Multispecific binding proteins and improvements thereon

Abstract

To provide multi-specific binding proteins and improvement thereof.SOLUTION: The invention provides improvement of single-chain variable fragment (scFv) antibodies, multi-specific binding proteins, pharmaceutical compositions comprising such proteins, and therapeutic methods using such proteins and pharmaceutical compositions, including treatment of cancer. In one aspect, the current invention provides improvement of a single-chain variable fragment (scFv) that is linked to an antibody constant domain via a hinge sequence. In some embodiments, the hinge comprises amino acids Ala-Ser.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of and priority to U.S. Provisional Patent Application No. 62 / 677,137, filed May 28, 2018, the disclosure of which is incorporated herein by reference in its entirety for all purposes.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The above ASCII copy, created on May 28, 2019, is named DFY-049WO_SL_ST25.txt and is 340,101 bytes in size.

[0003] FIELD OF THE INVENTION The present invention provides improved single-chain variable fragment (scFv) antibodies and multispecific binding proteins, pharmaceutical compositions comprising such proteins, and methods of treatment using such proteins and pharmaceutical compositions, including the treatment of cancer. [Background technology]

[0004] Cancer remains a serious health problem, despite considerable research efforts and scientific advances reported in the literature to treat this disease. Some of the most frequently diagnosed cancers include prostate cancer, breast cancer, and lung cancer. Prostate cancer is the most common form of cancer in men. Breast cancer remains the leading cause of death in women. Current treatment options for these cancers may not be effective for all patients and / or may have substantial adverse side effects. Other types of cancer also remain challenging to treat using existing treatment options.

[0005] Cancer immunotherapy is desirable because it is highly specific and can utilize the patient's own immune system to promote the destruction of cancer cells. Fusion proteins, such as bispecific T cell-engaging agents, are a cancer immunotherapy described in the literature that bind to tumor cells and T cells to promote tumor cell destruction. Antibodies that bind to specific tumor-associated antigens and specific immune cells have been described in the literature. See, for example, WO2016 / 134371 and WO2015 / 095412.

[0006] Natural killer (NK) cells are components of the innate immune system and account for approximately 15% of circulating lymphocytes. NK cells were originally characterized by their ability to infiltrate virtually all tissues and efficiently kill tumor cells without the need for prior sensitization. Activated NK cells kill target cells by means similar to cytotoxic T cells, i.e., via cytolytic granules containing perforin and granzymes and via death receptor pathways. Activated NK cells also secrete proinflammatory cytokines, such as IFN-γ and chemokines, which promote the recruitment of other leukocytes to target tissues. NK cells respond to signals through various activating and inhibitory receptors on their surface. For example, when NK cells encounter healthy autologous cells, their activity is inhibited through activation of killer cell immunoglobulin-like receptors (KIRs). Alternatively, when NK cells encounter foreign or cancer cells, they are activated through their activating receptors (e.g., NKG2D, NCR, DNAM1). NK cells are also activated by the constant regions of several immunoglobulins via the CD16 receptor on their surface. The overall sensitivity of NK cells to activation depends on the sum of stimulatory and inhibitory signals. NKG2D is a type II transmembrane protein expressed by essentially all natural killer cells, where it functions as an activating receptor. NKG2D is also found on T cells, where it functions as a costimulatory receptor. The ability to modulate NK cell function through NKG2D is useful in various therapeutic settings, including malignancies. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] International Publication No. 2016 / 134371 [Patent Document 2] International Publication No. 2015 / 095412 Summary of the Invention

[0008] In one aspect, the present invention provides improved single-chain variable fragments (scFvs) linked to an antibody constant domain via a hinge sequence. In some embodiments, the hinge comprises the amino acids Ala-Ser. In some other embodiments, the hinge comprises the amino acids Ala-Ser and Thr-Lys-Gly. The scFvs may comprise a heavy chain variable domain and a light chain variable domain. In some embodiments, the scFvs bind to NKG2D or a tumor-associated antigen. The hinge sequence provides flexibility in binding to the target antigen.

[0009] In some scFv embodiments, the heavy chain variable domain forms a disulfide bridge with the light chain variable domain. For example, a disulfide bridge can be formed between the C44 residue of the heavy chain variable domain and the C100 residue of the light chain variable domain. In some embodiments, the heavy chain variable domain is linked to the light chain variable domain via a flexible linker such as (G4S)4 (SEQ ID NO: 427). In some scFv embodiments, the heavy chain variable domain is located at the N-terminus of the light chain variable domain. In some scFv embodiments, the heavy chain variable domain is located at the C-terminus of the light chain variable domain.

[0010] In some embodiments, the antibody constant domain linked to the scFv is capable of binding to CD16. In some embodiments, the antibody constant domain comprises the CH2 and CH3 domains of an IgG antibody, e.g., a human IgG1 antibody. In some embodiments, mutations are introduced into the antibody constant domain to enable heterodimerization with another antibody constant domain. For example, if the antibody constant domain is derived from a human IgG1 constant domain, the antibody constant domain may comprise an amino acid sequence that is at least 90% identical to amino acids 234-332 of a human IgG1 antibody and differs at one or more positions selected from the group consisting of Q347, Y349, L351, S354, E356, E357, K360, Q362, S364, T366, L368, K370, N390, K392, T394, D399, S400, D401, F405, Y407, K409, T411, and K439. In some embodiments, the antibody constant domain may comprise an amino acid sequence at least 90% identical to amino acids 234-332 of a human IgG1 antibody, and may be selected from the group consisting of Q347E, Q347R, Y349S, Y349K, Y349T, Y349D, Y349E, Y349C, L351K, L351D, L351Y, S354C, E356K, E357Q, E357L, E357W, K360E, K360W, Q362E, S364K, S364E, S364H, S364D, T366V, T366I, T366L, T366M, and differing by one or more substitutions selected from the group consisting of T366K, T366W, T366S, L368E, L368A, L368D, K370S, N390D, N390E, K392L, K392M, K392V, K392F, K392D, K392E, T394F, D399R, D399K, D399V, S400K, S400R, D401K, F405A, F405T, Y407A, Y407I, Y407V, K409F, K409W, K409D, T411D, T411E, K439D, and K439E.

[0011] In another aspect, the invention provides a protein comprising an scFv linked to an antibody constant region as described above. In some embodiments, the protein comprises a first antigen-binding site comprising an scFv linked to an antibody constant domain, a second antigen-binding site which may be in the format of a Fab or scFv as described herein, and a second antibody constant domain linked to the second antigen-binding site. In some embodiments, the protein is multispecific, wherein the first antigen-binding site binds to NKG2D, the second antigen-binding site binds to a tumor-associated antigen, and the antibody constant region binds to CD16.

[0012] In some other embodiments, the protein is multispecific, with the first antigen-binding site binding to a tumor-associated antigen, the second antigen-binding site binding to NKG2D, and the antibody constant region binding to CD16. The antibody constant region linked to the scFv may heterodimerize with a second antibody constant region. The multispecific binding protein in these embodiments binds to the NKG2D and CD16 receptors on natural killer cells and to a tumor-associated antigen on cancer cells. Such proteins may engage multiple types of NK activating receptors and inhibit the binding of natural ligands to NKG2D. In certain embodiments, the protein may agonize human NK cells. In some embodiments, the protein may agonize NK cells in humans and / or in other species, such as rodents and / or cynomolgus monkeys.

[0013] In another embodiment, the present invention provides a multispecific binding protein comprising a first antigen-binding site that binds to a tumor-associated antigen, a second antigen-binding site that binds to the same tumor-associated antigen as the first antigen-binding site, a third antigen-binding site that binds to NKG2D, and a sufficient antibody constant region or portion thereof that binds to CD16, or a fourth antigen-binding site that binds to CD16. Any one of the antigen-binding sites can take the form of either a Fab or scFv, as described above. The multispecific binding proteins provided herein provide bivalent engagement of the tumor-associated antigen, thereby stabilizing the tumor-associated antigen on the surface of cancer cells and enhancing NK cell-mediated cytotoxicity against the cancer cells.

[0014] In some embodiments, bivalent engagement of tumor-associated antigens by multispecific binding proteins confers stronger binding of the multispecific binding protein to cancer cells, thereby promoting a stronger cytotoxic response of NK cells against cancer cells, particularly against cancer cells that express low levels of the tumor-associated antigen.

[0015] In some embodiments, the multispecific binding protein incorporates a sufficient portion of an antibody Fc domain to bind CD16, the antibody Fc domain comprising the hinge and CH2 domain and / or an amino acid sequence at least 90% identical to amino acid sequence 234-332 of a human IgG antibody. Mutations may be introduced into this antibody constant domain to allow heterodimerization with another antibody constant domain. For example, if the antibody constant domain is derived from the constant domain of human IgG1, the antibody constant domain may comprise an amino acid sequence that is at least 90% identical to amino acids 234 to 332 of a human IgG1 antibody and differs at one or more positions selected from the group consisting of Q347, Y349, L351, S354, E356, E357, K360, Q362, S364, T366, L368, K370, N390, K392, T394, D399, S400, D401, F405, Y407, K409, T411, and K439.

[0016] In some embodiments, the antibody constant domain may comprise an amino acid sequence at least 90% identical to amino acids 234-332 of a human IgG1 antibody, and may be selected from the group consisting of Q347E, Q347R, Y349S, Y349K, Y349T, Y349D, Y349E, Y349C, L351K, L351D, L351Y, S354C, E356K, E357Q, E357L, E357W, K360E, K360W, Q362E, S364K, S364E, S364H, S364D, T366V, T366I, T366L, T366M, and differing by one or more substitutions selected from the group consisting of T366K, T366W, T366S, L368E, L368A, L368D, K370S, N390D, N390E, K392L, K392M, K392V, K392F, K392D, K392E, T394F, D399R, D399K, D399V, S400K, S400R, D401K, F405A, F405T, Y407A, Y407I, Y407V, K409F, K409W, K409D, T411D, T411E, K439D, and K439E.

[0017] The tumor-associated antigen binding site can be any tumor-associated antigen, for example, ANO1, BCMA, EpCAM, CAIX, CEA, CCR4, CD2, CD123, CD133, CD19, CD20, CD22, CD25, CD30, CD33, CD37, CD38, CD40, CD52, CD70, CLAUDIN-18.2, DLL3, EGFR / ERBB1, GD2, IGF1R, HER2, HER3 / ERBB3, HER4 / ERBB4, MUC1, cMET, SLAMF7, PSMA, mesothelin, MICA, , MICB, TRAILR1, TRAILR2, TROP2, MAGE-A3, B7.1, B7.2, CTLA4, PD1, 5T4, GPNMB, FR-alpha, PAPP-A, FLT3, GPC3, CXCR4, ROR1, ROR2, HLA-E, P D-L1, VLA4, CD44, CD13, CD15, CD47, CLL1, CD81, CD23, CD79a, CD79b, CD80, CRLF2, SLAMF7, CD138, CA125, NaPi2b, Nectin 4, ADAM8, ADAM9, SL C44A4, CA19-9, LILRB1, LILRB2, LILRB3, LILRB4, LILRB5, LILRA1, LILRA2, LILRA3, LILRA4, LILRA5, and LILRA6, CCR8, CD7, CTLA4, CX3CR1, ENTPD1, HAVCR2, IL-1R2, PDCD1LG2, TIGIT, TNFRSF4, TNFRSF8, TNFRSF9, NT5E, TNFRSF18, MUC1, P-cadherin, Plexin-A1, TNFRSF10B, STEAP1, C The binding site may be DCP1, PTK7, Axl, erbB-3, EDNRB, Tyrp1, CD14, CD163, CSF3R, Siglec-9, ITGAM, VISTA, B7-H4 (VTCN1), CCR1, LRRC25, PTAFR, SIRPB1, TLR2, TLR4, CD300LB, ATP1A3, CCR5, MUC1 (or MUC1-C), Plexin-A1, TNFRSF10B, STERAP1, CDCP1, PTK7, AXL, EDNRB, OLR1, and TYRP1.

[0018] In another aspect, the invention provides a protein comprising: (a) an antigen-binding site that binds to NKG2D, (b) an antigen-binding T cell receptor (TCR) fragment, and (c) an antibody constant region or portion thereof sufficient to bind to CD 16, or an additional antigen-binding site that binds to CD 16. In certain embodiments, the protein is a multispecific binding protein that comprises an antigen-binding TCR fragment that binds to a tumor-associated antigen (TAA).

[0019] In some embodiments, the antigen-binding site is a Fab fragment, and the antigen-binding TCR fragment is a single-chain TCR (scTCR) fragment. In some embodiments, the scTCR fragment is linked to the polypeptide chain of the antibody constant region via a hinge comprising Ala-Ser. In some embodiments, the hinge further comprises the amino acid sequence Thr-Lys-Gly.

[0020] In some embodiments, the antigen-binding site is an scFv, and the antigen-binding TCR fragment is an extracellular TCR fragment. In some embodiments, the scFv is linked to the polypeptide chain of the antibody constant region via a hinge comprising Ala-Ser. In some embodiments, the hinge further comprises the amino acid sequence Thr-Lys-Gly.

[0021] In certain embodiments, the antigen-binding site is a Fab fragment and the antigen-binding TCR fragment is an extracellular TCR fragment.

[0022] In some embodiments, the multispecific binding protein further comprises an additional antigen-binding TCR fragment that binds to the same antigen as the antigen-binding TCR fragment. In some embodiments, the antigen-binding site is an scFv, and the antigen-binding TCR fragment and the additional antigen-binding TCR fragment are extracellular TCR fragments. In some embodiments, the antigen-binding site is an scFv, and the antigen-binding TCR fragment and the additional antigen-binding TCR fragment are scTCR fragments.

[0023] In certain embodiments of a multispecific binding protein comprising an scFv, the scFv comprises a heavy chain variable domain linked to a light chain variable domain via a flexible linker. In certain embodiments, the flexible linker comprises (G4S)4. In certain embodiments, the scFv comprises a heavy chain variable domain disposed N-terminal or C-terminal to the light chain variable domain.

[0024] In some embodiments, the antigen-binding site comprises a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain forms a disulfide bridge with the light chain variable domain. In some embodiments, the disulfide bridge is formed between Cys at position 44 of the heavy chain variable domain and Cys at position 100 of the light chain variable domain, these positions being defined under the Kabat numbering system. In some embodiments, the antigen-binding site comprising such a disulfide bridge is an scFv.

[0025] In certain embodiments, the antigen-binding site binds to NKG2D in humans.

[0026] In certain embodiments, the antigen-binding TCR fragment binds to a peptide derived from a tumor-associated antigen presented by the major histocompatibility complex (MHC).

[0027] In some embodiments, the antigen-binding TCR fragment binds to an ELAVL4 peptide presented by HLA-A*02:01:48, having the amino acid sequence of SEQ ID NO: 425. In some embodiments, the antigen-binding TCR fragment comprises an alpha chain variable domain related to SEQ ID NO: 351 and a beta chain variable domain related to SEQ ID NO: 352. For example, in some embodiments, the antigen-binding TCR fragment comprises an alpha chain variable domain that is at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 351 and / or incorporates an amino acid sequence identical to the CDR3α sequence of this alpha chain variable domain (SEQ ID NO: 353). Similarly, in some embodiments, the antigen-binding TCR fragment comprises a beta chain variable domain that is at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 352 and / or incorporates an amino acid sequence identical to the CDR3β sequence of this beta chain variable domain (SEQ ID NO: 354). In some embodiments, the antigen-binding TCR fragment comprises the alpha chain amino acid sequence set forth in SEQ ID NO: 349 and the beta chain amino acid sequence set forth in SEQ ID NO: 350.

[0028] In some embodiments, the antigen-binding TCR fragment binds to an insulin peptide presented by HLA-A*02:01:48 having the amino acid sequence of SEQ ID NO: 426. In some embodiments, the antigen-binding TCR fragment comprises an alpha chain variable domain related to SEQ ID NO: 357 and a beta chain variable domain related to SEQ ID NO: 358. For example, in some embodiments, the antigen-binding TCR fragment comprises an alpha chain variable domain that is at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 357 and / or incorporates an amino acid sequence identical to the CDR3α sequence of this alpha chain variable domain (SEQ ID NO: 359). Similarly, in some embodiments, the antigen-binding TCR fragment comprises a beta chain variable domain that is at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 358 and / or incorporates an amino acid sequence identical to the CDR3β sequence of this beta chain variable domain (SEQ ID NO: 360). In some embodiments, the antigen-binding TCR fragment comprises the alpha chain amino acid sequence set forth in SEQ ID NO: 355 and the beta chain amino acid sequence set forth in SEQ ID NO: 356.

[0029] In some embodiments, the antigen-binding TCR fragment binds to a TERT peptide presented by HLA-A*02:01:48 and having the amino acid sequence of SEQ ID NO: 340. In some embodiments, the antigen-binding TCR fragment comprises an alpha chain variable domain related to SEQ ID NO: 363 and a beta chain variable domain related to SEQ ID NO: 364. For example, in some embodiments, the antigen-binding TCR fragment comprises an alpha chain variable domain that is at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 363 and / or incorporates an amino acid sequence identical to the CDR3α sequence of this alpha chain variable domain (SEQ ID NO: 365). Similarly, in some embodiments, the antigen-binding TCR fragment comprises a beta chain variable domain that is at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 364 and / or incorporates an amino acid sequence identical to the CDR3β (SEQ ID NO: 366) of this beta chain variable domain. In some embodiments, the antigen-binding TCR fragment comprises the alpha chain amino acid sequence set forth in SEQ ID NO: 361 and the beta chain amino acid sequence set forth in SEQ ID NO: 362.

[0030] In some embodiments, the antigen-binding TCR fragment binds to an ERBB2 peptide presented by HLA-A*02 having the amino acid sequence of SEQ ID NO: 341. In some embodiments, the antigen-binding TCR fragment comprises an alpha chain variable domain related to SEQ ID NO: 430 and a beta chain variable domain related to SEQ ID NO: 431. For example, in some embodiments, the antigen-binding TCR fragment comprises an alpha chain variable domain that is at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 430 and / or incorporates an amino acid sequence identical to the CDR3α sequence of this alpha chain variable domain (SEQ ID NO: 367). Similarly, in some embodiments, the antigen-binding TCR fragment comprises a beta chain variable domain that is at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 431 and / or incorporates an amino acid sequence identical to the CDR3β (SEQ ID NO: 368) of this beta chain variable domain. In some embodiments, the antigen-binding TCR fragment comprises the alpha chain amino acid sequence set forth in SEQ ID NO: 428 and the beta chain amino acid sequence set forth in SEQ ID NO: 429.

[0031] In some embodiments, the antigen-binding TCR fragment binds to a WT1 peptide presented by HLA-A*02 having the amino acid sequence of SEQ ID NO: 342. In some embodiments, the antigen-binding TCR fragment comprises an alpha chain variable domain related to SEQ ID NO: 434 and a beta chain variable domain related to SEQ ID NO: 435. For example, in some embodiments, the antigen-binding TCR fragment comprises an alpha chain variable domain that is at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 434 and / or incorporates an amino acid sequence identical to the CDR3α sequence (SEQ ID NO: 369) of this alpha chain variable domain. Similarly, in some embodiments, the antigen-binding TCR fragment comprises a beta chain variable domain that is at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 435 and / or incorporates an amino acid sequence identical to the CDR3β (SEQ ID NO: 370) of this beta chain variable domain. In some embodiments, the antigen-binding TCR fragment comprises the alpha chain amino acid sequence set forth in SEQ ID NO: 432 and the beta chain amino acid sequence set forth in SEQ ID NO: 433.

[0032] In some embodiments, the antigen-binding TCR fragment binds to a WT1 peptide presented by HLA-A*02 having the amino acid sequence of SEQ ID NO: 342. In some embodiments, the antigen-binding TCR fragment comprises an alpha chain variable domain related to SEQ ID NO: 438 and a beta chain variable domain related to SEQ ID NO: 439. For example, in some embodiments, the antigen-binding TCR fragment comprises an alpha chain variable domain that is at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 438 and / or incorporates an amino acid sequence identical to the CDR3α sequence of this alpha chain variable domain (SEQ ID NO: 371). Similarly, in some embodiments, the antigen-binding TCR fragment comprises a beta chain variable domain that is at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 439 and / or incorporates an amino acid sequence identical to the CDR3β (SEQ ID NO: 372) of this beta chain variable domain. In some embodiments, the antigen-binding TCR fragment comprises the alpha chain amino acid sequence set forth in SEQ ID NO: 436 and the beta chain amino acid sequence set forth in SEQ ID NO: 437.

[0033] In some embodiments, the antigen-binding TCR fragment binds to a MAGE-A3 peptide presented by HLA-A1 having the amino acid sequence of SEQ ID NO: 343. In some embodiments, the antigen-binding TCR fragment comprises an alpha chain variable domain related to SEQ ID NO: 375 and a beta chain variable domain related to SEQ ID NO: 376. For example, in some embodiments, the antigen-binding TCR fragment comprises an alpha chain variable domain that is at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 375 and / or incorporates an amino acid sequence identical to the CDR3α sequence of this alpha chain variable domain (SEQ ID NO: 377). Similarly, in some embodiments, the antigen-binding TCR fragment comprises a beta chain variable domain that is at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 376 and / or incorporates an amino acid sequence identical to the CDR3β (SEQ ID NO: 378) of this beta chain variable domain. In some embodiments, the antigen-binding TCR fragment comprises the alpha chain amino acid sequence set forth in SEQ ID NO: 373 and the beta chain amino acid sequence set forth in SEQ ID NO: 374.

[0034] In some embodiments, the antigen-binding TCR fragment binds to a MART1 peptide presented by HLA-A2 having the amino acid sequence of SEQ ID NO: 344. In some embodiments, the antigen-binding TCR fragment comprises an alpha chain variable domain related to SEQ ID NO: 381 and a beta chain variable domain related to SEQ ID NO: 382. For example, in some embodiments, the antigen-binding TCR fragment comprises an alpha chain variable domain that is at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 381 and / or incorporates an amino acid sequence identical to the CDR3α sequence of this alpha chain variable domain (SEQ ID NO: 383). Similarly, in some embodiments, the antigen-binding TCR fragment comprises a beta chain variable domain that is at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 382 and / or incorporates an amino acid sequence identical to the CDR3β sequence of this beta chain variable domain (SEQ ID NO: 384). In some embodiments, the antigen-binding TCR fragment comprises the alpha chain amino acid sequence set forth in SEQ ID NO: 379 and the beta chain amino acid sequence set forth in SEQ ID NO: 380.

[0035] In some embodiments, the antigen-binding TCR fragment binds to a BIRC5 peptide presented by HLA-A2 having the amino acid sequence of SEQ ID NO: 346. In some embodiments, the antigen-binding TCR fragment comprises an alpha chain variable domain related to SEQ ID NO: 442 and a beta chain variable domain related to SEQ ID NO: 443. For example, in some embodiments, the antigen-binding TCR fragment comprises an alpha chain variable domain that is at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 442 and / or incorporates an amino acid sequence identical to the CDR3α sequence of this alpha chain variable domain (SEQ ID NO: 389). Similarly, in some embodiments, the antigen-binding TCR fragment comprises a beta chain variable domain that is at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 443 and / or incorporates an amino acid sequence identical to the CDR3β sequence (SEQ ID NO: 390) of this beta chain variable domain. In some embodiments, the antigen-binding TCR fragment comprises the alpha chain amino acid sequence set forth in SEQ ID NO: 440 and the beta chain amino acid sequence set forth in SEQ ID NO: 441.

[0036] In some embodiments, the antigen-binding TCR fragment binds to a BIRC5 peptide presented by HLA-A2 having the amino acid sequence of SEQ ID NO: 346. In some embodiments, the antigen-binding TCR fragment comprises an alpha chain variable domain related to SEQ ID NO: 444 and a beta chain variable domain related to SEQ ID NO: 445. For example, in some embodiments, the antigen-binding TCR fragment comprises an alpha chain variable domain that is at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 444 and / or incorporates an amino acid sequence identical to the CDR3α sequence of this alpha chain variable domain (SEQ ID NO: 391). Similarly, in some embodiments, the antigen-binding TCR fragment comprises a beta chain variable domain that is at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 445 and / or incorporates an amino acid sequence identical to the CDR3β sequence (SEQ ID NO: 392) of this beta chain variable domain. In some embodiments, the antigen-binding TCR fragment comprises the alpha chain amino acid sequence set forth in SEQ ID NO: 385 and the beta chain amino acid sequence set forth in SEQ ID NO: 386.

[0037] In some embodiments, the antigen-binding TCR fragment binds to a PRAME peptide presented by HLA-A2 having the amino acid sequence of SEQ ID NO: 347. In some embodiments, the antigen-binding TCR fragment comprises an alpha chain variable domain related to SEQ ID NO: 395 and a beta chain variable domain related to SEQ ID NO: 396. For example, in some embodiments, the antigen-binding TCR fragment comprises an alpha chain variable domain that is at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 395 and / or incorporates an amino acid sequence identical to the CDR3α sequence of this alpha chain variable domain (SEQ ID NO: 397). Similarly, in some embodiments, the antigen-binding TCR fragment comprises a beta chain variable domain that is at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 396 and / or incorporates an amino acid sequence identical to the CDR3β (SEQ ID NO: 398) of this beta chain variable domain. In some embodiments, the antigen-binding TCR fragment comprises the alpha chain amino acid sequence set forth in SEQ ID NO: 393 and the beta chain amino acid sequence set forth in SEQ ID NO: 394.

[0038] In some embodiments, the antigen-binding TCR fragment binds to a PRAME peptide presented by HLA-A2 having the amino acid sequence of SEQ ID NO: 347. In some embodiments, the antigen-binding TCR fragment comprises an alpha chain variable domain related to SEQ ID NO: 401 and a beta chain variable domain related to SEQ ID NO: 402. For example, in some embodiments, the antigen-binding TCR fragment incorporates an alpha chain variable domain that is at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 401 and / or an amino acid sequence identical to the CDR3α sequence of this alpha chain variable domain (SEQ ID NO: 403). Similarly, in some embodiments, the antigen-binding TCR fragment comprises a beta chain variable domain that is at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 402 and / or incorporates an amino acid sequence identical to the CDR3β (SEQ ID NO: 404) of this beta chain variable domain. In some embodiments, the antigen-binding TCR fragment comprises an alpha chain amino acid sequence set forth in SEQ ID NO: 399 and a beta chain amino acid sequence set forth in SEQ ID NO: 400.

[0039] In some embodiments, the antigen-binding TCR fragment binds to a PRAME peptide presented by HLA-A2 having the amino acid sequence of SEQ ID NO: 347. In some embodiments, the antigen-binding TCR fragment comprises an alpha chain variable domain related to SEQ ID NO: 407 and a beta chain variable domain related to SEQ ID NO: 408. For example, in some embodiments, the antigen-binding TCR fragment comprises an alpha chain variable domain that is at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 407 and / or incorporates an amino acid sequence identical to the CDR3α sequence of this alpha chain variable domain (SEQ ID NO: 409). Similarly, in some embodiments, the antigen-binding TCR fragment comprises a beta chain variable domain that is at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 408 and / or incorporates an amino acid sequence identical to the CDR3β (SEQ ID NO: 410) of this beta chain variable domain. In some embodiments, the antigen-binding TCR fragment comprises the alpha chain amino acid sequence set forth in SEQ ID NO: 405 and the beta chain amino acid sequence set forth in SEQ ID NO: 406.

[0040] In some embodiments, the antigen-binding TCR fragment binds to an HLA-A2-presented NY-ESO-1 peptide having the amino acid sequence of SEQ ID NO: 348. In some embodiments, the antigen-binding TCR fragment comprises an alpha chain variable domain related to SEQ ID NO: 413 and a beta chain variable domain related to SEQ ID NO: 414. For example, in some embodiments, the antigen-binding TCR fragment comprises an alpha chain variable domain that is at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 413 and / or incorporates an amino acid sequence identical to the CDR3α sequence of this alpha chain variable domain (SEQ ID NO: 415). Similarly, in certain embodiments, the antigen-binding TCR fragment comprises a beta chain variable domain that is at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 414 and / or incorporates an amino acid sequence identical to CDR3β (SEQ ID NO: 416) of the beta chain variable domain. In certain embodiments, the antigen-binding TCR fragment comprises the alpha chain amino acid sequence set forth in SEQ ID NO: 411 and the beta chain amino acid sequence set forth in SEQ ID NO: 412.

[0041] In some embodiments, the antigen-binding TCR fragment binds to an HLA-A2-presented NY-ESO-1 peptide having the amino acid sequence of SEQ ID NO: 348. In some embodiments, the antigen-binding TCR fragment comprises an alpha chain variable domain related to SEQ ID NO: 418 and a beta chain variable domain related to SEQ ID NO: 414. For example, in some embodiments, the antigen-binding TCR fragment comprises an alpha chain variable domain that is at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 418 and / or incorporates an amino acid sequence identical to the CDR3α sequence of the alpha chain variable domain (SEQ ID NO: 415). Similarly, in some embodiments, the antigen-binding TCR fragment comprises a beta chain variable domain that is at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 414 and / or incorporates an amino acid sequence identical to the CDR3β (SEQ ID NO: 416) of this beta chain variable domain. In some embodiments, the antigen-binding TCR fragment comprises the alpha chain amino acid sequence set forth in SEQ ID NO: 417 and the beta chain amino acid sequence set forth in SEQ ID NO: 412.

[0042] In some embodiments, the antigen-binding TCR fragment binds to an HLA-A2-presented NY-ESO-1 peptide having the amino acid sequence of SEQ ID NO: 348. In some embodiments, the antigen-binding TCR fragment comprises an alpha chain variable domain related to SEQ ID NO: 421 and a beta chain variable domain related to SEQ ID NO: 422. For example, in some embodiments, the antigen-binding TCR fragment comprises an alpha chain variable domain that is at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 421 and / or incorporates an amino acid sequence identical to the CDR3α sequence of this alpha chain variable domain (SEQ ID NO: 423). Similarly, in some embodiments, the antigen-binding TCR fragment comprises a beta chain variable domain that is at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 422 and / or incorporates an amino acid sequence identical to the CDR3β (SEQ ID NO: 424) of this beta chain variable domain. In some embodiments, the antigen-binding TCR fragment comprises the alpha chain amino acid sequence set forth in SEQ ID NO: 419 and the beta chain amino acid sequence set forth in SEQ ID NO: 420.

[0043] In certain embodiments, the antigen-binding TCR fragment binds to an SXX2 peptide presented by HLA-A2, having the amino acid sequence of SEQ ID NO: 345. In certain embodiments, the antigen-binding TCR fragment comprises an alpha chain variable domain incorporating the CDR3α sequence set forth in SEQ ID NO: 387. In certain embodiments, the antigen-binding TCR fragment comprises a beta chain variable domain incorporating the CDR3β sequence set forth in SEQ ID NO: 388.

[0044] In certain embodiments, the multispecific binding protein comprises an antibody constant region or portion thereof sufficient to bind to CD16, wherein the antibody constant region or portion thereof sufficient to bind to CD16 comprises the hinge and CH2 domains of a human IgG1 antibody. In certain embodiments, the antibody constant region or portion thereof sufficient to bind to CD16 comprises an amino acid sequence that is at least 90% identical to amino acids 234-332 of a human IgG1 antibody. Mutations may be introduced into this antibody constant domain to allow heterodimerization with another antibody constant domain. For example, if the antibody constant domain is derived from the constant domain of human IgG1, the antibody constant domain may comprise an amino acid sequence that is at least 90% identical to amino acids 234 to 332 of a human IgG1 antibody and differs at one or more positions selected from the group consisting of Q347, Y349, L351, S354, E356, E357, K360, Q362, S364, T366, L368, K370, N390, K392, T394, D399, S400, D401, F405, Y407, K409, T411, and K439. In some embodiments, the antibody constant domain may comprise an amino acid sequence at least 90% identical to amino acids 234-332 of a human IgG1 antibody, and may be selected from the group consisting of Q347E, Q347R, Y349S, Y349K, Y349T, Y349D, Y349E, Y349C, L351K, L351D, L351Y, S354C, E356K, E357Q, E357L, E357W, K360E, K360W, Q362E, S364K, S364E, S364H, S364D, T366V, T366I, T366L, T366M, and differing by one or more substitutions selected from the group consisting of T366K, T366W, T366S, L368E, L368A, L368D, K370S, N390D, N390E, K392L, K392M, K392V, K392F, K392D, K392E, T394F, D399R, D399K, D399V, S400K, S400R, D401K, F405A, F405T, Y407A, Y407I, Y407V, K409F, K409W, K409D, T411D, T411E, K439D, and K439E.

[0045] In certain embodiments of any one of the foregoing polypeptides or proteins comprising an antigen-binding site that binds to NKG2D (also referred to as an "NKG2D-binding site"), the NKG2D-binding site may have an amino acid sequence at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 1 and / or may incorporate a heavy chain variable domain related to SEQ ID NO: 1, such as by incorporating amino acid sequences identical to the sequences of CDR1 (SEQ ID NO: 3 or SEQ ID NO: 307), CDR2 (SEQ ID NO: 4), and CDR3 (SEQ ID NO: 5 or SEQ ID NO: 308) of SEQ ID NO: 1. A heavy chain variable domain related to SEQ ID NO: 1 may be combined with a different light chain variable domain to form an NKG2D-binding site. For example, an NKG2D binding site incorporating a heavy chain variable domain related to SEQ ID NO: 1 may further incorporate a light chain variable domain selected from any one of the sequences related to SEQ ID NOs: 2, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, and 43. For example, the NKG2D binding site incorporates a heavy chain variable domain having an amino acid sequence at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 1, and a light chain variable domain having an amino acid sequence at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to any one of the sequences selected from SEQ ID NOs: 2, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, and 43.

[0046] Alternatively, an NKG2D-binding site may incorporate a heavy chain variable domain related to SEQ ID NO: 44 and a light chain variable domain related to SEQ ID NO: 48. For example, the heavy chain variable domain of an NKG2D-binding site may be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 44 and / or may incorporate amino acid sequences that are identical to the sequences of CDR1 (SEQ ID NO: 45 or SEQ ID NO: 309), CDR2 (SEQ ID NO: 46), and CDR3 (SEQ ID NO: 47 or SEQ ID NO: 310) of SEQ ID NO: 44. Similarly, the light chain variable domain of the second antigen-binding site may be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 48 and / or may incorporate amino acid sequences that are identical to the sequences of CDR1 (SEQ ID NO: 49), CDR2 (SEQ ID NO: 50), and CDR3 (SEQ ID NO: 51) of SEQ ID NO: 48.

[0047] In other embodiments, an NKG2D-binding site may incorporate a heavy chain variable domain related to SEQ ID NO: 52 and a light chain variable domain related to SEQ ID NO: 56. For example, the heavy chain variable domain of the NKG2D-binding site may be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 52 and / or may incorporate amino acid sequences identical to the sequences of CDR1 (SEQ ID NO: 53 or SEQ ID NO: 311), CDR2 (SEQ ID NO: 54), and CDR3 (SEQ ID NO: 55 or SEQ ID NO: 312) of SEQ ID NO: 52. Similarly, the light chain variable domain of the NKG2D binding site may be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO:56 and / or may incorporate amino acid sequences identical to the sequences of CDR1 (SEQ ID NO:57), CDR2 (SEQ ID NO:58), and CDR3 (SEQ ID NO:59) of SEQ ID NO:56.

[0048] Alternatively, the NKG2D binding site may incorporate a heavy chain variable domain related to SEQ ID NO: 60 and a light chain variable domain related to SEQ ID NO: 61, such as by having an amino acid sequence that is at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 60 and at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 61, respectively.

[0049] In another embodiment, the NKG2D binding site may incorporate a heavy chain variable domain related to SEQ ID NO: 62 and a light chain variable domain related to SEQ ID NO: 66, e.g., the heavy chain variable domain of the NKG2D binding site may be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 62 and / or may incorporate amino acid sequences identical to the CDR1 (SEQ ID NO: 63), CDR2 (SEQ ID NO: 64), and CDR3 (SEQ ID NO: 65) sequences of SEQ ID NO: 62. Similarly, the light chain variable domain of the NKG2D binding site may be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO:66 and / or may incorporate amino acid sequences identical to the sequences of CDR1 (SEQ ID NO:67), CDR2 (SEQ ID NO:68), and CDR3 (SEQ ID NO:69) of SEQ ID NO:66.

[0050] An NKG2D-binding site, in some embodiments, may incorporate a heavy chain variable domain related to SEQ ID NO: 70 and a light chain variable domain related to SEQ ID NO: 74. For example, the heavy chain variable domain of an NKG2D-binding site may be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 70 and / or may incorporate amino acid sequences identical to the sequences of CDR1 (SEQ ID NO: 71 or SEQ ID NO: 313), CDR2 (SEQ ID NO: 72), and CDR3 (SEQ ID NO: 73 or SEQ ID NO: 314) of SEQ ID NO: 70. Similarly, the light chain variable domain of the NKG2D binding site may be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO:74 and / or may incorporate amino acid sequences identical to the sequences of CDR1 (SEQ ID NO:75), CDR2 (SEQ ID NO:76), and CDR3 (SEQ ID NO:77) of SEQ ID NO:74.

[0051] In some embodiments, an NKG2D-binding site may incorporate a heavy chain variable domain related to SEQ ID NO: 78 and a light chain variable domain related to SEQ ID NO: 82. For example, the heavy chain variable domain of an NKG2D-binding site may be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 78 and / or may incorporate amino acid sequences identical to the sequences of CDR1 (SEQ ID NO: 79 or SEQ ID NO: 315), CDR2 (SEQ ID NO: 80), and CDR3 (SEQ ID NO: 81 or SEQ ID NO: 316) of SEQ ID NO: 78. Similarly, the light chain variable domain of the NKG2D binding site may be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 82 and / or may incorporate amino acid sequences identical to the sequences of CDR1 (SEQ ID NO: 83), CDR2 (SEQ ID NO: 84), and CDR3 (SEQ ID NO: 85) of SEQ ID NO: 82.

[0052] In some embodiments, an NKG2D-binding site may incorporate a heavy chain variable domain related to SEQ ID NO: 86 and a light chain variable domain related to SEQ ID NO: 90. For example, the heavy chain variable domain of an NKG2D-binding site may be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 86 and / or may incorporate amino acid sequences identical to the sequences of CDR1 (SEQ ID NO: 87 or SEQ ID NO: 317), CDR2 (SEQ ID NO: 88), and CDR3 (SEQ ID NO: 89 or SEQ ID NO: 318) of SEQ ID NO: 86. Similarly, the light chain variable domain of the NKG2D binding site may be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO:90 and / or may incorporate amino acid sequences identical to the sequences of CDR1 (SEQ ID NO:91), CDR2 (SEQ ID NO:92), and CDR3 (SEQ ID NO:93) of SEQ ID NO:90.

[0053] In some embodiments, an NKG2D-binding site may incorporate a heavy chain variable domain related to SEQ ID NO: 94 and a light chain variable domain related to SEQ ID NO: 98. For example, the heavy chain variable domain of an NKG2D-binding site may be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 94 and / or may incorporate amino acid sequences identical to the sequences of CDR1 (SEQ ID NO: 95 or SEQ ID NO: 319), CDR2 (SEQ ID NO: 96), and CDR3 (SEQ ID NO: 97 or SEQ ID NO: 320) of SEQ ID NO: 94. Similarly, the light chain variable domain of the NKG2D binding site may be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO:98 and / or may incorporate amino acid sequences identical to the sequences of CDR1 (SEQ ID NO:99), CDR2 (SEQ ID NO:100), and CDR3 (SEQ ID NO:101) of SEQ ID NO:98.

[0054] In some embodiments, an NKG2D-binding site may incorporate a heavy chain variable domain related to SEQ ID NO: 102 and a light chain variable domain related to SEQ ID NO: 106. For example, the heavy chain variable domain of an NKG2D-binding site may be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 102 and / or may incorporate amino acid sequences identical to the sequences of CDR1 (SEQ ID NO: 103 or SEQ ID NO: 313), CDR2 (SEQ ID NO: 104), and CDR3 (SEQ ID NO: 105 or SEQ ID NO: 321) of SEQ ID NO: 102. Similarly, the light chain variable domain of the NKG2D binding site may be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 106 and / or may incorporate amino acid sequences identical to the sequences of CDR1 (SEQ ID NO: 107), CDR2 (SEQ ID NO: 108), and CDR3 (SEQ ID NO: 109) of SEQ ID NO: 106.

[0055] In some embodiments, an NKG2D-binding site may incorporate a heavy chain variable domain related to SEQ ID NO: 322 and a light chain variable domain related to SEQ ID NO: 98. For example, the heavy chain variable domain of an NKG2D-binding site may be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 322 and / or may incorporate amino acid sequences identical to the sequences of CDR1 (SEQ ID NO: 95 or SEQ ID NO: 319), CDR2 (SEQ ID NO: 96), and CDR3 (SEQ ID NO: 323 or SEQ ID NO: 324) of SEQ ID NO: 322. Similarly, the light chain variable domain of the NKG2D binding site may be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO:98 and / or may incorporate amino acid sequences identical to the sequences of CDR1 (SEQ ID NO:99), CDR2 (SEQ ID NO:100), and CDR3 (SEQ ID NO:101) of SEQ ID NO:98.

[0056] In some embodiments, an NKG2D-binding site may incorporate a heavy chain variable domain related to SEQ ID NO: 325 and a light chain variable domain related to SEQ ID NO: 98. For example, the heavy chain variable domain of the NKG2D-binding site may be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 325 and / or may incorporate amino acid sequences identical to the sequences of CDR1 (SEQ ID NO: 95 or SEQ ID NO: 319), CDR2 (SEQ ID NO: 96), and CDR3 (SEQ ID NO: 326 or SEQ ID NO: 327) of SEQ ID NO: 325. Similarly, the light chain variable domain of the NKG2D binding site may be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO:98 and / or may incorporate amino acid sequences identical to the sequences of CDR1 (SEQ ID NO:99), CDR2 (SEQ ID NO:100), and CDR3 (SEQ ID NO:101) of SEQ ID NO:98.

[0057] In some embodiments, an NKG2D-binding site may incorporate a heavy chain variable domain related to SEQ ID NO: 328 and a light chain variable domain related to SEQ ID NO: 98. For example, the heavy chain variable domain of an NKG2D-binding site may be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 328 and / or may incorporate amino acid sequences identical to the sequences of CDR1 (SEQ ID NO: 95 or SEQ ID NO: 319), CDR2 (SEQ ID NO: 96), and CDR3 (SEQ ID NO: 329 or SEQ ID NO: 330) of SEQ ID NO: 328. Similarly, the light chain variable domain of the NKG2D binding site may be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO:98 and / or may incorporate amino acid sequences identical to the sequences of CDR1 (SEQ ID NO:99), CDR2 (SEQ ID NO:100), and CDR3 (SEQ ID NO:101) of SEQ ID NO:98.

[0058] In some embodiments, an NKG2D-binding site may incorporate a heavy chain variable domain related to SEQ ID NO: 331 and a light chain variable domain related to SEQ ID NO: 98. For example, the heavy chain variable domain of an NKG2D-binding site may be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 331 and / or may incorporate amino acid sequences identical to the sequences of CDR1 (SEQ ID NO: 95 or SEQ ID NO: 319), CDR2 (SEQ ID NO: 96), and CDR3 (SEQ ID NO: 332 or SEQ ID NO: 333) of SEQ ID NO: 331. Similarly, the light chain variable domain of the NKG2D binding site may be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO:98 and / or may incorporate amino acid sequences identical to the sequences of CDR1 (SEQ ID NO:99), CDR2 (SEQ ID NO:100), and CDR3 (SEQ ID NO:101) of SEQ ID NO:98.

[0059] In some embodiments, an NKG2D-binding site may incorporate a heavy chain variable domain related to SEQ ID NO: 334 and a light chain variable domain related to SEQ ID NO: 98. For example, the heavy chain variable domain of an NKG2D-binding site may be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 334 and / or may incorporate amino acid sequences identical to the sequences of CDR1 (SEQ ID NO: 95 or SEQ ID NO: 319), CDR2 (SEQ ID NO: 96), and CDR3 (SEQ ID NO: 335 or SEQ ID NO: 336) of SEQ ID NO: 334. Similarly, the light chain variable domain of the NKG2D binding site may be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO:98 and / or may incorporate amino acid sequences identical to the sequences of CDR1 (SEQ ID NO:99), CDR2 (SEQ ID NO:100), and CDR3 (SEQ ID NO:101) of SEQ ID NO:98.

[0060] In some embodiments, an NKG2D-binding site may incorporate a heavy chain variable domain related to SEQ ID NO: 337 and a light chain variable domain related to SEQ ID NO: 98. For example, the heavy chain variable domain of an NKG2D-binding site may be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 337 and / or may incorporate amino acid sequences identical to the sequences of CDR1 (SEQ ID NO: 95 or SEQ ID NO: 319), CDR2 (SEQ ID NO: 96), and CDR3 (SEQ ID NO: 338 or SEQ ID NO: 339) of SEQ ID NO: 337. Similarly, the light chain variable domain of the NKG2D binding site may be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO:98 and / or may incorporate amino acid sequences identical to the sequences of CDR1 (SEQ ID NO:99), CDR2 (SEQ ID NO:100), and CDR3 (SEQ ID NO:101) of SEQ ID NO:98.

[0061] In some embodiments, the NKG2D binding site may incorporate a heavy chain variable domain related to SEQ ID NO: 110 and a light chain variable domain related to SEQ ID NO: 111, such as by having amino acid sequences at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 110 and at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 111, respectively. In some embodiments, the NKG2D binding site may incorporate a heavy chain variable domain related to SEQ ID NO: 112 and a light chain variable domain related to SEQ ID NO: 113, such as by having amino acid sequences at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 112 and at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 113, respectively.

[0062] Also provided are formulations comprising any one of the proteins described herein, cells comprising one or more nucleic acids that express the proteins, and methods of using the proteins to enhance tumor cell death.

[0063] Another aspect of the present invention provides a method of treating cancer in a patient, the method comprising administering to a patient in need thereof a therapeutically effective amount of a multispecific binding protein described herein. Cancers to be treated may include acute myeloid leukemia, acute myelomonocytic leukemia, B-cell lymphoma, bladder cancer, breast cancer, colorectal cancer, diffuse large B-cell lymphoma, esophageal cancer, Ewing's sarcoma, follicular lymphoma, gastric cancer, gastrointestinal cancer, gastrointestinal stromal tumor, glioblastoma, head and neck cancer, melanoma, mesothelioma, multiple myeloma, myelodysplastic syndrome, renal cell carcinoma, neuroblastoma, non-small cell lung cancer, neuroendocrine tumors, ovarian cancer, and pancreatic cancer, prostate cancer, sarcoma, small cell lung cancer, T-cell lymphoma, testicular cancer, thymic cancer, thyroid cancer, urothelial cancer, cancer infiltrated by myeloid-derived suppressor cells, cancer infiltrated by T regulatory cells, cancer with extracellular matrix deposition, cancer with high levels of reactive stroma, and cancer with angiogenesis. In certain embodiments, for example, the following are provided: (Item 1) A polypeptide comprising a single chain variable fragment (scFv) linked to an antibody constant domain via a hinge comprising Ala-Ser, said scFv comprising a heavy chain variable domain and a light chain variable domain. (Item 2) 2. The polypeptide of item 1, wherein the heavy chain variable domain forms a disulfide bridge with the light chain variable domain. (Item 3) 3. The polypeptide of item 2, wherein the disulfide bridge is formed between C44 from the heavy chain variable domain and C100 from the light chain variable domain. (Item 4) 4. The polypeptide of any one of items 1 to 3, wherein the heavy chain variable domain is linked to the light chain variable domain via a flexible linker. (Item 5) 5. The polypeptide of item 4, wherein the flexible linker comprises (G4S)4. (Item 6) 6. The polypeptide according to any one of items 1 to 5, wherein the heavy chain variable domain is located at the N-terminus or C-terminus of the light chain variable domain. (Item 7) 7. The polypeptide according to any one of items 1 to 6, wherein the hinge further comprises the amino acid sequence Thr-Lys-Gly. (Item 8) 8. The polypeptide of any one of items 1 to 7, wherein the antibody constant domain comprises an antibody Fc domain or a portion thereof sufficient to bind to CD16. (Item 9) 9. The polypeptide of item 8, wherein the antibody constant domain comprises the CH2 and CH3 domains of a human IgG1 antibody. (Item 10) 10. The polypeptide according to item 9, wherein the antibody constant domain comprises an amino acid sequence that is at least 90% identical to amino acids 234 to 332 of a human IgG1 antibody. (Item 11) 11. The polypeptide of item 10, wherein the antibody constant domain comprises an amino acid sequence at least 90% identical to the Fc domain of human IgG1 and differs at one or more positions selected from the group consisting of Q347, Y349, L351, S354, E356, E357, K360, Q362, S364, T366, L368, K370, N390, K392, T394, D399, S400, D401, F405, Y407, K409, T411, and K439. (Item 12) the antibody constant domain comprises an amino acid sequence at least 90% identical to the Fc domain of human IgG1, and is selected from the group consisting of Q347E, Q347R, Y349S, Y349K, Y349T, Y349D, Y349E, Y349C, L351K, L351D, L351Y, S354C, E356K, E357Q, E357L, E357W, K360E, K360W, Q362E, S364K, S364E, S364H, S364D, T366V, T366I, T366L, T366M, T366K, T366W, T366 12. The polypeptide of item 11, wherein the polypeptide differs by one or more substitutions selected from the group consisting of S, L368E, L368A, L368D, K370S, N390D, N390E, K392L, K392M, K392V, K392F, K392D, K392E, T394F, D399R, D399K, D399V, S400K, S400R, D401K, F405A, F405T, Y407A, Y407I, Y407V, K409F, K409W, K409D, T411D, T411E, K439D, and K439E. (Item 13) 13. The polypeptide of any one of items 1 to 12, wherein the scFv binds to NKG2D or a tumor-associated antigen. (Item 14) 14. The polypeptide of item 13, wherein the scFv binds to NKG2D and the heavy chain variable domain of the scFv comprises an amino acid sequence at least 90% identical to an amino acid sequence selected from SEQ ID NO:94, SEQ ID NO:1, SEQ ID NO:44, SEQ ID NO:52, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:70, SEQ ID NO:78, SEQ ID NO:86, SEQ ID NO:102, SEQ ID NO:322, SEQ ID NO:325, SEQ ID NO:328, SEQ ID NO:331, SEQ ID NO:334, and SEQ ID NO:337. (Item 15) 15. The polypeptide of item 14, wherein the heavy chain variable domain comprises an amino acid sequence at least 90% identical to SEQ ID NO: 94 and the light chain variable domain comprises an amino acid sequence at least 90% identical to SEQ ID NO: 98. (Item 16) 15. The polypeptide of item 14, wherein the heavy chain variable domain comprises an amino acid sequence at least 90% identical to SEQ ID NO: 322 and the light chain variable domain comprises an amino acid sequence at least 90% identical to SEQ ID NO: 98. (Item 17) 15. The polypeptide of item 14, wherein the heavy chain variable domain comprises an amino acid sequence at least 90% identical to SEQ ID NO: 325 and the light chain variable domain comprises an amino acid sequence at least 90% identical to SEQ ID NO: 98. (Item 18) 15. The polypeptide of item 14, wherein the heavy chain variable domain comprises an amino acid sequence at least 90% identical to SEQ ID NO: 328 and the light chain variable domain comprises an amino acid sequence at least 90% identical to SEQ ID NO: 98. (Item 19) 15. The polypeptide of item 14, wherein the heavy chain variable domain comprises an amino acid sequence at least 90% identical to SEQ ID NO: 331 and the light chain variable domain comprises an amino acid sequence at least 90% identical to SEQ ID NO: 98. (Item 20) 15. The polypeptide of item 14, wherein the heavy chain variable domain comprises an amino acid sequence at least 90% identical to SEQ ID NO: 334 and the light chain variable domain comprises an amino acid sequence at least 90% identical to SEQ ID NO: 98. (Item 21) 15. The polypeptide of item 14, wherein the heavy chain variable domain comprises an amino acid sequence at least 90% identical to SEQ ID NO: 337 and the light chain variable domain comprises an amino acid sequence at least 90% identical to SEQ ID NO: 98. (Item 22) 15. The polypeptide of item 14, wherein the heavy chain variable domain comprises an amino acid sequence at least 90% identical to SEQ ID NO: 44 and the light chain variable domain comprises an amino acid sequence at least 90% identical to SEQ ID NO: 48. (Item 23) 15. The polypeptide of item 14, wherein the heavy chain variable domain comprises an amino acid sequence at least 90% identical to SEQ ID NO: 52 and the light chain variable domain comprises an amino acid sequence at least 90% identical to SEQ ID NO: 56. (Item 24) 15. The polypeptide of item 14, wherein the heavy chain variable domain comprises an amino acid sequence at least 90% identical to SEQ ID NO: 60 and the light chain variable domain comprises an amino acid sequence at least 90% identical to SEQ ID NO: 61. (Item 25) 15. The polypeptide of item 14, wherein the heavy chain variable domain comprises an amino acid sequence at least 90% identical to SEQ ID NO: 62 and the light chain variable domain comprises an amino acid sequence at least 90% identical to SEQ ID NO: 66. (Item 26) 15. The polypeptide of item 14, wherein the heavy chain variable domain comprises an amino acid sequence at least 90% identical to SEQ ID NO: 70 and the light chain variable domain comprises an amino acid sequence at least 90% identical to SEQ ID NO: 74. (Item 27) 15. The polypeptide of item 14, wherein the heavy chain variable domain comprises an amino acid sequence at least 90% identical to SEQ ID NO: 78 and the light chain variable domain comprises an amino acid sequence at least 90% identical to SEQ ID NO: 82. (Item 28) 15. The polypeptide of item 14, wherein the heavy chain variable domain comprises an amino acid sequence at least 90% identical to SEQ ID NO: 86 and the light chain variable domain comprises an amino acid sequence at least 90% identical to SEQ ID NO: 90. (Item 29) 15. The polypeptide of item 14, wherein the heavy chain variable domain comprises an amino acid sequence at least 90% identical to SEQ ID NO: 102 and the light chain variable domain comprises an amino acid sequence at least 90% identical to SEQ ID NO: 106. (Item 30) 14. The polypeptide of item 13, wherein the scFv binds to NKG2D, and the heavy chain variable domain comprises an amino acid sequence at least 90% identical to SEQ ID NO: 110, and the light chain variable domain comprises an amino acid sequence at least 90% identical to SEQ ID NO: 111. (Item 31) 14. The polypeptide of item 13, wherein the scFv binds to NKG2D, and the heavy chain variable domain comprises an amino acid sequence at least 90% identical to SEQ ID NO: 112, and the light chain variable domain comprises an amino acid sequence at least 90% identical to SEQ ID NO: 113. (Item 32) Item 14. The polypeptide according to Item 13, wherein the scFv binds to a tumor-associated antigen, and the tumor-associated antigen is selected from the group consisting of ANO1, BCMA, EpCAM, CAIX, CEA, CCR4, CD2, CD123, CD133, CD19, CD20, CD22, CD25, CD30, CD33, CD37, CD38, CD40, CD52, CD70, CLAUDIN-18.2, DLL3, EGFR / ERBB1, GD2, IGF1R, and H ER2, HER3 / ERBB3, HER4 / ERBB4, MUC1, cMET, SLAMF7, PSMA, mesothelin, MICA, MICB, TRAILR1, TRAILR2, TROP2, MAGE-A3, B7.1, B7.2, CTLA4, PD1, 5T4, GPNMB, FR-alpha, PAPP-A, FLT3, GPC3, CXCR4, ROR1, ROR2, HLA-E, PD-L1, VLA4, CD44, CD 13, CD15, CD47, CLL1, CD81, CD23, CD79a, CD79b, CD80, CRLF2, SLAMF7, CD138, CA125, NaPi2b, nectin-4, ADAM8, ADAM9, SLC44A4, CA19-9, LILRB1, LILRB2, LILRB3, LILRB4, LILRB5, LILRA1, LILRA2, LILRA3, LILRA4, LILRA5, and LILRA6 , CCR8, CD7, CTLA4, CX3CR1, ENTPD1, HAVCR2, IL-1R2, PDCD1LG2, TIGIT, TNFRSF4, TNFRSF8, TNFRSF9, GEM, NT5E, TN FRSF18, MUC1, P-cadherin, Plexin-A1, TNFRSF10B, STEAP1, CDCP1, PTK7, Axl, erbB-3, EDNRB, Tyrp1, CD14, CD163, CSF3 The polypeptide is selected from the group consisting of R, Siglec-9, ITGAM, VISTA, B7-H4 (VTCN1), CCR1, LRRC25, PTAFR, SIRPB1, TLR2, TLR4, CD300LB, ATP1A3, CCR5, MUC1 (or MUC1-C), Plexin-A1, TNFRSF10B, STEAP1, CDCP1, PTK7, AXL, EDNRB, OLR1, and TYRP1. (Item 33) A protein comprising an scFv according to any one of the preceding items. (Item 34) 1. A protein comprising: (a) a first antigen-binding site comprising the polypeptide of any one of items 1 to 32; (b) a second antigen-binding site; (c) a second antibody constant domain. (Item 35) 35. The protein of item 34, wherein the first antigen-binding site binds to NKG2D and the second antigen-binding site binds to a tumor-associated antigen. (Item 36) 35. The protein of item 34, wherein the first antigen-binding site binds to a tumor-associated antigen and the second antigen-binding site binds to NKG2D. (Item 37) 37. The protein of any one of items 34 to 36, wherein the second antigen-binding site comprises an scFv or Fab. (Item 38) 38. The protein according to any one of items 34 to 37, wherein the second antibody constant domain comprises the hinge and CH2 domains of a human IgG1 antibody. (Item 39) 39. The protein according to any one of items 34 to 38, wherein the second antibody constant domain comprises an amino acid sequence that is at least 90% identical to amino acids 234 to 332 of a human IgG1 antibody. (Item 40) 40. The protein of item 39, wherein the second antibody constant domain comprises an amino acid sequence at least 90% identical to the human IgG1 Fc domain and differs at one or more positions selected from the group consisting of Q347, Y349, L351, S354, E356, E357, K360, Q362, S364, T366, L368, K370, N390, K392, T394, D399, S400, D401, F405, Y407, K409, T411, and K439. (Item 41) 1. A protein comprising: (a) a first antigen-binding site that binds to a tumor-associated antigen; (b) a second antigen-binding site that binds to the same tumor-associated antigen as the first antigen-binding site; (c) a third antigen-binding site that binds NKG2D; (d) an antibody constant region or portion thereof sufficient to bind to CD16, or CD16 and a fourth antigen-binding site that binds to (Item 42) 42. The protein of item 41, wherein the first antigen-binding site comprises an scFv or Fab. (Item 43) 43. The protein of item 41 or 42, wherein the second antigen-binding site comprises an scFv or Fab. (Item 44) 44. The protein of any one of items 41 to 43, wherein the third antigen-binding site comprises an scFv or Fab. (Item 45) The tumor-associated antigen is ANO1, BCMA, EpCAM, CAIX, CEA, CCR4, CD2, CD123, CD133, CD19, CD20, CD22, CD25, CD30, CD33, CD37, CD38, CD40, CD52, CD70, CLAUDIN-18.2, DLL3, EGFR / ERBB1, GD2, IGF1R, HER2, HER3 / ERBB3, HER4 / ERBB4, MUC1, cMET, SLAMF7, PSMA, mesothelin, MICA, MICB, TRAILR1, or TRAILR2 , TROP2, MAGE-A3, B7.1, B7.2, CTLA4, PD1, 5T4, GPNMB, FR-alpha, PAPP-A, FLT3, GPC3, CXCR4, ROR1, ROR2, HLA-E, PD-L1, VLA4, CD44, CD13, CD 15, CD47, CLL1, CD81, CD23, CD79a, CD79b, CD80, CRLF2, SLAMF7, CD138, CA125, NaPi2b, Nectin 4, ADAM8, ADAM9, SLC44A4, CA19-9, LILRB1, LIL RB2, LILRB3, LILRB4, LILRB5, LILRA1, LILRA2, LILRA3, LILRA4, LILRA5, and LILRA6, CCR8, CD7, CTLA4, CX3CR1, ENTPD1, HAVCR2, IL-1R2, PDC D1LG2, TIGIT, TNFRSF4, TNFSF8, TNFRSF9, GEM, NT5E, TNFRSF18, MUC1, P-cadherin, Plexin-A1, TNFRSF10B, STEAP1, CDCP1, PTK7, Axl, erbB-3, E 45. The protein of any one of Items 41 to 44, selected from the group consisting of DNRB, Tyrp1, CD14, CD163, CSF3R, Siglec-9, ITGAM, VISTA, B7-H4 (VTCN1), CCR1, LRRC25, PTAFR, SIRPB1, TLR2, TLR4, CD300LB, ATP1A3, CCR5, MUC1 (or MUC1-C), Plexin-A1, TNFRSF10B, STERAP1, CDCP1, PTK7, AXL, EDNRB, OLR1, and TYRP1. (Item 46) 46. The protein according to any one of Aspects 41 to 45, wherein the third antigen-binding site comprises a heavy chain variable domain comprising an amino acid sequence at least 90% identical to a sequence selected from SEQ ID NO: 94, SEQ ID NO: 1, SEQ ID NO: 44, SEQ ID NO: 52, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 70, SEQ ID NO: 78, SEQ ID NO: 86, SEQ ID NO: 102, SEQ ID NO: 322, SEQ ID NO: 325, SEQ ID NO: 328, SEQ ID NO: 331, SEQ ID NO: 334, and SEQ ID NO: 337. (Item 47) 47. The protein of item 46, wherein the third antigen-binding site comprises a heavy chain variable domain comprising an amino acid sequence at least 90% identical to SEQ ID NO: 94 and a light chain variable domain comprising an amino acid sequence at least 90% identical to SEQ ID NO: 98. (Item 48) 47. The protein of item 46, wherein the third antigen-binding site comprises a heavy chain variable domain comprising an amino acid sequence at least 90% identical to SEQ ID NO: 322 and a light chain variable domain comprising an amino acid sequence at least 90% identical to SEQ ID NO: 98. (Item 49) 47. The protein of item 46, wherein the third antigen-binding site comprises a heavy chain variable domain comprising an amino acid sequence at least 90% identical to SEQ ID NO: 325 and a light chain variable domain comprising an amino acid sequence at least 90% identical to SEQ ID NO: 98. (Item 50) 47. The protein of item 46, wherein the third antigen-binding site comprises a heavy chain variable domain comprising an amino acid sequence at least 90% identical to SEQ ID NO: 328 and a light chain variable domain comprising an amino acid sequence at least 90% identical to SEQ ID NO: 98. (Item 51) 47. The protein of item 46, wherein the third antigen-binding site comprises a heavy chain variable domain comprising an amino acid sequence at least 90% identical to SEQ ID NO: 331 and a light chain variable domain comprising an amino acid sequence at least 90% identical to SEQ ID NO: 98. (Item 52) 47. The protein of item 46, wherein the third antigen-binding site comprises a heavy chain variable domain comprising an amino acid sequence at least 90% identical to SEQ ID NO: 334 and a light chain variable domain comprising an amino acid sequence at least 90% identical to SEQ ID NO: 98. (Item 53) 47. The protein of item 46, wherein the third antigen-binding site comprises a heavy chain variable domain comprising an amino acid sequence at least 90% identical to SEQ ID NO: 337 and a light chain variable domain comprising an amino acid sequence at least 90% identical to SEQ ID NO: 98. (Item 54) 47. The protein of item 46, wherein the third antigen-binding site comprises a heavy chain variable domain comprising an amino acid sequence at least 90% identical to SEQ ID NO: 44 and a light chain variable domain comprising an amino acid sequence at least 90% identical to SEQ ID NO: 48. (Item 55) 47. The protein of item 46, wherein the third antigen-binding site comprises a heavy chain variable domain comprising an amino acid sequence at least 90% identical to SEQ ID NO: 52 and a light chain variable domain comprising an amino acid sequence at least 90% identical to SEQ ID NO: 56. (Item 56) 47. The protein of item 46, wherein the third antigen-binding site comprises a heavy chain variable domain comprising an amino acid sequence at least 90% identical to SEQ ID NO: 60 and a light chain variable domain comprising an amino acid sequence at least 90% identical to SEQ ID NO: 61. (Item 57) 47. The protein of item 46, wherein the third antigen-binding site comprises a heavy chain variable domain comprising an amino acid sequence at least 90% identical to SEQ ID NO: 62 and a light chain variable domain comprising an amino acid sequence at least 90% identical to SEQ ID NO: 66. (Item 58) 47. The protein of item 46, wherein the third antigen-binding site comprises a heavy chain variable domain comprising an amino acid sequence at least 90% identical to SEQ ID NO: 70 and a light chain variable domain comprising an amino acid sequence at least 90% identical to SEQ ID NO: 74. (Item 59) 47. The protein of item 46, wherein the third antigen-binding site comprises a heavy chain variable domain comprising an amino acid sequence at least 90% identical to SEQ ID NO: 78 and a light chain variable domain comprising an amino acid sequence at least 90% identical to SEQ ID NO: 82. (Item 60) 47. The protein of item 46, wherein the third antigen-binding site comprises a heavy chain variable domain comprising an amino acid sequence at least 90% identical to SEQ ID NO: 86 and a light chain variable domain comprising an amino acid sequence at least 90% identical to SEQ ID NO: 90. (Item 61) 47. The protein of item 46, wherein the third antigen-binding site comprises a heavy chain variable domain comprising an amino acid sequence at least 90% identical to SEQ ID NO: 102 and a light chain variable domain comprising an amino acid sequence at least 90% identical to SEQ ID NO: 106. (Item 62) 46. The protein according to any one of Aspects 41 to 45, wherein the third antigen-binding site comprises a heavy chain variable domain comprising an amino acid sequence at least 90% identical to SEQ ID NO: 110 and a light chain variable domain comprising an amino acid sequence at least 90% identical to SEQ ID NO: 111. (Item 63) 46. The protein according to any one of Items 41 to 45, wherein the third antigen-binding site comprises a heavy chain variable domain comprising an amino acid sequence at least 90% identical to SEQ ID NO: 112 and a light chain variable domain comprising an amino acid sequence at least 90% identical to SEQ ID NO: 113. (Item 64) 64. The protein of any one of items 41 to 63, wherein the antibody constant region or portion thereof sufficient to bind to CD16 comprises the hinge and CH2 domains of a human IgG1 antibody. (Item 65) 65. The protein of item 64, wherein the antibody constant region or portion thereof sufficient to bind to CD16 comprises an amino acid sequence that is at least 90% identical to amino acids 234 to 332 of a human IgG1 antibody. (Item 66) 66. The protein of item 65, wherein the antibody constant region or portion thereof sufficient to bind to CD16 comprises an amino acid sequence at least 90% identical to the Fc domain of human IgG1 and differs at one or more positions selected from the group consisting of Q347, Y349, L351, S354, E356, E357, K360, Q362, S364, T366, L368, K370, N390, K392, T394, D399, S400, D401, F405, Y407, K409, T411, and K439. (Item 67) 1. A protein comprising: (a) an antigen-binding site that binds to NKG2D; (b) an antigen-binding T cell receptor (TCR) fragment; (c) the protein comprising a sufficient antibody constant region or portion thereof to bind to CD16, or an additional antigen-binding site that binds to CD16. (Item 68) 68. The protein of item 67, wherein the antigen-binding site is a Fab fragment and the antigen-binding TCR fragment is a single-chain TCR (scTCR) fragment. (Item 69) 69. The protein of item 68, wherein the scTCR fragment is linked to the polypeptide chain of the antibody constant region via a hinge comprising Ala-Ser. (Item 70) 68. The protein of item 67, wherein the antigen-binding site is an scFv and the antigen-binding TCR fragment is an extracellular TCR fragment. (Item 71) 71. The protein of item 70, wherein the scFv is linked to the polypeptide chain of the antibody constant region via a hinge comprising Ala-Ser. (Item 72) 72. The protein of item 69 or 71, wherein the hinge further comprises the amino acid sequence Thr-Lys-Gly. (Item 73) 68. The protein of item 67, wherein the antigen-binding site is a Fab fragment and the antigen-binding TCR fragment is an extracellular TCR fragment. (Item 74) 68. The protein of item 67, further comprising an additional antigen-binding TCR fragment that binds to the same antigen as the antigen-binding TCR fragment. (Item 75) 75. The protein of item 74, wherein the antigen-binding site is an scFv, and the antigen-binding TCR fragment and the additional antigen-binding TCR fragment are extracellular TCR fragments. (Item 76) 75. The protein of item 74, wherein the antigen-binding site is an scFv, and the antigen-binding TCR fragment and the additional antigen-binding TCR fragment are scTCR fragments. (Item 77) 77. The protein according to any one of items 70 to 72 and 75 to 76, wherein the scFv comprises a heavy chain variable domain linked to a light chain variable domain via a flexible linker. (Item 78) 78. The protein of item 77, wherein the flexible linker comprises (G4S)4. (Item 79) 79. The protein according to any one of items 70 to 72 and 75 to 78, wherein the scFv comprises a heavy chain variable domain located at the N-terminus or the C-terminus of a light chain variable domain. (Item 80) 80. The protein according to any one of items 67 to 79, wherein the antigen-binding site comprises a heavy chain variable domain and a light chain variable domain, and the heavy chain variable domain forms a disulfide bridge with the light chain variable domain. (Item 81) 81. The protein according to item 80, wherein the disulfide bridge is formed between Cys at position 44 of the heavy chain variable domain and Cys at position 100 of the light chain variable domain, at positions defined under Kabat numbering. (Item 82) 82. The protein according to any one of items 67 to 81, wherein the antigen-binding site binds to human NKG2D. (Item 83) 83. The protein according to any one of Aspects 67 to 82, wherein the antigen-binding site comprises a heavy chain variable domain that is at least 90% identical to an amino acid sequence selected from SEQ ID NO: 94, SEQ ID NO: 1, SEQ ID NO: 44, SEQ ID NO: 52, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 70, SEQ ID NO: 78, SEQ ID NO: 86, SEQ ID NO: 102, SEQ ID NO: 322, SEQ ID NO: 325, SEQ ID NO: 328, SEQ ID NO: 331, SEQ ID NO: 334, and SEQ ID NO: 337. (Item 84) 84. The protein of item 83, wherein the antigen-binding site comprises a heavy chain variable domain comprising an amino acid sequence at least 90% identical to SEQ ID NO: 94 and a light chain variable domain comprising an amino acid sequence at least 90% identical to SEQ ID NO: 98. (Item 85) 84. The protein of item 83, wherein the antigen-binding site comprises a heavy chain variable domain comprising an amino acid sequence at least 90% identical to SEQ ID NO: 322 and a light chain variable domain comprising an amino acid sequence at least 90% identical to SEQ ID NO: 98. (Item 86) 84. The protein of item 83, wherein the antigen-binding site comprises a heavy chain variable domain comprising an amino acid sequence at least 90% identical to SEQ ID NO: 325 and a light chain variable domain comprising an amino acid sequence at least 90% identical to SEQ ID NO: 98. (Item 87) 84. The protein of item 83, wherein the antigen-binding site comprises a heavy chain variable domain comprising an amino acid sequence at least 90% identical to SEQ ID NO: 328 and a light chain variable domain comprising an amino acid sequence at least 90% identical to SEQ ID NO: 98. (Item 88) 84. The protein of item 83, wherein the antigen-binding site comprises a heavy chain variable domain comprising an amino acid sequence at least 90% identical to SEQ ID NO: 331 and a light chain variable domain comprising an amino acid sequence at least 90% identical to SEQ ID NO: 98. (Item 89) 84. The protein of item 83, wherein the antigen-binding site comprises a heavy chain variable domain comprising an amino acid sequence at least 90% identical to SEQ ID NO: 334 and a light chain variable domain comprising an amino acid sequence at least 90% identical to SEQ ID NO: 98. (Item 90) 84. The protein of item 83, wherein the antigen-binding site comprises a heavy chain variable domain comprising an amino acid sequence at least 90% identical to SEQ ID NO: 337 and a light chain variable domain comprising an amino acid sequence at least 90% identical to SEQ ID NO: 98. (Item 91) 84. The protein of item 83, wherein the antigen-binding site comprises a heavy chain variable domain comprising an amino acid sequence at least 90% identical to SEQ ID NO: 44 and a light chain variable domain comprising an amino acid sequence at least 90% identical to SEQ ID NO: 48. (Item 92) 84. The protein of item 83, wherein the antigen-binding site comprises a heavy chain variable domain comprising an amino acid sequence at least 90% identical to SEQ ID NO: 52 and a light chain variable domain comprising an amino acid sequence at least 90% identical to SEQ ID NO: 56. (Item 93) 84. The protein of item 83, wherein the antigen-binding site comprises a heavy chain variable domain comprising an amino acid sequence at least 90% identical to SEQ ID NO: 60 and a light chain variable domain comprising an amino acid sequence at least 90% identical to SEQ ID NO: 61. (Item 94) 84. The protein of item 83, wherein the antigen-binding site comprises a heavy chain variable domain comprising an amino acid sequence at least 90% identical to SEQ ID NO: 62 and a light chain variable domain comprising an amino acid sequence at least 90% identical to SEQ ID NO: 66. (Item 95) 84. The protein of item 83, wherein the antigen-binding site comprises a heavy chain variable domain comprising an amino acid sequence at least 90% identical to SEQ ID NO: 70 and a light chain variable domain comprising an amino acid sequence at least 90% identical to SEQ ID NO: 74. (Item 96) 84. The protein of item 83, wherein the antigen-binding site comprises a heavy chain variable domain comprising an amino acid sequence at least 90% identical to SEQ ID NO: 78 and a light chain variable domain comprising an amino acid sequence at least 90% identical to SEQ ID NO: 82. (Item 97) 84. The protein of item 83, wherein the antigen-binding site comprises a heavy chain variable domain comprising an amino acid sequence at least 90% identical to SEQ ID NO: 86 and a light chain variable domain comprising an amino acid sequence at least 90% identical to SEQ ID NO: 90. (Item 98) 84. The protein of item 83, wherein the antigen-binding site comprises a heavy chain variable domain comprising an amino acid sequence at least 90% identical to SEQ ID NO: 102 and a light chain variable domain comprising an amino acid sequence at least 90% identical to SEQ ID NO: 106. (Item 99) 83. The protein according to any one of Items 67 to 82, wherein the antigen-binding site comprises a heavy chain variable domain comprising an amino acid sequence at least 90% identical to SEQ ID NO: 110 and a light chain variable domain comprising an amino acid sequence at least 90% identical to SEQ ID NO: 111. (Item 100) 83. The protein according to any one of Items 67 to 82, wherein the antigen-binding site comprises a heavy chain variable domain comprising an amino acid sequence at least 90% identical to SEQ ID NO: 112 and a light chain variable domain comprising an amino acid sequence at least 90% identical to SEQ ID NO: 113. (Item 101) 101. The protein of any one of items 67 to 100, wherein the antigen-binding TCR fragment binds to a peptide derived from a tumor-associated antigen presented by the major histocompatibility complex (MHC). (Item 102) 102. The protein of claim 101, wherein the antigen-binding TCR fragment binds to an ELAVL4 peptide having the amino acid sequence of SEQ ID NO: 425, which is presented by HLA-A*02:01:48, and wherein the antigen-binding TCR fragment comprises an alpha chain variable domain that is at least 90% identical to SEQ ID NO: 351 and a beta chain variable domain that is at least 90% identical to SEQ ID NO: 352. (Item 103) 102. The protein of claim 101, wherein the antigen-binding TCR fragment binds to an insulin peptide having the amino acid sequence of SEQ ID NO: 426 that is presented by HLA-A*02:01:48, and the antigen-binding TCR fragment comprises an alpha chain variable domain that is at least 90% identical to SEQ ID NO: 357 and a beta chain variable domain that is at least 90% identical to SEQ ID NO: 358. (Item 104) 102. The protein of claim 101, wherein the antigen-binding TCR fragment binds to a TERT peptide having the amino acid sequence of SEQ ID NO: 340, which is presented by HLA-A*02:01:48, and wherein the antigen-binding TCR fragment comprises an alpha chain variable domain that is at least 90% identical to SEQ ID NO: 363 and a beta chain variable domain that is at least 90% identical to SEQ ID NO: 364. (Item 105) 102. The protein of claim 101, wherein the antigen-binding TCR fragment binds to an ERBB2 peptide presented by HLA-A*02 and having the amino acid sequence of SEQ ID NO: 341, and the antigen-binding TCR fragment comprises an alpha chain variable domain that is at least 90% identical to SEQ ID NO: 430 and a beta chain variable domain that is at least 90% identical to SEQ ID NO: 431. (Item 106) 102. The protein of claim 101, wherein the antigen-binding TCR fragment binds to a WT1 peptide having the amino acid sequence of SEQ ID NO: 342 presented by HLA-A*02, and the antigen-binding TCR fragment comprises an alpha chain variable domain at least 90% identical to SEQ ID NO: 434 and a beta chain variable domain at least 90% identical to SEQ ID NO: 435. (Item 107) 102. The protein of claim 101, wherein the antigen-binding TCR fragment binds to a WT1 peptide having the amino acid sequence of SEQ ID NO: 342 presented by HLA-A*02, and the antigen-binding TCR fragment comprises an alpha chain variable domain at least 90% identical to SEQ ID NO: 438 and a beta chain variable domain at least 90% identical to SEQ ID NO: 439. (Item 108) 102. The protein of claim 101, wherein the antigen-binding TCR fragment binds to a MAGE-A3 peptide presented by HLA-A1 having the amino acid sequence of SEQ ID NO: 343, and the antigen-binding TCR fragment comprises an alpha chain variable domain at least 90% identical to SEQ ID NO: 375 and a beta chain variable domain at least 90% identical to SEQ ID NO: 376. (Item 109) 102. The protein of claim 101, wherein the antigen-binding TCR fragment binds to a MART1 peptide presented by HLA-A2 and having the amino acid sequence of SEQ ID NO: 344, and the antigen-binding TCR fragment comprises an alpha chain variable domain that is at least 90% identical to SEQ ID NO: 381 and a beta chain variable domain that is at least 90% identical to SEQ ID NO: 382. (Item 110) 102. The protein of claim 101, wherein the antigen-binding TCR fragment binds to a BIRC5 peptide presented by HLA-A2 having the amino acid sequence of SEQ ID NO: 346, and the antigen-binding TCR fragment comprises an alpha chain variable domain at least 90% identical to SEQ ID NO: 442 and a beta chain variable domain at least 90% identical to SEQ ID NO: 443. (Item 111) 102. The protein of claim 101, wherein the antigen-binding TCR fragment binds to a BIRC5 peptide presented by HLA-A2 having the amino acid sequence of SEQ ID NO: 346, and the antigen-binding TCR fragment comprises an alpha chain variable domain at least 90% identical to SEQ ID NO: 444 and a beta chain variable domain at least 90% identical to SEQ ID NO: 445. (Item 112) 102. The protein of claim 101, wherein the antigen-binding TCR fragment binds to a PRAME peptide presented by HLA-A2 and having the amino acid sequence of SEQ ID NO: 347, and the antigen-binding TCR fragment comprises an alpha chain variable domain at least 90% identical to SEQ ID NO: 395 and a beta chain variable domain at least 90% identical to SEQ ID NO: 396. (Item 113) 102. The protein of claim 101, wherein the antigen-binding TCR fragment binds to a PRAME peptide presented by HLA-A2 and having the amino acid sequence of SEQ ID NO: 347, and the antigen-binding TCR fragment comprises an alpha chain variable domain that is at least 90% identical to SEQ ID NO: 401 and a beta chain variable domain that is at least 90% identical to SEQ ID NO: 402. (Item 114) 102. The protein of claim 101, wherein the antigen-binding TCR fragment binds to a PRAME peptide presented by HLA-A2 and having the amino acid sequence of SEQ ID NO: 347, and the antigen-binding TCR fragment comprises an alpha chain variable domain that is at least 90% identical to SEQ ID NO: 407 and a beta chain variable domain that is at least 90% identical to SEQ ID NO: 408. (Item 115) 102. The protein of claim 101, wherein the antigen-binding TCR fragment binds to an NY-ESO-1 peptide presented by HLA-A2 and has the amino acid sequence of SEQ ID NO: 348, and the antigen-binding TCR fragment comprises an alpha chain variable domain at least 90% identical to SEQ ID NO: 413 and a beta chain variable domain at least 90% identical to SEQ ID NO: 414. (Item 116) 102. The protein of claim 101, wherein the antigen-binding TCR fragment binds to an NY-ESO-1 peptide presented by HLA-A2 and having the amino acid sequence of SEQ ID NO: 348, and the antigen-binding TCR fragment comprises an alpha chain variable domain at least 90% identical to SEQ ID NO: 418 and a beta chain variable domain at least 90% identical to SEQ ID NO: 414. (Item 117) 102. The protein of claim 101, wherein the antigen-binding TCR fragment binds to an NY-ESO-1 peptide presented by HLA-A2 and has the amino acid sequence of SEQ ID NO: 348, and the antigen-binding TCR fragment comprises an alpha chain variable domain at least 90% identical to SEQ ID NO: 421 and a beta chain variable domain at least 90% identical to SEQ ID NO: 422. (Item 118) Item 119. The protein of Item 101, wherein the antigen-binding TCR fragment binds to an SSX2 peptide having the amino acid sequence of SEQ ID NO: 345, which is presented by HLA-A2. 119. The protein of any one of items 67 to 118, wherein the antibody constant region or portion thereof sufficient to bind to CD16 comprises the hinge and CH2 domains of a human IgG1 antibody. (Item 120) 119. The protein of item 119, wherein the antibody constant region or portion thereof sufficient to bind to CD16 comprises an amino acid sequence that is at least 90% identical to amino acids 234 to 332 of a human IgG1 antibody. (Item 121) 121. The protein of paragraph 120, wherein the antibody constant region or portion thereof sufficient to bind to CD16 comprises an amino acid sequence at least 90% identical to the Fc domain of human IgG1 and differs at one or more positions selected from the group consisting of Q347, Y349, L351, S354, E356, E357, K360, Q362, S364, T366, L368, K370, N390, K392, T394, D399, S400, D401, F405, Y407, K409, T411, and K439. (Item 122) A formulation comprising a protein according to any one of items 33 to 121 and a pharmaceutically acceptable carrier. (Item 123) 122. A cell comprising one or more nucleic acids encoding the protein according to any one of items 33 to 121. (Item 124) 122. A method for enhancing tumor cell death, comprising exposing tumor and natural killer cells to a protein according to any one of items 33 to 121. (Item 125) A method for treating cancer, comprising administering to a patient the protein according to any one of Items 33 to 121 or the formulation according to Item 122. (Item 126) 126. The method of claim 125, wherein the cancer is selected from the group consisting of acute myeloid leukemia, acute myelomonocytic leukemia, B-cell lymphoma, bladder cancer, breast cancer, colorectal cancer, diffuse large B-cell lymphoma, esophageal cancer, Ewing's sarcoma, follicular lymphoma, gastric cancer, gastrointestinal cancer, gastrointestinal stromal tumor, glioblastoma, head and neck cancer, melanoma, mesothelioma, multiple myeloma, myelodysplastic syndrome, renal cell carcinoma, neuroblastoma, non-small cell lung cancer, neuroendocrine tumor, ovarian cancer, and pancreatic cancer, prostate cancer, sarcoma, small cell lung cancer, T-cell lymphoma, testicular cancer, thymic cancer, thyroid cancer, urothelial cancer, cancer infiltrated by myeloid-derived suppressor cells, cancer with extracellular matrix deposition, cancer with high levels of reactive stroma, and cancer with angiogenesis. [Brief explanation of the drawings]

[0064] [Figure 1]Figures A-C show three exemplary formats of multispecific binding proteins comprising an scFv linked via a hinge to an antibody constant region / domain. Figure A represents a trispecific antibody (TriNKET) comprising a tumor-targeting scFv or single-chain TCR (scTCR) fragment, an NKG2D-targeting Fab, and a heterodimerized antibody constant region / domain ("CD domain") that binds CD16. This antibody format is referred to herein as F3'-TriNKET. Figure B represents an antibody comprising an NKG2D-targeting scFv, a tumor-targeting Fab or extracellular TCR fragment, and a heterodimerized antibody constant region. This antibody format is referred to herein as F3-TriNKET. Figure C represents an antibody comprising an NKG2D-targeting scFv, a tumor-targeting scFv or scTCR fragment, and a heterodimerized antibody constant region. In certain exemplary TriNKETs, heterodimerization mutations on the CD domain linked to the antigen-binding site that binds NKG2D include K360E and K409W, and heterodimerization mutations on the opposing CD include Q347R, D399V, and F405T. [Figure 2A]

[0013] Figure 1 shows a trispecific antibody (TriNKET) in which a first antigen-binding site or antigen-binding TCR fragment binds to a tumor-associated antigen (such as BCMA shown here) or a peptide thereof presented by the major histocompatibility complex (MHC). The second antigen-binding site binds to NKG2D. The heterodimerized antibody constant region binds to CD16. In certain exemplary TriNKETs, heterodimerization mutations on the CD domain linked to the antigen-binding site that binds to NKG2D include K360E and K409W. Heterodimerization mutations on the opposite CD include Q347R, D399V, and F405T. [Figure 2B]Figure 1 shows a trispecific antibody (TriNKET) in which the first and second antigen-binding sites, or the first and second antigen-binding TCR fragments, bind to the same tumor-associated antigen (such as BCMA shown here) or the same peptide derived from a tumor-associated antigen presented by the same MHC. The third antigen-binding site binds to NKG2D. And the heterodimerized antibody constant region binds to CD16. These antibody formats are referred to herein as F4-TriNKET. In certain exemplary TriNKETs, heterodimerization mutations on the CD domain linked to the antigen-binding site that binds to NKG2D include K360E and K409W. Heterodimerization mutations on the opposite CD include Q347R, D399V, and F405T. Figure 2 shows the first two antigen-binding sites of the Fab format. [Figure 2C]

[0013] Figure 1 shows a trispecific antibody (TriNKET) in which the first and second antigen-binding sites, or the first and second antigen-binding TCR fragments, bind to the same tumor-associated antigen (such as BCMA shown here) or the same peptide derived from a tumor-associated antigen presented by the same MHC. The third antigen-binding site binds to NKG2D. And the heterodimerized antibody constant region binds to CD16. These antibody formats are referred to herein as F4-TriNKET. In certain exemplary TriNKETs, heterodimerization mutations on the CD domain linked to the antigen-binding site that binds to NKG2D include K360E and K409W. Heterodimerization mutations on the opposite CD include Q347R, D399V, and F405T.

[0014] Figure 2 shows the first two antigen-binding sites in the scFv format. [Figure 3A] Results are presented from an accelerated stability study conducted at 37°C, which found that F3'-TriNKET was stable over a 4-week period. [Figure 3B] Results are presented from an accelerated stability study conducted at 37°C, which found that F3'-TriNKET was stable over a 4-week period. [Figure 3C] Results are presented from an accelerated stability study conducted at 37°C, which found that F3'-TriNKET was stable over a 4-week period. [Figure 4] This indicates that purified F3'-TrinKET was stable during low pH maintenance. [Figure 5] A and B show that purified F3'-TrinKET was stable after five freeze-thaw cycles, regardless of pH (A shows freeze-thaw cycles in PBS, and Figure 5B shows freeze-thaw cycles in citrate at pH 5.5). [Figure 6] A bar graph of the forced degradation conditions under which F3'-TrinKET remained stable is shown. [Figure 7] Binding of F3'-TrinKET-HER2 or trastuzumab to HER2+ Colo-201 cells is shown. [Figure 8] Binding of F3'-TrinKET-CD33 or CD33 monoclonal antibody to CD33 expressed on Molm-13 cells is shown. [Figure 9] 1 shows F3′-TrinKET-HER2-mediated cytotoxicity against the HER2-low cell line 786-O. [Figure 10] 1 shows F3′-TrinKET-HER2-mediated cytotoxicity against the HER2-high cell line SkBr-3. [Figure 11] Figure 1 shows F3'-TrinKET-CD33-mediated cytotoxicity against two CD33-positive human cell lines, EOL-1. [Figure 12] Figure 1 shows F3'-TrinKET-CD33-mediated cytotoxicity against two CD33-positive human cell lines, THP-1. [Figure 13A] This shows that F3'-TrinKET-HER2 binding to FcγRIa is similar to Herceptin. [Figure 13B] This shows that F3'-TrinKET-HER2 binding to FcγRIIa is similar to Herceptin. [Figure 13C]This shows that F3'-TrinKET-HER2 binding to FcγRIIIa 158V is similar to Herceptin. [Figure 14A] A shows that F3'-TrinKET-HER2, which is an scFv HER2 binder, binds to human HER2 similarly to Herceptin, which is an Fab HER2 binder. B shows that F3'TrinKET-CD33, which is an scFv CD33 binder, binds to human CD33 similarly to a CD33 monoclonal antibody, which is an Fab CD33 binder. [Figure 14B] A shows that F3'-TrinKET-HER2, which is an scFv HER2 binder, binds to human HER2 similarly to Herceptin, which is an Fab HER2 binder. B shows that F3'TrinKET-CD33, which is an scFv CD33 binder, binds to human CD33 similarly to a CD33 monoclonal antibody, which is an Fab CD33 binder. [Figure 15] Figure 1 shows that two-step purification of F3-TriNKET-BCMA achieves 99% purity. [Figure 16] Figure 1 shows simultaneous engagement of NKG2D and CD33 targets with high potency by F3-TriNKET-CD33. [Figure 17] Figure 1 shows simultaneous engagement of NKG2D and BCMA targets with high potency by F3-TriNKET-BCMA. [Figure 18A] This demonstrates that the F3-TriNKET format is stable for at least 14 days. [Figure 18B] This shows that the F3-TriNKET format is stable after low pH storage. [Figure 18C] This demonstrates that the F3-TriNKET format is stable after at least five freeze-thaw cycles. [Figure 19] 1 is a line graph showing that BCMA-targeted F4-TriNKETs with different NKG2D-binding domains enhance human NK cell lysis of KMS12-PE myeloma cells. [Figure 20] 1 is a line graph showing that BCMA-targeted F4-TriNKETs with different NKG2D-binding domains enhance human NK cell lysis of MM.1R myeloma cells. [Figure 21] 1 is a line graph showing binding of F4-TriNKET, duobody-TriNKET, and BCMA monoclonal antibodies to MM.1R myeloma cells. [Figure 22] FACS shows that incubation with F4-TriNKET increases surface BCMA expression over time. [Figure 23] 1 is a line graph showing that F4-TrinKET stabilizes surface BCMA. [Figure 24] 1 is a bar graph showing that BCMA-targeted F4-TriNKET with A49 binder mediates more potent killing of KMS12-PE myeloma cells at different concentrations over 30 hours than duobody-TriNKET. [Figure 25] 1 is a bar graph showing that BCMA-targeted F4-TriNKET with A49 binder mediates more potent killing of MM.1S myeloma cells at different concentrations over 30 hours than duobody-TriNKET. DETAILED DESCRIPTION OF THE INVENTION

[0065] The present invention provides improved single-chain variable fragments (scFvs) linked to antibody constant domains via a hinge sequence. This hinge sequence provides flexibility for the scFv in antigen binding. The present invention also provides multispecific binding proteins comprising one or more scFvs, wherein the multispecific binding proteins bind to the NKG2D and CD16 receptors on natural killer cells and a tumor-associated antigen. The present invention also provides multispecific binding proteins that contain two tumor-associated antigen-binding sites that bind to the same tumor-associated antigen and that bind to the NKG2D and CD16 receptors on natural killer cells. Pharmaceutical compositions comprising such multispecific binding proteins, as well as therapeutic methods using such multispecific binding proteins and pharmaceutical compositions for purposes such as the treatment of cancer, are also provided. Various aspects of the present invention are described in the following sections, although aspects of the present invention described in one particular section are not limited to any particular section.

[0066] To facilitate the understanding of this invention, a number of terms and phrases are defined below.

[0067] As used herein, the terms "a" and "an" mean "one or more" and include plurals, unless the context is inappropriate.

[0068] As used herein, the terms "subject" and "patient" refer to an organism treated by the methods and compositions described herein. Such organisms preferably include, but are not limited to, mammals (e.g., murine, simian, equine, bovine, porcine, canine, feline, etc.), and more preferably, humans.

[0069] As used herein, the term "antigen-binding site" refers to the portion of an immunoglobulin molecule involved in antigen binding. In human antibodies, the antigen-binding site is formed by amino acid residues from the N-terminal variable ("V") regions of the heavy ("H") and light ("L") chains. Three highly divergent stretches within the V regions of the heavy and light chains are called "hypervariable regions," which are sandwiched between more conserved adjacent stretches known as "framework regions," or "FRs." Thus, the term "FR" refers to the amino acid sequences naturally found adjacent to and between hypervariable regions in immunoglobulins. In human antibody molecules, the three hypervariable regions of the light chain and the three hypervariable regions of the heavy chain are positioned relative to one another in three-dimensional space to form an antigen-binding surface. The antigen-binding surface is complementary to the three-dimensional surface of a bound antigen, and the three hypervariable regions of each of the heavy and light chains are called "complementarity-determining regions," or "CDRs." In certain animals, such as camelids and cartilaginous fish, the antigen-binding site is formed by a single antibody chain, providing a "single domain antibody." The antigen-binding site may be present in an intact antibody, in an antigen-binding fragment of an antibody that retains the antigen-binding surface, or in a recombinant polypeptide such as an scFv, using a peptide linker to link the heavy chain variable domain to the light chain variable domain in a single polypeptide.

[0070] As used herein, the terms "antigen-binding T cell receptor fragment" and "antigen-binding TCR fragment" are used interchangeably and refer to a portion of a T cell receptor (TCR) that binds to a cognate antigen. In the human αβ TCR, the antigen-binding TCR fragment comprises an alpha chain variable domain (Vα) and a beta chain variable domain (Vβ), each of which contains three complementarity-determining regions (CDRs). The hypervariable loops, named CDR3α and CDR3β, occupy central positions for binding antigenic peptides. The germline-encoded CDR1α, CDR2α, CDR1β, and CDR2β loops make the most contact with MHC, which presents the antigenic peptide. In the human γδ TCR, the antigen-binding TCR fragment comprises a gamma chain variable domain (Vγ) and a delta chain variable domain (Vδ), each of which contains three CDRs. Human γδ TCRs recognize antigens (e.g., peptides or lipids) presented by MHC or MHC-associated molecules (e.g., CD1, endothelial protein C receptor (EPCR), or MHC class I polypeptide-related sequence A (MICA)). It is understood that other proteins, such as F1-ATPase, may also present antigens to γδ TCRs. Antigen-binding TCR fragments can be present in intact TCRs, in engineered TCRs with TCR chains linked by disulfide bonds, in antigen-binding fragments of intact or engineered TCRs that retain an antigen-binding surface, or in recombinant polypeptides with the variable domains of the TCR (e.g., Vα and Vβ) connected by a peptide linker within a single polypeptide. Non-limiting examples of antigen-binding TCR fragments include TCR fragments comprising variable domains (e.g., Vα and Vβ) and constant domains (e.g., Cα and Cβ), but lacking the connecting, transmembrane, and cytoplasmic regions of the TCR, referred to herein as "extracellular TCR fragments," and variable regions of the TCR (e.g., Vα and Vβ) connected by a peptide linker, referred to herein as "single-chain TCR (scTCR) fragments."

[0071] As used herein, the term "effective amount" refers to a sufficient amount of a compound (e.g., a compound of the present invention) to achieve a beneficial or desired result. An effective amount may be administered in one or more administrations, applications, or dosages, and is not intended to be limited to a particular formulation or route of administration. As used herein, the term "treating" includes any effect, e.g., resulting in the improvement of a condition, disease, disorder, etc., or the alleviation, reduction, reduction, modulation, amelioration, or elimination, or the amelioration of symptoms thereof.

[0072] As used herein, the term "pharmaceutical composition" refers to a combination of an active agent with an inert or active carrier that makes the composition particularly suitable for diagnostic or therapeutic use in vivo or ex vivo.

[0073] As used herein, the term "pharmaceutically acceptable carrier" refers to any of the standard pharmaceutical carriers, such as phosphate-buffered saline solution, water, emulsions (e.g., oil / water or water / oil emulsions), and various types of wetting agents. The composition may also contain stabilizers and preservatives. For examples of carriers, stabilizers, and adjuvants, see, for example, Martin, Remington's Pharmaceutical Sciences, 15th Ed., Mack Publ. Co., Easton, PA

[1975] .

[0074] Throughout this specification, where compositions are described as having, comprising, or consisting of particular ingredients, or processes and methods are described as having, comprising, or consisting of particular steps, it is contemplated that there are additionally compounds of the invention that consist essentially of or consist of the recited ingredients, and processes and methods of the invention that consist essentially of or consist of the recited processing steps.

[0075] As a general matter, compositions specifying percentages are by weight unless otherwise specified. Furthermore, if a variable is not accompanied by a definition, the previous definition of that variable takes precedence.

[0076] I. Protein The present invention provides improved single-chain variable fragments (scFv) linked to an antibody constant domain via a hinge sequence. In some embodiments, the hinge comprises the amino acids Ala-Ser. In some other embodiments, the hinge comprises the amino acids Ala-Ser and Thr-Lys-Gly. The scFv may comprise a heavy chain variable domain and a light chain variable domain. In some embodiments, the scFv binds to NKG2D or a tumor-associated antigen. The hinge sequence provides flexibility for the scFv to bind to the target antigen.

[0077] In some scFv embodiments, the heavy chain variable domain forms a disulfide bridge with the light chain variable domain to enhance the stability of the scFv. For example, a disulfide bridge can be formed between the C44 residue of the heavy chain variable domain and the C100 residue of the light chain variable domain. In some embodiments, the heavy chain variable domain is linked to the light chain variable domain via a flexible linker. Any suitable linker, such as a (G4S)4 linker, may be used. In some scFv embodiments, the heavy chain variable domain is located at the N-terminus of the light chain variable domain. In some scFv embodiments, the heavy chain variable domain is located at the C-terminus of the light chain variable domain.

[0078] In some embodiments, the antibody constant domain linked to the scFv can be derived from the constant region of any species of antibody that binds to CD16. In some embodiments, the amino acid sequence of the constant region is at least 90% identical to that of a human antibody constant region, such as a human IgG1 constant region, IgG2 constant region, IgG3 constant region, or IgG4 constant region. In some other embodiments, the amino acid sequence of the constant region is at least 90% identical to that of an antibody constant region derived from another mammal, such as a rabbit, dog, cat, mouse, or horse. In some embodiments, the antibody constant region comprises a hinge, a CH2 domain, a CH3 domain, and optionally a CH1 domain. In some embodiments, the antibody constant region comprising a hinge, a CH2 domain, a CH3 domain, and optionally a CH1 domain is derived from a human IgG1 antibody. In some embodiments, the antibody constant region comprises an amino acid sequence at least 90% identical to amino acids 234-332 of a human IgG1 antibody.

[0079] Within the Fc domain, CD16 binding is mediated by the hinge region and CH2 domain. For example, within human IgG1, interaction with CD16 is primarily focused on amino acid residues Asp265-Glu269, Asn297-Thr299, Ala327-Ile332, Leu234-Ser239, and the carbohydrate residue N-acetyl-D-glucosamine within the CH2 domain (see Sondermann et al., Nature, 406(6793):267-273). Based on the known domains, mutations can be selected to enhance or reduce binding affinity to CD16, such as by using a phage display library or a yeast surface-displayed cDNA library, or can be designed based on the known three-dimensional structure of the interaction.

[0080] In some embodiments, the antibody constant domain comprises the CH2 and CH3 domains of an IgG antibody, such as a human IgG1 antibody. In some embodiments, mutations are introduced into the antibody constant domain to enable heterodimerization with another antibody constant domain. For example, if the antibody constant domain is derived from a human IgG1 constant domain, the antibody constant domain may comprise an amino acid sequence that is at least 90% identical to amino acids 234-332 of a human IgG1 antibody and differs at one or more positions selected from the group consisting of Q347, Y349, L351, S354, E356, E357, K360, Q362, S364, T366, L368, K370, N390, K392, T394, D399, S400, D401, F405, Y407, K409, T411, and K439. In some embodiments, the antibody constant domain may comprise an amino acid sequence at least 90% identical to amino acids 234-332 of a human IgG1 antibody, and may be selected from the group consisting of Q347E, Q347R, Y349S, Y349K, Y349T, Y349D, Y349E, Y349C, L351K, L351D, L351Y, S354C, E356K, E357Q, E357L, E357W, K360E, K360W, Q362E, S364K, S364E, S364H, S364D, T366V, T366I, T366L, T366M, and differing by one or more substitutions selected from the group consisting of T366K, T366W, T366S, L368E, L368A, L368D, K370S, N390D, N390E, K392L, K392M, K392V, K392F, K392D, K392E, T394F, D399R, D399K, D399V, S400K, S400R, D401K, F405A, F405T, Y407A, Y407I, Y407V, K409F, K409W, K409D, T411D, T411E, K439D, and K439E.

[0081] Listed below are examples of scFvs linked to antibody constant regions that also contain mutations that allow heterodimerization of the two polypeptide chains. For example, the heavy chain variable domain (V) of trastuzumab is H ) and the light chain variable domain (V L) are used. Each sequence contains a V containing a heterodimerization mutation. L -(G4S)4-V H - Hinge (AS)-Fc (underlined). L and V H 44V H -100V L An S-S bridge (underlined) is included and can be derived from any tumor-targeting antibody or NKG2D-binding antibody. Ala-Ser (AS, underlined) is included in the elbow hinge region sequence to balance flexibility and optimal shape. In certain embodiments, an additional sequence, Thr-Lys-Gly, can be added to the AS sequence at the hinge. The (G4S)4 linker is underlined in the sequences listed in the following paragraph.

[0082] Trastuzumab-scFv-FcA1 and trastuzumab-scFv-Fc B1 preferentially pair to form heterodimers. Trastuzumab-scFv-FcA2 and trastuzumab-scFv-Fc B2 can preferentially pair to form heterodimers. Trastuzumab-scFv-Fc A1 [ka] (SEQ ID NO: 303) Trastuzumab-scFv-Fc B1 [ka] (SEQ ID NO: 304) Trastuzumab-scFv-Fc A2 [ka] (SEQ ID NO: 305) Trastuzumab-scFv-Fc B2 [ka] (SEQ ID NO: 306)

[0083] In another aspect, the present invention provides a protein comprising an scFv linked to an antibody constant region as described above. In some embodiments, the protein comprises a first antigen-binding site comprising an scFv linked to an antibody constant domain, a second antigen-binding site that can be in Fab or scFv format, and a second antibody constant domain linked to the second antigen-binding site. In some embodiments, the protein is multispecific, where the first antigen-binding site binds to NKG2D, the second antigen-binding site in the form of a Fab binds to a tumor-associated antigen, and the antibody constant region binds to CD16 (as shown in Figure 1B, herein referred to as F3-TriNKET). In some other embodiments, the protein is multispecific, where the first antigen-binding site binds to a tumor-associated antigen, the second antigen-binding site in the form of a Fab binds to NKG2D, and the antibody constant region binds to CD16 (as shown in Figure 1A, herein referred to as F3'-TriNKET). The antibody constant region linked to the scFv heterodimerizes with the antibody constant region of the second antigen-binding site of the protein described herein. The multispecific binding proteins, including scFvs, described herein can take a variety of formats, as shown in Figures 1A-1C.

[0084] The multispecific binding protein binds to NK cells, γδ T cells, and CD8 + The multispecific binding protein may bind to NKG2D receptor-expressing cells, which may include, but are not limited to, αβ T cells. Upon binding to NKG2D, the multispecific binding protein may block natural ligands such as ULBP6 and MICA from binding to NKG2D and activating the NKG2D receptor.

[0085] The multispecific binding protein binds to cells that express CD16, an Fc receptor on the surface of leukocytes, including natural killer cells, macrophages, neutrophils, eosinophils, mast cells, and follicular dendritic cells.

[0086] Upon binding to the NKG2D and CD16 receptors on natural killer cells and tumor-associated antigens on cancer cells, the multispecific binding protein can bind to multiple types of NK activating receptors and block the binding of natural ligands to NKG2D. In certain embodiments, the protein can agonize human NK cells. In some embodiments, the protein can agonize NK cells in humans as well as in other species, such as rodents and cynomolgus monkeys.

[0087] In certain embodiments of the invention, the multispecific binding protein comprises a first antigen-binding site that binds to a tumor-associated antigen, a second antigen-binding site that binds to the same tumor-associated antigen as the first antigen-binding site, a third antigen-binding site that binds to NKG2D, and an antibody constant region or portion thereof sufficient to bind to CD16, or a fourth antigen-binding site that binds to CD16. Any one of the antigen-binding sites can be in the form of either a Fab or an scFv. Exemplary formats are shown in Figures 2B-2C. The V of an scFv H- V L and V L -V H Both orientations are embodiments of the present disclosure.

[0088] In some embodiments, the first antigen-binding site or the second antigen-binding site that binds to the same tumor-associated antigen is an scFv, and the third antigen-binding site that binds to NKG2D is an scFv. In some embodiments, the first antigen-binding site and the second antigen-binding site that binds to the same tumor-associated antigen are each scFv, and the third antigen-binding site that binds to NKG2D is an scFv. In some embodiments, the first antigen-binding site or the second antigen-binding site that binds to the same tumor-associated antigen is a Fab, and the third antigen-binding site that binds to NKG2D is an scFv. In some embodiments, the first antigen-binding site and the second antigen-binding site that binds to the same tumor-associated antigen are each Fab, and the third antigen-binding site that binds to NKG2D is an scFv. In some embodiments, the first antigen-binding site and the second antigen-binding site that binds to the same tumor-associated antigen are each Fab, and the third antigen-binding site that binds to NKG2D is an scFv. In some embodiments, the first antigen-binding site and the second antigen-binding site of the F4-TrinKET of the present disclosure have identical amino acid sequences.

[0089] In other embodiments, the multispecific binding proteins disclosed herein comprise a first antigen-binding site that binds a tumor-associated antigen, a second antigen-binding site that binds a different antigen, a third antigen-binding site that binds NKG2D, and a sufficient antibody constant region or portion thereof to bind CD16, or a fourth antigen-binding site that binds CD16. Any one of the antigen-binding sites can be in the form of either a Fab or an scFv (in either a VH-VL or VL-VH orientation). In certain embodiments, the first antigen-binding site or the second antigen-binding site that binds two different antigens is an scFv, and the third antigen-binding site that binds NKG2D is an scFv. In certain embodiments, the first antigen-binding site and the second antigen-binding site that bind two different antigens are each scFv, and the third antigen-binding site that binds NKG2D is an scFv. In certain embodiments, the first antigen-binding site or the second antigen-binding site that binds two different antigens is a Fab, and the third antigen-binding site that binds NKG2D is an scFv. In certain embodiments, the first and second antigen-binding sites that bind to two different antigens are each a Fab, and the third antigen-binding site that binds to NKG2D is an scFv.

[0090] In certain embodiments, the multispecific binding protein provided herein (referred to herein as F4-TriNKET) provides bivalent engagement of tumor-associated antigens, thereby stabilizing and maintaining the tumor-associated antigens on the surface of cancer cells and enhancing cytotoxicity by NK cells against the cancer cells. In some embodiments, the bivalent engagement of tumor-associated antigens by the multispecific binding protein confers higher avidity of the multispecific binding protein to cancer cells, thereby promoting a stronger cytotoxic response from NK cells to cancer cells, particularly cancer cells that express low levels of tumor-associated antigens.

[0091] The present invention also provides multispecific binding proteins comprising (a) an antigen-binding site that binds to NKG2D, (b) an antigen-binding TCR fragment, and (c) a sufficient antibody constant region or portion thereof that binds to CD16, or an additional antigen-binding site that binds to CD16. In certain embodiments, the antigen-binding TCR fragment binds a peptide derived from a tumor-associated antigen (TAA) presented by MHC, e.g., a peptide derived from a human tumor-associated antigen presented by human leukocyte antigen (HLA). Element (b) can be present in various formats (e.g., soluble format), such as an extracellular TCR fragment or an scTCR fragment. Elements (a) and (c) may be present in various formats and / or may contain various mutations as disclosed above. For example, in certain embodiments, element (c) is an antibody constant region or portion thereof sufficient to bind to CD16, the antibody constant region or portion thereof comprising (i) a first antibody constant domain linked to an antigen-binding site that binds NKG2D, and (ii) a second antibody constant domain linked to an antigen-binding TCR fragment, wherein the first and second antibody constant domains are capable of heterodimerizing.

[0092] In certain embodiments, the antigen-binding site is a Fab fragment and the antigen-binding TCR fragment is an scTCR fragment. Provided that the NKG2D-binding portion is a Fab and the TAA-binding portion comprises variable domains linked by a single peptide linker (as with a multispecific binding protein comprising a first antigen-binding site comprising an scFv linked to an antibody constant domain; a second antigen-binding site in the form of a Fab; and a second antibody constant domain linked to the second antigen-binding site, where the first antigen-binding site binds a tumor-associated antigen, the second antigen-binding site binds NKG2D, and the antibody constant region binds CD16), this multispecific binding protein format is also referred to herein as F3'-TriNKET, as shown in Figure 1A.

[0093] In certain embodiments, the antigen-binding site is an scFv and the antigen-binding TCR fragment is an extracellular TCR fragment. Provided that the NKG2D-binding portion is an scFv and the TAA-binding portion comprises a variable domain and a constant domain (as in a multispecific binding protein comprising a first antigen-binding site comprising an scFv linked to an antibody constant domain; a second antigen-binding site in the form of an Fab; and a second antibody constant domain linked to the second antigen-binding site, where the first antigen-binding site binds NKG2D, the second antigen-binding site binds a tumor-associated antigen, and the antibody constant region binds CD16), this multispecific binding protein format is also referred to herein as F3-TriNKET, as shown in Figure 1B.

[0094] In certain embodiments, the antigen-binding site is an scFv and the antigen-binding TCR fragment is an scTCR fragment. Such a format is shown in Figure 1C. In certain embodiments, the antigen-binding site is a Fab and the antigen-binding TCR fragment is an extracellular TCR fragment. Such a format is shown in Figure 2A.

[0095] In certain embodiments, the multispecific binding protein comprises a first antigen-binding TCR fragment that binds to an antigen (e.g., a TAA peptide presented by MHC); a second antigen-binding TCR fragment that binds the same antigen as the first antigen-binding TCR fragment; an antigen-binding site that binds to NKG2D; and an antibody constant region or portion thereof sufficient to bind to CD16, or a fourth antigen-binding site that binds to CD16. Alternatively, the first antigen-binding TCR fragment and the second antigen-binding TCR fragment could bind to two different TAA peptides presented by the same or different MHC. Either one of the antigen-binding sites may take the form of either a Fab or an scFv. The first and second antigen-binding TCR fragments may take the form of either an extracellular TCR fragment or an scTCR fragment. An exemplary format that provides bivalent engagement of an antigen (e.g., a TAA peptide presented by MHC) is referred to herein as F4-TriNKET and is shown in Figures 2B-2C.

[0096] In some embodiments, the first antigen-binding TCR fragment and the second antigen-binding TCR fragment of the F4-TrinKET of the present disclosure bind to the same TAA peptide presented by the same MHC. In some embodiments, the first antigen-binding TCR fragment or the second antigen-binding TCR fragment is an scFv. In some embodiments, the first antigen-binding TCR fragment and the second antigen-binding TCR fragment are each scFv. In some embodiments, the first antigen-binding TCR fragment or the second antigen-binding TCR fragment is a Fab. In some embodiments, the first antigen-binding TCR fragment and the second antigen-binding TCR fragment are each a Fab.

[0097] The F4-TriNKET carrying the antigen-binding TCR fragment provided herein provides bivalent engagement of antigen (e.g., a TAA peptide presented by MHC), thereby stabilizing and maintaining the TAA peptide on the surface of cancer cells and enhancing NK cell cytotoxicity against the cancer cells. In some embodiments, bivalent engagement of the TAA peptide by the multispecific binding protein confers higher avidity of the multispecific binding protein to cancer cells, thereby promoting a stronger cytotoxic response from NK cells against cancer cells, particularly cancer cells that present low levels of the TAA peptide.

[0098] When the protein of the present invention comprises an scFv, V H is V L It is understood that Vα may be positioned at either the C-terminus or the N-terminus of Vβ. Similarly, when the protein of the invention comprises an scTCR fragment, Vα may be positioned at either the C-terminus or the N-terminus of Vβ.

[0099] Exemplary sequences of NKG2D-binding sites and tumor-associated antigen-binding sites or antigen-binding TCR fragments that can be incorporated into F3 / F3' and F4-TriNKET are listed herein.

[0100] NKG2D binding site Table 1 lists peptide sequences of heavy and light chain variable domains that can combine to bind to NKG2D. Unless otherwise indicated, the CDR sequences provided in Table 1 are as determined under Kabat. In some embodiments, the heavy and light chain variable domains are arranged in a Fab format. In some embodiments, the heavy and light chain variable domains are fused together to form an scFv.

[0101] NKG2D-binding domains may differ in their binding affinity to NKG2D, yet they all activate human NKG2D and NK cells. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7] [Table 1-8] [Table 1-9]

[0102] Alternatively, the heavy chain variable domain represented by SEQ ID NO: 110 may be paired with the light chain variable domain represented by SEQ ID NO: 111 to form an antigen-binding site capable of binding to NKG2D, as described in U.S. Patent No. 9,273,136. SEQ ID NO: 110 QVQLVESGGGLVKPGGSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAFIRYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDRGLGDGTYFDYWGQGTTVTVSS SEQ ID NO: 111 QSALTQPASVSGSPGQSITISCSGSSSNIGNNAVNWYQQLPGKAPKLLIYYDDLLPSGVSDRFSGSKSGTSAFLAISGLQSEDEADYYCAAWDDSLNGPVFGGGTKLTVL

[0103] Alternatively, the heavy chain variable domain set forth in SEQ ID NO: 112 may be paired with the light chain variable domain set forth in SEQ ID NO: 113 to form an antigen-binding site capable of binding to NKG2D, as set forth in U.S. Patent No. 7,879,985. SEQ ID NO: 112 QVHLQESGPGLVKPSETLSLTCTVSDDSISSYYWSWIRQPPGKGLEWIGHISYSGSANYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCANWDDAFNIWGQGTMVTVSS SEQ ID NO: 113 EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPWTFGQGTKVEIK Tumor-associated antigen binding site:

[0104] As used herein, "tumor-associated antigen" refers to any antigen, including but not limited to, a protein, glycoprotein, ganglioside, carbohydrate, or lipid, associated with cancer. Such antigens may be expressed on malignant cells or in the tumor microenvironment, such as on tumor-associated blood vessels, extracellular matrix, mesenchymal stroma, or immune infiltrate.For example, tumor-associated antigens include ANO1, BCMA, EpCAM, CAIX, CEA, CCR4, CD2, CD123, CD133, CD19, CD20, CD22, CD25, CD30, CD33, CD37, CD38, CD40, CD52, CD70, CLAUDIN-18.2, DLL3, EGFR / ERBB1, GD2, IGF1R, HER2, HER3 / ERBB3, HER4 / ERBB4, MUC1, cMET, SLAMF7, PSMA, mesothelin, MICA, and MI, which are expressed in cancer cells. CB, TRAILR1, TRAILR2, TROP2, MAGE-A3, B7.1, B7.2, CTLA4, PD1, 5T4, GPNMB, FR-alpha, PAPP-A, FLT3, GPC3, CXCR4, ROR1, ROR2, HLA-E, PD- L1, VLA4, CD44, CD13, CD15, CD47, CLL1, CD81, CD23, CD79a, CD79b, CD80, CRLF2, SLAMF7, CD138, CA125, NaPi2b, Nectin4, ADAM8, ADAM9, SL C44A4, CA19-9, LILRB1, LILRB2, LILRB3, LILRB4, LILRB5, LILRA1, LILRA2, LILRA3, LILRA4, LILRA5, and LILRA6, CCR8, CD7, CTLA4, CX3CR1 , ENTPD1, HAVCR2, IL-1R2, PDCD1LG2, TIGIT, TNFRSF4, TNFRSF8, TNFRSF9, GEM, NT5E, TNFRSF18, MUC1, P-cadherin, Plexin-A1, TNFRSF10B, ST These include EAP1, CDCP1, PTK7, Axl, erbB-3, EDNRB, Tyrp1, CD14, CD163, CSF3R, Siglec-9, ITGAM, VISTA, B7-H4 (VTCN1), CCR1, LRRC25, PTAFR, SIRPB1, TLR2, TLR4, CD300LB, ATP1A3, CCR5, MUC1 (or MUC1-C), Plexin-A1, TNFRSF10B, STEAP1, CDCP1, PTK7, AXL, EDNRB, OLR1, and TYRP1.

[0105] Tumor-associated antigen binding sites can be developed to bind to any tumor-associated antigen. In some embodiments, the tumor-associated antigen binding site comprises a heavy chain variable domain and a light chain variable domain, which can bind to the tumor-associated antigen in pairs. In some embodiments, the heavy chain variable domain and the light chain variable domain are arranged in a Fab format. In some embodiments, the heavy chain variable domain and the light chain variable domain are fused together to form an scFv. Exemplary tumor-associated antigen binding sites are listed below.

[0106] Table 2 lists peptide sequences of heavy and light chain variable domains that can combine to bind to BCMA. [Table 2-1] [Table 2-2] [Table 2-3]

[0107] Alternatively, the BCMA binding domain may comprise a heavy chain variable domain and a light chain variable domain, as listed in EM-801 and EM-901 below. EM-801 Heavy Chain Variable Domain (SEQ ID NO: 157): [ka] EM-801 Light Chain Variable Domain (SEQ ID NO: 158): [ka] [ka] EM-901 heavy chain variable domain (SEQ ID NO: 159) [ka] EM-901 Light Chain Variable Domain (SEQ ID NO: 160) [ka]

[0108] Alternatively, novel antigen binding sites capable of binding to BCMA may be identified by screening for binding to the amino acid sequence defined by SEQ ID NO:156. SEQ ID NO: 156 MLQMAGQCSQNEYFDSLLHACIPCQLRCSSNTPPLTCQRYCNASVTNSVKGTNAILWTCLGLSLIISLAVFVLMFLLRKINSEPLKDEFKNTGSGLLGMANIDLEKSRTGDEIILPRGLEYTVEECTCEDCIKSKPKVDSDHCFPLPAMEEGATILVTTKTNDYCKSLPAALSATEIEKSISAR

[0109] Table 3 lists peptide sequences of heavy and light chain variable domains that can combine to bind to CD33. The CD33 binding domains may differ in their binding affinity to CD33. [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4] [Table 3-5]

[0110] Alternatively, novel antigen-binding sites capable of binding to CD33 can be identified by screening for binding to the amino acid sequence defined by SEQ ID NO:273. SEQ ID NO: 273 MPLLLLLPLLWAGALAMDPNFWLQVQESVTVQEGLCVLVPCTFFHPIPYYDKNSPVHGYWFREGAIISRDSPVATNKLDQEVQEETQGRFRLLGDPSRNNCSLSIVDARRRDNGSYFFRMERGSTKYSYKSPQLSVHVTDLTHRPKILIPGTLEPGHSKNLTCSVSWACEQGTPPIFSWLSA APTSLGPRTTHSSVLIITPRPQDHGTNLTCQVKFAGAGVTTERTIQLNVTYVPQNPTTGIFPDGSGKQETRAGVVHGAIGGAGVTALLALCLCLIFFIVKTHRRKAARTAVGRNDTHPTTGSASPKHQKKSKLHGPTETSSCSGAAPTVEMDEELHYASLNFHGMNPSKDTSTEYSEVRTQ

[0111] Table 4 lists peptide sequences of heavy and light chain variable domains that can combine to bind to HER2. [Table 4-1] [Table 4-2]

[0112] Alternatively, novel antigen binding sites capable of binding to HER2 can be identified by screening for binding to the amino acid sequence defined by SEQ ID NO:298.

[0113] Antigen-binding TCR fragments can be developed to bind to tumor-associated antigen peptides presented by MHC. In some embodiments, the antigen-binding TCR fragment comprises an alpha chain variable domain and a beta chain variable domain, which can pair to bind to a TAA peptide presented by MHC. In some embodiments, the alpha chain variable domain and the beta chain variable domain are arranged in an extracellular TCR fragment format. In some embodiments, the alpha chain variable domain and the beta chain variable domain are fused together to form a scTCR fragment.

[0114] Non-limiting examples of proteins that can be processed into TCR-targetable TAA peptides include tissue differentiation antigens (e.g., MART-1, gp100, CEA, CD19, and tyrosinase), tumor germline antigens (e.g., NY-ESO-1, MAGE-A1, MAGE-A3, MAGE-A4, MAGE-A12, MAGE-C2, BAGE1, GAGE1, CTAG1, CTAG2, XAGE-1B, and SSX2), normal proteins overexpressed by cancer cells (e.g., hTERT, EGFR, ERBB2, WT1, MUC1, and mesothelin), viral proteins (e.g., HPV, EBV, and MCC), and tumor-specific mutated antigens. Exemplary tumor-associated antigen binding sites are listed below.

[0115] Table 5 lists the peptide sequences of the TCR targets and the corresponding TCR alpha and beta chain sequences. [Table 5-1] [Table 5-2] [Table 5-3] [Table 5-4] [Table 5-5] [Table 5-6] [Table 5-7] [Table 5-8] [Table 5-9] [Table 5-10]

[0116] Antibody constant regions and heterodimerization The antibody constant region (Fc domain) described herein can be derived from the constant region of any species of antibody that binds to CD16. In some embodiments, the amino acid sequence of the constant region is at least 90% identical to that of a human antibody constant region, such as a human IgG1 constant region, IgG2 constant region, IgG3 constant region, or IgG4 constant region. In some other embodiments, the amino acid sequence of the constant region is at least 90% identical to that of an antibody constant region derived from another mammal, such as a rabbit, dog, cat, mouse, or horse. In some embodiments, the antibody constant region comprises a hinge, a CH2 domain, a CH3 domain, and optionally a CH1 domain. In some embodiments, the antibody constant region comprising a hinge, a CH2 domain, a CH3 domain, and optionally a CH1 domain is derived from a human IgG1 antibody. In some embodiments, the antibody constant region comprises an amino acid sequence at least 90% identical to amino acids 234 to 332 of a human IgG1 antibody.

[0117] Within the Fc domain, CD16 binding is mediated by the hinge region and CH2 domain. For example, within human IgG1, interaction with CD16 is primarily focused on amino acid residues Asp265-Glu269, Asn297-Thr299, Ala327-Ile332, and Leu234-Ser239, as well as the carbohydrate residue N-acetyl-D-glucosamine within the CH2 domain (see Sondermann et al., Nature, 406(6793):267-273). Based on the known domains, mutations can be selected to enhance or reduce binding affinity to CD16, such as by using a phage display library or a yeast surface-displayed cDNA library, or can be designed based on the known three-dimensional structure of the interaction.

[0118] In certain embodiments, mutations that may be incorporated into CH1 of the human IgG1 constant region may be at amino acids V125, F126, P127, T135, T139, A140, F170, P171, and / or V173. In certain embodiments, mutations that may be incorporated into Cκ of the human IgG1 constant region may be at amino acids E123, F116, S176, V163, S174, and / or T164.

[0119] Assembly of heterodimeric antibody heavy chains can be achieved by expressing two different antibody heavy chain sequences in the same cell, which can result in the assembly of not only heterodimers but also homodimers of each antibody heavy chain. Promoting preferential assembly of heterodimers can be achieved by incorporating different mutations in the CH3 domain of each antibody constant region, as shown in US13 / 494870, US16 / 028850, US11 / 533709, US12 / 875015, US13 / 289934, US14 / 773418, US12 / 811207, US13 / 866756, US14 / 647480, and US14 / 830336. For example, based on human IgG1, mutations can be made in the CH3 domain to incorporate different pairs of amino acid substitutions in the first and second polypeptides, which allow these two chains to selectively heterodimerize with each other. All amino acid substitution positions shown below are numbered according to the EU index as per Kabat.

[0120] In one scenario, the amino acid substitutions in a first polypeptide replace the original amino acid with a larger amino acid selected from arginine (R), phenylalanine (F), tyrosine (Y), or tryptophan (W), and at least one amino acid substitution in a second polypeptide replaces the original amino acid(s) with a smaller amino acid(s) selected from alanine (A), serine (S), threonine (T), or valine (V) such that the larger amino acid substitution (protrusion) fits into the surface of the smaller amino acid substitution (cavity). For example, one polypeptide may incorporate a T366W substitution, while the other polypeptide may incorporate three substitutions including T366S, L368A, and Y407V.

[0121] Compared to the human IgG1 constant region, for example, one or more mutations at Q347, Y349, L351, S354, E356, E357, K360, Q362, S364, T366, L368, K370, N390, K392, T394, D399, S400, D401, F405, Y407, K409, T411 and / or K439 may be incorporated into the constant region. Exemplary substitutions include, for example, Q347E, Q347R, Y349S, Y349K, Y349T, Y349D, Y349E, Y349C, T350V, L351K, L351D, L351Y, S354C, E356K, E357Q, E357L, E357W, K360E, K360W, Q362E, S364K, S364E, S364H, S364D, T366V, T366I, T366L, T366M, T366K, T366W, and T366S. , L368E, L368A, L368D, K370S, N390D, N390E, K392L, K392M, K392V, K392F, K392D, K392E, T394F, T394W, D399R, D399K, D399V, S400K, S400R, D401K, F405A, F405T, Y407A, Y407I, Y407V, K409F, K409W, K409D, T411D, T411E, K439D, and K439E.

[0122] Alternatively, the amino acid substitutions may be selected from the following set of substitutions shown in Table 6. [Table 6]

[0123] Alternatively, the amino acid substitutions may be selected from the following set of substitutions shown in Table 7. [Table 7]

[0124] Alternatively, the amino acid substitutions may be selected from the following set of substitutions shown in Table 8. [Table 8]

[0125] Alternatively, at least one amino acid substitution in each polypeptide chain may be selected from Table 9. [Table 9]

[0126] Alternatively, at least one amino acid substitution can be selected from the following set of substitutions in Table 10, where the position(s) shown in the first polypeptide column are replaced by any known negatively charged amino acid, and the position(s) shown in the second polypeptide column are replaced by any known positively charged amino acid. [Table 10]

[0127] Alternatively, at least one amino acid substitution can be selected from the following set in Table 11, where the position(s) shown in the first polypeptide row are replaced by any known positively charged amino acid, and the position(s) shown in the second polypeptide row are replaced by any known negatively charged amino acid. [Table 11]

[0128] Alternatively, the amino acid substitutions may be selected from the following set shown in Table 12. [Table 12]

[0129] Alternatively, or in addition, the structural stability of a heteromultimeric protein can be increased by introducing S354C into either the first or second polypeptide chain and Y349C into the opposing polypeptide chain, forming an artificial disulfide bridge within the interface of the two polypeptides.

[0130] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region at position T366, and the amino acid sequence of the other polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region at one or more positions selected from the group consisting of T366, L368, and Y407.

[0131] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgG1 constant region at one or more positions selected from the group consisting of T366, L368, and Y407, and the amino acid sequence of the other polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgG1 constant region at position T366.

[0132] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgG1 constant region at one or more positions selected from the group consisting of E357, K360, Q362, S364, L368, K370, T394, D401, F405, and T411, and the amino acid sequence of the other polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgG1 constant region at one or more positions selected from the group consisting of Y349, E357, S364, L368, K370, T394, D401, F405, and T411.

[0133] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgG1 constant region at one or more positions selected from the group consisting of Y349, E357, S364, L368, K370, T394, D401, F405, and T411, and the amino acid sequence of the other polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgG1 constant region at one or more positions selected from the group consisting of E357, K360, Q362, S364, L368, K370, T394, D401, F405, and T411.

[0134] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgG1 constant region at one or more positions selected from the group consisting of L351, D399, S400, and Y407, and the amino acid sequence of the other polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgG1 constant region at one or more positions selected from the group consisting of T366, N390, K392, K409, and T411.

[0135] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgG1 constant region at one or more positions selected from the group consisting of T366, N390, K392, K409, and T411, and the amino acid sequence of the other polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgG1 constant region at one or more positions selected from the group consisting of L351, D399, S400, and Y407.

[0136] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgG1 constant region at one or more positions selected from the group consisting of Q347, Y349, K360, and K409, and the amino acid sequence of the other polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgG1 constant region at one or more positions selected from the group consisting of Q347, E357, D399, and F405.

[0137] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgG1 constant region at one or more positions selected from the group consisting of Q347, E357, D399, and F405, and the amino acid sequence of the other polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgG1 constant region at one or more positions selected from the group consisting of Y349, K360, Q347, and K409.

[0138] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgG1 constant region at one or more positions selected from the group consisting of K370, K392, K409, and K439, and the amino acid sequence of the other polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgG1 constant region at one or more positions selected from the group consisting of D356, E357, and D399.

[0139] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgG1 constant region at one or more positions selected from the group consisting of D356, E357, and D399, and the amino acid sequence of the other polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgG1 constant region at one or more positions selected from the group consisting of K370, K392, K409, and K439.

[0140] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgG1 constant region at one or more positions selected from the group consisting of L351, E356, T366, and D399, and the amino acid sequence of the other polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgG1 constant region at one or more positions selected from the group consisting of Y349, L351, L368, K392, and K409.

[0141] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgG1 constant region at one or more positions selected from the group consisting of Y349, L351, L368, K392, and K409, and wherein the amino acid sequence of the other polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgG1 constant region at one or more positions selected from the group consisting of L351, E356, T366, and D399.

[0142] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgG1 constant region by a S354C substitution, and wherein the amino acid sequence of the other polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgG1 constant region by a Y349C substitution.

[0143] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgG1 constant region by a Y349C substitution, and wherein the amino acid sequence of the other polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgG1 constant region by a S354C substitution.

[0144] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgG1 constant region by the substitutions K360E and K409W, and the amino acid sequence of the other polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgG1 constant region by the substitutions O347R, D399V, and F405T.

[0145] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgG1 constant region by the substitutions O347R, D399V, and F405T, and the amino acid sequence of the other polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgG1 constant region by the substitutions K360E and K409W.

[0146] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgG1 constant region by the substitution T366W, and the amino acid sequence of the other polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgG1 constant region by the substitutions T366S, T368A, and Y407V.

[0147] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgG1 constant region by the substitutions T366S, T368A, and Y407V, and wherein the amino acid sequence of the other polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgG1 constant region by the substitution T366W.

[0148] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgG1 constant region by the following substitutions: T350V, L351Y, F405A, and Y407V, and wherein the amino acid sequence of the other polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgG1 constant region by the following substitutions: T350V, T366L, K392L, and T394W.

[0149] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgG1 constant region by the following substitutions: T350V, T366L, K392L, and T394W, and wherein the amino acid sequence of the other polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgG1 constant region by the following substitutions: T350V, L351Y, F405A, and Y407V.

[0150] Listed below are examples of constant region mutations that allow heterodimerization of two polypeptide chains. Trastuzumab heavy chains A1, A2, B1, and B2 each contain the heavy chain variable domain of trastuzumab (sequence in bold) and a constant region derived from human IgG1 with mutations at the underlined amino acids. Trastuzumab heavy chain A1 and trastuzumab heavy chain B1 can preferentially pair to form heterodimers. Trastuzumab heavy chain A2 and trastuzumab heavy chain B2 can preferentially pair to form heterodimers. Trastuzumab heavy chain A [ka] (SEQ ID NO: 299). Trastuzumab heavy chain B [ka] (SEQ ID NO: 300). Trastuzumab heavy chain A1 [ka] Trastuzumab heavy chain B1 [ka]

[0151] The above-described proteins can be produced using recombinant DNA techniques well known to those skilled in the art. For example, a first nucleic acid sequence encoding a first immunoglobulin chain comprising an scFv, hinge, and antibody constant region can be cloned into a first expression vector; a second nucleic acid sequence encoding a second immunoglobulin heavy chain, for example comprising another scFv, hinge, and antibody constant region, or comprising an antibody heavy chain, can be cloned into a second expression vector; and a third nucleic acid sequence encoding an antibody light chain can be cloned into a third expression vector. The first, second, and optionally third expression vectors can be stably transfected together into a host cell to produce the multimeric proteins described herein.

[0152] To achieve the highest yield of multispecific binding protein, different ratios of the first, second, and third expression vectors may be explored to determine the optimal ratio for transfection into host cells. After transfection, single clones may be isolated for cell bank generation using methods known in the art, such as limiting dilution, ELISA, FACS, microscopy, or Clonepix.

[0153] The clones may be cultured under conditions suitable for scale-up in a bioreactor to maintain expression of the multispecific protein. The multispecific binding proteins may be isolated and purified using methods known in the art, including centrifugation, depth filtration, cell lysis, homogenization, freeze-thaw, affinity purification, gel filtration, ion exchange chromatography, hydrophobic interaction exchange chromatography, and mixed-mode chromatography.

[0154] II. Therapeutic Applications The present invention provides methods for treating cancer using the multispecific binding proteins described herein and / or the pharmaceutical compositions described herein. The methods can be used to treat a variety of cancers, including solid tumors, lymphomas, and leukemias. The type of cancer being treated desirably matches the type of cancer cells to which the protein binds. For example, treatment of HER2-expressing cancers, such as HER2-expressing breast cancer, is desirably treated using a protein described herein that binds to the protein. In some embodiments, the methods provide for treating various BCMA-expressing cancers by administering to a patient in need thereof a therapeutically effective amount of a multispecific binding protein described herein. In some embodiments, the methods provide for treating various CD33-expressing cancers by administering to a patient in need thereof a therapeutically effective amount of a multispecific binding protein described herein.

[0155] Accordingly, one aspect of the present invention provides a method of treating cancer in a patient, comprising administering to a patient in need thereof a therapeutically effective amount of a protein as described herein for treating cancer. Additional aspects and embodiments of this method of treatment are described below.

[0156] The treatment method can be characterized according to the cancer to be treated. For example, in some embodiments, the cancer is a solid tumor. In some other embodiments, the cancer is brain cancer, bladder cancer, breast cancer, cervical cancer, colon cancer, colorectal cancer, endometrial cancer, esophageal cancer, leukemia, lung cancer, liver cancer, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, rectal cancer, kidney cancer, stomach cancer, testicular cancer, or uterine cancer.In still other embodiments, the cancer is angiogenic tumor, squamous cell carcinoma, adenocarcinoma, small cell carcinoma, melanoma, neuroblastoma, sarcoma (e.g., angiosarcoma or chondrosarcoma), laryngeal carcinoma, parotid gland carcinoma, biliary tract carcinoma, thyroid carcinoma, acral lentiginous melanoma, actinic keratosis, acute lymphocytic leukemia, acute myeloid leukemia, adenoid cystic carcinoma, adenoma, adenosarcoma, adenosquamous carcinoma, anal canal carcinoma, anal carcinoma, anorectal carcinoma, astrocytic tumor, Bartholin's gland carcinoma, basal cell carcinoma, bile duct carcinoma, bone cancer, bone marrow carcinoma, bronchial carcinoma, bronchial adenocarcinoma, carcinoid, cholangiocarcinoma, chondrosarcoma, choriocarcinoma, sarcoma ... Plexiolar papilloma / cancer, chronic lymphocytic leukemia, chronic myeloid leukemia, clear cell carcinoma, connective tissue carcinoma, cystadenoma, digestive system cancer, duodenal cancer, endocrine system cancer, endodermal sinus tumor, endometrial hyperplasia, endometrial stromal sarcoma, endometrioid adenocarcinoma, endothelial cell carcinoma, ependymal carcinoma, epithelial cell carcinoma, Ing's sarcoma, eye and orbital cancer, female genital cancer, focal nodular hyperplasia, gallbladder cancer, gastric antrum cancer, gastric fundus cancer, gastrinoma, glioblastoma, glucagonoma, heart cancer, hemangioblastoma, hemangioendothelioma, hemangioma, hepatic adenoma, hepatic adenomatosis, hepatobiliary tract cancer, hepatocellular carcinoma, Hodgkin's disease, Ileal cancer, insulinoma, intraepithelial neoplasia, interepithelial squamous neoplasia, intrahepatic cholangiocarcinoma, invasive squamous cell carcinoma, jejunal cancer, joint cancer, Kaposi's sarcoma, pelvic cancer, large cell carcinoma, colon cancer, leiomyosarcoma, lentigo maligna melanoma, lymphoma, male genital cancer, malignant melanoma, malignant mesothelioma, medulloblastoma, medulloepithelioma, meningeal cancer, mesothelial carcinoma, metastatic cancer, oral cancer, mucoepidermoid carcinoma, multiple myeloma, muscle cancer, nasal passage cancer, nervous system cancer, neuroepithelial adenocarcinoma, nodular melanoma, nonepithelial skin cancer, oat cell carcinoma, oligodendroglial carcinoma, oral cancer, osteosarcoma, papillary serous adenocarcinoma, penis carcinoma, pharyngeal cancer, pituitary tumor, plasmacytoma, pseudosarcoma, pulmonary blastoma, rectal cancer, renal cell carcinoma, respiratory system cancer, retinoblastoma, rhabdomyosarcoma, sarcoma, serous carcinoma, sinonasal cancer, skin cancer, small cell carcinoma, small intestine cancer, smooth muscle carcinoma, soft tissue cancer, somatostatin-secreting tumor, spinal cancer, squamous cell carcinoma, rhabdomyosarcoma, submesothelial carcinoma, superficial spreading melanoma, T-cell leukemia, tongue cancer, undifferentiated carcinoma, ureteral cancer, urethral cancer, bladder cancer, urinary system cancer, cervical cancer, uterine cancer, uveal melanoma, vaginal cancer, verrucous carcinoma, vipoma, vulvar cancer, well-differentiated carcinoma, or Wilms' tumor.

[0157] In certain other embodiments, the cancer to be treated is a non-Hodgkin's lymphoma, such as a B-cell lymphoma or a T-cell lymphoma. In certain embodiments, the non-Hodgkin's lymphoma is a B-cell lymphoma, e.g., diffuse large B-cell lymphoma, primary mediastinal B-cell lymphoma, follicular lymphoma, small lymphocytic lymphoma, mantle cell lymphoma, marginal zone B-cell lymphoma, extranodal marginal zone B-cell lymphoma, nodal marginal zone B-cell lymphoma, splenic marginal zone B-cell lymphoma, Burkitt's lymphoma, lymphoplasmacytic lymphoma, hairy cell leukemia, or primary central nervous system (CNS) lymphoma. In certain other embodiments, the non-Hodgkin's lymphoma is a T-cell lymphoma, e.g., precursor T-lymphoblastic lymphoma, peripheral T-cell lymphoma, cutaneous T-cell lymphoma, angioimmunoblastic T-cell lymphoma, extranodal natural killer / T-cell lymphoma, enteropathy-type T-cell lymphoma, subcutaneous panniculitis-like T-cell lymphoma, anaplastic large cell lymphoma, or peripheral T-cell lymphoma.

[0158] In certain embodiments, the cancer is AML, myelodysplastic syndrome, chronic myelomonocytic leukemia, myeloid blast crisis of chronic myelogenous leukemia, and ALL.

[0159] In some embodiments, the cancer to be treated may be characterized according to the presence of specific antigens expressed on the surface of cancer cells, which may express, in addition to BCMA, one or more of the following: CD2, CD19, CD20, CD30, CD38, CD40, CD52, CD70, EGFR / ERBB1, IGF1R, HER3 / ERBB3, HER4 / ERBB4, MUC1, cMET, SLAMF7, PSCA, MICA, MICB, TRAILR1, TRAILR2, MAGE-A3, B7.1, B7.2, CTLA4, and PD-L1.

[0160] In some embodiments, the cancer cells may express, in addition to CD33, one or more of the following: CD2, CD19, CD20, CD30, CD38, CD40, CD52, CD70, EGFR / ERBB1, IGF1R, HER3 / ERBB3, HER4 / ERBB4, MUC1, TROP2, cMET, SLAMF7, PSCA, MICA, MICB, TRAILR1, TRAILR2, MAGE-A3, B7.1, B7.2, CTLA4, and PD-L1.

[0161] In some embodiments, the cancer cells express, in addition to HER2, one or more of the following: CD2, CD19, CD20, CD30, CD38, CD40, CD52, CD70, EGFR / ERBB1, IGF1R, HER3 / ERBB3, HER4 / ERBB4, MUC1, cMET, SLAMF7, PSCA, MICA, MICB, TRAILR1, TRAILR2, MAGE-A3, B7.1, B7.2, CTLA4, and PD-L1.

[0162] In certain embodiments, the cancer to be treated can be characterized according to the presence of specific TAA peptides by MHC on the surface of cancer cells. Non-limiting examples of proteins that can be processed into TCR-targetable TAA peptides include tissue differentiation antigens (e.g., MART-1, gp100, CEA, CD19, and tyrosinase), tumor germline antigens (e.g., NY-ESO-1, MAGE-A1, MAGE-A3, MAGE-A4, MAGE-A12, MAGE-C2, BAGE1, GAGE1, CTAG1, CTAG2, XAGE-1B, and SSX2), normal proteins overexpressed by cancer cells (e.g., hTERT, EGFR, ERBB2, WT1, MUC1, and mesothelin), viral proteins (e.g., HPV, EBV, and MCC), and tumor-specific mutated antigens. In some embodiments, the cancer cells comprise an ELAVL4 peptide having the amino acid sequence of SEQ ID NO: 425, which is presented by HLA-A*02:01:48. In some embodiments, the cancer cells comprise an insulin peptide having the amino acid sequence of SEQ ID NO: 426, which is presented by HLA-A*02:01:48. In some embodiments, the cancer cells comprise an hTERT peptide having the amino acid sequence of SEQ ID NO: 340, which is presented by HLA-A*02:01:48. In some embodiments, the cancer cells comprise an ERBB2 peptide having the amino acid sequence of SEQ ID NO: 341, which is presented by HLA-A*02. In some embodiments, the cancer cells comprise a WT1 peptide having the amino acid sequence of SEQ ID NO: 342, which is presented by HLA-A*02. In some embodiments, the cancer cells comprise a MAGE-A3 peptide having the amino acid sequence of SEQ ID NO: 343, which is presented by HLA-A1. In some embodiments, the cancer cells comprise a MART1 peptide having the amino acid sequence of SEQ ID NO: 344, which is presented by HLA-A2. In some embodiments, the cancer cells comprise a BIRC5 peptide that is presented by HLA-A2 and has the amino acid sequence of SEQ ID NO: 346. In some embodiments, the cancer cells comprise a PRAME peptide that is presented by HLA-A2 and has the amino acid sequence of SEQ ID NO: 347.In some embodiments, the cancer cells comprise an NY-ESO-1 peptide that is presented by HLA-A2 and has the amino acid sequence of SEQ ID NO: 348. In some embodiments, the cancer cells comprise an SSX2 peptide that is presented by HLA-A2 and has the amino acid sequence of SEQ ID NO: 345.

[0163] III. Combination Therapy Another aspect of the present invention provides combination therapy: the multispecific binding proteins described herein can be used in combination with additional therapeutic agents to treat cancer.

[0164] Exemplary therapeutic agents that may be used as part of a combination therapy in the treatment of cancer include, for example, radiation, mitomycin, tretinoin, ribomustine, gemcitabine, vincristine, etoposide, cladribine, mitobronitol, methotrexate, doxorubicin, carboquone, pentostatin, nitracrine, zinostatin, cetrorelix, letrozole, raltitrexed, daunorubicin, fadrozole, fotemustine, thymalfasin, sobuzoxane, nedaplatin, cytarabine, bicalutamide, vinorelbine, vesnarinone, aminoglutethimide, amsacrine, proglumide, elliptinium acetate, ketanserin, doxifluridine, etretinate, isotretinoin, streptozocin, nimustine, vindesine, fluoxet ... These include thamide, drogenil, butosin, carmofur, razoxane, sizofiran, carboplatin, mitolactol, tegafur, ifosfamide, prednimustine, picibanil, levamisole, teniposide, improsulfan, enocitabine, lisuride, oxymetholone, tamoxifen, progesterone, mepitiostane, epitiostanol, formestane, interferon-alpha, interferon-2alpha, interferon-beta, interferon-gamma, colony-stimulating factor-1, colony-stimulating factor-2, denileukin diftitox, interleukin-2, luteinizing hormone-releasing factor, and variations of the foregoing agents that exhibit different binding to their cognate receptors and may exhibit increased or decreased serum half-lives.

[0165] An additional class of drugs that can be used as part of a combination therapy in the treatment of cancer are immune checkpoint inhibitors. Exemplary immune checkpoint inhibitors include agents that inhibit one or more of: (i) cytotoxic T-lymphocyte-associated antigen 4 (CTLA4), (ii) programmed cell death protein 1 (PD1), (iii) PDL1, (iv) LAG3, (v) B7-H3, (vi) B7-H4, and (vii) TIM3. The CTLA4 inhibitor ipilimumab has been approved by the United States Food and Drug Administration for the treatment of melanoma.

[0166] Still other agents that may be used as part of a combination therapy in the treatment of cancer are monoclonal antibody agents that target non-checkpoint targets (e.g., Herceptin) and non-cytotoxic agents (e.g., tyrosine kinase inhibitors).

[0167] Further categories of anti-cancer drugs include, for example, (i) ALK inhibitors, ATR inhibitors, A2A antagonists, base excision repair inhibitors, Bcr-Abl tyrosine kinase inhibitors, Bruton's tyrosine kinase inhibitors, CDC7 inhibitors, CHK1 inhibitors, cyclin-dependent kinase inhibitors, DNA-PK inhibitors, inhibitors of both DNA-PK and mTOR, DNMT1 inhibitors, DNMT1 inhibitors and 2-chloro-deoxyadenosine, HDAC inhibitors, hedgehog signaling pathway inhibitors, IDO inhibitors, JAK inhibitors, mTOR inhibitors, MEK inhibitors, inhibitors, MELK inhibitors, MTH1 inhibitors, PARP inhibitors, phosphoinositide 3-kinase inhibitors, inhibitors of both PARP1 and DHODH, proteasome inhibitors, topoisomerase II inhibitors, tyrosine kinase inhibitors, VEGFR inhibitors, and WEE1 inhibitors; (ii) agonists of OX40, CD137, CD40, GITR, CD27, HVEM, TNFRSF25, or ICOS; and (iii) cytokines selected from IL-12, IL-15, GM-CSF, and G-CSF.

[0168] The proteins of the present invention may also be used as an adjunct to surgical removal of the primary lesion.

[0169] The amounts of the multispecific binding protein and additional therapeutic agent, as well as the relative timing of administration, can be selected to achieve a desired combined therapeutic effect. For example, when administering a combination therapy to a patient in need of such administration, the therapeutic agents in the combination, or pharmaceutical composition(s) comprising the therapeutic agents, can be administered in any order, e.g., sequentially, together, concurrently, simultaneously, etc. Furthermore, for example, the multispecific binding protein can be administered at the time when the additional therapeutic agent(s) would be exerting their prophylactic or therapeutic effect, or vice versa.

[0170] IV. Pharmaceutical Compositions The disclosure also features pharmaceutical compositions / formulations that include a therapeutically effective amount of the multispecific binding proteins described herein.

[0171] This composition can be formulated for use in various drug delivery systems.For suitable formulation, one or more physiologically acceptable excipients or carriers may also be included in the composition.Suitable formulations for use in the present disclosure can be found in Remington's Pharmaceutical Sciences, Mack Publishing Company, Philadelphia, Pa., 17th ed., 1985.For a brief review of methods for drug delivery, see, for example, Langer (Science 249:1527-1533, 1990).

[0172] The intravenous drug delivery formulation of the present disclosure may be contained in a bag, pen, or syringe. In certain embodiments, the bag may be connected to a channel containing tubing and / or a needle. In certain embodiments, the formulation may be a lyophilized formulation or a liquid formulation. In certain embodiments, the formulation may be freeze-dried (lyophilized) and contained in about 12 to 60 vials. In certain embodiments, the formulation may be freeze-dried, and 45 mg of the freeze-dried formulation may be contained in one vial. In certain embodiments, about 40 mg to about 100 mg of the freeze-dried formulation may be contained in one vial. In certain embodiments, freeze-dried formulations from 12, 27, or 45 vials are combined to obtain a therapeutic dose of protein in the intravenous drug formulation. In certain embodiments, the formulation may be a liquid formulation and may be stored at about 250 mg / vial to about 1000 mg / vial. In certain embodiments, the formulation may be a liquid formulation and may be stored at about 600 mg / vial. In certain embodiments, the formulation may be a liquid formulation and stored as about 250 mg per vial.

[0173] The proteins of the present disclosure may be present in a liquid, aqueous pharmaceutical formulation comprising a therapeutically effective amount of the protein in a buffered solution that forms the formulation.

[0174] These compositions may be sterilized by conventional sterilization techniques or sterile filtered. The resulting aqueous solutions may be packaged for immediate use or lyophilized, with the lyophilized preparation being combined with a sterile aqueous carrier prior to administration. The pH of the preparation is typically 3 to 11, more preferably 5 to 9 or 6 to 8, most preferably 7 to 8, e.g., 7 to 7.5. The resulting solid form compositions may be packaged in a plurality of single-dose units, each containing a fixed amount of the aforementioned agent(s). The solid form compositions may also be packaged in containers for varying amounts.

[0175] In certain embodiments, the present disclosure provides an extended shelf life formulation comprising a protein of the present disclosure in combination with mannitol, citric acid monohydrate, sodium citrate, disodium phosphate dihydrate, sodium dihydrogen phosphate dihydrate, sodium chloride, polysorbate 80, water, and sodium hydroxide.

[0176] In certain embodiments, aqueous formulations are prepared containing proteins of the present disclosure in a pH buffer solution. The buffers of the present invention may have a pH ranging from about 4 to about 8, e.g., from about 4.5 to about 6.0, or from about 4.8 to about 5.5, or from about 5.0 to about 5.2. Intermediate pH ranges are also intended to be part of the present disclosure. For example, ranges of values using any combination of the above values as upper and / or lower limits are intended to be included. Examples of buffers that control the pH within this range include acetate (e.g., sodium acetate), succinate (e.g., sodium succinate), gluconate, histidine, citrate, and other organic acid buffers.

[0177] In certain embodiments, the formulation includes a buffer system containing citrate and phosphate to maintain a pH in the range of about 4 to about 8. In certain embodiments, the pH range can be from about 4.5 to about 6.0, or from about 4.8 to about 5.5, or from about 5.0 to about 5.2. In certain embodiments, the buffer system includes citric acid monohydrate, sodium citrate, disodium phosphate dihydrate, and / or sodium dihydrogen phosphate dihydrate. In certain embodiments, the buffer system comprises about 1.3 mg / ml citric acid (e.g., 1.305 mg / ml), about 0.3 mg / ml sodium citrate (e.g., 0.305 mg / ml), about 1.5 mg / ml disodium phosphate dihydrate (e.g., 1.53 mg / ml), about 0.9 mg / ml sodium dihydrogen phosphate dihydrate (e.g., 0.86 mg / ml), and about 6.2 mg / ml sodium chloride (e.g., 6.165 mg / ml). In certain embodiments, the buffer system comprises 1-1.5 mg / ml citric acid, 0.25-0.5 mg / ml sodium citrate, 1.25-1.75 mg / ml disodium phosphate dihydrate, 0.7-1.1 mg / ml sodium dihydrogen phosphate dihydrate, and 6.0-6.4 mg / ml sodium chloride. In certain embodiments, the pH of the formulation is adjusted with sodium hydroxide.

[0178] Polyols, which can act as tonicity agents and stabilize antibodies, can also be included in the formulation. The polyol is added to the formulation in an amount that can vary depending on the desired tonicity of the formulation. In certain embodiments, the aqueous formulation can be isotonic. The amount of polyol added can also vary depending on the molecular weight of the polyol. For example, a smaller amount of a monosaccharide (e.g., mannitol) can be added compared to a disaccharide (e.g., trehalose). In certain embodiments, a polyol that can be used in the formulation as a tonicity agent is mannitol. In certain embodiments, the mannitol concentration can be about 5 to about 20 mg / ml. In certain embodiments, the mannitol concentration can be about 7.5 to 15 mg / ml. In certain embodiments, the mannitol concentration can be about 10 to 14 mg / ml. In certain embodiments, the mannitol concentration can be about 12 mg / ml. In certain embodiments, the polyol sorbitol can be included in the formulation.

[0179] Detergents or surfactants may also be added to the formulation. Exemplary detergents include non-ionic detergents such as polysorbates (e.g., polysorbate 20, 80, etc.) or poloxamers (e.g., poloxamer 188). The amount of detergent added is such that it reduces aggregation of the formulated antibody and / or minimizes the formation of particulates in the formulation and / or reduces adsorption. In certain embodiments, the formulation may include a surfactant that is a polysorbate. In certain embodiments, the formulation may contain the detergent polysorbate 80 or Tween 80. Tween 80 is a term used to describe polyoxyethylene (20) sorbitan monooleate (see Fiedler, Lexikon der Hifsstoffe, Editio Cantor Verlag Aulendorf, 4th edi., 1996). In certain embodiments, the formulation may contain from about 0.1 mg / mL to about 10 mg / mL, or from about 0.5 mg / mL to about 5 mg / mL, of polysorbate 80. In certain embodiments, about 0.1% polysorbate 80 may be added to the formulation.

[0180] In embodiments, the protein products of the present disclosure are formulated as liquid formulations. The liquid formulations may be provided at a concentration of 10 mg / mL in any USP / Ph Eur Type I 50R vial closed with a rubber stopper and sealed with an aluminum crimp seal closure. The stopper may be made from a USP and Ph Eur compliant elastomer. In certain embodiments, the vial may be filled with 61.2 mL of protein product solution to allow for an extractable volume of 60 mL. In certain embodiments, the liquid formulation may be diluted with 0.9% saline.

[0181] In some embodiments, a liquid formulation of a protein of the present disclosure may be prepared as a solution at a concentration of 10 mg / mL in combination with a sugar at a stabilizing level. In some embodiments, the liquid formulation may be prepared in an aqueous carrier. In some embodiments, the stabilizer may be added in an amount that does not exceed an amount that may result in an undesirable or unsuitable viscosity for intravenous administration. In some embodiments, the sugar may be a disaccharide, such as sucrose. In some embodiments, the liquid formulation may also include one or more of a buffer, a surfactant, and a preservative.

[0182] In certain embodiments, the pH of the liquid formulation may be set by the addition of a pharmaceutically acceptable acid and / or base. In certain embodiments, the pharmaceutically acceptable acid may be hydrochloric acid. In certain embodiments, the base may be sodium hydroxide.

[0183] In addition to aggregation, deamidation is a common product change of peptides and proteins that can occur during fermentation, harvesting / cell clarification, purification, drug substance / drug product storage, and sample analysis. Deamidation is the loss of NH3 from proteins to form a succinimide intermediate that can undergo hydrolysis. The succinimide intermediate results in a 17-dalton mass loss from the parent peptide. Subsequent hydrolysis results in an 18-dalton mass gain. Isolation of the succinimide intermediate is difficult due to its instability under aqueous conditions. Thus, deamidation is typically detectable as a 1-dalton mass gain. Deamidation of asparagine results in either aspartic acid or isoaspartic acid. Parameters that affect the rate of deamidation include pH, temperature, solvent dielectric constant, ionic strength, primary sequence, local polypeptide conformation, and tertiary structure. The amino acid residue adjacent to Asn in the peptide chain influences the deamidation rate. Gly and Ser following Asn in the protein sequence are more susceptible to deamidation.

[0184] In certain embodiments, liquid formulations of proteins from the present disclosure may be stored under conditions of pH and humidity to prevent deamination of the protein product.

[0185] Aqueous carriers of interest herein are those that are pharmaceutically acceptable (safe and non-toxic for human administration) and useful for preparing liquid formulations. Exemplary carriers include sterile water for injection (SWFI), bacteriostatic water for injection (BWFI), pH buffer solutions (e.g., phosphate-buffered saline), sterile saline, Ringer's solution, or dextrose solution.

[0186] Preservatives may optionally be added to the formulations herein to reduce bacterial action. The addition of a preservative may, for example, facilitate the production of multi-use (multi-dose) formulations.

[0187] Intravenous (IV) formulations may be the preferred route of administration in certain cases, such as when a patient receives all of their medications via the IV route after transplantation in a hospital. In certain embodiments, the liquid formulation is diluted with 0.9% sodium chloride solution before administration. In certain embodiments, the drug product diluted for injection is isotonic and suitable for administration by intravenous infusion.

[0188] In certain embodiments, salts or buffer components may be added in amounts of 10 mM to 200 mM. The salts and / or buffers are pharmaceutically acceptable and are derived from a variety of known acids (inorganic and organic) with "base-forming" metals or amines. In certain embodiments, the buffer may be a phosphate buffer. In certain embodiments, the buffer may be a glycinate, carbonate, or citrate buffer, in which case sodium, potassium, or ammonium ions may serve as counterions.

[0189] Preservatives may optionally be added to the formulations herein to reduce bacterial action. The addition of a preservative may, for example, facilitate the production of multi-use (multi-dose) formulations.

[0190] Aqueous carriers of interest herein are those that are pharmaceutically acceptable (safe and non-toxic for human administration) and useful for preparing liquid formulations. Exemplary carriers include sterile water for injection (SWFI), bacteriostatic water for injection (BWFI), pH buffer solutions (e.g., phosphate-buffered saline), sterile saline, Ringer's solution, or dextrose solution.

[0191] The protein of the present disclosure may be present in a lyophilized formulation comprising the protein and a cryoprotectant. The cryoprotectant may be a sugar, such as a disaccharide. In some embodiments, the cryoprotectant may be sucrose or maltose. The lyophilized formulation may also include one or more of a buffer, a surfactant, a bulking agent, and / or a preservative.

[0192] The amount of sucrose or maltose useful for stabilizing a lyophilized drug product can be a weight ratio of protein to sucrose or maltose of at least 1:2. In certain embodiments, the weight ratio of protein to sucrose or maltose can be from 1:2 to 1:5.

[0193] In certain embodiments, the pH of the formulation prior to lyophilization may be established by the addition of a pharmaceutically acceptable acid and / or base. In certain embodiments, the pharmaceutically acceptable acid may be hydrochloric acid. In certain embodiments, the pharmaceutically acceptable base may be sodium hydroxide.

[0194] Prior to lyophilization, the pH of the solution containing the protein of the present disclosure may be adjusted to between 6 and 8. In certain embodiments, the pH range of the lyophilized drug product may be between 7 and 8.

[0195] In certain embodiments, salts or buffer components may be added in amounts of 10 mM to 200 mM. The salts and / or buffers are pharmaceutically acceptable and are derived from a variety of known acids (inorganic and organic) with "base-forming" metals or amines. In certain embodiments, the buffer may be a phosphate buffer. In certain embodiments, the buffer may be a glycinate, carbonate, or citrate buffer, in which case sodium, potassium, or ammonium ions may serve as counterions.

[0196] In certain embodiments, a "bulking agent" may be added. A "bulking agent" is a compound that adds mass to the lyophilization mixture and contributes to the physical structure of the lyophilized cake (e.g., facilitating the production of an essentially uniform lyophilized cake that maintains an open pore structure). Exemplary bulking agents include mannitol, glycine, polyethylene glycol, and sorbitol. The lyophilized formulations of the present invention may contain such bulking agents.

[0197] Preservatives may optionally be added to the formulations herein to reduce bacterial action. The addition of a preservative may, for example, facilitate the production of multi-use (multi-dose) formulations.

[0198] In some embodiments, the lyophilized drug product can be comprised in an aqueous carrier. The aqueous carrier of interest herein is pharmaceutically acceptable (e.g., safe and non-toxic for human administration) and useful for preparing a liquid formulation after lyophilization. Exemplary diluents include sterile water for injection (SWFI), bacteriostatic water for injection (BWFI), pH buffer (e.g., phosphate-buffered saline), sterile saline, Ringer's solution, or dextrose solution.

[0199] In certain embodiments, the lyophilized drug product of the present disclosure is reconstituted with either Sterile Water for Injection, USP (SWFI) or 0.9% Sodium Chloride Injection, USP. During reconstitution, the lyophilized powder dissolves into solution.

[0200] In certain embodiments, the lyophilized protein product of the present disclosure is constituted in about 4.5 mL of water for injection and diluted with 0.9% saline (sodium chloride solution).

[0201] Actual dosage levels of the active ingredients in the pharmaceutical compositions of the present invention may be varied to obtain an amount of the active ingredient effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration without toxicity to the patient.

[0202] The specific dose may be a uniform dose for each patient, e.g., 50-5000 mg of protein. Alternatively, the patient's dose may be adjusted based on the patient's approximate body weight or surface area. Other factors in determining the appropriate dosage may include the disease or condition to be treated or prevented, the severity of the disease, the route of administration, and the patient's age, sex, and medical condition. Further refinement of the calculations necessary to determine the appropriate dosage for treatment can be routinely performed by those skilled in the art, particularly in light of the dosage information and assays disclosed herein. Dosages may also be determined by the use of known assays for determining dosages used in conjunction with appropriate dose-response data. Dosages for individual patients may be adjusted while monitoring disease progression. Blood levels of the target construct or complex in the patient may be measured to determine whether dosage adjustments are necessary to reach or maintain effective concentrations. Pharmacogenomics may be used to determine which targetable constructs and / or complexes, and their dosages, are likely to be most effective for a given individual (Schmitz et al., Clinica Chimica Acta 308:43-53, 2001; Steimer et al., Clinica Chimica Acta 308:33-41, 2001).

[0203] Generally, the dosage based on body weight is about 0.01 μg to about 100 mg per kg of body weight, for example, about 0.01 μg to about 100 mg / kg body weight, about 0.01 μg to about 50 mg / kg body weight, about 0.01 μg to about 10 mg / kg body weight, about 0.01 μg to about 1 mg / kg body weight, about 0.01 μg to about 100 μg / kg body weight, about 0.01 μg to about 50 μg / kg body weight, about 0.01 μg to about 10 μg / kg body weight, about 0.01 μg to about 1 μg / kg body weight, about 0.01 μg to about 0.1 μg / kg body weight, about 0.1 μg to about 100 mg / kg body weight, about 0.1 μg to about 50 mg / kg body weight, about 0.1 μg to about 10 mg / kg body weight, about 0.1 μg to about 1 mg / kg body weight, about 0.1 μg to about 100 μg / kg body weight, About 0.1 μg to about 10 μg / kg body weight, about 0.1 μg to about 1 μg / kg body weight, about 1 μg to about 100 mg / kg body weight, about 1 μg to about 50 mg / kg body weight, about 1 μg to about 10 mg / kg body weight, about 1 μg to about 1 mg / kg body weight, about 1 μg ~about 100μg / kg body weight, about 1μg to about 50μg / kg body weight, about 1μg to about 10μg / kg body weight, about 10μg to about 100mg / kg body weight, about 10μg to about 50mg / kg body weight, about 10μg to about 10mg / kg body weight, about 10μg ~1mg / kg body weight, approximately 10μg~100μg / kg body weight, approximately 10μg~50μg / kg body weight, approximately 50μg~100mg / kg body weight, approximately 50μg~approx. 50mg / kg body weight, approximately 50μg~approximately 10mg / kg body weight, approximately 50μg about 1 mg / kg body weight, about 50 μg to about 100 μg / kg body weight, about 100 μg to about 100 mg / kg body weight, about 100 μg to about 50 mg / kg body weight, about 100 μg to about 10 mg / kg body weight, about 100 μg to about 1 mg / kg body weight, about 1 mg to about 100 mg / kg body weight, about 1 mg to about 50 mg / kg body weight, about 1 mg to about 10 mg / kg body weight, about 10 mg to about 100 mg / kg body weight, about 10 mg to about 50 mg / kg body weight, and about 50 mg to about 100 mg / kg body weight.

[0204] Doses may be given one or more times daily, weekly, monthly, or yearly, or even once every 2 to 20 years. Those skilled in the art can readily estimate repetition rates for dosing based on measured residence times and concentrations of the targetable structure or complex in bodily fluids or tissues. Administration of the present invention may be intravenous, intraarterial, intraperitoneal, intramuscular, subcutaneous, intradural, intrathecal, intracavity, by perfusion via a catheter, or by direct intralesional injection. It may be administered one or more times daily, one or more times weekly, one or more times monthly, and one or more times yearly.

[0205] The above description describes several aspects and embodiments of the present invention, and this patent application specifically contemplates all combinations and permutations of those aspects and embodiments. [Example]

[0206] The invention generally described herein will be more readily understood by reference to the following examples, which are included solely for the purpose of illustrating certain aspects and embodiments of the invention and are not intended to limit the invention.

[0207] Example 1 - Purification of F3-TriNKET and F3'-TriNKET Both F3-TriNKET and F3'-TriNKET proteins were purified according to the steps used for purifying therapeutic monoclonal antibodies. Briefly, the purification method involved Protein A purification (to achieve approximately 80-90% monomer) followed by Poros HS CIEX salt step elution (to achieve approximately 97-99% monomer). In clinical manufacturing processes, an additional purification step using Q Sepharose or Q Mustang (flow-through mode) (to achieve approximately 99% monomer) may be added to further reduce total cell protein (WCP) and CHO DNA.

[0208] Purified F3'-TriNKET has high thermal stability (DSC), similar to that of therapeutic monoclonal antibodies. Furthermore, the stability of mutant Fc containing F3'-TrinKET is close to that of IgG1 Fc. See Table 13 below. [Table 13]

[0209] Example 2 - F3'-TriNKET is stable for at least 4 weeks In an accelerated stability study performed at 37°C, F3'-TriNKET was found to be stable for over 4 weeks. See Figures 3A-3C.

[0210] Purified F3'-TriNKET was stable during the low pH hold. 20 mg / mL of purified protein was incubated in glycine at pH 3.0 for 2 hours. Figure 4 shows that purified F3'-TrinKET was stable during the low pH hold.

[0211] Purified F3'-TriNKET was stable after five freeze-thaw cycles. Figures 5A and 5B show that purified F3'-TrinKET was stable after five freeze-thaw cycles, regardless of pH (Figure 5A shows freeze-thaw cycles in PBS, and Figure 5B shows freeze-thaw cycles in citrate at pH 5.5).

[0212] Purified F3'-TriNKET was stable in forced degradation studies. Figure 6 shows a bar graph of the forced degradation conditions under which F3'-TrinKET remained stable.

[0213] Example 3 - Evaluation of TriNKET binding to human cancer antigens expressed on cells Tumor antigen binding of TriNKET was assessed using human cancer cell lines expressing the cancer antigen of interest. The human AML cell line Molm-13 was used to assess binding of F3'-TriNKET-CD33 to CD33-expressing cells. The HER2+ Colo-201 cell line was used to assess binding of F3'-TriNKET-HER2 to HER2-expressing cells. F3'-TriNKET-HER2 and F3'-TriNKET-CD33 were diluted and incubated with the respective cells. Binding of F3'-TriNKET-HER2 and F3'-TriNKET-CD33 to HER2- and CD33-expressing cells, respectively, was detected using a fluorophore-conjugated anti-human IgG secondary antibody. Cells were analyzed by flow cytometry, and the binding MFI for HER2- and CD33-expressing cells, respectively, was normalized to the secondary antibody control to obtain fold-over-background values.

[0214] Example 4 - Primary human NK cell cytotoxicity assay Peripheral blood mononuclear cells (PBMCs) were isolated from human peripheral blood buffy coats using density gradient centrifugation. The isolated PBMCs were washed and prepared for NK cell isolation. NK cells were isolated using a negative selection technique using magnetic beads, and the purity of isolated NK cells was typically >90% CD3-CD56+. Isolated NK cells were rested overnight and used in cytotoxicity assays the following day.

[0215] DELFIA Cytotoxicity Assay: Human cancer cell lines expressing the target of interest were harvested from culture, the cells were washed with PBS, and the cells were incubated for 10 min for labeling with BATDA reagent (Perkin Elmer AD0116). 6 The target cells were resuspended in growth medium at 0.5–1.0 × 10 / mL. The manufacturer's instructions were followed for labeling of target cells. After labeling, the cells were washed three times with PBS and resuspended at 0.5–1.0 × 10 5The cells were resuspended in culture medium at 100 μl / mL. To prepare background wells, an aliquot of labeled cells was set aside and spun out of the medium. 100 μl of medium was carefully added to triplicate wells, avoiding disturbance of the pelleted cells. 100 μl of BATDA-labeled cells were added to each well of a 96-well plate. Wells were reserved for spontaneous release from target cells and prepared for maximum target cell lysis by adding 1% Triton-X. Monoclonal antibodies or TriNKET against the tumor target of interest were diluted in culture medium, and 50 μl of diluted mAb or TriNKET was added to each well. Resting and / or activated NK cells were harvested from the culture, washed, and diluted to 10 μl depending on the desired E:T ratio. 5 ~2.0×10 6 NK cells were resuspended in culture medium at 1 / mL. 50 μl of NK cells were added to each well of the plate for a total culture volume of 200 μl. Plates were incubated at 37°C with 5% CO2 for 2-3 hours before the assay was developed.

[0216] After 2-3 hours of incubation, the plates were removed from the incubator and the cells were pelleted by centrifugation at 200 g for 5 minutes. 20 μl of culture supernatant was transferred to a clean microplate provided by the manufacturer, and 200 μl of room temperature europium solution was added to each well. The plates were protected from light and incubated for 15 minutes at 250 rpm on a plate shaker. The plates were read using either a Victor3 or SpectraMax i3X instrument. % specific lysis was calculated as follows: % specific lysis = ((experimental release - spontaneous release) / (maximum release - spontaneous release)) * 100%.

[0217] Flow cytometry cytotoxicity assay Human cancer cell lines expressing BCMA after puromycin selection and transduced to stably express NucLight Green (Essen BioScience 4475) were harvested from culture, spun down, and resuspended in culture medium for 10 min. 5Target cells were resuspended at 4 × 10 / mL. 100 μl of target cells were added to each well of a 96-well plate. TriNKET against BCMA was diluted in culture medium, and 50 μl of each was added to duplicate wells. Purified human NK cells that had been rested overnight were harvested from culture, washed, and resuspended in culture medium at 4 × 10 5 NK cells were resuspended at 100 μl / mL. At a 2:1 E:T ratio, 50 μl of NK cells were added to all wells except for the target-only control, which received 100 μl of culture medium. The plates were incubated at 37°C with 5% CO for 30 hours.

[0218] After co-culture, cells were stained, fixed, and analyzed by flow cytometry. Remaining target cells were detected by a strong shift in the FITC channel, and dead cells were excluded with a viability stain. The number of green events was counted and the % kill was calculated by comparison with a target-only control sample. Counting beads were included to ensure comparable amounts were recorded.

[0219] Example 5 - F3'TriNKET binds to tumor antigens expressed on cells Figure 7 shows the binding of F3'-TrinKET-HER2 or trastuzumab to HER2+ Colo-201 cells. F3'-TriNKET-HER2 binds to Colo-201 cells to a high maximum fold over background, but only slightly decreases the EC 50 The binding value was reduced.

[0220] Figure 8 shows the binding of F3'-TrinKET-CD33 or CD33 monoclonal antibody to CD33 expressed on Molm-13 cells. F3'-TriNKET showed significantly higher binding to background and EC mAbs compared to CD33 mAb. 50 It bound to Molm-13 cells to a similar maximal fold excess binding value.

[0221] Example 6 - F3'-TriNKET mediates NK cytotoxicity against HER2+ and CD33+ target cells. Figures 9 and 10 show F3'-TrinKET-HER2-mediated cytotoxicity against the HER2-low cell line 786-O and the HER2-high cell line SkBr-3, respectively. F3'-TriNKET-HER2 exhibits potent EC2 activity for the induction of NK-mediated cytotoxicity against both HER2-low and HER2-high cell lines. 50 Compared with trastuzumab, F3'-TriNKET-HER2 exhibited greater maximum specific lysis and more potent EC 50 got the value.

[0222] Figures 11 and 12 show F3'-TrinKET-CD33-mediated cytotoxicity against two CD33-positive human cell lines, EOL-1 and THP-1, respectively. CD33 monoclonal antibodies can enhance NK cell lysis of EOL-1 target cells (Figure 11), but were found to have no effect on the FcR-high THP-1 cell line (Figure 12). F3'-TriNKET-CD33 showed subnanomolar EC for inducing NK-mediated cytotoxicity against both THP-1 and EOL-1 target cells. 50 values were obtained.

[0223] Example 7 - F4-TriNKET mediates NK cytotoxicity This example describes the efficacy of bivalent F4 format TriNKET-mediated enhancement of NK cell killing of target cancer cells. This example describes the binding of bivalent F4 format TriNKET to target cells. This example describes the effect of bivalent F4 format TriNKET in stabilizing and maintaining high cell surface expression of BCMA on the cell surface. This example demonstrates that the avidity of bivalent F4 format TriNKET to its target improves the affinity with which TriNKET binds to the target antigen, effectively stabilizing the expression and maintenance of high levels of the target antigen on the cell surface.

[0224] BCMA surface stabilization by F4-TriNKET BCMA-positive KMS12-PE myeloma cells were incubated with 10 μg / mL of F4-TriNKET or monoclonal antibody (EM-901). Samples were split into thirds, and aliquots of each sample were placed on ice for 20 minutes, at 37°C for 2 hours, or at 37°C for 24 hours. After the incubation period, cells were washed, and bound F4-TriNKET was detected using an anti-human IgG secondary antibody. After staining, cells were fixed and stored at 4°C. All samples were analyzed at the end of the study.

[0225] Evaluation of F4-TriNKET binding to human cancer antigens expressed on cells Tumor antigen binding of F4-TriNKET was assessed using human cancer cell lines expressing BCMA (e.g., A49-F4-TriNKET-BCMA, NKG2D-binding domain derived from clone ADI-27749 and BCMA-binding domain derived from EM-901). The human multiple myeloma cell line MM.1R, which expresses BCMA at higher levels than KMS12-PE myeloma cells, was used to assess TriNKET binding to cells expressing high levels of BCMA. F4-TriNKET was diluted and incubated with MM.1R cells. Binding of F4-TriNKET was detected using a fluorophore-conjugated anti-human IgG secondary antibody. Cells were analyzed by flow cytometry, and the MFI of binding to BCMA expressed on the cells was normalized to the secondary antibody control to obtain fold-over-background values.

[0226] Primary human NK cell cytotoxicity assay Peripheral blood mononuclear cells (PBMCs) were isolated from human peripheral blood buffy coats using density gradient centrifugation. The isolated PBMCs were washed and prepared for NK cell isolation. NK cells were isolated using a negative selection technique using magnetic beads, and the purity of isolated NK cells was typically >90% CD3-CD56+. Isolated NK cells were rested overnight and used in cytotoxicity assays the following day.

[0227] DELFIA cytotoxicity assay: Human cancer cell lines expressing the target of interest were harvested from culture, the cells were washed with PBS, and the cells were incubated for 10 min for labeling with BATDA reagent (Perkin Elmer AD0116). 6 The cells were resuspended in growth medium at 0.5–1.0 × 10 / mL. The manufacturer's instructions were followed for labeling of target cells. After labeling, the cells were washed three times with PBS and resuspended at 0.5–1.0 × 10 5 The cells were resuspended in culture medium at 100 μl / mL. To prepare background wells, an aliquot of labeled cells was set aside and spun out of the medium. 100 μl of medium was carefully added to triplicate wells, avoiding disturbance of the pelleted cells. 100 μl of BATDA-labeled cells were added to each well of a 96-well plate. Wells were reserved for spontaneous release from target cells and prepared for maximum target cell lysis by adding 1% Triton-X. Monoclonal antibodies or TriNKET against the tumor target of interest were diluted in culture medium, and 50 μl of diluted mAb or TriNKET was added to each well. Resting and / or activated NK cells were harvested from the culture, the cells were washed, and the cells were diluted to 10 μl depending on the desired E:T ratio. 5 ~2.0×10 6 The NK cells were resuspended in culture medium at 100 / mL. 50 μl of NK cells were added to each well of the plate for a total culture volume of 200 μl. The plates were incubated at 37°C with 5% CO2 for 2-3 hours before the assay was developed. After 2-3 hours of incubation, the plates were removed from the incubator and the cells were pelleted by centrifugation at 200 g for 5 minutes. 20 μl of culture supernatant was transferred to a clean microplate provided by the manufacturer, and 200 μl of room temperature europium solution was added to each well. The plate was protected from light and incubated for 15 minutes at 250 rpm on a plate shaker. The plate was read using either a Victor3 or SpectraMax i3X instrument. % specific lysis was calculated as follows: % specific lysis = ((experimental release - spontaneous release) / (maximum release - spontaneous release)) * 100%.

[0228] F4-TriNKET-mediated NK cytotoxicity F4-TriNKET-mediated lysis of BCMA-positive myeloma cells was assayed. Figure 19 shows F4-TriNKET-mediated lysis of BCMA-positive KMS12-PE myeloma cells by resting human NK effector cells. Figure 20 shows F4-TriNKET-mediated lysis of BCMA-positive MM.1R myeloma cells by resting human NK effector cells. EM-901 monoclonal antibody was used as a control in both experiments. In Figure 19, KMS12-PE cells (low BCMA expression) were used as target cells. In Figure 20, MM.1R (high BCMA expression) were used as target cells. F4-TriNKET showed subnanomolar EC activity against both high and low BCMA-expressing cells, MM.1R and KMS12-PE, respectively. 50 Compared to the BCMA monoclonal antibody EM-901, F4-TriNKET provided greater maximum specific lysis and potency against both cell lines.

[0229] Figure 21 shows the binding of F4-TriNKET (A49-F4-TriNKET-BCMA), duobody-TriNKET (A49-DB-TriNKET-BCMA), or a BCMA monoclonal antibody (EM-901) to MM.1R myeloma cells. All three proteins were able to bind to BCMA expressed on MM.1R cells in a dose-response manner. While tight binders were able to bind at a maximal fold over background that was slightly reduced compared to monovalent binders, tight binding was observed at EC 50 This resulted in improved binding values.

[0230] F4-TriNKET stabilizes surface BCMA Figure 22 shows staining of surface BCMA with F4-TrinKET or BCMA monoclonal antibody (EM-901) after incubation for the indicated times. Both BCMA mAb (EM-901) and F4-TriNKET were able to rapidly stabilize surface BCMA after incubation. BCMA mAb (EM-901) and F4-TriNKET were able to maintain increased BCMA surface expression over 24 hours.

[0231] Figure 23 shows the stabilization of surface BCMA on KMS12-PE cells after incubation with F4-TriNKET or a BCMA monoclonal antibody (EM-901). Initial stabilization of BCMA on the cell surface occurred rapidly after exposure to F4-TriNKET or the mAb (EM-901). The strong binding provided by F4-TriNKET and the mAb (EM-901) maintained high surface BCMA longer than monovalent BCMA binding, as indicated by the reduction in surface BCMA expression by duobody-TriNKET at 24 hours.

[0232] F4-TriNKET mediates substantial long-term cytotoxicity Flow cytometry cytotoxicity assay: Human cancer cell lines expressing BCMA after puromycin selection and transduced to stably express NucLight Green (Essen BioScience 4475) were harvested from culture, spun down, and resuspended in culture medium for 10 min. 5 Target cells were resuspended at 4 × 10 / mL. 100 μl of target cells were added to each well of a 96-well plate. TriNKET against BCMA was diluted in culture medium, and 50 μl of each was added to duplicate wells. Purified human NK cells that had been rested overnight were harvested from culture, washed, and resuspended in culture medium at 4 × 10 5 NK cells were resuspended at 100 μl / mL. At a 2:1 E:T ratio, 50 μl of NK cells were added to all wells except for the target-only control, which received 100 μl of culture medium. Plates were incubated at 37°C with 5% CO for 30 hours.

[0233] After co-culture, cells were stained, fixed, and analyzed by flow cytometry. Remaining target cells were detected by a strong shift in the FITC channel, and dead cells were excluded with a viability stain. The number of green events was exported and % killing was calculated relative to a target-only control sample. Counting beads were included to ensure comparable amounts were recorded.

[0234] F4-TriNKET mediates substantial long-term cytotoxicity Figures 24 and 25 show human NK cell lysis of BCMA-positive target cell lines at a 2:1 E:T ratio in the presence of F4 or DB-TrinKET after 30 hours. Figure 24 shows killing of KMS12-PE cells (low BCMA expression) over no killing of the protein control, while in Figure 25, MM.1S cells (higher BCMA expression) were used as target cells. Compared to monovalent duobody-TriNKET, F4-TriNKET demonstrated increased killing of both high and low BCMA-expressing cell lines at all concentrations tested.

[0235] Incorporation by Reference The entire disclosure of each of the patent documents and scientific articles referred to herein is incorporated by reference for all purposes.

[0236] equivalent The present invention may be embodied in other specific forms without departing from its spirit or essential characteristics. Therefore, the foregoing embodiments are to be considered in all respects as illustrative and not limiting of the invention described herein. The scope of the invention is, therefore, indicated by the appended claims, rather than by the foregoing description, and all changes that come within the meaning and range of equivalency of the claims are intended to be embraced therein.

Claims

1. A protein, (a) a single-chain variable fragment (scFv) that binds to a tumor-associated antigen, the scFv comprising a heavy chain variable domain and a light chain variable domain; (b) a Fab that binds to NKG2D, (i) an antibody heavy chain variable domain comprising a complementarity determining region 1 (CDR1) amino acid sequence of SEQ ID NO: 319, a complementarity determining region 2 (CDR2) amino acid sequence of SEQ ID NO: 96, and a complementarity determining region 3 (CDR3) amino acid sequence of SEQ ID NO: 320, 324, 327, 330, 333, or 336; and (ii) an antibody light chain variable domain comprising the CDR1 amino acid sequence of SEQ ID NO: 99, the CDR2 amino acid sequence of SEQ ID NO: 100, and the CDR3 amino acid sequence of SEQ ID NO: 101; and a Fab comprising: (c) a first antibody constant domain and a second antibody constant domain that form a heterodimer that binds to CD16, the first antibody constant domain and the second antibody constant domain comprising a heterodimerization mutation; wherein the scFv is linked to the N-terminus of the first antibody constant domain and the Fab is linked to the N-terminus of the second antibody constant domain; The scFv is linked to the N-terminus of the first antibody constant domain via a hinge comprising Ala-Ser.

2. The protein of claim 1, wherein the hinge further comprises the amino acid sequence Thr-Lys-Gly.

3. 3. The protein of claim 1, wherein the heavy chain variable domain of the scFv forms a disulfide bridge with the light chain variable domain of the scFv between C44 from the heavy chain variable domain of the scFv and C100 from the light chain variable domain of the scFv, this position being defined under Kabat numbering.

4. The protein of any one of claims 1 to 3, wherein the heavy chain variable domain of the scFv is linked to the light chain variable domain of the scFv via a flexible linker comprising (G 4 S) 4 .

5. The protein of any one of claims 1 to 4, wherein the heavy chain variable domain of the scFv is located C-terminal to the light chain variable domain of the scFv.

6. The protein of any one of claims 1 to 5, wherein the first antibody constant domain and the second antibody constant domain comprise the CH2 domain and CH3 domain of a human IgG1 antibody, respectively.

7. 7. The protein of claim 6, wherein the first antibody constant domain and the second antibody constant domain each comprise an amino acid sequence at least 90% identical to amino acids 234-332 of a human IgG1 antibody numbered according to the EU numbering system.

8. 8. The protein of claim 6 or 7, wherein the heterodimerization mutation is present at one or more positions selected from the group consisting of Q347, Y349, L351, S354, E356, E357, K360, Q362, S364, T366, L368, K370, N390, K392, T394, D399, S400, D401, F405, Y407, K409, T411, and K439, numbered according to the EU numbering system.

9. The heterodimerization mutations are Q347E, Q347R, Y349S, Y349K, Y349T, Y349D, Y349E, Y349C, L351K, L351D, L351Y, S354C, E356K, E357Q, E357L, E357W, K360E, K360W, Q362E, S364K, S364E, S364H, S364D, T366V, T366I, T366L, T366M, T366K, T366W, T366S, L368E, numbered according to the EU numbering system.

9. The protein of claim 8, comprising one or more substitutions selected from the group consisting of: L368A, L368D, K370S, N390D, N390E, K392L, K392M, K392V, K392F, K392D, K392E, T394F, D399R, D399K, D399V, S400K, S400R, D401K, F405A, F405T, Y407A, Y407I, Y407V, K409F, K409W, K409D, T411D, T411E, K439D, and K439E.

10. 10. The protein of claim 9, wherein the heterodimerization mutations comprise Q347R, D399V, and F405T substitutions in the first antibody constant domain and K360E and K409W substitutions in the second antibody constant domain, numbered according to the EU numbering system.

11. the antibody heavy chain variable domain of the Fab comprises the CDR1, CDR2, and CDR3 amino acid sequences of SEQ ID NOs: 319, 96, and 320; or 95, 96, and 97, respectively; and The protein of any one of claims 1 to 10, wherein the antibody light chain variable domain of the Fab comprises the CDR1, CDR2, and CDR3 amino acid sequences of SEQ ID NOs: 99, 100, and 101, respectively.

12. The protein described in claim 11, wherein the heavy chain variable domain of the Fab comprises an amino acid sequence at least 90% identical to SEQ ID NO: 94, and the light chain variable domain of the Fab comprises an amino acid sequence at least 90% identical to SEQ ID NO:

98.

13. the antibody heavy chain variable domain of the Fab comprises the CDR1, CDR2, and CDR3 amino acid sequences of SEQ ID NOs: 319, 96, and 324; or 95, 96, and 323, respectively; and The protein of any one of claims 1 to 10, wherein the antibody light chain variable domain of the Fab comprises the CDR1, CDR2, and CDR3 amino acid sequences of SEQ ID NOs: 99, 100, and 101, respectively.

14. The protein of claim 13, wherein the heavy chain variable domain of the Fab comprises an amino acid sequence at least 90% identical to SEQ ID NO: 322, and the light chain variable domain of the Fab comprises an amino acid sequence at least 90% identical to SEQ ID NO:

98.

15. A formulation comprising the protein of any one of claims 1 to 14 and a pharmaceutically acceptable carrier.

16. A cell comprising one or more nucleic acids encoding a protein according to any one of claims 1 to 14.

17. A composition comprising a protein according to any one of claims 1 to 14 or a formulation according to claim 15 for use in therapy.

18. 15. A composition comprising a protein according to any one of claims 1 to 14 for use in enhancing tumor cell death, said use comprising exposing tumor and natural killer cells to said protein.

19. 16. A composition comprising a protein according to any one of claims 1 to 14 or a formulation according to claim 15 for use in the treatment of cancer, said use comprising administering said protein or said formulation to a patient.

20. 20. The composition or formulation for use of claim 19, wherein the cancer is selected from the group consisting of acute myeloid leukemia, acute myelomonocytic leukemia, B-cell lymphoma, bladder cancer, breast cancer, colorectal cancer, diffuse large B-cell lymphoma, esophageal cancer, Ewing's sarcoma, follicular lymphoma, gastric cancer, gastrointestinal cancer, gastrointestinal stromal tumor, glioblastoma, head and neck cancer, melanoma, mesothelioma, multiple myeloma, myelodysplastic syndrome, renal cell carcinoma, neuroblastoma, non-small cell lung cancer, neuroendocrine tumor, ovarian cancer, and pancreatic cancer, prostate cancer, sarcoma, small cell lung cancer, T-cell lymphoma, testicular cancer, thymic cancer, thyroid cancer, urothelial cancer, cancer infiltrated by myeloid-derived suppressor cells, cancer with extracellular matrix deposition, cancer with high levels of reactive stroma, and cancer with angiogenesis.

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