Multifunctional molecules that bind to T cell-related cancer cells and uses thereof

Abstract

Multifunctional molecules are disclosed that include one, two, or all of: i) an antigen-binding domain that binds to T cell receptor beta chain constant domain 1 or T cell receptor beta chain constant domain 2; (ii) an immune cell engager (e.g., selected from an NK cell engager, a T cell engager, a B cell engager, a dendritic cell engager, or a macrophage cell engager); (iii) a cytokine molecule or a cytokine inhibitor molecule; (iv) a death receptor signal enhancer; and / or (v) a stromal-modifying moiety. Also disclosed are nucleic acids encoding the same, methods of producing the aforementioned molecules, and methods of treating cancer using the aforementioned molecules.

Description

Technical Field

[0001] Related Applications This application claims the benefit of U.S. Provisional Application No. 62 / 808,646, filed Feb. 21, 2019, the contents of which are hereby incorporated by reference in their entirety.

[0002] Sequence Listing This application includes a Sequence Listing that was electronically submitted in ASCII format and is hereby incorporated by reference in its entirety. The ASCII copy, created on Feb. 19, 2020, is named E2070-7022WO_SL.txt and is 810,998 bytes in size.

Background Art

[0003] Lymphoma is a cancer that originates from lymphocytes. T-cell lymphoma (TCL) is a lymphoma that originates from T cells and accounts for approximately 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. Given the current need for improved treatments for lymphomas such as TCL, new compositions and treatments targeting lymphoma, such as TCL, are highly desirable.

Summary of the Invention

Means for Solving the Problems

[0004] The present disclosure relates, inter alia, to novel multispecific, or multifunctional, molecules comprising one, two, or all of (i) an antigen-binding domain that binds to a tumor antigen on a lymphocytic cell (e.g., a T cell), such as a T cell receptor comprising the T cell receptor beta chain constant domain 1 (TRBC1) or a T cell receptor comprising the T cell receptor beta chain constant domain 2 (TRBC2); (ii) an immune cell engager (e.g., selected from an NK cell engager, a T cell engager, a B cell engager, a dendritic cell engager, or a macrophage cell engager); (iii) a cytokine molecule; and / or (iv) a stromal modifying moiety. The terms “multispecific” or “multifunctional” are used interchangeably herein.

[0005] Without being bound by theory, it is expected 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, such as cancer cells (e.g., lymphoma cells), that express a T cell receptor comprising TRBC1 or TRBC2, and / or modify the tumor stroma, e.g., modify the tumor microenvironment near the cancer site. Increasing the proximity and / or activity of immune cells using the multispecific molecules described herein is expected to enhance the immune response against target cells (e.g., cancer cells, e.g., lymphoma cells), thereby providing a more effective therapy (e.g., a more effective cancer therapy). Without being bound by theory, a targeted and localized immune response against target cells (e.g., cancer cells) is thought to reduce the systemic toxicity effects of the multispecific molecules described herein. Further, in the case where the target cancer cells are T cells (e.g., T cells that express a T cell receptor comprising TRBC1 or TRBC2), a targeted immune response against a cancerous T cell population that targets non-cancerous T cells to a lesser extent (e.g., does not target non-cancerous T cells) is thought to have less deleterious effects than a systemic ablation of all T cells.

[0006] Without being bound by theory, clonally-derived T cell lymphomas are positive for either TRBC1 or TRBC2, but not both. In the case of TRBC1+ T cell malignancies, the anti-TRBC1 molecules (e.g., multifunctional molecules that bind TRBC1 and NKp30) disclosed herein can deplete TRBC1+ cells while sparing TRBC2+ non-malignant T cells. Similarly, in the case of TRBC2+ T cell malignancies, the anti-TRBC2 molecules (e.g., multifunctional molecules that bind TRBC2 and NKp30) disclosed herein can deplete TRBC2+ cells while sparing TRBC1+ non-malignant T cells.

[0007] Without being bound by theory, in some embodiments, the multifunctional molecules (e.g., anti-TRBC1 / NKp30 antibodies) disclosed herein activate only NK cells in the presence of cells expressing TRBC1. Without being bound by theory, in some embodiments, the multifunctional molecules (e.g., anti-TRBC2 / NKp30 antibodies) disclosed herein activate only NK cells in the presence of cells expressing TRBC2.

[0008] Accordingly, multispecific molecules (e.g., multispecific or multifunctional antibody molecules) comprising the foregoing moieties, nucleic acids encoding them, methods of producing the foregoing molecules, and methods of using the foregoing molecules to treat cancer are provided herein, among other things.

[0009] In one aspect, provided herein is a multifunctional molecule comprising (i) a first antigen-binding domain that binds to the T cell receptor beta chain constant domain 1 (TRBC1) or the T cell receptor beta chain constant domain 2 (TRBC2), and (ii) a second antigen-binding domain that binds to NKp30.

[0010] 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 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 heavy chain complementarity determining region 1 (VHCDR1), VHCDR2, and VHCDR3, and a VL comprising light chain complementarity determining region 1 (VLCDR1), VLCDR2, and VLCDR3, wherein VHCDR1, VHCDR2, and VHCDR3 comprise the amino acid sequences of SEQ ID NO: 7346, 7355, and 202, respectively; SEQ ID NO: 7346, 201, and 202, respectively; SEQ ID NO: 7354, 201, and 202, respectively; or SEQ ID NO: 7354, 7355, and 202, respectively. In some embodiments, VLCDR1, VLCDR2, and VLCDR3 comprise the amino acid sequences of SEQ ID NO: 223, 224, and 225, respectively; SEQ ID NO: 7367, 224, and 225, respectively; SEQ ID NO: 223, 7368, and 225, respectively; SEQ ID NO: 223, 224, and 7369, respectively; or SEQ ID NO: 7367, 7368, and 7369, respectively.In some embodiments, VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2, and VLCDR3 each comprise the amino acid sequence of SEQ ID NO: 7346, 7355, 202, 223, 224, and 225; SEQ ID NO: 7346, 201, 202, 223, 224, and 225; SEQ ID NO: 7346, 7355, 202, 7367, 224, and 225; SEQ ID NO: 7346, 7355, 202, 223, 7368, and 225; SEQ ID NO: 7346, 7355, 202, 223, 224, and 7369; SEQ ID NO: 7346, 7355, 202, 7367, 7368, and 7369; SEQ ID NO: 7346, 201, 202, 7367, 224, and 225; SEQ ID NO: 7346, 201, 202, 223, 7368, and 225; SEQ ID NO: 7346, 201, 202, 223, 224, and 7369; SEQ ID NO: 7346, 201, 202, 7367, 7368, and 7369; SEQ ID NO: 7354, 201, 202, 223, 224, and 225; SEQ ID NO: 7354, 201, 202, 7367, 224, and 225; SEQ ID NO: 7354, 201, 202, 223, 7368, and 225; SEQ ID NO: 7354, 201, 202, 223, 224, and 7369; SEQ ID NO: 7354, 201, 202, 7367, 7368, and 7369; SEQ ID NO: 7354, 7355, 202, 223, 224, and 225; SEQ ID NO: 7354, 7355, 202, 7367, 224, and 225; SEQ ID NO: 7354, 7355, 202, 223, 7368, and 225; SEQ ID NO: 7354, 7355, 202, 223, 224, and 7369; or SEQ ID NO: 7354, 7355, 202, 7367, 7368, and 7369.In some embodiments, VH comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 7351, 253, 250 - 252, 254, 7343, 7344, 7350, and 7352 (or a sequence having at least 85%, 90%, 95%, or 99% identity thereto), and / or VL comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 258, 255 - 257, 259, 260, and 7357 - 7360 (or a sequence having at least 85%, 90%, 95%, or 99% identity thereto). In some embodiments, VH and VL each comprise the amino acid sequences of SEQ ID NO: 7351 and 258, respectively (or a sequence having at least 85%, 90%, 95%, or 99% identity thereto); or the amino acid sequences of SEQ ID NO: 253 and 258, respectively (or a sequence having at least 85%, 90%, 95%, or 99% identity thereto).

[0011] 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 not comprising TRBC1. Optionally, the K D for the binding between the first antigen - binding domain and TRBC1 is 40%, 30%, 20%, 10%, 1%, 0.1%, or 0.01% or less of the K D for the binding between the first antigen - binding domain and the T - cell receptor not comprising TRBC1. 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, the K D for the binding between the first antigen - binding domain and TRBC1 is the K Dis 40%, 30%, 20%, 10%, 1%, 0.1%, or 0.01% or less. In some embodiments, the binding of the first antigen-binding domain to TRBC1 in a lymphoma cell or lymphocyte, such as a T cell, activates the lymphoma cell or lymphocyte, such as a T cell, little, if at all, as measured by, for example, 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 or substantially activate NK cells in the absence of cells expressing TRBC1.

[0012] 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 comprising TRBC2 than for a T cell receptor not comprising TRBC2, and optionally, the K for the binding between the first antigen-binding domain and TRBC2 D is the K for the binding between the first antigen-binding domain and a T cell receptor not comprising TRBC2 D is 40%, 30%, 20%, 10%, 1%, 0.1%, or 0.01% or less. 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, and optionally, the K for the binding between the first antigen-binding domain and TRBC2 D is the K for the binding between the first antigen-binding domain and a T cell receptor comprising TRBC1 Dis 40%, 30%, 20%, 10%, 1%, 0.1%, or 0.01% or less. In some embodiments, the binding of the first antigen-binding domain to TRBC2 in lymphoma cells or lymphocytes, such as T cells, activates lymphoma cells or lymphocytes, such as T cells little, as measured by, for example, 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 or substantially activate NK cells in the absence of cells expressing TRBC2.

[0013] In some embodiments, the second antigen-binding domain comprises one or more CDRs, framework regions, variable regions, or antigen-binding domains disclosed in any of Tables 7-10, 18, and 19, or a sequence having at least 85%, 90%, 95%, or 99% identity thereto. In some embodiments, the second antigen-binding domain comprises a VH comprising heavy chain complementarity determining region 1 (VHCDR1), VHCDR2, and VHCDR3, and a VL comprising light chain complementarity determining region 1 (VLCDR1), VLCDR2, and VLCDR3, wherein the VHCDR1, VHCDR2, and VHCDR3 of the second antigen-binding domain comprise the amino acid sequences of SEQ ID NO: 7313, 6001, and 7315; SEQ ID NO: 7313, 6001, and 6002; SEQ ID NO: 7313, 6008, and 6009; SEQ ID NO: 7313, 7385, and 7315; or SEQ ID NO: 7313, 7318, and 6009, respectively. In some embodiments, the VLCDR1, VLCDR2, and VLCDR3 of the second antigen-binding domain comprise the amino acid sequences of SEQ ID NO: 7326, 7327, and 7329; SEQ ID NO: 6063, 6064, and 7293; SEQ ID NO: 6070, 6071, and 6072; or SEQ ID NO: 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 NO: 7313, 6001, 7315, 7326, 7327, and 7329; SEQ ID NO: 7313, 6001, 6002, 6063, 6064, and 7293; SEQ ID NO: 7313, 6008, 6009, 6070, 6071, and 6072; SEQ ID NO: 7313, 7385, 7315, 6070, 6064, and 7321; or SEQ ID NO: 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 the group consisting of SEQ ID NO: 7302, 7298, 7300, 7301, 7303, and 7304 (or a sequence having at least 85%, 90%, 95%, or 99% identity thereto), and / or the VL of the second antigen-binding domain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 7309, 7305, 7299, 7306 - 7308 (or a sequence having at least 85%, 90%, 95%, or 99% identity thereto). In some embodiments, the VH of the second antigen-binding domain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 6121 or 6123 - 6128 (or a sequence having at least 85%, 90%, 95%, or 99% identity thereto), and / or the VL of the second antigen-binding domain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 7294 or 6137 - 6141 (or a sequence having at least 85%, 90%, 95%, or 99% identity thereto). In some embodiments, the VH of the second antigen-binding domain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 6122 or 6129 - 6134 (or a sequence having at least 85%, 90%, 95%, or 99% identity thereto), and / or the VL of the second antigen-binding domain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 6136 or 6142 - 6147 (or a sequence having at least 85%, 90%, 95%, or 99% identity thereto). In some embodiments, the VH and VL of the second antigen-binding domain each comprise the amino acid sequences of SEQ ID NO: 7302 and 7309, respectively (or a sequence having at least 85%, 90%, 95%, or 99% identity thereto); or the amino acid sequences of SEQ ID NO: 7302 and 7305, respectively (or a sequence having at least 85%, 90%, 95%, or 99% identity thereto).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 thereto); SEQ ID NO: 6187 or 6188 (or a sequence having at least 85%, 90%, 95%, or 99% identity thereto); or SEQ ID NO: 6189 or 6190 (or a sequence having at least 85%, 90%, 95%, or 99% identity thereto).

[0014] In some embodiments, the multifunctional molecule binds monovalently to TRBC1 or TRBC2. In some embodiments, the multifunctional molecule comprises a configuration shown in any of FIGS. 29A - 29D, and optionally, (i) the multifunctional antibody molecule comprises an anti - TRBC1 Fab and an anti - NKp30 scFv, for example, comprises the configuration shown in FIG. 29A; (ii) the multifunctional antibody molecule comprises an anti - TRBC1 Fab and an anti - NKp30 Fab, for example, comprises the configuration shown in FIG. 29B; (iii) the multifunctional antibody molecule comprises an anti - NKp30 Fab and an anti - TRBC1 scFv, for example, comprises the configuration shown in FIG. 29C; or (iv) the multifunctional antibody molecule comprises an anti - TRBC1 scFv and an anti - NKp30 scFv, for example, comprises the configuration shown in FIG. 29D. In some embodiments, the multifunctional molecule comprises a configuration shown in any of FIGS. 30A - 30D, and optionally, (i) the multifunctional antibody molecule comprises an anti - TRBC2 Fab and an anti - NKp30 scFv, for example, comprises the configuration shown in FIG. 30A; (ii) the multifunctional antibody molecule comprises an anti - TRBC2 Fab and an anti - NKp30 Fab, for example, comprises the configuration shown in FIG. 30B; (iii) the multifunctional antibody molecule comprises an anti - NKp30 Fab and an anti - TRBC2 scFv, for example, comprises the configuration shown in FIG. 30C; or (iv) the multifunctional antibody molecule comprises an anti - TRBC2 scFv and an anti - NKp30 scFv, for example, comprises the configuration shown in FIG. 30D.

[0015] In some embodiments, the multifunctional molecules disclosed herein further comprise a dimerization module that includes one or more immunoglobulin heavy chain constant regions (e.g., Fc regions) that include one or more of paired holes and protrusions (“knob-in-hole”), electrostatic interactions, or strand exchange.

[0016] In some embodiments, the multifunctional molecule comprises an anti-TRBC1 amino acid sequence disclosed in any 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 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 thereto), an anti-TRBC1 VL of SEQ ID NO: 258 (or a sequence having at least 85%, 90%, 95%, or 99% identity thereto), an anti-NKp30 VH of SEQ ID NO: 7302 (or a sequence having at least 85%, 90%, 95%, or 99% identity thereto), and an anti-NKp30 VL of SEQ ID NO: 7309 (or a sequence having at least 85%, 90%, 95%, or 99% identity thereto); (ii) an anti-TRBC1 VH of SEQ ID NO: 7351 (or a sequence having at least 85%, 90%, 95%, or 99% identity thereto), an anti-TRBC1 VL of SEQ ID NO: 258 (or a sequence having at least 85%, 90%, 95%, or 99% identity thereto), and an anti-NKp30 scFv of SEQ ID NO: 7311 (or a sequence having at least 85%, 90%, 95%, or 99% identity thereto); or (iii) SEQ ID NOs: 7382, 7380, and 7383 (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: 253 (or a sequence having at least 85%, 90%, 95%, or 99% identity thereto), an anti-TRBC1 VL of SEQ ID NO: 258 (or a sequence having at least 85%, 90%, 95%, or 99% identity thereto), an anti-NKp30 VH of SEQ ID NO: 7302 (or a sequence having at least 85%, 90%, 95%, or 99% identity thereto), and an anti-NKp30 VL of SEQ ID NO: 7309 (or a sequence having at least 85%, 90%, 95%, or 99% identity thereto); (ii) an anti-TRBC1 VH of SEQ ID NO: 253 (or a sequence having at least 85%, 90%, 95%, or 99% identity thereto), an anti-TRBC1 VL of SEQ ID NO: 258 (or a sequence having at least 85%, 90%, 95%, or 99% identity thereto), and an anti-NKp30 scFv of SEQ ID NO: 7311 (or a sequence having at least 85%, 90%, 95%, or 99% identity thereto); or (iii) SEQ ID NOs: 7379, 7380, and 7383 (or sequences 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 thereto), an anti-TRBC1 VL of SEQ ID NO: 258 (or a sequence having at least 85%, 90%, 95%, or 99% identity thereto), an anti-NKp30 VH of SEQ ID NO: 7302 (or a sequence having at least 85%, 90%, 95%, or 99% identity thereto), and an anti-NKp30 VL of SEQ ID NO: 7305 (or a sequence having at least 85%, 90%, 95%, or 99% identity thereto); (ii) an anti-TRBC1 VH of SEQ ID NO: 7351 (or a sequence having at least 85%, 90%, 95%, or 99% identity thereto), an anti-TRBC1 VL of SEQ ID NO: 258 (or a sequence having at least 85%, 90%, 95%, or 99% identity thereto), and an anti-NKp30 scFv of SEQ ID NO: 7310 (or a sequence having at least 85%, 90%, 95%, or 99% identity thereto); or (iii) SEQ ID NOs: 7382, 7380, and 7384 (or sequences 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: 253 (or a sequence having at least 85%, 90%, 95%, or 99% identity thereto), an anti-TRBC1 VL of SEQ ID NO: 258 (or a sequence having at least 85%, 90%, 95%, or 99% identity thereto), an anti-NKp30 VH of SEQ ID NO: 7302 (or a sequence having at least 85%, 90%, 95%, or 99% identity thereto), and an anti-NKp30 VL of SEQ ID NO: 7305 (or a sequence having at least 85%, 90%, 95%, or 99% identity thereto); (ii) an anti-TRBC1 VH of SEQ ID NO: 253 (or a sequence having at least 85%, 90%, 95%, or 99% identity thereto), an anti-TRBC1 VL of SEQ ID NO: 258 (or a sequence having at least 85%, 90%, 95%, or 99% identity thereto), and an anti-NKp30 scFv of SEQ ID NO: 7310 (or a sequence having at least 85%, 90%, 95%, or 99% identity thereto); or (iii) SEQ ID NOs: 7379, 7380, and 7384 (or a sequence having at least 85%, 90%, 95%, or 99% identity thereto).

[0017] In some embodiments, the multifunctional molecule comprises a heavy chain constant region variant, e.g., an Fc region variant, comprising one or more mutations that result in a reduction or elimination of affinity for at least one Fc receptor. Optionally, the one or more mutations result in a reduction or elimination of antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell phagocytosis (ADCP), or complement-dependent cytotoxicity (CDC). In some embodiments, the Fc region variant comprises one or more mutations disclosed in Table 20. Optionally, the Fc region variant comprises the N297A mutation.

[0018] In one aspect, provided herein are antibody molecules that bind to TRBC1 and comprise one or more CDRs, framework regions, variable regions, or antigen-binding domains disclosed in any of Tables 2-6 and 19, or sequences having at least 85%, 90%, 95%, or 99% identity thereto.

[0019] In one aspect, provided herein are antibody molecules that bind to NKp30 and comprise one or more CDRs, framework regions, variable regions, or antigen-binding domains disclosed in any of Tables 7-10, 18, and 19, or sequences having at least 85%, 90%, 95%, or 99% identity thereto.

[0020] In some embodiments, the antibody molecule comprises a heavy chain constant region variant, e.g., an Fc region variant, that contains one or more mutations that result in reduced or eliminated affinity for at least one Fc receptor, and optionally, the one or more mutations result in reduced or eliminated antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell phagocytosis (ADCP), or complement-dependent cytotoxicity (CDC). In some embodiments, the Fc region variant contains one or more mutations disclosed in Table 20, and optionally, the Fc region variant contains the N297A mutation.

[0021] In one aspect, provided herein are nucleic acid molecules encoding the multifunctional molecules or antibody molecules disclosed herein. In one aspect, provided herein are vectors, e.g., expression vectors, comprising the nucleic acid molecules disclosed herein. In one aspect, provided herein are cells comprising the nucleic acid molecules or vectors disclosed herein. In one aspect, provided herein are pharmaceutical compositions comprising the multifunctional molecules or antibody molecules disclosed herein and a pharmaceutically acceptable carrier, excipient, or stabilizer.

[0022] In one aspect, provided herein is a method of making, e.g., producing, the multifunctional molecules disclosed herein or the antibody molecules disclosed herein, the method comprising culturing the cells disclosed herein under suitable conditions, e.g., conditions suitable for gene expression and / or homoor heterodimerization.

[0023] In one aspect, provided herein is a method of treating cancer, the method comprising administering to a subject in need thereof a multifunctional molecule disclosed herein or an antibody molecule disclosed herein, wherein the multifunctional molecule or antibody molecule is administered in an amount effective to treat cancer. In some embodiments, the method further comprises the step of identifying, evaluating, or selecting a subject in need of treatment, and the step of identifying, evaluating, or selecting comprises determining whether the subject has cancer cells expressing a T cell receptor comprising TRBC1 or TRBC2 (e.g., directly determining it or indirectly determining it, e.g., obtaining information regarding it). In some embodiments, in response to determining that the subject has cancer cells expressing a T cell receptor comprising TRBC1, the method further comprises, optionally, selecting the subject for treatment with a multifunctional molecule comprising an antigen-binding domain that binds to the T cell receptor comprising TRBC1, and administering a multifunctional molecule disclosed herein comprising an antigen-binding domain that binds to the T cell receptor comprising TRBC1. In some embodiments, in response to determining that the subject has cancer cells expressing a T cell receptor comprising TRBC2, the method further comprises, optionally, selecting the subject for treatment with a multifunctional molecule comprising an antigen-binding domain that binds to the T cell receptor comprising TRBC2, and administering a multifunctional molecule disclosed herein comprising an antigen-binding domain that binds to the T cell receptor comprising TRBC2.

[0024] In one aspect, provided herein is a method of treating cancer, such as lymphoma or leukemia, such as T cell lymphoma or leukemia, comprising administering to a subject a multifunctional molecule disclosed herein in response to determining that the subject has cancer cells expressing a T cell receptor comprising TRBC1, wherein a first antigen-binding domain of the multifunctional molecule binds to TRBC1, and the multifunctional molecule is administered in an amount effective to treat the cancer. In one aspect, provided herein is a method of treating cancer, such as lymphoma or leukemia, such as T cell lymphoma or leukemia, comprising administering to a subject a multifunctional molecule disclosed herein in response to determining that the subject has cancer cells expressing a T cell receptor comprising TRBC2, wherein a first antigen-binding domain of the multifunctional molecule binds to TRBC2, and the multifunctional molecule is administered in an amount effective to treat the cancer.

[0025] In one aspect, provided herein is a method of identifying a subject in need of treatment of cancer, such as lymphoma or leukemia, such as T cell lymphoma or leukemia, using a multifunctional molecule disclosed herein, the method comprising determining whether the subject has cancer cells expressing a T cell receptor comprising TRBC1 or TRBC2 (e.g., directly determining it or indirectly determining it, e.g., obtaining information regarding it), in response to determining that the subject has cancer cells expressing a T cell receptor comprising TRBC1, identifying the subject as a candidate for treatment using a multifunctional molecule comprising an antigen-binding domain that binds to TRBC1, and optionally not identifying the subject as a candidate for treatment using a multifunctional molecule comprising an antigen-binding domain that binds to TRBC2, or in response to determining that the subject has cancer cells expressing a T cell receptor comprising TRBC2, identifying the subject as a candidate for treatment using a multifunctional molecule comprising an antigen-binding domain that binds to TRBC2, and optionally not identifying the subject as a candidate for treatment using a multifunctional molecule comprising an antigen-binding domain that binds to TRBC1.

[0026] In some embodiments, the method responds to identifying a subject as a candidate for treatment using a multifunctional molecule comprising an antigen-binding domain that binds to TRBC1, and treating the subject with a multifunctional molecule comprising an antigen-binding domain that binds to TRBC1 (e.g., administering it to the subject), or responds to identifying a subject as a candidate for treatment using a multifunctional molecule comprising an antigen-binding domain that binds to TRBC2, and treating the subject with a multifunctional molecule comprising an antigen-binding domain that binds to TRBC2 (e.g., administering it to the subject). It further includes.

[0027] In some embodiments of the methods described above, the cancer is leukemia or lymphoma. In some embodiments, the cancer is selected from AIDS-related lymphoma, angioimmunoblastic T-cell lymphoma, adult T-cell leukemia / lymphoma, Burkitt lymphoma, central nervous system (CNS) lymphoma, diffuse large B-cell lymphoma (DLBCL), lymphoblastic lymphoma, mantle cell lymphoma (MCL), peripheral T-cell lymphoma (PTCL) (e.g., hepatosplenic γδ T-cell lymphoma (HSGDTCL), subcutaneous panniculitis-like T-cell lymphoma, or enteropathy-associated T-cell lymphoma), transformed follicular lymphoma and transformed mucosa-associated lymphoid tissue (MALT) lymphoma, cutaneous T-cell lymphoma (mycosis fungoides and Sézary syndrome), follicular lymphoma, lymphoplasmacytic lymphoma / Waldenström macroglobulinemia, marginal zone B-cell lymphoma, gastric mucosa-associated lymphoid tissue (MALT) lymphoma, chronic lymphocytic leukemia / small lymphocytic lymphoma (CLL / SLL), extranodal T / 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 lymphoma and is peripheral T-cell lymphoma (PTCL).

[0028] 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. Thus, in one aspect, the present disclosure (i) a first antigen-binding domain that selectively binds to T cell receptor beta chain constant domain 1 (TRBC1) or T cell receptor beta chain constant domain 2 (TRBC2); (ii) (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 cell engager; (b) a cytokine molecule or a cytokine inhibitor molecule; (c) a death receptor signal engager; and (d) a stromal modifying moiety characterized by a multifunctional molecule comprising one, two, or all of and.

[0029] In another aspect, the present disclosure (i) a first antigen-binding domain that selectively targets a T cell receptor comprising T cell receptor beta chain constant domain 1 (TRBC1), TRBC1, a T cell receptor comprising T cell receptor beta chain constant domain 2 (TRBC2), or a lymphocyte expressing (e.g., on its surface, e.g., presenting it) TRBC2; (ii) (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 cell engager; (b) a cytokine molecule or a cytokine inhibitor molecule; (c) a death receptor signal engager; and (c) a stromal modifying moiety characterized by a multifunctional molecule comprising one, two, or all of and.

[0030] In another aspect, the present disclosure (i) a tumor antigen on a lymphoma cell (e.g., a T cell), such as a T cell receptor containing the T cell receptor beta chain constant domain 1 (TRBC1), TRBC1, a T cell receptor containing the T cell receptor beta chain constant domain 2 (TRBC2), or a first antigen-binding domain that preferentially binds to TRBC2, and (ii) (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 cell engager; (b) a cytokine molecule or a cytokine inhibitor molecule; (c) a cell death receptor signal engager; and (d) a stromal modification moiety one, two, or all of which and characterized by a multifunctional molecule.

[0031] In another aspect, the present disclosure (i) a first antigen-binding domain that selectively binds to the T cell receptor beta chain constant domain 1 (TRBC1) or the T cell receptor beta chain constant domain 2 (TRBC2), and (ii) a complement activation domain that activates the complement pathway by binding to, for example, C1q characterized by an antibody molecule, such as an IgM antibody molecule.

[0032] In another aspect, the present disclosure A heavy chain variable region (VH) comprising the amino acid sequence of the heavy chain framework region 1 (VHFWR1) of SEQ ID NO: 215 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), the amino acid sequence of VHFWR2 of SEQ ID NO: 216 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), the amino acid sequence of VHFWR3 of SEQ ID NO: 217 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), and / or the amino acid sequence of VHFWR4 of SEQ ID NO: 218 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), and A light chain variable region (VL) comprising the amino acid sequence of the light chain framework region 1 (VLFWR1) of SEQ ID NO: 238 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), the amino acid sequence of VLFWR2 of SEQ ID NO: 239 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), the amino acid sequence of VLFWR3 of SEQ ID NO: 240 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), and / or the amino acid sequence of VLFWR4 of SEQ ID NO: 241 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions) characterized by an antibody molecule comprising the same.

[0033] 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 thereto), 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 thereto).

[0034] In another aspect, the present disclosure features a nucleic acid molecule encoding a multifunctional molecule disclosed herein. In another aspect, the present disclosure features a vector, e.g., an expression vector, comprising a nucleic acid molecule disclosed herein.

[0035] In another aspect, the present disclosure features a host cell comprising a nucleic acid molecule or vector disclosed herein. In another aspect, the present disclosure features a method of making, e.g., producing, a multifunctional molecule disclosed herein, the method comprising culturing a host cell disclosed herein under conditions appropriate for, e.g., gene expression and / or homoor heterodimerization.

[0036] In another aspect, the present disclosure features a pharmaceutical composition comprising a multifunctional molecule disclosed herein. In another aspect, the present disclosure is a method of treating cancer, comprising administering to a subject in need thereof a multifunctional molecule disclosed herein, wherein the multifunctional molecule is administered in an amount effective to treat cancer. In some embodiments, the cancer is a T cell malignancy, such as T cell lymphoma or T cell leukemia. In some embodiments, the cancer is anaplastic large cell lymphoma (ALCL); angioimmunoblastic T cell lymphoma; peripheral T cell lymphoma, not otherwise specified (NOS) (PTCL); cutaneous T cell lymphoma (CTCL); NKT cell lymphoma; Sézary 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, the expression of the TRBC subtype is analyzed, for example, by flow cytometry 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, such as 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, such as duvelisib. In some embodiments, the second agent is a farnesyltransferase inhibitor, such as tipifarnib. In some embodiments, the second agent is a SYK / JAK inhibitor, such as selumetinib. In some embodiments, the second agent is a chemotherapeutic agent.

[0037] In some embodiments, the second agent is. In another aspect, the present disclosure is a method of identifying a subject in need of treatment for cancer using a multifunctional molecule disclosed herein, comprising determining whether the subject has cancer cells expressing a T cell receptor comprising TRBC1 or TRBC2 (e.g., directly determining it or indirectly determining it, e.g., obtaining information regarding it). In response to determining that a subject has cancer cells expressing a T cell receptor that includes TRBC1, identifying the subject as a candidate for treatment using a multifunctional molecule that includes an antigen-binding domain that binds to TRBC1, and optionally not identifying the subject as a candidate for treatment using a multifunctional molecule that includes an antigen-binding domain that binds to TRBC2, In response to determining that a subject has cancer cells expressing a T cell receptor that includes TRBC2, identifying the subject as a candidate for treatment using a multifunctional molecule that includes an antigen-binding domain that binds to TRBC2, and optionally not identifying the subject as a candidate for treatment using a multifunctional molecule that includes an antigen-binding domain that binds to TRBC1, a method characterized thereby.

[0038] In another aspect, the disclosure is a method of evaluating a subject in need of treatment for cancer, such as lymphoma, the method comprising determining (e.g., directly determining it or indirectly determining it, e.g., obtaining information regarding it) whether the subject has cancer cells expressing a T cell receptor that includes TRBC1 or TRBC2.

[0039] Any additional features of the multifunctional molecule, nucleic acid, vector, host cell, or method described above include one or more of the following recited embodiments. One of ordinary skill in the art can recognize or confirm numerous equivalents to the specific embodiments of the invention described herein using only routine experimentation. Such equivalents are intended to be encompassed by the following recited embodiments.

[0040] Recited embodiments 1. (i) A first antigen-binding domain that preferentially binds to a tumor antigen on lymphoma cells (e.g., T cells), wherein the tumor antigen is T cell receptor beta chain constant domain 1 (TRBC1) or T cell receptor beta chain constant domain 2 (TRBC2), the first antigen-binding domain, (ii)(a) An immunocyte engager selected from an 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 cell engager; (b) A cytokine molecule or a cytokine inhibitor molecule; (c) A cell death receptor signal engager; and (d) A stromal modification moiety One, two, or all of A multifunctional molecule.

[0041] 1A. (i) A first antigen-binding domain that selectively binds to the T cell receptor beta chain constant domain 1 (TRBC1) or the T cell receptor beta chain constant domain 2 (TRBC2), and (ii)(a) An immunocyte 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 cell engager; (b) A cytokine molecule or a cytokine inhibitor molecule; (c) A cell death receptor signal engager; and (d) A stromal modification moiety One, two, or all of A multifunctional molecule.

[0042] 2. (i) A first antigen-binding domain that selectively targets lymphocytes expressing the T cell receptor beta chain constant domain 1 (TRBC1) or the T cell receptor beta chain constant domain 2 (TRBC2), and (ii)(a) An immunocyte engager selected from an 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 cell engager; (b) A cytokine molecule or a cytokine inhibitor molecule; (c) A cell death receptor signal engager; and (d) A stromal modification moiety One, two, or all of A multifunctional molecule comprising

[0043] 3. The multifunctional molecule specifically binds to (i) An epitope of TRBC1 or TRBC2, e.g., an epitope identical or similar to the epitope recognized by an anti-TRBC1 or anti-TRBC2 antibody molecule as described herein; (ii) Exhibits a binding affinity and / or specificity identical or similar to that of an anti-TRBC1 or anti-TRBC2 antibody molecule as described herein, or both; (iii) Inhibits the binding of an anti-TRBC1 or anti-TRBC2 antibody molecule as described herein, e.g., competitively inhibits; (iv) Binds to an epitope identical or overlapping with that of an anti-TRBC1 or anti-TRBC2 antibody molecule as described herein; or (v) A multifunctional molecule of any of the preceding embodiments that competes with the binding to an anti-TRBC1 or anti-TRBC2 antibody molecule and / or binds to the same epitope.

[0044] 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 or light chains, or antigen-binding domains selected from Tables 2-5, or a sequence substantially identical thereto.

[0045] 5. The multifunctional molecule of any of embodiments 1-4, wherein the antigen or tumor antigen is TRBC1. 6. The multifunctional molecule of any of embodiments 1-4, wherein the antigen or tumor antigen is TRBC2.

[0046] 7. The first antigen-binding domain is (i) A heavy chain variable region (VH) comprising the amino acid sequence of the heavy chain complementarity determining region 1 (VHCDR1) of SEQ ID NO: 200 (or a sequence having 1, 2, 3, or 4 or fewer mutations, such as substitutions, additions, or deletions), the amino acid sequence of VHCDR2 of SEQ ID NO: 201 (or a sequence having 1, 2, 3, or 4 or fewer mutations, such as substitutions, additions, or deletions), and / or the amino acid sequence of VHCDR3 of SEQ ID NO: 202 (or a sequence having 1, 2, 3, or 4 or fewer mutations, such as substitutions, additions, or deletions), and (ii) A light chain variable region (VL) comprising the amino acid sequence of the light chain complementarity determining region 1 (VLCDR1) of SEQ ID NO: 223 (or a sequence having 1, 2, 3, or 4 or fewer mutations, such as substitutions, additions, or deletions), the amino acid sequence of VLCDR2 of SEQ ID NO: 224 (or a sequence having 1, 2, 3, or 4 or fewer mutations, such as substitutions, additions, or deletions), and / or the amino acid sequence of VLCDR3 of SEQ ID NO: 225 (or a sequence having 1, 2, 3, or 4 or fewer mutations, such as substitutions, additions, or deletions) A multifunctional molecule according to any one of embodiments 1 to 5, comprising

[0047] 8. The first antigen-binding domain is (i) A heavy chain variable region (VH) comprising the amino acid sequence of the heavy chain complementarity determining region 1 (VHCDR1) of SEQ ID NO: 200, the amino acid sequence of VHCDR2 of SEQ ID NO: 201, and / or the amino acid sequence of VHCDR3 of SEQ ID NO: 202, and (ii) A light chain variable region (VL) comprising the amino acid sequence of the light chain complementarity determining region 1 (VLCDR1) of SEQ ID NO: 223, the amino acid sequence of VLCDR2 of SEQ ID NO: 224, and / or the amino acid sequence of VLCDR3 of SEQ ID NO: 225 A multifunctional molecule according to embodiment 7, comprising

[0048] 9. The first antigen-binding domain is (1) A heavy chain variable region (VH) comprising the amino acid sequence of the heavy chain framework region 1 (VHFWR1) of SEQ ID NO: 203 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), the amino acid sequence of VHFWR2 of SEQ ID NO: 204 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), the amino acid sequence of VHFWR3 of SEQ ID NO: 205 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), or the amino acid sequence of VHFWR4 of SEQ ID NO: 206 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), and / or (2) A light chain variable region (VL) comprising the amino acid sequence of the light chain framework region 1 (VLFWR1) of SEQ ID NO: 226 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), the amino acid sequence of VLFWR2 of SEQ ID NO: 227 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), the amino acid sequence of VLFWR3 of SEQ ID NO: 228 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), or the amino acid sequence of VLFWR4 of SEQ ID NO: 229 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions) A multifunctional molecule according to any one of Embodiments 1 to 5, 7, or 8.

[0049] 10. The first antigen-binding domain is (1) A heavy chain variable region (VH) comprising the amino acid sequence of the heavy chain framework region 1 (VHFWR1) of SEQ ID NO: 203, the amino acid sequence of VHFWR2 of SEQ ID NO: 204, the amino acid sequence of VHFWR3 of SEQ ID NO: 205, or the amino acid sequence of VHFWR4 of SEQ ID NO: 206, and / or (2) A light chain variable region (VL) comprising the amino acid sequence of light chain framework region 1 (VLFWR1) of SEQ ID NO: 226, the amino acid sequence of VLFWR2 of SEQ ID NO: 227, the amino acid sequence of VLFWR3 of SEQ ID NO: 228, or the amino acid sequence of VLFWR4 of SEQ ID NO: 229 The multifunctional molecule of embodiment 9 comprising

[0050] 11. The first antigen-binding domain is (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 with SEQ ID NO: 250), and / or (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 with SEQ ID NO: 255) The multifunctional molecule of any one of embodiments 1 to 5 or 7 to 10 comprising

[0051] 12. The first antigen-binding domain is 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), the multifunctional molecule of any one of embodiments 1 to 5 or 7 to 11

[0052] 13. The first antigen-binding domain is 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), the multifunctional molecule of any one of embodiments 1 to 5 or 7 to 12

[0053] 14. The first antigen-binding domain comprises a heavy-chain variable region (VH) comprising the amino acid sequence of the heavy-chain framework region 1 (VHFWR1) of SEQ ID NO: 207 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), the amino acid sequence of VHFWR2 of SEQ ID NO: 208 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), the amino acid sequence of VHFWR3 of SEQ ID NO: 209 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), or the amino acid sequence of VHFWR4 of SEQ ID NO: 210 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), a multifunctional molecule according to any one of embodiments 1 to 5, 7, or 8.

[0054] 15. The first antigen-binding domain comprises a heavy-chain variable region (VH) comprising the amino acid sequence of the heavy-chain framework region 1 (VHFWR1) of SEQ ID NO: 207, the amino acid sequence of VHFWR2 of SEQ ID NO: 208, the amino acid sequence of VHFWR3 of SEQ ID NO: 209, or the amino acid sequence of VHFWR4 of SEQ ID NO: 210, a multifunctional molecule according to embodiment 14.

[0055] 16. The first antigen-binding domain comprises a heavy-chain variable region (VH) comprising the amino acid sequence of the heavy-chain framework region 1 (VHFWR1) of SEQ ID NO: 211 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), the amino acid sequence of VHFWR2 of SEQ ID NO: 212 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), the amino acid sequence of VHFWR3 of SEQ ID NO: 213 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), or the amino acid sequence of VHFWR4 of SEQ ID NO: 214 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), a multifunctional molecule according to any one of embodiments 1 to 5, 7, or 8.

[0056] 17. The multifunctional molecule of embodiment 16, wherein the first antigen-binding domain comprises a heavy-chain variable region (VH) comprising the amino acid sequence of heavy-chain framework region 1 (VHFWR1) of SEQ ID NO: 211, the amino acid sequence of VHFWR2 of SEQ ID NO: 212, the amino acid sequence of VHFWR3 of SEQ ID NO: 213, or the amino acid sequence of VHFWR4 of SEQ ID NO: 214.

[0057] 18. The multifunctional molecule of any one of embodiments 1 to 5, 7, or 8, wherein the first antigen-binding domain comprises a heavy-chain variable region (VH) comprising the amino acid sequence of heavy-chain framework region 1 (VHFWR1) of SEQ ID NO: 215 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), the amino acid sequence of VHFWR2 of SEQ ID NO: 216 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), the amino acid sequence of VHFWR3 of SEQ ID NO: 217 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), or the amino acid sequence of VHFWR4 of SEQ ID NO: 218 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions).

[0058] 19. The multifunctional molecule of embodiment 18, wherein the first antigen-binding domain comprises a heavy-chain variable region (VH) comprising the amino acid sequence of heavy-chain framework region 1 (VHFWR1) of SEQ ID NO: 215, the amino acid sequence of VHFWR2 of SEQ ID NO: 216, the amino acid sequence of VHFWR3 of SEQ ID NO: 217, or the amino acid sequence of VHFWR4 of SEQ ID NO: 218.

[0059] 20. The first antigen-binding domain comprises a heavy chain variable region (VH) comprising the amino acid sequence of the heavy chain framework region 1 (VHFWR1) of SEQ ID NO: 219 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), the amino acid sequence of VHFWR2 of SEQ ID NO: 220 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), the amino acid sequence of VHFWR3 of SEQ ID NO: 221 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), or the amino acid sequence of VHFWR4 of SEQ ID NO: 222 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), and is a multifunctional molecule according to any one of embodiments 1 to 5, 7, or 8.

[0060] 21. The first antigen-binding domain comprises a heavy chain variable region (VH) comprising the amino acid sequence of the heavy chain framework region 1 (VHFWR1) of SEQ ID NO: 219, the amino acid sequence of VHFWR2 of SEQ ID NO: 220, the amino acid sequence of VHFWR3 of SEQ ID NO: 221, or the amino acid sequence of VHFWR4 of SEQ ID NO: 222, and is a multifunctional molecule according to embodiment 20.

[0061] 22. The first antigen-binding domain comprises a light chain variable region (VL) comprising the amino acid sequence of the light chain framework region 1 (VLFWR1) of SEQ ID NO: 230 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), the amino acid sequence of VLFWR2 of SEQ ID NO: 231 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), the amino acid sequence of VLFWR3 of SEQ ID NO: 232 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), or the amino acid sequence of VLFWR4 of SEQ ID NO: 233 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), and is a multifunctional molecule according to any one of embodiments 1 to 5, 7, 8, or 14 to 21.

[0062] 23. The multifunctional molecule of embodiment 22, wherein the first antigen-binding domain comprises a light chain variable region (VL) comprising the amino acid sequence of light chain framework region 1 (VLFWR1) of SEQ ID NO: 230, the amino acid sequence of VLFWR2 of SEQ ID NO: 231, the amino acid sequence of VLFWR3 of SEQ ID NO: 232, or the amino acid sequence of VLFWR4 of SEQ ID NO: 233.

[0063] 24. The multifunctional molecule of any one of embodiments 1 to 5, 7, 8, or 14 to 21, wherein the first antigen-binding domain comprises a light chain variable region (VL) comprising the amino acid sequence of light chain framework region 1 (VLFWR1) of SEQ ID NO: 234 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), the amino acid sequence of VLFWR2 of SEQ ID NO: 235 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), the amino acid sequence of VLFWR3 of SEQ ID NO: 236 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), or the amino acid sequence of VLFWR4 of SEQ ID NO: 237 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions).

[0064] 25. The multifunctional molecule of embodiment 24, wherein the first antigen-binding domain comprises a light chain variable region (VL) comprising the amino acid sequence of light chain framework region 1 (VLFWR1) of SEQ ID NO: 234, the amino acid sequence of VLFWR2 of SEQ ID NO: 235, the amino acid sequence of VLFWR3 of SEQ ID NO: 236, or the amino acid sequence of VLFWR4 of SEQ ID NO: 237.

[0065] 26. The first antigen-binding domain comprises a variable light chain (VL) region comprising the amino acid sequence of the variable light chain framework region 1 (VLFWR1) of SEQ ID NO: 238 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), the amino acid sequence of VLFWR2 of SEQ ID NO: 239 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), the amino acid sequence of VLFWR3 of SEQ ID NO: 240 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), or the amino acid sequence of VLFWR4 of SEQ ID NO: 241 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), a multifunctional molecule according to any of embodiments 1 to 5, 7, 8, or 14 to 21.

[0066] 27. The first antigen-binding domain comprises a variable light chain (VL) region comprising the amino acid sequence of the variable light chain framework region 1 (VLFWR1) of SEQ ID NO: 238, the amino acid sequence of VLFWR2 of SEQ ID NO: 239, the amino acid sequence of VLFWR3 of SEQ ID NO: 240, or the amino acid sequence of VLFWR4 of SEQ ID NO: 241, a multifunctional molecule according to embodiment 26.

[0067] 28. The first antigen-binding domain comprises a variable light chain (VL) region comprising the amino acid sequence of the variable light chain framework region 1 (VLFWR1) of SEQ ID NO: 242 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), the amino acid sequence of VLFWR2 of SEQ ID NO: 243 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), the amino acid sequence of VLFWR3 of SEQ ID NO: 244 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), or the amino acid sequence of VLFWR4 of SEQ ID NO: 245 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), a multifunctional molecule according to any of embodiments 1 to 5, 7, 8, or 14 to 21.

[0068] 29. The multifunctional molecule of embodiment 28, wherein the first antigen-binding domain comprises a variable light chain (VL) comprising the amino acid sequence of variable light chain framework region 1 (VLFWR1) of SEQ ID NO: 242, the amino acid sequence of VLFWR2 of SEQ ID NO: 243, the amino acid sequence of VLFWR3 of SEQ ID NO: 244, or the amino acid sequence of VLFWR4 of SEQ ID NO: 245.

[0069] 30. The multifunctional molecule according to any one of embodiments 1 to 5, 7, 8, or 14 to 21, wherein the first antigen-binding domain comprises a variable light chain (VL) comprising the amino acid sequence of variable light chain framework region 1 (VLFWR1) of SEQ ID NO: 246 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), the amino acid sequence of VLFWR2 of SEQ ID NO: 247 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), the amino acid sequence of VLFWR3 of SEQ ID NO: 248 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), or the amino acid sequence of VLFWR4 of SEQ ID NO: 249 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions).

[0070] 31. The multifunctional molecule of embodiment 30, wherein the first antigen-binding domain comprises a variable light chain (VL) comprising the amino acid sequence of variable light chain framework region 1 (VLFWR1) of SEQ ID NO: 246, the amino acid sequence of VLFWR2 of SEQ ID NO: 247, the amino acid sequence of VLFWR3 of SEQ ID NO: 248, or the amino acid sequence of VLFWR4 of SEQ ID NO: 249.

[0071] 32. The multifunctional molecule according to any one of embodiments 1 to 5, 7, or 8, wherein the first antigen-binding domain comprises a variable heavy chain (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).

[0072] 33. A multifunctional molecule according to any one of embodiments 1 to 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).

[0073] 34. A multifunctional molecule according to any one of embodiments 1 to 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).

[0074] 35. A multifunctional molecule according to any one of embodiments 1 to 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).

[0075] 36. A multifunctional molecule according to any one of embodiments 1 to 5, 7, 8, or 32 to 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).

[0076] 37. A multifunctional molecule according to any one of embodiments 1 to 5, 7, 8, or 32 to 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).

[0077] 38. A multifunctional molecule according to any one of embodiments 1 to 5, 7, 8, or 32 to 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).

[0078] 39. A multifunctional molecule according to any one of embodiments 1 to 5, 7, 8, or 32 to 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).

[0079] 40. A multifunctional molecule according to any one of embodiments 1 to 5, 7, 8, or 32 to 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).

[0080] 41. The first antigen-binding domain is (i) a heavy chain variable region (VH) comprising the amino acid sequence of heavy chain framework region 1 (VHFWR1) of SEQ ID NO: 215 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), the amino acid sequence of VHFWR2 of SEQ ID NO: 216 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), the amino acid sequence of VHFWR3 of SEQ ID NO: 217 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), or the amino acid sequence of VHFWR4 of SEQ ID NO: 218 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), and (ii) A variable light chain (VL) comprising the amino acid sequence of the light chain framework region 1 (VLFWR1) of SEQ ID NO: 238 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), the amino acid sequence of VLFWR2 of SEQ ID NO: 239 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), the amino acid sequence of VLFWR3 of SEQ ID NO: 240 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), or the amino acid sequence of VLFWR4 of SEQ ID NO: 241 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions). A multifunctional molecule according to any one of embodiments 1 to 5, 7, or 8, comprising .

[0081] 42. The first antigen-binding domain is (i) A variable heavy chain (VH) comprising the amino acid sequence of the heavy chain framework region 1 (VHFWR1) of SEQ ID NO: 215, the amino acid sequence of VHFWR2 of SEQ ID NO: 216, the amino acid sequence of VHFWR3 of SEQ ID NO: 217, or the amino acid sequence of VHFWR4 of SEQ ID NO: 218, and (ii) A variable light chain (VL) comprising the amino acid sequence of the light chain framework region 1 (VLFWR1) of SEQ ID NO: 238, the amino acid sequence of VLFWR2 of SEQ ID NO: 239, the amino acid sequence of VLFWR3 of SEQ ID NO: 240, or the amino acid sequence of VLFWR4 of SEQ ID NO: 241. A multifunctional molecule according to embodiment 41, comprising .

[0082] 43. The first antigen-binding domain is (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 (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). A multifunctional molecule according to any one of embodiments 41 or 42, comprising .

[0083] 44. The first antigen-binding domain has a higher affinity for a T cell receptor containing TRBC1 than for a T cell receptor not containing TRBC1. Optionally, the K for the binding between the first antigen-binding domain and TRBC1 D is 40%, 30%, 20%, 10%, 1%, 0.1%, or 0.01% or less of the K for the binding between the first antigen-binding domain and a T cell receptor not containing TRBC1. A multifunctional molecule according to any one of embodiments 1 to 43. D

[0084] 45. The first antigen-binding domain has a higher affinity for a T cell receptor containing TRBC2 than for a T cell receptor not containing TRBC2. Optionally, the K for the binding between the first antigen-binding domain and TRBC2 D is 40%, 30%, 20%, 10%, 1%, 0.1%, or 0.01% or less of the K for the binding between the first antigen-binding domain and a T cell receptor not containing TRBC2. A multifunctional molecule according to any one of embodiments 1 to 4 or 6. D

[0085] 46. The first antigen-binding domain has a higher affinity for a T cell receptor containing TRBC1 than for a T cell receptor containing TRBC2. Optionally, the K for the binding between the first antigen-binding domain and TRBC1 D is 40%, 30%, 20%, 10%, 1%, 0.1%, or 0.01% or less of the K for the binding between the first antigen-binding domain and a T cell receptor containing TRBC2. A multifunctional molecule according to any one of embodiments 1 to 44. D

[0086] 47. The first antigen-binding domain has a higher affinity for a T cell receptor containing TRBC2 than for a T cell receptor containing TRBC1. Optionally, the K for the binding between the first antigen-binding domain and TRBC2 D is 40%, 30%, 20%, 10%, 1%, 0.1%, or 0.01% or less of the K for the binding between the first antigen-binding domain and a T cell receptor containing TRBC1.​​​D A multifunctional molecule according to any one of Embodiments 1 to 4, 6, or 45, which is 40%, 30%, 20%, 10%, 1%, 0.1%, or 0.01% or less.

[0087] 48. A multifunctional molecule according to any preceding embodiment, wherein the binding of the first antigen-binding domain to TRBC1 or TRBC2 in a lymphoma cell or lymphocyte (e.g., a T cell), or to a tumor antigen in a lymphoma cell (e.g., a T cell), does not activate the lymphoma cell or lymphocyte, e.g., a T cell.

[0088] 49. A multifunctional molecule according to any preceding embodiment, wherein the binding of the first antigen-binding domain to TRBC1 or TRBC2 in a lymphoma cell or lymphocyte (e.g., a T cell), or to a tumor antigen in a lymphoma cell (e.g., a T cell), hardly activates the lymphoma cell or lymphocyte, e.g., a T cell, as measured by, for example, T cell proliferation, expression of T cell activation markers (e.g., CD69 or CD25), and / or expression of cytokines (e.g., TNFα and IFNγ).

[0089] 50. A multifunctional molecule according to any one of Embodiments 1 or 3 to 49, wherein the multifunctional molecule binds preferentially to lymphoma cells compared to non-lymphoma cells, and optionally, 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.

[0090] 51. (i) The binding between the multifunctional molecule and a lymphocyte expressing TRBC1 is greater than 10, 20, 30, 40, or 50 times greater than the binding between the multifunctional molecule and a lymphocyte not expressing TRBC1, or (ii) The binding between the multifunctional molecule and a lymphocyte expressing TRBC2 is greater than 10, 20, 30, 40, or 50 times greater than the binding between the multifunctional molecule and a lymphocyte not expressing TRBC2. A multifunctional molecule according to any one of Embodiments 2 to 47.

[0091] 52. The multifunctional molecule according to any one of embodiments 1 to 51, wherein the multifunctional molecule comprises an immune cell engager selected from an NK cell engager, a T cell engager, a B cell engager, a dendritic cell engager, or a macrophage cell engager.

[0092] 53. The multifunctional molecule according to embodiment 52, wherein the immune cell engager binds to and activates an immune cell, such as an effector cell. 54. The multifunctional molecule according to embodiment 52, wherein the immune cell engager binds to an immune cell, such as an effector cell, but does not activate it.

[0093] 55. The multifunctional molecule according to any one of embodiments 52 to 54, wherein the immune cell engager is a T cell engager, such as a T cell engager that mediates binding to and activation of T cells, or a T cell engager that mediates binding to T cells but does not mediate activation.

[0094] 56. The multifunctional molecule according to 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.

[0095] 57. The multifunctional molecule according to any one of embodiments 52 to 54, wherein the immune cell engager is an NK cell engager, such as an NK cell engager that mediates binding to and activation of NK cells, or an NK cell engager that mediates binding to NK cells but does not mediate activation.

[0096] 58. An NK cell engager that binds to (e.g., activates) NKp30, NKp40, NKp44, NKp46, NKG2D, DNAM1, DAP10, CD16 (e.g., CD16a, CD16b, or both), CRTAM, CD27, PSGL1, CD96, CD100 (SEMA4D), NKp80, CD244 (also known as SLAMF4 or 2B4), SLAMF6, SLAMF7, KIR2DS2, KIR2DS4, KIR3DS1, KIR2DS3, KIR2DS5, KIR2DS1, CD94, NKG2C, NKG2E, or CD160, an antibody molecule selected, for example, from an antigen-binding domain or a ligand, e.g., the NK cell engager is an antibody molecule or ligand that binds to (e.g., activates) NKp30, the multifunctional molecule of embodiment 57.

[0097] 59. An NK cell engager that is an antibody molecule, e.g., an antigen-binding domain, the multifunctional molecule of embodiment 57. 60. An NK cell engager that can engage with NK cells, the multifunctional molecule of any one of embodiments 58 or 59.

[0098] 61. An NK cell engager that is an antibody molecule that binds to NKp30, NKp46, NKG2D, or CD16, e.g., an antigen-binding domain, the multifunctional molecule of any one of embodiments 57 to 60.

[0099] 62. The multifunctional molecule is (i) specifically binds to an epitope of NKp30, NKp46, NKG2D, or CD16, e.g., an epitope identical or similar to the epitope recognized by an anti-NKp30, anti-NKp46, anti-NKG2D, or anti-CD16 antibody molecule as described herein; (ii) exhibits a binding affinity and / or specificity identical or similar to that of an anti-NKp30, anti-NKp46, anti-NKG2D, or anti-CD16 antibody molecule as described herein; (iii) As described herein, inhibit the binding of anti-NKp30, anti-NKp46, anti-NKG2D, or anti-CD16 antibody molecules, e.g., competitively inhibit; (iv) As described herein, bind to an epitope that is the same as or overlaps with the anti-NKp30, anti-NKp46, anti-NKG2D, or anti-CD16 antibody molecule; or (v) A multifunctional molecule of any of the preceding embodiments that competes for binding to and / or binds to the same epitope as an anti-NKp30, anti-NKp46, anti-NKG2D, or anti-CD16 molecule.

[0100] 63. A multifunctional molecule of any of embodiments 57 to 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 to 10 or 15, or a sequence substantially identical thereto.

[0101] 64. A multifunctional molecule of any of embodiments 57 to 63, wherein the NK cell engager is an antibody molecule that binds to NKp30, e.g., an antigen-binding domain. 65. A multifunctional molecule of any of embodiments 57 to 64, wherein the lysis of lymphoma cells or lymphocytes is mediated by NKp30.

[0102] 66. A multifunctional molecule of any of embodiments 57 to 65, wherein the multifunctional molecule does not activate NK cells when incubated with NK cells in the absence of tumor antigen in lymphoma cells or TRBC1 or TRBC2 on lymphocytes.

[0103] 67. A multifunctional molecule of any of embodiments 57 to 66, wherein the NK cells are NK cells expressing NKp30, and the multifunctional molecule activates the NK cells when either (1) the tumor antigen in the lymphoma cells is also present or (2) TRBC1 or TRBC2 on the lymphocytes is also present.

[0104] 68. In the case where the multifunctional molecule does not activate NK cells unless either (1) the tumor antigen in the lymphoma cells is also present or (2) TRBC1 or TRBC2 on the lymphocytes is also present, in NK cells that do not express NKp30, the multifunctional molecule according to any one of Embodiments 57 to 67.

[0105] 69. The NK cell engager is (i) a heavy chain variable region (VH) comprising the amino acid sequence of heavy chain complementarity determining region 1 (VHCDR1) of SEQ ID NO: 6000 (or a sequence having 1, 2, 3, or 4 or fewer mutations, such as substitutions, additions, or deletions), the amino acid sequence of VHCDR2 of SEQ ID NO: 6001 (or a sequence having 1, 2, 3, or 4 or fewer mutations, such as substitutions, additions, or deletions), and / or the amino acid sequence of VHCDR3 of SEQ ID NO: 6002 (or a sequence having 1, 2, 3, or 4 or fewer mutations, such as substitutions, additions, or deletions), and (ii) a light chain variable region (VL) comprising the amino acid sequence of light chain complementarity determining region 1 (VLCDR1) of SEQ ID NO: 6063 (or a sequence having 1, 2, 3, or 4 or fewer mutations, such as substitutions, additions, or deletions), the amino acid sequence of VLCDR2 of SEQ ID NO: 6064 (or a sequence having 1, 2, 3, or 4 or fewer mutations, such as substitutions, additions, or deletions), and / or the amino acid sequence of VLCDR3 of SEQ ID NO: 7293 (or a sequence having 1, 2, 3, or 4 or fewer mutations, such as substitutions, additions, or deletions) The multifunctional molecule according to any one of Embodiments 57 to 68.

[0106] 70. The NK cell engager is (i) a heavy chain variable region (VH) comprising the amino acid sequence of heavy chain complementarity determining region 1 (VHCDR1) of SEQ ID NO: 6000, the amino acid sequence of VHCDR2 of SEQ ID NO: 6001, and / or the amino acid sequence of VHCDR3 of SEQ ID NO: 6002, and (ii) A light chain variable region (VL) comprising the amino acid sequence of the light chain complementarity determining region 1 (VLCDR1) of SEQ ID NO: 6063, the amino acid sequence of VLCDR2 of SEQ ID NO: 6064, and / or the amino acid sequence of VLCDR3 of SEQ ID NO: 7293 The multifunctional molecule of embodiment 69, comprising

[0107] 71. The NK cell engager is (1) A heavy chain variable region (VH) comprising the amino acid sequence of the heavy chain framework region 1 (VHFWR1) of SEQ ID NO: 6003 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), the amino acid sequence of VHFWR2 of SEQ ID NO: 6004 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), the amino acid sequence of VHFWR3 of SEQ ID NO: 6005 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), or the amino acid sequence of VHFWR4 of SEQ ID NO: 6006 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), and / or (2) A light chain variable region (VL) comprising the amino acid sequence of the light chain framework region 1 (VLFWR1) of SEQ ID NO: 6066 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), the amino acid sequence of VLFWR2 of SEQ ID NO: 6067 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), the amino acid sequence of VLFWR3 of SEQ ID NO: 7292 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), or the amino acid sequence of VLFWR4 of SEQ ID NO: 6069 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions) The multifunctional molecule of any one of embodiments 57 to 70, comprising

[0108] 72. The NK cell engager is (1) A heavy chain variable region (VH) comprising the amino acid sequence of the heavy chain framework region 1 (VHFWR1) of SEQ ID NO: 6003, the amino acid sequence of VHFWR2 of SEQ ID NO: 6004, the amino acid sequence of VHFWR3 of SEQ ID NO: 6005, or the amino acid sequence of VHFWR4 of SEQ ID NO: 6006, and (3) A light chain variable region (VL) comprising the amino acid sequence of the light chain framework region 1 (VLFWR1) of SEQ ID NO: 6066, the amino acid sequence of VLFWR2 of SEQ ID NO: 6067, the amino acid sequence of VLFWR3 of SEQ ID NO: 7292, or the amino acid sequence of VLFWR4 of SEQ ID NO: 6069 The multifunctional molecule of embodiment 71, comprising

[0109] 73. The NK cell engager is (i) A VH comprising the amino acid sequence of SEQ ID NO: 6121 (or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with SEQ ID NO: 6121), and / or (ii) A VL comprising the amino acid sequence of SEQ ID NO: 7294 (or an amino acid sequence having at least about 93%, 95%, or 99% sequence identity with SEQ ID NO: 7294) The multifunctional molecule of any one of embodiments 57 to 72, comprising

[0110] 74. The multifunctional molecule of any one of embodiments 57 to 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).

[0111] 75. The multifunctional molecule of any one of embodiments 57 to 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).

[0112] 76. A multifunctional molecule according to any of embodiments 57 to 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 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).

[0113] 77. A multifunctional molecule according to any of embodiments 57 to 70, wherein the NK cell engager comprises a variable heavy chain (VH) comprising the amino acid sequence of SEQ ID NO: 6014 of the heavy chain framework region 1 (VHFWR1) (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations, such as substitutions, additions, or deletions therefrom), the amino acid sequence of SEQ ID NO: 6015 of VHFWR2 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations, such as substitutions, additions, or deletions therefrom), the amino acid sequence of SEQ ID NO: 6016 of VHFWR3 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations, such as substitutions, additions, or deletions therefrom), or the amino acid sequence of SEQ ID NO: 6017 of VHFWR4 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations, such as substitutions, additions, or deletions therefrom).

[0114] 78. A multifunctional molecule according to embodiment 77, wherein the NK cell engager comprises a variable heavy chain (VH) comprising the amino acid sequence of SEQ ID NO: 6014 of the heavy chain framework region 1 (VHFWR1), the amino acid sequence of SEQ ID NO: 6015 of VHFWR2, the amino acid sequence of SEQ ID NO: 6016 of VHFWR3, or the amino acid sequence of SEQ ID NO: 6017 of VHFWR4.

[0115] 79. A multifunctional molecule according to 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).

[0116] 80. An NK cell engager comprising a heavy chain variable region (VH) comprising the amino acid sequence of the heavy chain framework region 1 (VHFWR1) of SEQ ID NO: 6018 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations, such as substitutions, additions, or deletions therefrom), the amino acid sequence of VHFWR2 of SEQ ID NO: 6019 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations, such as substitutions, additions, or deletions therefrom), the amino acid sequence of VHFWR3 of SEQ ID NO: 6020 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations, such as substitutions, additions, or deletions therefrom), or the amino acid sequence of VHFWR4 of SEQ ID NO: 6021 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations, such as substitutions, additions, or deletions therefrom), a multifunctional molecule of any of embodiments 57 to 70.

[0117] 81. An NK cell engager comprising a heavy chain variable region (VH) comprising the amino acid sequence of the heavy chain framework region 1 (VHFWR1) of SEQ ID NO: 6018, the amino acid sequence of VHFWR2 of SEQ ID NO: 6019, the amino acid sequence of VHFWR3 of SEQ ID NO: 6020, or the amino acid sequence of VHFWR4 of SEQ ID NO: 6021, a multifunctional molecule of embodiment 80.

[0118] 82. An NK cell engager comprising a VH comprising the amino acid sequence of SEQ ID NO: 6124 (or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 6124), a multifunctional molecule of embodiment 81.

[0119] 83. An NK cell engager, which is a multifunctional molecule according to any one of embodiments 57 to 70, comprising a heavy chain variable region (VH) that includes the amino acid sequence of heavy chain framework region 1 (VHFWR1) of SEQ ID NO: 6022 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations, such as substitutions, additions, or deletions therefrom), the amino acid sequence of VHFWR2 of SEQ ID NO: 6023 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations, such as substitutions, additions, or deletions therefrom), the amino acid sequence of VHFWR3 of SEQ ID NO: 6024 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations, such as substitutions, additions, or deletions therefrom), or the amino acid sequence of VHFWR4 of SEQ ID NO: 6025 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations, such as substitutions, additions, or deletions therefrom).

[0120] 84. An NK cell engager, which is a multifunctional molecule according to embodiment 83, comprising a heavy chain variable region (VH) that includes the amino acid sequence of heavy chain framework region 1 (VHFWR1) of SEQ ID NO: 6022, the amino acid sequence of VHFWR2 of SEQ ID NO: 6023, the amino acid sequence of VHFWR3 of SEQ ID NO: 6024, or the amino acid sequence of VHFWR4 of SEQ ID NO: 6025.

[0121] 85. An NK cell engager, which is a multifunctional molecule according to embodiment 84, comprising a VH that includes the amino acid sequence of SEQ ID NO: 6125 (or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 6125).

[0122] 86. An NK cell engager, which is a multifunctional molecule according to any one of embodiments 57 to 70, comprising a heavy chain variable region (VH) that includes the amino acid sequence of the heavy chain framework region 1 (VHFWR1) of SEQ ID NO: 6026 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations, such as substitutions, additions, or deletions therefrom), the amino acid sequence of VHFWR2 of SEQ ID NO: 6027 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations, such as substitutions, additions, or deletions therefrom), the amino acid sequence of VHFWR3 of SEQ ID NO: 6028 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations, such as substitutions, additions, or deletions therefrom), or the amino acid sequence of VHFWR4 of SEQ ID NO: 6029 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations, such as substitutions, additions, or deletions therefrom).

[0123] 87. An NK cell engager, which is a multifunctional molecule according to embodiment 86, comprising a heavy chain variable region (VH) that includes the amino acid sequence of the heavy chain framework region 1 (VHFWR1) of SEQ ID NO: 6026, the amino acid sequence of VHFWR2 of SEQ ID NO: 6027, the amino acid sequence of VHFWR3 of SEQ ID NO: 6028, or the amino acid sequence of VHFWR4 of SEQ ID NO: 6029.

[0124] 88. An NK cell engager, which is a multifunctional molecule according to embodiment 87, comprising a VH that includes the amino acid sequence of SEQ ID NO: 6126 (or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 6126).

[0125] 89. An NK cell engager, which is a multifunctional molecule of any one of embodiments 57 to 70, comprising a heavy chain variable region (VH) that includes the amino acid sequence of the heavy chain framework region 1 (VHFWR1) of SEQ ID NO: 6030 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), the amino acid sequence of VHFWR2 of SEQ ID NO: 6032 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), the amino acid sequence of VHFWR3 of SEQ ID NO: 6033 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), or the amino acid sequence of VHFWR4 of SEQ ID NO: 6034 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions).

[0126] 90. An NK cell engager, which is a multifunctional molecule of embodiment 89, comprising a heavy chain variable region (VH) that includes the amino acid sequence of the heavy chain framework region 1 (VHFWR1) of SEQ ID NO: 6030, the amino acid sequence of VHFWR2 of SEQ ID NO: 6032, the amino acid sequence of VHFWR3 of SEQ ID NO: 6033, or the amino acid sequence of VHFWR4 of SEQ ID NO: 6034.

[0127] 91. An NK cell engager, which is a multifunctional molecule of embodiment 90, comprising a VH that includes the amino acid sequence of SEQ ID NO: 6127 (or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 6127).

[0128] 92. An NK cell engager, which is a multifunctional molecule according to any one of embodiments 57 to 70, comprising a heavy chain variable region (VH) that includes the amino acid sequence of the heavy chain framework region 1 (VHFWR1) of SEQ ID NO: 6035 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations, such as substitutions, additions, or deletions therefrom), the amino acid sequence of VHFWR2 of SEQ ID NO: 6036 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations, such as substitutions, additions, or deletions therefrom), the amino acid sequence of VHFWR3 of SEQ ID NO: 6037 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations, such as substitutions, additions, or deletions therefrom), or the amino acid sequence of VHFWR4 of SEQ ID NO: 6038 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations, such as substitutions, additions, or deletions therefrom).

[0129] 93. An NK cell engager, which is a multifunctional molecule according to embodiment 92, comprising a heavy chain variable region (VH) that includes the amino acid sequence of the heavy chain framework region 1 (VHFWR1) of SEQ ID NO: 6035, the amino acid sequence of VHFWR2 of SEQ ID NO: 6036, the amino acid sequence of VHFWR3 of SEQ ID NO: 6037, or the amino acid sequence of VHFWR4 of SEQ ID NO: 6038.

[0130] 94. An NK cell engager, which is a multifunctional molecule according to embodiment 93, comprising a VH that includes the amino acid sequence of SEQ ID NO: 6128 (or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 6128).

[0131] 95. An NK cell engager, which is a multifunctional molecule of any one of embodiments 57 to 70 or 77 to 94, comprising a variable light chain region (VL) that includes the amino acid sequence of the variable light chain framework region 1 (VLFWR1) of SEQ ID NO: 6077 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), the amino acid sequence of VLFWR2 of SEQ ID NO: 6078 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), the amino acid sequence of VLFWR3 of SEQ ID NO: 6079 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), or the amino acid sequence of VLFWR4 of SEQ ID NO: 6080 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions).

[0132] 96. An NK cell engager, which is a multifunctional molecule of embodiment 95, comprising a variable light chain region (VL) that includes the amino acid sequence of the variable light chain framework region 1 (VLFWR1) of SEQ ID NO: 6077, the amino acid sequence of VLFWR2 of SEQ ID NO: 6078, the amino acid sequence of VLFWR3 of SEQ ID NO: 6079, or the amino acid sequence of VLFWR4 of SEQ ID NO: 6080.

[0133] 97. An NK cell engager, which is a multifunctional molecule of embodiment 96, comprising a VL that includes 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).

[0134] 98. An NK cell engager, which is a multifunctional molecule of any one of embodiments 57 to 70 or 77 to 94, comprising a variable light chain region (VL) that includes the amino acid sequence of the variable light chain framework region 1 (VLFWR1) of SEQ ID NO: 6081 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), the amino acid sequence of VLFWR2 of SEQ ID NO: 6082 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), the amino acid sequence of VLFWR3 of SEQ ID NO: 6083 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), or the amino acid sequence of VLFWR4 of SEQ ID NO: 6084 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions).

[0135] 99. The multifunctional molecule of embodiment 98, wherein the NK cell engager comprises a variable light chain region (VL) that includes the amino acid sequence of the variable light chain framework region 1 (VLFWR1) of SEQ ID NO: 6081, the amino acid sequence of VLFWR2 of SEQ ID NO: 6082, the amino acid sequence of VLFWR3 of SEQ ID NO: 6083, or the amino acid sequence of VLFWR4 of SEQ ID NO: 6084.

[0136] 100. The multifunctional molecule of embodiment 99, wherein the NK cell engager comprises a VL that includes the amino acid sequence of SEQ ID NO: 6138 (or an amino acid sequence having at least about 93%, 95%, or 99% sequence identity to SEQ ID NO: 6138).

[0137] 101. An NK cell engager, which is a multifunctional molecule of any one of embodiments 57 to 70 or 77 to 94, comprising a variable light chain region (VL) that includes the amino acid sequence of the variable light chain framework region 1 (VLFWR1) of SEQ ID NO: 6085 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), the amino acid sequence of VLFWR2 of SEQ ID NO: 6086 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), the amino acid sequence of VLFWR3 of SEQ ID NO: 6087 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), or the amino acid sequence of VLFWR4 of SEQ ID NO: 6088 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions).

[0138] 102. The multifunctional molecule of embodiment 101, wherein the NK cell engager comprises a variable light chain region (VL) that includes the amino acid sequence of the variable light chain framework region 1 (VLFWR1) of SEQ ID NO: 6085, the amino acid sequence of VLFWR2 of SEQ ID NO: 6086, the amino acid sequence of VLFWR3 of SEQ ID NO: 6087, or the amino acid sequence of VLFWR4 of SEQ ID NO: 6088.

[0139] 103. The multifunctional molecule of embodiment 102, wherein the NK cell engager comprises a VL that includes 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).

[0140] 104. An NK cell engager, which is a multifunctional molecule of any one of embodiments 57 to 70 or 77 to 94, comprising a variable light chain region (VL) that includes the amino acid sequence of the variable light chain framework region 1 (VLFWR1) of SEQ ID NO: 6089 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations from it, such as substitutions, additions, or deletions), the amino acid sequence of VLFWR2 of SEQ ID NO: 6090 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations from it, such as substitutions, additions, or deletions), the amino acid sequence of VLFWR3 of SEQ ID NO: 6091 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations from it, such as substitutions, additions, or deletions), or the amino acid sequence of VLFWR4 of SEQ ID NO: 6092 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations from it, such as substitutions, additions, or deletions).

[0141] 105. The multifunctional molecule of embodiment 104, wherein the NK cell engager comprises a variable light chain region (VL) that includes the amino acid sequence of the variable light chain framework region 1 (VLFWR1) of SEQ ID NO: 6089, the amino acid sequence of VLFWR2 of SEQ ID NO: 6090, the amino acid sequence of VLFWR3 of SEQ ID NO: 6091, or the amino acid sequence of VLFWR4 of SEQ ID NO: 6092.

[0142] 106. The multifunctional molecule of embodiment 105, wherein the NK cell engager comprises a VL that includes 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).

[0143] 107. A multifunctional molecule of any of embodiments 57 to 70 or 77 to 94, wherein the NK cell engager comprises a variable light chain region (VL) comprising the amino acid sequence of the variable light chain framework region 1 (VLFWR1) of SEQ ID NO: 6093 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), the amino acid sequence of VLFWR2 of SEQ ID NO: 6094 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), the amino acid sequence of VLFWR3 of SEQ ID NO: 6095 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), or the amino acid sequence of VLFWR4 of SEQ ID NO: 6096 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions).

[0144] 108. The multifunctional molecule of embodiment 107, wherein the NK cell engager comprises a variable light chain region (VL) comprising the amino acid sequence of the variable light chain framework region 1 (VLFWR1) of SEQ ID NO: 6093, the amino acid sequence of VLFWR2 of SEQ ID NO: 6094, the amino acid sequence of VLFWR3 of SEQ ID NO: 6095, or the amino acid sequence of VLFWR4 of SEQ ID NO: 6096.

[0145] 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).

[0146] 110. The NK cell engager is (i) A heavy chain variable region (VH) comprising the amino acid sequence of the heavy chain complementarity determining region 1 (VHCDR1) of SEQ ID NO: 6007 (or a sequence having 1, 2, 3, or 4 or fewer mutations, such as substitutions, additions, or deletions), the amino acid sequence of VHCDR2 of SEQ ID NO: 6008 (or a sequence having 1, 2, 3, or 4 or fewer mutations, such as substitutions, additions, or deletions), and / or the amino acid sequence of VHCDR3 of SEQ ID NO: 6009 (or a sequence having 1, 2, 3, or 4 or fewer mutations, such as substitutions, additions, or deletions), and (ii) A light chain variable region (VL) comprising the amino acid sequence of the light chain complementarity determining region 1 (VLCDR1) of SEQ ID NO: 6070 (or a sequence having 1, 2, 3, or 4 or fewer mutations, such as substitutions, additions, or deletions), the amino acid sequence of VLCDR2 of SEQ ID NO: 6071 (or a sequence having 1, 2, 3, or 4 or fewer mutations, such as substitutions, additions, or deletions), and / or the amino acid sequence of VLCDR3 of SEQ ID NO: 6072 (or a sequence having 1, 2, 3, or 4 or fewer mutations, such as substitutions, additions, or deletions) A multifunctional molecule according to any one of embodiments 57 to 68, comprising

[0147] 111. The NK cell engager is (i) A heavy chain variable region (VH) comprising the amino acid sequence of the heavy chain complementarity determining region 1 (VHCDR1) of SEQ ID NO: 6007, the amino acid sequence of VHCDR2 of SEQ ID NO: 6008, and / or the amino acid sequence of VHCDR3 of SEQ ID NO: 6009, and (ii) A light chain variable region (VL) comprising the amino acid sequence of the light chain complementarity determining region 1 (VLCDR1) of SEQ ID NO: 6070, the amino acid sequence of VLCDR2 of SEQ ID NO: 6071, and / or the amino acid sequence of VLCDR3 of SEQ ID NO: 6072 A multifunctional molecule according to embodiment 110, comprising

[0148] 112. The NK cell engager is (1) A heavy chain variable region (VH) comprising the amino acid sequence of the heavy chain framework region 1 (VHFWR1) of SEQ ID NO: 6010 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), the amino acid sequence of VHFWR2 of SEQ ID NO: 6011 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), the amino acid sequence of VHFWR3 of SEQ ID NO: 6012 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), or the amino acid sequence of VHFWR4 of SEQ ID NO: 6013 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), and / or (2) A light chain variable region (VL) comprising the amino acid sequence of the light chain framework region 1 (VLFWR1) of SEQ ID NO: 6073 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), the amino acid sequence of VLFWR2 of SEQ ID NO: 6074 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), the amino acid sequence of VLFWR3 of SEQ ID NO: 6075 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), or the amino acid sequence of VLFWR4 of SEQ ID NO: 6076 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions) A multifunctional molecule according to any of embodiments 57 to 68, 110, or 111, comprising

[0149] 113. The NK cell engager is (1) A heavy chain variable region (VH) comprising the amino acid sequence of the heavy chain framework region 1 (VHFWR1) of SEQ ID NO: 6010, the amino acid sequence of VHFWR2 of SEQ ID NO: 6011, the amino acid sequence of VHFWR3 of SEQ ID NO: 6012, or the amino acid sequence of VHFWR4 of SEQ ID NO: 6013, and (3) A light chain variable region (VL) comprising the amino acid sequence of light chain framework region 1 (VLFWR1) of SEQ ID NO: 6073, the amino acid sequence of VLFWR2 of SEQ ID NO: 6074, the amino acid sequence of VLFWR3 of SEQ ID NO: 6075, or the amino acid sequence of VLFWR4 of SEQ ID NO: 6076 The multifunctional molecule of Embodiment 112, comprising

[0150] 114. The NK cell engager is (i) A VH comprising the amino acid sequence of SEQ ID NO: 6122 (or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with SEQ ID NO: 6122), and / or (ii) A VL comprising the amino acid sequence of SEQ ID NO: 6136 (or an amino acid sequence having at least about 93%, 95%, or 99% sequence identity with SEQ ID NO: 6136) The multifunctional molecule of any one of Embodiments 57 to 68 or 110 to 113, comprising

[0151] 115. The NK cell engager is the multifunctional molecule of any one of Embodiments 57 to 68 or 110 to 114, comprising 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).

[0152] 116. The NK cell engager is the multifunctional molecule of any one of Embodiments 57 to 68 or 110 to 115, comprising 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).

[0153] 117. An NK cell engager comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 6151 or 6152 (or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 6151 or 6152), and a light chain comprising the amino acid sequence of SEQ ID NO: 6153 (or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 6153), which is a multifunctional molecule of any one of embodiments 57 to 68 or 110 to 116.

[0154] 118. An NK cell engager comprising a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 6039 for heavy chain framework region 1 (VHFWR1) (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), the amino acid sequence of SEQ ID NO: 6040 for VHFWR2 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), the amino acid sequence of SEQ ID NO: 6041 for VHFWR3 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), or the amino acid sequence of SEQ ID NO: 6042 for VHFWR4 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), which is a multifunctional molecule of any one of embodiments 57 to 68, 110, or 111.

[0155] 119. An NK cell engager comprising a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 6039 for heavy chain framework region 1 (VHFWR1), the amino acid sequence of SEQ ID NO: 6040 for VHFWR2, the amino acid sequence of SEQ ID NO: 6041 for VHFWR3, or the amino acid sequence of SEQ ID NO: 6042 for VHFWR4, which is a multifunctional molecule of embodiment 118.

[0156] 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).

[0157] 121. The multifunctional molecule of any one of embodiments 57 to 68, 110, or 111, wherein the NK cell engager comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 6043 of the heavy chain framework region 1 (VHFWR1) (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), the amino acid sequence of SEQ ID NO: 6044 of VHFWR2 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), the amino acid sequence of SEQ ID NO: 6045 of VHFWR3 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), or the amino acid sequence of SEQ ID NO: 6046 of VHFWR4 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions).

[0158] 122. The multifunctional molecule of embodiment 121, wherein the NK cell engager comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 6043 of the heavy chain framework region 1 (VHFWR1), the amino acid sequence of SEQ ID NO: 6044 of VHFWR2, the amino acid sequence of SEQ ID NO: 6045 of VHFWR3, or the amino acid sequence of SEQ ID NO: 6046 of VHFWR4.

[0159] 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).

[0160] 124. An NK cell engager comprising a heavy chain variable region (VH) comprising the amino acid sequence of the heavy chain framework region 1 (VHFWR1) of SEQ ID NO: 6047 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), the amino acid sequence of VHFWR2 of SEQ ID NO: 6048 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), the amino acid sequence of VHFWR3 of SEQ ID NO: 6049 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), or the amino acid sequence of VHFWR4 of SEQ ID NO: 6050 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), and a multifunctional molecule of any of embodiments 57 to 68, 110, or 111.

[0161] 125. The multifunctional molecule of embodiment 124, wherein the NK cell engager comprises a heavy chain variable region (VH) comprising the amino acid sequence of the heavy chain framework region 1 (VHFWR1) of SEQ ID NO: 6047, the amino acid sequence of VHFWR2 of SEQ ID NO: 6048, the amino acid sequence of VHFWR3 of SEQ ID NO: 6049, or the amino acid sequence of VHFWR4 of SEQ ID NO: 6050.

[0162] 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 to SEQ ID NO: 6131).

[0163] 127. An NK cell engager comprising a heavy chain variable region (VH) comprising the amino acid sequence of the heavy chain framework region 1 (VHFWR1) of SEQ ID NO: 6051 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), the amino acid sequence of VHFWR2 of SEQ ID NO: 6052 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), the amino acid sequence of VHFWR3 of SEQ ID NO: 6053 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), or the amino acid sequence of VHFWR4 of SEQ ID NO: 6054 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), a multifunctional molecule of any of embodiments 57 to 68, 110, or 111.

[0164] 128. An NK cell engager comprising a heavy chain variable region (VH) comprising the amino acid sequence of the heavy chain framework region 1 (VHFWR1) of SEQ ID NO: 6051, the amino acid sequence of VHFWR2 of SEQ ID NO: 6052, the amino acid sequence of VHFWR3 of SEQ ID NO: 6053, or the amino acid sequence of VHFWR4 of SEQ ID NO: 6054, a multifunctional molecule of embodiment 127.

[0165] 129. An NK cell engager comprising a VH comprising the amino acid sequence of SEQ ID NO: 6132 (or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 6132), a multifunctional molecule of embodiment 128.

[0166] 130. An NK cell engager comprising a heavy chain variable region (VH) comprising the amino acid sequence of the heavy chain framework region 1 (VHFWR1) of SEQ ID NO: 6055 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), the amino acid sequence of VHFWR2 of SEQ ID NO: 6056 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), the amino acid sequence of VHFWR3 of SEQ ID NO: 6057 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), or the amino acid sequence of VHFWR4 of SEQ ID NO: 6058 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), a multifunctional molecule of any of embodiments 57 to 68, 110, or 111.

[0167] 131. A multifunctional molecule of embodiment 130, wherein the NK cell engager comprises a heavy chain variable region (VH) comprising the amino acid sequence of the heavy chain framework region 1 (VHFWR1) of SEQ ID NO: 6055, the amino acid sequence of VHFWR2 of SEQ ID NO: 6056, the amino acid sequence of VHFWR3 of SEQ ID NO: 6057, or the amino acid sequence of VHFWR4 of SEQ ID NO: 6058.

[0168] 132. A 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 to SEQ ID NO: 6133).

[0169] 133. An NK cell engager comprising a heavy chain variable region (VH) comprising the amino acid sequence of heavy chain framework region 1 (VHFWR1) of SEQ ID NO: 6059 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), the amino acid sequence of VHFWR2 of SEQ ID NO: 6060 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), the amino acid sequence of VHFWR3 of SEQ ID NO: 6061 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), or the amino acid sequence of VHFWR4 of SEQ ID NO: 6062 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), and being a multifunctional molecule of any of embodiments 57 to 68, 110, or 111.

[0170] 134. An NK cell engager comprising a heavy chain variable region (VH) comprising the amino acid sequence of heavy chain framework region 1 (VHFWR1) of SEQ ID NO: 6059, the amino acid sequence of VHFWR2 of SEQ ID NO: 6060, the amino acid sequence of VHFWR3 of SEQ ID NO: 6061, or the amino acid sequence of VHFWR4 of SEQ ID NO: 6062, and being a multifunctional molecule of embodiment 133.

[0171] 135. An NK cell engager comprising a VH comprising the amino acid sequence of SEQ ID NO: 6134 (or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 6134), and being a multifunctional molecule of embodiment 134.

[0172] 136. An NK cell engager, which is a multifunctional molecule of any one of embodiments 57 to 68, 110, 111, or 118 to 135, comprising a variable light chain (VL) region containing the amino acid sequence of the variable light chain framework region 1 (VLFWR1) of SEQ ID NO: 6097 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations from it, such as substitutions, additions, or deletions), the amino acid sequence of VLFWR2 of SEQ ID NO: 6098 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations from it, such as substitutions, additions, or deletions), the amino acid sequence of VLFWR3 of SEQ ID NO: 6099 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations from it, such as substitutions, additions, or deletions), or the amino acid sequence of VLFWR4 of SEQ ID NO: 6100 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations from it, such as substitutions, additions, or deletions).

[0173] 137. An NK cell engager, which is a multifunctional molecule of embodiment 136, comprising a variable light chain (VL) region containing the amino acid sequence of the variable light chain framework region 1 (VLFWR1) of SEQ ID NO: 6097, the amino acid sequence of VLFWR2 of SEQ ID NO: 6098, the amino acid sequence of VLFWR3 of SEQ ID NO: 6099, or the amino acid sequence of VLFWR4 of SEQ ID NO: 6100.

[0174] 138. An NK cell engager, which is a multifunctional molecule of embodiment 137, comprising a VL region containing 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).

[0175] 139. An NK cell engager comprising a variable light chain (VL) region comprising the amino acid sequence of the variable light chain framework region 1 (VLFWR1) of SEQ ID NO: 6101 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), the amino acid sequence of VLFWR2 of SEQ ID NO: 6102 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), the amino acid sequence of VLFWR3 of SEQ ID NO: 6103 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), or the amino acid sequence of VLFWR4 of SEQ ID NO: 6104 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), a multifunctional molecule of any of embodiments 57 to 68, 110, 111, or 118 to 135.

[0176] 140. An NK cell engager comprising a variable light chain (VL) region comprising the amino acid sequence of the variable light chain framework region 1 (VLFWR1) of SEQ ID NO: 6101, the amino acid sequence of VLFWR2 of SEQ ID NO: 6102, the amino acid sequence of VLFWR3 of SEQ ID NO: 6103, or the amino acid sequence of VLFWR4 of SEQ ID NO: 6104, a multifunctional molecule of embodiment 139.

[0177] 141. An NK cell engager comprising a VL comprising the amino acid sequence of SEQ ID NO: 6143 (or an amino acid sequence having at least about 93%, 95%, or 99% sequence identity to SEQ ID NO: 6143), a multifunctional molecule of embodiment 140.

[0178] 142. An NK cell engager comprising a variable light chain (VL) region comprising the amino acid sequence of the variable light chain framework region 1 (VLFWR1) of SEQ ID NO: 6105 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), the amino acid sequence of VLFWR2 of SEQ ID NO: 6106 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), the amino acid sequence of VLFWR3 of SEQ ID NO: 6107 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), or the amino acid sequence of VLFWR4 of SEQ ID NO: 6108 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), a multifunctional molecule of any of embodiments 57 to 68, 110, 111, or 118 to 135.

[0179] 143. The multifunctional molecule of embodiment 142, wherein the NK cell engager comprises a variable light chain (VL) region comprising the amino acid sequence of the variable light chain framework region 1 (VLFWR1) of SEQ ID NO: 6105, the amino acid sequence of VLFWR2 of SEQ ID NO: 6106, the amino acid sequence of VLFWR3 of SEQ ID NO: 6107, or the amino acid sequence of VLFWR4 of SEQ ID NO: 6108.

[0180] 144. The multifunctional molecule of embodiment 143, wherein the NK cell engager comprises a VL region comprising the amino acid sequence of SEQ ID NO: 6144 (or an amino acid sequence having at least about 93%, 95%, or 99% sequence identity to SEQ ID NO: 6144).

[0181] 145. An NK cell engager comprising a variable light chain region (VL) comprising the amino acid sequence of the variable light chain framework region 1 (VLFWR1) of SEQ ID NO: 6109 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations from it, such as substitutions, additions, or deletions), the amino acid sequence of VLFWR2 of SEQ ID NO: 6110 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations from it, such as substitutions, additions, or deletions), the amino acid sequence of VLFWR3 of SEQ ID NO: 6111 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations from it, such as substitutions, additions, or deletions), or the amino acid sequence of VLFWR4 of SEQ ID NO: 6112 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations from it, such as substitutions, additions, or deletions), a multifunctional molecule of any of embodiments 57 to 68, 110, 111, or 118 to 135.

[0182] 146. An NK cell engager comprising a variable light chain region (VL) comprising the amino acid sequence of the variable light chain framework region 1 (VLFWR1) of SEQ ID NO: 6109, the amino acid sequence of VLFWR2 of SEQ ID NO: 6110, the amino acid sequence of VLFWR3 of SEQ ID NO: 6111, or the amino acid sequence of VLFWR4 of SEQ ID NO: 6112, a multifunctional molecule of embodiment 145.

[0183] 147. An NK cell engager comprising a VL comprising the amino acid sequence of SEQ ID NO: 6145 (or an amino acid sequence having at least about 93%, 95%, or 99% sequence identity to SEQ ID NO: 6145), a multifunctional molecule of embodiment 146.

[0184] 148. An NK cell engager, comprising a variable light chain (VL) region comprising the amino acid sequence of the variable light chain framework region 1 (VLFWR1) of SEQ ID NO: 6113 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), the amino acid sequence of VLFWR2 of SEQ ID NO: 6114 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), the amino acid sequence of VLFWR3 of SEQ ID NO: 6115 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), or the amino acid sequence of VLFWR4 of SEQ ID NO: 6116 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), a multifunctional molecule of any of embodiments 57 to 68, 110, 111, or 118 to 135.

[0185] 149. An NK cell engager, comprising a variable light chain (VL) region comprising the amino acid sequence of the variable light chain framework region 1 (VLFWR1) of SEQ ID NO: 6113, the amino acid sequence of VLFWR2 of SEQ ID NO: 6114, the amino acid sequence of VLFWR3 of SEQ ID NO: 6115, or the amino acid sequence of VLFWR4 of SEQ ID NO: 6116, a multifunctional molecule of embodiment 148.

[0186] 150. An NK cell engager, comprising a VL region 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), a multifunctional molecule of embodiment 149.

[0187] 151. An NK cell engager comprising a variable light chain region (VL) comprising the amino acid sequence of the variable light chain framework region 1 (VLFWR1) of SEQ ID NO: 6117 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), the amino acid sequence of VLFWR2 of SEQ ID NO: 6118 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), the amino acid sequence of VLFWR3 of SEQ ID NO: 6119 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), or the amino acid sequence of VLFWR4 of SEQ ID NO: 6120 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), a multifunctional molecule of any of embodiments 57 to 68, 110, 111, or 118 to 135.

[0188] 152. An NK cell engager comprising a variable light chain region (VL) comprising the amino acid sequence of the variable light chain framework region 1 (VLFWR1) of SEQ ID NO: 6117, the amino acid sequence of VLFWR2 of SEQ ID NO: 6118, the amino acid sequence of VLFWR3 of SEQ ID NO: 6119, or the amino acid sequence of VLFWR4 of SEQ ID NO: 6120, a multifunctional molecule of embodiment 151.

[0189] 153. An NK cell engager comprising a VL comprising the amino acid sequence of SEQ ID NO: 6147 (or an amino acid sequence having at least about 93%, 95%, or 99% sequence identity to SEQ ID NO: 6147), a multifunctional molecule of embodiment 152.

[0190] 154. An NK cell engager that is an antibody molecule that binds to NKp46, e.g., an antigen-binding domain, a multifunctional molecule of any of embodiments 57 to 60. 155. A multifunctional molecule of embodiment 154, wherein lysis of lymphoma cells is mediated by NKp46.

[0191] When incubated with NK cells in the absence of tumor antigen in lymphoma cells, the multifunctional molecule of any one of embodiments 154 or 155 does not activate NK cells.

[0192] 157. The multifunctional molecule of any one of embodiments 154 to 156, wherein the NK cell is an NK cell expressing NKp46, and when a tumor antigen in lymphoma cells is present, the multifunctional molecule activates the NK cell.

[0193] 158. The multifunctional molecule of any one of embodiments 154 to 157, wherein the NK cell is not an NK cell expressing NKp46 and when a tumor antigen in lymphoma cells is also present, the multifunctional molecule does not activate the NK cell.

[0194] 159. The multifunctional molecule of any one of embodiments 154 to 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).

[0195] 160. The multifunctional molecule of any one of embodiments 154 to 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).

[0196] 161. The multifunctional molecule of 154 to 160, wherein the NK cell engager comprises an scFV comprising the amino acid sequence of SEQ ID NO: 6181 (or an amino acid sequence having at least about 93%, 95%, or 99% sequence identity with SEQ ID NO: 6181).

[0197] 162. The multifunctional molecule of any one of embodiments 57 to 60, wherein the NK cell engager is an antibody molecule that binds to NKG2D, for example, an antigen-binding domain. 163. The multifunctional molecule of embodiment 162, wherein the lysis of lymphoma cells is mediated by NKG2D.

[0198] 164. The multifunctional molecule according to any one of embodiments 162 or 163, wherein when incubated with NK cells in the absence of tumor antigen in lymphoma cells, the multifunctional molecule does not activate NK cells.

[0199] 165. The multifunctional molecule according to any one of embodiments 162 to 164, wherein the NK cells are NK cells expressing NKG2D, and when tumor antigen in lymphoma cells is also present, the multifunctional molecule activates NK cells.

[0200] 166. The multifunctional molecule according to any one of embodiments 162 to 165, wherein the NK cells are not NK cells expressing NKG2D, and when tumor antigen in lymphoma cells is also present, the multifunctional molecule does not activate NK cells.

[0201] 167. The multifunctional molecule according to any one of embodiments 162 to 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 to SEQ ID NO: 6176).

[0202] 168. The multifunctional molecule according to any one of embodiments 162 to 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 to SEQ ID NO: 6177).

[0203] 169. The multifunctional molecule according to any one of embodiments 162 to 168, wherein the NK cell engager comprises an scFV comprising the amino acid sequence of SEQ ID NO: 6175 (or an amino acid sequence having at least about 93%, 95%, or 99% sequence identity to SEQ ID NO: 6175).

[0204] 170. A multifunctional molecule according to any one of embodiments 162 to 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).

[0205] 171. A multifunctional molecule according to any one of embodiments 162 to 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).

[0206] 172. A multifunctional molecule according to any of embodiments 162 to 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).

[0207] 173. A multifunctional molecule according to any one of embodiments 57 to 60, wherein the NK cell engager is an antibody molecule that binds to CD16, for example, an antigen-binding domain. 174. A multifunctional molecule according to embodiment 162, wherein the lysis of lymphoma cells is mediated by CD16.

[0208] 175. A multifunctional molecule according to 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 antigen in lymphoma cells.

[0209] 176. A multifunctional molecule according to any one of embodiments 173 to 175, wherein the NK cells are NK cells expressing CD16, and when tumor antigen in lymphoma cells is also present, the multifunctional molecule activates NK cells.

[0210] In the case where the tumor antigen in lymphoma cells is also present not in NK cells expressing CD16 but in NK cells, any one of the multifunctional molecules of Embodiments 173 to 176 that do not activate NK cells.

[0211] Any multifunctional molecule of Embodiments 173 to 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 to SEQ ID NO: 6185).

[0212] Any one of the multifunctional molecules of Embodiments 173 to 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 to SEQ ID NO: 6186).

[0213] Any one of the multifunctional molecules of Embodiments 173 to 179, wherein the NK cell engager comprises an scFV comprising the amino acid sequence of SEQ ID NO: 6184 (or an amino acid sequence having at least about 93%, 95%, or 99% sequence identity to SEQ ID NO: 6184).

[0214] Any multifunctional molecule of Embodiment 57, wherein the NK cell engager is a ligand, and optionally the ligand further comprises an immunoglobulin constant region, such as an Fc region. Any multifunctional molecule of Embodiment 181, wherein the NK cell engager is a ligand of NKp44 or NKp46, such as viral HA.

[0215] Any multifunctional molecule of Embodiment 181, wherein the NK cell engager is a ligand of DAP10, such as a co-receptor of NKG2D. Any multifunctional molecule of Embodiment 181, wherein the NK cell engager is a ligand of CD16, such as a CD16a / b ligand, such as a CD16a / b ligand further comprising an antibody Fc region.

[0216] 185. A multifunctional molecule according to any one of embodiments 52 to 54, wherein the immune cell engager mediates binding to, activation of, or both, one or more of B cells, macrophages, and / or dendritic cells.

[0217] 186. A multifunctional molecule according to embodiment 185, wherein the immune cell engager is selected from one or more of CD40 ligand (CD40L) or CD70 ligand; an antibody molecule that binds to CD40 or CD70; an antibody molecule against OX40; OX40 ligand (OX40L); an agonist of a Toll-like receptor (e.g., TLR4, e.g., constitutively active TLR4 (caTLR4) or TLR9 agonist); 41BB; a CD2 agonist; CD47; or a STING agonist, or a combination thereof, and is a B cell, macrophage, and / or dendritic cell engager.

[0218] 187. A multifunctional molecule according to any one of embodiments 52 to 54, wherein the immune cell engager is a B cell engager, such as CD40L, OX40L, or CD70 ligand, or an antibody molecule that binds to OX40, CD40, or CD70.

[0219] 188. A multifunctional molecule according to any one of embodiments 52 to 54, wherein the immune cell engager is a macrophage cell engager, such as a CD2 agonist; CD40L; OX40L; an antibody molecule that binds to OX40, CD40, or CD70; an agonist of a Toll-like receptor (TLR) (e.g., TLR4, e.g., constitutively active TLR4 (caTLR4) or TLR9 agonist); CD47; or a STING agonist.

[0220] 189. The immunocyte engager is a multifunctional molecule of any one of embodiments 52 to 54, which is a dendritic cell engager, such as a CD2 agonist, an OX40 antibody, OX40L, a 4-1BB 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.

[0221] 190. The STING agonist includes, together with, a cyclic nucleotide, such as cyclic di-GMP (cdGMP), cyclic di-AMP (cdAMP), or a combination thereof, and optionally has a 2′,5′ or 3′,5′ phosphate linkage. For example, the STING agonist is covalently coupled to the multifunctional molecule. The multifunctional molecule of embodiment 188 or 189.

[0222] 191. The multifunctional molecule of any one of embodiments 1 to 51 includes a cytokine molecule. 192. 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 foregoing cytokines. The multifunctional molecule of embodiment 191.

[0223] 192A. The cytokine molecule is interleukin-2 (IL-2). The multifunctional molecule of embodiment 192. 193. The cytokine molecule is a monomer or a dimer. The multifunctional molecule of embodiment 191 or 192.

[0224] 194. The cytokine molecule further includes a receptor dimerization domain, such as an IL15R alpha dimerization domain. The multifunctional molecule of any one of embodiments 191 to 193.

[0225] 195. The multifunctional molecule of embodiment 194, wherein the cytokine molecule (e.g., IL-15) and the receptor dimerization domain (e.g., the IL15R alpha dimerization domain) are not covalently linked, e.g., non-covalently associated.

[0226] 196. The multifunctional molecule according to any one of embodiments 1 to 51, wherein the multifunctional molecule comprises a cytokine inhibitor molecule. 197. The multifunctional molecule of embodiment 196, wherein the cytokine inhibitor molecule is a TGF-beta inhibitor.

[0227] 198. The multifunctional molecule according to any one of embodiments 196 or 197, wherein the TGF-beta inhibitor inhibits (e.g., reduces its activity) (i) TGF-beta 1; (ii) TGF-beta 2; (iii) TGF-beta 3; (iv) (i) and (ii); (v) (i) and (iii); (vi) (ii) and (iii); or (vii) (i), (ii), and (iii).

[0228] 199. The multifunctional molecule according to any one of embodiments 196 to 198, wherein the TGF-beta inhibitor comprises a part of a TGF-beta receptor (e.g., the extracellular domain of a TGF-beta receptor) that can inhibit (e.g., reduce its activity) TGF-beta, or a functional fragment or variant thereof.

[0229] 200. The multifunctional molecule of embodiment 199, wherein the TGF-beta inhibitor comprises a part of (i) TGFBR1; (ii) TGFBR2; (iii) TGFBR3; (iv) (i) and (ii); (v) (i) and (iii); (vi) (ii) and (iii); or (vii) (i), (ii), and (iii).

[0230] 201. The multifunctional molecule according to any one of embodiments 196 to 200, wherein the TGF-beta inhibitor comprises an amino acid sequence selected from Table 16, or an amino acid sequence having at least about 93%, 95%, or 99% sequence identity thereto.

[0231] The multifunctional molecule according to any one of embodiments 1 to 51, comprising a cell death receptor signal engager selected from a TNF-related apoptosis-inducing ligand (TRAIL) molecule, a cell death receptor molecule, or an antigen-binding domain that specifically binds to a cell death receptor.

[0232] 203. The multifunctional molecule according to embodiment 202, wherein the cell death receptor signal engager activates cell death receptor signaling in, for example, lymphoma cells (e.g., T cells) or lymphocytes that express TRBC1 or TRBC2, and induces, for example, apoptosis or cell death in said cells.

[0233] 204. The multifunctional molecule according to any one of embodiments 202 or 203, wherein the cell death receptor signal engager does not activate cell death receptor signaling in non-lymphoma cells and lymphocytes that do not express TRBC1 or do not express TRBC2.

[0234] 205. The multifunctional molecule according to any one of embodiments 202 to 204, wherein the cell death receptor signal engager comprises a TRAIL molecule, for example, one or more TRAIL polypeptides or fragments thereof.

[0235] 206. The multifunctional molecule according to embodiment 205, wherein the TRAIL molecule specifically binds to cell death receptor 4 (DR4) or cell death receptor 5 (DR5). 207. The multifunctional molecule according to any one of embodiments 205 or 206, wherein the TRAIL molecule comprises a shortened TRAIL polypeptide, for example, relative to a wild-type TRAIL polypeptide.

[0236] 208. The multifunctional molecule according to embodiment 207, wherein the TRAIL molecule comprises a shortened TRAIL molecule comprising residues corresponding to at least amino acids 95 to 281 of human TRAIL, for example, residues corresponding to amino acids 95 to 281 of human TRAIL.

[0237] The multifunctional molecule of embodiment 208, wherein the TRAIL molecule comprises a truncated TRAIL polypeptide that includes amino acids 95-281 of human TRAIL and, for example, does not include amino acids 1-94 of human TRAIL.

[0238] The multifunctional molecule of embodiment 207, wherein the TRAIL molecule comprises a truncated TRAIL molecule that includes residues corresponding to at least amino acids 122-281 of human TRAIL, for example, residues corresponding to amino acids 122-281 of human TRAIL.

[0239] The multifunctional molecule of embodiment 210, wherein the TRAIL molecule comprises a truncated TRAIL polypeptide that includes amino acids 122-281 of human TRAIL and, for example, does not include amino acids 1-121 of human TRAIL.

[0240] The multifunctional molecule of any one of embodiments 205-211, wherein the cell death receptor signal engager comprises one, two, or three TRAIL molecules. The multifunctional molecule of any one of embodiments 202-204, wherein the cell death receptor signal engager comprises an antigen-binding domain that specifically binds to a cell death receptor, for example, cell death receptor 4 (DR4) or cell death receptor 5 (DR5).

[0241] The multifunctional molecule of embodiment 213, wherein the cell death receptor signal engager comprises one, two, or three antigen-binding domains that specifically bind to a cell death receptor. The multifunctional molecule of any one of embodiments 213 or 214, wherein the antigen-binding domain that specifically binds to a cell death receptor binds to DR5.

[0242] The multifunctional molecule of any one of embodiments 213-215, wherein the antigen-binding domain that specifically binds to a cell death receptor comprises tigatuzumab, drozitumab, or conatumumab.

[0243] 217. A multifunctional molecule according to any one of embodiments 202 to 216, wherein the cell death receptor signal engager comprises an amino acid sequence selected from Table 11, or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto.

[0244] 218. A multifunctional molecule according to any one of embodiments 202 to 217, wherein the cell death receptor signal engager comprises the amino acid sequence of SEQ ID NO: 6157, or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto.

[0245] 219. A multifunctional molecule according to any one of embodiments 202 to 217, wherein the cell death receptor signal engager comprises the amino acid sequence of SEQ ID NO: 6158, or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto.

[0246] 220. A multifunctional molecule according to any one of embodiments 202 to 217, wherein the cell death receptor signal engager comprises the amino acid sequence of SEQ ID NO: 6159, or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto.

[0247] 221. A multifunctional molecule according to any one of embodiments 202 to 217, wherein the cell death receptor signal engager comprises the amino acid sequence of SEQ ID NO: 6160, or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto.

[0248] 222. A multifunctional molecule according to any one of embodiments 202 to 217, wherein the cell death receptor signal engager comprises the amino acid sequence of SEQ ID NO: 6161, or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto.

[0249] 223. A multifunctional molecule according to any one of embodiments 202 to 217, wherein the cell death receptor signal engager comprises the amino acid sequence of SEQ ID NO: 6162, or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto.

[0250] 224. A multifunctional molecule according to any one of embodiments 202 to 217, wherein the cell death receptor signal engager comprises the amino acid sequence of SEQ ID NO: 6163, or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto.

[0251] 225. A multifunctional molecule according to any one of embodiments 202 to 217, wherein the cell death receptor signal engager comprises the amino acid sequence of SEQ ID NO: 6164, or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto.

[0252] 226. A multifunctional molecule according to any one of embodiments 202 to 217, wherein the cell death receptor signal engager comprises the amino acid sequence of SEQ ID NO: 6165, or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto.

[0253] 227. A multifunctional molecule according to embodiment 56, wherein the T cell engager binds to TCRβ, such as the TCR beta V chain (TCRBV). 228. A multifunctional molecule according to embodiment 227, wherein the T cell engager comprises an antigen-binding domain (such as an antibody molecule or a fragment thereof) that binds to (e.g., activates in some embodiments) TCRβ.

[0254] 229. A multifunctional molecule according to any one of embodiments 227 or 228, wherein the T cell engager comprises an anti-TCRβV antibody molecule, such as an anti-TCR beta V chain (TCRβV) antibody molecule that specifically binds to the human TCR beta V chain (TCRβV).

[0255] The multifunctional molecule of any one of embodiments 227 to 229, wherein the T cell engager does not bind to lymphoma cells or lymphocytes expressing TRBC1 or TRBC2. The multifunctional molecule of any one of embodiments 227 to 229, wherein the T cell engager is capable of binding to, or binds to, lymphoma cells or lymphocytes expressing TRBC1 or TRBC2.

[0256] The multifunctional molecule of any one of embodiments 227 to 231, wherein the T cell engager does not activate lymphoma cells or lymphocytes expressing TRBC1 or TRBC2.

[0257] The multifunctional molecule of any one of embodiments 227 to 232, wherein the T cell engager comprises an anti-TCRβV antibody molecule that specifically binds to a TCRβV subfamily or a subfamily member in Table 12.

[0258] 234. The anti-TCRβV antibody molecule is TCRβV6, e.g., 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 * The multifunctional molecule of embodiment 233, which specifically binds to the TCRβV6 subfamily comprising 01.

[0259] 235. The multifunctional molecule of embodiment 234, wherein the anti-TCRβV antibody molecule comprises one or more CDRs, framework regions, or variable heavy and / or light chain regions provided in Table 13 or having at least about 93%, 95%, or 99% sequence identity thereto.

[0260] 236. The anti-TCRβV antibody molecule binds specifically to the TCRβV12 subfamily containing TCRβV12, such as TCRβV12-4 * 01, TCRβV12-3 * 01 or TCRβV12-5 * 01, the multifunctional molecule of embodiment 233.

[0261] 237. The anti-TCRβV antibody molecule of the multifunctional molecule of embodiment 236, comprising one or more CDRs, framework regions, or variable heavy and / or light chain regions provided in Table 14 or having at least about 93%, 95%, or 99% sequence identity thereto.

[0262] 238. The multifunctional molecule of any one of embodiments 1 to 51, wherein the multifunctional molecule comprises a stromal modifying moiety. 239. The stromal modifying moiety results in one or more of: reducing the level or production of stromal or extracellular matrix (ECM) components; reducing tumor fibrosis; increasing tumor transport in the stroma; improving tumor perfusion; dilating the tumor microvasculature; reducing the interstitial fluid pressure (IFP) in the tumor; or decreasing or enhancing the penetration or diffusion of an agent, such as a cancer therapeutic agent or a cell therapy agent, into the tumor or tumor vasculature, the multifunctional molecule of embodiment 238.

[0263] 240. The multifunctional molecule of embodiment 239, wherein the reduced stromal or ECM component is selected from glycosaminoglycans or extracellular proteins, or combinations thereof. 241. The multifunctional molecule is (i) an immune cell engager (e.g., a T cell engager, an NK cell engager, a B cell engager, a dendritic cell engager, or a macrophage cell engager) and a cytokine molecule, (ii) an immune cell engager (e.g., a T cell engager, an NK cell engager, a B cell engager, a dendritic cell engager, or a macrophage cell engager) and a cytokine inhibitor molecule, (iii) An immune cell engager (e.g., a T cell engager, an NK cell engager, a B cell engager, a dendritic cell engager, or a macrophage cell engager) and a cell death receptor signal engager, (iv) An immune cell engager (e.g., a T cell engager, an NK cell engager, a B cell engager, a dendritic cell engager, or a macrophage cell engager) and a stromal modification moiety, (v) A cytokine molecule and a stromal modification moiety, (vi) A cytokine molecule and a cell death receptor signal engager, (vii) A cytokine inhibitor molecule and a stromal modification moiety, (viii) A cytokine inhibitor molecule and a cell death receptor signal engager, (ix) An immune cell engager (e.g., a T cell engager, an NK cell engager, a B cell engager, a dendritic cell engager, or a macrophage cell engager), a cytokine molecule, a cell death receptor signal engager, and a stromal modification moiety, or (x) An immune cell engager (e.g., a T cell engager, an NK cell engager, a B cell engager, a dendritic cell engager, or a macrophage cell engager), a cytokine inhibitor molecule, a cell death receptor signal engager, and a stromal modification moiety The multifunctional molecule according to any one of Embodiments 1 to 240, comprising.

[0264] 242. The multifunctional molecule has the following structure: A,B - [Dimerization module] - C, - D wherein: (a) The dimerization module comprises an immunoglobulin constant domain, e.g., a heavy chain constant domain (e.g., a homodimeric or heterodimeric heavy chain constant region, e.g., the Fc region), or a constant domain of an immunoglobulin variable region (e.g., the Fab region); (b) A, B, C, and D are each independently absent or are (i) an antigen-binding domain that preferentially binds to TRBC1 or TRBC2; (ii) an immune cell engager selected from a T cell engager, an NK cell engager, a B cell engager, a dendritic cell engager, or a macrophage cell engager; (iii) a cytokine molecule or a cytokine inhibitor molecule; (iv) a cell death receptor signal engager; or (v) a stromal modifying moiety, provided that at least one, two, or three of A, B, C, and D contain an antigen-binding domain that preferentially binds to TRBC1 or TRBC2, and any of the remaining A, B, C, and D is absent or contains one of an immune cell engager, a cytokine molecule, a cytokine inhibitor molecule, a cell death receptor signal engager, or a stromal modifying moiety, a multifunctional molecule of any one of embodiments 1 to 241.

[0265] 243. (1) A contains an antigen-binding domain that preferentially binds to a T cell receptor comprising TRBC1 or TRBC2, and B, C, or D contains an immune cell engager, for example, a T cell engager, for example, an anti-TCRβV antibody molecule; (2) A contains an antigen-binding domain that preferentially binds to TRBC1 or TRBC2, and B, C, or D contains an immune cell engager, for example, an NK cell engager, for example, an anti-NKp30 or anti-NKp46 antibody molecule; (3) A contains an antigen-binding domain that preferentially binds to TRBC1 or TRBC2, and B, C, or D contains a cytokine molecule; (4) A contains an antigen-binding domain that preferentially binds to TRBC1 or TRBC2, and B, C, or D contains a cytokine inhibitor molecule; (5) A contains an antigen-binding domain that preferentially binds to TRBC1 or TRBC2, and B, C, or D contains a cell death receptor signal engager; (6) A comprises an antigen-binding domain that preferentially binds to TRBC1 or TRBC2, and B, C, or D comprises a stromal modifying moiety; (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; (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; (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; (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; (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 cell death receptor signal engager; (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 stromal modifying moiety; (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, such as a T cell engager, such as an anti-TCRβV antibody molecule; (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, such as an NK cell engager, such as an anti-NKp30, anti-NKp46, anti-NKG2D, or anti-CD16 antibody molecule; (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; (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; (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 cell death receptor signal engager; (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 stromal modification moiety; (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, such as an NK cell engager, such as an anti-NKp30, anti-NKp46, anti-NKG2D, or anti-CD16 antibody molecule, and (b) a cytokine molecule; (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, such as an NK cell engager, such as an anti-NKp30, anti-NKp46, anti-NKG2D, or anti-CD16 antibody molecule, and (b) a cytokine inhibitor molecule; (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, such as an NK cell engager, such as an anti-NKp30, anti-NKp46, anti-NKG2D, or anti-CD16 antibody molecule, and (b) a cell death receptor signal engager; (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, such as an NK cell engager, such as an anti-NKp30, anti-NKp46, anti-NKG2D, or anti-CD16 antibody molecule, and (b) a stromal modification moiety; (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, such as a T cell engager, such as an anti-TCRβV antibody molecule, and (b) a cytokine molecule; (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, such as a T cell engager, such as an anti-TCRβV antibody molecule, and (b) a cytokine inhibitor molecule; (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, such as a T cell engager, such as an anti-TCRβV antibody molecule, and (b) a cell death receptor signal engager; (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, such as a T cell engager, such as an anti-TCRβV antibody molecule, and (b) a stromal modification moiety; (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 stroma-modifying moiety; (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 cell death receptor signal engager; (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 stroma-modifying moiety; (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 cell death receptor signal engager; (31) A comprises a first antigen-binding domain that preferentially binds to TRBC1 or TRBC2, and B, C, or D comprises (a) a cell death receptor signal engager and (b) a stroma-modifying moiety; (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; (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; (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, such as an NK cell engager, such as an anti-NKp30, anti-NKp46, anti-NKG2D, or anti-CD16 antibody molecule, and (b) a cell death receptor signal engager; (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, such as an NK cell engager, such as an anti-NKp30, anti-NKp46, anti-NKG2D, or anti-CD16 antibody molecule, and (b) a stromal modification moiety; (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, such as a T cell engager, such as an anti-TCRβV antibody molecule, and (b) a cytokine molecule; (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, such as a T cell engager, such as an anti-TCRβV antibody molecule, and (b) a cytokine inhibitor molecule; (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, such as a T cell engager, such as an anti-TCRβV antibody molecule, and (b) a cell death receptor signal engager; (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, such as a T cell engager, such as an anti-TCRβV antibody molecule, and (b) a stroma-modifying moiety; (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 stroma-modifying moiety; (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 cell death receptor signal engager; (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 stroma-modifying moiety; (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 cell death receptor signal engager; (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 stroma-modifying moiety and (b) a cell death receptor signal engager; (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, such as an NK cell engager, such as an anti-NKp30, anti-NKp46, anti-NKG2D, or anti-CD16 antibody molecule, and (b) a cytokine molecule; (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, such as an NK cell engager, such as an anti-NKp30, anti-NKp46, anti-NKG2D, or anti-CD16 antibody molecule, and (b) a cytokine inhibitor molecule; (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, such as an NK cell engager, such as an anti-NKp30, anti-NKp46, anti-NKG2D, or anti-CD16 antibody molecule, and (b) a cell death receptor signal engager; (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, such as an NK cell engager, such as an anti-NKp30, anti-NKp46, anti-NKG2D, or anti-CD16 antibody molecule, and (b) a stromal modification moiety; (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, such as a T cell engager, such as an anti-TCRβV antibody molecule, and (b) a cytokine molecule; (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, such as a T cell engager, such as an anti-TCRβV antibody molecule, and (b) a cytokine inhibitor molecule; (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, such as a T cell engager, such as an anti-TCRβV antibody molecule, and (b) a cell death receptor signal engager; (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, such as a T cell engager, such as an anti-TCRβV antibody molecule, and (b) a stromal modification moiety; (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 stromal modification moiety; (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 cell death receptor signal engager; (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 stromal modification moiety; (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 cell death receptor signal engager; or (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 stroma-modifying moiety and (b) a cell death receptor signal engager, the multifunctional molecule of embodiment 242.

[0266] 244. The multifunctional molecule of embodiment 242 or 243, wherein the dimerization module comprises one or more immunoglobulin heavy chain constant regions (e.g., Fc region) comprising one or more of paired holes and protrusions (“knob-in-hole”), electrostatic interactions, or strand exchange.

[0267] 245. The multifunctional molecule of embodiment 244, wherein one or more immunoglobulin heavy chain constant regions (e.g., Fc region) comprise amino acid substitutions at positions selected from one or more of 347, 349, 350, 351, 366, 368, 370, 392, 394, 395, 397, 398, 399, 405, 407, or 409 of, for example, the Fc region of human IgG1, and optionally one or more immunoglobulin heavy chain constant regions (e.g., Fc region) comprise amino acid substitutions selected from T366S, L368A, or Y407V (e.g., corresponding to a hole or a hall), or T366W (e.g., corresponding to a protrusion or a knob), or combinations thereof.

[0268] 246. A multifunctional molecule according to any one of embodiments 1 to 245, further comprising a linker between one or more of: a linker, for example, an antigen-binding domain and an immune cell engager, an antigen-binding domain and a cytokine molecule, an antigen-binding domain and a stromal modifying moiety, an immune cell engager and a cytokine molecule, an immune cell engager and a stromal modifying moiety, a cytokine molecule and a stromal modifying moiety, an antigen-binding domain and a dimerization module, an immune cell engager and a dimerization module, a cytokine molecule and a dimerization module, or a stromal modifying moiety and a dimerization module.

[0269] 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.

[0270] 248. The multifunctional molecule of embodiment 246 or 247, wherein the linker is a peptide linker. 249. The multifunctional molecule of 248, wherein the peptide linker comprises Gly and Ser.

[0271] 250. The multifunctional molecule of 249, wherein the peptide linker comprises an amino acid sequence selected from SEQ ID NOs: 7249 to 7252 or 75 to 78. 251. (i) a first antigen-binding domain that preferentially binds to TRBC1, (ii) an NK cell engager, for example, an anti-NKp30 antibody molecule, an anti-NKp46 antibody molecule, an anti-NKG2D antibody molecule, or an anti-CD16 antibody molecule and a multifunctional molecule comprising the same.

[0272] 252. The multifunctional molecule of embodiment 251, wherein the NK cell engager comprises an anti-NKp30 antibody molecule. 254. The multifunctional molecule of embodiment 251, wherein the NK cell engager comprises an anti-NKp46 antibody molecule.

[0273] 255. The NK cell engager is the multifunctional molecule of claim 251, comprising an anti-NKG2D antibody molecule. 255A. The NK cell engager is the multifunctional molecule of claim 251, comprising an anti-CD16 antibody molecule.

[0274] 256. (i) A first antigen-binding domain that preferentially binds to TRBC1, and (ii) a cell death receptor signal engager and a multifunctional molecule.

[0275] 257. (i) A first antigen-binding domain that preferentially binds to TRBC1, and (ii) a T cell engager, for example, an antigen-binding domain that binds to the TCR beta V chain (TCRBV) and a multifunctional molecule.

[0276] 259. (i) A first antigen-binding domain that preferentially binds to TRBC1, and (ii) a cytokine inhibitor molecule, for example, a TGF-beta inhibitor and a multifunctional molecule.

[0277] 262. The multifunctional molecule of any one of embodiments 1 or 3 to 261, wherein the multifunctional molecule binds monovalently to TRBC1, TRBC2, or a tumor antigen. 263. The multifunctional molecule of any one of embodiments 1 or 3 to 261, wherein the multifunctional molecule binds multivalently, for example, divalently, trivalently, tetravalently, pentavalently, hexavalently, heptavalently, octavalently, nonavalently, or decavalently, to TRBC1, TRBC2, or a tumor antigen.

[0278] 264. The multifunctional molecule of any one of embodiments 2 to 261, wherein the multifunctional molecule binds monovalently to TRBC1, TRBC2, or a lymphocyte expressing TRBC1 or TRBC2.

[0279] The multifunctional molecule according to any one of embodiments 2 to 261, wherein the multifunctional molecule is multivalent, for example, divalent, trivalent, tetravalent, pentavalent, hexavalent, heptavalent, octavalent, nonavalent, or decavalent, and binds to lymphocytes expressing TRBC1 or TRBC2.

[0280] The multifunctional molecule according to any one of the preceding embodiments, wherein the multifunctional molecule binds monovalently to immune cells, for example, via an immune cell engager. The multifunctional molecule according to any one of embodiments 1 to 265, wherein the multifunctional molecule binds multivalently, for example, divalent, trivalent, tetravalent, pentavalent, hexavalent, heptavalent, octavalent, nonavalent, or decavalent, to immune cells, for example, via an immune cell engager.

[0281] The multifunctional molecule according to any one of the preceding embodiments, further comprising a heavy chain constant region that mediates antibody-dependent cell cytotoxicity (ADCC), for example, an Fc region. The multifunctional molecule according to any one of the preceding embodiments, further comprising a heavy chain constant region that mediates antibody-dependent cell phagocytosis (ADCP), for example, an Fc region.

[0282] The multifunctional molecule of embodiment 268A, wherein the first antigen-binding domain that binds to TRBC1 comprises an IgG2 heavy chain constant region, or the immune cell engager, cytokine inhibitor molecule, or cell death receptor signal engager comprises an IgG2 heavy chain constant region.

[0283] The multifunctional molecule according to any one of the preceding embodiments, further comprising a heavy chain constant region that mediates complement-dependent cell cytotoxicity (e.g., via C1q), for example, an Fc region. An antibody molecule that binds to TRBC1 and comprises one or more CDRs, framework regions, variable domains, heavy or light chains, or antigen-binding domains selected from Tables 2 to 5, or a sequence substantially identical thereto.

[0284] An antibody molecule of Embodiment 269A, comprising a heavy chain variable region (VH) comprising the amino acid sequence of heavy chain framework region 1 (VHFWR1) of SEQ ID NO: 215 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), the amino acid sequence of VHFWR2 of SEQ ID NO: 216 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), the amino acid sequence of VHFWR3 of SEQ ID NO: 217 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), or the amino acid sequence of VHFWR4 of SEQ ID NO: 218 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions).

[0285] An antibody molecule of either Embodiment 269A or 269B, comprising a light chain variable region (VL) comprising the amino acid sequence of light chain framework region 1 (VLFWR1) of SEQ ID NO: 238 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), the amino acid sequence of VLFWR2 of SEQ ID NO: 239 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), the amino acid sequence of VLFWR3 of SEQ ID NO: 240 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions), or the amino acid sequence of VLFWR4 of SEQ ID NO: 241 (or a sequence having 1, 2, 3, 4, 5, or 6 or fewer mutations therefrom, such as substitutions, additions, or deletions).

[0286] An antibody molecule of any one of Embodiments 269A to 269C, comprising 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).

[0287] An antibody molecule according to any one of embodiments 269A to 269D, comprising 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).

[0288] 270. A nucleic acid molecule encoding a multifunctional molecule or an antibody molecule according to any one of embodiments 1 to 269E. 271. A vector, such as an expression vector, comprising the nucleic acid molecule of embodiment 270.

[0289] 272. A host cell comprising the nucleic acid molecule of embodiment 270 or the vector of embodiment 271. 273. A method of making, e.g., producing, a multifunctional molecule or an antibody molecule according to any one of embodiments 1 to 269E, the method comprising culturing the host cell of embodiment 272 under suitable conditions, e.g., conditions suitable for gene expression and / or homoor heterodimerization.

[0290] 274. A pharmaceutical composition comprising a multifunctional molecule according to any one of embodiments 1 to 269 and a pharmaceutically acceptable carrier, excipient, or stabilizer. 275. A method of treating cancer, the method comprising administering to a subject in need thereof a multifunctional molecule according to any one of embodiments 1 to 269, wherein the multifunctional molecule is administered in an amount effective to treat cancer.

[0291] 276. The method of embodiment 275, further comprising the step of identifying, evaluating, or selecting a subject in need of treatment, the step of identifying, evaluating, or selecting comprising determining whether the subject has cancer cells expressing a T cell receptor comprising TRBC1 or TRBC2 (e.g., directly determining it or indirectly determining it, e.g., obtaining information regarding it).

[0292] 277. In response to determining that the subject has cancer cells expressing a T cell receptor comprising TRBC1 Selecting a subject for treatment with a multifunctional molecule comprising an antigen-binding domain that binds to a T cell receptor comprising TRBC1, as needed, and administering a multifunctional molecule comprising an antigen-binding domain that binds to a T cell receptor comprising TRBC1 The method of embodiment 276, further comprising.

[0293] 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. 278A. A method of treating cancer, such as lymphoma or leukemia, responding to the determination that the subject has cancer cells expressing a T cell receptor comprising TRBC1, administering to the subject in need thereof a multifunctional molecule of any one of claims 1 to 269, wherein the multifunctional molecule is administered in an amount effective to treat the cancer.

[0294] 279. Responding to the determination that the subject has cancer cells expressing a T cell receptor comprising TRBC2, selecting a subject for treatment with a multifunctional molecule comprising an antigen-binding domain that binds to a T cell receptor comprising TRBC2, as needed, and administering a multifunctional molecule comprising an antigen-binding domain that binds to a T cell receptor comprising TRBC2 The method of embodiment 276, further comprising.

[0295] 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. 281. The method of any one of embodiments 275 to 278, wherein the subject has cancer cells expressing a T cell receptor comprising TRBC1.

[0296] 282. The method of any one of embodiments 275, 276, 279, or 280, wherein the subject has cancer cells expressing a T cell receptor comprising TRBC2. A method for identifying a subject in need of treatment for cancer, comprising determining whether the subject has cancer cells expressing a T cell receptor comprising TRBC1 or TRBC2 (e.g., directly determining it or indirectly determining it, e.g., obtaining information regarding it), using a multifunctional molecule or an antibody molecule according to any one of Embodiments 1 to 269E, in response to determining that the subject has cancer cells expressing a T cell receptor comprising TRBC1, identifying the subject as a candidate for treatment using a multifunctional molecule comprising an antigen-binding domain that binds to TRBC1, and optionally not identifying the subject as a candidate for treatment using a multifunctional molecule comprising an antigen-binding domain that binds to TRBC2, in response to determining that the subject has cancer cells expressing a T cell receptor comprising TRBC2, identifying the subject as a candidate for treatment using a multifunctional molecule comprising an antigen-binding domain that binds to TRBC2, and optionally not identifying the subject as a candidate for treatment using a multifunctional molecule comprising an antigen-binding domain that binds to TRBC1.

[0297] In response to identifying the subject as a candidate for treatment using a multifunctional molecule comprising an antigen-binding domain that binds to TRBC1, treating (e.g., administering it to) the subject with a multifunctional molecule comprising an antigen-binding domain that binds to TRBC1, or in response to identifying the subject as a candidate for treatment using a multifunctional molecule comprising an antigen-binding domain that binds to TRBC2, treating (e.g., administering it to) the subject with a multifunctional molecule comprising an antigen-binding domain that binds to TRBC2 The method according to Embodiment 283, further comprising.

[0298] A method for evaluating a subject in need of treatment for cancer, such as lymphoma, comprising determining whether the subject has cancer cells expressing a T cell receptor comprising TRBC1 or TRBC2 (e.g., directly determining it or indirectly determining it, e.g., obtaining information regarding it).

[0299] 286. A method according to embodiment 285, further comprising treating (e.g., administering to) a subject with 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 in response to the evaluation.

[0300] 287. A method according to any one of embodiments 275 to 286, wherein the cancer is a blood cancer. 288. A method according to embodiment 287, wherein the blood cancer is leukemia or lymphoma. 289. The blood cancer is 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 (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, Sézary syndrome, Waldenström macroglobulinemia), chronic myeloproliferative neoplasms, Langerhans cell histiocytosis, multiple myeloma / plasma cell neoplasms, myelodysplastic syndromes, or myelodysplastic / myeloproliferative neoplasms, a method according to embodiment 288, selected from the group consisting of

[0301] 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 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 γδ T-cell lymphoma (HSGDTCL), subcutaneous panniculitis-like T-cell lymphoma, or enteropathy-associated T-cell lymphoma), transformed follicular lymphoma and transformed mucosa-associated lymphoid tissue (MALT) lymphoma, cutaneous T-cell lymphoma (mycosis fungoides and Sézary syndrome), follicular lymphoma, lymphoplasmacytic lymphoma / Waldenström macroglobulinemia, marginal zone B-cell lymphoma, gastric mucosa-associated lymphoid tissue (MALT) lymphoma, chronic lymphocytic leukemia / small lymphocytic lymphoma (CLL / SLL), extranodal T / NK-cell lymphoma (nasal type), or anaplastic large cell lymphoma (e.g., primary cutaneous anaplastic large cell lymphoma or systemic anaplastic large cell lymphoma).

[0302] 291. The method of any one of embodiments 275 to 286, wherein the cancer is a solid tumor cancer. 292. The method of any of embodiments 275 to 291, further comprising the step of administering a second therapeutic treatment.

[0303] 293. The method of embodiment 292, wherein the second therapeutic treatment comprises a therapeutic agent (e.g., a chemotherapeutic agent, a biologic agent, hormonal therapy), radiation, or surgery. 294. The method of embodiment 293, wherein the therapeutic agent is selected from a chemotherapeutic agent or a biologic agent.

[0304] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

[0305] Other features and advantages of the present invention will be apparent from the following detailed description and the claims.

Brief Description of the Drawings

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Mode for Carrying Out the Invention

[0307] Multifunctional molecules (also referred to herein as "bispecific molecules") that include a plurality of (e.g., two or more) functionalities (or binding specificities), comprising (i) an antigen-binding domain that preferentially binds to TRBC1 or TRBC2, and (ii) one, two, or all of (a) an immune cell engager selected from a T cell engager, an NK cell engager (e.g., a molecule that binds to NKp30, NKp46, NKG2D, or CD16), a B cell engager, a dendritic cell engager, or a macrophage cell engager, (b) a cytokine molecule, and (c) a stromal modifying moiety, are disclosed herein. Antibody molecules that include an antigen-binding domain that preferentially binds to TRBC1 or TRBC2 are also disclosed herein. In some embodiments, the antigen-binding domain that binds to TRBC1 or TRBC2 includes the sequences or portions of sequences found in Tables 2 to 5. In some embodiments, the immune cell engager includes an NK cell engager that includes the sequences or portions of sequences found in Tables 7 to 10. In some embodiments, the antigen-binding domain includes the sequences or portions of sequences found in Tables 2 to 5, and the immune cell engager includes an NK cell engager that includes the sequences or portions of sequences found in Tables 7 to 10.

[0308] In one embodiment, the multispecific or multifunctional molecule is a bispecific (or bifunctional) molecule, a trispecific (or trifunctional) molecule, or a tetra-specific (or tetra-functional) molecule. In some embodiments, the multifunctional molecule comprises an antigen-binding domain that binds, in the presence of a tumor antigen on the surface of a T cell receptor comprising TRBC1, to a lymphoma cell (e.g., a T cell) expressing a T cell receptor comprising TRBC1 (e.g., via an immune cell engager).

[0309] Without being bound by theory, the multispecific or multifunctional molecules disclosed herein are expected to localize (e.g., crosslink) and / or activate immune cells (e.g., immune effector cells selected from T cells, NK cells, B cells, dendritic cells, or macrophages) in the presence of cells expressing a T cell receptor comprising TRBC1 or TRBC2 on their surface (e.g., cancer cells, e.g., lymphoma cells, e.g., T cells). It is expected that the proximity and / or activity of immune cells will be increased in the presence of cells expressing a T cell receptor comprising TRBC1 or TRBC2 (e.g., cancer cells, e.g., lymphoma cells, e.g., T cells), and that the use of the multispecific or multifunctional molecules described herein will enhance the immune response against target cells, thereby providing a more effective therapy.

[0310] Without being bound by theory, T cells are typically thought not to express T cell receptors containing TRBC1 and T cell receptors containing TRBC2. In some embodiments, by using multispecific or multifunctional molecules that are specific for T cell receptors containing TRBC1 or T cell receptors containing TRBC2, but not specific for both types of T cell receptors, the adverse effects of increasing the proximity or activity of immune cells to T cells can generally be reduced. Thus, the use of the multispecific or multifunctional molecules disclosed herein is thought to be able to increase the proximity or activity of immune cells to cancer cells (e.g., lymphoma cells, e.g., T cells) without necessarily increasing the proximity or activity of immune cells to T cells.

[0311] (i) novel multifunctional, e.g., multispecific molecules are disclosed that comprise a stromal modifying moiety and (ii) an antigen-binding domain that preferentially binds to tumor antigens on lymphoma cells (e.g., T cells), e.g., T cell receptors containing TRBC1 or T cell receptors containing TRBC2. Without being bound by theory, the multifunctional molecules disclosed herein are thought to, inter alia, target (e.g., localize to) the cancer site and modify the tumor stroma, e.g., modify the tumor microenvironment near the cancer site. The multifunctional molecule can further comprise one or both of an immune cell engager (e.g., selected from one, two, three, or all of a T cell engager, an NK cell engager, a B cell engager, a dendritic cell engager, or a macrophage cell engager) and / or a cytokine molecule. Accordingly, inter alia, multifunctional, e.g., multispecific molecules comprising the foregoing moieties, nucleic acids encoding the same, methods of producing the foregoing molecules, and methods of using the foregoing molecules to treat cancer are provided herein.

[0312] Accordingly, provided herein are, inter alia, multispecific or multifunctional molecules (e.g., multispecific or multifunctional antibody molecules) that include the foregoing moieties, nucleic acids encoding the same, methods of producing the foregoing molecules, and methods of using the foregoing molecules to treat a disease or disorder, such as cancer.

[0313] Definitions In some embodiments, the multifunctional molecule includes an immune cell engager. An “immune cell engager” refers to one or more binding specificities that bind and / or activate an immune cell, e.g., a cell involved in an immune response. In embodiments, the immune cell is selected from T cells, NK cells, B cells, dendritic cells, and / or macrophage cells. The immune cell engager can be an antibody molecule, a receptor molecule (e.g., a full-length receptor, a receptor fragment, or a fusion thereof (e.g., a receptor-Fc fusion)), or a ligand molecule (e.g., a full-length ligand, a ligand fragment, or a fusion thereof (e.g., a ligand-Fc fusion)) that binds to an immune cell antigen (e.g., a T cell, NK cell antigen, B cell antigen, dendritic cell antigen, and / or macrophage cell antigen). In embodiments, the immune cell engager specifically binds, e.g., preferentially binds, to a target immune cell. For example, if the immune cell engager is an antibody molecule, it binds to an immune cell antigen (e.g., a T cell antigen, NK cell antigen, B cell antigen, dendritic cell antigen, and / or macrophage cell antigen) with a dissociation constant of less than about 10 nM.

[0314] In some embodiments, the multifunctional molecule comprises a cytokine molecule. As used herein, the term "cytokine molecule" refers to the full length, fragment or variant of a cytokine; further, a cytokine comprising a receptor domain, such as a cytokine receptor dimerization domain; or an agonist of a cytokine receptor that induces activation of at least one of the naturally occurring cytokines, such as an antibody molecule (e.g., an agonist antibody) against a cytokine receptor. In some embodiments, the cytokine molecule is selected from interleukin-2 (IL-2), interleukin-7 (IL-7), interleukin-12 (IL-12), interleukin-15 (IL-15), interleukin-18 (IL-18), interleukin-21 (IL-21), or interferon γ, or fragments or variants thereof, or any combination of the aforementioned cytokines. The cytokine molecule can be a monomer or a dimer. In embodiments, the cytokine molecule can further comprise a cytokine receptor dimerization domain. In other embodiments, the cytokine molecule is an agonist of a cytokine receptor, such as an antibody molecule (e.g., an agonist antibody) against a cytokine receptor selected from IL-15Ra or IL-21R.

[0315] As used herein, for example, the term "molecule" as used in antibody molecules, cytokine molecules, receptor molecules includes full-length naturally occurring molecules as well as variants, such as functional variants (e.g., truncations, fragments, mutations (e.g., substantially similar sequences) or derivatives thereof), as long as at least one function and / or activation of the unmodified (e.g., naturally occurring) molecule remains.

[0316] In some embodiments, the multifunctional molecule comprises a stromal modifying moiety. As used herein, a "stromal modifying moiety" refers to an agent, such as a protein (e.g., an enzyme), that can modify, e.g., degrade, a component of the stroma. In embodiments, the components of the stroma are selected from, e.g., ECM components such as glycosaminoglycans such as hyaluronan (also known as hyaluronic acid or HA), chondroitin sulfate, chondroitin, dermatan sulfate, heparan sulfate, heparin, entactin, tenascin, aggrecan and keratin sulfate; or extracellular proteins such as collagen, laminin, elastin, fibrinogen, fibronectin, and vitronectin.

[0317] Define certain terms as follows. As used herein, the articles "a" and "an" refer to one or more, e.g., at least one, of the grammatical objects of the article. The use of the term "a" or "an" can mean "one" when used in conjunction with the term "comprising" herein, but is also consistent with the meaning of "one or more", "at least one" and "one or more than one".

[0318] As used herein, "about" and "substantially" generally mean the degree of acceptable error for a measured quantity given the nature or precision of the measurement. Exemplary degrees of error are within 20 percent (%) of a given range of values, typically within 10%, and more typically within 5%.

[0319] As used herein, the term "antibody molecule" refers to a protein, such as an immunoglobulin chain or a fragment thereof, that includes at least one immunoglobulin variable domain sequence. Antibody molecules include antibodies (e.g., full-length antibodies) and antibody fragments. In certain embodiments, an antibody molecule comprises an antigen-binding or functional fragment of a full-length antibody or a full-length immunoglobulin chain. For example, a full-length antibody is an immunoglobulin (Ig) molecule (e.g., an IgG antibody) that occurs naturally or is formed by a recombinant process of normal immunoglobulin gene fragments. In embodiments, an antibody molecule refers to an immunologically active antigen-binding portion of an immunoglobulin molecule, such as an antibody fragment. Antibody fragments, such as functional fragments, are part of an antibody, e.g., Fab, Fab’, F(ab’)2, F(ab)2, variable fragment (Fv), domain antibody (dAb), or single-chain variable fragment (scFv). Functional antibody fragments bind to the same antigen recognized by an intact (e.g., full-length) antibody. The terms "antibody fragment" or "functional fragment" also include isolated fragments consisting of variable regions, such as an "Fv" fragment consisting of the variable regions of the heavy and light chains, or a recombinant single-chain polypeptide molecule in which the variable regions of the light and heavy chains are linked by a peptide linker ("scFv protein"). In some embodiments, an antibody fragment includes a portion of an antibody that does not have antigen-binding activity, such as an Fc fragment or does not include a single amino acid residue. Exemplary antibody molecules include full-length antibodies and antibody fragments, such as dAb (domain antibody), single-chain, Fab, Fab’, and F(ab’)2 fragments, and single-chain variable fragments (scFv).

[0320] As used herein, the term "immunoglobulin variable domain sequence" refers to an amino acid sequence capable of forming the structure of an immunoglobulin variable domain. For example, the sequence can include all or part of the amino acid sequence of a naturally occurring variable domain. For example, the sequence may or may not include one, two, or more N-terminal or C-terminal amino acids, or may include other modifications compatible with protein structure formation.

[0321] In embodiments, the antibody molecule is monospecific and includes, for example, binding specificity for a single epitope. In some embodiments, the antibody molecule is multispecific; for example, it includes multiple immunoglobulin variable domain sequences, wherein the first immunoglobulin variable domain sequence has binding specificity for a first epitope and the second immunoglobulin variable domain sequence has binding specificity for a second epitope. In some embodiments, the antibody molecule is a bispecific antibody molecule. As used herein, the term "bispecific antibody molecule" refers to an antibody molecule having specificity for more than one (e.g., two, three, four, or more) epitopes and / or antigens.

[0322] As used herein, the term "antigen" (Ag) refers to a molecule capable of eliciting an immune response, for example, one that is associated with activation of certain immune cells and / or antibody production. Virtually any macromolecule, including most proteins or peptides, can be an antigen. Antigens can also be derived from genomic recombinants or DNA. For example, any DNA that includes a nucleotide sequence or partial nucleotide sequence encoding a protein capable of inducing an immune response encodes an "antigen." In embodiments, an antigen need not be encoded only by the full-length nucleotide sequence of a gene, nor need an antigen be encoded by a gene at all. In embodiments, an antigen can be synthesized or derived from a biological sample, such as a tissue sample, tumor sample, cell, or fluid having other biological components. As used herein, the term "tumor antigen" or, synonymously, "cancer antigen" includes any molecule present on or associated with a tumor, such as a cancer cell, or the tumor microenvironment that is capable of eliciting an immune response. As used herein, the term "immune cell antigen" includes any molecule present on or associated with an immune cell that is capable of eliciting an immune response.

[0323] The "antigen-binding site" or "binding site" of an antibody molecule refers to a part of the antibody molecule involved in antigen binding, such as a part of an immunoglobulin (Ig) molecule. In embodiments, the antigen-binding site is formed by amino acid residues in the variable regions (V) of the heavy chain (H) and light chain (L). Three highly divergent stretches within the variable regions of the heavy and light chains, called hypervariable regions, are positioned between more conserved adjacent regions called "framework regions" (FR). The FR is the amino acid sequence naturally found between and adjacent to the hypervariable regions in immunoglobulins. In embodiments, in an antibody molecule, the three hypervariable regions of the light chain and the three hypervariable regions of the heavy chain are arranged relative to each other in three-dimensional space to form an antigen-binding surface complementary to the three-dimensional surface of the binding antigen. Each of the three hypervariable regions of the heavy and light chains is called a "complementary determining region" or "CDR". The framework regions and CDRs are defined and described, for example, in Kabat, E.A. et al. (1991) Sequences of Proteins of Immunological Interest, 5th ed., U.S. Department of Health and Human Services, NIH Publication No. 91-3242, and Chothia, C. et al. (1987) J. Mol. Biol. 196:901-917. Each variable chain (e.g., variable heavy chain and variable light chain) typically consists of three CDRs and four FRs, arranged in amino-terminal to carboxy-terminal order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.

[0324] As used herein, "cancer" can encompass all types of carcinogenic processes and / or cancerous growths. In embodiments, cancer includes primary tumors, as well as metastatic tissues, or cells, tissues, or organs that have been malignantly transformed. In embodiments, cancer encompasses all histopathologies and stages of cancer, for example, invasive / severity stages. In embodiments, cancer includes recurrent cancer and / or resistant cancer. The terms "cancer" and "tumor" can be used interchangeably. For example, both terms encompass solid tumors and liquid tumors. As used herein, the term "cancer" or "tumor" includes precancer, as well as malignant cancers and tumors.

[0325] As used herein, "immune cell" refers to any of various cells that function in the immune system, for example, defend against infectious and foreign agents. In embodiments, the term includes white blood cells, such as neutrophils, eosinophils, basophils, lymphocytes, and monocytes. Innate white blood cells include phagocytes (e.g., macrophages, neutrophils, and dendritic cells), mast cells, eosinophils, basophils, and natural killer cells. Innate white blood cells identify and eliminate pathogens by attacking larger pathogens through contact or by engulfing and killing microorganisms and are mediators of activation of the adaptive immune response. Cells of the adaptive immune system are a special type of white blood cell called lymphocytes. B cells and T cells are important types of lymphocytes and are derived from hematopoietic stem cells in the bone marrow. B cells are involved in the humoral immune response, and T cells are involved in the cellular immune response. The term "immune cell" includes immune effector cells.

[0326] The term "immune effector cell," as used herein, refers to a cell involved in promoting an immune response, for example, an immune effector response. Examples of immune effector cells include, but are not limited to, T cells, such as alpha / beta T cells and gamma / delta T cells, B cells, natural killer (NK) cells, natural killer T cells, and mast cells.

[0327] The term "effector function" or "effector response" refers to the specialized functions of a cell. The effector functions of T cells can be, for example, cytolytic activity or helper activity including the secretion of cytokines.

[0328] The compositions and methods of the present invention include polypeptides and nucleic acids having a specific sequence, or a sequence that is substantially identical or similar thereto, for example, a sequence that is at least 80%, 85%, 90%, 95% identical to a specific sequence, or more identical. In the context of amino acid sequences, the term "substantially identical" is used herein such that the first and second amino acid sequences may have a common structural domain and / or a common functional activity, i) being identical to the second amino acid sequence, or ii) the first amino acid containing a sufficient or minimal number of amino acid residues that are conservative substitutions of the aligned amino acid residues in the second amino acid sequence. For example, an amino acid sequence containing a common structural domain has at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to a reference sequence, such as the sequences provided herein.

[0329] In the context of nucleotide sequences, the term "substantially identical" is used herein to refer to a first nucleic acid sequence containing a sufficient or minimal number of nucleotides that are identical to the aligned nucleotides in a second nucleic acid sequence such that the first and second nucleotide sequences encode a polypeptide having a common functional activity, or encode a common polypeptide structural domain or a common polypeptide functional activity. For example, a nucleotide sequence having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to a reference sequence, such as the sequences provided herein.

[0330] The term "variant" refers to a polypeptide having an amino acid sequence that is 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.

[0331] The term "functional variant" refers to a polypeptide having an amino acid sequence that is substantially identical to a reference amino acid sequence or encoded by a substantially identical nucleotide sequence and that may have one or more activities of the reference amino acid sequence.

[0332] The calculation of homology or sequence identity between sequences (these terms are used interchangeably herein) is performed as follows. To determine the percent identity between two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., for optimal alignment, gaps may be introduced into one or both of the first and second amino acid or nucleic acid sequences, and non-homologous sequences may be disregarded for comparison purposes). In preferred embodiments, the length of the reference sequence aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%, 60%, even more preferably at least 70%, 80%, 90%, 100% of the length of the reference sequence. Next, the amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are compared. If the position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, the molecules are identical at that position (as used herein, amino acid or nucleic acid "identity" is equal to amino acid or nucleic acid "homology").

[0333] The percent identity between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps that need to be introduced for optimal alignment of the two sequences and the length of each gap.

[0334] The comparison of sequences and determination of the percent identity between two sequences can be accomplished using a mathematical algorithm. In a preferred embodiment, the percent identity between two amino acid sequences is determined using the Needleman and Wunsch ((1970) J. Mol. Biol. 48:444-453) algorithm incorporated into the GAP program of the GCG software package (available at http: / / www.gcg.com) using either the Blossum62 matrix or PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. In yet another preferred embodiment, the percent identity between two nucleotide sequences is determined using the GAP program of the GCG software package (available at http: / / www.gcg.com) with the NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. A particularly preferred set of parameters (and those to be used unless otherwise specified) is the Blossum62 score matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5.

[0335] 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 the PAM120 residue weight table, a gap length penalty of 12, and a gap penalty of 4.

[0336] The nucleic acid and protein sequences described herein can be used, for example, as "query sequences" for performing searches against public databases to identify other family members or related sequences. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul et al. (1990) J. Mol. Biol. 215:403-10. BLAST nucleotide searches can be performed using the NBLAST program, score = 100, wordlength = 12, to obtain nucleotide sequences homologous to the nucleic acid molecules of the invention. BLAST protein searches can be performed using the XBLAST program, score = 50, wordlength = 3, to obtain amino acid sequences homologous to the protein molecules of the invention. To obtain gapped alignments for comparison purposes, gapped BLAST can be utilized as described in Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402. When utilizing the BLAST and gapped BLAST programs, the default parameters of each program (e.g., XBLAST and NBLAST) can be used. See http: / / www.ncbi.nlm.nih.gov.

[0337] It is understood that the molecules of the present invention can have additional conservative or non-essential amino acid substitutions that do not substantially affect their function. The term "amino acid" is intended to include both natural and synthetic molecules that contain both amino functionality and acid functionality and all molecules that can be included in a polymer of naturally occurring amino acids. Exemplary amino acids include naturally occurring amino acids; their analogs, derivatives and homologs; amino acid analogs with variant side chains; and all stereoisomers of any of the foregoing. As used herein, the term "amino acid" includes both D or L optical isomers and peptidomimetics.

[0338] "Conservative amino acid substitution" means that an amino acid residue is substituted with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains are defined in the art. These families include amino acids having basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[0339] The terms "polypeptide", "peptide" and "protein" (in the case of a single chain) are used interchangeably herein and refer to a polymer of amino acids of any length. The polymer can be linear or branched, can contain modified amino acids, and can be interrupted by non-amino acids. These terms also include amino acid polymers that have been modified; for example, any other manipulation such as disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or conjugation with a labeling component. A polypeptide can be isolated from a natural source, produced by recombinant techniques from a eukaryotic or prokaryotic host, or be the product of synthetic methods.

[0340] The terms "nucleic acid", "nucleic acid sequence", "nucleotide sequence", or "polynucleotide sequence", and "polynucleotide" are used interchangeably. They refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. A polynucleotide can be either single-stranded or double-stranded, and if single-stranded, can be either the coding strand or the non-coding (antisense) strand. A polynucleotide can include modified nucleotides such as methylated nucleotides and nucleotide analogs. The sequence of nucleotides can be interrupted by non-nucleotide components. A polynucleotide can be further modified after polymerization, for example, by conjugation with a labeling component. A nucleic acid can be a recombinant polynucleotide, or a polynucleotide of genomic, cDNA, semi-synthetic, or synthetic origin that is ligated to another polynucleotide in a non-native or unnatural sequence.

[0341] The term "isolated", as used herein, refers to a material that has been removed from its original environment or natural environment (e.g., the natural environment if it occurs naturally). For example, a naturally-occurring polynucleotide or polypeptide present in a living animal is not isolated, but the same polynucleotide or polypeptide separated by human intervention from some or all of the coexisting materials in the natural system is isolated. Such a polynucleotide can be part of a vector, and / or such a polynucleotide or polypeptide can be part of a composition, and such a vector or composition can still be considered isolated in that it is not part of the environment in which it is found naturally.

[0342] Various aspects of the invention are described in further detail below. Additional definitions are set forth throughout the specification. Antibody molecule In one embodiment, the antibody molecule binds to a cancer antigen, such as a tumor antigen or a stromal antigen. In some embodiments, the cancer antigen is, for example, a mammalian, such as a human, cancer antigen. In other embodiments, the antibody molecule binds to an immune cell antigen, such as a mammalian, such as a human, immune cell antigen. For example, the antibody molecule specifically binds to an epitope on a cancer antigen or an immune cell antigen, such as a linear epitope or a conformational epitope.

[0343] In one embodiment, the antibody molecule is a monospecific antibody molecule that binds to a single epitope. For example, the monospecific antibody molecule has a plurality of immunoglobulin variable domain sequences, each of which binds to the same epitope.

[0344] In one embodiment, the antibody molecule is a multispecific or multifunctional antibody molecule. For example, it includes a plurality of immunoglobulin variable domain sequences, wherein the first of the plurality of immunoglobulin variable domain sequences has binding specificity for a first epitope, and the second of the plurality of immunoglobulin variable domain sequences has binding specificity for a second epitope. In one embodiment, the first and second epitopes are on the same antigen, such as the same protein (or a subunit of a multimeric protein). In one embodiment, the first and second epitopes overlap. In one embodiment, the first and second epitopes do not overlap. In one embodiment, the first and second epitopes are on different antigens, such as different proteins (or different subunits of a multimeric protein). In one embodiment, the multispecific antibody molecule includes a third, fourth, or fifth immunoglobulin variable domain. In one embodiment, the multispecific antibody molecule is a bispecific antibody molecule, a trispecific antibody molecule, or a tetravalent antibody molecule.

[0345] In one embodiment, the multispecific antibody molecule is a bispecific antibody molecule. The bispecific antibody has specificity for only two antigens. The bispecific antibody molecule is characterized by a first immunoglobulin variable domain sequence having binding specificity for a first epitope and a second immunoglobulin variable domain sequence having binding specificity for a second epitope. In one embodiment, the first and second epitopes are on the same antigen, for example, the same protein (or a subunit of a multimeric protein). In one embodiment, the first and second epitopes overlap. In one embodiment, the first and second epitopes do not overlap. In one embodiment, the first and second epitopes are on different antigens, for example, different proteins (or different subunits of a multimeric protein). In one embodiment, the bispecific antibody molecule comprises a heavy chain variable domain sequence and a light chain variable domain sequence having binding specificity for a first epitope and a heavy chain variable domain sequence and a light chain variable domain sequence having binding specificity for a second epitope. In one embodiment, the bispecific antibody molecule comprises a half antibody having binding specificity for a first epitope and a half antibody having binding specificity for a second epitope. In one embodiment, the bispecific antibody molecule comprises a half antibody having binding specificity for a first epitope, or a fragment thereof, and a half antibody having binding specificity for a second epitope, or a fragment thereof. In one embodiment, the bispecific antibody molecule comprises a scFv or Fab having binding specificity for a first epitope, or a fragment thereof, and a scFv or Fab having binding specificity for a second epitope, or a fragment thereof.

[0346] In one embodiment, antibody molecules include antigen-binding fragments of antibodies (e.g., Fab, F(ab’)2, and Fv), in addition to diabodies and single-chain molecules. For example, an antibody molecule can include a heavy (H) chain variable domain sequence (abbreviated herein as VH) and a light (L) chain variable domain sequence (abbreviated herein as VL). In one embodiment, an antibody molecule comprises or consists of a heavy chain and a light chain (referred to herein as a half antibody). In another example, an antibody molecule includes two heavy (H) chain variable domain sequences and two light (L) chain variable domain sequences, thereby forming two antigen-binding sites, e.g., Fab, Fab’, F(ab’)2, Fc, Fd, Fd’, Fv, single-chain antibody (e.g., scFv), single variable domain antibody, diabody (Dab) (bivalent and bispecific), and chimeric (e.g., humanized) antibodies, which may be produced by modification of a full-length antibody or synthesized de novo using recombinant DNA technology. These functional antibody fragments retain their ability to selectively bind to their respective antigen or receptor. Antibodies and antibody fragments can be from any class of antibodies including, but not limited to, IgG, IgA, IgM, IgD, and IgE, and from any subclass of antibodies (e.g., IgG1, IgG2, IgG3, and IgG4). Preparations of antibody molecules can be monoclonal or polyclonal. Antibody molecules can also be human, humanized, CDR-grafted, or in vitro-generated antibodies. An antibody can have a heavy chain constant region selected, for example, from IgG1, IgG2, IgG3, or IgG4. An antibody can also have a light chain selected, for example, from kappa or lambda. The term “immunoglobulin” (Ig) is used interchangeably herein with the term “antibody”.

[0347] Examples of antigen-binding fragments of antibody molecules include: (i) Fab fragments, which are monovalent fragments consisting of VL, VH, CL, and CH1 domains; (ii) F(ab’)2 fragments, which are divalent fragments containing two Fab fragments linked by disulfide bridges in the hinge region; (iii) Fd fragments, which consist of VH and CH1 domains; (iv) Fv fragments, which consist of the VL and VH domains of a single arm of an antibody; (v) dAb (domain antibody) fragments, which consist of VH domains; (vi) camelid or camelized 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; and (viii) single-domain antibodies. These antibody fragments can be obtained using conventional techniques known to those skilled in the art, and the fragments are screened for utility in the same manner as intact antibodies.

[0348] Antibody molecules include, in addition to intact molecules, functional fragments thereof. The constant region of an antibody molecule can be modified, e.g., mutated, to modify the properties of the antibody (e.g., to increase or decrease one or more of Fc receptor binding, antibody glycosylation, number of cysteine residues, effector cell function, or complement function).

[0349] The antibody molecule can also be a single-domain antibody. A single-domain antibody can include an antibody in which its complementary determining region is part of a single-domain polypeptide. Examples include, but are not limited to, heavy-chain antibodies, antibodies that are naturally lacking in light chains, single-domain antibodies derived from conventional four-chain antibodies, engineered antibodies, and single-domain scaffolds other than those derived from antibodies. The single-domain antibody can be any one in the art or any future single-domain antibody. The single-domain antibody can be derived from any species including, but not limited to, mouse, human, camel, llama, fish, shark, goat, rabbit, and cow. According to another aspect of the invention, the single-domain antibody is a naturally occurring single-domain antibody known as a heavy-chain antibody lacking a light chain. Such single-domain antibodies are disclosed, for example, in WO9404678. For reasons of clarity, this variable domain derived from a heavy-chain antibody that is naturally lacking in a light chain is known herein as VHH or nanobody to distinguish it from the conventional VH of a four-chain immunoglobulin. Such VHH molecules can be derived from antibodies produced in camelid species such as camel, llama, alpaca, and guanaco. Other species other than camelids may produce heavy-chain antibodies that are naturally lacking in light chains, and such VHHs are within the scope of the invention.

[0350] The VH and VL regions can be further divided into hypervariable regions called "complementary determining regions" (CDRs) flanked by more conserved regions called "framework regions" (FR or FW).

[0351] The framework regions and the CDR ranges are precisely defined in a number of ways (see Kabat, E. A. et al., (1991) Sequences of Proteins of Immunological Interest, 5th Ed., U.S. Department of Health and Human Services, NIH Publication No. 91-3242; Chothia, C. et al., (1987) J. Mol. Biol. 196:901-917; and the AbM definition used by Oxford Molecular's AbM antibody modeling software. Generally, see, for example, "Protein Sequence and Structure Analysis of Antibody Variable Domains", Antibody Engineering Lab Manual (Duebel, S. and Kontermann, R., eds., Springer-Verlag, Heidelberg)).

[0352] The terms "complementary determining region" and "CDR", as used herein, refer to the sequences of amino acids within the antibody variable regions that confer antigen specificity and binding affinity. Generally, there are three CDRs (HCDR1, HCDR2, HCDR3) in each heavy chain variable region and three CDRs (LCDR1, LCDR2, LCDR3) in each light chain variable region.

[0353] The precise amino acid sequence boundaries of a given CDR can be determined using any of a number of well-known schemes, including those described in Kabat et al., (1991), "Sequences of Proteins of Immunological Interest", 5th Edition, Public Health Service, National Institutes of Health, Bethesda, MD ("Kabat" numbering scheme), Al-Lazikani et al., (1997) JMB 273, pages 927-948 ("Chothia" numbering scheme). As used herein, a CDR defined according to the "Chothia" numbering scheme may also be referred to as a "hypervariable loop".

[0354] For example, under Kabat, the CDR amino acid residues in the heavy chain variable domain (VH) are numbered 31-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3), and the CDR amino acid residues in the light chain variable domain (VL) are numbered 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3). Under Chothia, the CDR amino acids in VH are numbered 26-32 (HCDR1), 52-56 (HCDR2), and 95-102 (HCDR3), and the amino acid residues in VL are numbered 26-32 (LCDR1), 50-52 (LCDR2), and 91-96 (LCDR3).

[0355] Each VH and VL typically contains three CDRs and four FRs arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from the amino terminus to the carboxy terminus.

[0356] The antibody molecule can be a polyclonal or monoclonal antibody. 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 produced by hybridoma technology or by methods that do not use hybridoma technology (e.g., recombinant methods).

[0357] Antibodies can be produced recombinantly, e.g., by phage display or combinatorial methods. Phage display and combinatorial methods for generating antibodies are known in the art (e.g., Ladner et al., U.S. Patent No. 5,223,409; Kang et al., International Publication WO92 / 18619; Dower et al., International Publication WO91 / 17271; Winter et al., International Publication WO92 / 20791; Markland et al., International Publication WO92 / 15679; Breitling et al., International Publication WO93 / 01288; McCafferty et al., International Publication WO92 / 01047; Garrard et al., International Publication WO92 / 09690; Ladner et al., International Publication WO90 / 02809; Fuchs et al., (1991) Bio / Technology 9:1370-1372; Hay et al., (1992) Hum Antibod Hybridomas 3:81-85; Huse et al., (1989) Science 246:1275-1281; Griffiths et al., (1993) EMBO J 12:725-734; Hawkins et al., (1992) J Mol Biol 226:889-896; Clackson et al., (1991) Nature 352:624-628; Gram et al., (1992) PNAS 89:3576-3580; Garrad et al., (1991) Bio / Technology 9:1373-1377; Hoogenboom et al., (1991) Nuc Acid Res 19:4133-4137; and Barbas et al., (1991) PNAS 88:7978-7982, all of which are hereby incorporated by reference).

[0358] In one embodiment, the antibody is a fully human antibody (e.g., an antibody made in a mouse genetically engineered to produce antibodies from human immunoglobulin sequences), or a non-human antibody, e.g., a rodent (mouse or rat), goat, primate (e.g., monkey), camelid antibody. Preferably, the non-human antibody is a rodent (mouse or rat antibody). Methods for producing rodent antibodies are known in the art.

[0359] Human monoclonal antibodies can be produced using transgenic mice that have human immunoglobulin genes instead of mouse strains. Using spleen cells from these transgenic mice immunized with the antigen of interest, hybridomas are produced that secrete human mAbs having specific affinity for epitopes from human proteins (see, for example, Wood et al., International Application WO91 / 00906; Kucherlapati et al., PCT International Publication WO91 / 10741; Lonberg et al., International Application WO92 / 03918; Kay et al., International Application 92 / 03917; Lonberg, N. et al., 1994 Nature 368:856-859; Green, L.L. et al., 1994 Nature Genet. 7:13-21; Morrison, S.L. et al., 1994 Proc. Natl. Acad. Sci. USA 81:6851-6855; Bruggeman et al., 1993 Year Immunol 7:33-40; Tuaillon et al., 1993 PNAS 90:3720-3724; Bruggeman et al., 1991 Eur J Immunol 21:1323-1326).

[0360] Antibody molecules can have variable regions, or portions thereof, such as CDRs, that are produced in non-human organisms such as rats or mice. Chimeric, CDR-grafted, and humanized antibodies are within the scope of the invention. Antibody molecules that are produced in non-human organisms such as rats or mice and then modified, for example, in the variable framework or constant region to reduce antigenicity in humans are within the scope of the invention.

[0361] The "effective human" protein is a protein that does not substantially induce a neutralizing antibody response, such as a human anti-mouse antibody (HAMA) response. HAMA can be a problem in a number of situations, for example, in the treatment of chronic or relapsing disease states where antibody molecules are repeatedly administered. The HAMA response can render repeated antibody administration potentially ineffective due to increased antibody clearance from the serum (see, for example, Saleh et al., Cancer Immunol. Immunother., 32:180-190 (1990)) and also due to potential allergic reactions (see, for example, LoBuglio et al., Hybridoma, 5:5117-5123 (1986)).

[0362] Chimeric antibodies can be produced by recombinant DNA techniques known in the art (see Robinson et al., International Publication PCT / US86 / 02269; Akira et al., European Patent Application No. 184,187; Taniguchi, M., European Patent Application No. 171,496; Morrison et al., European Patent Application No. 173,494; Neuberger et al., International Application WO86 / 01533; Cabilly et al., U.S. Patent No. 4,816,567; Cabilly et al., European Patent Application No. 125,023; Better et al., (1988 Science 240:1041-1043); Liu et al., (1987) PNAS 84:3439-3443; Liu et al., 1987, J. Immunol. 139:3521-3526; Sun et al., (1987) PNAS 84:214-218; Nishimura et al., 1987, Canc. Res. 47:999-1005; Wood et al., (1985) Nature 314:446-449; and Shaw et al., 1988, J. Natl Cancer Inst. 80:1553-1559).

[0363] Humanized or CDR-grafted antibodies have at least one or two (of the heavy and / or light immunoglobulin chains), and generally all three recipient CDRs replaced with donor CDRs. The antibody may be replaced with at least a portion of the non-human CDRs, or only a portion of the CDRs may be replaced with non-human CDRs. It is only necessary to replace the number of CDRs required for binding to the antigen. Preferably, the donor is a rodent antibody, such as a rat or mouse antibody, and the recipient is a human framework or human consensus framework. Typically, the immunoglobulin providing the CDRs is referred to as the "donor", and the immunoglobulin providing the framework is referred to as the "acceptor". In one embodiment, the donor immunoglobulin is non-human (e.g., rodent). The acceptor framework is a naturally occurring (e.g., human) framework or consensus framework, or a sequence that is about 85% or higher, preferably 90%, 95%, 99% or more identical thereto.

[0364] As used herein, the term "consensus sequence" refers to a sequence formed from the amino acids (or nucleotides) that occur most frequently in a family of related sequences (see, e.g., Winnaker, From Genes to Clones (Verlagsgesellschaft, Weinheim, Germany 1987)). In a family of proteins, each position in the consensus sequence is occupied by the amino acid that occurs most frequently at that position in the family. If two amino acids occur equally frequently, either can be included in the consensus sequence. A "consensus framework" refers to the framework region in a consensus immunoglobulin sequence.

[0365] Antibody molecules can be humanized by methods known in the art (see, for example, Morrison, S. L., 1985, Science 229:1202-1207, Oi et al., 1986, BioTechniques 4:214, and Queen et al., U.S. Patent Nos. 5,585,089, 5,693,761, and 5,693,762, the entire contents of which are hereby incorporated by reference).

[0366] Humanized or CDR-grafted antibody molecules can be produced by CDR grafting or CDR replacement, in which one, two, or all of the CDRs of an immunoglobulin chain can be replaced. See, 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, U.S. Patent No. 5,225,539 (the entire contents of which are hereby expressly incorporated by reference). Winter describes a CDR grafting method that can be used to prepare the humanized antibodies of the present invention (British Patent Application No. 2188638A, filed Mar. 26, 1987; Winter, U.S. Patent No. 5,225,539), the contents of which are hereby incorporated by reference.

[0367] Humanized antibody molecules with specific amino acid substitutions, deletions, or additions are also within the scope of the present invention. The criteria for selecting amino acids from the donor are described in U.S. Patent No. 5,585,089, for example, columns 12-16 of U.S. Patent No. 5,585,089 (the contents of which are hereby incorporated by reference). Other techniques for humanizing antibodies are described in Padlan et al., EP519596A1, published Dec. 23, 1992.

[0368] The antibody molecule can be a single-chain antibody. The single-chain antibody (scFv) may be engineered (see, for example, Colcher, D. et al., (1999) Ann N Y Acad Sci 880:263-280; and Reiter, Y., (1996) Clin Cancer Res 2:245-252). The single-chain antibody can dimerize or multimerize to produce a multivalent antibody having specificity for different epitopes of the same target protein.

[0369] In still other embodiments, the antibody molecule has a heavy chain constant region selected from, for example, the heavy chain constant regions of IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE, particularly, for example, the (e.g., human) heavy chain constant regions of IgG1, IgG2, IgG3, and IgG4. In another embodiment, the antibody molecule has a light chain constant region selected from, for example, the (e.g., human) kappa or lambda light chain constant regions. The constant region can be altered, for example, mutated, to modify the properties of the antibody (e.g., to increase or decrease one or more of Fc receptor binding, antibody glycosylation, number of cysteine residues, effector cell function, and / or complement function). In one embodiment, the antibody has effector function and can fix complement. In other embodiments, the antibody does not recruit effector cells and does not fix complement. In another embodiment, the antibody has a reduced or no ability to bind to Fc receptors. For example, it is an isotype or subtype, fragment or other mutant that does not support binding to Fc receptors, e.g., it has a mutagenized Fc receptor-binding region or lacks it.

[0370] Methods for altering the constant region of an antibody are known in the art. Antibodies having altered functions, such as altered affinity for an effector ligand, e.g., an FcR on a cell, 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., EP388,151A1, U.S. Patent No. 5,624,821, and U.S. Patent No. 5,648,260, the entire contents of all of which are hereby incorporated by reference). Similar types of alterations that reduce or eliminate these functions when applied to mouse, or other species, immunoglobulins can be described.

[0371] Antibody molecules can be derivatized or conjugated to another functional molecule (e.g., another peptide or protein). As used herein, a “derivatized” antibody molecule is an antibody molecule that has been modified. Methods of derivatization include, but are not limited to, the addition of a fluorescent moiety, a radioactive nucleotide, a toxin, an enzyme, or an affinity ligand, e.g., biotin. Thus, the antibody molecules of the invention are intended to include derivatives and other modified forms of the antibodies described herein, including immunoadhesion molecules. For example, an antibody molecule can be functionally linked (by chemical coupling, genetic fusion, non-covalent association, or otherwise) to one or more other molecular entities, such as another antibody (e.g., a bispecific antibody or diabody), a detectable agent, a cytotoxic agent, a pharmaceutical agent, and / or a protein or peptide that can mediate association with another molecule of the antibody or antibody portion (e.g., a streptavidin core region or polyhistidine tag).

[0372] One type of derivatized antibody molecule is produced by crosslinking two or more antibodies (e.g., of the same or different types, such as to create a bispecific antibody). Suitable crosslinkers include heterobifunctional ones having two separately reactive groups separated by an appropriate spacer (e.g., m-maleimidobenzoyl-N-hydroxysuccinimide ester) or homobifunctional ones (e.g., disuccinimidyl suberate). Such linkers are available from Pierce Chemical Company, Rockford, Ill.

[0373] Multispecific or multifunctional antibody molecules Exemplary structures of the multispecific and multifunctional molecules defined herein are described throughout. Exemplary structures are further described in Weidle U et al., (2013) The Intriguing Options of Multispecific Antibody Formats for Treatment of Cancer. Cancer Genomics & Proteomics 10: 1-18 (2013); and Spiess C et al., (2015) Alternative molecular formats and therapeutic applications for bispecific antibodies. Molecular Immunology 67: 95-106 (the entire contents of each of which are hereby incorporated by reference herein).

[0374] In embodiments, the multispecific antibody molecule can include more than one antigen-binding site where different sites are specific for different antigens. In embodiments, the multispecific antibody molecule can bind to more than one (e.g., two or more) epitopes on the same antigen. In embodiments, the multispecific antibody molecule includes an antigen-binding site specific for a target cell (e.g., a cancer cell) and a different antigen-binding site specific for an immune effector cell. In one embodiment, the multispecific antibody molecule is a bispecific antibody molecule. Bispecific antibody molecules can be classified into five different structural groups: (i) bispecific immunoglobulin G (BsIgG), (ii) IgG with an additional antigen-binding moiety attached, (iii) bispecific antibody fragments, (iv) bispecific fusion proteins, and (v) bispecific antibody conjugates.

[0375] BsIgG is a monovalent format for each antigen. Exemplary BsIgG formats include, but are not limited to, crossMab, DAF (two-in-one), DAF (four-in-one), DutaMab, DT-IgG, knobs-in-holes common LC, knobs-in-holes assembly, charge pair, Fab arm exchange, SEED body, triomab, LUZ-Y, Fcab, κλ body, and orthogonal Fab. See Spiess et al., Mol. Immunol. 67 (2015): 95-106. Exemplary BsIgGs include catumaxomab (Fresenius Biotech, Trion Pharma, Neopharm) containing an anti-CD3 arm and an anti-EpCAM arm, and ertumaxomab (Neovii Biotech, Fresenius Biotech) targeting CD3 and HER2. In some embodiments, BsIgG comprises heavy chains engineered for heterodimerization. For example, the heavy chains can be engineered for heterodimerization using the "knobs-into-holes" strategy, the SEED platform, a common heavy chain (e.g., in a κλ body), and the use of a heterodimeric Fc region. See Spiess et al., Mol. Immunol. 67 (2015): 95-106. Strategies that have been used to avoid homodimeric heavy chain pairing in BsIgG include knobs-in-holes, duobody, azymetric, charge pair, HA-TF, SEED body, and differential protein A affinity. See the same reference. BsIgG can be produced by separate expression of the component antibodies in different host cells and subsequent purification / assembly into BsIgG. BsIgG can also be produced by expression of the component antibodies in a single host cell. BsIgG can be purified, for example, using affinity chromatography using protein A and sequential pH elution.

[0376] IgG with an added additional antigen-binding part is another format of bispecific antibody molecules. For example, a monospecific IgG can be engineered to have bispecificity by adding an additional antigen-binding unit to the monospecific IgG, for example, at the N or C terminus of either the heavy or light chain. Exemplary additional antigen-binding units include single-domain antibodies (e.g., variable heavy or variable light chains), engineered protein scaffolds, and paired antibody variable domains (e.g., single-chain variable fragments or variable fragments). See the same reference. Examples of the added IgG formats include dual variable domain IgG (DVD-Ig), IgG(H)-scFv, scFv-(H)IgG, IgG(L)-scFv, scFv-(L)IgG, IgG(L,H)-Fv, IgG(H)-V, V(H)-IgG, IgG(L)-V, V(L)-IgG, KIH IgG-scFab, 2scFv-IgG, IgG-2scFv, scFv4-Ig, zybody, and DVI-IgG (4-in-1). See Spiess et al., Mol. Immunol. 67 (2015): 95-106. An example of IgG-scFv is MM-141 (Merrimack Pharmaceuticals) that binds to IGF-1R and HER3. Examples of DVD-Ig include ABT-981 (AbbVie) that binds to IL-1α and IL-1β, and ABT-122 (AbbVie) that binds to TNF and IL-17A.

[0377] Bispecific antibody fragments (BsAbs) are formats of bispecific antibody molecules that lack some or all of the antibody constant domains. For example, some BsAbs lack the Fc region. In embodiments, the bispecific antibody fragment comprises heavy and light chain regions connected by a peptide linker that permits efficient expression of the BsAb in a single host cell. Exemplary bispecific antibody fragments include, but are not limited to, nanobodies, nanobody-HAS, BiTE, diabodies, DART, TandAb, scDiabody, scDiabody-CH3, diabody-CH3, tribodies, miniantibodies, minibodies, TriBi minibodies, scFv-CH3 KIH, Fab-scFv, scFv-CH-CL-scFv, F(ab’)2, F(ab’)2-scFv2, scFv-KIH, Fab-scFv-Fc, tetravalent HCAb, scDiabody-Fc, diabody-Fc, tandem scFv-Fc, and intrabodies. See the same reference. For example, the BiTE format comprises a tandem scFv, and the component scFvs bind to CD3 on T cells and a surface antigen on cancer cells.

[0378] Examples of bispecific fusion proteins include, for example, antibody fragments conjugated to other proteins to add additional specificity and / or functionality. An example of a bispecific fusion protein is immTAC, which comprises an anti-CD3 scFv conjugated to an affinity matured T cell receptor that recognizes an HLA-presented peptide. In embodiments, the dock-and-lock (DNL) method can be used to generate bispecific antibody molecules with higher valency. Also, fusion to an albumin-binding protein or human serum albumin can extend the serum half-life of the antibody fragment. See the same reference.

[0379] In embodiments, chemical conjugation, e.g., chemical conjugation of an antibody and / or an antibody fragment, can be used to generate a BsAb molecule. See the same reference. Exemplary bispecific antibody conjugates include the CovX body format, in which a low molecular weight drug is site-specifically conjugated to each Fab arm or a single reactive lysine in an antibody or fragment thereof. In embodiments, the conjugation improves the serum half-life of the low molecular weight drug. An exemplary CovX body is CVX-241 (NCT01004822), which comprises an antibody conjugated to two short peptides that inhibit either VEGF or Ang2. See the same reference.

[0380] Antibody molecules can be produced, for example, by recombinant expression of at least one or more components in a host system. Exemplary host systems include eukaryotic cells (e.g., mammalian cells, e.g., CHO cells, or insect cells, e.g., SF9 or S2 cells) and prokaryotic cells (e.g., E. coli). Bispecific antibody molecules can be produced by separate expression of the components in different host cells and subsequent purification / assembly. Alternatively, antibody molecules can be produced by expression of the components in a single host cell. Purification of bispecific antibody molecules can be performed by various methods, such as affinity chromatography, e.g., using Protein A and sequential pH elution. In other embodiments, affinity tags, e.g., histidine-containing tags, myc tags, or streptavidin tags, can be used for purification.

[0381] CDR grafted scaffold In an embodiment, the antibody molecule is a CDR-grafted scaffold domain. In an embodiment, the scaffold domain is based on a fibronectin domain, for example, a fibronectin type III domain. The overall fold of the fibronectin type III (Fn3) domain is closely related to that of the minimal functional antibody fragment, the variable domain of the antibody heavy chain. There are three loops at the ends of Fn3, and the positions of the BC, DE, and FG loops roughly correspond to those of CDR1, 2, and 3 of the VH domain of the antibody. Fn3 does not have a disulfide bond and thus, unlike antibodies and their fragments, is stable under reducing conditions (see, for example, WO98 / 56915, WO01 / 64942, WO00 / 34784). The Fn3 domain can be modified or altered (for example, using the CDRs or hypervariable loops described herein) to select a domain that binds to an antigen / marker / cell described herein, for example.

[0382] In embodiments, the scaffold domain, e.g., the folded domain, is based on a "minibody" scaffold created by deleting three beta strands from the heavy chain variable domain of an antibody, e.g., a monoclonal antibody (see, e.g., Tramontano et al., 1994, J Mol. Recognit. 7:9, and Martin et al., 1994, EMBO J. 13:5303-5309). The "minibody" can be used to present two hypervariable loops. In embodiments, the scaffold domain is a V-like domain (see, e.g., Coia et al., WO99 / 45110), or a domain derived from tendamistatin, a 74-residue six-stranded beta sheet sandwich held together by two disulfide bonds (see, e.g., McConnell and Hoess, 1995, J Mol. Biol. 250:460). For example, the loops of tendamistatin can be modified or altered (e.g., using CDRs or hypervariable loops) to select domains that bind to, e.g., the markers / antigens / cells described herein. Another exemplary scaffold domain is a beta-sandwich structure derived from the extracellular domain of CTLA-4 (see, e.g., WO00 / 60070).

[0383] 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 domain, echistatin, and BPTI, etc.), TPR repeats, the trifoil structure, zinc finger domains, DNA-binding proteins, particularly monomeric DNA-binding proteins, RNA-binding proteins, enzymes, e.g., proteases (particularly inactivated proteases), RNases, chaperones, e.g., thioredoxin, and heat shock proteins, as well as intracellular signaling domains (e.g., SH2 and SH3 domains). See, for example, U.S. Patent Application Publication No. 20040009530 and U.S. Patent No. 7,501,121, which are incorporated herein by reference.

[0384] In embodiments, the scaffold domain is evaluated and selected by, for example, one or more of the following criteria: (1) amino acid sequence, (2) the sequences of several homologous domains, (3) three-dimensional structure, and / or (4) stability data over a range of pH, temperature, salt, organic solvent, oxidant concentration. In embodiments, the scaffold domain is a small, stable protein domain, e.g., a protein of less than 100, 70, 50, 40 or 30 amino acids. The domain may contain one or more disulfide bonds or may chelate a metal, e.g., zinc.

[0385] Antibody-based fusions A variety of formats can be generated that contain additional binding entities attached to the N or C terminus of the antibody. These fusions having single-chain or disulfide-stabilized Fv or Fab result in the production of tetravalent molecules having a bivalent binding specificity for each antigen. Combinations of scFv and scFab with IgG allow the production of molecules that can recognize three or more different antigens.

[0386] Antibody-Fab fusions An antibody-Fab fusion is a bispecific antibody that includes a conventional antibody against a first target and a Fab against a second target fused to the C-terminus of the antibody heavy chain. Generally, the antibody and the Fab have a common light chain. The antibody fusion can be produced by (1) manipulating the DNA sequence of the target fusion and (2) transfecting the target DNA into a suitable host cell to express the fusion protein. As described by Coloma, J et al., (1997) Nature Biotech 15:159, the antibody-scFv fusion may be linked by a (Gly)-Ser linker between the C-terminus of the CH3 domain and the N-terminus of the scFv. antibody-scFv fusion An antibody-scFv fusion is a bispecific antibody that includes a conventional antibody and an scFv of a unique specificity fused to the C-terminus of the antibody heavy chain. The scFv can be fused to the C-terminus directly or through the heavy chain of the scFv through a linker peptide. The antibody fusion can be produced by (1) manipulating the DNA sequence of the target fusion and (2) transfecting the target DNA into a suitable host cell to express the fusion protein. As described by Coloma, J et al., (1997) Nature Biotech 15:159, the antibody-scFv fusion may be linked by a (Gly)-Ser linker between the C-terminus of the CH3 domain and the N-terminus of the scFv.

[0387] Variable domain immunoglobulin DVD A related format is the dual variable domain immunoglobulin (DVD) composed of VH and VL domains of a second specificity located at the N-terminus of the V domain by a shorter linker sequence.

[0388] Other exemplary bispecific antibody formats include, for example, the following: U.S. Patent Application Publication No. 20160114057A1, U.S. Patent Application Publication No. 20130243775A1, U.S. Patent Application Publication No. 20140051833, U.S. Patent Application Publication No. 20130022601, U.S. Patent Application Publication No. 20150017187A1, U.S. Patent Application Publication No. 20120201746A1, U.S. Patent Application Publication No. 20150133638A1, U.S. Patent Application Publication No. 20130266568A1, U.S. Patent Application Publication No. 20160145340A1, WO2015127158A1, U.S. Patent Application Publication No. 20150203591A1, U.S. Patent Application Publication No. 20140322221A1, U.S. Patent Application Publication No. 20130303396A1, U.S. Patent Application Publication No. 20110293613, U.S. Patent Application Publication No. 20130017200A1, U.S. Patent Application Publication No. 20160102135A1, WO2015197598A2, WO2015197582A1, U.S. Patent No. 9359437, U.S. Patent Application Publication No. 20150018529, WO2016115274A1, WO2016087416A1, U.S. Patent Application Publication No. 20080069820A1, U.S. Patent No. 9145588B, U.S. Patent No. 7919257, and U.S. Patent Application Publication No. 20150232560A1. Exemplary bispecific molecules utilizing the full-length antibody-Fab / scFab format include the following: U.S. Patent No. 9382323B2, U.S. Patent Application Publication No. 20140072581A1, U.S. Patent Application Publication No. 20140308285A1, U.S. Patent Application Publication No. 20130165638A1, U.S. Patent Application Publication No. 20130267686A1, U.S. Patent Application Publication No. 20140377269A1, U.S. Patent No. 7741446B2, and WO1995009917A1.Exemplary multispecific molecules that utilize a domain exchange format include, among others: those described in US Patent Application Publication No. 20150315296A1, WO2016087650A1, US Patent Application Publication No. 20160075785A1, WO2016016299A1, US Patent Application Publication No. 20160130347A1, US Patent Application Publication No. 20150166670, US Patent No. 8703132B2, US Patent Application Publication No. 20100316645, US Patent No. 8227577B2, US Patent Application Publication No. 20130078249.

[0389] Fc-containing entity (minibody) Fc-containing entities, also known as minibodies, can be generated by fusing scFv to the C-terminus of the constant heavy chain domain 3 (CH3-scFv) and / or the hinge region of an antibody with a different specificity (scFv-hinge-Fc). Trivalent entities with disulfide-stabilized variable domains fused to the C-terminus of the CH3 domain of IgG (without a peptide linker) can also be produced.

[0390] Fc-containing multispecific molecule In some embodiments, the multispecific molecules disclosed herein include an immunoglobulin constant region (e.g., an Fc region). Exemplary Fc regions can be selected from the heavy chain constant regions of IgG1, IgG2, IgG3, or IgG4, more particularly, the heavy chain constant regions of human IgG1, IgG2, IgG3, or IgG4.

[0391] In some embodiments, the immunoglobulin chain constant region (e.g., an Fc region) is modified, e.g., mutated, to increase or decrease one or more of Fc receptor binding, antibody glycosylation, the number of cysteine residues, effector cell function, or complement function.

[0392] In other embodiments, the boundary between the first and second immunoglobulin heavy chain constant regions (e.g., the first and second Fc regions) is altered, e.g., mutated, to increase or decrease dimerization compared to, e.g., an unmanipulated boundary, e.g., a naturally occurring boundary. For example, dimerization of the immunoglobulin heavy chain constant region (e.g., the Fc region) can be enhanced by providing one or more of paired holes and protrusions ("knobs-in-holes"), electrostatic interactions, or strand exchange at the Fc boundary of the first and second Fc regions, thereby forming a higher ratio of hetero-oligomers:homo-oligomers compared to, e.g., an unmanipulated boundary.

[0393] In some embodiments, the multispecific molecule comprises paired amino acid substitutions at one or more positions selected from, e.g., 347, 349, 350, 351, 366, 368, 370, 392, 394, 395, 397, 398, 399, 405, 407, or 409 of the Fc region of human IgG1. For example, the immunoglobulin heavy chain constant region (e.g., the Fc region) can comprise paired amino acid substitutions selected from T366S, L368A, or Y407V (e.g., corresponding to a hole or a cavity), and T366W (e.g., corresponding to a protrusion or a knob).

[0394] In other embodiments, the multifunctional molecule comprises a half-life extender, e.g., human serum albumin or an antibody molecule against human serum albumin. Heterodimeric antibody molecules and methods of making the same A variety of methods have been disclosed for producing multispecific antibodies to address the problem of incorrect heavy chain pairing. Exemplary methods are described below. Exemplary multispecific antibody formats and methods of making said multispecific antibodies are also disclosed, e.g., in Speiss et al., Molecular Immunology 67 (2015) pages 95-106, and Klein et al., mAb 4:6, pages 653-663, November / December 2012 (the respective contents of which are hereby incorporated by reference herein).

[0395] Heterodimerizing bispecific antibodies are based on the native IgG structure, with two binding arms recognizing different antigens. The IgG-derived format that enables defined monovalent (and simultaneous) antigen binding is generated by forced heavy chain heterodimerization combined with techniques that minimize light chain mispairing (e.g., common light chain). Forced heavy chain heterodimerization can be achieved, for example, using a knob-in-hole or a strand exchange engineering domain (SEED).

[0396] Knob-in-hole The knob-in-hole described in U.S. Patent No. 5,731,116, U.S. Patent No. 7,476,724, and Ridgway, J et al., (1996) Prot. Engineering 9(7):617-621 generally involves (1) mutating the CH3 domain of one or both antibodies to promote heterodimerization and (2) combining the mutated antibodies under conditions that promote heterodimerization. The "knob" or "protrusion" is typically created by replacing a small amino acid in the parental antibody with a larger amino acid (e.g., T366Y or T366W), and the "hole" or "pore" is created by replacing a larger residue in the parental antibody with a smaller amino acid (e.g., Y407T, T366S, L368A, and / or Y407V).

[0397] For bispecific antibodies containing an Fc domain, the introduction of specific mutations into the constant region of the heavy chain can be utilized to promote the correct heterodimerization of the Fc portion. Some such techniques are reviewed in Klein et al. (mAb (2012) 4:6, pages 1-11) (the content of which is hereby incorporated by reference in its entirety). These techniques include the "knob-into-hole" (KiH) approach involving the introduction of a bulky residue into one of the CH3 domains of one of the antibody heavy chains. This bulky residue fits into a complementary "hole" in the other CH3 domain of the paired heavy chain, thereby promoting the correct pairing of the heavy chains (see, for example, U.S. Patent No. 7,642,228).

[0398] Exemplary KiH mutations include S354C, T366W in the "knob" heavy chain and Y349C, T366S, L368A, Y407V in the "hole" heavy chain. Other exemplary KiH mutations are provided in Table 1, along with stabilizing Fc cysteine mutations as needed.

[0399]

Table 1

[0400] Other Fc mutations have been provided by Igawa and Tsunoda, who identified three negatively charged residues in the CH3 domain of one chain that pair with three positively charged residues in the CH3 domain of the other chain. These specific charged residue pairs are E356-K439, E357-K370, D399-K409 and their reverse. In addition to the following three mutations in chain A: E356K, E357K and D399K, alone or in combination with newly identified disulfide bridges, by introducing at least two of K370E, K409D, K439E in chain B, they were able to act favorably on very efficient heterodimerization while simultaneously suppressing homodimerization (Martens T et al., "A novel one-armed antic-Met antibody inhibits glioblastoma growth in vivo.", Clin Cancer Res 2006;12:6144-52; PMID:17062691). Xencor defined 41 variant pairs based on a combination of structural calculations and sequence information, which were then screened for maximum heterodimerization, defining the combination of S364H, F405A (HA) on chain A and Y349T, T394F (TF) on chain B (Moore GL et al., "A novel bispecific antibody format enables simultaneous bivalent and monovalent co-engagement of distinct target antigens.", MAbs 2011;3:546-57; PMID:22123055).

[0401] Other exemplary Fc mutations for promoting heterodimerization of bispecific antibodies include the following references (the contents of each of which are hereby incorporated by reference into this specification): WO2016071377A1, US Patent Application Publication No. 20140079689A1, US Patent Application Publication No. 20160194389A1, US Patent Application Publication No. 20160257763, WO2016071376A2, WO2015107026A1, WO2015107025A1, WO2015107015A1, US Patent Application Publication No. 20150353636A1, US Patent Application Publication No. 20140199294A1, US Patent No. 7750128B2, US Patent Application Publication No. 20160229915A1, US Patent Application Publication No. 20150344570A1, US Patent No. 8003774A1, US Patent Application Publication No. 20150337049A1, US Patent Application Publication No. 20150175707A1, US Patent Application Publication No. 20140242075A1, US Patent Application Publication No. 20130195849A1, US Patent Application Publication No. 20120149876A1, US Patent Application Publication No. 20140200331A1, US Patent No. 9309311B2, US Patent No. 8586713, US Patent Application Publication No. 20140037621A1, US Patent Application Publication No. 20130178605A1, US Patent Application Publication No. 20140363426A1, US Patent Application Publication No. 20140051835A1, and US Patent Application Publication No. 20110054151A1.

[0402] Stabilizing cysteine mutations have also been used in combination with KiH and other Fc heterodimerization-promoting variants (see, e.g., US Patent No. 7183076). Other exemplary cysteine modifications include, for example, those disclosed in US Patent Application Publication No. 20140348839A1, US Patent No. 7855275B2, and US Patent No. 9000130B2.

[0403] Chain Exchange Operation Domain (SEED) Heterodimeric Fc platforms are known that support the design of bispecific and asymmetric fusion proteins by engineering lock-exchange operation domains (SEED) C(H)3 heterodimers. These derivatives of human IgG and IgA C(H)3 domains create complementary human SEED C(H)3 heterodimers composed of alternating segments of the human IgA and IgG C(H)3 sequences. The resulting pair of SEED C(H)3 domains preferentially associate to form heterodimers when expressed in mammalian cells. SEED body (Sb) fusion proteins may be genetically linked to one or more fusion partners and consist of [IgG1 hinge]-C(H)2-[SEED C(H)3] (see, for example, Davis JH et al., "SEEDbodies: fusion proteins based on strand exchange engineered domain (SEED) CH3 heterodimers in an Fc analogue platform for asymmetric binders or immunofusions and bispecific antibodies.", Protein Eng Des Sel 2010;23:195-202; PMID:20299542 and U.S. Patent No. 8,871,912, the contents of each of which are hereby incorporated by reference herein). Duobody The "duobody" technology for producing bispecific antibodies with correct heavy chain pairing is known. The duobody technology involves three basic steps for generating stable bispecific human IgG1 antibodies in a post-production exchange reaction. In the first step, two IgG1s each containing a single matched mutation in the third constant (CH3) domain are produced separately using standard mammalian recombinant cell lines. Subsequently, these IgG1 antibodies are purified according to standard methods for recovery and purification. After production and purification (post-production), the two antibodies are recombined under controlled laboratory conditions to yield bispecific antibody products with very high yields (typically >95%) (see, for example, Labrijn et al., PNAS 2013;110(13):5145-5150 and Labrijn et al., Nature Protocols 2014;9(10):2450-63, the contents of each of which are hereby incorporated by reference herein).

[0404] Electrostatic interaction Methods for making multispecific antibodies using CH3 amino acid changes with charged amino acids such that homodimer formation is electrostatically unfavorable are disclosed. EP1870459 and WO2009089004 describe other strategies that favor heterodimer formation during co-expression of different antibody domains in host cells. In these methods, the heavy chain constant domain 3 (CH3), one or more residues that make up the CH3-CH3 interface in both CH3 domains, are replaced with charged amino acids such that homodimer formation is electrostatically unfavorable and heterodimerization is electrostatically favorable. Additional methods for making multispecific molecules using electrostatic interactions are described in references including US Patent Application Publication No. 20100015133, US Patent No. 8592562B2, US Patent No. 9200060B2, US Patent Application Publication No. 20140154254A1, and US Patent No. 9358286A1, the contents of each of which are hereby incorporated by reference herein).

[0405] Common light chain Light chain mispairing needs to be avoided in order to generate a homogeneous preparation of bispecific IgG. One way to achieve this is through the use of the common light chain principle, i.e., by combining two binders that share one light chain but still have separate specificities. An exemplary method for enhancing the formation of the desired bispecific antibody from a mixture of monomers is by providing a common variable light chain for interacting with each of the heteromeric variable heavy chain regions of the bispecific antibody. Compositions of bispecific antibodies having a common light chain and methods for their production are disclosed, for example, in U.S. Patent No. 7183076 B2, U.S. Patent Application Publication No. 20110177073 A1, EP2847231 A1, WO2016079081 A1, and EP3055329 A1 (the contents of each of which are hereby incorporated by reference herein).

[0406] CrossMab Another option for reducing light chain mispairing is CrossMab technology, which avoids non-specific L-chain mispairing by exchanging the CH1 and CL domains in the Fab of one half of a bispecific antibody. Such crossover variants retain binding specificity and affinity but make the two arms different so that L-chain mispairing is prevented. (As reviewed by Klein et al., supra) The CrossMab technology involves domain swapping between the heavy and light chains to facilitate the formation of correct pairings. Briefly, a two-step modification process is applied to construct a bispecific IgG-like CrossMab antibody that can bind to two antigens by using two separate light chain-heavy chain pairs. First, the dimerization interface is engineered into the C-terminus of each heavy chain using a heterodimerization approach, such as the knob-into-hole (KiH) technology, to ensure that only heterodimers of two separate heavy chains from one antibody (e.g., antibody A) and a second antibody (e.g., antibody B) are efficiently formed. Next, the constant heavy chain 1 (CH1) and constant light chain (CL) domains of one antibody (antibody A) are exchanged while keeping the variable heavy chain (VH) and variable light chain (VL) domains consistent. The exchange of the CH1 and CL domains ensures that only the desired bispecific CrossMab is efficiently formed because the modified antibody (antibody A) light chain dimerizes efficiently only with the modified antibody (antibody A) heavy chain and the unmodified antibody (antibody B) light chain dimerizes efficiently only with the unmodified antibody (antibody B) heavy chain (see, for example, Cain, C., SciBX 4(28); doi:10.1038 / scibx.2011.783, the content of which is hereby incorporated by reference herein).

[0407] Common heavy chain Exemplary methods for enhancing the formation of a desired bispecific antibody from a mixture of monomers involve providing a common variable heavy chain for interacting with each of the heteromeric variable light chain regions of the bispecific antibody. Compositions of bispecific antibodies having a common heavy chain and methods for their production are disclosed, for example, in U.S. Patent Application Publication No. 20120184716, U.S. Patent Application Publication No. 20130317200, and U.S. Patent Application Publication No. 20160264685A1, the contents of each of which are hereby incorporated by reference herein.

[0408] Amino acid modifications Alternative compositions of multispecific antibodies having correct light chain pairing and methods for their production include various amino acid modifications. For example, Zymeworks describes one or more amino acid modifications in the CH1 and / or CL domains, in the VH and / or VL domains, or a combination thereof, which are part of the interface between the light and heavy chains and create preferential pairing of each heavy chain with a desired light chain, such that when two heavy chains and two light chains of a heterodimer pair are co-expressed in a cell, the heavy chain of the first heterodimer preferentially pairs with one of the light chains over the others (see, for example, WO2015181805). Other exemplary methods are described in WO2016026943 (Argen-X), U.S. Patent Application Publication No. 20150211001, U.S. Patent Application Publication No. 20140072581A1, U.S. Patent Application Publication No. 20160039947A1, and U.S. Patent Application Publication No. 20150368352.

[0409] Lambda / kappa format Multispecific molecules (e.g., multispecific antibody molecules) comprising lambda light chain polypeptides and kappa light chain polypeptides can be used to enable heterodimerization. Methods for generating bispecific antibody molecules comprising lambda light chain polypeptides and kappa light chain polypeptides are disclosed in PCT / US17 / 53053, filed September 22, 2017, the entire contents of which are hereby incorporated by reference herein.

[0410] In an embodiment, the multispecific molecule includes a multispecific antibody molecule, for example, an antibody molecule having two binding specificities, for example, a bispecific antibody molecule. The multispecific antibody molecule includes a lambda light chain polypeptide 1 (LLCP1) specific for a first epitope, a heavy chain polypeptide 1 (HCP1) specific for the first epitope, a kappa light chain polypeptide 2 (KLCP2) specific for a second epitope, and a heavy chain polypeptide 2 (HCP2) specific for the second epitope. It includes.

[0411] "Lambda light chain polypeptide 1 (LLCP1)", as the term is used herein, refers to a polypeptide that, when combined with its cognate heavy chain variable region, mediates specific binding to its epitope and can complex with HCP1, and includes a light chain (LC) sequence sufficient to do so. In one embodiment, it includes all or a fragment of the CH1 region. In one embodiment, LLCP1 includes LC-CDR1, LC-CDR2, LC-CDR3, FR1, FR2, FR3, FR4, and CH1, or sequences therefrom sufficient to mediate specific binding to its epitope and complex with HCP1. LLCP1, together with its HCP1, provides specificity for the first epitope (KLCP2, together with its HCP2, provides specificity for the second epitope). As described elsewhere herein, LLCP1 has a higher affinity for HCP1 than for HCP2.

[0412] "Kappa light chain polypeptide 2 (KLCP2)", as the term is used herein, refers to a polypeptide that, when combined with its cognate heavy chain variable region, contains a light chain (LC) sequence sufficient to mediate specific binding to its epitope and to complex with HCP2. In one embodiment, it includes all or a fragment of the CH1 region. In one embodiment, KLCP2 includes LC-CDR1, LC-CDR2, LC-CDR3, FR1, FR2, FR3, FR4, and CH1, or sequences therefrom sufficient to mediate specific binding to its epitope and to complex with HCP2. KLCP2, together with its HCP2, provides specificity for a second epitope (LLCP1, together with its HCP1, provides specificity for a first epitope).

[0413] "Heavy chain polypeptide 1 (HCP1)", as the term is used herein, refers to a polypeptide that, when combined with its cognate LLCP1, contains a heavy chain (HC) sequence sufficient to mediate specific binding to its epitope and to complex with HCP1, e.g., an HC variable region sequence. In one embodiment, it includes all or a fragment of the CH1 region. In one embodiment, it includes all or a fragment of the CH2 and / or CH3 regions. In one embodiment, HCP1 includes HC-CDR1, HC-CDR2, HC-CDR3, FR1, FR2, FR3, FR4, CH1, CH2, and CH3, or (i) sequences therefrom sufficient to mediate specific binding to its epitope and to complex with LLCP1, (ii) to preferentially complex with LLCP1 as contrasted with KLCP2 as described herein, and (iii) to preferentially complex with HCP2 as contrasted with another molecule of HCP1 as described herein. HCP1, together with its LLCP1, provides specificity for a first epitope (KLCP2, together with its HCP2, provides specificity for a second epitope).

[0414] "Heavy chain polypeptide 2 (HCP2)", as the term is used herein, refers to a polypeptide comprising a heavy chain (HC) sequence, e.g., an HC variable region sequence, that is sufficient to mediate specific binding to its epitope and complex with HCP1 when combined with cognate LLCP1. In one embodiment, it comprises all or a fragment of the CH1 region. In one embodiment, it comprises all or a fragment of the CH2 and / or CH3 r...

Claims

**Claim 1** (i) a first antigen-binding domain that binds to a tumor antigen, wherein the tumor antigen is the T cell receptor beta chain constant domain 1 (TRBC1), (a) a VH comprising a VHCDR1 sequence, a VHCDR2 sequence, and a VHCDR3 sequence comprising the sequences of SEQ ID NOs: 7346, 7355, and 202, respectively, and a VL comprising a VLCDR1 sequence, a VLCDR2 sequence, and a VLCDR3 sequence comprising the sequences of SEQ ID NOs: 223, 224, and 225, respectively; or (b) a VH comprising a VHCDR1 sequence, a VHCDR2 sequence, and a VHCDR3 sequence comprising the sequences of SEQ ID NOs: 7346, 201, and 202, respectively, and a VL comprising a VLCDR1 sequence, a VLCDR2 sequence, and a VLCDR3 sequence comprising the sequences of SEQ ID NOs: 223, 224, and 225, respectively comprising the first antigen-binding domain, and (ii) a second antigen-binding domain that binds to NKp30 A multispecific molecule that binds to NK cells and activates NK cells. **Claim 2** The multispecific molecule comprises a dimerization module comprising a first immunoglobulin chain constant region and a second immunoglobulin chain constant region, and The first antigen-binding domain binds to the first immunoglobulin chain constant region, and the second antigen-binding domain binds to the first immunoglobulin chain constant region or the second immunoglobulin chain constant region. The multispecific molecule according to claim 1. **Claim 3** (i) a first antigen-binding domain that binds to a tumor antigen, wherein the tumor antigen is the T cell receptor beta chain constant domain 1 (TRBC1), (a) a VH comprising a VHCDR1 sequence, a VHCDR2 sequence, and a VHCDR3 sequence comprising the sequences of SEQ ID NOs: 7346, 7355, and 202, respectively, and a VL comprising a VLCDR1 sequence, a VLCDR2 sequence, and a VLCDR3 sequence comprising the sequences of SEQ ID NOs: 223, 224, and 225, respectively; (b) a VH comprising a VHCDR1 sequence, a VHCDR2 sequence, and a VHCDR3 sequence comprising the sequences of SEQ ID NOs: 7346, 201, and 202, respectively, and a VL comprising a VLCDR1 sequence, a VLCDR2 sequence, and a VLCDR3 sequence comprising the sequences of SEQ ID NOs: 223, 224, and 225, respectively comprising the first antigen-binding domain, and (ii) a second antigen-binding domain that binds to NKp30, A VH comprising a VH CDR1 sequence, a VH CDR2 sequence, and a VH CDR3 sequence, each comprising the sequences of SEQ ID NOs: 7313, 6001, and 7315, respectively, and a VL comprising a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, each comprising the sequences of SEQ ID NOs: 7326, 7327, and 7329, respectively and a second antigen-binding domain comprising A multispecific molecule comprising, which binds to NK cells and activates NK cells

4. The first antigen-binding domain is (i) a VH comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 7351 and a VL comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 258; or (ii) a VH comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 253 and a VL comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 258 The multispecific molecule according to any one of claims 1 to 3, comprising

5. The first antigen-binding domain is (i) a VH comprising the amino acid sequence of SEQ ID NO: 7351 and a VL comprising the amino acid sequence of SEQ ID NO: 258; or (ii) a VH comprising the amino acid sequence of SEQ ID NO: 253 and a VL comprising the amino acid sequence of SEQ ID NO: 258 The multispecific molecule according to any one of claims 1 to 4, comprising

6. The first antigen-binding domain comprises a VH comprising an amino acid sequence having at least 90% sequence identity with the sequence of SEQ ID NO: 7351 and a VL comprising an amino acid sequence having at least 90% sequence identity with the sequence of SEQ ID NO:

258. The multispecific molecule according to any one of claims 1 to 3

7. The first antigen-binding domain comprises a VH comprising the sequence of SEQ ID NO: 7351 and a VL comprising the sequence of SEQ ID NO:

258. The multispecific molecule according to any one of claims 1 to 6

8. The first antigen-binding domain is (i) a heavy chain comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 7379 and a light chain comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 7380; or (ii) a heavy chain comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 7382 and a light chain comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 7380 The multispecific molecule according to any one of claims 1 to 7, comprising

9. The first antigen-binding domain is (i) 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; or (ii) a heavy chain comprising the amino acid sequence of SEQ ID NO: 7382 and a light chain comprising the amino acid sequence of SEQ ID NO: 7380 The multispecific molecule according to any one of claims 1 to 8, comprising.

10. The second antigen-binding domain is a VH comprising a VHCDR1 sequence, a VHCDR2 sequence, and a VHCDR3 sequence each comprising the sequences of SEQ ID NOs: 7313, 6001, and 7315, and a VL comprising a VLCDR1 sequence, a VLCDR2 sequence, and a VLCDR3 sequence each comprising the sequences of SEQ ID NOs: 7326, 7327, and 7329, the multispecific molecule according to claim 1 or 2.

11. The second antigen-binding domain is a VH comprising an amino acid sequence having at least 90% sequence identity with the sequence of SEQ ID NO: 7302 and a VL comprising an amino acid sequence having at least 90% sequence identity with the sequence of SEQ ID NO: 7309 The multispecific molecule according to claim 3 or 10, comprising.

12. The second antigen-binding domain is a VH comprising the amino acid sequence of SEQ ID NO: 7302 and a VL comprising the amino acid sequence of SEQ ID NO: 7309, the multispecific molecule according to claim 3, 10 or 11.

13. The second antigen-binding domain is an amino acid sequence having at least 90% sequence identity with any one of the sequences of SEQ ID NOs: 7310, 7311, 7383 and 7384 The multispecific molecule according to any one of claims 3 and 10 to 12, comprising.

14. The second antigen-binding domain is an amino acid sequence selected from the group consisting of SEQ ID NOs: 7310, 7311, 7383 and 7384 The multispecific molecule according to any one of claims 3 and 10 to 13, comprising.

15. The first antigen-binding domain comprises a Fab or scFv, the second antigen-binding domain comprises a Fab or scFv, or a combination of any of them, the multispecific molecule according to any one of claims 1 to 14.

16. The multispecific molecule is (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 sequences of SEQ ID NO: 7302 and SEQ ID NO: 7309; (ii) 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: 7311; (iii) a first antigen-binding domain comprising the sequences of SEQ ID NO: 253 and SEQ ID NO: 258, and a second antigen-binding domain comprising the sequences of SEQ ID NO: 7302 and SEQ ID NO: 7309; (iv) a first antigen-binding domain comprising the sequences of SEQ ID NO: 253 and SEQ ID NO: 258, and a second antigen-binding domain comprising the sequence of SEQ ID NO: 7311; (v) 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: 7310; or (vi) a first antigen-binding domain comprising the sequences of SEQ ID NO: 253 and SEQ ID NO: 258, and a second antigen-binding domain comprising the sequence of SEQ ID NO: 7310 The multispecific molecule according to any one of claims 1 to 15, comprising.

17. The multispecific molecule is (i) a first antigen-binding domain comprising the sequences of SEQ ID NO: 7379 and SEQ ID NO: 7380, and a second antigen-binding domain comprising the sequence of SEQ ID NO: 7381; (ii) a first antigen-binding domain comprising the sequences of SEQ ID NO: 7382 and SEQ ID NO: 7380, and a second antigen-binding domain comprising the sequence of SEQ ID NO: 7383; (iii) a first antigen-binding domain comprising the sequences of SEQ ID NO: 7379 and SEQ ID NO: 7380, and a second antigen-binding domain comprising the sequence of SEQ ID NO: 7383; (iv) a first antigen-binding domain comprising the sequences of SEQ ID NO: 7382 and SEQ ID NO: 7380, and a second antigen-binding domain comprising the sequence of SEQ ID NO: 7384; or (v) a first antigen-binding domain comprising the sequences of SEQ ID NO: 7379 and SEQ ID NO: 7380, and a second antigen-binding domain comprising the sequence of SEQ ID NO: 7384 The multispecific molecule according to any one of claims 1 to 16, comprising.

18. The multispecific molecule according to any one of claims 1 to 17, further comprising a cytokine molecule.

19. The multispecific molecule according to claim 18, wherein the cytokine molecule is interleukin-2 (IL-2) or a functional variant thereof, interleukin-7 (IL-7) or a functional variant thereof, interleukin-12 (IL-12) or a functional variant thereof, interleukin-15 (IL-15) or a functional variant thereof, interleukin-18 (IL-18) or a functional variant thereof, interleukin-21 (IL-21) or a functional variant thereof, interferon γ or a functional variant thereof, or any combination thereof.

20. The first immunoglobulin heavy chain constant region, the second immunoglobulin heavy chain constant region, or a combination thereof is (i) one or more of paired holes and protrusions, electrostatic interactions, or strand exchange; (ii) one or more mutations that result in a reduction or elimination of affinity for at least one Fc receptor; or (iii) any combination thereof The multispecific molecule according to any one of claims 2 to 19, comprising.

21. A polynucleotide comprising a sequence encoding the multispecific molecule according to any one of claims 1 to 20.

22. A method for producing the multispecific molecule according to any one of claims 1 to 20, comprising culturing a cell comprising the polynucleotide according to claim 21 under conditions suitable for gene expression and / or homo- or heterodimerization.

23. A pharmaceutical composition comprising the multispecific molecule according to any one of claims 1 to 20 and a pharmaceutically acceptable carrier, excipient, or stabilizer.

24. A pharmaceutical composition comprising the multispecific molecule according to any one of claims 1 to 20 for use in the treatment of cancer in a subject in need thereof.

25. The pharmaceutical composition according to claim 24, wherein (i) the cancer is leukemia or lymphoma; or A pharmaceutical composition, wherein the cancer is selected from the group consisting of acquired immunodeficiency syndrome (AIDS)-related lymphoma, angioimmunoblastic T-cell lymphoma, adult T-cell leukemia / lymphoma, Burkitt lymphoma, central nervous system (CNS) lymphoma, diffuse large B-cell lymphoma (DLBCL), lymphoblastic lymphoma, mantle cell lymphoma (MCL), peripheral T-cell lymphoma (PTCL), transformed follicular lymphoma and transformed mucosa-associated lymphoid tissue (MALT) lymphoma, cutaneous T-cell lymphoma (mycosis fungoides and Sézary syndrome), follicular lymphoma, lymphoplasmacytic lymphoma / Waldenström macroglobulinemia, marginal zone B-cell lymphoma, gastric mucosa-associated lymphoid tissue (MALT) lymphoma, chronic lymphocytic leukemia / small lymphocytic lymphoma (CLL / SLL), extranodal T / NK-cell lymphoma (nasal type), and anaplastic large cell lymphoma.

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