224 and 225, respectively; SEQ ID NOs: 223, 7368 and 225, respectively; SEQ ID NOs: 223, 224 and 7369, respectively; or SEQ ID NOs: 7367, 7368 and 7369, respectively.In some embodiments, the VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2 and VLCDR3 comprise the amino acid sequences of: SEQ ID NOs: 7346, 7355, 202, 223, 224 and 225, respectively; SEQ ID NOs: 7346, 201, 202, 223, 224 and 225, respectively; SEQ ID NOs: 7346, 7355, 202, 7367, 224 and 225, respectively; SEQ ID NOs: 7346, 7355, 202, 223, 7368 and 225, respectively; SEQ ID NOs: 7346, 7355, 202, 223, 224 and 7369, respectively; SEQ ID NOs: 7346, 7355, 202, 7367, 7368 and 7369, respectively; NO:7346, 201, 202, 7367, 224 and 225; SEQ ID NO:7346, 201, 202, 223, 7368 and 225, respectively; SEQ ID NO:7346, 201, 202, 223, 224 and 7369, respectively; SEQ ID NO:7346, 201, 202, 7367, 7368 and 7369, respectively; SEQ ID NO:7354, 201, 202, 223, 224 and 225, respectively; SEQ ID NO:7354, 201, 202, 223, 7368 and 225, respectively; SEQ ID NO:7354, 201, 202, 223, 7368 and 225, respectively; NO:7354, 201, 202, 223, 224 and 7369; SEQ ID NO:7354, 201, 202, 7367, 7368 and 7369, respectively; SEQ ID NO:7354, 7355, 202, 223, 224 and 225, respectively; SEQ ID NO:7354, 7355, 202, 7367, 224 and 225, respectively; SEQ ID NO:7354, 7355, 202, 223, 7368 and 225, respectively; SEQ ID NO:7354, 7355, 202, 223, 224 and 7369, respectively; or SEQ ID NO:7354, 7355, 202, 7367, 7368 and 7369, respectively.In some embodiments, the VH comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 7351, 253, 250-252, 254, 7343, 7344, 7350, and 7352 (or a sequence having at least 85%, 90%, 95%, or 99% identity thereof) and / or the VL comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 258, 255-257, 259, 260, and 7357-7360 (or a sequence having at least 85%, 90%, 95%, or 99% identity thereof). In some embodiments, the VH and VL comprise the amino acid sequences of: SEQ ID NOs: 7351 and 258, respectively (or a sequence having at least 85%, 90%, 95%, or 99% identity thereof); or SEQ ID NOs: 253 and 258, respectively (or a sequence having at least 85%, 90%, 95%, or 99% identity thereof).

[0013] In some embodiments, the first antigen binding domain has a higher affinity for a T cell receptor that includes TRBC1 than for a T cell receptor that does not include TRBC1, optionally wherein the K affinity of the binding between the first antigen binding domain and TRBC1 is D does not exceed the K of binding between the first antigen binding domain and the T cell receptor excluding TRBC1 D In some embodiments, the first antigen binding domain has a higher affinity for a T cell receptor comprising TRBC1 than for a T cell receptor comprising TRBC2, optionally wherein the K of the binding between the first antigen binding domain and TRBC1 is D does not exceed the K of binding between the first antigen binding domain and the T cell receptor including TRBC2 D In some embodiments, the binding of the first antigen binding domain to TRBC1 on lymphoma cells or lymphocytes, such as T cells, does not significantly activate lymphoma cells or lymphocytes, such as T cells (e.g., as measured by T cell proliferation, expression of T cell activation markers (e.g., CD69 or CD25), and / or expression of cytokines (e.g., TNFα and IFNγ)). In some embodiments, the multifunctional molecule does not activate NK cells or substantially does not activate NK cells in the absence of cells expressing TRBC1.

[0014] In some embodiments, the first antigen binding domain binds to TRBC2. In some embodiments, the first antigen binding domain has a higher affinity for a T cell receptor that includes TRBC2 than a T cell receptor that does not include TRBC2, optionally wherein the K of the binding between the first antigen binding domain and TRBC2 isD does not exceed the K of binding between the first antigen binding domain and the T cell receptor excluding TRBC2 D In some embodiments, the first antigen binding domain has a higher affinity for a T cell receptor comprising TRBC2 than for a T cell receptor comprising TRBC1, optionally wherein the K of the binding between the first antigen binding domain and TRBC2 is greater than 40%, 30%, 20%, 10%, 1%, 0.1% or 0.01%. D does not exceed the K of binding between the first antigen binding domain and the T cell receptor including TRBC1 D In some embodiments, the binding of the first antigen binding domain to TRBC2 on lymphoma cells or lymphocytes, such as T cells, does not significantly activate lymphoma cells or lymphocytes, such as T cells (e.g., as measured by T cell proliferation, expression of T cell activation markers (e.g., CD69 or CD25), and / or expression of cytokines (e.g., TNFα and IFNγ)). In some embodiments, the multifunctional molecule does not activate NK cells or substantially activate NK cells in the absence of cells expressing TRBC2.

[0015] In some embodiments, the second antigen binding domain comprises one or more CDRs, framework regions, variable regions, or antigen binding domains disclosed in any one of Tables 7-10, 18, and 19, or a sequence at least 85%, 90%, 95%, or 99% identical thereto. In some embodiments, the second antigen binding domain comprises a VH comprising a heavy chain complementary determining region 1 (VHCDR1), a VHCDR2 and a VHCDR3; and a VL comprising a light chain complementary determining region 1 (VLCDR1), a VLCDR2 and a VLCDR3, wherein VHCDR1, VHCDR2 and VHCDR3 of the second antigen binding domain comprise the amino acid sequences of: SEQ ID NOs: 7313, 6001 and 7315, respectively; SEQ ID NOs: 7313, 6001 and 6002, respectively; SEQ ID NOs: 7313, 6008 and 6009, respectively; SEQ ID NOs: 7313, 7385 and 7315, respectively; or SEQ ID NOs: 7313, 7318 and 6009, respectively. In some embodiments, VLCDR1, VLCDR2 and VLCDR3 of the second antigen binding domain comprise the amino acid sequences of: SEQ ID NOs: 7326, 7327 and 7329, respectively; SEQ ID NOs: 6063, 6064 and 7293, respectively; SEQ ID NOs: 6070, 6071 and 6072, respectively; or SEQ ID NOs: 6070, 6064 and 7321, respectively. In some embodiments, the VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2 and VLCDR3 of the second antigen binding domain comprise the amino acid sequences of: SEQ ID NOs: 7313, 6001, 7315, 7326, 7327 and 7329, respectively; SEQ ID NOs: 7313, 6001, 6002, 6063, 6064 and 7293, respectively; SEQ ID NOs: 7313, 6008, 6009, 6070, 6071 and 6072, respectively; SEQ ID NOs: 7313, 7385, 7315, 6070, 6064 and 7321, respectively; or SEQ ID NOs: 7313, 7318, 6009, 6070, 6064 and 7321, respectively.In some embodiments, the VH of the second antigen binding domain comprises an amino acid sequence selected from SEQ ID NOs: 7302, 7298, 7300, 7301, 7303, and 7304 (or a sequence with at least 85%, 90%, 95%, or 99% identity thereof) and / or the VL of the second antigen binding domain comprises an amino acid sequence selected from SEQ ID NOs: 7309, 7305, 7299, 7306-7308 (or a sequence with at least 85%, 90%, 95%, or 99% identity thereof). In some embodiments, the VH of the second antigen binding domain comprises an amino acid sequence selected from SEQ ID NO: 6121 or 6123-6128 (or a sequence having at least 85%, 90%, 95% or 99% identity thereof) and / or the VL of the second antigen binding domain comprises an amino acid sequence selected from SEQ ID NO: 7294 or 6137-6141 (or a sequence having at least 85%, 90%, 95% or 99% identity thereof). In some embodiments, the VH of the second antigen binding domain comprises an amino acid sequence selected from SEQ ID NO: 6122 or 6129-6134 (or a sequence having at least 85%, 90%, 95% or 99% identity thereof) and / or the VL of the second antigen binding domain comprises an amino acid sequence selected from SEQ ID NO: 6136 or 6142-6147 (or a sequence having at least 85%, 90%, 95% or 99% identity thereof). In some embodiments, the VH and VL of the second antigen binding domain comprise the amino acid sequence of SEQ ID NO: 7302 and 7309, respectively (or a sequence having at least 85%, 90%, 95% or 99% identity thereof); or SEQ ID NO: 7302 and 7305, respectively (or a sequence having at least 85%, 90%, 95% or 99% identity thereof). In some embodiments, the second antigen binding domain comprises the amino acid sequence of SEQ ID NO: 7311 or 7310 (or a sequence having at least 85%, 90%, 95% or 99% identity thereof); SEQ ID NO: 6187 or 6188 (or a sequence having at least 85%, 90%, 95% or 99% identity thereof); or SEQ ID NO: 6189 or 6190 (or a sequence having at least 85%, 90%, 95% or 99% identity thereof).

[0016] In some embodiments, the multifunctional molecule monovalently binds to TRBC1 or TRBC2. In some embodiments, the multifunctional molecule comprises Figures 29A-29D Any of the configurations shown in , optionally wherein: (i) the multifunctional antibody molecule comprises anti-TRBC1 Fab and anti-NKp30 scFv, for example, comprising Fig.29A (ii) the multifunctional antibody molecule comprises anti-TRBC1 Fab and anti-NKp30 Fab, for example, comprising Fig.29B (iii) the multifunctional antibody molecule comprises anti-NKp30 Fab and anti-TRBC1scFv, for example, comprising Fig.29C or (iv) the multifunctional antibody molecule comprises anti-TRBC1 scFv and anti-NKp30 scFv, for example, comprising Fig.29D In some embodiments, the multifunctional molecule comprises Figures 30A-30D The configuration shown in any one of the above, optionally wherein: (i) the multifunctional antibody molecule comprises anti-TRBC2 Fab and anti-NKp30 scFv, for example, comprising Fig. 30A (ii) the multifunctional antibody molecule comprises anti-TRBC2 Fab and anti-NKp30 Fab, for example, comprising Fig. 30B (iii) the multifunctional antibody molecule comprises anti-NKp30 Fab and anti-TRBC2 scFv, for example, comprising Fig. 30C or (iv) the multifunctional antibody molecule comprises anti-TRBC2 scFv and anti-NKp30 scFv, for example, comprising Fig.30D Configuration shown.

[0017] In some embodiments, the multifunctional molecules disclosed herein further include a dimerization module, which includes one or more immunoglobulin chain constant regions (e.g., Fc regions), which include one or more of the following items: paired cavity-protrusions ("knob-in-a hole"), electrostatic interactions, or chain exchange.

[0018] In some embodiments, the multifunctional molecule comprises an anti-TRBC1 amino acid sequence disclosed in any one of Tables 2-6 and 19, or a sequence having at least 85%, 90%, 95% or 99% identity thereto; and / or an anti-NKp30 amino acid sequence disclosed in any one of Tables 7-10, 18 and 19, or a sequence having at least 85%, 90%, 95% or 99% identity thereto. In some embodiments, the multifunctional molecule comprises: (i) an anti-TRBC1 VH of SEQ ID NO: 7351 (or a sequence having at least 85%, 90%, 95% or 99% identity thereof), an anti-TRBC1 VL of SEQ ID NO: 258 (or a sequence having at least 85%, 90%, 95% or 99% identity thereof), an anti-NKp30 VH of SEQ ID NO: 7302 (or a sequence having at least 85%, 90%, 95% or 99% identity thereof), and an anti-NKp30 VL of SEQ ID NO: 7309 (or a sequence having at least 85%, 90%, 95% or 99% identity thereof); (ii) an anti-TRBC1 VH of SEQ ID NO: 7351 (or a sequence having at least 85%, 90%, 95% or 99% identity thereof), an anti-TRBC1 VL of SEQ ID NO: 258 (or a sequence having at least 85%, 90%, 95% or 99% identity thereof), an anti-NKp30 VH of SEQ ID NO: 7302 (or a sequence having at least 85%, 90%, 95% or 99% identity thereof), and an anti-NKp30 VL of SEQ ID NO: 7309 (or a sequence having at least 85%, 90%, 95% or 99% identity thereof). or (iii) SEQ ID NOs: 7382, 7380 and 7383 (or sequences at least 85%, 90%, 95% or 99% identical thereto).In some embodiments, the multifunctional molecule comprises: (i) an anti-TRBC1 VH of SEQ ID NO: 253 (or a sequence having at least 85%, 90%, 95% or 99% identity thereof), an anti-TRBC1 VL of SEQ ID NO: 258 (or a sequence having at least 85%, 90%, 95% or 99% identity thereof), an anti-NKp30 VH of SEQ ID NO: 7302 (or a sequence having at least 85%, 90%, 95% or 99% identity thereof), and an anti-NKp30 VL of SEQ ID NO: 7309 (or a sequence having at least 85%, 90%, 95% or 99% identity thereof); (ii) an anti-TRBC1 VH of SEQ ID NO: 253 (or a sequence having at least 85%, 90%, 95% or 99% identity thereof), an anti-TRBC1 VL of SEQ ID NO: 258 (or a sequence having at least 85%, 90%, 95% or 99% identity thereof), an anti-NKp30 VH of SEQ ID NO: 7302 (or a sequence having at least 85%, 90%, 95% or 99% identity thereof), and an anti-NKp30 VL of SEQ ID NO: 7309 (or a sequence having at least 85%, 90%, 95% or 99% identity thereof). or (iii) SEQ ID NOs: 7379, 7380 and 7383 (or sequences at least 85%, 90%, 95% or 99% identical thereto). In some embodiments, the multifunctional molecule comprises: (i) an anti-TRBC1 VH of SEQ ID NO: 7351 (or a sequence having at least 85%, 90%, 95% or 99% identity thereof), an anti-TRBC1 VL of SEQ ID NO: 258 (or a sequence having at least 85%, 90%, 95% or 99% identity thereof), an anti-NKp30 VH of SEQ ID NO: 7302 (or a sequence having at least 85%, 90%, 95% or 99% identity thereof), and an anti-NKp30 VL of SEQ ID NO: 7305 (or a sequence having at least 85%, 90%, 95% or 99% identity thereof); (ii) an anti-TRBC1 VH of SEQ ID NO: 7351 (or a sequence having at least 85%, 90%, 95% or 99% identity thereof), an anti-TRBC1 VL of SEQ ID NO: 258 (or a sequence having at least 85%, 90%, 95% or 99% identity thereof), an anti-NKp30 VH of SEQ ID NO: 7302 (or a sequence having at least 85%, 90%, 95% or 99% identity thereof), and an anti-NKp30 VL of SEQ ID NO: 7305 (or a sequence having at least 85%, 90%, 95% or 99% identity thereof). or (iii) SEQ ID NOs: 7382, 7380 and 7384 (or sequences at least 85%, 90%, 95% or 99% identical thereto).In some embodiments, the multifunctional molecule comprises: (i) an anti-TRBC1 VH of SEQ ID NO: 253 (or a sequence having at least 85%, 90%, 95% or 99% identity thereof), an anti-TRBC1 VL of SEQ ID NO: 258 (or a sequence having at least 85%, 90%, 95% or 99% identity thereof), an anti-NKp30 VH of SEQ ID NO: 7302 (or a sequence having at least 85%, 90%, 95% or 99% identity thereof), and an anti-NKp30 VL of SEQ ID NO: 7305 (or a sequence having at least 85%, 90%, 95% or 99% identity thereof); (ii) an anti-TRBC1 VH of SEQ ID NO: 253 (or a sequence having at least 85%, 90%, 95% or 99% identity thereof), an anti-TRBC1 VL of SEQ ID NO: 258 (or a sequence having at least 85%, 90%, 95% or 99% identity thereof), an anti-NKp30 VH of SEQ ID NO: 7302 (or a sequence having at least 85%, 90%, 95% or 99% identity thereof), and an anti-NKp30 VL of SEQ ID NO: 7305 (or a sequence having at least 85%, 90%, 95% or 99% identity thereof). or (iii) SEQ ID NOs: 7379, 7380 and 7384 (or sequences at least 85%, 90%, 95% or 99% identical thereto).

[0019] In some embodiments, the multifunctional molecule includes: a heavy chain constant region variant, e.g., an Fc region variant, comprising one or more mutations that result in reduced or eliminated affinity for at least one Fc receptor, optionally wherein the one or more mutations result in reduced or eliminated antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP) or complement-dependent cytotoxicity (CDC). In some embodiments, the Fc region variant includes one or more mutations disclosed in Table 20, optionally wherein the Fc region variant includes an N297A mutation.

[0020] In one aspect, provided herein is an antibody molecule that binds to TRBC1, comprising one or more CDRs, framework regions, variable regions, or antigen binding domains disclosed in any one of Tables 2-6 and 19, or a sequence having at least 85%, 90%, 95% or 99% identity thereto.

[0021] In one aspect, provided herein is an antibody molecule that binds to NKp30, comprising one or more CDRs, framework regions, variable regions, or antigen binding domains disclosed in any one of Tables 7-10, 18 and 19, or a sequence having at least 85%, 90%, 95% or 99% identity thereto.

[0022] In some embodiments, the antibody molecule includes a heavy chain constant region variant, e.g., an Fc region variant, comprising one or more mutations resulting in reduced or eliminated affinity for at least one Fc receptor, optionally wherein the one or more mutations result in antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP) or complement-dependent cytotoxicity (CDC) reduction or elimination. In some embodiments, the Fc region variant includes one or more mutations disclosed in Table 20, optionally wherein the Fc region variant includes an N297A mutation.

[0023] In one aspect, a nucleic acid molecule is provided herein, encoding a multifunctional molecule disclosed herein or an antibody molecule disclosed herein. In one aspect, a vector is provided herein, e.g., an expression vector, comprising a nucleic acid molecule disclosed herein. In one aspect, a cell is provided herein, comprising a nucleic acid molecule disclosed herein or a vector disclosed herein. In one aspect, a pharmaceutical composition is provided herein, comprising a multifunctional molecule disclosed herein or an antibody molecule disclosed herein and a pharmaceutically acceptable carrier, excipient or stabilizer.

[0024] In one aspect, provided herein is a method for preparing, e.g., producing, a multifunctional molecule disclosed herein or an antibody molecule disclosed herein, comprising culturing a cell disclosed herein under suitable conditions, e.g., conditions suitable for gene expression and / or homodimerization or heterodimerization.

[0025] On the one hand, there is provided herein a method for treating cancer, comprising administering a multifunctional molecule disclosed herein or an antibody molecule disclosed herein to a subject in need thereof, wherein the multifunctional molecule or antibody molecule is administered in an amount effective to treat the cancer. In some embodiments, the method further comprises identifying, evaluating or selecting a subject in need of treatment, wherein identifying, evaluating or selecting comprises determining (e.g., directly determining or indirectly determining, e.g., obtaining information about the following) whether the subject has cancer cells expressing a T cell receptor including TRBC1 or TRBC2. In some embodiments, the method further comprises: in response to determining that the subject has cancer cells expressing a T cell receptor including TRBC1: optionally, selecting the subject for treatment with a multifunctional molecule, the multifunctional molecule comprising an antigen binding domain that binds to a T cell receptor including TRBC1; and administering a multifunctional molecule disclosed herein comprising an antigen binding domain that binds to a T cell receptor including TRBC1. In some embodiments, the method further comprises: in response to determining that the subject has cancer cells expressing a T cell receptor comprising TRBC2: optionally, selecting the subject for treatment with a multifunctional molecule comprising an antigen binding domain that binds to a T cell receptor comprising TRBC2; and administering a multifunctional molecule disclosed herein comprising an antigen binding domain that binds to a T cell receptor comprising TRBC2.

[0026] In one aspect, provided herein is a method for treating cancer, e.g., lymphoma or leukemia, e.g., T-cell lymphoma or leukemia, comprising: in response to determining that a subject has cancer cells expressing a T-cell receptor comprising TRBC1, administering to the subject a multifunctional molecule disclosed herein, wherein the first antigen binding domain of the multifunctional molecule binds to TRBC1, wherein the multifunctional molecule is administered in an amount effective to treat the cancer. In one aspect, provided herein is a method for treating cancer, e.g., lymphoma or leukemia, e.g., T-cell lymphoma or leukemia, comprising: in response to determining that a subject has cancer cells expressing a T-cell receptor comprising TRBC2, administering to the subject a multifunctional molecule disclosed herein, wherein the first antigen binding domain of the multifunctional molecule binds to TRBC2, wherein the multifunctional molecule is administered in an amount effective to treat the cancer.

[0027] In one aspect, provided herein is a method of identifying a subject in need of treatment for cancer, e.g., lymphoma or leukemia, e.g., T-cell lymphoma or leukemia, using a multifunctional molecule disclosed herein, the method comprising determining (e.g., directly determining or indirectly determining, e.g., obtaining information about) whether the subject has cancer cells expressing a T cell receptor comprising TRBC1 or TRBC2, wherein:

[0028] responsive to determining that the subject has cancer cells expressing a T cell receptor that includes TRBC1, identifying the subject as a candidate for treatment with a multifunctional molecule that includes an antigen binding domain that binds to TRBC1, and optionally identifying the subject as not a candidate for treatment with a multifunctional molecule that includes an antigen binding domain that binds to TRBC2, or

[0029] In response to determining that the subject has cancer cells expressing a T cell receptor that includes TRBC2, the subject is identified as a candidate for treatment with a multifunctional molecule that includes an antigen binding domain that binds to TRBC2, and optionally the subject is identified as not a candidate for treatment with a multifunctional molecule that includes an antigen binding domain that binds to TRBC1.

[0030] In some embodiments, the method further comprises:

[0031] In response to identifying the subject as a candidate for treatment with a multifunctional molecule comprising an antigen binding domain that binds to TRBC1, treating the subject with (e.g., administering to the subject) a multifunctional molecule comprising an antigen binding domain that binds to TRBC1, or

[0032] In response to identifying the subject as a candidate for treatment with a multifunctional molecule comprising an antigen binding domain that binds to TRBC2, the subject is treated with (eg, administered to the subject) a multifunctional molecule comprising an antigen binding domain that binds to TRBC2.

[0033] In some embodiments of the above methods, the cancer is a leukemia or lymphoma. In some embodiments, the cancer is selected from acquired immunodeficiency syndrome (AIDS)-related lymphoma, angioimmunoblastic T-cell lymphoma, adult T-cell leukemia / lymphoma, Burkitt's lymphoma, central nervous system (CNS) lymphoma, diffuse large B-cell lymphoma (DLBCL), lymphoblastic lymphoma, mantle cell lymphoma (MCL), peripheral T-cell lymphoma (PTCL) (e.g., hepatosplenic T-cell lymphoma (HSGDTCL), subcutaneous panniculitis-like T-cell lymphoma or enteropathy-associated T-cell lymphoma), transformed follicular lymphoma (PTCL), or leukemia / lymphoma. and transformed mucosa-associated lymphoid tissue (MALT) lymphoma, cutaneous T-cell lymphoma (mycosis fungoides and Sezary syndrome), follicular lymphoma, lymphoplasmacytic lymphoma / Waldenstrom's macroglobulinemia, marginal zone B-cell lymphoma, gastric mucosa-associated lymphoid tissue (MALT) lymphoma, chronic lymphocytic leukemia / small cell lymphocytic lymphoma (CLL / SLL), extranodal T-cell / NK-cell lymphoma (nasal type), and anaplastic large cell lymphoma (e.g., primary cutaneous anaplastic large cell lymphoma or systemic anaplastic large cell lymphoma). In some embodiments, the cancer is a lymphoma, which is a peripheral T-cell lymphoma (PTCL).

[0034] In one aspect, the invention provides a composition comprising a multifunctional molecule or an antibody molecule disclosed herein for use in a method of treating a subject having cancer.

[0035] Thus, in one aspect, the disclosure features a multifunctional molecule comprising:

[0036] (i) a first antigen binding domain that selectively binds to T cell receptor β chain constant domain 1 (TRBC1) or T cell receptor β chain constant domain 2 (TRBC2),

[0037] and

[0038] (ii) one, two or all of the following:

[0039] (a) an immune cell engager selected from an NK cell engager (e.g., a molecule that binds to NKp30, NKp46, NKG2D, or CD16), a T cell engager (e.g., one that binds to a T cell antigen other than CD3), a B cell engager, a dendritic cell engager, or a macrophage engager;

[0040] (b) cytokine molecules or cytokine inhibitor molecules;

[0041] (c) death receptor signaling adaptors; and

[0042] (d) Matrix modification part.

[0043] In another aspect, the disclosure features a multifunctional molecule including:

[0044] (i) a first antigen binding domain that selectively targets a lymphocyte expressing (e.g., on the cell surface, e.g., displays) a T cell receptor comprising a T cell receptor beta chain constant domain 1 (TRBC1), TRBC1, a T cell receptor comprising a T cell receptor beta chain constant domain 2 (TRBC2), or TRBC2,

[0045] and

[0046] (ii) one, two or all of the following:

[0047] (a) an immune cell engager selected from an NK cell engager (e.g., a molecule that binds to NKp30, NKp46, NKG2D, or CD16), a T cell engager (e.g., one that binds to a T cell antigen other than CD3), a B cell engager, a dendritic cell engager, or a macrophage engager;

[0048] (b) cytokine molecules or cytokine inhibitor molecules;

[0049] (c) death receptor signaling adaptors; and

[0050] (c) Matrix modification part.

[0051] In another aspect, the disclosure features a multifunctional molecule including:

[0052] (i) a first antigen binding domain that preferentially binds to a tumor antigen (e.g., a T cell receptor including T cell receptor β chain constant domain 1 (TRBC1), TRBC1, a T cell receptor including T cell receptor β chain constant domain 2 (TRBC2), or TRBC2) on a lymphoma cell (e.g., a T cell),

[0053] and

[0054] (ii) one, two or all of the following:

[0055] (a) an immune cell engager selected from an NK cell engager (e.g., a molecule that binds to NKp30, NKp46, NKG2D, or CD16), a T cell engager (e.g., one that binds to a T cell antigen other than CD3), a B cell engager, a dendritic cell engager, or a macrophage engager;

[0056] (b) cytokine molecules or cytokine inhibitor molecules;

[0057] (c) death receptor signaling adaptors; and

[0058] (d) Matrix modification part.

[0059] In another aspect, the disclosure features an antibody molecule, e.g., an IgM antibody molecule, including:

[0060] (i) a first antigen binding domain that selectively binds to T cell receptor β chain constant domain 1 (TRBC1) or T cell receptor β chain constant domain 2 (TRBC2),

[0061] and

[0062] (ii) The complement activation domain, which activates the complement pathway, for example, by binding to C1q.

[0063] In another aspect, the disclosure features an antibody molecule comprising:

[0064] a heavy chain variable region (VH), comprising a heavy chain framework region 1 (VHFWR1) amino acid sequence of SEQ ID NO: 215 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations (e.g., substitutions, additions or deletions) thereof), a VHFWR2 amino acid sequence of SEQ ID NO: 216 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations (e.g., substitutions, additions or deletions) thereof), a VHFWR3 amino acid sequence of SEQ ID NO: 217 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations (e.g., substitutions, additions or deletions) thereof) and / or a VHFWR4 amino acid sequence of SEQ ID NO: 218 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations (e.g., substitutions, additions or deletions) thereof), and

[0065] A light chain variable region (VL), which includes a light chain framework region 1 (VLFWR1) amino acid sequence of SEQ ID NO: 238 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations (e.g., substitutions, additions or deletions) thereof), a VLFWR2 amino acid sequence of SEQ ID NO: 239 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations (e.g., substitutions, additions or deletions) thereof), a VLFWR3 amino acid sequence of SEQ ID NO: 240 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations (e.g., substitutions, additions or deletions) thereof) and / or a VLFWR4 amino acid sequence of SEQ ID NO: 241 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations (e.g., substitutions, additions or deletions) thereof).

[0066] In some embodiments, the antibody molecule or fragment thereof comprises a VH comprising the amino acid sequence of SEQ ID NO: 253 (or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95% or 99% sequence identity thereof), and / or a VL comprising the amino acid sequence of SEQ ID NO: 258 (or an amino acid sequence having at least about 93%, 95% or 99% sequence identity thereof).

[0067] In another aspect, the disclosure features a nucleic acid molecule encoding a multifunctional molecule disclosed herein.

[0068] In another aspect, the disclosure features a vector, e.g., an expression vector, that includes a nucleic acid molecule disclosed herein.

[0069] In another aspect, the disclosure features a host cell including a nucleic acid molecule or a vector disclosed herein.

[0070] In another aspect, the disclosure features a method of making, e.g., producing, a multifunctional molecule disclosed herein, comprising culturing a host cell disclosed herein under suitable conditions, e.g., conditions suitable for gene expression and / or homodimerization or heterodimerization.

[0071] In another aspect, the disclosure features a pharmaceutical composition including a multifunctional molecule disclosed herein.

[0072] On the other hand, the present disclosure features a method for treating cancer, comprising administering a multifunctional molecule disclosed herein to a subject in need thereof, wherein the multifunctional molecule is administered in an amount effective to treat the cancer. In some embodiments, the cancer is a T-cell malignancy, e.g., a T-cell lymphoma or a T-cell leukemia. In some embodiments, the cancer is selected from: anaplastic large cell lymphoma (ALCL); angioimmunoblastic T-cell lymphoma; peripheral T-cell lymphoma (PTCL), not specifically designated (NOS); cutaneous T-cell lymphoma (CTCL); NKT-cell lymphoma; Sezary syndrome; T acute lymphoblastic leukemia or lymphoma; adult T-cell leukemia or lymphoma; T prolymphocytic leukemia; and T large granular leukemia. In some embodiments, the cancer is PTCL. In some embodiments, TRBC subtype expression is analyzed by, for example, flow cytometric analysis of fresh tumor tissue. In some embodiments, the multifunctional molecule is used in combination with a second agent. In some embodiments, the second agent is a histone deacetylase (HDAC) inhibitor, for example, romidepsin or belinostat. In some embodiments, the second agent is a kinase or enzyme inhibitor. In some embodiments, the second agent is a PI3K inhibitor, for example, duvelisib. In some embodiments, the second agent is a farnesyl transferase inhibitor, for example, tipifarnib. In some embodiments, the second agent is a SYK / JAK inhibitor, for example, cerdulatinib. In some embodiments, the second agent is chemotherapy.

[0073] In some embodiments, the second agent is

[0074] In another aspect, the disclosure features a method of identifying a subject in need of treatment for cancer using a multifunctional molecule disclosed herein, the method comprising determining (e.g., directly determining or indirectly determining, e.g., obtaining information about) whether the subject has cancer cells that express a T cell receptor that includes TRBC1 or TRBC2, wherein:

[0075] responsive to determining that the subject has cancer cells expressing a T cell receptor that includes TRBC1, identifying the subject as a candidate for treatment with a multifunctional molecule that includes an antigen binding domain that binds to TRBC1, and optionally identifying the subject as not a candidate for treatment with a multifunctional molecule that includes an antigen binding domain that binds to TRBC2, and

[0076] In response to determining that the subject has cancer cells expressing a T cell receptor that includes TRBC2, the subject is identified as a candidate for treatment with a multifunctional molecule that includes an antigen binding domain that binds to TRBC2, and optionally the subject is identified as not a candidate for treatment with a multifunctional molecule that includes an antigen binding domain that binds to TRBC1.

[0077] In another aspect, the disclosure features a method of evaluating a subject in need of treatment for cancer, e.g., lymphoma, comprising determining (e.g., directly determining or indirectly determining, e.g., obtaining information about) whether the subject has cancer cells expressing a T cell receptor comprising TRBC1 or TRBC2.

[0078] Additional features of any of the above multifunctional molecules, nucleic acids, vectors, host cells or methods include one or more of the embodiments listed below.

[0079] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the embodiments listed below.

[0080] List of implementation plans

[0081] 1. A multifunctional molecule comprising:

[0082] (i) a first antigen binding domain that preferentially binds to a tumor antigen on a lymphoma cell (e.g., a T cell), wherein the tumor antigen is T cell receptor β chain constant domain 1 (TRBC1) or T cell receptor β chain constant domain 2 (TRBC2),

[0083] and

[0084] (ii) one, two or all of the following:

[0085] (a) an immune cell engager selected from a NK cell engager (e.g., a molecule that binds to NKp30, NKp46, NKG2D, or CD16), a T cell engager, a B cell engager, a dendritic cell engager, or a macrophage engager;

[0086] (b) cytokine molecules or cytokine inhibitor molecules;

[0087] (c) death receptor signaling adaptors; and

[0088] (d) Matrix modification part.

[0089] 1A. A multifunctional molecule comprising:

[0090] (i) a first antigen binding domain that selectively binds to T cell receptor β chain constant domain 1 (TRBC1) or T cell receptor β chain constant domain 2 (TRBC2),

[0091] and

[0092] (ii) one, two or all of the following:

[0093] (a) an immune cell engager selected from an NK cell engager (e.g., a molecule that binds to NKp30, NKp46, NKG2D, or CD16), a T cell engager that binds to a T cell antigen other than CD3, a B cell engager, a dendritic cell engager, or a macrophage engager;

[0094] (b) cytokine molecules or cytokine inhibitor molecules;

[0095] (c) death receptor signaling adaptors; and

[0096] (d) Matrix modification part.

[0097] 2. A multifunctional molecule comprising:

[0098] (i) a first antigen binding domain that selectively targets lymphocytes expressing T cell receptor β chain constant domain 1 (TRBC1) or T cell receptor β chain constant domain 2 (TRBC2),

[0099] and

[0100] (ii) one, two or all of the following:

[0101] (a) an immune cell engager selected from a NK cell engager (e.g., a molecule that binds to NKp30, NKp46, NKG2D, or CD16), a T cell engager, a B cell engager, a dendritic cell engager, or a macrophage engager;

[0102] (b) cytokine molecules or cytokine inhibitor molecules;

[0103] (c) death receptor signaling adaptors; and

[0104] (d) Matrix modification part.

[0105] 3. The multifunctional molecule according to any one of the preceding embodiments, wherein the multifunctional molecule:

[0106] (i) specifically binds to an epitope of TRBC1 or TRBC2 (e.g., an epitope that is the same as or similar to an epitope recognized by an anti-TRBC1 or anti-TRBC2 antibody molecule described herein);

[0107] (ii) exhibit the same or similar binding affinity or specificity, or both, as the anti-TRBC1 or anti-TRBC2 antibody molecules described herein;

[0108] (iii) inhibiting, e.g., competitively inhibiting, the binding of an anti-TRBC1 or anti-TRBC2 antibody molecule described herein;

[0109] (iv) binds to the same or overlapping epitope as the anti-TRBC1 or anti-TRBC2 antibody molecules described herein; or

[0110] (v) competes for binding and / or binds to the same epitope as an anti-TRBC1 or anti-TRBC2 antibody molecule described herein.

[0111] 4. The multifunctional molecule of embodiment 3, wherein the anti-TRBC1 or anti-TRBC2 antibody molecule comprises one or more CDRs, framework regions, variable domains, heavy chains or light chains, or antigen binding domains selected from Tables 2-5, or sequences substantially identical thereto.

[0112] 5. The multifunctional molecule according to any one of embodiments 1 to 4, wherein the antigen or tumor antigen is TRBC1.

[0113] 6. The multifunctional molecule according to any one of embodiments 1 to 4, wherein the antigen or tumor antigen is TRBC2.

[0114] 7. The multifunctional molecule of any one of embodiments 1-5, wherein the first antigen binding domain comprises:

[0115] (i) a heavy chain variable region (VH) comprising a heavy chain complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 200 (or a sequence having no more than 1, 2, 3 or 4 mutations (e.g., substitutions, additions or deletions)), a VHCDR2 amino acid sequence of SEQ ID NO: 201 (or a sequence having no more than 1, 2, 3 or 4 mutations (e.g., substitutions, additions or deletions)) and / or a VHCDR3 amino acid sequence of SEQ ID NO: 202 (or a sequence having no more than 1, 2, 3 or 4 mutations (e.g., substitutions, additions or deletions)); and

[0116] (ii) a light chain variable region (VL), which includes a light chain complementary determining region 1 (VLCDR1) amino acid sequence of SEQ ID NO: 223 (or a sequence having no more than 1, 2, 3 or 4 mutations (e.g., substitutions, additions or deletions)), a VLCDR2 amino acid sequence of SEQ ID NO: 224 (or a sequence having no more than 1, 2, 3 or 4 mutations (e.g., substitutions, additions or deletions)) and / or a VLCDR3 amino acid sequence of SEQ ID NO: 225 (or a sequence having no more than 1, 2, 3 or 4 mutations (e.g., substitutions, additions or deletions)).

[0117] 8. The multifunctional molecule of embodiment 7, wherein the first antigen binding domain comprises:

[0118] (i) a heavy chain variable region (VH), comprising a heavy chain complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 200, a VHCDR2 amino acid sequence of SEQ ID NO: 201, and / or a VHCDR3 amino acid sequence of SEQ ID NO: 202, and

[0119] (ii) a light chain variable region (VL), which includes a light chain complementary determining region 1 (VLCDR1) amino acid sequence of SEQ ID NO: 223, a VLCDR2 amino acid sequence of SEQ ID NO: 224 and / or a VLCDR3 amino acid sequence of SEQ ID NO: 225.

[0120] 9. The multifunctional molecule of any one of embodiments 1-5, 7 or 8, wherein the first antigen binding domain comprises:

[0121] (1) a heavy chain variable region (VH), comprising a heavy chain framework region 1 (VHFWR1) amino acid sequence of SEQ ID NO: 203 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations (e.g., substitutions, additions or deletions) thereof), a VHFWR2 amino acid sequence of SEQ ID NO: 204 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations (e.g., substitutions, additions or deletions) thereof), a VHFWR3 amino acid sequence of SEQ ID NO: 205 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations (e.g., substitutions, additions or deletions) thereof), or a VHFWR4 amino acid sequence of SEQ ID NO: 206 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations (e.g., substitutions, additions or deletions) thereof), and / or

[0122] (2) a light chain variable region (VL), comprising a light chain framework region 1 (VLFWR1) amino acid sequence of SEQ ID NO: 226 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations (e.g., substitutions, additions or deletions) thereof), a VLFWR2 amino acid sequence of SEQ ID NO: 227 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations (e.g., substitutions, additions or deletions) thereof), a VLFWR3 amino acid sequence of SEQ ID NO: 228 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations (e.g., substitutions, additions or deletions) thereof), or a VLFWR4 amino acid sequence of SEQ ID NO: 229 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations (e.g., substitutions, additions or deletions) thereof).

[0123] 10. The multifunctional molecule of embodiment 9, wherein the first antigen binding domain comprises:

[0124] (1) a heavy chain variable region (VH), the heavy chain variable region comprising the heavy chain framework region 1 (VHFWR1) amino acid sequence of SEQ ID NO: 203, the VHFWR2 amino acid sequence of SEQ ID NO: 204, the VHFWR3 amino acid sequence of SEQ ID NO: 205, or the VHFWR4 amino acid sequence of SEQ ID NO: 206, and / or

[0125] (2) a light chain variable region (VL), comprising a light chain framework region 1 (VLFWR1) amino acid sequence of SEQ ID NO: 226, a VLFWR2 amino acid sequence of SEQ ID NO: 227, a VLFWR3 amino acid sequence of SEQ ID NO: 228, or a VLFWR4 amino acid sequence of SEQ ID NO: 229.

[0126] 11. The multifunctional molecule of any one of embodiments 1-5 or 7-10, wherein the first antigen binding domain comprises:

[0127] (i) a VH comprising the amino acid sequence of SEQ ID NO: 250 (or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NO: 250), and / or

[0128] (ii) a VL comprising the amino acid sequence of SEQ ID NO: 255 (or an amino acid sequence having at least about 93%, 95%, or 99% sequence identity to SEQ ID NO: 255).

[0129] 12. The multifunctional molecule of any one of embodiments 1-5 or 7-11, wherein the first antigen binding domain comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 6154, 6155, 6167 or 6168 (or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95% or 99% sequence identity with SEQ ID NO: 6154, 6155, 6167 or 6168).

[0130] 13. The multifunctional molecule of any one of embodiments 1-5 or 7-12, wherein the first antigen binding domain comprises a light chain comprising the amino acid sequence of SEQ ID NO: 6156 or 6169 (or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95% or 99% sequence identity with SEQ ID NO: 6156 or 6169).

[0131] 14. The multifunctional molecule of any one of embodiments 1-5, 7 or 8, wherein the first antigen binding domain comprises a heavy chain variable region (VH), the heavy chain variable region comprising the heavy chain framework region 1 (VHFWR1) amino acid sequence of SEQ ID NO: 207 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations (e.g., substitutions, additions or deletions) thereof), the VHFWR2 amino acid sequence of SEQ ID NO: 208 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations (e.g., substitutions, additions or deletions) thereof), the VHFWR3 amino acid sequence of SEQ ID NO: 209 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations (e.g., substitutions, additions or deletions) thereof), or the VHFWR4 amino acid sequence of SEQ ID NO: 210 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations (e.g., substitutions, additions or deletions) thereof).

[0132] 15. The multifunctional molecule of embodiment 14, wherein the first antigen binding domain comprises a heavy chain variable region (VH), the heavy chain variable region comprising the heavy chain framework region 1 (VHFWR1) amino acid sequence of SEQ ID NO: 207, the VHFWR2 amino acid sequence of SEQ ID NO: 208, the VHFWR3 amino acid sequence of SEQ ID NO: 209, or the VHFWR4 amino acid sequence of SEQ ID NO: 210.

[0133] 16. The multifunctional molecule of any one of embodiments 1-5, 7 or 8, wherein the first antigen binding domain comprises a heavy chain variable region (VH), the heavy chain variable region comprising the heavy chain framework region 1 (VHFWR1) amino acid sequence of SEQ ID NO: 211 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations (e.g., substitutions, additions or deletions) thereof), the VHFWR2 amino acid sequence of SEQ ID NO: 212 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations (e.g., substitutions, additions or deletions) thereof), the VHFWR3 amino acid sequence of SEQ ID NO: 213 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations (e.g., substitutions, additions or deletions) thereof), or the VHFWR4 amino acid sequence of SEQ ID NO: 214 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations (e.g., substitutions, additions or deletions) thereof).

[0134] 17. The multifunctional molecule of embodiment 16, wherein the first antigen binding domain comprises a heavy chain variable region (VH), the heavy chain variable region comprising the heavy chain framework region 1 (VHFWR1) amino acid sequence of SEQ ID NO:211, the VHFWR2 amino acid sequence of SEQ ID NO:212, the VHFWR3 amino acid sequence of SEQ ID NO:213, or the VHFWR4 amino acid sequence of SEQ ID NO:214.

[0135] 18. The multifunctional molecule of any one of embodiments 1-5, 7 or 8, wherein the first antigen binding domain comprises a heavy chain variable region (VH), the heavy chain variable region comprising the heavy chain framework region 1 (VHFWR1) amino acid sequence of SEQ ID NO: 215 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations (e.g., substitutions, additions or deletions) thereof), the VHFWR2 amino acid sequence of SEQ ID NO: 216 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations (e.g., substitutions, additions or deletions) thereof), the VHFWR3 amino acid sequence of SEQ ID NO: 217 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations (e.g., substitutions, additions or deletions) thereof), or the VHFWR4 amino acid sequence of SEQ ID NO: 218 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations (e.g., substitutions, additions or deletions) thereof).

[0136] 19. The multifunctional molecule of embodiment 18, wherein the first antigen binding domain comprises a heavy chain variable region (VH), the heavy chain variable region comprising the heavy chain framework region 1 (VHFWR1) amino acid sequence of SEQ ID NO:215, the VHFWR2 amino acid sequence of SEQ ID NO:216, the VHFWR3 amino acid sequence of SEQ ID NO:217, or the VHFWR4 amino acid sequence of SEQ ID NO:218.

[0137] 20. The multifunctional molecule of any one of embodiments 1-5, 7 or 8, wherein the first antigen binding domain comprises a heavy chain variable region (VH), the heavy chain variable region comprising the heavy chain framework region 1 (VHFWR1) amino acid sequence of SEQ ID NO: 219 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations (e.g., substitutions, additions or deletions) thereof), the VHFWR2 amino acid sequence of SEQ ID NO: 220 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations (e.g., substitutions, additions or deletions) thereof), the VHFWR3 amino acid sequence of SEQ ID NO: 221 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations (e.g., substitutions, additions or deletions) thereof), or the VHFWR4 amino acid sequence of SEQ ID NO: 222 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations (e.g., substitutions, additions or deletions) thereof).

[0138] 21. The multifunctional molecule of embodiment 20, wherein the first antigen binding domain comprises a heavy chain variable region (VH), the heavy chain variable region comprising the heavy chain framework region 1 (VHFWR1) amino acid sequence of SEQ ID NO:219, the VHFWR2 amino acid sequence of SEQ ID NO:220, the VHFWR3 amino acid sequence of SEQ ID NO:221, or the VHFWR4 amino acid sequence of SEQ ID NO:222.

[0139] 22. The multifunctional molecule of any one of embodiments 1-5, 7, 8 or 14-21, wherein the first antigen binding domain comprises a light chain variable region (VL), the light chain variable region comprising the light chain framework region 1 (VLFWR1) amino acid sequence of SEQ ID NO: 230 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations (e.g., substitutions, additions or deletions) thereof), the VLFWR2 amino acid sequence of SEQ ID NO: 231 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations (e.g., substitutions, additions or deletions) thereof), the VLFWR3 amino acid sequence of SEQ ID NO: 232 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations (e.g., substitutions, additions or deletions) thereof), or the VLFWR4 amino acid sequence of SEQ ID NO: 233 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations (e.g., substitutions, additions or deletions) thereof).

[0140] 23. A multifunctional molecule of embodiment 22, wherein the first antigen binding domain comprises a light chain variable region (VL), and the light chain variable region comprises a light chain framework region 1 (VLFWR1) amino acid sequence of SEQ ID NO:230, a VLFWR2 amino acid sequence of SEQ ID NO:231, a VLFWR3 amino acid sequence of SEQ ID NO:232, or a VLFWR4 amino acid sequence of SEQ ID NO:233.

[0141] 24. The multifunctional molecule of any one of embodiments 1-5, 7, 8 or 14-21, wherein the first antigen binding domain comprises a light chain variable region (VL), the light chain variable region comprising the light chain framework region 1 (VLFWR1) amino acid sequence of SEQ ID NO: 234 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations (e.g., substitutions, additions or deletions) thereof), the VLFWR2 amino acid sequence of SEQ ID NO: 235 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations (e.g., substitutions, additions or deletions) thereof), the VLFWR3 amino acid sequence of SEQ ID NO: 236 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations (e.g., substitutions, additions or deletions) thereof), or the VLFWR4 amino acid sequence of SEQ ID NO: 237 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations (e.g., substitutions, additions or deletions) thereof).

[0142] 25. A multifunctional molecule according to embodiment 24, wherein the first antigen binding domain comprises a light chain variable region (VL), the light chain variable region comprising a light chain framework region 1 (VLFWR1) amino acid sequence of SEQ ID NO:234, a VLFWR2 amino acid sequence of SEQ ID NO:235, a VLFWR3 amino acid sequence of SEQ ID NO:236, or a VLFWR4 amino acid sequence of SEQ ID NO:237.

[0143] 26. The multifunctional molecule of any one of embodiments 1-5, 7, 8 or 14-21, wherein the first antigen binding domain comprises a light chain variable region (VL), the light chain variable region comprising the light chain framework region 1 (VLFWR1) amino acid sequence of SEQ ID NO: 238 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations (e.g., substitutions, additions or deletions) thereof), the VLFWR2 amino acid sequence of SEQ ID NO: 239 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations (e.g., substitutions, additions or deletions) thereof), the VLFWR3 amino acid sequence of SEQ ID NO: 240 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations (e.g., substitutions, additions or deletions) thereof), or the VLFWR4 amino acid sequence of SEQ ID NO: 241 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations (e.g., substitutions, additions or deletions) thereof).

[0144] 27. A multifunctional molecule of embodiment 26, wherein the first antigen binding domain comprises a light chain variable region (VL), the light chain variable region comprising a light chain framework region 1 (VLFWR1) amino acid sequence of SEQ ID NO:238, a VLFWR2 amino acid sequence of SEQ ID NO:239, a VLFWR3 amino acid sequence of SEQ ID NO:240, or a VLFWR4 amino acid sequence of SEQ ID NO:241.

[0145] 28. The multifunctional molecule of any one of embodiments 1-5, 7, 8 or 14-21, wherein the first antigen binding domain comprises a light chain variable region (VL), the light chain variable region comprising the light chain framework region 1 (VLFWR1) amino acid sequence of SEQ ID NO: 242 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations (e.g., substitutions, additions or deletions) thereof), the VLFWR2 amino acid sequence of SEQ ID NO: 243 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations (e.g., substitutions, additions or deletions) thereof), the VLFWR3 amino acid sequence of SEQ ID NO: 244 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations (e.g., substitutions, additions or deletions) thereof), or the VLFWR4 amino acid sequence of SEQ ID NO: 245 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations (e.g., substitutions, additions or deletions) thereof).

[0146] 29. A multifunctional molecule according to embodiment 28, wherein the first antigen binding domain comprises a light chain variable region (VL), the light chain variable region comprising a light chain framework region 1 (VLFWR1) amino acid sequence of SEQ ID NO:242, a VLFWR2 amino acid sequence of SEQ ID NO:243, a VLFWR3 amino acid sequence of SEQ ID NO:244, or a VLFWR4 amino acid sequence of SEQ ID NO:245.

[0147] 30. The multifunctional molecule of any one of embodiments 1-5, 7, 8 or 14-21, wherein the first antigen binding domain comprises a light chain variable region (VL), the light chain variable region comprising the light chain framework region 1 (VLFWR1) amino acid sequence of SEQ ID NO: 246 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations (e.g., substitutions, additions or deletions) thereof), the VLFWR2 amino acid sequence of SEQ ID NO: 247 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations (e.g., substitutions, additions or deletions) thereof), the VLFWR3 amino acid sequence of SEQ ID NO: 248 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations (e.g., substitutions, additions or deletions) thereof), or the VLFWR4 amino acid sequence of SEQ ID NO: 249 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations (e.g., substitutions, additions or deletions) thereof).

[0148] 31. A multifunctional molecule according to embodiment 30, wherein the first antigen binding domain comprises a light chain variable region (VL), the light chain variable region comprising a light chain framework region 1 (VLFWR1) amino acid sequence of SEQ ID NO:246, a VLFWR2 amino acid sequence of SEQ ID NO:247, a VLFWR3 amino acid sequence of SEQ ID NO:248, or a VLFWR4 amino acid sequence of SEQ ID NO:249.

[0149] 32. The multifunctional molecule of any one of embodiments 1-5, 7 or 8, wherein the first antigen binding domain comprises a VH comprising the amino acid sequence of SEQ ID NO: 251 (or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95% or 99% sequence identity thereto).

[0150] 33. The multifunctional molecule of any one of embodiments 1-5, 7 or 8, wherein the first antigen binding domain comprises a VH comprising the amino acid sequence of SEQ ID NO: 252 (or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95% or 99% sequence identity thereto).

[0151] 34. The multifunctional molecule of any one of embodiments 1-5, 7 or 8, wherein the first antigen binding domain comprises a VH comprising the amino acid sequence of SEQ ID NO: 253 (or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95% or 99% sequence identity thereto).

[0152] 35. The multifunctional molecule of any one of embodiments 1-5, 7 or 8, wherein the first antigen binding domain comprises a VH comprising the amino acid sequence of SEQ ID NO: 254 (or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95% or 99% sequence identity thereto).

[0153] 36. The multifunctional molecule of any one of embodiments 1-5, 7, 8 or 32-35, wherein the first antigen binding domain comprises a VL comprising the amino acid sequence of SEQ ID NO: 256 (or an amino acid sequence having at least about 93%, 95% or 99% sequence identity thereto).

[0154] 37. The multifunctional molecule of any one of embodiments 1-5, 7, 8 or 32-35, wherein the first antigen binding domain comprises a VL comprising the amino acid sequence of SEQ ID NO: 257 (or an amino acid sequence having at least about 93%, 95% or 99% sequence identity thereto).

[0155] 38. The multifunctional molecule of any one of embodiments 1-5, 7, 8 or 32-35, wherein the first antigen binding domain comprises a VL comprising the amino acid sequence of SEQ ID NO: 258 (or an amino acid sequence having at least about 93%, 95% or 99% sequence identity thereto).

[0156] 39. The multifunctional molecule of any one of embodiments 1-5, 7, 8 or 32-35, wherein the first antigen binding domain comprises a VL comprising the amino acid sequence of SEQ ID NO: 259 (or an amino acid sequence having at least about 93%, 95% or 99% sequence identity thereto).

[0157] 40. The multifunctional molecule of any one of embodiments 1-5, 7, 8 or 32-35, wherein the first antigen binding domain comprises a VL comprising the amino acid sequence of SEQ ID NO: 260 (or an amino acid sequence having at least about 93%, 95% or 99% sequence identity thereto).

[0158] 41. The multifunctional molecule of any one of embodiments 1-5, 7 or 8, wherein the first antigen binding domain comprises:

[0159] (i) a heavy chain variable region (VH), comprising a heavy chain framework region 1 (VHFWR1) amino acid sequence of SEQ ID NO: 215 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations (e.g., substitutions, additions or deletions) thereof), a VHFWR2 amino acid sequence of SEQ ID NO: 216 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations (e.g., substitutions, additions or deletions) thereof), a VHFWR3 amino acid sequence of SEQ ID NO: 217 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations (e.g., substitutions, additions or deletions) thereof), or a VHFWR4 amino acid sequence of SEQ ID NO: 218 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations (e.g., substitutions, additions or deletions) thereof), and

[0160] (ii) a light chain variable region (VL), which comprises a light chain framework region 1 (VLFWR1) amino acid sequence of SEQ ID NO: 238 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations (e.g., substitutions, additions or deletions) thereof), a VLFWR2 amino acid sequence of SEQ ID NO: 239 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations (e.g., substitutions, additions or deletions) thereof), a VLFWR3 amino acid sequence of SEQ ID NO: 240 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations (e.g., substitutions, additions or deletions) thereof), or a VLFWR4 amino acid sequence of SEQ ID NO: 241 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations (e.g., substitutions, additions or deletions) thereof).

[0161] 42. The multifunctional molecule of embodiment 41, wherein the first antigen binding domain comprises:

[0162] (i) a heavy chain variable region (VH), the heavy chain variable region comprising the heavy chain framework region 1 (VHFWR1) amino acid sequence of SEQ ID NO: 215, the VHFWR2 amino acid sequence of SEQ ID NO: 216, the VHFWR3 amino acid sequence of SEQ ID NO: 217, or the VHFWR4 amino acid sequence of SEQ ID NO: 218, and

[0163] (ii) a light chain variable region (VL), which includes a light chain framework region 1 (VLFWR1) amino acid sequence of SEQ ID NO:238, a VLFWR2 amino acid sequence of SEQ ID NO:239, a VLFWR3 amino acid sequence of SEQ ID NO:240, or a VLFWR4 amino acid sequence of SEQ ID NO:241.

[0164] 43. The multifunctional molecule of any one of embodiments 41 or 42, wherein the first antigen binding domain comprises:

[0165] (i) a VH comprising the amino acid sequence of SEQ ID NO: 253 (or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto), and

[0166] (ii) a VL comprising the amino acid sequence of SEQ ID NO: 258 (or an amino acid sequence having at least about 93%, 95%, or 99% sequence identity thereto).

[0167] 44. The multifunctional molecule of any one of embodiments 1-43, wherein the first antigen binding domain has a higher affinity for a T cell receptor that includes TRBC1 than for a T cell receptor that does not include TRBC1, optionally wherein the K affinity of the binding between the first antigen binding domain and TRBC1 is D does not exceed the K of binding between the first antigen binding domain and the T cell receptor excluding TRBC1 D 40%, 30%, 20%, 10%, 1%, 0.1% or 0.01%.

[0168] 45. The multifunctional molecule of any one of embodiments 1-4 or 6, wherein the first antigen binding domain has a higher affinity for a T cell receptor that includes TRBC2 than for a T cell receptor that does not include TRBC2, optionally wherein the K of the binding between the first antigen binding domain and TRBC2 is D does not exceed the K of binding between the first antigen binding domain and the T cell receptor excluding TRBC2 D 40%, 30%, 20%, 10%, 1%, 0.1% or 0.01%.

[0169] 46. ​​The multifunctional molecule of any one of embodiments 1-44, wherein the first antigen binding domain has a higher affinity for a T cell receptor comprising TRBC1 than for a T cell receptor comprising TRBC2, optionally wherein the K affinity of the binding between the first antigen binding domain and TRBC1 is D does not exceed the K of binding between the first antigen binding domain and the T cell receptor including TRBC2 D 40%, 30%, 20%, 10%, 1%, 0.1% or 0.01%.

[0170] 47. The multifunctional molecule of any one of embodiments 1-4, 6 or 45, wherein the first antigen binding domain has a higher affinity for a T cell receptor comprising TRBC2 than for a T cell receptor comprising TRBC1, optionally wherein the K affinity of the binding between the first antigen binding domain and TRBC2 is D No more than the K of the binding between the first antigen binding domain and the T cell receptor including TRBC1 D 40%, 30%, 20%, 10%, 1%, 0.1% or 0.01%.

[0171] 48. The multifunctional molecule of any preceding embodiment, wherein binding of the first antigen binding domain to TRBC1 or TRBC2 on a lymphoma cell or a lymphocyte (e.g., a T cell) or to a tumor antigen on a lymphoma cell (e.g., a T cell) does not activate the lymphoma cell or the lymphocyte, e.g., a T cell.

[0172] 49. The multifunctional molecule of any preceding embodiment, wherein binding of the first antigen binding domain to TRBC1 or TRBC2 on lymphoma cells or lymphocytes (e.g., T cells) or to a tumor antigen on lymphoma cells (e.g., T cells) does not significantly activate lymphoma cells or lymphocytes such as T cells (e.g., as measured by T cell proliferation, expression of T cell activation markers (e.g., CD69 or CD25), and / or expression of cytokines (e.g., TNFα and IFNγ)).

[0173] 50. The multifunctional molecule of any one of embodiments 1 or 3-49, wherein the multifunctional molecule preferentially binds to lymphoma cells compared to non-lymphoma cells, optionally wherein the binding between the multifunctional molecule and lymphoma cells is greater than 10, 20, 30, 40 or 50 times greater than the binding between the multifunctional molecule and non-lymphoma cells.

[0174] 51. The multifunctional molecule of any one of embodiments 2-47, wherein:

[0175] (i) the binding of the multifunctional molecule to lymphocytes expressing TRBC1 is greater than 10, 20, 30, 40 or 50 times greater than the binding of the multifunctional molecule to lymphocytes not expressing TRBC1, or

[0176] (ii) the binding of the multifunctional molecule to lymphocytes expressing TRBC2 is greater than 10, 20, 30, 40 or 50 times greater than the binding of the multifunctional molecule to lymphocytes not expressing TRBC2.

[0177] 52. The multifunctional molecule of any one of embodiments 1-51, wherein the multifunctional molecule comprises an immune cell engager selected from a NK cell engager, a T cell engager, a B cell engager, a dendritic cell engager or a macrophage engager.

[0178] 53. The multifunctional molecule of embodiment 52, wherein the immune cell engager binds to and activates an immune cell, such as an effector cell.

[0179] 54. The multifunctional molecule of embodiment 52, wherein the immune cell engager binds to but does not activate immune cells, such as effector cells.

[0180] 55. The multifunctional molecule of any one of embodiments 52-54, wherein the immune cell engager is a T cell engager, e.g., a T cell engager that mediates binding and activation of T cells, or a T cell engager that mediates binding but not activation of T cells.

[0181] 56. The multifunctional molecule of embodiment 55, wherein the T cell engager binds to TCRα, TCRβ, TCRγ, TCRζ, ICOS, CD28, CD27, HVEM, LIGHT, CD40, 4-1BB, OX40, DR3, GITR, CD30, TIM1, SLAM, CD2 or CD226, for example, the T cell engager is an anti-TCRβ antibody molecule.

[0182] 57. The multifunctional molecule of any one of embodiments 52-54, wherein the immune cell engager is a NK cell engager, e.g., a NK cell engager that mediates binding and activation of NK cells, or a NK cell engager that mediates binding but does not activate NK cells.

[0183] 58. The multifunctional molecule of embodiment 57, wherein the NK cell engager is selected from an antibody molecule (e.g., an antigen binding domain) or a ligand, which binds to (e.g., activates): NKp30, NKp40, NKp44, NKp46, NKG2D, DNAM1, DAP10, CD16 (e.g., CD16a, CD16b or both), CRTAM, CD27, PSGL1, CD96, CD100 (SEMA4D), NKp80, CD244 (also known as SLAMF4 or 2B4), SLAMF6, SLAMF7, KIR2DS2, KIR2DS4, KIR3DS1, KIR2DS3, KIR2DS5, KIR2DS1, CD94, NKG2C, NKG2E or CD160, for example, the NK cell engager is an antibody molecule or ligand that binds to (e.g., activates) NKp30.

[0184] 59. The multifunctional molecule of embodiment 57, wherein the NK cell engager is an antibody molecule, e.g., an antigen binding domain.

[0185] 60. The multifunctional molecule of any one of embodiments 58 or 59, wherein the NK cell engager is capable of engaging a NK cell.

[0186] 61. The multifunctional molecule of any one of embodiments 57-60, wherein the NK cell engager is an antibody molecule, e.g., an antigen binding domain, that binds NKp30, NKp46, NKG2D or CD16.

[0187] 62. The multifunctional molecule of any preceding embodiment, wherein the multifunctional molecule:

[0188] (i) specifically binds to an epitope of NKp30, NKp46, NKG2D or CD16, e.g., an epitope that is the same or similar to an epitope recognized by an anti-NKp30, anti-NKp46, anti-NKG2D or anti-CD16 antibody molecule described herein;

[0189] (ii) exhibit the same or similar binding affinity or specificity, or both, as the anti-NKp30, anti-NKp46, anti-NKG2D or anti-CD16 antibody molecules described herein;

[0190] (iii) inhibiting, e.g., competitively inhibiting, the binding of an anti-NKp30, anti-NKp46, anti-NKG2D or anti-CD16 antibody molecule described herein;

[0191] (iv) binds to the same or overlapping epitope as the anti-NKp30, anti-NKp46, anti-NKG2D or anti-CD16 antibody molecules described herein; or

[0192] (v) compete for binding, and / or bind to the same epitope as the anti-NKp30, anti-NKp46, anti-NKG2D or -CD16 molecules described herein.

[0193] 63. The multifunctional molecule of any one of embodiments 57-62, wherein the anti-NKp30, anti-NKp46, anti-NKG2D or anti-CD16 antibody molecule comprises one or more CDRs, framework regions, variable domains, heavy or light chains, or antigen binding domains selected from Tables 7-10 or 15, or a sequence substantially identical thereto.

[0194] 64. The multifunctional molecule of any one of embodiments 57-63, wherein the NK cell engager is an antibody molecule, e.g., an antigen binding domain, that binds NKp30.

[0195] 65. The multifunctional molecule according to any one of embodiments 57-64, wherein lysis of said lymphoma cells or lymphocytes is mediated by NKp30.

[0196] 66. The multifunctional molecule of any one of embodiments 57-65, wherein the multifunctional molecule does not activate NK cells when incubated with NK cells in the absence of tumor antigens on lymphoma cells or in the absence of TRBC1 or TRBC2 on lymphocytes.

[0197] 67. The multifunctional molecule of any one of embodiments 57-66, wherein the multifunctional molecule activates NK cells when the NK cells are NK cells expressing NKp30 and: (1) a tumor antigen on a lymphoma cell is also present or (2) TRBC1 or TRBC2 on a lymphocyte is also present.

[0198] 68. The multifunctional molecule of any one of embodiments 57-67, wherein the multifunctional molecule does not activate NK cells when the NK cells are not NK cells expressing NKp30 and: (1) a tumor antigen on a lymphoma cell is also present or (2) TRBC1 or TRBC2 on a lymphocyte is also present.

[0199] 69. The multifunctional molecule of any one of embodiments 57-68, wherein the NK cell engager comprises:

[0200] (i) a heavy chain variable region (VH), comprising a heavy chain complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 6000 (or a sequence having no more than 1, 2, 3 or 4 mutations (e.g., substitutions, additions or deletions) thereof), a VHCDR2 amino acid sequence of SEQ ID NO: 6001 (or a sequence having no more than 1, 2, 3 or 4 mutations (e.g., substitutions, additions or deletions) thereof) and / or a VHCDR3 amino acid sequence of SEQ ID NO: 6002 (or a sequence having no more than 1, 2, 3 or 4 mutations (e.g., substitutions, additions or deletions) thereof), and

[0201] (ii) a light chain variable region (VL), which includes a light chain complementary determining region 1 (VLCDR1) amino acid sequence of SEQ ID NO: 6063 (or a sequence having no more than 1, 2, 3 or 4 mutations (e.g., substitutions, additions or deletions)), a VLCDR2 amino acid sequence of SEQ ID NO: 6064 (or a sequence having no more than 1, 2, 3 or 4 mutations (e.g., substitutions, additions or deletions)) and / or a VLCDR3 amino acid sequence of SEQ ID NO: 7293 (or a sequence having no more than 1, 2, 3 or 4 mutations (e.g., substitutions, additions or deletions)).

[0202] 70. The multifunctional molecule of embodiment 69, wherein the NK cell engager comprises:

[0203] (i) a heavy chain variable region (VH), comprising a heavy chain complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 6000, a VHCDR2 amino acid sequence of SEQ ID NO: 6001 and / or a VHCDR3 amino acid sequence of SEQ ID NO: 6002, and

[0204] (ii) a light chain variable region (VL), which includes a light chain complementary determining region 1 (VLCDR1) amino acid sequence of SEQ ID NO: 6063, a VLCDR2 amino acid sequence of SEQ ID NO: 6064 and / or a VLCDR3 amino acid sequence of SEQ ID NO: 7293.

[0205] 71. The multifunctional molecule of any one of embodiments 57-70, wherein the NK cell engager comprises:

[0206] (1) a heavy chain variable region (VH), comprising a heavy chain framework region 1 (VHFWR1) amino acid sequence of SEQ ID NO: 6003 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations (e.g., substitutions, additions or deletions) thereof), a VHFWR2 amino acid sequence of SEQ ID NO: 6004 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations (e.g., substitutions, additions or deletions) thereof), a VHFWR3 amino acid sequence of SEQ ID NO: 6005 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations (e.g., substitutions, additions or deletions) thereof), or a VHFWR4 amino acid sequence of SEQ ID NO: 6006 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations (e.g., substitutions, additions or deletions) thereof), and / or

[0207] (2) a light chain variable region (VL), comprising a light chain framework region 1 (VLFWR1) amino acid sequence of SEQ ID NO: 6066 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations (e.g., substitutions, additions or deletions) thereof), a VLFWR2 amino acid sequence of SEQ ID NO: 6067 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations (e.g., substitutions, additions or deletions) thereof), a VLFWR3 amino acid sequence of SEQ ID NO: 7292 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations (e.g., substitutions, additions or deletions) thereof), or a VLFWR4 amino acid sequence of SEQ ID NO: 6069 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations (e.g., substitutions, additions or deletions) thereof).

[0208] 72. The multifunctional molecule of embodiment 71, wherein the NK cell engager comprises:

[0209] (1) a heavy chain variable region (VH), the heavy chain variable region comprising the heavy chain framework region 1 (VHFWR1) amino acid sequence of SEQ ID NO: 6003, the VHFWR2 amino acid sequence of SEQ ID NO: 6004, the VHFWR3 amino acid sequence of SEQ ID NO: 6005, or the VHFWR4 amino acid sequence of SEQ ID NO: 6006, and

[0210] (3) a light chain variable region (VL), comprising a light chain framework region 1 (VLFWR1) amino acid sequence of SEQ ID NO: 6066, a VLFWR2 amino acid sequence of SEQ ID NO: 6067, a VLFWR3 amino acid sequence of SEQ ID NO: 7292 or a VLFWR4 amino acid sequence of SEQ ID NO: 6069.

[0211] 73. The multifunctional molecule of any one of embodiments 57-72, wherein the NK cell engager comprises:

[0212] (i) a VH comprising the amino acid sequence of SEQ ID NO:6121 (or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NO:6121), and / or

[0213] (ii) a VL comprising the amino acid sequence of SEQ ID NO: 7294 (or an amino acid sequence having at least about 93%, 95%, or 99% sequence identity to SEQ ID NO: 7294).

[0214] 74. The multifunctional molecule of any one of embodiments 57-73, wherein the NK cell engager comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:6148 or 6149 (or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95% or 99% sequence identity with SEQ ID NO:6148 or 6149).

[0215] 75. The multifunctional molecule of any one of embodiments 57-74, wherein the NK cell engager comprises a light chain comprising the amino acid sequence of SEQ ID NO:6150 (or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95% or 99% sequence identity with SEQ ID NO:6150).

[0216] 76. The multifunctional molecule of any one of embodiments 57-75, wherein the NK cell engager comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:6148 or 6149 (or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95% or 99% sequence identity with SEQ ID NO:6148 or 6149), and a light chain comprising the amino acid sequence of SEQID NO:6150 (or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95% or 99% sequence identity with SEQ ID NO:6150).

[0217] 77. The multifunctional molecule of any one of embodiments 57-70, wherein the NK cell engager comprises a heavy chain variable region (VH), the heavy chain variable region comprising the heavy chain framework region 1 (VHFWR1) amino acid sequence of SEQ ID NO:6014 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations (e.g., substitutions, additions or deletions) thereof), the VHFWR2 amino acid sequence of SEQ ID NO:6015 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations (e.g., substitutions, additions or deletions) thereof), the VHFWR3 amino acid sequence of SEQ ID NO:6016 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations (e.g., substitutions, additions or deletions) thereof), or the VHFWR4 amino acid sequence of SEQ ID NO:6017 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations (e.g., substitutions, additions or deletions) thereof).

[0218] 78. The multifunctional molecule of embodiment 77, wherein the NK cell engager comprises a heavy chain variable region (VH), wherein the heavy chain variable region comprises a heavy chain framework region 1 (VHFWR1) amino acid sequence of SEQ ID NO:6014, a VHFWR2 amino acid sequence of SEQ ID NO:6015, a VHFWR3 amino acid sequence of SEQ ID NO:6016, or a VHFWR4 amino acid sequence of SEQ ID NO:6017.

[0219] 79. The multifunctional molecule of embodiment 78, wherein the NK cell engager comprises a VH comprising the amino acid sequence of SEQ ID NO:6123 (or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95% or 99% sequence identity with SEQ ID NO:6123).

[0220] 80. The multifunctional molecule of any one of embodiments 57-70, wherein the NK cell engager comprises a heavy chain variable region (VH), the heavy chain variable region comprising the heavy chain framework region 1 (VHFWR1) amino acid sequence of SEQ ID NO:6018 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations (e.g., substitutions, additions or deletions) thereof), the VHFWR2 amino acid sequence of SEQ ID NO:6019 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations (e.g., substitutions, additions or deletions) thereof), the VHFWR3 amino acid sequence of SEQ ID NO:6020 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations (e.g., substitutions, additions or deletions) thereof), or the VHFWR4 amino acid sequence of SEQ ID NO:6021 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations (e.g., substitutions, additions or deletions) thereof).

[0221] 81. The multifunctional molecule of embodiment 80, wherein the NK cell engager comprises a heavy chain variable region (VH), wherein the heavy chain variable region comprises a heavy chain framework region 1 (VHFWR1) amino acid sequence of SEQ ID NO:6018, a VHFWR2 amino acid sequence of SEQ ID NO:6019, a VHFWR3 amino acid sequence of SEQ ID NO:6020, or a VHFWR4 amino acid sequence of SEQ ID NO:6021.

[0222] 82. The multifunctional molecule of embodiment 81, wherein the NK cell engager comprises a VH comprising the amino acid sequence of SEQ ID NO:6124 (or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95% or 99% sequence identity with SEQ ID NO:6124).

[0223] 83. The multifunctional molecule of any one of embodiments 57-70, wherein the NK cell engager comprises a heavy chain variable region (VH), the heavy chain variable region comprising the heavy chain framework region 1 (VHFWR1) amino acid sequence of SEQ ID NO:6022 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations (e.g., substitutions, additions or deletions) thereof), the VHFWR2 amino acid sequence of SEQ ID NO:6023 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations (e.g., substitutions, additions or deletions) thereof), the VHFWR3 amino acid sequence of SEQ ID NO:6024 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations (e.g., substitutions, additions or deletions) thereof), or the VHFWR4 amino acid sequence of SEQ ID NO:6025 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations (e.g., substitutions, additions or deletions) thereof).

[0224] 84. The multifunctional molecule of embodiment 83, wherein the NK cell engager comprises a heavy chain variable region (VH), wherein the heavy chain variable region comprises a heavy chain framework region 1 (VHFWR1) amino acid sequence of SEQ ID NO:6022, a VHFWR2 amino acid sequence of SEQ ID NO:6023, a VHFWR3 amino acid sequence of SEQ ID NO:6024, or a VHFWR4 amino acid sequence of SEQ ID NO:6025.

[0225] 85. The multifunctional molecule of embodiment 84, wherein the NK cell engager comprises a VH comprising the amino acid sequence of SEQ ID NO:6125 (or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95% or 99% sequence identity with SEQ ID NO:6125).

[0226] 86. The multifunctional molecule of any one of embodiments 57-70, wherein the NK cell engager comprises a heavy chain variable region (VH), the heavy chain variable region comprising the heavy chain framework region 1 (VHFWR1) amino acid sequence of SEQ ID NO:6026 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations (e.g., substitutions, additions or deletions) thereof), the VHFWR2 amino acid sequence of SEQ ID NO:6027 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations (e.g., substitutions, additions or deletions) thereof), the VHFWR3 amino acid sequence of SEQ ID NO:6028 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations (e.g., substitutions, additions or deletions) thereof), or the VHFWR4 amino acid sequence of SEQ ID NO:6029 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations (e.g., substitutions, additions or deletions) thereof).

[0227] 87. The multifunctional molecule of embodiment 86, wherein the NK cell engager comprises a heavy chain variable region (VH), wherein the heavy chain variable region comprises a heavy chain framework region 1 (VHFWR1) amino acid sequence of SEQ ID NO:6026, a VHFWR2 amino acid sequence of SEQ ID NO:6027, a VHFWR3 amino acid sequence of SEQ ID NO:6028, or a VHFWR4 amino acid sequence of SEQ ID NO:6029.

[0228] 88. The multifunctional molecule of embodiment 87, wherein the NK cell engager comprises a VH comprising the amino acid sequence of SEQ ID NO:6126 (or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95% or 99% sequence identity with SEQ ID NO:6126).

[0229] 89. The multifunctional molecule of any one of embodiments 57-70, wherein the NK cell engager comprises a heavy chain variable region (VH), the heavy chain variable region comprising the heavy chain framework region 1 (VHFWR1) amino acid sequence of SEQ ID NO:6030 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations (e.g., substitutions, additions or deletions) thereof), the VHFWR2 amino acid sequence of SEQ ID NO:6032 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations (e.g., substitutions, additions or deletions) thereof), the VHFWR3 amino acid sequence of SEQ ID NO:6033 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations (e.g., substitutions, additions or deletions) thereof), or the VHFWR4 amino acid sequence of SEQ ID NO:6034 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations (e.g., substitutions, additions or deletions) thereof).

[0230] 90. The multifunctional molecule of embodiment 89, wherein the NK cell engager comprises a heavy chain variable region (VH), wherein the heavy chain variable region comprises a heavy chain framework region 1 (VHFWR1) amino acid sequence of SEQ ID NO:6030, a VHFWR2 amino acid sequence of SEQ ID NO:6032, a VHFWR3 amino acid sequence of SEQ ID NO:6033, or a VHFWR4 amino acid sequence of SEQ ID NO:6034.

[0231] 91. The multifunctional molecule of embodiment 90, wherein the NK cell engager comprises a VH comprising the amino acid sequence of SEQ ID NO:6127 (or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95% or 99% sequence identity with SEQ ID NO:6127).

[0232] 92. The multifunctional molecule of any one of embodiments 57-70, wherein the NK cell engager comprises a heavy chain variable region (VH), the heavy chain variable region comprising the heavy chain framework region 1 (VHFWR1) amino acid sequence of SEQ ID NO:6035 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations (e.g., substitutions, additions or deletions) thereof), the VHFWR2 amino acid sequence of SEQ ID NO:6036 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations (e.g., substitutions, additions or deletions) thereof), the VHFWR3 amino acid sequence of SEQ ID NO:6037 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations (e.g., substitutions, additions or deletions) thereof), or the VHFWR4 amino acid sequence of SEQ ID NO:6038 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations (e.g., substitutions, additions or deletions) thereof).

[0233] 93. A multifunctional molecule according to embodiment 92, wherein the NK cell engager comprises a heavy chain variable region (VH), wherein the heavy chain variable region comprises a heavy chain framework region 1 (VHFWR1) amino acid sequence of SEQ ID NO:6035, a VHFWR2 amino acid sequence of SEQ ID NO:6036, a VHFWR3 amino acid sequence of SEQ ID NO:6037, or a VHFWR4 amino acid sequence of SEQ ID NO:6038.

[0234] 94. The multifunctional molecule of embodiment 93, wherein the NK cell engager comprises a VH comprising the amino acid sequence of SEQ ID NO:6128 (or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95% or 99% sequence identity with SEQ ID NO:6128).

[0235] 95. The multifunctional molecule of any one of embodiments 57-70 or 77-94, wherein the NK cell engager comprises a light chain variable region (VL), the light chain variable region comprising the light chain framework region 1 (VLFWR1) amino acid sequence of SEQ ID NO:6077 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations (e.g., substitutions, additions or deletions) thereof), the VLFWR2 amino acid sequence of SEQ ID NO:6078 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations (e.g., substitutions, additions or deletions) thereof), the VLFWR3 amino acid sequence of SEQ ID NO:6079 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations (e.g., substitutions, additions or deletions) thereof), or the VLFWR4 amino acid sequence of SEQ ID NO:6080 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations (e.g., substitutions, additions or deletions) thereof).

[0236] 96. A multifunctional molecule according to embodiment 95, wherein the NK cell engager comprises a light chain variable region (VL), wherein the light chain variable region comprises a light chain framework region 1 (VLFWR1) amino acid sequence of SEQ ID NO:6077, a VLFWR2 amino acid sequence of SEQ ID NO:6078, a VLFWR3 amino acid sequence of SEQ ID NO:6079, or a VLFWR4 amino acid sequence of SEQ ID NO:6080.

[0237] 97. The multifunctional molecule of embodiment 96, wherein the NK cell engager comprises a VL comprising the amino acid sequence of SEQ ID NO:6137 (or an amino acid sequence having at least about 93%, 95% or 99% sequence identity with SEQ ID NO:6137).

[0238] 98. The multifunctional molecule of any one of embodiments 57-70 or 77-94, wherein the NK cell engager comprises a light chain variable region (VL), the light chain variable region comprising the light chain framework region 1 (VLFWR1) amino acid sequence of SEQ ID NO:6081 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations (e.g., substitutions, additions or deletions) thereof), the VLFWR2 amino acid sequence of SEQ ID NO:6082 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations (e.g., substitutions, additions or deletions) thereof), the VLFWR3 amino acid sequence of SEQ ID NO:6083 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations (e.g., substitutions, additions or deletions) thereof), or the VLFWR4 amino acid sequence of SEQ ID NO:6084 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations (e.g., substitutions, additions or deletions) thereof).

[0239] 99. A multifunctional molecule according to embodiment 98, wherein the NK cell engager comprises a light chain variable region (VL), wherein the light chain variable region comprises a light chain framework region 1 (VLFWR1) amino acid sequence of SEQ ID NO:6081, a VLFWR2 amino acid sequence of SEQ ID NO:6082, a VLFWR3 amino acid sequence of SEQ ID NO:6083, or a VLFWR4 amino acid sequence of SEQ ID NO:6084.

[0240] 100. The multifunctional molecule of embodiment 99, wherein the NK cell engager comprises a VL comprising the amino acid sequence of SEQ ID NO:6138 (or an amino acid sequence having at least about 93%, 95% or 99% sequence identity with SEQ ID NO:6138).

[0241] 101. The multifunctional molecule of any one of embodiments 57-70 or 77-94, wherein the NK cell engager comprises a light chain variable region (VL), comprising a light chain framework region 1 (VLFWR1) amino acid sequence of SEQ ID NO:6085 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations (e.g., substitutions, additions or deletions) thereof), a VLFWR2 amino acid sequence of SEQ ID NO:6086 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations (e.g., substitutions, additions or deletions) thereof), a VLFWR3 amino acid sequence of SEQ ID NO:6087 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations (e.g., substitutions, additions or deletions) thereof), or a VLFWR4 amino acid sequence of SEQ ID NO:6088 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations (e.g., substitutions, additions or deletions) thereof).

[0242] 102. A multifunctional molecule according to embodiment 101, wherein the NK cell engager comprises a light chain variable region (VL), wherein the light chain variable region comprises a light chain framework region 1 (VLFWR1) amino acid sequence of SEQ ID NO:6085, a VLFWR2 amino acid sequence of SEQ ID NO:6086, a VLFWR3 amino acid sequence of SEQ ID NO:6087, or a VLFWR4 amino acid sequence of SEQ ID NO:6088.

[0243] 103. The multifunctional molecule of embodiment 102, wherein the NK cell engager comprises a VL comprising the amino acid sequence of SEQ ID NO:6139 (or an amino acid sequence having at least about 93%, 95% or 99% sequence identity with SEQ ID NO:6139).

[0244] 104. The multifunctional molecule of any one of embodiments 57-70 or 77-94, wherein the NK cell engager comprises a light chain variable region (VL), the light chain variable region comprising the light chain framework region 1 (VLFWR1) amino acid sequence of SEQ ID NO:6089 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations (e.g., substitutions, additions or deletions) thereof), the VLFWR2 amino acid sequence of SEQ ID NO:6090 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations (e.g., substitutions, additions or deletions) thereof), the VLFWR3 amino acid sequence of SEQ ID NO:6091 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations (e.g., substitutions, additions or deletions) thereof), or the VLFWR4 amino acid sequence of SEQ ID NO:6092 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations (e.g., substitutions, additions or deletions) thereof).

[0245] 105. A multifunctional molecule of embodiment 104, wherein the NK cell engager comprises a light chain variable region (VL), wherein the light chain variable region comprises a light chain framework region 1 (VLFWR1) amino acid sequence of SEQ ID NO:6089, a VLFWR2 amino acid sequence of SEQ ID NO:6090, a VLFWR3 amino acid sequence of SEQ ID NO:6091, or a VLFWR4 amino acid sequence of SEQ ID NO:6092.

[0246] 106. The multifunctional molecule of embodiment 105, wherein the NK cell engager comprises a VL comprising the amino acid sequence of SEQ ID NO:6140 (or an amino acid sequence having at least about 93%, 95% or 99% sequence identity with SEQ ID NO:6140).

[0247] 107. The multifunctional molecule of any one of embodiments 57-70 or 77-94, wherein the NK cell engager comprises a light chain variable region (VL), the light chain variable region comprising the light chain framework region 1 (VLFWR1) amino acid sequence of SEQ ID NO:6093 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations (e.g., substitutions, additions or deletions) thereof), the VLFWR2 amino acid sequence of SEQ ID NO:6094 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations (e.g., substitutions, additions or deletions) thereof), the VLFWR3 amino acid sequence of SEQ ID NO:6095 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations (e.g., substitutions, additions or deletions) thereof), or the VLFWR4 amino acid sequence of SEQ ID NO:6096 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations (e.g., substitutions, additions or deletions) thereof).

[0248] 108. A multifunctional molecule of embodiment 107, wherein the NK cell engager comprises a light chain variable region (VL), wherein the light chain variable region comprises a light chain framework region 1 (VLFWR1) amino acid sequence of SEQ ID NO:6093, a VLFWR2 amino acid sequence of SEQ ID NO:6094, a VLFWR3 amino acid sequence of SEQ ID NO:6095, or a VLFWR4 amino acid sequence of SEQ ID NO:6096.

[0249] 109. The multifunctional molecule of embodiment 108, wherein the NK cell engager comprises a VL comprising the amino acid sequence of SEQ ID NO:6141 (or an amino acid sequence having at least about 93%, 95% or 99% sequence identity with SEQ ID NO:6141).

[0250] 110. The multifunctional molecule of any one of embodiments 57-68, wherein the NK cell engager comprises:

[0251] (i) a heavy chain variable region (VH) comprising a heavy chain complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 6007 (or a sequence having no more than 1, 2, 3 or 4 mutations (e.g., substitutions, additions or deletions)), a VHCDR2 amino acid sequence of SEQ ID NO: 6008 (or a sequence having no more than 1, 2, 3 or 4 mutations (e.g., substitutions, additions or deletions)) and / or a VHCDR3 amino acid sequence of SEQ ID NO: 6009 (or a sequence having no more than 1, 2, 3 or 4 mutations (e.g., substitutions, additions or deletions)), and

[0252] (ii) a light chain variable region (VL), which includes a light chain complementary determining region 1 (VLCDR1) amino acid sequence of SEQ ID NO: 6070 (or a sequence having no more than 1, 2, 3 or 4 mutations (e.g., substitutions, additions or deletions)), a VLCDR2 amino acid sequence of SEQ ID NO: 6071 (or a sequence having no more than 1, 2, 3 or 4 mutations (e.g., substitutions, additions or deletions)) and / or a VLCDR3 amino acid sequence of SEQ ID NO: 6072 (or a sequence having no more than 1, 2, 3 or 4 mutations (e.g., substitutions, additions or deletions)).

[0253] 111. The multifunctional molecule of embodiment 110, wherein the NK cell engager comprises:

[0254] (i) a heavy chain variable region (VH), comprising a heavy chain complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 6007, a VHCDR2 amino acid sequence of SEQ ID NO: 6008 and / or a VHCDR3 amino acid sequence of SEQ ID NO: 6009, and

[0255] (ii) a light chain variable region (VL), which includes a light chain complementary determining region 1 (VLCDR1) amino acid sequence of SEQ ID NO: 6070, a VLCDR2 amino acid sequence of SEQ ID NO: 6071 and / or a VLCDR3 amino acid sequence of SEQ ID NO: 6072.

[0256] 112. The multifunctional molecule of any one of embodiments 57-68, 110 or 111, wherein the NK cell engager comprises:

[0257] (1) a heavy chain variable region (VH), comprising a heavy chain framework region 1 (VHFWR1) amino acid sequence of SEQ ID NO: 6010 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations (e.g., substitutions, additions or deletions) thereof), a VHFWR2 amino acid sequence of SEQ ID NO: 6011 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations (e.g., substitutions, additions or deletions) thereof), a VHFWR3 amino acid sequence of SEQ ID NO: 6012 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations (e.g., substitutions, additions or deletions) thereof), or a VHFWR4 amino acid sequence of SEQ ID NO: 6013 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations (e.g., substitutions, additions or deletions) thereof), and / or

[0258] (2) a light chain variable region (VL), comprising a light chain framework region 1 (VLFWR1) amino acid sequence of SEQ ID NO: 6073 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations (e.g., substitutions, additions or deletions) thereof), a VLFWR2 amino acid sequence of SEQ ID NO: 6074 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations (e.g., substitutions, additions or deletions) thereof), a VLFWR3 amino acid sequence of SEQ ID NO: 6075 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations (e.g., substitutions, additions or deletions) thereof), or a VLFWR4 amino acid sequence of SEQ ID NO: 6076 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations (e.g., substitutions, additions or deletions) thereof).

[0259] 113. The multifunctional molecule of embodiment 112, wherein the NK cell engager comprises:

[0260] (1) a heavy chain variable region (VH), comprising a heavy chain framework region 1 (VHFWR1) amino acid sequence of SEQ ID NO: 6010, a VHFWR2 amino acid sequence of SEQ ID NO: 6011, a VHFWR3 amino acid sequence of SEQ ID NO: 6012, or a VHFWR4 amino acid sequence of SEQ ID NO: 6013, and

[0261] (3) a light chain variable region (VL), comprising a light chain framework region 1 (VLFWR1) amino acid sequence of SEQ ID NO: 6073, a VLFWR2 amino acid sequence of SEQ ID NO: 6074, a VLFWR3 amino acid sequence of SEQ ID NO: 6075, or a VLFWR4 amino acid sequence of SEQ ID NO: 6076.

[0262] 114. The multifunctional molecule of any one of embodiments 57-68 or 110-113, wherein the NK cell engager comprises:

[0263] (i) a VH comprising the amino acid sequence of SEQ ID NO:6122 (or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NO:6122), and / or

[0264] (ii) a VL comprising the amino acid sequence of SEQ ID NO: 6136 (or an amino acid sequence having at least about 93%, 95%, or 99% sequence identity to SEQ ID NO: 6136).

[0265] 115. The multifunctional molecule of any one of embodiments 57-68 or 110-114, wherein the NK cell engager comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:6151 or 6152 (or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95% or 99% sequence identity with SEQ ID NO:6151 or 6152).

[0266] 116. The multifunctional molecule of any one of embodiments 57-68 or 110-115, wherein the NK cell engager comprises a light chain comprising the amino acid sequence of SEQ ID NO:6153 (or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95% or 99% sequence identity with SEQ ID NO:6153).

[0267] 117. The multifunctional molecule of any one of embodiments 57-68 or 110-116, wherein the NK cell engager comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:6151 or 6152 (or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95% or 99% sequence identity with SEQ ID NO:6151 or 6152), and a light chain comprising the amino acid sequence of SEQ ID NO:6153 (or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95% or 99% sequence identity with SEQ ID NO:6153).

[0268] 118. The multifunctional molecule of any of embodiments 57-68, 110 or 111, wherein the NK cell engager comprises a heavy chain variable region (VH), the heavy chain variable region comprising the heavy chain framework region 1 (VHFWR1) amino acid sequence of SEQ ID NO:6039 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations (e.g., substitutions, additions or deletions) thereof), the VHFWR2 amino acid sequence of SEQ ID NO:6040 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations (e.g., substitutions, additions or deletions) thereof), the VHFWR3 amino acid sequence of SEQ ID NO:6041 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations (e.g., substitutions, additions or deletions) thereof), or the VHFWR4 amino acid sequence of SEQ ID NO:6042 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations (e.g., substitutions, additions or deletions) thereof).

[0269] 119. The multifunctional molecule of embodiment 118, wherein the NK cell engager comprises a heavy chain variable region (VH), wherein the heavy chain variable region comprises a heavy chain framework region 1 (VHFWR1) amino acid sequence of SEQ ID NO:6039, a VHFWR2 amino acid sequence of SEQ ID NO:6040, a VHFWR3 amino acid sequence of SEQ ID NO:6041, or a VHFWR4 amino acid sequence of SEQ ID NO:6042.

[0270] 120. The multifunctional molecule of embodiment 119, wherein the NK cell engager comprises a VH comprising the amino acid sequence of SEQ ID NO:6129 (or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95% or 99% sequence identity with SEQ ID NO:6129).

[0271] 121. The multifunctional molecule of any of embodiments 57-68, 110 or 111, wherein the NK cell engager comprises a heavy chain variable region (VH), the heavy chain variable region comprising the heavy chain framework region 1 (VHFWR1) amino acid sequence of SEQ ID NO:6043 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations (e.g., substitutions, additions or deletions) thereof), the VHFWR2 amino acid sequence of SEQ ID NO:6044 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations (e.g., substitutions, additions or deletions) thereof), the VHFWR3 amino acid sequence of SEQ ID NO:6045 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations (e.g., substitutions, additions or deletions) thereof), or the VHFWR4 amino acid sequence of SEQ ID NO:6046 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations (e.g., substitutions, additions or deletions) thereof).

[0272] 122. The multifunctional molecule of embodiment 121, wherein the NK cell engager comprises a heavy chain variable region (VH), wherein the heavy chain variable region comprises a heavy chain framework region 1 (VHFWR1) amino acid sequence of SEQ ID NO:6043, a VHFWR2 amino acid sequence of SEQ ID NO:6044, a VHFWR3 amino acid sequence of SEQ ID NO:6045, or a VHFWR4 amino acid sequence of SEQ ID NO:6046.

[0273] 123. The multifunctional molecule of embodiment 122, wherein the NK cell engager comprises a VH comprising the amino acid sequence of SEQ ID NO:6130 (or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95% or 99% sequence identity with SEQ ID NO:6130).

[0274] 124. The multifunctional molecule of any of embodiments 57-68, 110 or 111, wherein the NK cell engager comprises a heavy chain variable region (VH), the heavy chain variable region comprising the heavy chain framework region 1 (VHFWR1) amino acid sequence of SEQ ID NO:6047 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations (e.g., substitutions, additions or deletions) thereof), the VHFWR2 amino acid sequence of SEQ ID NO:6048 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations (e.g., substitutions, additions or deletions) thereof), the VHFWR3 amino acid sequence of SEQ ID NO:6049 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations (e.g., substitutions, additions or deletions) thereof), or the VHFWR4 amino acid sequence of SEQ ID NO:6050 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations (e.g., substitutions, additions or deletions) thereof).

[0275] 125. The multifunctional molecule of embodiment 124, wherein the NK cell engager comprises a heavy chain variable region (VH), wherein the heavy chain variable region comprises a heavy chain framework region 1 (VHFWR1) amino acid sequence of SEQ ID NO:6047, a VHFWR2 amino acid sequence of SEQ ID NO:6048, a VHFWR3 amino acid sequence of SEQ ID NO:6049, or a VHFWR4 amino acid sequence of SEQ ID NO:6050.

[0276] 126. The multifunctional molecule of embodiment 125, wherein the NK cell engager comprises a VH comprising the amino acid sequence of SEQ ID NO:6131 (or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95% or 99% sequence identity with SEQ ID NO:6131).

[0277] 127. The multifunctional molecule of any one of experimental schemes 57-68, 110 or 111, wherein the NK cell engager comprises a heavy chain variable region (VH), wherein the heavy chain variable region comprises the heavy chain framework region 1 (VHFWR1) amino acid sequence of SEQ ID NO:6051 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations (e.g., substitutions, additions or deletions) thereof), the VHFWR2 amino acid sequence of SEQ ID NO:6052 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations (e.g., substitutions, additions or deletions) thereof), the VHFWR3 amino acid sequence of SEQ ID NO:6053 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations (e.g., substitutions, additions or deletions) thereof), or the VHFWR4 amino acid sequence of SEQ ID NO:6054 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations (e.g., substitutions, additions or deletions) thereof).

[0278] 128. The multifunctional molecule of embodiment 127, wherein the NK cell engager comprises a heavy chain variable region (VH), wherein the heavy chain variable region comprises a heavy chain framework region 1 (VHFWR1) amino acid sequence of SEQ ID NO:6051, a VHFWR2 amino acid sequence of SEQ ID NO:6052, a VHFWR3 amino acid sequence of SEQ ID NO:6053, or a VHFWR4 amino acid sequence of SEQ ID NO:6054.

[0279] 129. The multifunctional molecule of embodiment 128, wherein the NK cell engager comprises a VH comprising the amino acid sequence of SEQ ID NO:6132 (or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95% or 99% sequence identity with SEQ ID NO:6132).

[0280] 130. The multifunctional molecule of any of embodiments 57-68, 110 or 111, wherein the NK cell engager comprises a heavy chain variable region (VH), the heavy chain variable region comprising the heavy chain framework region 1 (VHFWR1) amino acid sequence of SEQ ID NO:6055 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations (e.g., substitutions, additions or deletions) thereof), the VHFWR2 amino acid sequence of SEQ ID NO:6056 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations (e.g., substitutions, additions or deletions) thereof), the VHFWR3 amino acid sequence of SEQ ID NO:6057 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations (e.g., substitutions, additions or deletions) thereof), or the VHFWR4 amino acid sequence of SEQ ID NO:6058 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations (e.g., substitutions, additions or deletions) thereof).

[0281] 131. The multifunctional molecule of embodiment 130, wherein the NK cell engager comprises a heavy chain variable region (VH), wherein the heavy chain variable region comprises a heavy chain framework region 1 (VHFWR1) amino acid sequence of SEQ ID NO:6055, a VHFWR2 amino acid sequence of SEQ ID NO:6056, a VHFWR3 amino acid sequence of SEQ ID NO:6057, or a VHFWR4 amino acid sequence of SEQ ID NO:6058.

[0282] 132. The multifunctional molecule of embodiment 131, wherein the NK cell engager comprises a VH comprising the amino acid sequence of SEQ ID NO:6133 (or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95% or 99% sequence identity with SEQ ID NO:6133).

[0283] 133. The multifunctional molecule of any of embodiments 57-68, 110 or 111, wherein the NK cell engager comprises a heavy chain variable region (VH), the heavy chain variable region comprising the heavy chain framework region 1 (VHFWR1) amino acid sequence of SEQ ID NO:6059 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations (e.g., substitutions, additions or deletions) thereof), the VHFWR2 amino acid sequence of SEQ ID NO:6060 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations (e.g., substitutions, additions or deletions) thereof), the VHFWR3 amino acid sequence of SEQ ID NO:6061 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations (e.g., substitutions, additions or deletions) thereof), or the VHFWR4 amino acid sequence of SEQ ID NO:6062 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations (e.g., substitutions, additions or deletions) thereof).

[0284] 134. The multifunctional molecule of embodiment 133, wherein the NK cell engager comprises a heavy chain variable region (VH), wherein the heavy chain variable region comprises a heavy chain framework region 1 (VHFWR1) amino acid sequence of SEQ ID NO:6059, a VHFWR2 amino acid sequence of SEQ ID NO:6060, a VHFWR3 amino acid sequence of SEQ ID NO:6061, or a VHFWR4 amino acid sequence of SEQ ID NO:6062.

[0285] 135. The multifunctional molecule of embodiment 134, wherein the NK cell engager comprises a VH comprising the amino acid sequence of SEQ ID NO:6134 (or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95% or 99% sequence identity with SEQ ID NO:6134).

[0286] 136. The multifunctional molecule of any one of embodiments 57-68, 110, 111 or 118-135, wherein the NK cell engager comprises a light chain variable region (VL), the light chain variable region comprising the light chain framework region 1 (VLFWR1) amino acid sequence of SEQ ID NO:6097 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations (e.g., substitutions, additions or deletions) thereof), the VLFWR2 amino acid sequence of SEQ ID NO:6098 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations (e.g., substitutions, additions or deletions) thereof), the VLFWR3 amino acid sequence of SEQ ID NO:6099 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations (e.g., substitutions, additions or deletions) thereof), or the VLFWR4 amino acid sequence of SEQ ID NO:6100 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations (e.g., substitutions, additions or deletions) thereof).

[0287] 137. A multifunctional molecule of embodiment 136, wherein the NK cell engager comprises a light chain variable region (VL), wherein the light chain variable region comprises a light chain framework region 1 (VLFWR1) amino acid sequence of SEQ ID NO:6097, a VLFWR2 amino acid sequence of SEQ ID NO:6098, a VLFWR3 amino acid sequence of SEQ ID NO:6099, or a VLFWR4 amino acid sequence of SEQ ID NO:6100.

[0288] 138. The multifunctional molecule of embodiment 137, wherein the NK cell engager comprises a VL comprising the amino acid sequence of SEQ ID NO:6142 (or an amino acid sequence having at least about 93%, 95% or 99% sequence identity with SEQ ID NO:6142).

[0289] 139. The multifunctional molecule of any one of embodiments 57-68, 110, 111 or 118-135, wherein the NK cell engager comprises a light chain variable region (VL), the light chain variable region comprising the light chain framework region 1 (VLFWR1) amino acid sequence of SEQ ID NO:6101 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations (e.g., substitutions, additions or deletions) thereof), the VLFWR2 amino acid sequence of SEQ ID NO:6102 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations (e.g., substitutions, additions or deletions) thereof), the VLFWR3 amino acid sequence of SEQ ID NO:6103 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations (e.g., substitutions, additions or deletions) thereof), or the VLFWR4 amino acid sequence of SEQ ID NO:6104 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations (e.g., substitutions, additions or deletions) thereof).

[0290] 140. A multifunctional molecule of embodiment 139, wherein the NK cell engager comprises a light chain variable region (VL), wherein the light chain variable region comprises a light chain framework region 1 (VLFWR1) amino acid sequence of SEQ ID NO:6101, a VLFWR2 amino acid sequence of SEQ ID NO:6102, a VLFWR3 amino acid sequence of SEQ ID NO:6103, or a VLFWR4 amino acid sequence of SEQ ID NO:6104.

[0291] 141. The multifunctional molecule of embodiment 140, wherein the NK cell engager comprises a VL comprising the amino acid sequence of SEQ ID NO:6143 (or an amino acid sequence having at least about 93%, 95% or 99% sequence identity with SEQ ID NO:6143).

[0292] 142. The multifunctional molecule of any one of embodiments 57-68, 110, 111 or 118-135, wherein the NK cell engager comprises a light chain variable region (VL), the light chain variable region comprising the light chain framework region 1 (VLFWR1) amino acid sequence of SEQ ID NO:6105 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations (e.g., substitutions, additions or deletions) thereof), the VLFWR2 amino acid sequence of SEQ ID NO:6106 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations (e.g., substitutions, additions or deletions) thereof), the VLFWR3 amino acid sequence of SEQ ID NO:6107 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations (e.g., substitutions, additions or deletions) thereof), or the VLFWR4 amino acid sequence of SEQ ID NO:6108 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations (e.g., substitutions, additions or deletions) thereof).

[0293] 143. A multifunctional molecule of embodiment 142, wherein the NK cell engager comprises a light chain variable region (VL), wherein the light chain variable region comprises a light chain framework region 1 (VLFWR1) amino acid sequence of SEQ ID NO:6105, a VLFWR2 amino acid sequence of SEQ ID NO:6106, a VLFWR3 amino acid sequence of SEQ ID NO:6107, or a VLFWR4 amino acid sequence of SEQ ID NO:6108.

[0294] 144. The multifunctional molecule of embodiment 143, wherein the NK cell engager comprises a VL comprising the amino acid sequence of SEQ ID NO:6144 (or an amino acid sequence having at least about 93%, 95% or 99% sequence identity with SEQ ID NO:6144).

[0295] 145. The multifunctional molecule of any one of embodiments 57-68, 110, 111 or 118-135, wherein the NK cell engager comprises a light chain variable region (VL), the light chain variable region comprising the light chain framework region 1 (VLFWR1) amino acid sequence of SEQ ID NO:6109 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations (e.g., substitutions, additions or deletions) thereof), the VLFWR2 amino acid sequence of SEQ ID NO:6110 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations (e.g., substitutions, additions or deletions) thereof), the VLFWR3 amino acid sequence of SEQ ID NO:6111 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations (e.g., substitutions, additions or deletions) thereof), or the VLFWR4 amino acid sequence of SEQ ID NO:6112 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations (e.g., substitutions, additions or deletions) thereof).

[0296] 146. A multifunctional molecule of embodiment 145, wherein the NK cell engager comprises a light chain variable region (VL), wherein the light chain variable region comprises a light chain framework region 1 (VLFWR1) amino acid sequence of SEQ ID NO:6109, a VLFWR2 amino acid sequence of SEQ ID NO:6110, a VLFWR3 amino acid sequence of SEQ ID NO:6111, or a VLFWR4 amino acid sequence of SEQ ID NO:6112.

[0297] 147. The multifunctional molecule of embodiment 146, wherein the NK cell engager comprises a VL comprising the amino acid sequence of SEQ ID NO:6145 (or an amino acid sequence having at least about 93%, 95% or 99% sequence identity with SEQ ID NO:6145).

[0298] 148. The multifunctional molecule of any one of embodiments 57-68, 110, 111 or 118-135, wherein the NK cell engager comprises a light chain variable region (VL), the light chain variable region comprising the light chain framework region 1 (VLFWR1) amino acid sequence of SEQ ID NO:6113 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations (e.g., substitutions, additions or deletions) thereof), the VLFWR2 amino acid sequence of SEQ ID NO:6114 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations (e.g., substitutions, additions or deletions) thereof), the VLFWR3 amino acid sequence of SEQ ID NO:6115 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations (e.g., substitutions, additions or deletions) thereof), or the VLFWR4 amino acid sequence of SEQ ID NO:6116 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations (e.g., substitutions, additions or deletions) thereof).

[0299] 149. A multifunctional molecule of embodiment 148, wherein the NK cell engager comprises a light chain variable region (VL), wherein the light chain variable region comprises a light chain framework region 1 (VLFWR1) amino acid sequence of SEQ ID NO:6113, a VLFWR2 amino acid sequence of SEQ ID NO:6114, a VLFWR3 amino acid sequence of SEQ ID NO:6115, or a VLFWR4 amino acid sequence of SEQ ID NO:6116.

[0300] 150. The multifunctional molecule of embodiment 149, wherein the NK cell engager comprises a VL comprising the amino acid sequence of SEQ ID NO:6146 (or an amino acid sequence having at least about 93%, 95% or 99% sequence identity with SEQ ID NO:6146).

[0301] 151. The multifunctional molecule of any one of embodiments 57-68, 110, 111 or 118-135, wherein the NK cell engager comprises a light chain variable region (VL), the light chain variable region comprising the light chain framework region 1 (VLFWR1) amino acid sequence of SEQ ID NO:6117 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations (e.g., substitutions, additions or deletions) thereof), the VLFWR2 amino acid sequence of SEQ ID NO:6118 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations (e.g., substitutions, additions or deletions) thereof), the VLFWR3 amino acid sequence of SEQ ID NO:6119 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations (e.g., substitutions, additions or deletions) thereof), or the VLFWR4 amino acid sequence of SEQ ID NO:6120 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations (e.g., substitutions, additions or deletions) thereof).

[0302] 152. A multifunctional molecule of embodiment 151, wherein the NK cell engager comprises a light chain variable region (VL), wherein the light chain variable region comprises a light chain framework region 1 (VLFWR1) amino acid sequence of SEQ ID NO:6117, a VLFWR2 amino acid sequence of SEQ ID NO:6118, a VLFWR3 amino acid sequence of SEQ ID NO:6119, or a VLFWR4 amino acid sequence of SEQ ID NO:6120.

[0303] 153. The multifunctional molecule of embodiment 152, wherein the NK cell engager comprises a VL comprising the amino acid sequence of SEQ ID NO:6147 (or an amino acid sequence having at least about 93%, 95% or 99% sequence identity with SEQ ID NO:6147).

[0304] 154. The multifunctional molecule of any one of embodiments 57-60, wherein the NK cell engager is an antibody molecule, e.g., an antigen binding domain, that binds to NKp46.

[0305] 155. The multifunctional molecule of embodiment 154, wherein lysis of said lymphoma cells is mediated by NKp46.

[0306] 156. The multifunctional molecule of any one of embodiments 154 or 155, wherein the multifunctional molecule does not activate NK cells when incubated with NK cells in the absence of tumor antigens on lymphoma cells.

[0307] 157. The multifunctional molecule of any one of embodiments 154-156, wherein the multifunctional molecule activates NK cells when the NK cells are NK cells expressing NKp46 and a tumor antigen on a lymphoma cell is also present.

[0308] 158. The multifunctional molecule of any one of embodiments 154-157, wherein the multifunctional molecule does not activate NK cells when the NK cells are not NK cells expressing NKp46 and tumor antigens on lymphoma cells are also present.

[0309] 159. The multifunctional molecule of any one of embodiments 154-158, wherein the NK cell engager comprises a VH comprising the amino acid sequence of SEQ ID NO:6182 (or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95% or 99% sequence identity with SEQ ID NO:6182).

[0310] 160. The multifunctional molecule of any one of embodiments 154-159, wherein the NK cell engager comprises a VL comprising the amino acid sequence of SEQ ID NO:6183 (or an amino acid sequence having at least about 93%, 95% or 99% sequence identity with SEQ ID NO:6183).

[0311] 161. The multifunctional molecule of 154-160, wherein the NK cell engager comprises a scFV comprising the amino acid sequence of SEQ ID NO:6181 (or an amino acid sequence having at least about 93%, 95% or 99% sequence identity with SEQ ID NO:6181).

[0312] 162. The multifunctional molecule of any one of embodiments 57-60, wherein the NK cell engager is an antibody molecule, e.g., an antigen binding domain, that binds to NKG2D.

[0313] 163. The multifunctional molecule of embodiment 162, wherein the lysis of the lymphoma cells is mediated by NKG2D.

[0314] 164. The multifunctional molecule of any one of embodiments 162 or 163, wherein the multifunctional molecule does not activate NK cells when incubated with NK cells in the absence of tumor antigens on lymphoma cells.

[0315] 165. The multifunctional molecule of any one of embodiments 162-164, wherein the multifunctional molecule activates NK cells when the NK cells are NK cells expressing NKG2D and a tumor antigen on a lymphoma cell is also present.

[0316] 166. The multifunctional molecule of any one of embodiments 162-165, wherein the multifunctional molecule does not activate NK cells when the NK cells are not NK cells expressing NKG2D and tumor antigens on lymphoma cells are also present.

[0317] 167. The multifunctional molecule of any one of embodiments 162-166, wherein the NK cell engager comprises a VH comprising the amino acid sequence of SEQ ID NO:6176 (or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95% or 99% sequence identity with SEQ ID NO:6176).

[0318] 168. The multifunctional molecule of any one of embodiments 162-167, wherein the NK cell engager comprises a VL comprising the amino acid sequence of SEQ ID NO:6177 (or an amino acid sequence having at least about 93%, 95% or 99% sequence identity with SEQ ID NO:6177).

[0319] 169. The multifunctional molecule of any one of embodiments 162-168, wherein the NK cell engager comprises a scFV comprising the amino acid sequence of SEQ ID NO:6175 (or an amino acid sequence having at least about 93%, 95% or 99% sequence identity with SEQ ID NO:6175).

[0320] 170. The multifunctional molecule of any one of embodiments 162-166, wherein the NK cell engager comprises a VH comprising the amino acid sequence of SEQ ID NO:6179 (or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95% or 99% sequence identity with SEQ ID NO:6179).

[0321] 171. The multifunctional molecule of any one of embodiments 162-166 or 170, wherein the NK cell engager comprises a VL comprising the amino acid sequence of SEQ ID NO:6180 (or an amino acid sequence having at least about 93%, 95% or 99% sequence identity with SEQ ID NO:6180).

[0322] 172. The multifunctional molecule of any one of embodiments 162-166, 170 or 171, wherein the NK cell engager comprises a scFV comprising the amino acid sequence of SEQ ID NO:6178 (or an amino acid sequence having at least about 93%, 95% or 99% sequence identity with SEQ ID NO:6178).

[0323] 173. The multifunctional molecule of any one of embodiments 57-60, wherein the NK cell engager is an antibody molecule, e.g., an antigen binding domain, that binds to CD16.

[0324] 174. The multifunctional molecule of embodiment 162, wherein the lysis of the lymphoma cells is mediated by CD16.

[0325] 175. The multifunctional molecule of any one of embodiments 173 or 174, wherein the multifunctional molecule does not activate NK cells when incubated with NK cells in the absence of tumor antigens on lymphoma cells.

[0326] 176. The multifunctional molecule of any one of embodiments 173-175, wherein the multifunctional molecule activates NK cells when the NK cells are CD16 expressing NK cells and a tumor antigen on a lymphoma cell is also present.

[0327] 177. The multifunctional molecule of any one of embodiments 173-176, wherein the multifunctional molecule does not activate NK cells when the NK cells are not CD16 expressing NK cells and tumor antigens on lymphoma cells are also present.

[0328] 178. The multifunctional molecule of any one of embodiments 173-177, wherein the NK cell engager comprises a VH comprising the amino acid sequence of SEQ ID NO:6185 (or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95% or 99% sequence identity with SEQ ID NO:6185).

[0329] 179. The multifunctional molecule of any one of embodiments 173-178, wherein the NK cell engager comprises a VL comprising the amino acid sequence of SEQ ID NO:6186 (or an amino acid sequence having at least about 93%, 95% or 99% sequence identity with SEQ ID NO:6186).

[0330] 180. The multifunctional molecule of any one of embodiments 173-179, wherein the NK cell engager comprises a scFV comprising the amino acid sequence of SEQ ID NO:6184 (or an amino acid sequence having at least about 93%, 95%, 99% sequence identity with SEQ ID NO:6184).

[0331] 181. The multifunctional molecule of embodiment 57, wherein the NK cell engager is a ligand, optionally, the ligand further comprises an immunoglobulin constant region, e.g., an Fc region.

[0332] 182. The multifunctional molecule of embodiment 181, wherein the NK cell engager is a ligand of NKp44 or NKp46, e.g., viral HA.

[0333] 183. The multifunctional molecule of embodiment 181, wherein the NK cell engager is a ligand of DAP10, e.g., a co-receptor of NKG2D.

[0334] 184. The multifunctional molecule of embodiment 181, wherein the NK cell engager is a ligand of CD16, e.g., a CD16a / b ligand, e.g., a CD16a / b ligand further comprising an antibody Fc region.

[0335] 185. The multifunctional molecule of any one of embodiments 52-54, wherein the immune cell engager mediates binding to one or more of B cells, macrophages and / or dendritic cells or mediates activation of one or more of B cells, macrophages and / or dendritic cells, or both.

[0336] 186. The multifunctional molecule of embodiment 185, wherein the immune cell engager comprises a B cell, macrophage and / or dendritic cell engager selected from one or more of the following: a CD40 ligand (CD40L) or a CD70 ligand; an antibody molecule that binds to CD40 or CD70; an antibody molecule of OX40; an OX40 ligand (OX40L); a Toll-like receptor agonist (e.g., TLR4, e.g., a constitutively active TLR4 (caTLR4) or a TLR9 agonist); 41BB; a CD2 agonist; CD47; or a STING agonist, or a combination thereof.

[0337] 187. The multifunctional molecule of any one of embodiments 52-54, wherein the immune cell engager is a B cell engager, e.g., a CD40L, OX40L or CD70 ligand, or an antibody molecule that binds OX40, CD40 or CD70.

[0338] 188. The multifunctional molecule of any one of embodiments 52-54, wherein the immune cell engager is a macrophage engager, e.g., a CD2 agonist; CD40L; OX40L; an antibody molecule that binds to OX40, CD40, or CD70; a Toll-like receptor (TLR) agonist (e.g., TLR4, e.g., a constitutively active TLR4 (caTLR4) or TLR9 agonist); CD47; or a STING agonist.

[0339] 189. The multifunctional molecule of any one of embodiments 52-54, wherein the immune cell engager is a dendritic cell engager, e.g., a CD2 agonist, an OX40 antibody, OX40L, a 41BB agonist, a Toll-like receptor agonist or a fragment thereof (e.g., TLR4, e.g., constitutively active TLR4 (caTLR4)), a CD47 agonist, or a STING agonist.

[0340] 190. The multifunctional molecule of embodiment 188 or 189, wherein the STING agonist comprises a cyclic dinucleotide, e.g., cyclic di-GMP (cdGMP), cyclic di-AMP (cdAMP), or a combination thereof, optionally having a 2',5' phosphoester bond or a 3',5' phosphoester bond, e.g., wherein the STING agonist is covalently coupled to the multifunctional molecule.

[0341] 191. The multifunctional molecule of any one of embodiments 1-51, wherein the multifunctional molecule comprises a cytokine molecule.

[0342] 192. A multifunctional molecule according to embodiment 191, wherein the cytokine molecule is selected from interleukin-2 (IL-2), interleukin-7 (IL-7), interleukin-12 (IL-12), interleukin-15 (IL-15), interleukin-18 (IL-18), interleukin-21 (IL-21) or interferon γ, or a fragment or variant thereof, or a combination of any of the above cytokines.

[0343] 192A. The multifunctional molecule of embodiment 192, wherein the cytokine molecule is interleukin-2 (IL-2).

[0344] 193. The multifunctional molecule of embodiment 191 or 192, wherein the cytokine molecule is a monomer or a dimer.

[0345] 194. The multifunctional molecule of any one of embodiments 191-193, wherein the cytokine molecule further comprises a receptor dimerization domain, e.g., an IL15Rα dimerization domain.

[0346] 195. The multifunctional molecule of embodiment 194, wherein the cytokine molecule (e.g., IL-15) and the receptor dimerization domain (e.g., IL15Rα dimerization domain) are not covalently linked, e.g., non-covalently associated.

[0347] 196. The multifunctional molecule of any one of embodiments 1-51, wherein the multifunctional molecule comprises a cytokine inhibitor molecule.

[0348] 197. The multifunctional molecule of embodiment 196, wherein the cytokine inhibitor molecule is a TGF-β inhibitor.

[0349] 198. A multifunctional molecule according to any one of embodiments 196 or 197, wherein the TGF-β inhibitor inhibits (e.g., reduces the activity of): (i) TGF-β1; (ii) TGF-β2; (iii) TGF-β3; (iv) (i) and (ii); (v) (i) and (iii); (vi) (ii) and (iii); or (vii) (i), (ii) and (iii).

[0350] 199. The multifunctional molecule of any one of embodiments 196-198, wherein the TGF-β inhibitor comprises a portion of a TGF-β receptor (e.g., the extracellular domain of a TGF-β receptor) that is capable of inhibiting (e.g., reducing the activity of) TGF-β, or a functional fragment or variant thereof.

[0351] 200. A multifunctional molecule of embodiment 199, wherein the TGF-β inhibitor comprises a portion of: (i) TGFBR1; (ii) TGFBR2; (iii) TGFBR3; (iv) (i) and (ii); (v) (i) and (iii); (vi) (ii) and (iii); or (vii) (i), (ii) and (iii).

[0352] 201. The multifunctional molecule of any one of embodiments 196-200, wherein the TGF-β inhibitor comprises an amino acid sequence selected from Table 16, or an amino acid sequence having at least about 93%, 95% or 99% sequence identity thereto.

[0353] 202. The multifunctional molecule of any one of embodiments 1-51, wherein the multifunctional molecule comprises a death receptor signaling linker, wherein the death receptor signaling linker is selected from a TNF-related apoptosis-inducing ligand (TRAIL) molecule, a death receptor molecule, or an antigen binding domain that specifically binds to a death receptor.

[0354] 203. The multifunctional molecule of embodiment 202, wherein the death receptor signaling adaptor activates death receptor signaling in lymphoma cells (e.g., T cells) or lymphocytes expressing TRBC1 or TRBC2, for example, and induces apoptosis or cell death in the cells.

[0355] 204. The multifunctional molecule of any one of embodiments 202 or 203, wherein the death receptor signaling adaptor does not activate death receptor signaling on non-lymphoma cells and lymphocytes that do not express TRBC1 or do not express TRBC2.

[0356] 205. The multifunctional molecule of any one of embodiments 202-204, wherein the death receptor signaling adaptor comprises a TRAIL molecule, eg, one or more TRAIL polypeptides or fragments thereof.

[0357] 206. The multifunctional molecule of embodiment 205, wherein the TRAIL molecule specifically binds to death receptor 4 (DR4) or death receptor 5 (DR5).

[0358] 207. The multifunctional molecule of any one of embodiments 205 or 206, wherein the TRAIL molecule comprises a truncated TRAIL polypeptide, e.g., relative to a wild-type TRAIL polypeptide.

[0359] 208. The multifunctional molecule of embodiment 207, wherein the TRAIL molecule comprises at least the residues corresponding to amino acids 95-281 of human TRAIL, for example, a truncated TRAIL molecule comprising residues corresponding to amino acids 95-281 of human TRAIL.

[0360] 209. The multifunctional molecule of embodiment 208, wherein the TRAIL molecule comprises a truncated TRAIL polypeptide comprising amino acids 95-281 of human TRAIL, for example, and not amino acids 1-94 of human TRAIL.

[0361] 210. The multifunctional molecule of embodiment 207, wherein the TRAIL molecule comprises at least the residues corresponding to amino acids 122-281 of human TRAIL, for example, a truncated TRAIL molecule comprising residues corresponding to amino acids 122-281 of human TRAIL.

[0362] 211. The multifunctional molecule of embodiment 210, wherein the TRAIL molecule comprises a truncated TRAIL polypeptide comprising amino acids 122-281 of human TRAIL, for example, and not amino acids 1-121 of human TRAIL.

[0363] 212. The multifunctional molecule of any one of embodiments 205-211, wherein the death receptor signaling adaptor comprises one, two or three TRAIL molecules.

[0364] 213. The multifunctional molecule of any one of embodiments 202-204, wherein the death receptor signaling adaptor comprises an antigen binding domain that specifically binds to a death receptor (e.g., death receptor 4 (DR4) or death receptor 5 (DR5)).

[0365] 214. The multifunctional molecule of embodiment 213, wherein the death receptor signaling adaptor comprises one, two or three antigen binding domains that specifically bind to a death receptor.

[0366] 215. The multifunctional molecule of any of embodiments 213 or 214, wherein the antigen binding domain that specifically binds to a death receptor binds to DR5.

[0367] 216. The multifunctional molecule of any one of embodiments 213-215, wherein the antigen binding domain that specifically binds to a death receptor comprises tigatuzumab, drozitumab, or conatumumab.

[0368] 217. The multifunctional molecule of any one of embodiments 202-216, wherein the death receptor signaling adaptor comprises an amino acid sequence selected from Table 11, or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95% or 99% sequence identity thereto.

[0369] 218. The multifunctional molecule of any one of embodiments 202-217, wherein the death receptor signaling adaptor comprises the amino acid sequence of SEQ ID NO: 6157, or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95% or 99% sequence identity thereto.

[0370] 219. The multifunctional molecule of any one of embodiments 202-217, wherein the death receptor signaling adaptor comprises the amino acid sequence of SEQ ID NO: 6158, or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95% or 99% sequence identity thereto.

[0371] 220. The multifunctional molecule of any one of embodiments 202-217, wherein the death receptor signaling adaptor comprises the amino acid sequence of SEQ ID NO: 6159, or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95% or 99% sequence identity thereto.

[0372] 221. The multifunctional molecule of any one of embodiments 202-217, wherein the death receptor signaling adaptor comprises the amino acid sequence of SEQ ID NO: 6160, or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95% or 99% sequence identity thereto.

[0373] 222. The multifunctional molecule of any one of embodiments 202-217, wherein the death receptor signaling adaptor comprises the amino acid sequence of SEQ ID NO: 6161, or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95% or 99% sequence identity thereto.

[0374] 223. The multifunctional molecule of any one of embodiments 202-217, wherein the death receptor signaling adaptor comprises the amino acid sequence of SEQ ID NO: 6162, or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95% or 99% sequence identity thereto.

[0375] 224. The multifunctional molecule of any one of embodiments 202-217, wherein the death receptor signaling adaptor comprises the amino acid sequence of SEQ ID NO: 6163, or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95% or 99% sequence identity thereto.

[0376] 225. The multifunctional molecule of any one of embodiments 202-217, wherein the death receptor signaling adaptor comprises the amino acid sequence of SEQ ID NO: 6164, or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95% or 99% sequence identity thereto.

[0377] 226. The multifunctional molecule of any one of embodiments 202-217, wherein the death receptor signaling adaptor comprises the amino acid sequence of SEQ ID NO: 6165, or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95% or 99% sequence identity thereto.

[0378] 227. The multifunctional molecule of embodiment 56, wherein the T cell engager binds to TCRβ, for example, to the TCRβV chain (TCRBV).

[0379] 228. The multifunctional molecule of embodiment 227, wherein the T cell engager comprises an antigen binding domain (e.g., an antibody molecule or fragment thereof) that binds to (e.g., and in some embodiments activates) TCRβ.

[0380] 229. The multifunctional molecule of any one of embodiments 227 or 228, wherein the T cell engager comprises, for example, an anti-TCRβV antibody molecule that specifically binds to the human TCRβV chain (TCRβV).

[0381] 230. The multifunctional molecule of any one of embodiments 227-229, wherein the T cell engager does not bind to lymphoma cells and lymphocytes expressing TRBC1 or TRBC2.

[0382] 231. The multifunctional molecule of any one of embodiments 227-229, wherein the T cell engager is capable of binding to or to lymphoma cells or lymphocytes expressing TRBC1 or TRBC2.

[0383] 232. The multifunctional molecule of any one of embodiments 227-231, wherein the T cell engager does not activate lymphoma cells and lymphocytes expressing TRBC1 or TRBC2.

[0384] 233. The multifunctional molecule of any one of embodiments 227-232, wherein the T cell engager comprises an anti-TCRβV antibody molecule that specifically binds to the TCRβV subfamily or subfamily member of Table 12.

[0385] 234. A multifunctional molecule of embodiment 233, wherein the anti-TCRβV antibody molecule specifically binds to TCRβV6, for example, the TCRβV6 subfamily, which includes: TCRβV6-4*01, TCRβV6-4*02, TCRβV6-9*01, TCRβV6-8*01, TCRβV6-5*01, TCRβV6-6*02, TCRβV6-6*01, TCRβV6-2*01, TCRβV6-3*01 or TCRβV6-1*01.

[0386] 235. A multifunctional molecule of embodiment 234, wherein the anti-TCRβV antibody molecule comprises one or more CDRs, framework regions, or variable heavy chain regions and / or variable light chain regions provided in Table 13, or a sequence having at least about 93%, 95% or 99% sequence identity thereto.

[0387] 236. The multifunctional molecule of embodiment 233, wherein the anti-TCRβV antibody molecule specifically binds to TCRβV12, for example, the TCRβV12 subfamily, which includes: TCRβV12-4*01, TCRβV12-3*01 or TCRβV12-5*01.

[0388] 237. A multifunctional molecule of embodiment 236, wherein the anti-TCRβV antibody molecule comprises one or more CDRs, framework regions, or variable heavy chain regions and / or variable light chain regions provided in Table 14, or a sequence having at least about 93%, 95% or 99% sequence identity thereto.

[0389] 238. The multifunctional molecule of any one of embodiments 1-51, wherein the multifunctional molecule comprises a matrix modification portion.

[0390] 239. The multifunctional molecule of embodiment 238, wherein the matrix modification portion results in one or more of the following: reducing the level or production of matrix or extracellular matrix (ECM) components; reducing tumor fibrosis; increasing interstitial tumor transport; improving tumor perfusion; expanding tumor microvasculature; reducing interstitial fluid pressure (IFP) in the tumor; or reducing or enhancing the penetration or diffusion of an agent (e.g., a cancer therapeutic or a cell therapy) into a tumor or tumor vasculature.

[0391] 240. The multifunctional molecule of embodiment 239, wherein the reduced matrix or ECM component is selected from glycosaminoglycans or extracellular proteins, or a combination thereof.

[0392] 241. The multifunctional molecule of any one of embodiments 1-240, wherein the multifunctional molecule comprises:

[0393] (i) an immune cell engager (e.g., a T cell engager, a NK cell engager, a B cell engager, a dendritic cell engager or a macrophage engager) and a cytokine molecule,

[0394] (ii) an immune cell engager (e.g., a T cell engager, a NK cell engager, a B cell engager, a dendritic cell engager or a macrophage engager) and a cytokine inhibitor molecule,

[0395] (iii) an immune cell engager (e.g., a T cell engager, a NK cell engager, a B cell engager, a dendritic cell engager or a macrophage engager) and a death receptor signaling engager,

[0396] (iv) an immune cell engager (e.g., a T cell engager, a NK cell engager, a B cell engager, a dendritic cell engager, or a macrophage engager) and a matrix modifying moiety,

[0397] (v) cytokine molecules and matrix modification moieties,

[0398] (vi) cytokine molecules and death receptor signaling adaptors,

[0399] (vii) a cytokine inhibitor molecule and a matrix modifying moiety,

[0400] (viii) cytokine inhibitor molecules and death receptor signaling adaptors,

[0401] (ix) an immune cell engager (e.g., a T cell engager, a NK cell engager, a B cell engager, a dendritic cell engager, or a macrophage engager), a cytokine molecule, a death receptor signaling engager, and a matrix modifying moiety, or

[0402] (x) immune cell engagers (e.g., T cell engagers, NK cell engagers, B cell engagers, dendritic cell engagers, or macrophage engagers), cytokine inhibitor molecules, death receptor signaling engagers, and matrix modification moieties.

[0403] 242. The multifunctional molecule of any one of embodiments 1-241, wherein the multifunctional molecule comprises the following configuration:

[0404] A,B-[dimerization module]-C,-D, where:

[0405] (a) the dimerization module comprises an immunoglobulin constant domain, e.g., a heavy chain constant region (e.g., a homodimer or heterodimer heavy chain constant region, e.g., an Fc region) or a constant domain of an immunoglobulin variable region (e.g., a Fab region); and

[0406] (b) A, B, C and D are independently absent; (i) an antigen binding domain that preferentially binds to TRBC1 or TRBC2; (ii) an immune cell engager selected from a T cell engager, a NK cell engager, a B cell engager, a dendritic cell engager or a macrophage engager; (iii) a cytokine molecule or a cytokine inhibitor molecule; (iv) a death receptor signaling engager; or (v) a matrix modifying moiety, provided that:

[0407] At least one, two or three of A, B, C and D comprise an antigen binding domain that preferentially binds to TRBC1 or TRBC2, and

[0408] Any of the remaining A, B, C and D is absent or includes one of an immune cell engager, a cytokine molecule, a cytokine inhibitor molecule, a death receptor signaling engager or a matrix modification moiety.

[0409] 243. The multifunctional molecule of embodiment 242, wherein:

[0410] (1) A comprises an antigen binding domain that preferentially binds to a T cell receptor comprising TRBC1 or TRBC2, and B, C or D comprises an immune cell engager, e.g., a T cell engager, e.g., an anti-TCRβV antibody molecule;

[0411] (2) A comprises an antigen binding domain that preferentially binds to TRBC1 or TRBC2, and B, C or D comprises an immune cell engager, e.g., an NK cell engager, e.g., an anti-NKp30 or anti-NKp46 antibody molecule;

[0412] (3) A comprises an antigen binding domain that preferentially binds to TRBC1 or TRBC2, and B, C, or D comprises a cytokine molecule;

[0413] (4) A comprises an antigen binding domain that preferentially binds to TRBC1 or TRBC2, and B, C or D comprises a cytokine inhibitor molecule;

[0414] (5) A comprises an antigen binding domain that preferentially binds to TRBC1 or TRBC2, and B, C, or D comprises a death receptor signaling adaptor;

[0415] (6) A comprises an antigen binding domain that preferentially binds to TRBC1 or TRBC2, and B, C or D comprises a matrix modifying portion;

[0416] (7) A comprises a first antigen binding domain that binds to TRBC1 or TRBC2, B comprises a second antigen binding domain that preferentially binds to TRBC1 or TRBC2, and C or D comprises an immune cell engager, e.g., a T cell engager, e.g., an anti-TCRβV antibody molecule;

[0417] (8) A comprises a first antigen binding domain that binds to TRBC1 or TRBC2, B comprises a second antigen binding domain that preferentially binds to TRBC1 or TRBC2, and C or D comprises an immune cell engager, e.g., an NK cell engager, e.g., an anti-NKp30, anti-NKp46, anti-NKG2D or anti-CD16 antibody molecule;

[0418] (9) A comprises a first antigen binding domain that preferentially binds to TRBC1 or TRBC2, B comprises a second antigen binding domain that preferentially binds to TRBC1 or TRBC2, and C or D comprises a cytokine molecule;

[0419] (10) A comprises a first antigen binding domain that preferentially binds to TRBC1 or TRBC2, B comprises a second antigen binding domain that preferentially binds to TRBC1 or TRBC2, and C or D comprises a cytokine inhibitor molecule;

[0420] (11) A comprises a first antigen binding domain that preferentially binds to TRBC1 or TRBC2, B comprises a second antigen binding domain that preferentially binds to TRBC1 or TRBC2, and C or D comprises a death receptor signaling adaptor;

[0421] (12) A comprises a first antigen binding domain that preferentially binds to TRBC1 or TRBC2, B comprises a second antigen binding domain that preferentially binds to TRBC1 or TRBC2, and C or D comprises a matrix modification portion;

[0422] (13) A comprises a first antigen binding domain that preferentially binds to TRBC1 or TRBC2, C comprises a second antigen binding domain that preferentially binds to TRBC1 or TRBC2, and B or D comprises an immune cell engager, e.g., a T cell engager, e.g., an anti-TCRβV antibody molecule;

[0423] (14) A comprises a first antigen binding domain that preferentially binds to TRBC1 or TRBC2, C comprises a second antigen binding domain that preferentially binds to TRBC1 or TRBC2, and B or D comprises an immune cell engager, e.g., an NK cell engager, e.g., an anti-NKp30, anti-NKp46, anti-NKG2D or anti-CD16 antibody molecule;

[0424] (15) A comprises a first antigen binding domain that preferentially binds to TRBC1 or TRBC2, C comprises a second antigen binding domain that preferentially binds to TRBC1 or TRBC2, and B or D comprises a cytokine molecule;

[0425] (16) A comprises a first antigen binding domain that preferentially binds to TRBC1 or TRBC2, C comprises a second antigen binding domain that preferentially binds to TRBC1 or TRBC2, and B or D comprises a cytokine inhibitor molecule;

[0426] (17) A comprises a first antigen binding domain that preferentially binds to TRBC1 or TRBC2, C comprises a second antigen binding domain that preferentially binds to TRBC1 or TRBC2, and B or D comprises a death receptor signaling adaptor;

[0427] (18) A comprises a first antigen binding domain that preferentially binds to TRBC1 or TRBC2, C comprises a second antigen binding domain that preferentially binds to TRBC1 or TRBC2, and B or D comprises a matrix modification portion;

[0428] (19) A comprises a first antigen binding domain that preferentially binds to TRBC1 or TRBC2, and B, C or D comprises (a) an immune cell engager, e.g., an NK cell engager, e.g., an anti-NKp30, anti-NKp46, anti-NKG2D or anti-CD16 antibody molecule, and (b) a cytokine molecule;

[0429] (20) A comprises a first antigen binding domain that preferentially binds to TRBC1 or TRBC2, and B, C or D comprises (a) an immune cell engager, e.g., an NK cell engager, e.g., an anti-NKp30, anti-NKp46, anti-NKG2D or anti-CD16 antibody molecule, and (b) a cytokine inhibitor molecule;

[0430] (21) A comprises a first antigen binding domain that preferentially binds to TRBC1 or TRBC2, and B, C or D comprises (a) an immune cell engager, e.g., an NK cell engager, e.g., an anti-NKp30, anti-NKp46, anti-NKG2D or anti-CD16 antibody molecule, and (b) a death receptor signaling engager;

[0431] (22) A comprises a first antigen binding domain that preferentially binds to TRBC1 or TRBC2, and B, C or D comprises (a) an immune cell engager, e.g., an NK cell engager, e.g., an anti-NKp30, anti-NKp46, anti-NKG2D or anti-CD16 antibody molecule, and (b) a matrix modifying portion;

[0432] (23) A comprises a first antigen binding domain that preferentially binds to TRBC1 or TRBC2, and B, C or D comprises (a) an immune cell engager, e.g., a T cell engager, e.g., an anti-TCRβV antibody molecule, and (b) a cytokine molecule;

[0433] (24) A comprises a first antigen binding domain that preferentially binds to TRBC1 or TRBC2, and B, C or D comprises (a) an immune cell engager, e.g., a T cell engager, e.g., an anti-TCRβV antibody molecule, and (b) a cytokine inhibitor molecule;

[0434] (25) A comprises a first antigen binding domain that preferentially binds to TRBC1 or TRBC2, and B, C or D comprises (a) an immune cell engager, e.g., a T cell engager, e.g., an anti-TCRβV antibody molecule, and (b) a death receptor signaling engager;

[0435] (26) A comprises a first antigen binding domain that preferentially binds to TRBC1 or TRBC2, and B, C or D comprises (a) an immune cell engager, e.g., a T cell engager, e.g., an anti-TCRβV antibody molecule, and (b) a matrix modifying portion;

[0436] (27) A comprises a first antigen binding domain that preferentially binds to TRBC1 or TRBC2, and B, C or D comprises (a) a cytokine molecule and (b) a matrix modifying moiety;

[0437] (28) A comprises a first antigen binding domain that preferentially binds to TRBC1 or TRBC2, and B, C or D comprises (a) a cytokine molecule and (b) a death receptor signaling adaptor;

[0438] (29) A comprises a first antigen binding domain that preferentially binds to TRBC1 or TRBC2, and B, C or D comprises (a) a cytokine inhibitor molecule and (b) a matrix modifying moiety;

[0439] (30) A comprises a first antigen binding domain that preferentially binds to TRBC1 or TRBC2, and B, C or D comprises (a) a cytokine inhibitor molecule and (b) a death receptor signaling adaptor;

[0440] (31) A comprises a first antigen binding domain that preferentially binds to TRBC1 or TRBC2, and B, C or D comprises (a) a death receptor signaling adaptor and (b) a substrate modification moiety;

[0441] (32) A comprises a first antigen binding domain that preferentially binds to TRBC1 or TRBC2, B comprises a second antigen binding domain that preferentially binds to TRBC1 or TRBC2, and C or D comprises (a) an immune cell engager, e.g., an NK cell engager, e.g., an anti-NKp30, anti-NKp46, anti-NKG2D or anti-CD16 antibody molecule, and (b) a cytokine molecule;

[0442] (33) A comprises a first antigen binding domain that preferentially binds to TRBC1 or TRBC2, B comprises a second antigen binding domain that preferentially binds to TRBC1 or TRBC2, and C or D comprises (a) an immune cell engager, e.g., an NK cell engager, e.g., an anti-NKp30, anti-NKp46, anti-NKG2D or anti-CD16 antibody molecule, and (b) a cytokine inhibitor molecule;

[0443] (34) A comprises a first antigen binding domain that preferentially binds to TRBC1 or TRBC2, B comprises a second antigen binding domain that preferentially binds to TRBC1 or TRBC2, and C or D comprises (a) an immune cell engager, e.g., an NK cell engager, e.g., an anti-NKp30, anti-NKp46, anti-NKG2D or anti-CD16 antibody molecule, and (b) a death receptor signaling engager;

[0444] (35) A comprises a first antigen binding domain that preferentially binds to TRBC1 or TRBC2, B comprises a second antigen binding domain that preferentially binds to TRBC1 or TRBC2, and C or D comprises (a) an immune cell engager, e.g., an NK cell engager, e.g., an anti-NKp30, anti-NKp46, anti-NKG2D or anti-CD16 antibody molecule, and (b) a matrix modification portion;

[0445] (36) A comprises a first antigen binding domain that preferentially binds to TRBC1 or TRBC2, B comprises a second antigen binding domain that preferentially binds to TRBC1 or TRBC2, and C or D comprises (a) an immune cell engager, e.g., a T cell engager, e.g., an anti-TCRβV antibody molecule, and (b) a cytokine molecule;

[0446] (37) A comprises a first antigen binding domain that preferentially binds to TRBC1 or TRBC2, B comprises a second antigen binding domain that preferentially binds to TRBC1 or TRBC2, and C or D comprises (a) an immune cell engager, e.g., a T cell engager, e.g., an anti-TCRβV antibody molecule, and (b) a cytokine inhibitor molecule;

[0447] (38) A comprises a first antigen binding domain that preferentially binds to TRBC1 or TRBC2, B comprises a second antigen binding domain that preferentially binds to TRBC1 or TRBC2, and C or D comprises (a) an immune cell engager, e.g., a T cell engager, e.g., an anti-TCRβV antibody molecule, and (b) a death receptor signaling engager;

[0448] (39) A comprises a first antigen binding domain that preferentially binds to TRBC1 or TRBC2, B comprises a second antigen binding domain that preferentially binds to TRBC1 or TRBC2, and C or D comprises (a) an immune cell engager, e.g., a T cell engager, e.g., an anti-TCRβV antibody molecule, and (b) a matrix modification portion;

[0449] (40) A comprises a first antigen binding domain that preferentially binds to TRBC1 or TRBC2, B comprises a second antigen binding domain that preferentially binds to TRBC1 or TRBC2, and C or D comprises (a) a cytokine molecule and (b) a matrix modifying portion;

[0450] (41) A comprises a first antigen binding domain that preferentially binds to TRBC1 or TRBC2, B comprises a second antigen binding domain that preferentially binds to TRBC1 or TRBC2, and C or D comprises (a) a cytokine molecule and (b) a death receptor signaling adaptor;

[0451] (42) A comprises a first antigen binding domain that preferentially binds to TRBC1 or TRBC2, B comprises a second antigen binding domain that preferentially binds to TRBC1 or TRBC2, and C or D comprises (a) a cytokine inhibitor molecule and (b) a matrix modification moiety;

[0452] (43) A comprises a first antigen binding domain that preferentially binds to TRBC1 or TRBC2, B comprises a second antigen binding domain that preferentially binds to TRBC1 or TRBC2, and C or D comprises (a) a cytokine inhibitor molecule and (b) a death receptor signaling adaptor;

[0453] (44) A comprises a first antigen binding domain that preferentially binds to TRBC1 or TRBC2, B comprises a second antigen binding domain that preferentially binds to TRBC1 or TRBC2, and C or D comprises (a) a substrate modification portion and (b) a death receptor signaling adaptor;

[0454] (45) A comprises a first antigen binding domain that preferentially binds to TRBC1 or TRBC2, C comprises a second antigen binding domain that preferentially binds to TRBC1 or TRBC2, and B or D comprises (a) an immune cell engager, e.g., an NK cell engager, e.g., an anti-NKp30, anti-NKp46, anti-NKG2D or anti-CD16 antibody molecule, and (b) a cytokine molecule;

[0455] (46) A comprises a first antigen binding domain that preferentially binds to TRBC1 or TRBC2, C comprises a second antigen binding domain that preferentially binds to TRBC1 or TRBC2, and B or D comprises (a) an immune cell engager, e.g., an NK cell engager, e.g., an anti-NKp30, anti-NKp46, anti-NKG2D or anti-CD16 antibody molecule, and (b) a cytokine inhibitor molecule;

[0456] (47) A comprises a first antigen binding domain that preferentially binds to TRBC1 or TRBC2, C comprises a second antigen binding domain that preferentially binds to TRBC1 or TRBC2, and B or D comprises (a) an immune cell engager, e.g., an NK cell engager, e.g., an anti-NKp30, anti-NKp46, anti-NKG2D or anti-CD16 antibody molecule, and (b) a death receptor signaling engager;

[0457] (48) A comprises a first antigen binding domain that preferentially binds to TRBC1 or TRBC2, C comprises a second antigen binding domain that preferentially binds to TRBC1 or TRBC2, and B or D comprises (a) an immune cell engager, e.g., an NK cell engager, e.g., an anti-NKp30, anti-NKp46, anti-NKG2D or anti-CD16 antibody molecule, and (b) a matrix modification moiety;

[0458] (49) A comprises a first antigen binding domain that preferentially binds to TRBC1 or TRBC2, C comprises a second antigen binding domain that preferentially binds to TRBC1 or TRBC2, and B or D comprises (a) an immune cell engager, e.g., a T cell engager, e.g., an anti-TCRβV antibody molecule, and (b) a cytokine molecule;

[0459] (50) A comprises a first antigen binding domain that preferentially binds to TRBC1 or TRBC2, C comprises a second antigen binding domain that preferentially binds to TRBC1 or TRBC2, and B or D comprises (a) an immune cell engager, e.g., a T cell engager, e.g., an anti-TCRβV antibody molecule, and (b) a cytokine inhibitor molecule;

[0460] (51) A comprises a first antigen binding domain that preferentially binds to TRBC1 or TRBC2, C comprises a second antigen binding domain that preferentially binds to TRBC1 or TRBC2, and B or D comprises (a) an immune cell engager, e.g., a T cell engager, e.g., an anti-TCRβV antibody molecule, and (b) a death receptor signaling engager;

[0461] (52) A comprises a first antigen binding domain that preferentially binds to TRBC1 or TRBC2, C comprises a second antigen binding domain that preferentially binds to TRBC1 or TRBC2, and B or D comprises (a) an immune cell engager, e.g., a T cell engager, e.g., an anti-TCRβV antibody molecule, and (b) a matrix modification moiety;

[0462] (53) A comprises a first antigen binding domain that preferentially binds to TRBC1 or TRBC2, C comprises a second antigen binding domain that preferentially binds to TRBC1 or TRBC2, and B or D comprises (a) a cytokine molecule and (b) a matrix modifying portion;

[0463] (54) A comprises a first antigen binding domain that preferentially binds to TRBC1 or TRBC2, C comprises a second antigen binding domain that preferentially binds to TRBC1 or TRBC2, and B or D comprises (a) a cytokine molecule and (b) a death receptor signaling adaptor;

[0464] (55) A comprises a first antigen binding domain that preferentially binds to TRBC1 or TRBC2, C comprises a second antigen binding domain that preferentially binds to TRBC1 or TRBC2, and B or D comprises (a) a cytokine inhibitor molecule and (b) a matrix modification moiety;

[0465] (56) A comprises a first antigen binding domain that preferentially binds to TRBC1 or TRBC2, C comprises a second antigen binding domain that preferentially binds to TRBC1 or TRBC2, and B or D comprises (a) a cytokine inhibitor molecule and (b) a death receptor signaling adaptor; or

[0466] (57) A comprises a first antigen binding domain that preferentially binds to TRBC1 or TRBC2, C comprises a second antigen binding domain that preferentially binds to TRBC1 or TRBC2, and B or D comprises (a) a substrate modification portion and (b) a death receptor signaling adaptor.

[0467] 244. A multifunctional molecule of embodiment 242 or 243, wherein the dimerization module comprises one or more immunoglobulin chain constant regions (e.g., Fc regions), wherein the immunoglobulin chain constant regions comprise one or more of the following: paired cavity-protrusions ("knob-in-hole structures"), electrostatic interactions, or chain exchange.

[0468] 245. The multifunctional molecule of embodiment 244, wherein the one or more immunoglobulin chain constant regions (e.g., Fc regions) comprise an amino acid replacement at a position selected from one or more of 347, 349, 350, 351, 366, 368, 370, 392, 394, 395, 397, 398, 399, 405, 407 or 409 of the Fc region of, e.g., human IgG1, optionally wherein the one or more immunoglobulin chain constant regions (e.g., Fc regions) comprise an amino acid replacement selected from T366S, L368A or Y407V (e.g., corresponding to a cavity or hole), or T366W (e.g., corresponding to a protrusion or knob), or a combination thereof.

[0469] 246. The multifunctional molecule of any one of embodiments 1-245 further comprises a linker, for example, a linker between one or more of the following: an antigen binding domain and an immune cell connector, an antigen binding domain and a cytokine molecule, an antigen binding domain and a matrix modification portion, an immune cell connector and a cytokine molecule, an immune cell connector and a matrix modification portion, a cytokine molecule and a matrix modification portion, an antigen binding domain and a dimerization module, an immune cell connector and a dimerization module, a cytokine molecule and a dimerization module, or a matrix modification portion and a dimerization module.

[0470] 247. The multifunctional molecule of embodiment 246, wherein the linker is selected from: a cleavable linker, a non-cleavable linker, a peptide linker, a flexible linker, a rigid linker, a helical linker or a non-helical linker.

[0471] 248. The multifunctional molecule of embodiment 246 or 247, wherein the linker is a peptide linker.

[0472] 249. The multifunctional molecule of 248, wherein the peptide linker comprises Gly and Ser.

[0473] 250. The multifunctional molecule of 249, wherein the peptide linker comprises an amino acid sequence selected from SEQ ID NO: 7249-7252 or 75-78.

[0474] 251. A multifunctional molecule comprising:

[0475] (i) a first antigen binding domain that preferentially binds to TRBC1, and

[0476] (ii) NK cell engagers, for example, anti-NKp30 antibody molecules, anti-NKp46 antibody molecules, anti-NKG2D antibody molecules or anti-CD16 antibody molecules.

[0477] 252. The multifunctional molecule of embodiment 251, wherein the NK cell engager comprises an anti-NKp30 antibody molecule.

[0478] 254. The multifunctional molecule of embodiment 251, wherein the NK cell engager comprises an anti-NKp46 antibody molecule.

[0479] 255. The multifunctional molecule of embodiment 251, wherein the NK cell engager comprises an anti-NKG2D antibody molecule.

[0480] 255A. The multifunctional molecule of embodiment 251, wherein the NK cell engager comprises an anti-CD16 antibody molecule.

[0481] 256. A multifunctional molecule comprising:

[0482] (i) a first antigen binding domain that preferentially binds to TRBC1, and

[0483] (ii) Death receptor signaling adaptors.

[0484] 257. A multifunctional molecule comprising:

[0485] (i) a first antigen binding domain that preferentially binds to TRBC1, and

[0486] (ii) T cell engager, e.g., an antigen binding domain that binds to the TCRβ V chain (TCRBV).

[0487] 259. A multifunctional molecule comprising:

[0488] (i) a first antigen binding domain that preferentially binds to TRBC1, and

[0489] (ii) Cytokine inhibitor molecules, for example, TGF-β inhibitors.

[0490] 262. The multifunctional molecule of any one of embodiments 1 or 3-261, wherein the multifunctional molecule monovalently binds to TRBC1, TRBC2 or a tumor antigen.

[0491] 263. The multifunctional molecule of any one of embodiments 1 or 3-261, wherein the multifunctional molecule binds to TRBC1, TRBC2 or a tumor antigen multivalently, for example, bivalently, trivalently, tetravalently, pentavalently, hexavalently, heptavalently, octavalently, nonavalently or decavalently.

[0492] 264. The multifunctional molecule of any one of embodiments 2-261, wherein the multifunctional molecule monovalently binds to TRBC1, TRBC2, or a lymphocyte expressing TRBC1 or TRBC2.

[0493] 265. The multifunctional molecule of any one of embodiments 2-261, wherein the multifunctional molecule binds multivalently, for example, bivalently, trivalently, tetravalently, pentavalently, hexavalently, heptavalently, octavalently, nonavalently or decavalently, to lymphocytes expressing TRBC1 or TRBC2.

[0494] 266. The multifunctional molecule of any preceding embodiment, wherein the multifunctional molecule monovalently binds to an immune cell, e.g., via an immune cell engager.

[0495] 267. The multifunctional molecule of any one of embodiments 1-265, wherein the multifunctional molecule is multivalently bound to an immune cell, for example, via an immune cell connector, for example, bivalent, trivalent, tetravalent, pentavalent, hexavalent, heptavalent, octavalent, nonavalent or decavalent.

[0496] 268. The multifunctional molecule of any preceding embodiment, further comprising a heavy chain constant region, eg, an Fc region, that mediates antibody-dependent cellular cytotoxicity (ADCC).

[0497] 268A. The multifunctional molecule of any preceding embodiment, further comprising a heavy chain constant region that mediates antibody-dependent cellular phagocytosis (ADCP), eg, an Fc region.

[0498] 268B. The multifunctional molecule of embodiment 268A, wherein the first antigen binding domain that binds TRBC1 or TRBC2 comprises an IgG2 heavy chain constant region or the immune cell engager, cytokine inhibitor molecule or death receptor signaling engager comprises an IgG2 heavy chain constant region.

[0499] 269. The multifunctional molecule of any preceding embodiment, further comprising a heavy chain constant region, eg, an Fc region, that mediates complement dependent cytotoxicity (eg, via C1q).

[0500] 269A. An antibody molecule that binds to TRBC1, comprising one or more CDRs, framework regions, variable domains, heavy chains or light chains, or antigen binding domains selected from Tables 2-5, or sequences substantially identical thereto.

[0501] 269B. The antibody molecule of embodiment 269A, comprising a heavy chain variable region (VH), said heavy chain variable region comprising the heavy chain framework region 1 (VHFWR1) amino acid sequence of SEQ ID NO:215 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations (e.g., substitutions, additions or deletions) thereof), the VHFWR2 amino acid sequence of SEQ ID NO:216 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations (e.g., substitutions, additions or deletions) thereof), the VHFWR3 amino acid sequence of SEQ ID NO:217 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations (e.g., substitutions, additions or deletions) thereof), or the VHFWR4 amino acid sequence of SEQ ID NO:218 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations (e.g., substitutions, additions or deletions) thereof).

[0502] 269C. The antibody molecule of any one of embodiments 269A or 269B, comprising a light chain variable region (VL), comprising a light chain framework region 1 (VLFWR1) amino acid sequence of SEQ ID NO: 238 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations (e.g., substitutions, additions or deletions) thereof), a VLFWR2 amino acid sequence of SEQ ID NO: 239 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations (e.g., substitutions, additions or deletions) thereof), a VLFWR3 amino acid sequence of SEQ ID NO: 240 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations (e.g., substitutions, additions or deletions) thereof), or a VLFWR4 amino acid sequence of SEQ ID NO: 241 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations (e.g., substitutions, additions or deletions) thereof).

[0503] 269D. The antibody molecule of any one of embodiments 269A-269C, wherein the antibody molecule comprises a VH comprising the amino acid sequence of SEQ ID NO: 253 (or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95% or 99% sequence identity thereto).

[0504] 269E. The antibody molecule of any one of embodiments 269A-269D, wherein the antibody molecule comprises a VL comprising the amino acid sequence of SEQ ID NO: 258 (or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95% or 99% sequence identity thereto).

[0505] 270. A nucleic acid molecule encoding the multifunctional molecule or antibody molecule of any one of embodiments 1-269E.

[0506] 271. A vector, e.g., an expression vector, comprising the nucleic acid molecule of embodiment 270.

[0507] 272. A host cell comprising the nucleic acid molecule of embodiment 270 or the vector of embodiment 271.

[0508] 273. A method for preparing, e.g., producing, a multifunctional molecule or antibody molecule of any one of embodiments 1-269E, comprising culturing the host cell of embodiment 272 under suitable conditions, e.g., conditions suitable for gene expression and / or homodimerization or heterodimerization.

[0509] 274. A pharmaceutical composition comprising the multifunctional molecule of any one of embodiments 1-269 and a pharmaceutically acceptable carrier, excipient or stabilizer.

[0510] 275. A method of treating cancer, comprising administering to a subject in need thereof a multifunctional molecule of any one of embodiments 1-269, wherein the multifunctional molecule is administered in an amount effective to treat the cancer.

[0511] 276. The method of embodiment 275 further includes identifying, evaluating or selecting a subject in need of treatment, wherein the identification, evaluation or selection includes determining (e.g., directly determining or indirectly determining, e.g., obtaining information about) whether the subject has cancer cells that express T cell receptors including TRBC1 or TRBC2.

[0512] 277. The method of embodiment 276, further comprising, in response to determining that the subject has cancer cells expressing a T cell receptor comprising TRBC1:

[0513] Optionally, the subject is selected for treatment with a multifunctional molecule comprising an antigen binding domain that binds to a T cell receptor comprising TRBC1, and

[0514] A multifunctional molecule comprising an antigen binding domain that binds to a T cell receptor comprising TRBC1 is administered.

[0515] 278. The method of embodiment 277, further comprising not administering a multifunctional molecule comprising an antigen binding domain that binds to a T cell receptor comprising TRBC2.

[0516] 278A. A method of treating cancer, such as lymphoma or leukemia, comprising:

[0517] In response to determining that the subject has cancer cells expressing a T cell receptor comprising TRBC1, administering the multifunctional molecule of any one of embodiments 1-269 to a subject in need thereof, wherein the multifunctional molecule is administered in an amount effective to treat the cancer.

[0518] 279. The method of embodiment 276, further comprising, in response to determining that the subject has cancer cells expressing a T cell receptor comprising TRBC2:

[0519] Optionally, the subject is selected for treatment with a multifunctional molecule comprising an antigen binding domain that binds to a T cell receptor comprising TRBC2, and

[0520] A multifunctional molecule comprising an antigen binding domain that binds to a T cell receptor comprising TRBC2 is administered.

[0521] 280. The method of embodiment 279, further comprising not administering a multifunctional molecule comprising an antigen binding domain that binds to a T cell receptor comprising TRBC1.

[0522] 281. The method of any one of embodiments 275-278, wherein the subject has cancer cells expressing T cell receptors including TRBC1.

[0523] 282. The method of any one of embodiments 275, 276, 279 or 280, wherein the subject has cancer cells that express T cell receptors including TRBC2.

[0524] 283. A method of identifying a subject in need of treatment for cancer using a multifunctional molecule or antibody molecule of any one of embodiments 1-269E, comprising determining (e.g., directly determining or indirectly determining, e.g., obtaining information about) whether the subject has cancer cells that express a T cell receptor that includes TRBC1 or TRBC2, wherein:

[0525] responsive to determining that the subject has cancer cells expressing a T cell receptor that includes TRBC1, identifying the subject as a candidate for treatment with a multifunctional molecule that includes an antigen binding domain that binds to TRBC1, and optionally identifying the subject as not a candidate for treatment with a multifunctional molecule that includes an antigen binding domain that binds to TRBC2, and

[0526] In response to determining that the subject has cancer cells expressing a T cell receptor that includes TRBC2, the subject is identified as a candidate for treatment with a multifunctional molecule that includes an antigen binding domain that binds to TRBC2, and optionally the subject is identified as not a candidate for treatment with a multifunctional molecule that includes an antigen binding domain that binds to TRBC1.

[0527] 284. The method of embodiment 283, further comprising:

[0528] In response to identifying the subject as a candidate for treatment with a multifunctional molecule comprising an antigen binding domain that binds to TRBC1, treating the subject with (e.g., administering to the subject) a multifunctional molecule comprising an antigen binding domain that binds to TRBC1, or

[0529] In response to identifying the subject as a candidate for treatment with a multifunctional molecule comprising an antigen binding domain that binds to TRBC2, the subject is treated with (eg, administered to the subject) a multifunctional molecule comprising an antigen binding domain that binds to TRBC2.

[0530] 285. A method for evaluating a subject in need of treatment for cancer, such as lymphoma, comprising determining (e.g., directly determining or indirectly determining, e.g., obtaining information about) whether the subject has cancer cells that express a T cell receptor comprising TRBC1 or TRBC2.

[0531] 286. The method of embodiment 285 further comprises, in response to the evaluation, treating the subject with (e.g., administering to the subject) a multifunctional molecule comprising an antigen binding domain that binds to TRBC1 or a multifunctional molecule comprising an antigen binding domain that binds to TRBC2.

[0532] 287. The method of any one of embodiments 275-286, wherein the cancer is a hematological cancer.

[0533] 288. The method of embodiment 287, wherein the blood cancer is leukemia or lymphoma.

[0534] 289. The method of embodiment 288, wherein the blood cancer is selected from leukemia (e.g., acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), hairy cell leukemia, acute monocytic leukemia (AMoL), chronic myelomonocytic leukemia (CMML), juvenile myelomonocytic leukemia (JMML), or large granular lymphocytic leukemia), lymphoma (e.g., AIDS-related lymphoma, cutaneous T-cell lymphoma, Hodgkin lymphoma (e.g., classical Hodgkin lymphoma or nodular lymphocyte-predominant Hodgkin lymphoma), mycosis fungoides, non-Hodgkin lymphoma lymphoma (e.g., B-cell non-Hodgkin lymphoma (e.g., Burkitt lymphoma, small lymphocytic lymphoma (CLL / SLL), diffuse large B-cell lymphoma, follicular lymphoma, immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma, or mantle cell lymphoma) or T-cell non-Hodgkin lymphoma (mycosis fungoides, anaplastic large cell lymphoma, or precursor T-lymphoblastic lymphoma)), primary central nervous system lymphoma, Sezary syndrome, Waldenstrom's macroglobulinemia), chronic myeloproliferative neoplasm, Langerhans cell histiocytosis, multiple myeloma / plasmacytoma, myelodysplastic syndrome, or myelodysplastic / myeloproliferative neoplasm.

[0535] 290. The method of embodiment 288, wherein the lymphoma is selected from acquired immunodeficiency syndrome (AIDS)-related lymphoma, angioimmunoblastic T-cell lymphoma, adult T-cell leukemia / lymphoma, Burkitt's lymphoma, central nervous system (CNS) lymphoma, diffuse large B-cell lymphoma (DLBCL), lymphoblastic lymphoma, mantle cell lymphoma (MCL), peripheral T-cell lymphoma (PTCL) (e.g., hepatosplenic T-cell lymphoma (HSGDTCL), subcutaneous panniculitis-like T-cell lymphoma, or enteropathy-associated T-cell lymphoma), transgenic T-cell lymphoma (T-cell lymphoma), Variant follicular and transformed mucosa-associated lymphoid tissue (MALT) lymphoma, cutaneous T-cell lymphoma (mycosis fungoides and Sézary syndrome), follicular lymphoma, lymphoplasmacytic lymphoma / Waldenstrom's macroglobulinemia, marginal zone B-cell lymphoma, gastric mucosa-associated lymphoid tissue (MALT) lymphoma, chronic lymphocytic leukemia / small cell lymphocytic lymphoma (CLL / SLL), extranodal T-cell / NK-cell lymphoma (nasal type), and anaplastic large cell lymphoma (e.g., primary cutaneous anaplastic large cell lymphoma or systemic anaplastic large cell lymphoma).

[0536] 291. The method of any one of embodiments 275-286, wherein the cancer is a solid tumor cancer.

[0537] 292. The method of any of embodiments 275-291, further comprising administering a second therapeutic treatment.

[0538] 293. The method of embodiment 292, wherein the second therapeutic treatment comprises a therapeutic agent (e.g., a chemotherapeutic agent, a biologic agent, a hormonal therapy), radiation, or surgery.

[0539] 294. The method of embodiment 293, wherein the therapeutic agent is selected from: a chemotherapeutic agent or a biologic agent.

[0540] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those of ordinary skill in the art to which the invention belongs. Suitable methods and materials are described below, but methods and materials similar to or equivalent to the methods and materials described herein can be used in the practice or testing of the present invention. All publications, patent applications, patents and other references mentioned herein are incorporated herein by reference in their entirety. In the event of a conflict, the present specification including definitions shall prevail. In addition, materials, methods and examples are illustrative only and are not intended to be limiting.

[0541] Other features and advantages of the invention will be apparent from the following detailed description, and from the claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0542] Figures 1A-1D is a schematic diagram of exemplary formats and configurations of multispecific antibodies (eg, bispecific antibodies) that bind to TRBC1 and NKp30. Figure 1A Depicted is an anti-TRBC1 antibody fused to an anti-NKp30 scFv. The anti-TRBC1 antibody comprises two heavy chains and two light chains. The anti-NKp30 scFv is fused to the N-terminus of one heavy chain of the anti-TRBC1 antibody. Figure 1B Depicted is an antibody molecule comprising anti-TRBC1 Fab, anti-NKp30 scFv and Fc dimer. The Fc dimer comprises two Fc chains. The C-terminus of the anti-TRBC1 Fab heavy chain is fused to the N-terminus of one Fc chain. The anti-NKp30 scFv is fused to the N-terminus of the other Fc chain. Figure 1C and 1D Depicted is an anti-TRBC1 antibody fused to two anti-NKp30 scFvs. The anti-TRBC1 antibody comprises two heavy chains and two light chains. Figure 1C As shown, two anti-NKp30 scFvs were fused to the C-termini of the two light chains of the anti-TRBC1 antibody. Figure 1D As shown, two anti-NKp30 scFvs were fused to the N-termini of the two heavy chains of the anti-TRBC1 antibody, respectively.

[0543] Figures 2A-2Fare schematic diagrams of exemplary forms and configurations of antibody molecules including moieties that bind to TRBC1 and a TRAIL molecule (eg, a trimer, dimer, or monomeric TRAIL molecule). Figure 2A and 2D Antibody molecules including anti-TRBC1 Fab, trimeric TRAIL molecules and Fc dimers are depicted. Figure 2B and 2E Antibody molecules including anti-TRBC1 Fab, dimeric TRAIL molecules and Fc dimers are depicted. Figure 2C and 2F Depicted is an antibody molecule comprising an anti-TRBC1 Fab, a monomeric TRAIL molecule, and an Fc dimer. The Fc dimer comprises two Fc chains. The C-terminus of the anti-TRBC1 Fab heavy chain is fused to the N-terminus of one Fc chain. The trimer, dimer, or monomeric TRAIL molecule is fused to the N-terminus of another Fc chain. In some embodiments, Figure 2A The antibody molecule depicted in comprises the amino acid sequences of SEQ ID NOs: 6169, 6167 and 6159. In some embodiments, Figure 2B The antibody molecule depicted in comprises the amino acid sequences of SEQ ID NOs: 6169, 6167 and 6158. In some embodiments, Figure 2C The antibody molecule depicted in includes the amino acid sequences of SEQ ID NOs: 6169, 6167, and 6157. In some embodiments, then Figure 2D The antibody molecule depicted in includes the amino acid sequences of SEQ ID NOs: 6169, 6167, and 6162. In some embodiments, Figure 2E The antibody molecule depicted in comprises the amino acid sequences of SEQ ID NOs: 6169, 6167 and 6161. In some embodiments, Figure 2F The antibody molecules depicted in include the amino acid sequences of SEQ ID NOs: 6169, 6167 and 6160.

[0544] Figure 3A and 3B is a schematic diagram of exemplary formats and configurations of multispecific antibodies (eg, bispecific antibodies) that bind to TRBC1 and DR5. Figure 3A Depicted is a multispecific antibody (e.g., a bispecific antibody) comprising an anti-TRBC1 Fab, an anti-DR5 scFv, and an Fc dimer. The Fc dimer comprises two Fc chains. The C-terminus of the anti-TRBC1 Fab heavy chain is fused to the N-terminus of one Fc chain. The anti-DR5 scFv is fused to the N-terminus of the other Fc chain. Figure 3BAn anti-TRBC1 antibody fused to two anti-DR5 scFvs is depicted. The anti-TRBC1 antibody comprises two heavy chains and two light chains. The two anti-DR5 scFvs are fused to the C-termini of the two light chains of the anti-TRBC1 antibody, respectively. In some embodiments, Figure 3A The multispecific antibody depicted in SEQ ID NO: 6169, 6167 and 6163 comprises the amino acid sequences. In some embodiments, Figure 3B The multispecific antibody depicted in comprises the amino acid sequences of SEQ ID NOs: 6170 and 6168.

[0545] Figure 4A-4B Alignment of the H131 derived mouse VH and VL framework 1, CDR 1, framework 2, CDR 2, framework 3, CDR3 and framework 4 regions with their respective humanized sequences is shown. Kabat CDRs are shown in bold, Chothia CDRs are shown in italics, and combined CDRs are shown in boxes. Backmutated framework positions are shown in double underlines. Figure 4A The VH sequences of murine H131 (SEQ ID NO: 1) and humanized H131 (SEQ ID NO: 9) are shown. Figure 4B The VL sequences of murine H131 (SEQ ID NO: 2) and humanized H131 (SEQ ID NO: 10 and SEQ ID NO: 11) are shown.

[0546] Figure 5A-5B Alignment of 16G8 derived mouse VH and VL framework 1, CDR 1, framework 2, CDR 2, framework 3, CDR3 and framework 4 regions with their respective humanized sequences is shown. Kabat CDRs are shown in bold, Chothia CDRs are shown in italics, and combined CDRs are shown in boxes. Backmutated framework positions are shown in double underlines. Figure 5A The VH sequences of murine 16G8 (SEQ ID NO: 15) and humanized 16G8 (SEQ ID NO: 23-25) are shown. Figure 5B The VL sequences of murine 16G8 (SEQ ID NO: 16) and humanized 16G8 (SEQ ID NOs: 26-30) are shown.

[0547] Figure 6A phylogenetic tree of the TCRBV gene families and subfamilies with mapped corresponding antibodies is depicted. The subfamily identities are as follows: Subfamily A: TCRβV6; Subfamily B: TCRβV10; Subfamily C: TCRβV12; Subfamily D: TCRβV5; Subfamily E: TCRβV7; Subfamily F: TCRβV11; Subfamily G: TCRβV14; Subfamily H: TCRβV16; Subfamily I: TCRβV18; Subfamily J: TCRβV9; Subfamily K: TCRβV13; Subfamily L: TCRβV4; subfamily M: TCRβV3; subfamily N: TCRβV2; subfamily O: TCRβV15; subfamily P: TCRβV30; subfamily Q: TCRβV19; subfamily R: TCRβV27; subfamily S: TCRβV28; subfamily T: TCRβV24; subfamily U: TCRβV20; subfamily V: TCRβV25; and subfamily W: TCRβV29 subfamily. Subfamily members are described in detail in the section entitled "TCR beta V (TCRβV)" herein.

[0548] Figure 7 It is a figure which shows the binding of JOVI.1 and humanized JOVI.1 to human TRBC1.

[0549] Figure 8 is a set of graphs showing the binding of JOVI.1 Fab (left) and humanized JOVI.1 Fab to human TRBC1 (right).

[0550] Fig. 9 is a graph showing the binding of NKp30 antibody to NK92 cells. Data are calculated as the percentage of the AF747 positive population.

[0551] Fig.10 is a graph showing activation of NK92 cells by NKp30 antibodies. The data were generated using hamster anti-NKp30 mAb.

[0552] Figures 11A-11E is a schematic diagram of anti-TRBC1 / NKp30 antibodies and control molecules.

[0553] Figures 12A-12B This is a diagram showing the binding of antibodies to THP1 cells expressing Fcγ receptors.

[0554] Figures 13A-13D are graphs showing T cell activation after incubation with the indicated antibodies. Fig.13A is a graph showing the fractionated CD4+ %. Fig. 13B is a graph showing the fractionated CD8+ %. Fig. 13C It is a graph showing the CD69-CD25+% of CD4+. Fig.13D It is a graph showing the CD69-CD25+% of CD8+.

[0555] Figures 14A-14D is a schematic diagram of anti-TRBC1 / NKp30 antibodies. Fig. 14B and 14D "460" indicates Fab based on BIM0460; "578" indicates Fab based on BJM0578; "407" indicates scFv based on BJM0407 ( Fig.18B ) or Fab( Fig.14D ); "411" indicates scFv based on BJM0411 ( Fig.18B ) or Fab( Fig.14D ); and “N297A” indicates that the antibody includes an N297A mutation in the Fc region.

[0556] Figures 15A-15D is to show that the specified antibody binds to the NK cell line KHYG-1 ( Fig.15A ) and TRBC1+Jurkat cells ( Fig. 15B )’s combined graph. Fig. 15C is a table that provides information about the antibodies tested. Fig.15D is a table providing the EC50 for binding to KHYG-1 cells or TRBC1+Jurkat cells.

[0557] Figures 16A-16C is a graph showing the killing of TRBC1+ target cells in the presence of NK-92 effector cells. The target cells are TRBC1+ Jurkat cells ( Fig.16A ) or H9 cells ( Fig. 16B TRBC2+HPB-ALL cells were used as control ( Fig. 16C ).

[0558] Figures 17A-17C is a graph showing the killing of TRBC1+ target cells in the presence of primary NK cells. The target cells are TRBC1+ Jurkat cells ( Fig.17A ) or H9 cells ( Fig. 17B TRBC2+HPB-ALL cells were used as control ( Fig. 17C ).

[0559] Figures 18A-18C is a graph showing NK cell activation after co-culture with TRBC1+Jurkat cells in the presence of anti-TRBC1 / NKp30 antibodies. Fig.18A The % CD69+CD107a+ NK cells are shown. Fig.18B The levels of IFNγ are shown. Fig.18C Levels of TNFα are shown.

[0560] Figures 19A-19Bis a graph showing the levels of cytokines produced by NK cells in the presence or absence of TRBC1+Jurkat cells. Fig.19A The levels of IFNγ are shown. Fig.19B Levels of TNFα are shown.

[0561] Fig. 20 is a graph showing the % NK cell death induced by the indicated antibodies in the presence of TRBC1+Jurkat cells.

[0562] Fig.21A and 21B Schematic diagrams of the single-arm anti-TRBC1 antibody and the bispecific anti-TRBC1 / NKp30 antibody, respectively.

[0563] Figures 22A-22D are graphs showing NK cell-mediated killing of TRBC1+PDX in the presence of the indicated antibodies.

[0564] Fig.23 is a set of graphs showing killing of TRBC1+Jurkat cells in the presence of the indicated antibodies. The NK cells tested were isolated from healthy donors (upper graph) or PTCL patients (lower graph).

[0565] Fig.24 It is displayed in Fig.23 A set of graphs showing the activation of NK cells during the killing assay shown in. The NK cells tested were isolated from healthy donors (upper graph) or PTCL patients (lower graph).

[0566] Fig.25A and 25B Figure 2 shows the expression of IFNγ ( Fig.25A ) or TNFα( Fig.25B ) A set of graphs showing secretion levels of NK cells. The tested NK cells were isolated from healthy donors (upper graph) or PTCL patients (lower graph).

[0567] Figures 26A-26C is a graph showing the measurement of NKp30 binding in ELISA. Fig.26A Binding of B7-H6 to NKp30 is shown. Fig.26B The binding of BJM1042 to NKp30 is shown. Fig.26C Binding of B7-H6 to NKp30 in the presence of various concentrations of the indicated antibodies is shown.

[0568] Figures 27A-27C is a graph from an in vivo TRBC1+ tumor study. Fig.27A The study design is shown. Fig.27B Tumor volumes for indicated treatments are shown. Fig.27Cis a waterfall plot showing the percentage change in tumor volume on day 3 after treatment. Fig.27C The following treatment groups are shown from left to right: no NK, PBS; no NK, TRBC1×NKp30; NK, PBS; NK, TRBC1; NK, NKp30; and NK+1 mpk BJM1042.

[0569] Fig.28A -28C is a graph from an in vivo TRBC2+ tumor study. Fig.28A The study design is shown. Fig.28B Tumor volumes for indicated treatments are shown.

[0570] Figures 29A-29C is a schematic diagram of anti-TRBC1 / NKp30 antibodies.

[0571] Figures 30A-30C is a schematic diagram of anti-TRBC1 / NKp30 antibodies. DETAILED DESCRIPTION

[0572] Disclosed herein are multifunctional molecules (also referred to herein as "multispecific molecules") that include multiple (e.g., two or more) functions (or binding specificities), including (i) an antigen binding domain that preferentially binds to TRBC1 or TRBC2, and (ii) one, two, or all of the following: (a) an immune cell engager selected from a T cell engager, an NK cell engager (e.g., a molecule that binds to NKp30, NKp46, NKG2D, or CD16), a B cell engager, a dendritic cell engager, or a macrophage engager; (b) a cytokine molecule; and (c) a matrix modification portion. Also disclosed herein are antibody molecules that include an antigen binding domain that preferentially binds to TRBC1 or TRBC2. In some embodiments, the antigen binding domain that binds to TRBC1 or TRBC2 includes a sequence or a portion of a sequence that can be found in Tables 2-5. In some embodiments, the immune cell engager includes: an NK cell engager that includes a sequence or a portion of a sequence that can be found in Tables 7-10. In some embodiments, the antigen binding domain comprises a sequence or a portion of a sequence found in Tables 2-5, and the immune cell engager comprises: a NK cell engager comprising a sequence or a portion of a sequence found in Tables 7-10.

[0573] In an embodiment, the multispecific or multifunctional molecule is a bispecific (or bifunctional) molecule, a trispecific (or trifunctional) molecule, or a tetraspecific (or tetrafunctional) molecule.

[0574] In some embodiments, the multifunctional molecule comprises an antigen binding domain that binds a tumor antigen on the surface of a T cell receptor comprising TRBC1, and the multifunctional molecule targets an immune cell (e.g., via an immune cell engager) to a lymphoma cell (e.g., a T cell) that exhibits a T cell receptor comprising TRBC1.

[0575] Without being bound by theory, in the presence of cells (e.g., cancer cells, e.g., lymphoma cells, e.g., T cells) that express, for example, T cell receptors comprising TRBC1 or TRBC2 on their surfaces, it is expected that the multispecific or multifunctional molecules disclosed herein will localize (e.g., bridge) and / or activate immune cells (e.g., immune effector cells selected from T cells, NK cells, B cells, dendritic cells, or macrophages). It is expected that in the presence of cells (e.g., cancer cells, e.g., lymphoma cells, e.g., T cells) that express T cell receptors comprising TRBC1 or TRBC2, increasing the proximity and / or activity of immune cells using the multispecific or multifunctional molecules described herein will enhance the immune response to the target cells, thereby providing a more effective therapy.

[0576] Without being bound by theory, it is believed that T cells do not normally express a T cell receptor that includes TRBC1 and a T cell receptor that includes TRBC2. In some embodiments, by utilizing a multispecific or multifunctional molecule that is specific for a T cell receptor that includes TRBC1 or a T cell receptor that includes TRBC2, but not for both types of T cell receptors, it is expected that the deleterious effects of increasing the proximity or activity of immune cells to T cells can generally be mitigated. In this way, it is believed that the use of the multispecific or multifunctional molecules disclosed herein can increase the proximity or activity of immune cells to cancer cells (e.g., lymphoma cells, e.g., T cells) without necessarily generally increasing the proximity or activity of immune cells to T cells.

[0577] A novel multifunctional (e.g., multispecific) molecule is disclosed, comprising (i) a matrix modification portion and (ii) an antigen binding domain that preferentially binds to a tumor antigen (e.g., a T cell receptor comprising TRBC1 or a T cell receptor comprising TRBC2) on a lymphoma cell (e.g., a T cell receptor). Without being bound by theory, the multifunctional molecules disclosed herein are believed to particularly target (e.g., localize to) a cancer site and alter the tumor matrix, e.g., alter the tumor microenvironment near the cancer site. The multifunctional molecule may further include one or both of the following items: an immune cell connector (e.g., selected from one, two, three or all of a T cell connector, an NK cell connector, a B cell connector, a dendritic cell connector or a macrophage connector); and / or a cytokine molecule. Therefore, the present invention particularly provides a multifunctional, e.g., multispecific molecule comprising the above-mentioned portion, a nucleic acid encoding the multispecific molecule, a method for producing the above-mentioned molecule, and a method for treating cancer using the above-mentioned molecule.

[0578] Thus, provided herein, inter alia, are multispecific or multifunctional molecules (e.g., multispecific or multifunctional antibody molecules) comprising the above-described portions, nucleic acids encoding the multispecific molecules, methods of producing the above-described molecules, and methods of using the above-described molecules to treat a disease or disorder (e.g., cancer).

[0579] definition

[0580] In some embodiments, the multifunctional molecule includes an immune cell adapter. "Immune cell adapter" refers to one or more binding specificities of binding and / or activating immune cells (e.g., cells participating in immune responses). In embodiments, immune cells are selected from T cells, NK cells, B cells, dendritic cells and / or macrophages. Immune cell adapters can be antibody molecules, receptor molecules (e.g., full-length receptors, receptor fragments or their fusions (e.g., receptor-Fc fusions)) or ligand molecules (e.g., full-length ligands, ligand fragments or their fusions (e.g., ligand-Fc fusions)), which bind to immune cell antigens (e.g., T cells, NK cell antigens, B cell antigens, dendritic cell antigens and / or macrophage antigens). In embodiments, immune cell adapters specifically bind to target immune cells, for example, preferentially bind to target immune cells. For example, when the immune cell adapter is an antibody molecule, it binds to immune cell antigens (e.g., T cell antigens, NK cell antigens, B cell antigens, dendritic cell antigens and / or macrophage antigens) with a dissociation constant less than about 10 nM.

[0581] In some embodiments, the multifunctional molecule includes a cytokine molecule. As used herein, "cytokine molecule" refers to the full length, fragment or variant of a cytokine; It also includes a receptor domain, for example, a cytokine of a cytokine receptor dimer domain; Or an agonist of a cytokine receptor, for example, an antibody molecule (e.g., an agonist antibody) directed against a cytokine receptor, which stimulates at least one activity of a naturally occurring cytokine. In some embodiments, the cytokine molecule is selected from interleukin 2 (IL-2), interleukin 7 (IL-7), interleukin 12 (IL-12), interleukin 15 (IL-15), interleukin -18 (IL-18), interleukin -21 (IL-21) or interferon γ, or a fragment or variant thereof, or a combination of any of the above cytokines. The cytokine molecule can be a monomer or a dimer. In embodiments, the cytokine molecule can further include a cytokine receptor dimer domain. In other embodiments, the cytokine molecule is an agonist of a cytokine receptor, for example, an antibody molecule (e.g., an agonist antibody) directed against a cytokine receptor selected from IL-15Ra or IL-21R.

[0582] As used herein, the term "molecule" as used in, e.g., antibody molecules, cytokine molecules, receptor molecules, includes full-length, naturally occurring molecules, as well as variants, e.g., functional variants (e.g., truncations, fragments, mutations (e.g., substantially similar sequences) or derivative forms thereof), so long as at least one function and / or activity of the unmodified (e.g., naturally occurring) molecule is retained.

[0583] In some embodiments, the multifunctional molecule includes a matrix modification portion. As used herein, a "matrix modification portion" refers to an agent that can change, for example, degrade a matrix component, for example, a protein (e.g., an enzyme). In embodiments, the components of the matrix are selected from, for example, ECM components, for example, glycosaminoglycans, for example, acetic hyaluronic acid (also referred to as hyaluronic acid or HA), chondroitin sulfate, chondroitin, dermatan sulfate, heparin sulfate, heparin, nidogen, tenascin, aggrecan, and keratin sulfate; or extracellular proteins, for example, collagen, laminin, elastin, fibrinogen, fibronectin, and vitronectin.

[0584] Certain terms are defined below.

[0585] As used herein, the articles "a" and "an" refer to one or more than one (e.g., at least one) of the grammatical object of the article. When used with the term "comprising," the use of the terms "a" or "an" can mean "one," but is also consistent with the meaning of "one or more," "at least one," and "one or more than one."

[0586] As used herein, "about" and "approximately" generally refer to an acceptable degree of error in the measured quantity given the nature or precision of the measurement. Exemplary degrees of error are within 20%, typically within 10%, and more typically within 5% of a given value range.

[0587] As used herein, "antibody molecule" refers to a protein including at least one immunoglobulin variable domain sequence, for example, an immunoglobulin chain or a fragment thereof. Antibody molecules include antibodies (e.g., full-length antibodies) and antibody fragments. In embodiments, antibody molecules include antigen binding or functional fragments of full-length antibodies, or full-length immunoglobulin chains. For example, full-length antibodies are immunoglobulin (Ig) molecules (e.g., IgG antibodies) that occur naturally or are formed by normal immunoglobulin gene fragment recombination processes. In embodiments, antibody molecules refer to immunologically active antigen-binding portions of immunoglobulin molecules, such as antibody fragments. Antibody fragments, for example, functional fragments, are parts of antibodies, for example, Fab, Fab', F(ab')2, F(ab)2, variable fragments (Fv), domain antibodies (dAb) or single-chain variable fragments (scFv). Functional antibody fragments bind to the same antigen recognized by complete (e.g., full-length) antibodies. The term "antibody fragment" or "functional fragment" also includes isolated fragments consisting of variable regions (such as "Fv" fragments consisting of variable regions of heavy and light chains), or recombinant single-chain polypeptide molecules in which the light and heavy chain variable regions are connected by a peptide linker ("scFv protein"). In some embodiments, the antibody fragment does not include antibody portions without antigen binding activity, such as Fc fragments or single amino acid residues. Exemplary antibody molecules include full-length antibodies and antibody fragments, for example, dAb (domain antibodies), single chains, Fab, Fab' and F(ab')2 fragments, and single-chain variable fragments (scFv).

[0588] As used herein, "immunoglobulin variable domain sequence" refers to an amino acid sequence that can form the structure of an immunoglobulin variable domain. For example, the sequence may include all or part of the amino acid sequence of a naturally occurring variable domain. For example, the sequence may or may not include one, two or more N- or C-terminal amino acids, or may include other changes that are compatible with the formation of protein structure.

[0589] In embodiments, the antibody molecule is monospecific, for example, it comprises a binding specificity to a single epitope. In some embodiments, the antibody molecule is multispecific, for example, it comprises a plurality of immunoglobulin variable domain sequences, wherein the first immunoglobulin variable domain sequence has a binding specificity to a first epitope, and the second immunoglobulin variable domain sequence has a binding specificity to a second epitope. In some embodiments, the antibody molecule is a bispecific antibody molecule. As used herein, a "bispecific antibody molecule" refers to an antibody molecule that has specificity for more than one (e.g., two, three, four or more) epitopes and / or antigens.

[0590] As used herein, "antigen" (Ag) refers to a molecule that can cause an immune response (e.g., involving the activation of certain immune cells and / or antibody production). Any macromolecule, including almost all proteins or peptides, can be an antigen. Antigens can also be derived from genomic recombinants or DNA. For example, any DNA containing a nucleotide sequence or a partial nucleotide sequence encoding a protein that can cause an immune response encodes an "antigen". In an embodiment, an antigen need not be encoded only by the full-length nucleotide sequence of a gene, nor does an antigen need to be encoded by a gene. In an embodiment, an antigen can be synthesized or can be derived from a biological sample, such as a tissue sample, a tumor sample, a cell, or a fluid with other biological components. As used herein, "tumor antigens" or interchangeably, "cancer antigens" include any molecules present on or associated with cancer (e.g., cancer cells or tumor microenvironments that can cause an immune response). As used herein, "immune cell antigens" include any molecules present on or associated with immune cells that can cause an immune response.

[0591] The "antigen binding site" or "binding portion" of an antibody molecule refers to a portion of an antibody molecule, such as an immunoglobulin (Ig) molecule, that participates in antigen binding. In an embodiment, the antigen binding site is formed by amino acid residues in the variable (V) region of the heavy (H) chain and the light (L) chain. The three highly divergent sequence segments within the variable region of the heavy and light chains are called hypervariable regions, located between more conservative flanking sequence segments (called "framework regions" (FR)). FR is an amino acid sequence that is naturally present between and adjacent to the hypervariable regions in immunoglobulins. In an embodiment, in an antibody molecule, the three hypervariable regions of the light chain and the three hypervariable regions of the heavy chain are arranged relative to each other in three-dimensional space to form an antigen binding surface that is complementary to the three-dimensional surface of the bound antigen. The three hypervariable regions in each heavy and light chain are called "complementarity determining regions" or "CDRs". Framework regions and CDRs have been defined and described in, for example, 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 Chothia, C. et al., (1987) J. Mol. Biol. 196: 901-917. Each variable chain (e.g., variable heavy chain and variable light chain) is typically composed of three CDRs and four FRs, which are arranged from amino terminus to carboxyl terminus in the following amino acid sequence: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.

[0592] As used herein, "cancer" can encompass all types of carcinogenic processes and / or cancerous growths. In embodiments, cancer includes primary tumors and metastatic tissues or malignantly transformed cells, tissues or organs. In embodiments, cancer encompasses all histopathologies and stages, such as the stage of invasiveness / severity of cancer. In embodiments, cancer includes recurrent and / or drug-resistant cancers. The terms "cancer" and "tumor" can be used interchangeably. For example, both terms include solid tumors and liquid tumors. As used herein, the terms "cancer" or "tumor" include pre-malignant and malignant cancers and tumors.

[0593] As used herein, "immune cell" refers to any of the various cells that work in the immune system, such as to protect against infection and foreign bodies. In embodiments, the term includes leukocytes, such as neutrophils, eosinophils, basophils, lymphocytes and monocytes. Intrinsic leukocytes include phagocytes (such as macrophages, neutrophils and dendritic cells), mast cells, eosinophils, basophils and natural killer cells. Intrinsic leukocytes attack larger pathogens by contact or identify and eliminate pathogens by engulfing and killing microorganisms, and are mediators that activate adaptive immune responses. The cells of the adaptive immune system are special types of leukocytes, referred to as lymphocytes. B cells and T cells are important lymphocyte types, which are derived from hematopoietic stem cells in the bone marrow. B cells participate in humoral immune responses, while T cells participate in cell-mediated immune responses. The term "immune cell" includes immune effector cells.

[0594] As used herein, the term "immune effector cell" refers to a cell that participates in an immune response, e.g., promotes an immune effector response. Examples of immune effector cells include, but are not limited to, T cells (e.g., α / β T cells and γ / δ T cells), B cells, natural killer (NK) cells, natural killer T (NK T) cells, and mast cells.

[0595] The term "effector function" or "effector response" refers to a specialized function of a cell. The effector function of a T cell may be, for example, cytolytic activity or helper activity, including the secretion of cytokines.

[0596] The compositions and methods of the present invention include polypeptides and nucleic acids having a specified sequence or a sequence substantially identical or similar thereto (e.g., a sequence at least 80%, 85%, 90%, 95% identical or more identical to a specified sequence). In the context of amino acid sequences, the term "substantially identical" as used herein refers to a first amino acid comprising a sufficient or minimum number of amino acid residues that are i) identical to the alignment amino acid residues in the second amino acid sequence, or ii) have conservative substitutions such that the first amino acid sequence and the second amino acid sequence may have a common domain and / or a common functional activity. For example, an amino acid sequence contains a common domain with at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to a reference sequence (e.g., a sequence provided herein).

[0597] In the context of nucleotide sequences, the term "substantially identical" as used herein refers to a first nucleic acid sequence comprising sufficient or a minimum number of nucleotides identical to the aligned nucleotides in a second nucleic acid sequence, such that the first nucleotide sequence and the second nucleotide sequence encode a polypeptide having a common functional activity, or a polypeptide encoding a common structural polypeptide domain or a common functional polypeptide activity. For example, a nucleotide sequence having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to a reference sequence (e.g., a sequence provided herein).

[0598] The term "variant" refers to a polypeptide having an amino acid sequence substantially identical to a reference amino acid sequence, or encoded by a substantially identical nucleotide sequence. In some embodiments, the variant is a functional variant.

[0599] The term "functional variant" refers to a polypeptide having an amino acid sequence substantially identical to a reference amino acid sequence, or encoded by a nucleotide sequence substantially identical to a reference amino acid sequence, and capable of having one or more activities of the reference amino acid sequence.

[0600] Calculations of homology or sequence identity (the terms are used interchangeably herein) between sequences are performed as follows.

[0601] To determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of the first and second amino acid or nucleic acid sequences for optimal alignment, and non-homologous sequences can be ignored for comparison purposes). In a preferred embodiment, the length of the reference sequence compared for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%, 60%, and even more preferably at least 70%, 80%, 90%, 100% of the length of the reference sequence. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, the molecules are identical at that position (as used herein, "identity" of an amino acid or nucleic acid is equivalent to "homology" of an amino acid or nucleic acid).

[0602] The percent identity between the 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, which need to be introduced for optimal alignment of the two sequences.

[0603] Mathematical algorithms can be used to complete the comparison of sequences and the determination of the percent identity between two sequences. In a preferred embodiment, the Needleman and Wunsch ((1970) J. Mol. Biol. 48: 444-453) algorithm (incorporated into the GAP program in the GCG software package (available from http: / / www.gcg.com)) is used, using a Blossum 62 matrix or a PAM250 matrix and a gap weight of 16, 14, 12, 10, 8, 6 or 4 and a length weight of 1, 2, 3, 4, 5 or 6 to determine the percent identity between two amino acid sequences. In another preferred embodiment, the GAP program in the GCG software package (available from http: / / www.gcg.com) is used, using the NWSgapdna.CMP matrix, a gap weight of 40, 50, 60, 70 or 80 and a length weight of 1, 2, 3, 4, 5 or 6 to determine the percent identity between two nucleotide sequences. A particularly preferred set of parameters (the set of parameters that should be used unless otherwise stated) is the Blossum 62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5.

[0604] The percent identity between two amino acid or nucleotide sequences can be determined using the algorithm of E. Meyers and W. Miller ((1989) CABIOS, 4: 11-17) (incorporated into the ALIGN program (version 2.0)) using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.

[0605] The nucleic acid and protein sequences described herein can be used as a "query sequence" to search public databases to, for example, identify other family members or related sequences. The NBLAST and XBLAST programs (version 2.0) of Altschul et al., (1990) J. Mol. Biol. 215:403-10 can be used for searches. BLAST nucleotide searches can be performed with the NBLAST program (score = 100, word length = 12) to obtain nucleotide sequences homologous to the nucleic acid molecules of the invention. BLAST protein searches can be performed with the XBLAST program (score = 50, word length = 3) to obtain amino acid sequences homologous to the protein molecules of the invention. To obtain gapped alignments for comparison purposes, the Gapped BLAST described in Altschul et al., (1997) Nucleic Acids Res. 25:3389-3402 can be utilized. When using BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. See http: / / www.ncbi.nlm.nih.gov .

[0606] It will be appreciated that the molecules of the invention may have additional conservative or non-essential amino acid substitutions which have no substantial effect on their function.

[0607] The term "amino acid" is intended to encompass all molecules, whether natural or synthetic, that include both an amino functional group and an acid functional group and that are capable of being included in a polymer of naturally occurring amino acids. Exemplary amino acids include naturally occurring amino acids; analogs, derivatives, and congeners; amino acid analogs with variant side chains; and all stereoisomers of any of the foregoing. As used herein, the term "amino acid" includes D- or L- optical isomers and peptidomimetics.

[0608] "Conservative amino acid substitution" is a substitution in which an amino acid residue is replaced by an amino acid residue with a similar side chain. Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), amino acids with non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), amino acids with β-branched side chains (e.g., threonine, valine, isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[0609] The terms "polypeptide", "peptide" and "protein" (if single chain) are used interchangeably herein to refer to polymers of amino acids of any length. The polymer may be linear or branched, it may contain modified amino acids, and may be interrupted by non-amino acids. The term also encompasses amino acid polymers that have been modified, for example, by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation, such as conjugation with a labeling component. Polypeptides may be isolated from natural sources, may be produced by recombinant techniques from eukaryotic or prokaryotic hosts, or may be the product of synthetic methods.

[0610] The terms "nucleic acid", "nucleic acid sequence", "nucleotide sequence" or "polynucleotide sequence" and "polynucleotide" are used interchangeably. They refer to a polymeric form of nucleotides of any length, i.e., deoxyribonucleotides or ribonucleotides or their analogs. Polynucleotides can be single-stranded or double-stranded, and if single-stranded, can be a coding strand or a non-coding (antisense) strand. Polynucleotides can contain modified nucleotides, such as methylated nucleotides and nucleotide analogs. The sequence of nucleotides can be interrupted by non-nucleotide components. Polynucleotides can be further modified after polymerization, for example, by conjugation with a labeling component. Nucleic acids can be recombinant polynucleotides, or polynucleotides of genomic, cDNA, semisynthetic or synthetic origin, which do not occur naturally, or are linked to another polynucleotide in a non-natural manner.

[0611] As used herein, the term "isolated" refers to material removed from its source or natural environment (e.g., the natural environment, if it occurs naturally). For example, a naturally occurring polynucleotide or polypeptide present in a living animal is not isolated, but the same polynucleotide or polypeptide separated from some or all of the coexisting materials in the natural system by human intervention is isolated. Such a polynucleotide can be part of a vector and / or such a polynucleotide or polypeptide can be part of a composition and still be isolated because such a vector or composition is not part of the environment in which it is found in nature.

[0612] Various aspects of the invention are described in further detail below. Additional definitions are set forth throughout the specification.

[0613] Antibody molecules

[0614] In one embodiment, the antibody molecule binds to a cancer antigen, for example, a tumor antigen or a matrix antigen. In some embodiments, the cancer antigen is, for example, a mammalian (e.g., human) cancer antigen. In other embodiments, the antibody molecule binds to an immune cell antigen, for example, a mammalian (e.g., human) immune cell antigen. For example, the antibody molecule specifically binds to an epitope on a cancer antigen or immune cell antigen, for example, a linear or conformational epitope.

[0615] In an embodiment, the antibody molecule is a monospecific antibody molecule and binds a single epitope. For example, a monospecific antibody molecule having multiple immunoglobulin variable domain sequences, each of which binds to the same epitope.

[0616] In an embodiment, the antibody molecule is a multispecific or multifunctional antibody molecule, for example, it includes multiple immunoglobulin variable domain sequences, wherein the first immunoglobulin variable domain sequence of the multiple immunoglobulin variable domain sequences has binding specificity to the first epitope, and the second immunoglobulin variable domain sequence of the multiple immunoglobulin variable domain sequences has binding specificity to the second epitope. In an embodiment, the first and second epitopes are on the same antigen, for example, the same protein (or the subunit of a multimeric protein). In an embodiment, the first and second epitopes overlap. In an embodiment, the first and second epitopes do not overlap. In an embodiment, the first and second epitopes are on different antigens, for example, different proteins (or different subunits of a multimeric protein). In an embodiment, the multispecific antibody molecule includes the third, fourth or fifth immunoglobulin variable domain. In an embodiment, the multispecific antibody molecule is a bispecific antibody molecule, a trispecific antibody molecule or a tetraspecific antibody molecule.

[0617] In an embodiment, a multispecific antibody molecule is a bispecific antibody molecule. Bispecific antibodies have specificity for no more than two antigens. The bispecific antibody molecule is characterized by a first immunoglobulin variable domain sequence having binding specificity to a first epitope and a second immunoglobulin variable domain sequence having binding specificity to a second epitope. In an embodiment, the first and second epitopes are on the same antigen, for example, the same protein (or subunit of a multimeric protein). In an embodiment, the first and second epitopes overlap. In an embodiment, the first and second epitopes do not overlap. In an embodiment, the first and second epitopes are on different antigens, for example, different proteins (or different subunits of a multimeric protein). In an embodiment, a bispecific antibody molecule includes a heavy chain variable domain sequence and a light chain variable domain sequence having binding specificity to a first epitope and a heavy chain variable domain sequence and a light chain variable domain sequence having binding specificity to a second epitope. In an embodiment, a bispecific antibody molecule includes a half antibody having binding specificity to a first epitope and a half antibody having binding specificity to a second epitope. In an embodiment, a bispecific antibody molecule includes a half antibody or a fragment thereof having binding specificity to a first epitope and a half antibody or a fragment thereof having binding specificity to a second epitope. In an embodiment, the bispecific antibody molecule comprises a scFv or Fab or fragment thereof having binding specificity for a first epitope and a scFv or Fab or fragment thereof having binding specificity for a second epitope.

[0618] In an embodiment, the antibody molecules include diabodies and single chain molecules, as well as antigen-binding fragments of antibodies (e.g., Fab, F(ab') 2and Fv). For example, an antibody molecule may include a heavy (H) chain variable domain sequence (abbreviated herein as VH) and a light (L) chain variable domain sequence (abbreviated herein as VL). In an embodiment, an antibody molecule includes or consists of a heavy chain and a light chain (referred to herein as a half antibody). In another example, an antibody molecule includes two heavy (H) chain variable domain sequences and two light (L) chain variable domain sequences, thereby forming two antigen binding sites, such as Fab, Fab', F(ab') 2 , Fc, Fd, Fd', Fv, single-chain antibodies (e.g., scFv), single variable domain antibodies, diabodies (Dab) (divalent and bispecific), and chimeric (e.g., humanized) antibodies, which can be produced by modifying complete antibodies or antibodies synthesized de novo using recombinant DNA technology. These functional antibody fragments retain the ability to selectively bind to their respective antigens or receptors. Antibodies and antibody fragments can be from any class of antibodies, including but not limited to, IgG, IgA, IgM, IgD and IgE, and from any subclass of antibodies (e.g., IgG1, IgG2, IgG3 and IgG4). The preparation of antibody molecules can be monoclonal or polyclonal. Antibody molecules can also be human antibodies, humanized antibodies, CDR-transplanted antibodies, or antibodies produced in vitro. Antibodies can have a heavy chain constant region selected from, for example, IgG1, IgG2, IgG3 or IgG4. Antibodies can also have a light chain selected from, for example, κ or λ. The term "immunoglobulin" (Ig) is used interchangeably with the term "antibody" herein.

[0619] Examples of antigen-binding fragments of antibody molecules include: (i) Fab fragments, monovalent fragments consisting of VL, VH, CL and CH1 domains; (ii) F(ab')2 fragments, bivalent fragments comprising two Fab fragments connected by a disulfide bond in the hinge region; (iii) Fd fragments consisting of VH and CH1 domains; (iv) Fv fragments consisting of the VL and VH domains of a single arm of an antibody, (v) diabody (dAb) fragments consisting of VH domains; (vi) camelid or camelidized variable domains; (vii) single-chain Fv (scFv), see, e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883); (viii) single-domain antibodies. These antibody fragments are obtained using conventional techniques known to those skilled in the art, and the utility of the fragments is screened in the same manner as intact antibodies.

[0620] Antibody molecules include complete molecules and functional fragments thereof. The constant region of an antibody molecule may be altered, for example, mutated, to modify the properties of the antibody (e.g., to increase or decrease one or more of the following: Fc receptor binding, antibody glycosylation, the number of cysteine ​​residues, effector cell function, or complement function).

[0621] The antibody molecule can also be a single domain antibody. A single domain antibody can include an antibody whose complementary determining region is a part of a single domain polypeptide. Examples include, but are not limited to, heavy chain antibodies, antibodies naturally lacking light chains, single domain antibodies derived from conventional 4-chain antibodies, engineered antibodies, and single domain scaffolds other than those derived from antibodies. A single domain antibody can be an antibody in any art, or any future single domain antibody. Single domain antibodies can be derived from any species, including but not limited to mice, humans, camels, llamas, fish, sharks, sheep, rabbits, and cattle. According to another aspect of the present invention, a single domain antibody is a naturally occurring single domain antibody, referred to as a heavy chain antibody lacking a light chain. For example, such single domain antibodies are disclosed in WO 9404678. For clarity, the variable domains derived from the heavy chain antibodies naturally lacking light chains are referred to herein as VHH or nano antibodies, to distinguish them from the conventional VH of four-chain immunoglobulins. Such VHH molecules can be derived from antibodies produced in camelid species (e.g., in camels, llamas, dromedaries, alpacas, and guanacos). Species other than Camelidae may produce heavy chain antibodies that naturally lack light chains; such VHHs are within the scope of the present invention.

[0622] The VH and VL regions can be subdivided into regions of hypervariability, termed "complementarity determining regions" (CDRs), interspersed with regions that are more conserved, termed "framework regions" (FR or FW).

[0623] The extent of the framework regions and CDRs has been precisely defined by a variety of methods (see, Kabat, EA, et al. (1991) Sequences of Proteins of Immunological Interest, 5th ed., US Department of Health and Human Services, NIH Publication No. 91-3242; Chothia, C. et al. (1987) J. Mol. Biol. 196:901-917; and the AbM definition used by Oxford Molecular's AbM antibody modeling software. See generally, e.g., Protein Sequence and Structure Analysis of Antibody Variable Domains. In: Antibody Engineering Lab Manual (Editors: Duebel, S. and Kontermann, R., Springer-Verlag, Heidelberg).

[0624] As used herein, the terms "complementarity determining region" and "CDR" refer to the amino acid sequence within the variable region of an antibody that confers antigen specificity and binding affinity. Typically, there are 3 CDRs (HCDR1, HCDR2, HCDR3) in each heavy chain variable region, and 3 CDRs (LCDR1, LCDR2, LCDR3) in each light chain variable region.

[0625] The precise amino acid sequence boundaries of a given CDR can be determined using any of a number of known schemes, including Kabat et al. (1991), "Sequences of Proteins of Immunological Interest," 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD ("Kabat" numbering scheme), Al-Lazikani et al., (1997) JMB 273, 927-948 ("Chothia" numbering scheme). As used herein, CDRs defined according to the "Chothia" numbering scheme are sometimes also referred to as "hypervariable loops."

[0626] For example, according to Kabat, the CDR amino acid residues in the heavy chain variable domain (VH) are numbered 31-35 (HCDR1), 50-65 (HCDR2) and 95-102 (HCDR3); while the CDR amino acid residues in the light chain variable domain (VL) are numbered 24-34 (LCDR1), 50-56 (LCDR2) and 89-97 (LCDR3). According to Chothia, the CDR amino acid residues in VH are numbered 26-32 (HCDR1), 52-56 (HCDR2) and 95-102 (HCDR3); while the amino acid residues in VL are numbered 26-32 (LCDR1), 50-52 (LCDR2) and 91-96 (LCDR3).

[0627] Each VH and VL typically includes three CDRs and four FRs, arranged in the following order from amino terminus to carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0628] Antibody molecules can be polyclonal or monoclonal.

[0629] As used herein, the term "monoclonal antibody" or "monoclonal antibody composition" refers to a preparation of antibody molecules of a single molecular composition. A monoclonal antibody composition exhibits a single binding specificity and affinity for a particular epitope. Monoclonal antibodies can be prepared by hybridoma technology or by methods that do not use hybridoma technology (e.g., recombinant methods).

[0630] Antibodies can be produced recombinantly, for example, by phage display, or by combinatorial methods.

[0631] Phage display and combinatorial methods for generating antibodies are known in the art (as described in, e.g., Ladner et al., U.S. Pat. No. 5,223,409; Kang et al., International Publication No. WO 92 / 18619; Dower et al., International Publication No. WO 91 / 17271; Winter et al., International Publication No. WO 92 / 20791; Markland et al., International Publication No. WO 92 / 15679; Breitling et al., International Publication No. WO 93 / 01288; McCafferty et al., International Publication No. WO 92 / 01047; Garrard et al., International Publication No. WO 92 / 09690; Ladner et al., International Publication No. WO 90 / 02809; Fuchs et al., (1991) Bio / Technology 9:1370-1372; Hay et al., (1992) Hum Antibod Hybridomas 3:81-85; Huse et al., (1989) Science 246:1275-1281; Griffths et al., (1993) EMBO J 12:725-734; Hawkins et al., (1992) J Mol Biol 226:889-896; Clackson et al., (1991) Nature 352:624-628; Gram et al., (1992) PNAS 89:3576-3580; Garrad et al., (1991) Bio / Technology 9:1373-1377; Hoogenboom et al., (1991) Nuc Acid Res 19:4133-4137; and Barbas et al., (1991) PNAS 88:7978-7982, all of which are incorporated herein by reference).

[0632] In one embodiment, the antibody is a fully human antibody (e.g., an antibody prepared in a mouse that has been genetically engineered to produce antibodies from human immunoglobulin sequences), or a non-human antibody, e.g., a rodent (mouse or rat), sheep, primate (e.g., monkey), camel antibody. Preferably, the non-human antibody is a rodent (mouse or rat antibody). Methods for producing rodent antibodies are known in the art.

[0633] Human monoclonal antibodies can be generated using transgenic mice carrying human immunoglobulin genes rather than mouse systems. Splenocytes from these transgenic mice immunized with the antigen of interest are used to generate hybridomas that secrete human mAbs with specific affinity for epitopes from human proteins (see, e.g., Wood et al., International Application WO 91 / 00906, Kucherlapati et al., PCT Publication WO 91 / 10741; Lonberg et al., International Application WO 92 / 03918; Kay et al., International Application 92 / 03917; Lonberg, N. et al., 1994 Nature 368:856-859; Green, LL et al., 1994 Nature Genet. 7:13-21; Morrison, SL et al., 1994 Proc. Natl. Acad. Sci. USA 81:6851-6855; Bruggeman et al., 1993 Year Immunol 7:33-40; Tuaillon et al., 1993 PNAS 90:3720-3724; Bruggeman et al., 1991 Eur J Immunol 21:1323-1326).

[0634] The antibody molecule can be an antibody molecule in which the variable region or a portion thereof (e.g., CDR) is produced in a non-human organism (e.g., rat or mouse). Chimeric, CDR-transplanted and humanized antibodies are within the scope of the invention. Antibody molecules produced in a non-human organism (e.g., rat or mouse) and then modified in, for example, the variable framework or constant region to reduce antigenicity in the human body are also within the scope of the invention.

[0635] An "effectively human" protein is one that does not substantially elicit a neutralizing antibody response, e.g., a human anti-mouse antibody (HAMA) response. For example, if the antibody molecule is administered repeatedly, e.g., in the treatment of chronic or recurrent diseases, HAMA may be problematic in many cases. The HAMA response may potentially render repeated antibody administration ineffective because of increased clearance of the antibody from serum (see, e.g., Saleh et al., Cancer Immunol. Immunother., 32: 180-190 (1990)), and also because of potential allergic reactions (see, e.g., LoBuglio et al., Hybridoma, 5: 5117-5123 (1986)).

[0636] Chimeric antibodies can be produced by recombinant DNA techniques known in the art (see Robinson et al., International Patent Publication No. PCT / US86 / 02269; Akira, et al., European Patent Application No. 184,187; Taniguchi, M., European Patent Application No. 171,496; Morrison et al., European Patent Application No. 173,494; Neuberger et al., International Application No. WO 86 / 01533; Cabilly et al., U.S. Patent No. 4,816,567; Cabilly et al., European Patent Application No. 125,023; Better et al. (1988 Science 240:1041-1043); Liu et al. (1987) PNAS 84:3439-3443; Liu et al., 1987, J. Immunol. 139:3521-3526; Sun et al. (1987) PNAS 84:214-218; Nishimura et al., 1987, Canc. Res. 47:999-1005; Wood et al. (1985) Nature 314:446-449; and Shaw et al., 1988, J. Natl Cancer Inst. 80:1553-1559).

[0637] Humanized or CDR-grafted antibodies will have at least one or two but usually all three (of the immunoglobulin heavy and / or light chains) recipient CDRs replaced by donor CDRs. Antibodies can be replaced with at least a portion of non-human CDRs or only a portion of CDRs can be replaced with non-human CDRs. It is only necessary to replace the number of CDRs required for binding to the antigen. Preferably, the donor will be a rodent antibody, such as a rat or mouse antibody, and the recipient will be a human framework or a human consensus framework. Typically, the immunoglobulin providing the CDR is called a "donor," and the immunoglobulin providing the framework is called an "acceptor." In one embodiment, the donor immunoglobulin is non-human (e.g., rodent). The acceptor framework is a naturally occurring (e.g., human) framework or a consensus framework, or a sequence with about 85% or more, preferably 90%, 95%, 99% or more identity thereto.

[0638] As used herein, the term "consensus sequence" refers to a sequence formed by the most frequently occurring amino acids (or nucleotides) in a family of related sequences (see, e.g., Winnaker, From Genes to Clones (Verlagsgesellschaft, Weinheim, Germany 1987)). In a family of proteins, each position in the consensus sequence is occupied by the most frequently occurring amino acid at that position in the family. If two amino acids occur equally, both can be included in the consensus sequence. "Consensus framework" refers to the framework region in the consensus immunoglobulin sequence.

[0639] Antibody molecules can be humanized by methods known in the art (see, e.g., Morrison, SL, 1985, Science 229: 1202-1207, Oi et al., 1986, BioTechniques 4: 214, and Queen et al. US 5,585,089, US 5,693,761 and US 5,693,762, all of which are incorporated herein by reference).

[0640] Humanized or CDR-grafted antibody molecules can be produced by CDR grafting or CDR replacement, wherein one, two or all CDRs of an immunoglobulin chain can be replaced. See, for example, U.S. Patent No. 5,225,539; Jones et al., 1986 Nature 321:552-525; Verhoeyan et al., 1988 Science 239:1534; Beidler et al., 1988 J. Immunol. 141:4053-4060; Winter US 5,225,539, all of which are expressly incorporated herein by reference. Winter describes a CDR grafting method that can be used to prepare humanized antibodies of the present invention (British patent application GB2188638A, filed March 26, 1987; Winter US 5,225,539), the contents of which are expressly incorporated by reference.

[0641] Humanized antibody molecules in which specific amino acids have been replaced, lacked or added are also within the scope of the present invention. The standard for selecting amino acids from donors is described in US 5,585,089, for example, and hurdles 12-16 of US 5,585,089, for example, and hurdles 12-16 of US 5,585,089, the contents of which are incorporated herein by reference. The people such as Padlan disclosed on December 23, 1992, EP 519596A1 have described other techniques for humanized antibodies.

[0642] The antibody molecule can be a single-chain antibody. Single-chain antibodies (scFV) can be engineered (see, e.g., Colcher, D. et al., (1999) Ann NY Acad Sci 880:263-80; and Reiter, Y. (1996) Clin Cancer Res 2:245-52). Single-chain antibodies can be dimerized or multimerized to produce multivalent antibodies specific for different epitopes of the same target protein.

[0643] In other embodiments, the antibody molecule has a heavy chain constant region selected from the following, for example, IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD and IgE heavy chain constant region; in particular, selected from, for example, IgG1, IgG2, IgG3 and IgG4 (e.g., human) heavy chain constant region. In another embodiment, the antibody molecule has a light chain constant region selected from the following, for example, κ or λ (e.g., human) light chain constant region. The constant region can be changed, for example, mutated, to modify the characteristics of the antibody (e.g., increase or decrease one or more of the following items: Fc receptor binding, antibody glycosylation, the number of cysteine ​​residues, effector cell function and / or complement function). In one embodiment, the antibody has: effector function; and can fix complement. In other embodiments, the antibody will not; recruit effector cells; or fix complement. In another embodiment, the ability of the antibody to bind to Fc receptors is reduced or there is no ability to bind to Fc receptors. For example, it is an isoform or subtype, fragment or other mutant that does not support binding to an Fc receptor, for example, it has a mutagenized or deleted Fc receptor binding region.

[0644] Methods for changing the constant region of an antibody are known in the art. Antibodies with altered functions (e.g., altered affinity for effector ligands (e.g., FcR on cells or the C1 component of complement)) can be produced by replacing at least one amino acid residue in the constant portion of the antibody with a different residue (see, e.g., EP 388,151 A1, U.S. Pat. No. 5,624,821 and U.S. Pat. No. 5,648,260, the entire contents of which are incorporated herein by reference). Similar types of changes can be described, and if they are applied to mice or other species, immunoglobulins will reduce or eliminate these functions.

[0645] Antibody molecules can be derived or connected to another functional molecule (e.g., another peptide or protein). As used herein, a "derived" antibody molecule is a modified antibody molecule. Derivatization methods include, but are not limited to, adding fluorescent moieties, radionucleotides, toxins, enzymes or affinity ligands such as biotin. Therefore, antibody molecules of the present invention are intended to include derivative forms and other modified forms of antibodies described herein, including immunoadhesion molecules. For example, antibody molecules can be functionally connected (by chemical coupling, genetic fusion, non-covalent association or other means) to one or more other molecular entities, such as another antibody (e.g., bispecific antibody or double antibody), a detectable agent, a cytotoxic agent, a pharmaceutical agent, and / or a protein or peptide (e.g., a streptavidin core region or a polyhistidine tag) that can mediate the binding of an antibody or antibody portion to another molecule.

[0646] One type of derivatized antibody molecule is produced by cross-linking two or more antibodies (same or different types, e.g., to produce bispecific antibodies). Suitable cross-linking agents include heterobifunctional cross-linking agents, having two different reactive groups separated by an appropriate spacer (e.g., m-maleimidobenzoyl-N-hydroxysuccinimide ester), or homobifunctional cross-linking agents (e.g., disuccinimidyl suberate). Such linkers are available from Pierce Chemical Company, Rockford, Ill.

[0647] Multispecific or multifunctional antibody molecules

[0648] Exemplary structures of multispecific and multifunctional molecules defined herein are described in full. Exemplary structures are further described in the following literature: Weidle U et al., (2013) The Intriguing Options of Multispecific Antibody Formats for Treatment of Cancer, Cancer Genomics & Proteomics 10: 1-18 (2013); and Spiess C et al. (2015) Alternative molecular formats and therapeutic applications for bispecific antibodies, Molecular Immunology 67: 95-106; the entire contents of which are incorporated herein by reference.

[0649] In an embodiment, a multispecific antibody molecule may comprise more than one antigen binding site, wherein different sites are specific for different antigens. In an embodiment, a multispecific antibody molecule may bind to more than one (e.g., two or more) epitopes on the same antigen. In an embodiment, a multispecific antibody molecule comprises an antigen binding site that is specific for a target cell (e.g., a cancer cell) and different antigen binding sites that are specific for immune effector cells. In one embodiment, a multispecific antibody molecule is a bispecific antibody molecule. Bispecific antibody molecules can be divided into five different structural groups: (i) bispecific immunoglobulin G (BsIgG); (ii) IgG with additional antigen binding moieties attached; (iii) bispecific antibody fragments; (iv) bispecific fusion proteins; and (v) bispecific antibody conjugates.

[0650] BsIgG is a monovalent form for each antigen. Exemplary BsIgG forms include, but are not limited to, crossMab, DAF (two-in-one), DAF (four-in-one), DutaMab, DT-IgG, common LC of a knob-in-hole structure, a knob-in-hole structure assembly, a charge pair, Fab arm exchange, SEEDbody, triomab, LUZ-Y, Fcab, κλ antibody, orthogonal Fab. See Spiess et al., Mol. Immunol. 67 (2015): 95-106. Exemplary BsIgG include catumaxomab (Fresenius Biotech, Trion Pharma, Neopharm), which comprises an anti-CD3 arm and an anti-EpCAM arm; and ertumaxomab (Neovii Biotech, Fresenius Biotech), which targets CD3 and HER2. In some embodiments, BsIgG comprises a heavy chain engineered for heterodimerization. For example, the heavy chain can be engineered for heterodimerization using a "knob-in-hole" strategy, a SEED platform, a common heavy chain (e.g., in kλ antibodies), and the use of a heterodimer Fc region. See Spiess et al., Mol. Immunol. 67 (2015): 95-106. Strategies used to avoid heavy chain pairing of homodimers in BsIgG include knob-in-hole, Duobody, azymetric, charge pairs, HA-TF, SEEDbody, and differential A protein affinity. See the previous document. BsIgG can be produced by expressing the component antibodies separately in different host cells and then purifying / assembling into BsIgG. BsIgG can also be produced by expressing the component antibodies in a single host cell. BsIgG can be purified using affinity chromatography, for example, using A protein and continuous pH elution.

[0651] IgG with additional antigen binding moieties is another form of bispecific antibody molecule. For example, monospecific IgG can be engineered to have bispecificity by attaching additional antigen binding units on monospecific IgG (e.g., at the N-terminus or C-terminus of heavy chain or light chain). Exemplary additional antigen binding units include single domain antibodies (e.g., variable heavy chain or variable light chain), engineered protein scaffolds and paired antibody variable domains (e.g., single chain variable fragments or variable fragments). Referring to the previous document. Additional IgG forms include dual variable domain IgG (DVD-Ig), IgG (H)-scFv, scFv-(H) IgG, IgG (L)-scFv, scFv-(L) IgG, IgG (L, H)-Fv, IgG (H)-V, V (H)-IgG, IgG (L)-V, V (L)-IgG, KIH IgG-scFab, 2scFv-IgG, IgG-2scFv, scFv4-Ig, zybody and D VI-IgG (four in one). See Spiess et al., Mol. Immunol. 67 (2015): 95-106. An example of an IgG-scFv is MM-141 (Merrimack Pharmaceuticals), which binds to IGF-1R and HER3. Examples of DVD-Igs include ABT-981 (AbbVie), which binds to IL-1α and IL-1β; and ABT-122 (AbbVie), which binds to TNF and IL-17A.

[0652] Bispecific antibody fragment (BsAb) is the form of bispecific antibody molecule lacking some or all antibody constant domains.For example, some BsAb lack Fc region.In embodiments, bispecific antibody fragment includes heavy chain and light chain region connected by peptide linker, and described peptide linker allows BsAb to be effectively expressed in single host cell.Exemplary bispecific antibody fragment includes but is not limited to nano antibody, nano antibody-HAS, BiTE, double antibody, DART, TandAb, sc double antibody, sc double antibody-CH3, double antibody-CH3, three antibodies (triple body), micro antibody, micro body, TriBi micro body, scFv-CH3 KIH, Fab-scFv, scFv-CH-CL-scFv, F (ab ') 2, F (ab ') 2-scFv2, scFv-KIH, Fab-scFv-Fc, tetravalent HCAb, sc double antibody-Fc, double antibody-Fc, series connection scFv-Fc and intracellular antibody.Referring to previous document. For example, the BiTE format includes tandem scFvs, where the component scFvs bind to CD3 on T cells and to a surface antigen on a cancer cell.

[0653] Bispecific fusion proteins include antibody fragments linked to other proteins, for example, to add additional specificity and / or function. An example of a bispecific fusion protein is immTAC, which comprises an anti-CD3 scFv linked to an affinity-matured T cell receptor that recognizes an HLA-presented peptide. In embodiments, a dock-and-lock (DNL) approach can be used to generate bispecific antibody molecules with higher valence. Moreover, fusions with albumin-binding proteins or human serum albumin can extend the serum half-life of antibody fragments. See previous document.

[0654] In an embodiment, chemical conjugation, such as chemical conjugation of antibodies and / or antibody fragments, can be used to produce BsAb molecules. See the previous document. Exemplary bispecific antibody conjugates include CovX body forms, in which low molecular weight drugs are site-specifically conjugated to a single reactive lysine in each Fab arm or antibody or fragment thereof. In an embodiment, conjugation improves the serum half-life of low molecular weight drugs. An exemplary CovX body is CVX-241 (NCT01004822), which comprises antibodies conjugated to two short peptides that inhibit VEGF or Ang2. See the previous document.

[0655] Antibody molecules can be produced by recombinant expression of at least one or more components in a host system. Exemplary host systems include eukaryotic cells (e.g., mammalian cells, such as CHO cells, or insect cells, such as SF9 or S2 cells) and prokaryotic cells (e.g., Escherichia coli). Bispecific antibody molecules can be produced by the individual expression of components in different host cells and subsequent purification / assembly. Alternatively, antibody molecules can be produced by expressing components in a single host cell. Purification of bispecific antibody molecules can be carried out by various methods, such as affinity chromatography, for example, using protein A and continuous pH elution. In other embodiments, affinity tags can be used for purification, such as histidine-containing tags, myc tags or streptavidin tags.

[0656] CDR grafted scaffold

[0657] In embodiments, the antibody molecule is a scaffold domain of CDR transplantation. In embodiments, the scaffold domain is based on a fibronectin domain, such as a fibronectin type III domain. The overall folding of the fibronectin type III (Fn3) domain is closely related to the folding of the smallest functional antibody fragment (the variable domain of the antibody heavy chain). There are three loops at the end of Fn3; the positions of the BC, DE and FG loops correspond roughly to the positions of the CDR1, 2 and 3 of the VH domain of the antibody. Fn3 does not have a disulfide bond; therefore, unlike antibodies and their fragments, Fn3 is stable under reducing conditions (see, e.g., WO 98 / 56915; WO 01 / 64942; WO 00 / 34784). The Fn3 domain can be modified (e.g., using CDR or hypervariable loops as described herein) or changed, for example, to select a domain that binds to an antigen / marker / cell as described herein.

[0658] In an embodiment, the scaffold domain, such as a folded domain, is based on an antibody, such as a "mini-antibody" scaffold generated by deleting three β-chains from the heavy chain variable domain of a monoclonal antibody (see, e.g., Tramontano et al., 1994, J Mol. Recognit. 7:9; and Martin et al., 1994, EMBO J. 13:5303-5309). "Mini-antibodies" can be used to present two hypervariable loops. In an embodiment, the scaffold domain is a V-like domain (see, e.g., Coia et al., WO 99 / 45110) or a domain derived from tendamistatin, which is a 74-residue six-stranded β-sheet sandwich structure held together by two disulfide bonds (see, e.g., McConnell and Hoess, 1995, J Mol. Biol. 250:460). For example, the loops of tendamistatin can be modified or altered (e.g., using CDRs or hypervariable loops), for example, to select a domain that binds to a marker / antigen / cell described herein. Another exemplary scaffold domain is the β-sandwich structure derived from the extracellular domain of CTLA-4 (see, e.g., WO 00 / 60070).

[0659] 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, colicin, BPTI, etc.); TPR repeats; trifoil structures; zinc finger domains; DNA binding proteins; in particular, monomeric DNA binding proteins; RNA binding proteins; enzymes, such as proteases (in particular, inactivated proteases), RNA enzymes; protein chaperones, such as thioredoxin and heat shock proteins; and intracellular signaling domains (e.g., SH2 and SH3 domains). See, e.g., US 20040009530 and US 7,501,121, which are incorporated herein by reference.

[0660] In embodiments, scaffold domains are evaluated and selected, for example, by one or more of the following criteria: (1) amino acid sequence, (2) sequence of several homologous domains, (3) 3-dimensional structure, and / or (4) stability data over a range of pH, temperature, salinity, organic solvents, oxidant concentrations. In embodiments, scaffold domains are small, stable protein domains, such as proteins of less than 100, 70, 50, 40, or 30 amino acids. The domains may include one or more disulfide bonds or may chelate metals, such as zinc.

[0661] Antibody-based fusions

[0662] Various formats can be produced that contain additional binding entities attached to the N or C termini of the antibody. These fusions with single-chain or disulfide-stabilized Fv or Fab result in tetravalent molecules with divalent binding specificity for each antigen. The combination of scFv and scFab with IgG can produce molecules that can recognize three or more different antigens.

[0663] Antibody-Fab fusion

[0664] Antibody-Fab fusions are bispecific antibodies that include a conventional antibody for a first target and a Fab for a second target fused to the C-terminus of an antibody heavy chain. Typically, the antibody and Fab will have a common light chain. Antibody fusions can be produced by (1) engineering the DNA sequence of the target fusion, and (2) transfecting the target DNA into a suitable host cell to express the fusion protein. As described in Coloma, J. et al. (1997) Nature Biotech 15:159, it appears that antibody-scFv fusions can be connected via a (Gly)-Ser linker between the C-terminus of the CH3 domain and the N-terminus of the scFv.

[0665] Antibody-scFv fusion

[0666] Antibody-scFv fusions are bispecific antibodies that include a traditional antibody fused to the C-terminus of an antibody heavy chain and an scFv with unique specificity. The scFv can be fused to the C-terminus directly through the scFv heavy chain or through a linker peptide. Antibody fusions can be produced by (1) engineering the DNA sequence of the target fusion, and (2) transfecting the target DNA into a suitable host cell to express the fusion protein. As described in Coloma, J. et al. (1997) Nature Biotech 15:159, it appears that antibody-scFv fusions can be connected via a (Gly)-Ser linker between the C-terminus of the CH3 domain and the N-terminus of the scFv.

[0667] Variable Domain Immunoglobulin DVD

[0668] A related format is the dual variable domain immunoglobulin (DVD), which consists of a VH and a VL domain at a second specific position N-terminal to the V domain via a short linker sequence.

[0669] Other exemplary multispecific antibody formats include, for example, those described in the following patents: US20160114057A1, US20130243775A1, US20140051833, US20130022601, US20150017187A1, US20120201746A1, US20150133638A1, US20130266568A1, US20160145340A1, WO2015127158A1, US20150203591A1, US2014 0322221A1, US20130303396A1, US20110293613, US20130017200A1, US20160102135A1, WO2015197598A2, WO2015197582A1, US9359437, US20150018529, WO2016115274A1, WO2016087416A1, US20080069820A1, US9145588B, US7919257 and US20150232560A1. Exemplary multispecific molecules in the form of whole antibody-Fab / scFab include those described in the following patents: US9382323B2, US20140072581A1, US20140308285A1, US20130165638A1, US20130267686A1, US20140377269A1, US7741446B2 and WO1995009917A1. Exemplary multispecific molecules employing domain swapping formats include those described in the following patents: US20150315296A1, WO2016087650A1, US20160075785A1, WO2016016299A1, US20160130347A1, US20150166670, US8703132B2, US20100316645, US8227577B2, US20130078249.

[0670] Fc-containing entities (minibodies)

[0671] Fc-containing entities (also called minibodies) can be generated by fusing scFv to the C-terminus of constant heavy chain domain 3 (CH3-scFv) and / or to the hinge region of an antibody with a different specificity (scFv-hinge-Fc). Trivalent entities with disulfide-stabilized variable domains fused to the C-terminus of the CH3 domain of IgG (without a peptide linker) can also be prepared.

[0672] Fc-containing multispecific molecules

[0673] In some embodiments, the multispecific molecules disclosed herein include an immunoglobulin constant region (e.g., an Fc region). Exemplary Fc regions can be selected from the heavy chain constant regions of IgG1, IgG2, IgG3, or IgG4; more particularly, the heavy chain constant regions of human IgG1, IgG2, IgG3, or IgG4.

[0674] In some embodiments, the immunoglobulin chain constant region (e.g., Fc region) is altered, e.g., mutated, to increase or decrease one or more of: Fc receptor binding, antibody glycosylation, number of cysteine ​​residues, effector cell function, or complement function.

[0675] In other embodiments, the interface of the first immunoglobulin chain constant region and the second immunoglobulin chain constant region (e.g., the first Fc region and the second Fc region) is altered (e.g., mutated) to, for example, increase or decrease dimerization relative to a non-engineered interface (e.g., a naturally occurring interface). For example, dimerization of immunoglobulin chain constant regions (e.g., Fc regions) can be enhanced by providing the Fc interface of the first Fc region and the second Fc region with one or more of the following: paired protrusions-cavities ("knobs-and-holes"), electrostatic interactions, or chain exchange, so that, for example, relative to a non-engineered interface, the ratio of heteromultimers to homomultimers is greater.

[0676] In some embodiments, the multispecific molecules include paired amino acid replacements at positions selected from one or more of 347, 349, 350, 351, 366, 368, 370, 392, 394, 395, 397, 398, 399, 405, 407, or 409 of the Fc region of, e.g., human IgGl. For example, an immunoglobulin chain constant region (e.g., Fc region) can include paired amino acid replacements selected from: T366S, L368A, or Y407V (e.g., corresponding to a cavity or hole) and T366W (e.g., corresponding to a protrusion or knob).

[0677] In other embodiments, the multifunctional molecule includes a half-life extender, such as human serum albumin, or an antibody molecule directed against human serum albumin.

[0678] Heterodimerized antibody molecule and preparation method thereof

[0679] Various methods for generating multispecific antibodies have been disclosed to address the problem of incorrect heavy chain pairing. Exemplary methods are described below. Exemplary multispecific antibody formats and methods for preparing the same are also disclosed, for example, in Speiss et al., Molecular Immunology 67 (2015) 95–106; and Klein et al., mAbs 4:6, 653–663; November / December 2012; the entire contents of each of which are incorporated herein by reference.

[0680] Heterodimerized bispecific antibodies are based on natural IgG structures, in which two binding arms recognize different antigens. By forcing heavy chain heterodimerization, combined with the technology of minimizing light chain (e.g., shared light chain) mispairing, an IgG-derived form capable of achieving determined monovalent (and simultaneous) antigen binding is produced. Forcing heavy chain heterodimerization can be obtained using, for example, a knob-and-hole structure or a chain exchange engineered domain (SEED).

[0681] Pestle and Mortar Structure

[0682] Knob-hole structures are described in US 5,731,116, US 7,476,724 and Ridgway, J. et al. (1996) Prot. Engineering 9(7):617-621 and generally involve: (1) mutating the CH3 domain of one or both antibodies to promote heterodimerization; and (2) combining the mutated antibodies under conditions that promote heterodimerization. The "knob" or "protrusion" is typically generated by replacing a small amino acid in a parent antibody with a larger amino acid (e.g., T366Y or T366W); the "hole" or "cavity" is generated by replacing a larger residue in a parent antibody with a smaller amino acid (e.g., Y407T, T366S, L368A and / or Y407V).

[0683] For bispecific antibodies including Fc domains, specific mutations can be introduced into the constant region of the heavy chain to promote the correct heterodimerization 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 the "knob-in-hole" (KiH) method, which involves introducing bulky residues into a CH3 domain of an antibody heavy chain. The bulky residue fits into the complementary "hole" in the other CH3 domain of the paired heavy chain, thereby promoting the correct pairing of the heavy chain (see, e.g., US7642228).

[0684] Exemplary KiH mutations include S354C, T366W in the "knob" heavy chain and Y349C, T366S, L368A, Y407V in the "hole" heavy chain. Table 1 provides other exemplary KiH mutations, as well as additional optional stabilizing Fc cysteine ​​mutations.

[0685] Table 1. Exemplary Fc KiH mutations and optional cysteine ​​mutations

[0686]

[0687] Other Fc mutations were provided by Igawa and Tsunoda, who identified three negatively charged residues in the CH3 domain of one chain that paired with three positively charged residues in the CH3 domain of the other chain. These specific pairs of charged residues were: E356-K439, E357-K370, D399-K409, and vice versa. By introducing at least two of the following three mutations in chain A: E356K, E357K and D399K, and at least two of the following three mutations in chain B: K370E, K409D, K439E, either alone or in combination with the newly discovered disulfide bridge, very efficient heterodimerization can be promoted while homodimerization is inhibited (Martens T et al., A novel one-armed antic-Met antibody inhibits glioblastoma growth in vivo. Clin Cancer Res 2006; 12: 6144-52; PMID: 17062691). Xencor defined 41 variant pairs based on a combination of structural calculations and sequence information, and subsequently screened for maximum heterodimerization, defining a combination of S364H, F405A (HA) on chain A and Y349T, T394F (TF) on chain B (Moore GL et al., A novel bispecific antibody format enables simultaneous bivalent and monovalent co-engagement of distinct target antigens. MAbs 2011; 3: 546-57; PMID: 22123055).

[0688] Other exemplary Fc mutations that promote heterodimerization of multispecific antibodies include those described in the following references, each of which is incorporated herein by reference: WO2016071377A1, US20140079689A1, US20160194389A1, US20160257763, WO2016071376A2, WO2015107026A1, WO2015107025A1, WO2015107015A1, US20150353636A1, US20140199294A1, US7750128B2, US201602299 15A1, US20150344570A1, US8003774A1, US20150337049A1, US20150175707A1, US20140242075A1, US20130195849A1, US20120149876A1, US20140200331A1, US9309311B2, US8586713, US20140037621A1, US20130178605A1, US20140363426A1, US20140051835A1 and US20110054151A1.

[0689] Stabilizing cysteine ​​mutations have also been used in combination with KiH and other variants that promote Fc heterodimerization, see, for example, US7183076. Other exemplary cysteine ​​modifications include, for example, those disclosed in US20140348839A1, US7855275B2, and US9000130B2.

[0690] Chain Exchange Engineered Domain (SEED)

[0691] Heteromeric Fc platforms that support bispecific and asymmetric fusion protein design by designing strand exchange engineered domain (SEED) C(H)3 heterodimers are known. These derivatives of human IgG and IgA C(H)3 domains can form complementary human SEED C(H)3 heterodimers, which are composed of alternating segments of human IgA and IgG C(H)3 sequences. When expressed in mammalian cells, the resulting pair of SEED C(H)3 domains preferentially associate to form heterodimers. SEEDbody (Sb) fusion proteins consist of [IgG1 hinge]-C(H)2-[SEED C(H)3] and can be genetically linked to one or more fusion partners (e.g., see Davis JH et al., SEEDbodies: fusion proteins based on strand exchange engineereddomain (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. The contents of each of which are incorporated herein by reference).

[0692] Duobody

[0693] The "Duobody" technology for producing bispecific antibodies with correct heavy chain pairing is known. DuoBody technology involves three basic steps for generating stable bispecific human IgG1 antibodies in a post-production exchange reaction. In the first step, two IgG1s are produced separately using a standard mammalian recombinant cell line, each of which contains a single matching mutation in the third constant (CH3) domain. Subsequently, these IgG1 antibodies are purified according to standard methods for recovery and purification. After production and (post-production) purification, the two antibodies are recombined under customized laboratory conditions to produce bispecific antibody products with very high yields (usually >95%) (see, for example, Labrijn et al., PNAS 2013; 110 (13): 5145-5150 and Labrijn et al., Nature Protocols 2014; 9 (10): 2450-63, the contents of each of which are incorporated herein by reference).

[0694] Electrostatic interactions

[0695] Disclosed is a method for preparing multispecific antibodies using charged amino acids and CH3 amino acid changes, so that the formation of homodimers is disadvantageous in electrostatics. EP1870459 and WO 2009089004 describe other strategies that are conducive to heterodimer formation when co-expressing different antibody domains in host cells. In these methods, one or more residues constituting heavy chain constant domain 3 (CH3) and CH3-CH3 interface in two CH3 domains are replaced with charged amino acids, so that homodimer formation is disadvantageous in electrostatics and heterodimerization is favorable in electrostatics. Other methods for preparing multispecific molecules using electrostatic interactions are described in the following references, and the contents of each reference are incorporated herein by reference, including US20100015133, US8592562B2, US9200060B2, US20140154254A1 and US9358286A1.

[0696] Common light chain

[0697] It is necessary to avoid light chain mispairing to produce a homogeneous preparation of bispecific IgG. One of its implementation methods is by using a common light chain principle, that is, sharing a light chain but still having two combinations of binding substances with different specificities. An exemplary method of enhancing the formation of a desired bispecific antibody by a monomer mixture is to interact with each heteropolymeric variable heavy chain region of a bispecific antibody by providing a common variable light chain. The composition and method for producing a bispecific antibody with a common light chain are disclosed in, for example, US7183076B2, US20110177073A1, EP2847231A1, WO2016079081A1 and EP3055329A1, and their respective contents are incorporated herein by reference.

[0698] CrossMab

[0699] Another option for reducing light chain mispairing is CrossMab technology, which avoids nonspecific L chain mispairing by exchanging the CH1 and CL domains in the Fab of half of the bispecific antibody. Such staggered variants retain binding specificity and affinity, but make the two arms so different that L chain mispairing is prevented. CrossMab technology (as outlined in Klein et al., above) involves domain swaps between heavy and light chains to promote the formation of correct pairing. In short, in order to construct a bispecific IgG-like CrossMab antibody that can bind to two antigens by using two different light chain-heavy chain pairs, a two-step modification process is used. First, a heterodimerization method (e.g., knob-in-hole (KiH) technology) is used to engineer the dimerization interface to the C-terminus of each heavy chain to ensure that only heterodimers of two different heavy chains from one antibody (e.g., antibody A) and a second antibody (e.g., antibody B) are effectively formed. Next, the constant heavy chain 1 domain (CH1) and the constant light chain domain (CL) of one antibody (antibody A) are exchanged, while the variable heavy chain (VH) and the variable light chain (VL) domains are kept the same. The exchange of CH1 and CL domains ensures that the modified antibody (antibody A) light chain can only effectively dimerize with the modified antibody (antibody A) heavy chain, while the unmodified antibody (antibody B) light chain can only effectively dimerize with the unmodified antibody (antibody B) heavy chain; therefore, only the desired bispecific CrossMab is effectively formed (see, e.g., Cain, C. SciBX 4 (28); doi: 10.1038 / scibx.2011.783, the contents of which are incorporated herein by reference).

[0700] Common heavy chain

[0701] An exemplary method for enhancing the formation of a desired bispecific antibody by a monomer mixture is to interact with each heteropolymeric variable light chain region of a bispecific antibody by providing a common variable heavy chain. Compositions and methods for producing bispecific antibodies with a common heavy chain are disclosed in, for example, US20120184716, US20130317200, and US20160264685A1, each of which is incorporated herein by reference.

[0702] Amino acid modification

[0703] Producing alternative compositions and methods for multispecific antibodies with correct light chain pairing includes various amino acid modifications. For example, Zymeworks describes heterodimers having one or more amino acid modifications in CH1 and / or CL domains, one or more amino acid modifications in VH and / or VL domains, or combinations thereof, which are part of the interface between the light chain and the heavy chain, and establish 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 the cell, the heavy chain of the first heterodimer is preferentially paired with a light chain rather than with each other (e.g., see WO2015181805). Other exemplary methods are described in WO2016026943 (Argen-X), US20150211001, US20140072581A1, US20160039947A1, and US20150368352.

[0704] λ / κ form

[0705] Multispecific molecules (e.g., multispecific antibody molecules) including λ light chain polypeptides and κ light chain polypeptides can be used to allow heterodimerization. Methods for producing bispecific antibody molecules comprising λ light chain polypeptides and κ light chain polypeptides are disclosed in PCT / US17 / 53053, filed September 22, 2017, which is incorporated herein by reference in its entirety.

[0706] In an embodiment, the multispecific molecule comprises a multispecific antibody molecule, e.g., an antibody molecule having two binding specificities, e.g., a bispecific antibody molecule. The multispecific antibody molecule comprises:

[0707] lambda light chain polypeptide 1 (LLCP1) specific for the first epitope;

[0708] heavy chain polypeptide 1 (HCP1) specific for the first epitope;

[0709] kappa light chain polypeptide 2 (KLCP2) specific for a second epitope; and

[0710] Heavy chain polypeptide 2 (HCP2) specific for the second epitope.

[0711] As used herein, the term "λ light chain polypeptide 1 (LLCP1)" refers to a polypeptide comprising sufficient light chain (LC) sequence so that when combined with a cognate heavy chain variable region, it can mediate specific binding to its epitope and complex with HCP1. In one embodiment, LLCP1 comprises all or a fragment of the CH1 region. In one embodiment, LLCP1 comprises LC-CDR1, LC-CDR2, LC-CDR3, FR1, FR2, FR3, FR4 and CH1, or a sequence sufficient therefrom to mediate specific binding to its epitope and complex with HCP1. LLCP1 provides specificity to the first epitope with its HCP1 (while KLCP2 provides specificity to the second epitope with its HCP2). As described elsewhere herein, LLCP1 has a higher affinity for HCP1 than for HCP2.

[0712] As used herein, the term "κ light chain polypeptide 2 (KLCP2)" refers to a polypeptide comprising sufficient light chain (LC) sequences so that when combined with a cognate heavy chain variable region, it can mediate specific binding to its epitope and complexing with HCP2. In one embodiment, LLCP1 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 sequences therefrom sufficient to mediate specific binding to its epitope and complexing with HCP2. KLCP2, together with its HCP2, provides specificity for the second epitope (while LLCP1 and its HCP1 provide specificity for the first epitope).

[0713] As used herein, the term "heavy chain polypeptide 1 (HCP1)" refers to a polypeptide comprising sufficient heavy chain (HC) sequence (e.g., HC variable region sequence) to mediate specific binding to its epitope and complexing with HCP1 when bound to a homologous LLCP1. In one embodiment, HCP1 comprises all or a fragment of the CH1 region. In one embodiment, HCP1 comprises all or a fragment of the CH2 and / or CH 3 regions. In one embodiment, HCP1 comprises HC-CDR1, HC-CDR2, HC-CDR3, FR1, FR2, FR3, FR4, CH1, CH2, and CH3, or sequences therefrom sufficient to achieve the following: (i) mediate specific binding to its epitope and complexing with LLCP1, (ii) preferentially complex with LLCP1 rather than KLCP2 as described herein; and (iii) preferentially complex with HCP2 rather than another HCP1 molecule as described herein. HCP1 together with its LLCP1 provides specificity for the first epitope (while KLCP2 together with its HCP2 provides specificity for the second epitope).

[0714] As used herein, the term "heavy chain polypeptide 2 (HCP2)" refers to a polypeptide comprising sufficient heavy chain (HC) sequence (e.g., HC variable region sequence) so that when bound to a homologous LLCP1, it can mediate specific binding to its epitope and complex with HCP1. In one embodiment, HCP2 comprises all or a fragment of the CH1 region. In one embodiment, HCP2 comprises all or a fragment of the CH2 and / or CH3 regions. In one embodiment, HCP1 comprises HC-CDR1, HC-CDR2, HC-CDR3, FR1, FR2, FR3, FR4, CH1, CH2, and CH3, or sequences therefrom sufficient to achieve the following: (i) mediate specific binding to its epitope and complex with KLCP2, (ii) preferentially complex with KLCP2 rather than LLCP1 as described herein; and (iii) preferentially complex with HCP1 rather than another HCP2 molecule as described herein. HCP2 together with its KLCP2 provides specificity for the second epitope (while LLCP1 together with its HCP1 provides specificity for the first epitope).

[0715] In some embodiments of the multispecific antibody molecules disclosed herein: LLCP1 has a higher affinity for HCP1 than for HCP2; and / or KLCP2 has a higher affinity for HCP2 than for HCP1.

[0716] In an embodiment, the affinity of LLCP1 for HCP1 is sufficiently greater than its affinity for HCP2 such that under preselected conditions, e.g., in an aqueous buffer (e.g., at pH 7), in saline (e.g., at pH 7), or under physiological conditions, at least 75%, 80%, 90%, 95%, 98%, 99%, 99.5% or 99.9% of the multispecific antibody molecules have LLCP1 complexed or engaged with HCP1.

[0717] In some embodiments of the multispecific antibody molecules disclosed herein: HCP1 has a greater affinity for HCP2 than for a second molecule of HCP1; and / or HCP2 has a greater affinity for HCP1 than for a second molecule of HCP2.

[0718] In embodiments, the affinity of HCP1 for HCP2 is sufficiently greater than its affinity for a second HCP1 molecule such that under preselected conditions, e.g., in an aqueous buffer (e.g., at pH 7), in saline (e.g., at pH 7), or under physiological conditions, at least 75%, 80%, 90%, 95%, 98%, 99%, 99.5% or 99.9% of the multispecific antibody molecules have HCP1 complexed or engaged with HCP2.

[0719] In another aspect, the present invention discloses a method for preparing or generating a multispecific antibody molecule. The method comprises:

[0720] (i) providing a first heavy chain polypeptide (e.g., a heavy chain polypeptide comprising one, two, three or all of a first heavy chain variable region (first VH), a first CH1, a first heavy chain constant region (e.g., a first CH2, a first CH3 or both);

[0721] (ii) providing a second heavy chain polypeptide (e.g., a heavy chain polypeptide comprising one, two, three or all of a second heavy chain variable region (second VH), a second CH1, a second heavy chain constant region (e.g., a second CH2, a second CH3 or both);

[0722] (iii) providing a lambda chain polypeptide (e.g., a lambda light variable region (VLλ), a lambda light constant chain (VLλ), or both) that preferentially associates with a first heavy chain polypeptide (e.g., a first VH); and

[0723] (iv) providing a kappa chain polypeptide (eg, a kappa light variable region (VLκ), a kappa light constant chain (VLκ), or both) that preferentially associates with a second heavy chain polypeptide (eg, a second VH).

[0724] In an embodiment, the first heavy chain and the second heavy chain polypeptide form an Fc interface that enhances heterodimerization.

[0725] In an embodiment, (i)-(iv) (eg, nucleic acids encoding (i)-(iv)) are introduced into a single cell, eg, a single mammalian cell, eg, a CHO cell. In an embodiment, (i)-(iv) are expressed in the cell.

[0726] In an embodiment, (i)-(iv) (e.g., nucleic acids encoding (i)-(iv)) are introduced into different cells, e.g., different mammalian cells, e.g., two or more CHO cells. In an embodiment, (i)-(iv) are expressed in the cell.

[0727] In one embodiment, the method further comprises purifying the cell-expressed antibody molecule, e.g., using lambda-specific and / or kappa-specific purification, e.g., affinity chromatography.

[0728] In embodiments, the method further comprises evaluating the multispecific antibody molecules expressed by the cells. For example, the multispecific antibody molecules expressed by the purified cells can be analyzed by techniques known in the art (including mass spectrometry). In one embodiment, the antibody molecules expressed by the purified cells are cleaved, for example, digested with papain to produce Fab portions, and evaluated using mass spectrometry.

[0729] In embodiments, the method produces correctly paired kappa / lambda multispecific (e.g., bispecific) antibody molecules in high yield, e.g., at least 75%, 80%, 90%, 95%, 98%, 99%, 99.5% or 99.9%.

[0730] In other embodiments, the multispecific (e.g., bispecific) antibody molecule comprises:

[0731] (i) a first heavy chain polypeptide (HCP1) (e.g., a heavy chain polypeptide comprising one, two, three, or all of a first heavy chain variable region (first VH), a first CH1, a first heavy chain constant region (e.g., a first CH2, a first CH3, or both), e.g., wherein HCP1 binds to a first epitope;

[0732] (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 CHI, a second heavy chain constant region (e.g., a second CH2, a second CH3, or both), e.g., wherein HCP2 binds to a second epitope;

[0733] (iii) a lambda light chain polypeptide (LLCP1) (e.g., a lambda light variable region (VL1), a lambda light constant chain (VL1), or both) that preferentially associates with a first heavy chain polypeptide (e.g., a first VH), e.g., wherein LLCP1 binds to a first epitope; and

[0734] (iv) a kappa light chain polypeptide (KLCP2) (e.g., a kappa light variable region (VLk), a kappa light constant chain (VLk), or both) that preferentially associates with a second heavy chain polypeptide (e.g., a second VH), e.g., wherein the KLCP2 binds to a second epitope.

[0735] In embodiments, the first heavy chain and the second heavy chain polypeptide form an Fc interface that enhances heterodimerization. In embodiments, the multispecific antibody molecule has a first binding specificity comprising a hybrid VL1-CL1 that heterodimerizes to a first heavy chain variable region (with a knob modification) connected to an Fc constant CH2-CH3 domain, and a second binding specificity comprising a hybrid VLk-CLk that heterodimerizes to a second heavy chain variable region (with a hole modification) connected to an Fc constant CH2-CH3 domain.

[0736] TRBC1 and TRBC2 antigen binding domains

[0737] The present disclosure provides, among other things, antibody molecules, e.g., multispecific (e.g., bispecific, trispecific, tetraspecific) or multifunctional molecules, comprising (e.g., engineered to contain) one or more antigen binding domains that bind to tumor antigens on lymphoma cells (e.g., T cells). 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., relative 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., relative to a T cell receptor comprising TRBC1). In some embodiments, the multifunctional molecule comprises (e.g., engineered to contain) 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 comprising TRBC1 or a T cell receptor comprising TRBC2.

[0738] T cell receptor (TCR) is a receptor found on the surface of lymphocytes, particularly on T lymphocytes (T cells). TCR is responsible for identifying antigen fragments presented by major histocompatibility complex (MHC) molecules on other immune cells (e.g., B cells) by signal transduction and activating T cells via related CD3. The vast majority of human TCRs are heterodimers including α chains and β chains. The α and β chains of TCR both include variable regions and constant regions. The variable regions of α and β chains are encoded by different DNA elements (V, D and J elements of β chains; V and J elements of α chains). The recombination between these elements produces changes in TCR antigen binding specificity to a large extent. TCR β chain constant region is selected from two different domains, β constant domain 1 and β constant domain 2. Without wishing to be bound by theory, it is believed that most TCRs including β chains include β chains including β constant domain 1 or β constant domain 2, rather than β chains including constant domain 1 and constant domain 2 simultaneously.

[0739] In some embodiments, the multifunctional or multispecific molecules or antibody molecules of the present application include antigen binding domains that bind to tumor antigens (e.g., T cell receptors including TRBC1, TRBC1, T cell receptors including TRBC2, or TRBC2) on lymphoma cells (e.g., T cells). In some embodiments, the multifunctional or multispecific molecules or antibody molecules of the present application include antigen binding domains that selectively target lymphocytes expressing T cell receptors including TRBC1, TRBC1, T cell receptors including TRBC2, or TRBC2. Although lymphocytes or lymphoma cells presenting T cell receptors including TRBC1 or TRBC2 are T cells in the most typical cases, cancer causes the destruction of many non-disease expression patterns. Therefore, in some embodiments, lymphoma cells or lymphocytes may not be T cells. In some embodiments, lymphoma cells or lymphocytes are B cells. In some embodiments, lymphoma cells or lymphocytes are natural killer cells.

[0740] In some embodiments, the antigen binding domain (e.g., the first antigen binding domain) includes 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.

[0741] In some embodiments, the antigen binding domain that binds to TRBC1 includes one or more CDRs disclosed in Table 2, Table 6, or Table 3 (e.g., VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2, and / or VLCDR3), or a sequence having at least 85%, 90%, 95%, or 99% identity thereto. In some embodiments, the antigen binding domain that binds to TRBC1 includes one or more framework regions disclosed in Table 2, Table 6, or Table 3 (e.g., VHFWR1, VHFWR2, VHFWR3, VHFWR4, VLFWR1, VLFWR2, VLFWR3, and / or VLFWR4), or a sequence having at least 85%, 90%, 95%, or 99% identity thereto. In some embodiments, the antigen binding domain that binds to TRBC1 includes VH and / or VL disclosed in Table 4, or a sequence having at least 85%, 90%, 95%, or 99% identity thereto. In some embodiments, the antigen binding domain that binds to TRBC1 comprises an amino acid sequence disclosed in Table 5, or a sequence at least 85%, 90%, 95%, or 99% identical thereto.

[0742] In some embodiments, the antigen binding domain that binds to TRBC1 includes: VH including heavy chain complementary determining region 1 (VHCDR1), VHCDR2, and VHCDR3, and VL including light chain complementary determining region 1 (VLCDR1), VLCDR2, and VLCDR3.

[0743] In some embodiments, VHCDR1, VHCDR2 and VHCDR3 include the amino acid sequences of SEQ ID NOs: 7346, 7355 and 202, respectively (or sequences having at least 85%, 90%, 95% or 99% identity thereof). In some embodiments, VHCDR1, VHCDR2 and VHCDR3 include the amino acid sequences of SEQ ID NOs: 7346, 201 and 202, respectively (or sequences having at least 85%, 90%, 95% or 99% identity thereof). In some embodiments, VHCDR1, VHCDR2 and VHCDR3 include the amino acid sequences of SEQ ID NOs: 7354, 201 and 202, respectively (or sequences having at least 85%, 90%, 95% or 99% identity thereof). In some embodiments, VHCDR1, VHCDR2 and VHCDR3 comprise the amino acid sequences of SEQ ID NOs: 7354, 7355 and 202, respectively (or sequences at least 85%, 90%, 95% or 99% identical thereto).

[0744] In some embodiments, VLCDR1, VLCDR2 and VLCDR3 include the amino acid sequences of SEQ ID NOs: 223, 224 and 225, respectively (or sequences having at least 85%, 90%, 95% or 99% identity thereof). In some embodiments, VLCDR1, VLCDR2 and VLCDR3 include the amino acid sequences of SEQ ID NOs: 7367, 224 and 225, respectively (or sequences having at least 85%, 90%, 95% or 99% identity thereof). In some embodiments, VLCDR1, VLCDR2 and VLCDR3 include the amino acid sequences of SEQ ID NOs: 223, 7368 and 225, respectively (or sequences having at least 85%, 90%, 95% or 99% identity thereof). In some embodiments, VLCDR1, VLCDR2 and VLCDR3 include the amino acid sequences of SEQ ID NOs: 223, 224 and 7369, respectively (or sequences having at least 85%, 90%, 95% or 99% identity thereof). In some embodiments, VLCDR1, VLCDR2 and VLCDR3 include the amino acid sequences of SEQ ID NOs: 7367, 7368 and 7369, respectively (or sequences having at least 85%, 90%, 95% or 99% identity thereof).

[0745] In some embodiments, VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2 and VLCDR3 comprise the amino acid sequences of SEQ ID NOs: 7346, 7355, 202, 223, 224 and 225, respectively (or sequences having at least 85%, 90%, 95% or 99% identity thereof). In some embodiments, VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2 and VLCDR3 comprise the amino acid sequences of SEQ ID NOs: 7346, 201, 202, 223, 224 and 225, respectively (or sequences having at least 85%, 90%, 95% or 99% identity thereof). In some embodiments, VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2 and VLCDR3 comprise the amino acid sequences of: SEQ ID NOs: 7346, 7355, 202, 7367, 224 and 225, respectively (or sequences having at least 85%, 90%, 95% or 99% identity thereof); SEQ ID NOs: 7346, 7355, 202, 223, 7368 and 225, respectively (or sequences having at least 85%, 90%, 95% or 99% identity thereof); SEQ ID NOs: 7346, 7355, 202, 223, 224 and 7369, respectively (or sequences having at least 85%, 90%, 95% or 99% identity thereof); NO: 7346, 7355, 202, 7367, 7368 and 7369 (or sequences with at least 85%, 90%, 95% or 99% identity thereof); SEQ ID NO: 7346, 201, 202, 7367, 224 and 225, respectively (or sequences with at least 85%, 90%, 95% or 99% identity thereof); SEQ ID NO: 7346, 201, 202, 223, 7368 and 225, respectively (or sequences with at least 85%, 90%, 95% or 99% identity thereof); SEQ ID NO: 7346, 201, 202, 223, 224 and 7369, respectively (or sequences with at least 85%, 90%, 95% or 99% identity thereof); NO:7346, 201, 202, 7367, 7368 and 7369 (or sequences at least 85%, 90%, 95% or 99% identical thereto); SEQ ID NO:7354, 201, 202, 223, 224 and 225 (or sequences at least 85%, 90%, 95% or 99% identical thereto), respectively;SEQ ID NOs: 7354, 201, 7367, 224 and 225, respectively (or sequences having at least 85%, 90%, 95% or 99% identity thereof); SEQ ID NOs: 7354, 201, 202, 223, 7368 and 225, respectively (or sequences having at least 85%, 90%, 95% or 99% identity thereof); SEQ ID NOs: 7354, 201, 202, 223, 224 and 7369, respectively (or sequences having at least 85%, 90%, 95% or 99% identity thereof); SEQ ID NOs: 7354, 201, 202, 7367, 7368 and 7369, respectively (or sequences having at least 85%, 90%, 95% or 99% identity thereof); NO: 7354, 7355, 202, 223, 224 and 225 (or sequences with at least 85%, 90%, 95% or 99% identity thereof); SEQ ID NO: 7354, 7355, 202, 7367, 224 and 225, respectively (or sequences with at least 85%, 90%, 95% or 99% identity thereof); SEQ ID NO: 7354, 7355, 202, 223, 7368 and 225, respectively (or sequences with at least 85%, 90%, 95% or 99% identity thereof); SEQ ID NO: 7354, 7355, 202, 223, 224 and 7369, respectively (or sequences with at least 85%, 90%, 95% or 99% identity thereof); or SEQ ID NO: 7354, 7355, 202, 223, 224 and 7369, respectively (or sequences with at least 85%, 90%, 95% or 99% identity thereof); NO:7354, 7355, 202, 7367, 7368 and 7369 (or sequences having at least 85%, 90%, 95% or 99% identity thereto). ;

[0746] In some embodiments, VH comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 7351, 253, 250-252, 254, 7343, 7344, 7350, and 7352 (or a sequence having at least 85%, 90%, 95%, or 99% identity thereof) and / or VL comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 258, 255-257, 259, 260, and 7357-7360 (or a sequence having at least 85%, 90%, 95%, or 99% identity thereof). In some embodiments, VH and VL comprise the amino acid sequences of SEQ ID NOs: 7351 and 258, respectively (or a sequence having at least 85%, 90%, 95%, or 99% identity thereof). In some embodiments, VH and VL comprise the amino acid sequences of SEQ ID NOs: 253 and 258, respectively (or sequences at least 85%, 90%, 95%, or 99% identical thereto).

[0747] In some embodiments, an antigen binding domain (e.g., a first antigen binding domain) that binds to a tumor antigen (e.g., a T cell receptor including TRBC1, TRBC1, a T cell receptor including TRBC2, or TRBC2) on a lymphoma cell (e.g., a T cell receptor) on a lymphoma cell (e.g., a T cell receptor) on a lymphoma cell (e.g., a T cell receptor) on a T cell receptor) includes any CDR amino acid sequence, framework region (FWR) amino acid sequence, or variable region amino acid sequence of Tables 2-6. In some embodiments, an antigen binding domain (e.g., a first antigen binding domain) that binds to a tumor antigen (e.g., a T cell receptor including TRBC1, TRBC1, a T cell receptor including TRBC2, or TRBC2) on a lymphoma cell (e.g., a T cell receptor) on a T cell receptor) on a T cell receptor) includes a heavy chain and / or light chain amino acid sequence of Table 5. In some embodiments, an antigen binding domain (e.g., a first antigen binding domain) that selectively targets lymphocytes expressing a T cell receptor including TRBC1, TRBC1, a T cell receptor including TRBC2, or TRBC2 includes any CDR amino acid sequence, framework region (FWR) amino acid sequence, or variable region amino acid sequence disclosed in Tables 2-6. In some embodiments, an antigen binding domain (e.g., a first antigen binding domain) that selectively targets a T cell receptor that expresses TRBC1, TRBC1, a T cell receptor that expresses TRBC2, or a lymphocyte that expresses TRBC2 comprises a heavy chain and / or light chain amino acid sequence of Table 5. An antigen binding domain that binds to a tumor antigen that comprises TRBC1 or selectively targets a lymphocyte that expresses TRBC1 may be referred to as targeting TRBC1 (i.e., an antigen binding domain that targets TRBC1). An antigen binding domain that binds to a tumor antigen that comprises TRBC2 or selectively targets a lymphocyte that expresses TRBC2 may be referred to as targeting TRBC2 (i.e., an antigen binding domain that targets TRBC2).

[0748] In some embodiments, the antigen binding domain targeting TRBC1 includes a VH, the VH including the heavy chain complementary determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 200 (or a sequence having no more than 1, 2, 3 or 4 mutations (e.g., substitutions, additions or deletions) thereof), the VHCDR2 amino acid sequence of SEQ ID NO: 201 (or a sequence having no more than 1, 2, 3 or 4 mutations (e.g., substitutions, additions or deletions) thereof), and / or the VHCDR3 amino acid sequence of SEQ ID NO: 202 (or a sequence having no more than 1, 2, 3 or 4 mutations (e.g., substitutions, additions or deletions) thereof). In some embodiments, the TRBC1 antigen binding domain includes a VH, the VH including the VHCDR1 amino acid sequence of SEQ ID NO: 200, the VHCDR2 amino acid sequence of SEQ ID NO: 201, and / or the VHCDR3 amino acid sequence of SEQ ID NO: 202.

[0749] In some embodiments, the antigen binding domain targeting TRBC1 includes a VL comprising the light chain complementary determining region 1 (VLCDR1) amino acid sequence of SEQ ID NO: 223 (or a sequence having no more than 1, 2, 3, or 4 mutations (e.g., substitutions, additions, or deletions) thereof), the VLCDR2 amino acid sequence of SEQ ID NO: 224 (or a sequence having no more than 1, 2, 3, or 4 mutations (e.g., substitutions, additions, or deletions) thereof), and / or the VLCDR3 amino acid sequence of SEQ ID NO: 225 (or a sequence having no more than 1, 2, 3, or 4 mutations (e.g., substitutions, additions, or deletions) thereof). In some embodiments, the antigen binding domain targeting TRBC1 includes a VL comprising the VLCDR1 amino acid sequence of SEQ ID NO: 223, the VLCDR2 amino acid sequence of SEQ ID NO: 224, and the VLCDR3 amino acid sequence of SEQ ID NO: 225.

[0750] In some embodiments, the antigen binding domain targeting TRBC1 includes a VH comprising a heavy chain framework region 1 (VHFWR1) amino acid sequence of SEQ ID NO:203, a VHFWR2 amino acid sequence of SEQ ID NO:204, a VHFWR3 amino acid sequence of SEQ ID NO:205, and / or a VHFWR4 amino acid sequence of SEQ ID NO:206.

[0751] In some embodiments, the antigen binding domain targeting TRBC1 includes a VL comprising a light chain framework region 1 (VLFWR1) amino acid sequence of SEQ ID NO:226, a VLFWR2 amino acid sequence of SEQ ID NO:227, a VLFWR3 amino acid sequence of SEQ ID NO:228, and / or a VLFWR4 amino acid sequence of SEQ ID NO:229.

[0752] In some embodiments, the antigen binding domain targeting TRBC1 includes a VH, wherein the VH includes the VHFWR1 amino acid sequence of SEQ ID NO:203 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations (e.g., substitutions, additions or deletions) thereof), the VHFWR2 amino acid sequence of SEQ ID NO:204 (or a sequence having no more than 1, 2, 3, 4, 5 or 6 mutations (e.g., substitutions, additions or deletions) thereof), the VHFWR3 amino acid sequence of SEQ ID NO:205 (or a sequence having no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 mutations (e.g., substitutions, additions or deletions) thereof) and / or the VHFWR4 amino acid sequence of SEQ ID NO:206.

[0753] In some embodiments, the antigen binding domain targeting TRBC1 includes a VL, wherein the VL includes the VLFWR1 amino acid sequence of SEQ ID NO:226 (or a sequence having no more than 1, 2 or 3 mutations (e.g., substitutions, additions or deletions) thereof), the VLFWR2 amino acid sequence of SEQ ID NO:227 (or a sequence having no more than 1 mutation (e.g., substitutions, additions or deletions) thereof), the VLFWR3 amino acid sequence of SEQ ID NO:228 (or a sequence having no more than 1 mutation (e.g., substitutions, additions or deletions) thereof) and / or the VLFWR4 amino acid sequence of SEQ ID NO:229.

[0754] In some embodiments, the antigen binding domain targeting TRBC1 includes a VH comprising a heavy chain framework region 1 (VHFWR1) amino acid sequence of SEQ ID NO:207, a VHFWR2 amino acid sequence of SEQ ID NO:208, a VHFWR3 amino acid sequence of SEQ ID NO:209, and / or a VHFWR4 amino acid sequence of SEQ ID NO:210.

[0755] In some embodiments, the ant...

Claims

1. A multifunctional molecule comprising: (i) a first antigen binding domain that binds to T cell receptor β chain constant domain 1 (TRBC1), wherein the first antigen binding domain comprises: (a) a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises a heavy chain complementary determining region 1 (VHCDR1), a heavy chain complementary determining region 2 (VHCDR2) and a heavy chain complementary determining region 3 (VHCDR3) of SEQ ID NO: 7346, SEQ ID NO: 7355 and SEQ ID NO: 202, respectively; and the VL comprises a light chain complementary determining region 1 (VLCDR1), a light chain complementary determining region 2 (VLCDR2) and a light chain complementary determining region 3 (VLCDR3) of SEQ ID NO: 223, SEQ ID NO: 224 and SEQ ID NO: 225, respectively, or (b) VH and VL, wherein the VH comprises VHCDR1, VHCDR2 and VHCDR3 of SEQ ID NO: 7346, SEQ ID NO: 201 and SEQ ID NO: 202, respectively, and the VL comprises VLCDR1, VLCDR2 and VLCDR3 of SEQ ID NO: 223, SEQ ID NO: 224 and SEQ ID NO: 225, respectively, and (ii) a second antigen binding domain that selectively binds to NKp30, wherein the second antigen binding domain comprises a VH and a VL, wherein the VH comprises a VHCDR1, a VHCDR2 and a VHCDR3 of SEQ ID NO: 7313, SEQ ID NO: 6001 and SEQ ID NO: 6002, respectively, and the VL comprises a VLCDR1, a VLCDR2 and a VLCDR3 of SEQ ID NO: 7326, SEQ ID NO: 7327 and SEQ ID NO: 7329, respectively, wherein the VHCDR1-3 and VLCDR1-3 are conveniently numbered by Kabat; and The multifunctional molecule binds to and activates NK cells.

2. The multifunctional molecule of claim 1, wherein the first antigen binding domain comprises: A VH comprising a sequence having at least 85% identity to an amino acid sequence selected from SEQ ID NO: 7351 or 253.

3. The multifunctional molecule of claim 2, wherein the first antigen binding domain comprises: VH, which includes the amino acid sequence of SEQ ID NO:7351 or 253.

4. The multifunctional molecule of claim 1, wherein the first antigen binding domain comprises: A VL comprising a sequence having at least 85% identity to an amino acid sequence selected from SEQ ID NO:

258.

5. The multifunctional molecule of claim 4, wherein the first antigen binding domain comprises: VL comprising the amino acid sequence of SEQ ID NO: 258; or (iii) any combination thereof.

6. The multifunctional molecule of claim 1, wherein the first antigen binding domain comprises: VH and VL comprising amino acid sequences at least 85% identical to SEQ ID NOs: 7351 and 258, respectively; or A VH and a VL comprising an amino acid sequence at least 85% identical to SEQ ID NOs: 253 and 258, respectively.

7. The multifunctional molecule of claim 6, wherein the first antigen binding domain comprises: VH and VL comprising the sequences of SEQ ID NO: 7351 and 258, respectively; or VH and VL comprising the sequences of SEQ ID NO: 253 and 258, respectively.

8. The multifunctional molecule of claim 1, wherein the first antigen binding domain comprises: (i) a heavy chain comprising an amino acid sequence that is at least 75% identical to SEQ ID NO: 7382 or 7379; (ii) a light chain comprising a sequence that is at least 75% identical to SEQ ID NO: 7380; or (iii) any combination thereof.

9. The multifunctional molecule of claim 8, wherein the first antigen binding domain comprises: (i) a heavy chain comprising the amino acid sequence of SEQ ID NO: 7382 or 7379; (ii) a light chain comprising the amino acid sequence of SEQ ID NO: 7380; or (iii) any combination thereof.

10. The multifunctional molecule of claim 1, wherein the second antigen binding domain comprises: A VH comprising an amino acid sequence having at least 85% sequence identity to the sequence of SEQ ID NO:7302.

11. The multifunctional molecule of claim 10, wherein the second antigen binding domain comprises: VH, which comprises an amino acid sequence selected from SEQ ID NO:7302.

12. The multifunctional molecule of claim 11, wherein the second antigen binding domain comprises a VL comprising an amino acid sequence having at least 85% sequence identity to the sequence of SEQ ID NO: 7309.

13. The multifunctional molecule of claim 12, wherein the second antigen binding domain comprises a VL comprising the amino acid sequence of SEQ ID NO: 7309.

14. The multifunctional molecule of claim 1, wherein the VH and VL of the second antigen binding domain comprise the amino acid sequence of: comprising amino acid sequences that are at least 85% identical to SEQ ID NOs: 7302 and 7309, respectively.

15. The multifunctional molecule of claim 14, wherein the VH and VL of the second antigen binding domain comprise the amino acid sequence of: Containing the amino acid sequences of SEQ ID NO: 7302 and 7309 respectively.

16. The multifunctional molecule of claim 1, wherein the second antigen binding domain comprises the amino acid sequence of: An amino acid sequence that is at least 85% identical to SEQ ID NO:7383.

17. The multifunctional molecule of claim 16, wherein the second antigen binding domain comprises the amino acid sequence of SEQ ID NO: 7383.

18. The multifunctional molecule of claim 1, wherein the first antigen binding domain comprises a Fab or a scFv, the second antigen binding domain comprises a Fab or a scFv, or a combination thereof.

19. The multifunctional molecule according to claim 18, comprising: (i) a first antigen binding domain comprising the sequences of SEQ ID NO: 7351 and SEQ ID NO: 258, and a second antigen binding domain comprising the sequence of SEQ ID NO: 7302; or (ii) a first antigen binding domain comprising the sequence of SEQ ID NO:253 and SEQ ID NO:258, and a second antigen binding domain comprising the sequence of SEQ ID NO:7302.

20. The multifunctional molecule according to claim 19, comprising: (i) a first antigen binding domain comprising the sequence of SEQ ID NO: 7382 and SEQ ID NO: 7380, and a second antigen binding domain comprising the sequence of SEQ ID NO: 7383; or (ii) a first antigen binding domain comprising the sequence of SEQ ID NO: 7379 and SEQ ID NO: 7380, and a second antigen binding domain comprising the sequence of SEQ ID NO: 7383.

21. The multifunctional molecule according to any one of claims 1-20, wherein the multifunctional molecule further comprises a cytokine molecule.

22. The multifunctional molecule of claim 21, wherein the cytokine molecule comprises: Interleukin-2 (IL-2) or its functional variant, interleukin-12 (IL-12) or its functional variant, interleukin-15 (IL-15) or its functional variant, interleukin-18 (IL-18) or its functional variant, interleukin-21 (IL-21) or its functional variant, interleukin-7 (IL-7) or its functional variant, interferon γ or its functional variant, or any combination thereof.

23. The multifunctional molecule according to claim 22, wherein the multifunctional molecule binds monovalently to TRBC1 or TRBC2.

24. An antibody molecule that binds to TRBC1, comprising: (a) a VH comprising the following VHCDR1, VHCDR2 and VHCDR3: SEQ ID NOs: 7346, 201 and 202, respectively; and Includes the following VLs for VLCDR1, VLCDR2, and VLCDR3: SEQ ID NO: 223, 224 and 225, respectively; or (b) a VH comprising the following VHCDR1, VHCDR2 and VHCDR3: SEQ ID NOs: 7346, 7355 and 202, respectively; and Includes the following VLs for VLCDR1, VLCDR2, and VLCDR3: They are SEQ ID NO: 223, 224 and 225 respectively.

25. An antibody molecule according to claim 24, comprising: (i) a heavy chain comprising an amino acid sequence that is at least 75% identical to SEQ ID NO: 7382; A light chain comprising a sequence having at least 75% identity to SEQ ID NO: 7380; or any combination thereof; or (ii) a heavy chain comprising the amino acid sequence of SEQ ID NO: 7379; and a light chain comprising the amino acid sequence of SEQ ID NO: 7380.

26. A polynucleotide comprising a nucleic acid molecule encoding the multifunctional molecule according to any one of claims 1 to 23 or the antibody molecule according to claim 24 or 25.

27. A method for preparing a multifunctional molecule according to any one of claims 1 to 23 or an antibody molecule according to claim 24 or 25, comprising culturing a cell comprising a polynucleotide according to claim 26 under conditions suitable for gene expression and / or homodimerization or heterodimerization.

28. A medicament comprising the multifunctional molecule according to any one of claims 1 to 23 or the antibody molecule according to claim 24 or 25 and a pharmaceutically acceptable carrier, excipient or stabilizer.

29. Use of a multifunctional molecule according to any one of claims 1 to 23 in the preparation of a medicament for treating cancer in a subject in need thereof.

30. The use according to claim 29, wherein the subject has cancer cells expressing T cell receptors including TRBC1.

31. The use according to claim 29 or 30, wherein the cancer is leukemia or lymphoma.

32. The use according to claim 29 or 30, wherein the cancer is selected from acquired immunodeficiency syndrome (AIDS)-related lymphoma, angioimmunoblastic T-cell lymphoma, adult T-cell leukemia / lymphoma, Burkitt's 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 Sezary syndrome), follicular lymphoma, lymphoplasmacytic lymphoma / Waldenstrom's macroglobulinemia, marginal zone B-cell lymphoma, gastric MALT lymphoma, chronic lymphocytic leukemia / small cell lymphocytic lymphoma (CLL / SLL), extranodal T-cell / NK-cell lymphoma (nasal type), and anaplastic large cell lymphoma.

33. The use according to claim 32, wherein the PTCL is hepatosplenic T-cell lymphoma (HSGDTCL), subcutaneous panniculitis-like T-cell lymphoma or enteropathy-associated T-cell lymphoma.

34. The use according to claim 32, wherein the anaplastic large cell lymphoma is primary cutaneous anaplastic large cell lymphoma or systemic anaplastic large cell lymphoma.

35. The use according to claim 31, wherein the lymphoma is peripheral T-cell lymphoma (PTCL).

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