Multifunctional molecules that bind to TCRs and uses thereof

Abstract

Provided herein are multifunctional molecules comprising a T cell receptor variable beta binding portion and a cytokine and methods of using same to treat a condition or disease in a subject.

Description

[Technical Field]

[0001] cross reference

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 379,243, filed October 12, 2022, the entire contents of which are incorporated herein by reference. [Background technology]

[0002] Currently available molecules designed to redirect T cells to promote tumor cell lysis for cancer immunotherapy typically target the CD3 epsilon (CD3e) subunit of the T cell receptor (TCR). However, this approach has limitations. Previous studies have shown, for example, that low doses of anti-CD3e monoclonal antibodies (mAbs) can cause T cell dysfunction and produce immunosuppressive effects. In addition, anti-CD3e mAbs bind to all T cells and thus activate large numbers of T cells. Such non-physiological, massive activation of T cells by these anti-CD3e mAbs can lead to the production of pro-inflammatory cytokines, such as IFN-gamma, IL-1-beta, IL-6, IL-10, and TNF-alpha, causing a "cytokine storm," also known as cytokine release syndrome (CRS) and associated with neurotoxicity (NT). Therefore, improved T cell receptor binding molecules are needed to redirect T cells for cancer immunotherapy. Summary of the Invention

[0003]

[0003] In one aspect, provided herein is a multifunctional molecule comprising, inter alia: (a) a tumor-associated antigen binding portion; (b) at least one cytokine molecule or a functional fragment or functional variant thereof; and (c) a TCRβV binding portion covalently linked to at least one cytokine molecule or a functional fragment or functional variant thereof.

[0004]

[0004] In some embodiments, the multifunctional molecule comprises a first polypeptide chain comprising a first portion of a dimerization module and a second polypeptide chain comprising a second portion of a dimerization module; the first polypeptide chain and the second polypeptide chain are non-contiguous, the tumor-associated antigen binding portion is linked to the first portion of the dimerization module, and at least one cytokine molecule or a functional fragment or functional variant thereof is linked to the first portion of the dimerization module, the second portion of the dimerization module, or a combination thereof.

[0005]

[0005] In some embodiments, (i) the tumor-associated antigen binding portion is linked to the N-terminus of the first portion of the dimerization module, and at least one cytokine molecule or a functional fragment or functional variant thereof is linked to the C-terminus of the first portion of the dimerization module, the N-terminus of the second portion of the dimerization module, the C-terminus of the second portion of the dimerization module, or any combination thereof; or (ii) the tumor-associated antigen binding portion is linked to the C-terminus of the first portion of the dimerization module, and at least one cytokine molecule or a functional fragment or functional variant thereof is linked to the N-terminus of the first portion of the dimerization module, the N-terminus of the second portion of the dimerization module, the C-terminus of the second portion of the dimerization module, or any combination thereof.

[0006]

[0006] In some embodiments, the TCRβV binding portion and at least one cytokine molecule or a functional fragment or functional variant thereof are present within a single continuous polypeptide chain of the first polypeptide chain or the second polypeptide chain.

[0007]

[0007] In some embodiments, the tumor-associated antigen-binding portion, the TCRβV-binding portion, or a combination thereof comprises an antibody or an antigen-binding fragment thereof, and the antigen-binding fragment comprises any one selected from the group consisting of Fab, F(ab')2, Fv, single-chain Fv (scFv), single-domain antibody, diabody (dAb), camelid antibody, and any combination thereof.

[0008] In some embodiments, the TCRβV binding portion comprises a heavy chain variable domain (VH) and a light chain variable domain (VL), or a single domain antibody.

[0009] In some embodiments, the TCRβV binding portion comprises a first portion of the TCRβV binding portion, and the multifunctional molecule further comprises a third polypeptide chain comprising a second portion of the TCRβV binding portion, wherein the third polypeptide chain is non-contiguous with the first polypeptide chain and the second polypeptide chain.

[0009]

[0010] In some embodiments, the first portion of the TCRβV binding portion comprises the VH of the TCRβV binding portion and the second portion of the TCRβV binding portion comprises the VL of the TCRβV binding portion, or the first portion of the TCRβV binding portion comprises the VL of the TCRβV binding portion and the second portion of the TCRβV binding portion comprises the VH of the TCRβV binding portion.

[0010]

[0011] In some embodiments, the tumor-associated antigen-binding portion comprises a VH and a VL, or a single domain antibody.

[0012] In some embodiments, the tumor-associated antigen-binding portion comprises a first portion of the tumor-associated antigen-binding portion, and the multifunctional molecule further comprises a fourth polypeptide chain comprising a second portion of the tumor-associated antigen-binding portion, wherein the fourth polypeptide chain is non-contiguous with the first polypeptide chain, the second polypeptide chain, and the third polypeptide chain.

[0011]

[0013] In some embodiments, the first portion of the tumor-associated antigen-binding moiety comprises the VH of the tumor-associated antigen-binding moiety and the second portion of the tumor-associated antigen-binding moiety comprises the VL of the tumor-associated antigen-binding moiety, or the first portion of the tumor-associated antigen-binding moiety comprises the VL of the tumor-associated antigen-binding moiety and the second portion of the tumor-associated antigen-binding moiety comprises the VH of the tumor-associated antigen-binding moiety.

[0012]

[0014] In some embodiments, the first portion of the dimerization module and the second portion of the dimerization module are dimerized.

[0015] In some embodiments, (i) the tumor-associated antigen-binding portion further comprises a heavy chain constant domain 1 (CH1) linked to the VH of the tumor-associated antigen-binding portion; (ii) the TCRβV-binding portion further comprises a heavy chain constant domain 1 (CH1) linked to the VH of the TCRβV-binding portion; or (iii) a combination thereof.

[0013]

[0016] In some embodiments, (i) the tumor-associated antigen-binding portion further comprises a light chain constant domain (CL) linked to the VL of the tumor-associated antigen-binding portion; (ii) the TCRβV-binding portion further comprises a light chain constant domain (CL) linked to the VL of the TCRβV-binding portion; or (iii) a combination thereof.

[0014]

[0017] In some embodiments, (i) the CL linked to the VL of the tumor-associated antigen-binding portion comprises a kappa chain constant domain or a lambda chain constant domain; (ii) the CL linked to the VL of the TCRβV-binding portion comprises a kappa chain constant domain or a lambda chain constant domain; or (iii) a combination thereof.

[0015]

[0018] In some embodiments, the kappa or lambda chain constant domain comprises any one of the light chain constant region sequences listed in Tables 3, 21, or 22.

[0019] In some embodiments, the multifunctional molecules provided herein comprise: (i) a linker between a first portion of a dimerization module and a tumor-associated antigen binding portion or a first portion of a tumor-associated antigen; (ii) a linker between at least one cytokine molecule, or a functional fragment or functional variant thereof, and the first portion of the dimerization module, a linker between at least one cytokine molecule, or a functional fragment or functional variant thereof, and a second portion of the dimerization module, or any combination thereof; (iii) at least one cytokine molecule, or a functional fragment thereof or a linker between the functional variant and the TCRβV binding portion or the first portion of the TCRβV binding portion; (iv) a linker between the VH and the VL of the tumor-associated antigen binding portion; (v) a linker between the VH and the VL of the TCRβV binding portion; (vi) a linker between the CH1 and the VH of the tumor-associated antigen binding portion; (vii) a linker between the CH1 and the VH of the TCRβV binding portion; (viii) a linker between the CL and the VL of the tumor-associated antigen binding portion; (ix) a linker between the CL and the VL of the TCRβV binding portion; or (x) any combination thereof.

[0016]

[0020] In some embodiments, the linker is selected from the group consisting of a cleavable linker, a non-cleavable linker, a peptide linker, a flexible linker, a rigid linker, a helical linker, and a non-helical linker.

[0017]

[0021] In some embodiments, the linker is a peptide linker comprising the sequence of SEQ ID NO:3308 or SEQ ID NO:3643.

[0022] In some embodiments, the multifunctional molecule is an isolated multifunctional molecule.

[0018]

[0023] In some embodiments, the tumor-associated antigen-binding portion, the TCRβV-binding portion, or a combination thereof comprises a Fab or scFv.

[0024] In some embodiments, the at least one cytokine molecule or functional fragment or functional variant thereof is selected from the group consisting of interleukin-2 (IL-2) or a functional fragment or functional variant thereof, interleukin-7 (IL-7) or a functional fragment or functional variant thereof, interleukin-12 (IL-12) or a functional fragment or functional variant thereof, interleukin-15 (IL-15) or a functional fragment or functional variant thereof, interleukin-18 (IL-18) or a functional fragment or functional variant thereof, interleukin-21 (IL-21) or a functional fragment or functional variant thereof, or interferon gamma or a functional fragment or functional variant thereof, or any combination thereof.

[0019]

[0025] In some embodiments, the at least one cytokine molecule or functional fragment or variant thereof comprises interleukin-2 (IL-2) or a functional fragment or variant thereof.

[0020]

[0026] In some embodiments, at least one cytokine molecule or functional fragment or variant thereof is an IL-2 variant comprising a substitution mutation.

[0027] In some embodiments, the at least one cytokine molecule or functional fragment or variant thereof is an IL-2 variant comprising a C125A mutation.

[0021]

[0028] In some embodiments, at least one cytokine molecule or functional fragment or functional variant thereof comprises a sequence having at least 75% sequence identity to the sequence of SEQ ID NO: 2270 or the sequence of SEQ ID NO: 2191.

[0022]

[0029] In some embodiments, at least one cytokine molecule or functional fragment or functional variant thereof comprises the sequence of SEQ ID NO: 2270 or the sequence of SEQ ID NO: 2191.

[0023]

[0030] In some embodiments, the first portion of the dimerization module comprises a first immunoglobulin constant region (Fc region) and the second portion of the dimerization module comprises a second Fc region.

[0024]

[0031] In some embodiments, the first Fc region, the second Fc region, or a combination thereof is selected from the group consisting of an IgG1 Fc region or fragment thereof, an IgG2 Fc region or fragment thereof, an IgG3 Fc region or fragment thereof, an IgGA1 Fc region or fragment thereof, an IgGA2 Fc region or fragment thereof, an IgGA4 Fc region or fragment thereof, an IgJ Fc region or fragment thereof, an IgM Fc region or fragment thereof, an IgD Fc region or fragment thereof, and an IgE Fc region or fragment thereof.

[0025]

[0032] In some embodiments, the first Fc region, the second Fc region, or a combination thereof is selected from the group consisting of a human IgG1 Fc region or fragment thereof, a human IgG2 Fc region or fragment thereof, and a human IgG4 Fc region or fragment thereof.

[0026]

[0033] In some embodiments, the first Fc region, the second Fc region, or a combination thereof, comprises an Fc interface having one or more of paired holes and protrusions, electrostatic interactions, or strand exchange, and dimerization of the first Fc region and the second Fc region is enhanced as indicated by a higher ratio of heteromultimer:homomultimer forms compared to dimerization of Fc regions having an unengineered interface.

[0027]

[0034] In some embodiments, the first Fc region, the second Fc region, or a combination thereof comprises an amino acid substitution listed in Table 14, 21, or 22.

[0035] In some embodiments, the first Fc region, the second Fc region, or a combination thereof comprises an Asn297Ala (N297A) mutation or a Leu234Ala / Leu235Ala (LALA) mutation.

[0028]

[0036] In some embodiments, the first Fc region, the second Fc region, or a combination thereof comprises a sequence having at least 75% sequence identity to the sequence of SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:3645, SEQ ID NO:3646, SEQ ID NO:3647, SEQ ID NO:3648, SEQ ID NO:3649, SEQ ID NO:3792, or SEQ ID NO:3794.

[0029]

[0037] In some embodiments, the first Fc region, the second Fc region, or a combination thereof comprises the sequence of SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:3645, SEQ ID NO:3646, SEQ ID NO:3647, SEQ ID NO:3648, SEQ ID NO:3649, SEQ ID NO:3792, or SEQ ID NO:3794.

[0030]

[0038] In some embodiments, the first Fc region comprises a sequence having at least 75% sequence identity to the sequence of SEQ ID NO: 3649 or SEQ ID NO: 3792 and the second Fc region comprises a sequence having at least 75% sequence identity to the sequence of SEQ ID NO: 3648 or SEQ ID NO: 3794, or the first Fc region comprises a sequence having at least 75% sequence identity to the sequence of SEQ ID NO: 3648 or SEQ ID NO: 3794 and the second Fc region comprises a sequence having at least 75% sequence identity to the sequence of SEQ ID NO: 3648 or SEQ ID NO: 3794.

[0031]

[0039] In some embodiments, the first Fc region comprises a sequence having the sequence of SEQ ID NO: 3649 or SEQ ID NO: 3792, and the second Fc region comprises a sequence of SEQ ID NO: 3648 or SEQ ID NO: 3794, or the first Fc region comprises a sequence of SEQ ID NO: 3648 or SEQ ID NO: 3794, and the second Fc region comprises a sequence of SEQ ID NO: 3648 or SEQ ID NO: 3794.

[0032]

[0040] In some embodiments, the TCRβ V binding moiety binds to one or more TCRβ V subfamilies selected from the group consisting of TCRβ V2 subfamily, TCRβ V3 subfamily, TCRβ V4 subfamily, TCRβ V5 subfamily, TCRβ V6 subfamily, TCRβ V9 subfamily, TCRβ V10 subfamily, TCRβ V11 subfamily, TCRβ V12 subfamily, TCRβ V13 subfamily, TCRβ V16 subfamily, TCRβ V19, TCRβ V20 subfamily, TCRβ V21 subfamily, TCRβ V23 subfamily, TCRβ V27 subfamily, and TCRβ V28 subfamily.

[0033]

[0041] In some embodiments, the TCRβ V-binding portion is selected from the group consisting of: (i) the TCRβ V2 subfamily, which includes TCRβ V2*01; (ii) the TCRβ V3 subfamily, which includes TCRβ V3-1*01; (iii) the TCRβ V4 subfamily, which includes one or more selected from TCRβ V4-1, TCRβ V4-2, and TCRβ V4-3; (iv) the TCRβ V5 subfamily, which includes one or more selected from TCRβ V5-6*01, TCRβ V5-4*01, TCRβ V5-1*01, and TCRβ V5-8*01; (v) the TCRβ V6-4*01, TCRβ V6-4*02, TCRβ V6-9*01, TCRβ V6-8*01, TCRβ V6-5*01, TCRβ V6-6*02, TCRβ V6-6*01, TCRβ (vi) the TCRβ V10 subfamily, including one or more selected from TCRβ V10-1*01, TCRβ V10-1*02, TCRβ V10-3*01, and TCRβ V10-2*01; (vii) the TCRβ V11 subfamily, including TCRβ V11-2; (viii) the TCRβ V12 subfamily, including one or more selected from TCRβ V12-4*01, TCRβ V12-3*01, and TCRβ V12-5*01; (ix) the TCRβ V13 subfamily, including TCRβ V13*01; (x) the TCRβ V16 subfamily, including TCRβ V16*01; (xi) the TCRβ (xii) the TCRβ V19 subfamily, including one or more selected from TCRβ V19*01 and TCRβ V19*02; or (xiii) the TCRβ V20 subfamily, including TCRβ V20-1*01, or TCRβ V20-1*02.

[0034]

[0042] In some embodiments, the TCRβV binding moiety binds to the TCRβ V6 subfamily or the TCRβ V20 subfamily.

[0043] In some embodiments, the TCRβV-binding portion comprises (i) a VH comprising a combination of HC CDR1, HC CDR2, and HC CDR3 listed in Table 1, 2, 10B, 11, 12, 13, 21, or 22; (ii) a VL comprising a combination of LC CDR1, LC CDR2, and LC CDR3 listed in Table 1, 2, 10B, 11, 12, 13, 21, or 22; or (iii) a combination thereof.

[0035]

[0044] In some embodiments, the TCRβV-binding portion comprises (i) a VH comprising the HC CDR1, HC CDR2, and HC CDR3 of any one of the amino acid sequences of a heavy chain variable domain listed in Table 1, 2, 10B, 11, 12, 13, 21, or 22; (ii) a VL comprising the LC CDR1, LC CDR2, and LC CDR3 of any one of the amino acid sequences of a light chain variable domain listed in Table 1, 2, 10B, 11, 12, 13, 21, or 22; or (iii) a combination thereof.

[0036]

[0045] In some embodiments, the TCRβV-binding portion comprises: (i) a VH comprising a sequence having at least 70% sequence identity to any one of the amino acid sequences of a heavy chain variable domain listed in Table 1, 2, 10B, 11, 12, 13, 21, or 22; (ii) a VL comprising a sequence having at least 70% sequence identity to any one of the amino acid sequences of a light chain variable domain listed in Table 1, 2, 10B, 11, 12, 13, 21, or 22; or (iii) a combination thereof.

[0037]

[0046] In some embodiments, the TCRβV-binding portion comprises: (i) a VH comprising any one of the amino acid sequences of a heavy chain variable domain listed in Table 1, 2, 10B, 11, 12, 13, 21, or 22; (ii) a VL comprising any one of the amino acid sequences of a light chain variable domain listed in Table 1, 2, 10B, 11, 12, 13, 21, or 22; or (iii) a combination thereof.

[0038]

[0047] In some embodiments, the second polypeptide chain comprises a sequence having at least 70% sequence identity to the sequence of SEQ ID NO: 1346, a sequence having at least 70% sequence identity to the sequence of SEQ ID NO: 1349, a sequence having at least 70% sequence identity to the sequence of SEQ ID NO: 2270, and a sequence having at least 70% sequence identity to the sequence of SEQ ID NO: 3648.

[0039]

[0048] In some embodiments, the second polypeptide chain comprises the sequence of SEQ ID NO:1346, the sequence of SEQ ID NO:1349, the sequence of SEQ ID NO:2270, and the sequence of SEQ ID NO:3648.

[0049] In some embodiments, the second polypeptide chain further comprises the sequence of SEQ ID NO: 3801, the sequence of SEQ ID NO: 3309, the sequence of SEQ ID NO: 3308, or any combination thereof.

[0040]

[0050] In some embodiments, the second polypeptide chain comprises a sequence having at least 70% sequence identity to the sequence of SEQ ID NO: 1346, wherein the sequence having at least 70% sequence identity to the sequence of SEQ ID NO: 1346 is operably linked to a sequence having at least 70% sequence identity to the sequence of SEQ ID NO: 1349, wherein the sequence having at least 70% sequence identity to the sequence of SEQ ID NO: 1349 is operably linked to a sequence having at least 70% sequence identity to the sequence of SEQ ID NO: 2270, wherein the sequence having at least 70% sequence identity to the sequence of SEQ ID NO: 2270 is operably linked to a sequence having at least 70% sequence identity to the sequence of SEQ ID NO: 3648.

[0041]

[0051] In some embodiments, a sequence having at least 70% sequence identity to the sequence of SEQ ID NO: 1346 is operably linked to a sequence having at least 70% sequence identity to the sequence of SEQ ID NO: 1349 via the sequence of SEQ ID NO: 3801.

[0042]

[0052] In some embodiments, a sequence having at least 70% sequence identity to the sequence of SEQ ID NO: 1349 is operably linked to a sequence having at least 70% sequence identity to the sequence of SEQ ID NO: 2270 via the sequence of SEQ ID NO: 3309.

[0043]

[0053] In some embodiments, a sequence having at least 70% sequence identity to the sequence of SEQ ID NO: 2270 is operably linked to a sequence having at least 70% sequence identity to the sequence of SEQ ID NO: 3648 via the sequence of SEQ ID NO: 3308.

[0044]

[0054] In some embodiments, the second polypeptide chain comprises the sequence of SEQ ID NO: 1346, which is operably linked to the sequence of SEQ ID NO: 1349, which is operably linked to the sequence of SEQ ID NO: 2270, which is operably linked to the sequence of SEQ ID NO: 3648.

[0045]

[0055] In some embodiments, the sequence of SEQ ID NO: 1346 is operably linked to the sequence of SEQ ID NO: 1349 via the sequence of SEQ ID NO: 3801.

[0056] In some embodiments, the sequence of SEQ ID NO: 1349 is operably linked to the sequence of SEQ ID NO: 2270 via the sequence of SEQ ID NO: 3309.

[0046]

[0057] In some embodiments, the sequence of SEQ ID NO: 2270 is operably linked to the sequence of SEQ ID NO: 3648 via the sequence of SEQ ID NO: 3308.

[0058] In some embodiments, the second polypeptide chain comprises a sequence having at least 70% sequence identity to the sequence of SEQ ID NO:3800.

[0047]

[0059] In some embodiments, the second polypeptide chain comprises the sequence of SEQ ID NO:3800.

[0060] In some embodiments, the multifunctional molecule consists of a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain comprises a tumor-associated antigen binding moiety linked to an Fc region, wherein the tumor-associated antigen binding moiety is an scFv that binds to the tumor-associated antigen, and the second polypeptide chain comprises the sequence of SEQ ID NO: 3800.

[0048]

[0061] In some embodiments, the multifunctional molecule consists of a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain comprises a tumor-associated antigen-binding moiety linked to a sequence having at least 75% sequence identity to the sequence of SEQ ID NO: 3649 or SEQ ID NO: 3792, wherein the tumor-associated antigen-binding moiety is an scFv that binds to the tumor-associated antigen, and the second polypeptide chain comprises the sequence of SEQ ID NO: 3800.

[0049]

[0062] In some embodiments, the multifunctional molecule consists of a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain comprises a tumor-associated antigen-binding moiety linked to the sequence of SEQ ID NO: 3649 or SEQ ID NO: 3792, wherein the tumor-associated antigen-binding moiety is an scFv that binds to the tumor-associated antigen, and the second polypeptide chain comprises the sequence of SEQ ID NO: 3800.

[0050]

[0063] In some embodiments, the multifunctional molecule consists of a first polypeptide chain, a second polypeptide chain, and a third polypeptide chain, wherein the first polypeptide chain comprises a first portion of a tumor-associated antigen binding moiety linked to an Fc region, wherein the tumor-associated antigen binding moiety is a Fab that binds to the tumor-associated antigen; the second polypeptide chain comprises the sequence of SEQ ID NO: 3800; and the third polypeptide chain comprises a second portion of the tumor-associated antigen binding moiety.

[0051]

[0064] In some embodiments, the multifunctional molecule consists of a first polypeptide chain, a second polypeptide chain, and a third polypeptide chain, wherein the first polypeptide chain comprises a first portion of a tumor-associated antigen binding moiety linked to a sequence having at least 75% sequence identity to the sequence of SEQ ID NO: 3649 or SEQ ID NO: 3792, wherein the tumor-associated antigen binding moiety is a Fab that binds to the tumor-associated antigen; the second polypeptide chain comprises the sequence of SEQ ID NO: 3800; and the third polypeptide chain comprises a second portion of the tumor-associated antigen binding moiety.

[0052]

[0065] In some embodiments, the multifunctional molecule consists of a first polypeptide chain, a second polypeptide chain, and a third polypeptide chain, wherein the first polypeptide chain comprises a first portion of a tumor-associated antigen binding moiety linked to the sequence of SEQ ID NO: 3649 or SEQ ID NO: 3792, wherein the tumor-associated antigen binding moiety is a Fab that binds to the tumor-associated antigen; the second polypeptide chain comprises the sequence of SEQ ID NO: 3800; and the third polypeptide chain comprises a second portion of the tumor-associated antigen binding moiety.

[0053]

[0066] In some embodiments, the tumor-associated antigen binding portion is selected from the group consisting of CD19, CD123, CD22, CD30, CD171, CS-1, C-type lectin-like molecule-1, CD33, epidermal growth factor receptor variant III (EGFRvIII), ganglioside G2 (GD2), ganglioside GD3, TNF receptor family member B-cell maturation (BCMA), Tn antigen (TnAg) or (GalNAcα-Ser / Thr)), prostate-specific membrane antigen (PSMA), receptor tyrosine kinase-like orphan receptor 1 (ROR1), Fms-like tyrosine kinase 3 (FLT3), tumor-associated glycoprotein 72 (TAG72), CD38, CD44v6, carcinoembryonic antigen (CEA), epithelial cell adhesion molecule (EPCAM), B7H3 (CD276), KIT (CD117), interleukin-13 receptor subunit alpha-2, mesothelin, interleukin-11 receptor alpha (IL-11Ra), prostate stem cell antigen (PSCA), protease serine 21, vascular endothelial growth factor receptor 2 (VEGFR2), Lewis (Y) antigen, CD24, platelet-derived growth factor receptor beta (PDGFR-beta), stage-specific embryonic antigen-4 (SSEA-4), CD20, folate receptor alpha, receptor tyrosine-protein kinase ERBB2 (Her2 / neu), cell surface-associated mucin 1 (MUC1), epidermal growth factor receptor (EGFR), neural cell adhesion molecule (NCAM), prostase, prostatic acid phosphatase (PAP), mutant elongation factor 2 (ELF2M) ), ephrin B2, fibroblast activation protein alpha (FAP), insulin-like growth factor 1 receptor (IGF-I receptor), carbonic anhydrase IX (CAIX), proteasome (prosome, macropein) subunit, beta type 9 (LMP2), glycoprotein 100 (gp100 / pmel17), oncogene fusion protein (bcr-abl) consisting of breakpoint cluster region (BCR) and Abelson murine leukemia viral oncogene homolog 1 (Abl), tyrosinase, ephrin type A receptor 2 (EphA2) ), fucosyl GM1, sialyl Lewis adhesion molecule (sLe), ganglioside GM3, transglutaminase 5 (TGS5), high molecular weight melanoma-associated antigen (HMWMAA), o-acetyl-GD2 ganglioside (OAcGD2), folate receptor beta, tumor endothelial marker 1 (TEM1 / CD248), tumor endothelial marker 7-related (TEM7R), claudin 6 (CLDN6), thyroid-stimulating hormone receptor (TSHR), G protein-coupled receptor class C group 5, member D (GPRC5D), chromosome X open reading frame61 (CXORF61), CD97, CD179a, anaplastic lymphoma kinase (ALK), polysialic acid, placenta-specific 1 (PLAC1), hexasaccharide moiety of globoH glycoceramide (GloboH), mammary differentiation antigen (NY-BR-1), uroplakin 2 (UPK2), hepatitis A virus cellular receptor 1 (HAVCR1), adrenoceptor beta 3 (ADRB3), pannexin 3 (PANX3), G protein-coupled receptor 20 (GPR20), lymphocyte antigen 6 complex, locus K9 (LY6K), olfactory receptor 5 1E2 (OR51E2), TCR gamma alternate reading frame protein (TARP), Wilms tumor protein (WT1), cancer / testis antigen 1 (NY-ESO-1 / LAGE-1), cancer / testis antigen 2 (LAGE-1a), melanoma-associated antigen 1 (MAGE-A1), ETS translocation variant gene 6 located on chromosome 12p (ETV6-AML), sperm protein 17 (SPA17), X antigen family, member 1A (XAGE1), angiopoietin-binding cell surface receptor 2 (Tie 2), melanoma cancer testis antigen-1 (MAD-CT-1), melanoma cancer testis antigen-2 (MAD-CT-2), Fos-related antigen 1, tumor protein p53 (p53), p53 mutant, prostein, survivin, telomerase, prostate cancer tumor antigen-1, melanoma antigen 1 recognized by T cells, rat sarcoma (Ras) mutant, human telomerase reverse transcriptase (hTERT), sarcoma translocation breakpoint, melanoma inhibitor of apoptosis (ML-IAP), ER G (transmembrane protease, serine 2 (TMPRSS2) ETS fusion gene), N-acetylglucosaminyl-transferase V (NA17), paired box protein Pax-3 (PAX3), androgen receptor, cyclin B1, v-myc avian myelocytomatosis viral oncogene neuroblastoma-derived homolog (MYCN), Ras homolog family member C (RhoC), tyrosinase-related protein 2 (TRP-2), cytochrome P4501B1 (CYP1B1), CCCTC-binding factor (zinc finger protein)-like, squamous cell carcinoma antigen 3 recognized by T cells (SART3), paired box protein Pax-5 (PAX5), proacrosin-binding protein sp32 (OY-TES1), lymphocyte-specific protein tyrosine kinase (LCK), A-kinase anchor protein 4 (AKAP-4), synovial sarcoma X breakpoint 2 (SSX2), receptor for advanced glycation end products (RAGE-1), renal ubiquitous 1 (RU1), renal ubiquitous 2 (RU2), legumain, human papillomavirus E6 (HPV E6), human papillomavirus E7 (HPV E7), intestinal carboxylesterase, mutant heat shock protein 70-2 (muthsp70-2), CD79a, CD79b, CD72, leukocyte-associated immunoglobulin-like receptor 1 (LAIR1), Fc fragment of IgA receptor (FCAR or CD89), leukocyte immunoglobulin-like receptor subfamily A member 2 (LILRA2), CD300 molecule-like family member f (CD300LF), C-type lectin domain family 12 member A (CLEC12A), bone marrow stromal cell antigen 2 (BST2), EGF -like module-containing mucin-like hormone receptor-like 2 (EMR2), lymphocyte antigen 75 (LY75), glypican-3 (GPC3), Fc receptor-like 5 (FCRL5), FcRH5, PDL1, CD47, prostate-specific membrane antigen (PMSA), prostate-specific antigen (PSA), Ron kinase, c-Met, immature laminin receptor, TAG-72, BING-4, calcium-activated chloride channel 2, cyclin-B1, 9D7, Ep-CA M, EphA3, SAP-1, PRAME, SSX-2, Melan-A / MART-1, TRP1 / gp75, MC1R, β-catenin, BRCA1 / 2, CDK4, CML66, fibronectin, Ras, TGF-B receptor, AFP, ETA, MAGE, CA-125, BAGE, GAGE, CDC27, α-actinin-4, ganglioside, MART-2, MUC2, MUM1, MUM2, MUM3, NA88-1, NP The antibody binds to a cancer antigen selected from the group consisting of M, OA1, OGT, RCC, RU11, RU12, SAGE, TRG, TSTA, L1-CAM, gpA33, GM2, VEGFR, integrins, carbohydrates, TRAILR1, TRAILR2, RANKL, TGF-beta, hyaluronic acid, collagen, tenascin-C, tenascin-W, and immunoglobulin lambda-like polypeptide 1 (IGLL1).

[0054]

[0067] In some embodiments, the tumor-associated antigen-binding portion binds to a cancer antigen selected from the group consisting of CD20, MSLN, gp75 (Tryp1), or any combination thereof.

[0055]

[0068] In some embodiments, the multifunctional molecule is a polypeptide molecule.

[0069] In some embodiments, the tumor-associated antigen-binding portion comprises a VH comprising HC CDR1, HC CDR2 and HC CDR3 comprising the sequences of SEQ ID NO: 524, SEQ ID NO: 525 and SEQ ID NO: 526, respectively, or a VH comprising HC CDR1, HC CDR2 and HC CDR3 comprising the sequences of SEQ ID NO: 538, SEQ ID NO: 539 and SEQ ID NO: 540, respectively.

[0056]

[0070] In some embodiments, the tumor-associated antigen-binding portion comprises a VL comprising LC CDR1, LC CDR2 and LC CDR3 comprising the sequences of SEQ ID NO: 1168, SEQ ID NO: 1295, and SEQ ID NO: 528, respectively, or a VL comprising LC CDR1, LC CDR2 and LC CDR3 comprising the sequences of SEQ ID NO: 562, SEQ ID NO: 563, and SEQ ID NO: 564, respectively.

[0057]

[0071] In some embodiments, the tumor-associated antigen-binding portion comprises a VH comprising HC CDR1, HC CDR2 and HC CDR3 comprising the sequences of SEQ ID NO: 524, SEQ ID NO: 525 and SEQ ID NO: 526, respectively, and a VL comprising LC CDR1, LC CDR2 and LC CDR3 comprising the sequences of SEQ ID NO: 1168, SEQ ID NO: 1295 and SEQ ID NO: 528, respectively.

[0058]

[0072] In some embodiments, the tumor-associated antigen-binding portion comprises a VH comprising HC CDR1, HC CDR2 and HC CDR3 comprising the sequences of SEQ ID NO: 538, SEQ ID NO: 539 and SEQ ID NO: 540, respectively, and a VL comprising LC CDR1, LC CDR2 and LC CDR3 comprising the sequences of SEQ ID NO: 562, SEQ ID NO: 563 and SEQ ID NO: 564, respectively.

[0059]

[0073] In some embodiments, the tumor-associated antigen-binding portion comprises a VH comprising a sequence having at least 70% sequence identity to the sequence of SEQ ID NO:523 or the sequence of SEQ ID NO:537.

[0060]

[0074] In some embodiments, the tumor-associated antigen-binding portion comprises a VL comprising a sequence having at least 70% sequence identity to the sequence of SEQ ID NO:527 or the sequence of SEQ ID NO:561.

[0061]

[0075] In some embodiments, the tumor-associated antigen-binding portion comprises a VH comprising the sequence of SEQ ID NO: 523 or the sequence of SEQ ID NO: 537.

[0076] In some embodiments, the tumor-associated antigen-binding portion comprises a VL comprising the sequence of SEQ ID NO: 527 or the sequence of SEQ ID NO: 561.

[0062]

[0077] In some embodiments, the tumor-associated antigen-binding portion comprises a VH comprising a sequence having at least 70% sequence identity to the sequence of SEQ ID NO: 523, and a VL comprising a sequence having at least 70% sequence identity to the sequence of SEQ ID NO: 527.

[0063]

[0078] In some embodiments, the tumor-associated antigen-binding portion comprises a VH comprising the sequence of SEQ ID NO:523 and a VL comprising the sequence of SEQ ID NO:527.

[0079] In some embodiments, the tumor-associated antigen-binding portion comprises a VH comprising a sequence having at least 70% sequence identity to the sequence of SEQ ID NO: 537, and a VL comprising a sequence having at least 70% sequence identity to the sequence of SEQ ID NO: 561.

[0064]

[0080] In some embodiments, the tumor-associated antigen-binding portion comprises a VH comprising the sequence of SEQ ID NO:537 and a VL comprising the sequence of SEQ ID NO:561.

[0081] In some embodiments, the tumor-associated antigen-binding portion comprises a VH comprising HC CDR1, HC CDR2 and HC CDR3 comprising the sequences of SEQ ID NO:581, SEQ ID NO:582 and SEQ ID NO:583, respectively.

[0065]

[0082] In some embodiments, the tumor-associated antigen-binding portion comprises a VL comprising LC CDR1, LC CDR2 and LC CDR3 comprising the sequences of SEQ ID NO:586, SEQ ID NO:587 and SEQ ID NO:588, respectively.

[0066]

[0083] In some embodiments, the tumor-associated antigen-binding portion comprises a VH comprising HC CDR1, HC CDR2 and HC CDR3 comprising the sequences of SEQ ID NO: 581, SEQ ID NO: 582 and SEQ ID NO: 583, respectively, and a VL comprising LC CDR1, LC CDR2 and LC CDR3 comprising the sequences of SEQ ID NO: 586, SEQ ID NO: 587 and SEQ ID NO: 588, respectively.

[0067]

[0084] In some embodiments, the tumor-associated antigen-binding portion comprises a VH comprising a sequence having at least 70% sequence identity to the sequence of SEQ ID NO:580.

[0085] In some embodiments, the tumor-associated antigen-binding portion comprises a VL comprising a sequence having at least 70% sequence identity to the sequence of SEQ ID NO:585.

[0068]

[0086] In some embodiments, the tumor-associated antigen-binding portion comprises a VH comprising the sequence of SEQ ID NO:580.

[0087] In some embodiments, the tumor-associated antigen-binding portion comprises a VL comprising the sequence of SEQ ID NO:585.

[0069]

[0088] In some embodiments, the tumor-associated antigen-binding portion comprises a VH comprising a sequence having at least 70% sequence identity to the sequence of SEQ ID NO: 580, and a VL comprising a sequence having at least 70% sequence identity to the sequence of SEQ ID NO: 585.

[0070]

[0089] In some embodiments, the tumor-associated antigen-binding portion comprises a VH comprising the sequence of SEQ ID NO:580 and a VL comprising the sequence of SEQ ID NO:585.

[0090] In some embodiments, the TCRβV binding portion comprises a VH comprising HC CDR1, HC CDR2 and HC CDR3 comprising the sequences of SEQ ID NO: 3650, SEQ ID NO: 3651, and SEQ ID NO: 5, respectively, or a VH comprising HC CDR1, HC CDR2 and HC CDR3 comprising the sequences of SEQ ID NO: 542, SEQ ID NO: 543, and SEQ ID NO: 544, respectively.

[0071]

[0091] In some embodiments, the TCRβV binding portion comprises a VL comprising LC CDR1, LC CDR2 and LC CDR3 comprising the sequences of SEQ ID NO: 3655, SEQ ID NO: 3653, and SEQ ID NO: 8, respectively, or a VL comprising LC CDR1, LC CDR2 and LC CDR3 comprising the sequences of SEQ ID NO: 545, SEQ ID NO: 546, and SEQ ID NO: 547, respectively.

[0072]

[0092] In some embodiments, the TCRβV binding portion comprises a VH comprising HC CDR1, HC CDR2 and HC CDR3 comprising the sequences of SEQ ID NO: 3650, SEQ ID NO: 3651 and SEQ ID NO: 5, respectively, and a VL comprising LC CDR1, LC CDR2 and LC CDR3 comprising the sequences of SEQ ID NO: 3655, SEQ ID NO: 3653 and SEQ ID NO: 8, respectively.

[0073]

[0093] In some embodiments, the TCRβV binding portion comprises a VH comprising HC CDR1, HC CDR2 and HC CDR3 comprising the sequences of SEQ ID NO: 542, SEQ ID NO: 543 and SEQ ID NO: 544, respectively, and a VL comprising LC CDR1, LC CDR2 and LC CDR3 comprising the sequences of SEQ ID NO: 545, SEQ ID NO: 546 and SEQ ID NO: 547, respectively.

[0074]

[0094] In some embodiments, the TCRβV-binding portion comprises a VH comprising a sequence having at least 70% sequence identity to the sequence of SEQ ID NO: 1346 or the sequence of SEQ ID NO: 541.

[0075]

[0095] In some embodiments, the TCRβV-binding portion comprises a VL comprising a sequence having at least 70% sequence identity to the sequence of SEQ ID NO: 1349 or the sequence of SEQ ID NO: 3527.

[0076]

[0096] In some embodiments, the TCRβV-binding portion comprises a VH comprising the sequence of SEQ ID NO: 1346 or the sequence of SEQ ID NO:541.

[0097] In some embodiments, the TCRβV-binding portion comprises a VL comprising the sequence of SEQ ID NO: 1349 or the sequence of SEQ ID NO: 3527.

[0077]

[0098] In some embodiments, the TCRβV binding portion comprises a VH comprising a sequence having at least 70% sequence identity to the sequence of SEQ ID NO: 1346, and a VL comprising a sequence having at least 70% sequence identity to the sequence of SEQ ID NO: 1349.

[0078]

[0099] In some embodiments, the TCRβV binding portion comprises a VH comprising the sequence of SEQ ID NO:1346 and a VL comprising the sequence of SEQ ID NO:1349.

[0100] In some embodiments, the TCRβV binding portion comprises a VH comprising a sequence having at least 70% sequence identity to the sequence of SEQ ID NO: 541, and a VL comprising a sequence having at least 70% sequence identity to the sequence of SEQ ID NO: 3527.

[0079]

[0101] In some embodiments, the TCRβV binding portion comprises a VH comprising the sequence of SEQ ID NO:541 and a VL comprising the sequence of SEQ ID NO:3527.

[0102] In some embodiments, the TCRβV binding portion comprises an scFv comprising the sequence of SEQ ID NO: 1346 operably linked to the sequence of SEQ ID NO: 1349 via a linker comprising the sequence of SEQ ID NO: 3801.

[0080]

[0103] In some embodiments, the TCRβV binding portion comprises an scFv comprising the sequence of SEQ ID NO: 541 operably linked to the sequence of SEQ ID NO: 3527 via a linker comprising the sequence of SEQ ID NO: 3801.

[0081]

[0104] In some embodiments, the TCRβV binding portion comprises an scFv comprising a sequence having at least 70% sequence identity to the sequence of SEQ ID NO:1331 or the sequence of SEQ ID NO:1376.

[0082]

[0105] In some embodiments, the TCRβV binding portion comprises an scFv comprising the sequence of SEQ ID NO:1331 or the sequence of SEQ ID NO:1376.

[0106] In some embodiments, the multifunctional molecule consists of a first polypeptide chain, a second polypeptide chain, and a third polypeptide chain; (i) the first polypeptide chain comprises the sequence of SEQ ID NO: 523 and the sequence of SEQ ID NO: 3649; (ii) the second polypeptide chain comprises the sequence of SEQ ID NO: 1331, the sequence of SEQ ID NO: 2270, and the sequence of SEQ ID NO: 3648; (iii) the third polypeptide chain comprises the sequence of SEQ ID NO: 527 and the sequence of SEQ ID NO: 3644.

[0083]

[0107] In some embodiments, the multifunctional molecule consists of a first polypeptide chain, a second polypeptide chain, and a third polypeptide chain; (i) the first polypeptide chain comprises the sequence of SEQ ID NO: 523 operably linked to the sequence of SEQ ID NO: 3649; (ii) the second polypeptide chain comprises the sequence of SEQ ID NO: 1331 operably linked to the sequence of SEQ ID NO: 2270, which is operably linked to the sequence of SEQ ID NO: 3648; (iii) the third polypeptide chain comprises the sequence of SEQ ID NO: 527 operably linked to the sequence of SEQ ID NO: 3644.

[0084]

[0108] In some embodiments, the multifunctional molecule consists of a first polypeptide chain, a second polypeptide chain, and a third polypeptide chain; (i) the first polypeptide chain comprises a sequence having at least 70% sequence identity to the sequence of SEQ ID NO: 501; (ii) the second polypeptide chain comprises a sequence having at least 70% sequence identity to the sequence of SEQ ID NO: 3800; (iii) the third polypeptide chain comprises a sequence having at least 70% sequence identity to the sequence of SEQ ID NO: 502.

[0085]

[0109] In some embodiments, the multifunctional molecule consists of a first polypeptide chain, a second polypeptide chain, and a third polypeptide chain; (i) the first polypeptide chain comprises the sequence of SEQ ID NO: 501; (ii) the second polypeptide chain comprises the sequence of SEQ ID NO: 3800; (iii) the third polypeptide chain comprises the sequence of SEQ ID NO: 502.

[0086]

[0110] In some embodiments, the multifunctional molecule consists of a first polypeptide chain, a second polypeptide chain, and a third polypeptide chain; (i) the first polypeptide chain comprises a first sequence of SEQ ID NO: 523, a second sequence of SEQ ID NO: 523, and a sequence of SEQ ID NO: 3649; (ii) the second polypeptide chain comprises the sequence of SEQ ID NO: 1331, the sequence of SEQ ID NO: 2270, and the sequence of SEQ ID NO: 3648; (iii) the third polypeptide chain comprises the sequence of SEQ ID NO: 527 and the sequence of SEQ ID NO: 3644.

[0087]

[0111] In some embodiments, the multifunctional molecule consists of a first polypeptide chain, a second polypeptide chain, and a third polypeptide chain; (i) the first polypeptide chain comprises a first sequence of SEQ ID NO: 523 operably linked to a second sequence of SEQ ID NO: 523 operably linked to a sequence of SEQ ID NO: 3649; (ii) the second polypeptide chain comprises the sequence of SEQ ID NO: 1331 operably linked to the sequence of SEQ ID NO: 2270, which is operably linked to the sequence of SEQ ID NO: 3648; (iii) the third polypeptide chain comprises the sequence of SEQ ID NO: 527 operably linked to the sequence of SEQ ID NO: 3644.

[0088]

[0112] In some embodiments, the multifunctional molecule consists of a first polypeptide chain, a second polypeptide chain, and a third polypeptide chain; (i) the first polypeptide chain comprises a sequence having at least 70% sequence identity to the sequence of SEQ ID NO: 590; (ii) the second polypeptide chain comprises a sequence having at least 70% sequence identity to the sequence of SEQ ID NO: 3800; (iii) the third polypeptide chain comprises a sequence having at least 70% sequence identity to the sequence of SEQ ID NO: 502.

[0089]

[0113] In some embodiments, the multifunctional molecule consists of a first polypeptide chain, a second polypeptide chain, and a third polypeptide chain; (i) the first polypeptide chain comprises the sequence of SEQ ID NO: 590; (ii) the second polypeptide chain comprises the sequence of SEQ ID NO: 3800; (iii) the third polypeptide chain comprises the sequence of SEQ ID NO: 502.

[0090]

[0114] In some embodiments, the multifunctional molecule consists of a first polypeptide chain, a second polypeptide chain, and a third polypeptide chain; (i) the first polypeptide chain comprises the sequence of SEQ ID NO: 537 and the sequence of SEQ ID NO: 559; (ii) the second polypeptide chain comprises the sequence of SEQ ID NO: 1376, the sequence of SEQ ID NO: 2191, and the sequence of SEQ ID NO: 3533; (iii) the third polypeptide chain comprises the sequence of SEQ ID NO: 561 and the sequence of SEQ ID NO: 558.

[0091]

[0115] In some embodiments, the multifunctional molecule consists of a first polypeptide chain, a second polypeptide chain, and a third polypeptide chain; (i) the first polypeptide chain comprises the sequence of SEQ ID NO: 537 operably linked to the sequence of SEQ ID NO: 559; (ii) the second polypeptide chain comprises the sequence of SEQ ID NO: 1376 operably linked to the sequence of SEQ ID NO: 2191, which is operably linked to the sequence of SEQ ID NO: 3533; (iii) the third polypeptide chain comprises the sequence of SEQ ID NO: 561 operably linked to the sequence of SEQ ID NO: 558.

[0092]

[0116] In some embodiments, the multifunctional molecule consists of a first polypeptide chain, a second polypeptide chain, and a third polypeptide chain; (i) the first polypeptide chain comprises a sequence having at least 70% sequence identity to the sequence of SEQ ID NO: 509; (ii) the second polypeptide chain comprises a sequence having at least 70% sequence identity to the sequence of SEQ ID NO: 511; (iii) the third polypeptide chain comprises a sequence having at least 70% sequence identity to the sequence of SEQ ID NO: 510.

[0093]

[0117] In some embodiments, the multifunctional molecule consists of a first polypeptide chain, a second polypeptide chain, and a third polypeptide chain; (i) the first polypeptide chain comprises the sequence of SEQ ID NO: 509; (ii) the second polypeptide chain comprises the sequence of SEQ ID NO: 511; (iii) the third polypeptide chain comprises the sequence of SEQ ID NO: 510.

[0094]

[0118] In some embodiments, the multifunctional molecule consists of a first polypeptide chain, a second polypeptide chain, and a third polypeptide chain; (i) the first polypeptide chain comprises a first sequence of SEQ ID NO: 537, a second sequence of SEQ ID NO: 537, and a sequence of SEQ ID NO: 559; (ii) the second polypeptide chain comprises the sequence of SEQ ID NO: 1376, the sequence of SEQ ID NO: 2191, and the sequence of SEQ ID NO: 3533; (iii) the third polypeptide chain comprises the sequence of SEQ ID NO: 561 and the sequence of SEQ ID NO: 558.

[0095]

[0119] In some embodiments, the multifunctional molecule consists of a first polypeptide chain, a second polypeptide chain, and a third polypeptide chain; (i) the first polypeptide chain comprises a first sequence of SEQ ID NO: 537 operably linked to a second sequence of SEQ ID NO: 537 operably linked to a sequence of SEQ ID NO: 559; (ii) the second polypeptide chain comprises the sequence of SEQ ID NO: 1376 operably linked to the sequence of SEQ ID NO: 2191, which is operably linked to the sequence of SEQ ID NO: 3533; (iii) the third polypeptide chain comprises the sequence of SEQ ID NO: 561 operably linked to the sequence of SEQ ID NO: 558.

[0096]

[0120] In some embodiments, the multifunctional molecule consists of a first polypeptide chain, a second polypeptide chain, and a third polypeptide chain; (i) the first polypeptide chain comprises a sequence having at least 70% sequence identity to the sequence of SEQ ID NO: 512; (ii) the second polypeptide chain comprises a sequence having at least 70% sequence identity to the sequence of SEQ ID NO: 511; (iii) the third polypeptide chain comprises a sequence having at least 70% sequence identity to the sequence of SEQ ID NO: 510.

[0097]

[0121] In some embodiments, the multifunctional molecule consists of a first polypeptide chain, a second polypeptide chain, and a third polypeptide chain; (i) the first polypeptide chain comprises the sequence of SEQ ID NO: 512; (ii) the second polypeptide chain comprises the sequence of SEQ ID NO: 511; (iii) the third polypeptide chain comprises the sequence of SEQ ID NO: 510.

[0098]

[0122] In some embodiments, the multifunctional molecule consists of a first polypeptide chain, a second polypeptide chain, and a third polypeptide chain; (i) the first polypeptide chain comprises the sequence of SEQ ID NO: 580 and the sequence of SEQ ID NO: 584; (ii) the second polypeptide chain comprises the sequence of SEQ ID NO: 1376, the sequence of SEQ ID NO: 2191, and the sequence of SEQ ID NO: 3533; (iii) the third polypeptide chain comprises the sequence of SEQ ID NO: 585 and the sequence of SEQ ID NO: 3528.

[0099]

[0123] In some embodiments, the multifunctional molecule consists of a first polypeptide chain, a second polypeptide chain, and a third polypeptide chain; (i) the first polypeptide chain comprises the sequence of SEQ ID NO: 580 operably linked to the sequence of SEQ ID NO: 584; (ii) the second polypeptide chain comprises the sequence of SEQ ID NO: 1376 operably linked to the sequence of SEQ ID NO: 2191, which is operably linked to the sequence of SEQ ID NO: 3533; (iii) the third polypeptide chain comprises the sequence of SEQ ID NO: 585 operably linked to the sequence of SEQ ID NO: 3528.

[0100]

[0124] In some embodiments, the multifunctional molecule consists of a first polypeptide chain, a second polypeptide chain, and a third polypeptide chain; (i) the first polypeptide chain comprises a sequence having at least 70% sequence identity to the sequence of SEQ ID NO: 519; (ii) the second polypeptide chain comprises a sequence having at least 70% sequence identity to the sequence of SEQ ID NO: 511; (iii) the third polypeptide chain comprises a sequence having at least 70% sequence identity to the sequence of SEQ ID NO: 520.

[0101]

[0125] In some embodiments, the multifunctional molecule consists of a first polypeptide chain, a second polypeptide chain, and a third polypeptide chain; (i) the first polypeptide chain comprises the sequence of SEQ ID NO: 519; (ii) the second polypeptide chain comprises the sequence of SEQ ID NO: 511; (iii) the third polypeptide chain comprises the sequence of SEQ ID NO: 520.

[0102]

[0126] In some embodiments, the multifunctional molecule is a multispecific molecule.

[0127] In another aspect, provided herein is a polynucleotide comprising a sequence encoding a multifunctional molecule provided herein.

[0103]

[0128] In some embodiments, the polynucleotide is an isolated nucleic acid molecule.

[0129] In another aspect, provided herein are vectors comprising one or more of the polynucleotides provided herein.

[0104]

[0130] In another aspect, provided herein is a cell comprising a polynucleotide provided herein, or a vector provided herein.

[0131] In another aspect, provided herein is a pharmaceutical composition comprising a multifunctional molecule provided herein, a polynucleotide provided herein, a vector provided herein, or a cell provided herein, and a pharmaceutically acceptable carrier, excipient, or diluent.

[0105]

[0132] In another aspect, provided herein is a method of treating a condition or disease in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a multifunctional molecule provided herein, a polynucleotide provided herein, a vector provided herein, a cell provided herein, a pharmaceutical composition provided herein, or any combination thereof, wherein the administering is effective to treat the condition or disease in the subject.

[0106]

[0133] In some aspects, the condition or disease is cancer.

[0134] In some embodiments, the cancer is a solid tumor, a blood cancer, a metastatic cancer, a soft tissue tumor, or any combination thereof.

[0107]

[0135] In some embodiments, the cancer is a solid tumor, and the solid tumor is selected from the group consisting of melanoma, pancreatic cancer, breast cancer, colorectal cancer, lung cancer, skin cancer, ovarian cancer, liver cancer, and any combination thereof.

[0108]

[0136] In some embodiments, the cancer is a blood cancer, and the blood cancer is selected from the group consisting of Hodgkin's lymphoma, non-Hodgkin's lymphoma, acute myeloid leukemia (AML), chronic myeloid leukemia, myelodysplastic syndrome, multiple myeloma, T-cell lymphoma, acute lymphocytic leukemia, and any combination thereof.

[0109]

[0137] In some embodiments, the non-Hodgkin's lymphoma is selected from the group consisting of B-cell lymphoma, diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, chronic lymphocytic leukemia (B-CLL), mantle cell lymphoma, marginal zone B-cell lymphoma, Burkitt lymphoma, lymphoplasmacytic lymphoma, hairy cell leukemia, and any combination thereof.

[0110]

[0138] In some embodiments, the T-cell lymphoma is peripheral T-cell lymphoma.

[0139] In some embodiments, the cancer is characterized by a cancer antigen present on the cancer.

[0111]

[0140] In some embodiments, the cancer antigen is a tumor antigen, a stromal antigen, or a blood antigen.

[0141] In some embodiments, the cancer antigen is CD19, CD123, CD22, CD30, CD171, CS-1, C-type lectin-like molecule-1, CD33, epidermal growth factor receptor variant III (EGFRvIII), ganglioside G2 (GD2), ganglioside GD3, TNF receptor family member B-cell maturation (BCMA), Tn antigen (TnAg) or (GalNAcα-Ser / Thr)), prostate-specific membrane antigen (PSMA), receptor tyrosine kinase-like orphan receptor 1 (ROR1), Fms-like tyrosine kinase 3 (FLT3), tumor-associated glycoprotein 72 (TAG72), CD38, CD44v6, carcinoembryonic antigen (CEA), epithelial cell adhesion molecule (EPCAM), B7H3 (CD276), KIT (CD117), interleukin-13 receptor subunit alpha-2, mesothelin, interleukin-11 receptor alpha (IL-11Ra), prostate stem cell antigen (PSCA), protease serine 21, vascular endothelial growth factor receptor 2 (VEGFR2), Lewis (Y) antigen, CD24, platelet-derived growth factor receptor beta (PDGFR-beta), stage-specific embryonic antigen-4 (SSEA-4), CD20, folate receptor alpha, receptor tyrosine-protein kinase ERBB2 (Her2 / neu), cell surface-associated mucin 1 (MUC1), epidermal growth factor receptor (EGFR), neural cell adhesion molecule (NCAM), prostase, prostatic acid phosphatase (PAP), mutant elongation factor 2 (ELF2M) ), ephrin B2, fibroblast activation protein alpha (FAP), insulin-like growth factor 1 receptor (IGF-I receptor), carbonic anhydrase IX (CAIX), proteasome (prosome, macropein) subunit, beta type 9 (LMP2), glycoprotein 100 (gp100 / pmel17), oncogene fusion protein (bcr-abl) consisting of breakpoint cluster region (BCR) and Abelson murine leukemia viral oncogene homolog 1 (Abl), tyrosinase, ephrin type A receptor 2 (EphA2) ), fucosyl GM1, sialyl Lewis adhesion molecule (sLe), ganglioside GM3, transglutaminase 5 (TGS5), high molecular weight melanoma-associated antigen (HMWMAA), o-acetyl-GD2 ganglioside (OAcGD2), folate receptor beta, tumor endothelial marker 1 (TEM1 / CD248), tumor endothelial marker 7-related (TEM7R), claudin 6 (CLDN6), thyroid-stimulating hormone receptor (TSHR), G protein-coupled receptor class C group 5, member D (GPRC5D), chromosome X open reading frame61 (CXORF61), CD97, CD179a, anaplastic lymphoma kinase (ALK), polysialic acid, placenta-specific 1 (PLAC1), hexasaccharide moiety of globoH glycoceramide (GloboH), mammary differentiation antigen (NY-BR-1), uroplakin 2 (UPK2), hepatitis A virus cellular receptor 1 (HAVCR1), adrenoceptor beta 3 (ADRB3), pannexin 3 (PANX3), G protein-coupled receptor 20 (GPR20), lymphocyte antigen 6 complex, locus K9 (LY6K), olfactory receptor 5 1E2 (OR51E2), TCR gamma alternate reading frame protein (TARP), Wilms tumor protein (WT1), cancer / testis antigen 1 (NY-ESO-1 / LAGE-1), cancer / testis antigen 2 (LAGE-1a), melanoma-associated antigen 1 (MAGE-A1), ETS translocation variant gene 6 located on chromosome 12p (ETV6-AML), sperm protein 17 (SPA17), X antigen family, member 1A (XAGE1), angiopoietin-binding cell surface receptor 2 (Tie 2), melanoma cancer testis antigen-1 (MAD-CT-1), melanoma cancer testis antigen-2 (MAD-CT-2), Fos-related antigen 1, tumor protein p53 (p53), p53 mutant, prostein, survivin, telomerase, prostate cancer tumor antigen-1, melanoma antigen 1 recognized by T cells, rat sarcoma (Ras) mutant, human telomerase reverse transcriptase (hTERT), sarcoma translocation breakpoint, melanoma inhibitor of apoptosis (ML-IAP), ER G (transmembrane protease, serine 2 (TMPRSS2) ETS fusion gene), N-acetylglucosaminyl-transferase V (NA17), paired box protein Pax-3 (PAX3), androgen receptor, cyclin B1, v-myc avian myelocytomatosis viral oncogene neuroblastoma-derived homolog (MYCN), Ras homolog family member C (RhoC), tyrosinase-related protein 2 (TRP-2), cytochrome P4501B1 (CYP1B1), CCCTC-binding factor (zinc finger protein)-like, squamous cell carcinoma antigen 3 recognized by T cells (SART3), paired box protein Pax-5 (PAX5), proacrosin-binding protein sp32 (OY-TES1), lymphocyte-specific protein tyrosine kinase (LCK), A-kinase anchor protein 4 (AKAP-4), synovial sarcoma X breakpoint 2 (SSX2), receptor for advanced glycation end products (RAGE-1), renal ubiquitous 1 (RU1), renal ubiquitous 2 (RU2), legumain, human papillomavirus E6 (HPVE6), human papillomavirus E7 (HPV E7), intestinal carboxylesterase, mutant heat shock protein 70-2 (muthsp70-2), CD79a, CD79b, CD72, leukocyte-associated immunoglobulin-like receptor 1 (LAIR1), Fc fragment of IgA receptor (FCAR or CD89), leukocyte immunoglobulin-like receptor subfamily A member 2 (LILRA2), CD300 molecule-like family member f (CD300LF), C-type lectin domain family 12 member A (CLEC12A), bone marrow stromal cell antigen 2 (BST2), E GF-like module-containing mucin-like hormone receptor-like 2 (EMR2), lymphocyte antigen 75 (LY75), glypican-3 (GPC3), Fc receptor-like 5 (FCRL5), FcRH5, PDL1, CD47, prostate-specific membrane antigen (PMSA), prostate-specific antigen (PSA), Ron kinase, c-Met, immature laminin receptor, TAG-72, BING-4, calcium-activated chloride channel 2, cyclin-B1, 9D7, E p-CAM, EphA3, SAP-1, PRAME, SSX-2, Melan-A / MART-1, TRP1 / gp75, MC1R, β-catenin, BRCA1 / 2, CDK4, CML66, fibronectin, Ras, TGF-B receptor, AFP, ETA, MAGE, CA-125, BAGE, GAGE, CDC27, α-actinin-4, ganglioside, MART-2, MUC2, MUM1, MUM2, MUM3, NA8 8-1, NPM, OA1, OGT, RCC, RU11, RU12, SAGE, TRG, TSTA, L1-CAM, gpA33, GM2, VEGFR, integrins, carbohydrates, TRAILR1, TRAILR2, RANKL, TGF-beta, hyaluronic acid, collagen, tenascin-C, tenascin-W, and immunoglobulin lambda-like polypeptide 1 (IGLL1).

[0112]

[0142] In some embodiments, the methods provided herein further include administering a second therapeutic agent or treatment to the subject.

[0143] In some embodiments, the second therapeutic agent or treatment comprises a chemotherapeutic agent, a biologic agent, hormone therapy, radiation, or surgery.

[0113]

[0144] In some embodiments, the second therapeutic agent or treatment is administered in combination, sequentially, simultaneously, or concurrently with a multifunctional molecule provided herein, a polynucleotide provided herein, a vector provided herein, a cell provided herein, or a pharmaceutical composition provided herein.

[0114] Incorporation by Reference

[0145] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0115]

[0146] The novel features of the present disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings. [Brief explanation of the drawings]

[0116] [Figure 1A]

[0147] Figures 1A-1T show exemplary embodiments of multifunctional molecules described herein. Figures 1A, 1B, and 1C show exemplary embodiments of multifunctional molecules containing multiple, e.g., two, molecules of an exemplary cytokine, interleukin-2 (IL-2), linked to an antibody molecule that binds to a T cell receptor beta variable region (TCRβV) ("anti-TCRβV antibody molecule"). Figures 1D, 1E, and 1F show exemplary embodiments of multifunctional molecules containing a single molecule of an exemplary cytokine, IL-2, linked to an anti-TCRβV antibody molecule. Figures 1G, 1H, 1I, and 1J show exemplary embodiments of multifunctional molecules containing an exemplary cytokine, IL-2, linked to a first dimerization module. Figures 1K, 1L, and 1M show exemplary embodiments of multifunctional molecules containing an exemplary dimerization module, e.g., an Fc region comprising an N297A mutation, and multiple, e.g., two, molecules of an exemplary cytokine, IL-2, linked to an anti-TCRβV antibody molecule. Figures 1N, 1O, and 1P show exemplary embodiments of multifunctional molecules containing an exemplary dimerization module, e.g., an Fc region (knobs-in-hole) comprising an N297A mutation, and a single molecule of an exemplary cytokine, IL-2, linked to an anti-TCRβV antibody molecule. Figures 1Q, 1R, 1S, and 1T show exemplary embodiments of multifunctional molecules containing an exemplary dimerization module, e.g., an Fc region (knobs-in-hole) comprising an N297A mutation, and an exemplary cytokine, IL-2, linked to an exemplary dimerization module. [Figure 1B] 1A, 1B, and 1C show exemplary embodiments of multifunctional molecules containing multiple, e.g., two, molecules of an exemplary cytokine, interleukin-2 (IL-2), linked to an antibody molecule that binds to the T cell receptor beta variable region (TCRβV) (an "anti-TCRβV antibody molecule"). [Figure 1C]1A, 1B, and 1C show exemplary embodiments of multifunctional molecules containing multiple, e.g., two, molecules of an exemplary cytokine, interleukin-2 (IL-2), linked to an antibody molecule that binds to the T cell receptor beta variable region (TCRβV) (an "anti-TCRβV antibody molecule"). [Figure 1D] 1D, 1E, and 1F show exemplary embodiments of multifunctional molecules containing a single molecule of an exemplary cytokine, IL-2, linked to an anti-TCRβV antibody molecule. [Figure 1E] 1D, 1E, and 1F show exemplary embodiments of multifunctional molecules containing a single molecule of an exemplary cytokine, IL-2, linked to an anti-TCRβV antibody molecule. [Figure 1F] 1D, 1E, and 1F show exemplary embodiments of multifunctional molecules containing a single molecule of an exemplary cytokine, IL-2, linked to an anti-TCRβV antibody molecule. [Figure 1G] 1G, 1H, 1I, and 1J show exemplary embodiments of multifunctional molecules containing an exemplary cytokine, IL-2, linked to a first dimerization module. [Figure 1H] 1G, 1H, 1I, and 1J show exemplary embodiments of multifunctional molecules containing an exemplary cytokine, IL-2, linked to a first dimerization module. [Figure 1I] 1G, 1H, 1I, and 1J show exemplary embodiments of multifunctional molecules containing an exemplary cytokine, IL-2, linked to a first dimerization module. [Figure 1J] 1G, 1H, 1I, and 1J show exemplary embodiments of multifunctional molecules containing an exemplary cytokine, IL-2, linked to a first dimerization module. [Figure 1K] Figures 1K, 1L, and 1M show exemplary embodiments of multifunctional molecules containing multiple, e.g., two, molecules of an exemplary cytokine, IL-2, linked to an exemplary dimerization module, e.g., an Fc region comprising an N297A mutation, and an anti-TCRβV antibody molecule. [Figure 1L] Figures 1K, 1L, and 1M show exemplary embodiments of multifunctional molecules containing multiple, e.g., two, molecules of an exemplary cytokine, IL-2, linked to an exemplary dimerization module, e.g., an Fc region comprising an N297A mutation, and an anti-TCRβV antibody molecule. [Figure 1M] Figures 1K, 1L, and 1M show exemplary embodiments of multifunctional molecules containing multiple, e.g., two, molecules of an exemplary cytokine, IL-2, linked to an exemplary dimerization module, e.g., an Fc region comprising an N297A mutation, and an anti-TCRβV antibody molecule. [Figure 1N] Figures 1N, 1O, and 1P show exemplary embodiments of multifunctional molecules containing an exemplary dimerization module, e.g., an Fc region containing an N297A mutation (knobs-in-holes), and a single molecule of an exemplary cytokine, IL-2, linked to an anti-TCRβV antibody molecule. [Figure 1O] Figures 1N, 1O, and 1P show exemplary embodiments of multifunctional molecules containing an exemplary dimerization module, e.g., an Fc region containing an N297A mutation (knobs-in-holes), and a single molecule of an exemplary cytokine, IL-2, linked to an anti-TCRβV antibody molecule. [Figure 1P] Figures 1N, 1O, and 1P show exemplary embodiments of multifunctional molecules containing an exemplary dimerization module, e.g., an Fc region containing an N297A mutation (knobs-in-holes), and a single molecule of an exemplary cytokine, IL-2, linked to an anti-TCRβV antibody molecule. [Figure 1Q] Figures 1Q, 1R, 1S, and 1T show exemplary embodiments of multifunctional molecules containing an exemplary dimerization module, e.g., an Fc region comprising an N297A mutation (knobs-in-holes), and an exemplary cytokine, IL-2, linked to an exemplary dimerization module. [Figure 1R]Figures 1Q, 1R, 1S, and 1T show exemplary embodiments of multifunctional molecules containing an exemplary dimerization module, e.g., an Fc region comprising an N297A mutation (knobs-in-holes), and an exemplary cytokine, IL-2, linked to an exemplary dimerization module. [Figure 1S] Figures 1Q, 1R, 1S, and 1T show exemplary embodiments of multifunctional molecules containing an exemplary dimerization module, e.g., an Fc region comprising an N297A mutation (knobs-in-holes), and an exemplary cytokine, IL-2, linked to an exemplary dimerization module. [Figure 1T] Figures 1Q, 1R, 1S, and 1T show exemplary embodiments of multifunctional molecules containing an exemplary dimerization module, e.g., an Fc region comprising an N297A mutation (knobs-in-holes), and an exemplary cytokine, IL-2, linked to an exemplary dimerization module. [Figure 2A]

[0148] Figures 2A-2B show alignments of the mouse VH and VL framework 1, CDR1, framework 2, CDR2, framework 3, CDR3, and framework 4 regions of the antibody A source with their respective humanized sequences. Kabat CDRs are shown in bold, Chothia CDRs in italics, and combined CDRs in boxes. Backmutated framework positions are double underlined. Figure 2A shows the VH sequences of mouse antibody A (SEQ ID NO: 1) and humanized antibody AH (SEQ ID NO: 9). Figure 2B shows the VL sequences of mouse antibody A (SEQ ID NO: 2) and humanized antibody AH (SEQ ID NO: 10 and SEQ ID NO: 11). [Figure 2B] FIG. 2B shows the VL sequences of murine antibody A (SEQ ID NO: 2) and humanized antibody AH (SEQ ID NO: 10 and SEQ ID NO: 11). [Figure 3A]

[0149] Figures 3A-3B show alignments of the mouse VH and VL framework 1, CDR1, framework 2, CDR2, framework 3, CDR3, and framework 4 regions of antibody B source with their respective humanized sequences. Kabat CDRs are shown in bold, Chothia CDRs in italics, and combined CDRs in boxes. Backmutated framework positions are double underlined. Figure 3A shows the VH sequences of mouse antibody B (SEQ ID NO: 15) and humanized VH sequences BH.1A-BH.1C (SEQ ID NOs: 23-25). Figure 3B shows the VL sequences of mouse antibody B (SEQ ID NO: 16) and humanized VL sequences BH.1D-BH.1H (SEQ ID NOs: 26-30). [Figure 3B-1] FIG. 3B shows the VL sequences of mouse antibody B (SEQ ID NO: 16) and humanized VL sequences BH.1D to BH.1H (SEQ ID NOs: 26 to 30). [Figure 3B-2] Same as above. [Figure 4]

[0150] FIG. 1 shows a phylogenetic tree of TCRBV gene families and subfamilies to which the corresponding antibodies were mapped. The subfamily identities are as follows: Subfamily A: TCRβ V6; Subfamily B: TCRβ V10; Subfamily C: TCRβ V12; Subfamily D: TCRβ V5; Subfamily E: TCRβ V7; Subfamily F: TCRβ V11; Subfamily G: TCRβ V14; Subfamily H: TCRβ V16; Subfamily I: TCRβ V18; Subfamily J: TCRβ V9; Subfamily K: TCRβ V13; Subfamily L: TCRβ V4; Subfamily M: TCRβ V3; Subfamily N: TCRβ V2; Subfamily O: TCRβ V15; Subfamily P: TCRβ V30; Subfamily Q: TCRβ V19; Subfamily R: TCRβ V27; Subfamily S: TCRβ V28; Subfamily T: TCRβ V24; Subfamily U: TCRβ V20; Subfamily V: TCRβ V25; and subfamily W: TCRβ V29 subfamily. Subfamily members are described in detail herein in the section entitled "TCR beta V (TCRβV)." [Figure 5A]

[0151] Figures 5A-5C show human CD3+ T cells activated with anti-TCR Vβ13.1 antibody (AH.1) for 6 days. Human CD3+ T cells were isolated using magnetic bead separation (negative selection) and activated with immobilized (plate-coated) anti-TCR Vβ13.1 (AH.1) or anti-CD3ε (OKT3) antibodies at 100 nM for 6 days. Figure 5A shows two scatter plots (left: activated with OKT3; right: activated with AH.1) of expanded T cells assessed for TCR Vβ13.1 surface expression using anti-TCR Vβ13.1 (AH.1) followed by a secondary fluorochrome-conjugated antibody for flow cytometry analysis. Figure 5B shows the percentage (%) of TCR Vβ13.1-positive T cells activated by anti-TCR Vβ13.1 (AH.1) or anti-CD3e (OKT3) plotted against total T cells (CD3+). Figure 5C shows the relative cell count obtained by counting the number of events in each T cell subset gate (CD3 or TCR Vβ13.1) at a constant rate of 60 μl / min for 20 seconds. Data are shown as average values ​​from three donors. [Figure 5B] FIG. 5B is a plot of the percentage (%) of TCR Vβ13.1 positive T cells activated by anti-TCR Vβ13.1 (AH.1) or anti-CD3e (OKT3) versus total T cells (CD3+). [Figure 5C] Figure 5C shows relative cell counts obtained by counting the number of events in each T cell subset gate (CD3 or TCR Vβ13.1) for 20 seconds at a constant rate of 60 μl / min. Data are shown as the average from three donors. [Figure 6A]

[0152] Figures 6A-6B show the cytolytic activity of human CD3+ T cells activated with anti-TCR Vβ13.1 antibody (AH.1) against the transformed cell line RPMI8226. Figure 6A shows target cytolysis of human CD3+ T cells activated with AH.1 or OKT3. Human CD3+ T cells were isolated using magnetic bead separation (negative selection) and activated with immobilized (plate-coated) AH.1 or OKT3 at the indicated concentrations for 4 days before coculture with RPMI8226 cells at a 5:1 (E:T) ratio for 2 days. Samples were then analyzed for cytolysis of RPMI8226 cells using flow cytometry analysis by FACS staining for CFSE / CD138-labeled and membrane-impermeable DNA dye (DRAQ7). Figure 6B shows target cell lysis of human CD3+ T cells activated with AH.1 or OKT3 incubated with RPMI-8226 at a 5:1 (E:T) ratio for 6 days, followed by cytolysis analysis of RPMI8226 cells as described above. The percentage (%) of target cell lysis was determined by normalizing to basal target cell lysis (i.e., without antibody treatment) using the following formula: [(x-basal) / (100%-basal), where x is the cytolysis of the sample]. Data shown are representative of n=1 donor. [Figure 6B] Figure 6B shows target cell lysis of human CD3+ T cells activated with AH.1 or OKT3 incubated with RPMI-8226 at a 5:1 (E:T) ratio for 6 days, followed by cytolysis analysis of RPMI8226 cells as described above. The percentage (%) of target cell lysis was determined by normalizing to basal target cell lysis (i.e., without antibody treatment) using the following formula: [(x-basal) / (100%-basal), where x is the cytolysis of the sample]. Data shown are representative of n=1 donor. [Figure 7A]

[0153] Figures 7A-7B show IFNγ production by human PBMCs activated with the indicated antibodies. Human PBMCs were isolated from whole blood from the indicated number of donors, followed by solid-phase (plate-coated) stimulation with the indicated antibodies at 100 Nm. Supernatants were collected on days 1, 2, 3, 5, or 6. Figure 7A is a graph comparing IFNγ production in human PBMCs activated with the indicated antibodies, activated with an anti-TCR Vβ13.1 antibody (AH.1 or AH.2) or an anti-CD3e antibody (OKT3 or SP34-2) on days 1, 2, 3, 5, or 6 after activation. Figure 7B shows IFNγ production in human PBMCs activated with the indicated antibodies, activated with the indicated anti-TCR Vβ13.1 antibody or anti-CD3e antibody (OKT3) on days 1, 2, 3, 5, or 6 after activation. [Figure 7B] Figure 7B shows IFNγ production in human PBMCs activated with the indicated anti-TCR Vβ13.1 antibodies or anti-CD3e antibody (OKT3) on days 1, 2, 3, 5, or 6 after activation. [Figure 8A]

[0154] Figures 8A-8B show IL-2 production by human PBMCs activated with the indicated antibodies. A similar experimental setup as described in Figures 7A-7B was used. [Figure 8B] Figures 8A-8B show IL-2 production by human PBMCs activated with the indicated antibodies. A similar experimental setup as described in Figures 7A-7B was used. [Figure 9A]

[0155] Figures 9A-9B show IL-6 production by human PBMCs activated with the indicated antibodies. A similar experimental setup as described in Figures 7A-7B was used. [Figure 9B] Figures 9A-9B show IL-6 production by human PBMCs activated with the indicated antibodies. A similar experimental setup as described in Figures 7A-7B was used. [Figure 10A]

[0156] Figures 10A-10B show TNF-α production by human PBMCs activated with the indicated antibodies. A similar experimental setup as described in Figures 7A-7B was used. [Figure 10B] Figures 10A-10B show TNF-α production by human PBMCs activated with the indicated antibodies. A similar experimental setup as described in Figures 7A-7B was used. [Figure 11A]

[0157] 11A-11B show IL-1β production by human PBMCs activated with the indicated antibodies. A similar experimental setup as described in FIGS. 7A-7B was used. [Figure 11B] 11A-11B show IL-1β production by human PBMCs activated with the indicated antibodies. A similar experimental setup as described in FIGS. 7A-7B was used. [Figure 12A]

[0158] Figures 12A-12B are graphs showing the delayed kinetics of IFNγ secretion in human PBMCs activated with the anti-TCR Vβ13.1 antibody AH.1 compared to PBMCs activated with the anti-CD3e antibody OKT3. Figure 12A shows IFNγ secretion data from four donors. Figure 12B shows IFNγ secretion data from an additional four donors. Data shown are representative of n=8 donors. [Figure 12B] Figure 12B shows IFNγ secretion data from four additional donors. Data shown are representative of n=8 donors. [Figure 13]

[0159] FIG. 1 shows an increase in CD8+ TSCM and Tempra T cell subsets in human PBMCs activated with anti-TCR Vβ13.1 antibodies (AH.1 or AH.2) compared to PBMCs activated with anti-CD3e antibodies (OKT3 or SP34-2). [Figure 14A]

[0160] Figures 14A-14F show the characteristics of anti-TCRVb antibodies. Figure 14A is a graph showing the proliferation of T cells activated with anti-CD3 (OKT3) or anti-TCRVb antibodies. Figure 14B shows the selective expansion of CD45RA+ effector memory CD8+ and CD4+ T cells (TEMRA) using anti-TCRVb antibodies. Tn = naive T cells; Tscm = stem cell memory T cells; Tcm = central memory T cells; Tem = effector memory T cells; TEMRA = effector memory CD45RA+ T cells. Figure 14C is a graph showing IFN-γ secretion by PBMCs stimulated with anti-TCRVb or anti-CD3 antibodies. Figure 14D shows target cell lysis by T cells stimulated with anti-TCRVb or anti-CD3 antibodies. Cells were stimulated for 4 days, followed by 2 days of incubation with multiple myeloma target cells to assess cell killing. Figure 14E is a graph showing perforin secretion by T cells stimulated with anti-TCRVb antibody or anti-CD3 antibody. Perforin was analyzed by FACS staining in TCRVB-positive and TCRVB-negative T cells in PBMCs after stimulation with 100 ng / ml plate-bound antibody for 5 days. Figure 14F is a graph showing granzyme B secretion by T cells stimulated with anti-TCRVb antibody or anti-CD3 antibody. Granzyme B was analyzed by FACS staining in TCRVB-positive and TCRVB-negative T cells in PBMCs after stimulation with 100 ng / ml plate-bound antibody for 5 days. [Figure 14B] Figure 14B shows the selective expansion of CD45RA+ effector memory CD8+ and CD4+ T cells (TEMRA) cells using anti-TCRVb antibodies. Tn = naive T cells; Tscm = stem cell memory T cells; Tcm = central memory T cells; Tem = effector memory T cells; Temra = effector memory CD45RA+ T cells. [Figure 14C] Figure 14C is a graph showing IFN-g secretion by PBMCs stimulated with anti-TCRVb antibody or anti-CD3 antibody. [Figure 14D]Figure 14D shows target cell lysis by T cells stimulated with anti-TCRVb or anti-CD3 antibodies. Cells were stimulated for 4 days and then incubated with multiple myeloma target cells for 2 days to assess cell killing. [Figure 14E] Figure 14E is a graph showing perforin secretion by T cells stimulated with anti-TCRVb or anti-CD3 antibodies. Perforin was analyzed by FACS staining in TCRVb-positive and TCRVb-negative T cells in PBMCs after 5 days of stimulation with 100 ng / ml plate-bound antibody. [Figure 14F] Figure 14F is a graph showing granzyme B by T cells stimulated with anti-TCRVb or anti-CD3 antibodies. Granzyme B was analyzed by FACS staining in TCRVb-positive and TCRVb-negative T cells in PBMCs after stimulation with 100 ng / ml plate-bound antibody for 5 days. [Figure 15A]

[0161] Figures 15A-15B show the production of IL-2 and IL-15 and the expansion of human NK cells following stimulation of PBMCs with anti-TCRVb antibody at a dose of 100 nM for 6 days. Figure 15A shows the secretion of IL-2 or IL-15 in T cells stimulated with anti-TCRVb antibody or anti-CD3 antibody. Figure 15B shows flow cytometry dot plots showing NKp46 staining versus CD56 antibody staining in cells stimulated with anti-TCRVb antibody or anti-CD3 antibody, or a control sample. [Figure 15B-1] Figure 15B shows flow cytometry dot plots showing NKp46 staining versus CD56 antibody staining in cells stimulated with anti-TCRVb or anti-CD3 antibodies, or control samples. [Figure 15B-2] Figure 15B shows flow cytometry dot plots showing NKp46 staining versus CD56 antibody staining in cells stimulated with anti-TCRVb or anti-CD3 antibodies, or control samples. [Figure 16A]

[0162] 16A to 16C are diagrams showing cytokine secretion in PBMCs stimulated with anti-TCRVb antibody or anti-CD3 antibody. [Figure 16B] 16A to 16C are diagrams showing cytokine secretion in PBMCs stimulated with anti-TCRVb antibody or anti-CD3 antibody. [Figure 16C] 16A to 16C are diagrams showing cytokine secretion in PBMCs stimulated with anti-TCRVb antibody or anti-CD3 antibody. [Figure 17A]

[0163] Figures 17A-17B show killing of MM cells by dual-targeting BCMA-TCRvb antibody molecules. Figure 17A shows in vitro killing by one of the following dual-targeting antibody molecules: BCMA-TCRVb (molecule I), BCMA-CD3, or control-TCRVb; or an isotype control. Figure 17B shows in vivo killing of MM cells by dual-targeting BCMA-TCRVb antibody (molecule I). [Figure 17B] Figures 17A-17B show killing of MM cells by dual-targeting BCMA-TCRvb antibody molecules. Figure 17A shows in vitro killing by one of the following dual-targeting antibody molecules: BCMA-TCRVb (molecule I), BCMA-CD3, or control-TCRVb; or an isotype control. Figure 17B shows in vivo killing of MM cells by dual-targeting BCMA-TCRVb antibody (molecule I). [Figure 18]

[0164] Figure 1 shows lysis of MM target cells using a dual-targeting antibody (molecule E) that recognizes FcRH5 on one arm and TCRVb on the other arm. [Figure 19A]

[0165] Figures 19A-19B show cytokine production from human PBMCs activated with anti-TCR Vβ8a antibody (BH.1) compared to those activated with anti-CD3ε antibody (OKT3 or SP34-2). Figure 19A shows that human PBMCs activated with anti-TCR Vβ8a antibody (BH.1) produce similar or reduced levels of IFNγ. Figure 19B shows that human PBMCs activated with anti-TCR Vβ8a antibody (BH.1) produce higher levels of IL-2 compared to those activated with anti-CD3ε antibody (OKT3 or SP34-2). Data shown are representative of n=6 donors. [Figure 19B] Figure 19B shows that human PBMCs activated with an anti-TCR Vβ8a antibody (BH.1) produce higher levels of IL-2 when compared to those activated with an anti-CD3ε antibody (OKT3 or SP34-2). Data shown are representative of n=6 donors. [Figure 20A]

[0166] Figures 20A-20C show cytokine production from human PBMCs activated with anti-TCR Vβ8a antibody (BH.1). Human PBMCs activated with anti-TCR Vβ8a antibody (BH.1) produced significantly less IL-6 (Figure 20A), IL1β (Figure 20B), and less TNFα (Figure 20C) compared to PBMCs activated with anti-CD3ε antibodies (OKT3 or SP34-2). Data shown are representative of n=6 donors. [Figure 20B] Figures 20A-20C show cytokine production from human PBMCs activated with anti-TCR Vβ8a antibody (BH.1). Human PBMCs activated with anti-TCR Vβ8a antibody (BH.1) produced significantly less IL-6 (Figure 20A), IL1β (Figure 20B), and less TNFα (Figure 20C) compared to PBMCs activated with anti-CD3ε antibodies (OKT3 or SP34-2). Data shown are representative of n=6 donors. [Figure 20C]Figures 20A-20C show cytokine production from human PBMCs activated with anti-TCR Vβ8a antibody (BH.1). Human PBMCs activated with anti-TCR Vβ8a antibody (BH.1) produced significantly less IL-6 (Figure 20A), IL1β (Figure 20B), and less TNFα (Figure 20C) compared to PBMCs activated with anti-CD3ε antibodies (OKT3 or SP34-2). Data shown are representative of n=6 donors. [Figure 21A]

[0167] Figures 21A-21E show cytokine production from human PBMCs activated with anti-TCRβVD antibody compared to a control anti-CD3e antibody (OKT3). Figure 21A shows that human PBMCs activated with anti-TCRβVD antibody produce similar or reduced levels of IFNγ. Figure 21B shows that human PBMCs activated with anti-TCRβVD antibody produce higher levels of IL-2 compared to those activated with anti-CD3ε antibody (OKT3). Human PBMCs activated with anti-TCRβVD antibody do not significantly produce IL-1β (Figure 21C), IL-6 (Figure 21D), or TNF-alpha (Figure 21E). Data shown are representative of n=4 donors. [Figure 21B] Figure 21B shows that human PBMCs activated with anti-TCRβVD antibody produce higher levels of IL-2 compared to those activated with anti-CD3ε antibody (OKT3). Human PBMCs activated with anti-TCRβVD antibody do not significantly produce IL-1β (Figure 21C), IL-6 (Figure 21D), or TNF-alpha (Figure 21E). Data shown are representative of n=4 donors. [Figure 21C]Figure 21B shows that human PBMCs activated with anti-TCRβVD antibody produce higher levels of IL-2 compared to those activated with anti-CD3ε antibody (OKT3). Human PBMCs activated with anti-TCRβVD antibody do not significantly produce IL-1β (Figure 21C), IL-6 (Figure 21D), or TNF-alpha (Figure 21E). Data shown are representative of n=4 donors. [Figure 21D] Figure 21B shows that human PBMCs activated with anti-TCRβVD antibody produce higher levels of IL-2 compared to those activated with anti-CD3ε antibody (OKT3). Human PBMCs activated with anti-TCRβVD antibody do not significantly produce IL-1β (Figure 21C), IL-6 (Figure 21D), or TNF-alpha (Figure 21E). Data shown are representative of n=4 donors. [Figure 21E] Figure 21B shows that human PBMCs activated with anti-TCRβVD antibody produce higher levels of IL-2 compared to those activated with anti-CD3ε antibody (OKT3). Human PBMCs activated with anti-TCRβVD antibody do not significantly produce IL-1β (Figure 21C), IL-6 (Figure 21D), or TNF-alpha (Figure 21E). Data shown are representative of n=4 donors. [Figure 22A]

[0168] Figures 22A-22B show cytokine production from human PBMCs activated with an anti-TCR Vβ5 antibody (Antibody E). Figure 22A shows that human PBMCs activated with an anti-TCR Vβ5 antibody produce similar or reduced levels of IFNγ compared to PBMCs activated with an anti-CD3ε antibody (OKT3 or SP34-2). Figure 22B shows that human PBMCs activated with an anti-TCR Vβ5 1 antibody produce higher levels of IL-2 when compared to those activated with an anti-CD3ε antibody (OKT3 or SP34-2). Data shown are representative of n=4 donors. [Figure 22B]Figure 22B shows that human PBMCs activated with anti-TCR Vβ5 1 antibodies produce higher levels of IL-2 when compared to those activated with anti-CD3ε antibodies (OKT3 or SP34-2). Data shown are representative of n=4 donors. [Figure 23A]

[0169] Figures 23A-23D show cytokine production from human PBMCs activated with an anti-TCR Vβ5 antibody (Antibody E). Human PBMCs activated with anti-TCR Vβ5 antibodies produce significantly less IL-1β (Figure 23A), IL-6 (Figure 23B), TNF-alpha (Figure 23C), or IL-10 (Figure 23D) compared to PBMCs activated with anti-CD3ε antibodies (OKT3 or SP34-2). Data shown are representative of n=4 donors. [Figure 23B] Figures 23A-23D show cytokine production from human PBMCs activated with an anti-TCR Vβ5 antibody (Antibody E). Human PBMCs activated with anti-TCR Vβ5 antibodies produce significantly less IL-1β (Figure 23A), IL-6 (Figure 23B), TNF-alpha (Figure 23C), or IL-10 (Figure 23D) compared to PBMCs activated with anti-CD3ε antibodies (OKT3 or SP34-2). Data shown are representative of n=4 donors. [Figure 23C] Figures 23A-23D show cytokine production from human PBMCs activated with an anti-TCR Vβ5 antibody (Antibody E). Human PBMCs activated with anti-TCR Vβ5 antibodies produce significantly less IL-1β (Figure 23A), IL-6 (Figure 23B), TNF-alpha (Figure 23C), or IL-10 (Figure 23D) compared to PBMCs activated with anti-CD3ε antibodies (OKT3 or SP34-2). Data shown are representative of n=4 donors. [Figure 23D]Figures 23A-23D show cytokine production from human PBMCs activated with an anti-TCR Vβ5 antibody (Antibody E). Human PBMCs activated with anti-TCR Vβ5 antibodies produce significantly less IL-1β (Figure 23A), IL-6 (Figure 23B), TNF-alpha (Figure 23C), or IL-10 (Figure 23D) compared to PBMCs activated with anti-CD3ε antibodies (OKT3 or SP34-2). Data shown are representative of n=4 donors. [Figure 24A]

[0170] Figures 24A-24F show cytokine production from human PBMCs activated with dual-targeting (bispecific molecules) containing an anti-TCRβV-binding moiety and a BCMA-binding moiety. Figure 24A shows that human PBMCs activated with the bispecific molecules produce similar or reduced levels of IFNγ as PBMCs activated with an anti-CD3ε antibody (OKT3). Figure 24B shows that human PBMCs activated with the bispecific molecules produce higher levels of IL-2 compared to PBMCs activated with an anti-CD3ε antibody (OKT3). Human PBMCs activated with the bispecific molecules do not significantly produce IL-1 beta (Figure 24C), IL-6 (Figure 24D), TNF alpha (Figure 24E), or IL-10 (Figure 24F). Data shown are representative of n=3 donors. [Figure 24B] Figure 24B shows that human PBMCs activated with the bispecific molecule produce higher levels of IL-2 when compared to PBMCs activated with an anti-CD3ε antibody (OKT3). Human PBMCs activated with the bispecific molecule do not significantly produce IL-1 beta (Figure 24C), IL-6 (Figure 24D), TNF alpha (Figure 24E), or IL-10 (Figure 24F). Data shown are representative of n=3 donors. [Figure 24C] Human PBMCs activated with the bispecific molecules do not significantly produce IL-1 beta (Figure 24C), IL-6 (Figure 24D), TNF alpha (Figure 24E), or IL-10 (Figure 24F). Data shown are representative of n=3 donors. [Figure 24D] Human PBMCs activated with the bispecific molecules do not significantly produce IL-1 beta (Figure 24C), IL-6 (Figure 24D), TNF alpha (Figure 24E), or IL-10 (Figure 24F). Data shown are representative of n=3 donors. [Figure 24E] Human PBMCs activated with the bispecific molecules do not significantly produce IL-1 beta (Figure 24C), IL-6 (Figure 24D), TNF alpha (Figure 24E), or IL-10 (Figure 24F). Data shown are representative of n=3 donors. [Figure 24F] Human PBMCs activated with the bispecific molecules do not significantly produce IL-1 beta (Figure 24C), IL-6 (Figure 24D), TNF alpha (Figure 24E), or IL-10 (Figure 24F). Data shown are representative of n=3 donors. [Figure 25A]

[0171] Figures 25A-25B show the structures and sequences of eight TCRβV proteins from seven different subfamilies: the TCRβV6 subfamily (showing TCRβV6-5 and TCRβV6-4), the TCRβV28 subfamily, the TCRβV19 subfamily, the TCRβV9 subfamily, the TCRβV5 subfamily, the TCRβV20 subfamily, and the TCRβV12 subfamily. Figure 25A shows a structural alignment of the different TCRβV proteins. The circled regions represent the outward-facing regions containing the proposed binding sites for the anti-TCRβV antibodies described herein. Figure 25B shows an amino acid sequence alignment of the proteins shown in Figure 25A (SEQ ID NOS: 3449-3456, respectively, in order of appearance). The various TCRβV proteins (from the seven different TCRβV subfamilies) have diverse sequences but share conserved (similar) structure and function. [Figure 25B]Figure 25B shows an amino acid sequence alignment of the proteins shown in Figure 25A (SEQ ID NOS: 3449-3456, respectively, in order of appearance). The various TCRβV proteins (from seven different TCRβV subfamilies) have diverse sequences but share conserved (similar) structure and function. [Figure 26A]

[0172] Figures 26A-26J show cytokine or chemokine secretion in PBMCs activated with anti-TCRVb antibodies (molecule H), a control isotype (122), or bispecific anti-TCRVb antibodies (AH.1, BH.1) containing anti-CD3e antibodies (OKT3). Data shown are representative of n=2 donors and are representative of two independent experiments. [Figure 26B] Figures 26A-26J show cytokine or chemokine secretion in PBMCs activated with anti-TCRVb antibodies (molecule H), a control isotype (122), or bispecific anti-TCRVb antibodies (AH.1, BH.1) containing anti-CD3e antibodies (OKT3). Data shown are representative of n=2 donors and are representative of two independent experiments. [Figure 26C] Figures 26A-26J show cytokine or chemokine secretion in PBMCs activated with anti-TCRVb antibodies (molecule H), a control isotype (122), or bispecific anti-TCRVb antibodies (AH.1, BH.1) containing anti-CD3e antibodies (OKT3). Data shown are representative of n=2 donors and are representative of two independent experiments. [Figure 26D] Figures 26A-26J show cytokine or chemokine secretion in PBMCs activated with anti-TCRVb antibodies (molecule H), a control isotype (122), or bispecific anti-TCRVb antibodies (AH.1, BH.1) containing anti-CD3e antibodies (OKT3). Data shown are representative of n=2 donors and are representative of two independent experiments. [Figure 26E]Figures 26A-26J show cytokine or chemokine secretion in PBMCs activated with anti-TCRVb antibodies (molecule H), a control isotype (122), or bispecific anti-TCRVb antibodies (AH.1, BH.1) containing anti-CD3e antibodies (OKT3). Data shown are representative of n=2 donors and are representative of two independent experiments. [Figure 26F] Figures 26A-26J show cytokine or chemokine secretion in PBMCs activated with anti-TCRVb antibodies (molecule H), a control isotype (122), or bispecific anti-TCRVb antibodies (AH.1, BH.1) containing anti-CD3e antibodies (OKT3). Data shown are representative of n=2 donors and are representative of two independent experiments. [Figure 26G] Figures 26A-26J show cytokine or chemokine secretion in PBMCs activated with anti-TCRVb antibodies (molecule H), a control isotype (122), or bispecific anti-TCRVb antibodies (AH.1, BH.1) containing anti-CD3e antibodies (OKT3). Data shown are representative of n=2 donors and are representative of two independent experiments. [Figure 26H] Figures 26A-26J show cytokine or chemokine secretion in PBMCs activated with anti-TCRVb antibodies (molecule H), a control isotype (122), or bispecific anti-TCRVb antibodies (AH.1, BH.1) containing anti-CD3e antibodies (OKT3). Data shown are representative of n=2 donors and are representative of two independent experiments. [Figure 26I] Figures 26A-26J show cytokine or chemokine secretion in PBMCs activated with anti-TCRVb antibodies (molecule H), a control isotype (122), or bispecific anti-TCRVb antibodies (AH.1, BH.1) containing anti-CD3e antibodies (OKT3). Data shown are representative of n=2 donors and are representative of two independent experiments. [Figure 26J]Figures 26A-26J show cytokine or chemokine secretion in PBMCs activated with anti-TCRVb antibodies (molecule H), a control isotype (122), or bispecific anti-TCRVb antibodies (AH.1, BH.1) containing anti-CD3e antibodies (OKT3). Data shown are representative of n=2 donors and are representative of two independent experiments. [Figure 27A]

[0173] Figures 27A-27H show cytokine or chemokine secretion in PBMCs activated with anti-TCRVb antibodies (molecule H), a control isotype (122), or bispecific anti-TCRVb antibodies (AH.1, BH.1) containing an anti-CD3e antibody (OKT3). Data shown are representative of n=2 donors and are representative of two independent experiments. [Figure 27B] Figures 27A-27H show cytokine or chemokine secretion in PBMCs activated with anti-TCRVb antibodies (molecule H), a control isotype (122), or bispecific anti-TCRVb antibodies (AH.1, BH.1) containing an anti-CD3e antibody (OKT3). Data shown are representative of n=2 donors and are representative of two independent experiments. [Figure 27C] Figures 27A-27H show cytokine or chemokine secretion in PBMCs activated with anti-TCRVb antibodies (molecule H), a control isotype (122), or bispecific anti-TCRVb antibodies (AH.1, BH.1) containing an anti-CD3e antibody (OKT3). Data shown are representative of n=2 donors and are representative of two independent experiments. [Figure 27D] Figures 27A-27H show cytokine or chemokine secretion in PBMCs activated with anti-TCRVb antibodies (molecule H), a control isotype (122), or bispecific anti-TCRVb antibodies (AH.1, BH.1) containing an anti-CD3e antibody (OKT3). Data shown are representative of n=2 donors and are representative of two independent experiments. [Figure 27E]Figures 27A-27H show cytokine or chemokine secretion in PBMCs activated with anti-TCRVb antibodies (molecule H), a control isotype (122), or bispecific anti-TCRVb antibodies (AH.1, BH.1) containing an anti-CD3e antibody (OKT3). Data shown are representative of n=2 donors and are representative of two independent experiments. [Figure 27F] Figures 27A-27H show cytokine or chemokine secretion in PBMCs activated with anti-TCRVb antibodies (molecule H), a control isotype (122), or bispecific anti-TCRVb antibodies (AH.1, BH.1) containing an anti-CD3e antibody (OKT3). Data shown are representative of n=2 donors and are representative of two independent experiments. [Figure 27G] Figures 27A-27H show cytokine or chemokine secretion in PBMCs activated with anti-TCRVb antibodies (molecule H), a control isotype (122), or bispecific anti-TCRVb antibodies (AH.1, BH.1) containing an anti-CD3e antibody (OKT3). Data shown are representative of n=2 donors and are representative of two independent experiments. [Figure 27H] Figures 27A-27H show cytokine or chemokine secretion in PBMCs activated with anti-TCRVb antibodies (molecule H), a control isotype (122), or bispecific anti-TCRVb antibodies (AH.1, BH.1) containing an anti-CD3e antibody (OKT3). Data shown are representative of n=2 donors and are representative of two independent experiments. [Figure 28A]

[0174] Figures 28A-28L show cytokine or chemokine secretion in PBMCs activated with anti-TCRVb antibodies (molecule H), a control isotype (122), or bispecific anti-TCRVb antibodies (AH.1, BH.1) containing anti-CD3e antibodies (OKT3). Data shown are representative of n=2 donors and are representative of two independent experiments. [Figure 28B]Figures 28A-28L show cytokine or chemokine secretion in PBMCs activated with anti-TCRVb antibodies (molecule H), a control isotype (122), or bispecific anti-TCRVb antibodies (AH.1, BH.1) containing anti-CD3e antibodies (OKT3). Data shown are representative of n=2 donors and are representative of two independent experiments. [Figure 28C] Figures 28A-28L show cytokine or chemokine secretion in PBMCs activated with anti-TCRVb antibodies (molecule H), a control isotype (122), or bispecific anti-TCRVb antibodies (AH.1, BH.1) containing anti-CD3e antibodies (OKT3). Data shown are representative of n=2 donors and are representative of two independent experiments. [Figure 28D] Figures 28A-28L show cytokine or chemokine secretion in PBMCs activated with anti-TCRVb antibodies (molecule H), a control isotype (122), or bispecific anti-TCRVb antibodies (AH.1, BH.1) containing anti-CD3e antibodies (OKT3). Data shown are representative of n=2 donors and are representative of two independent experiments. [Figure 28E] Figures 28A-28L show cytokine or chemokine secretion in PBMCs activated with anti-TCRVb antibodies (molecule H), a control isotype (122), or bispecific anti-TCRVb antibodies (AH.1, BH.1) containing anti-CD3e antibodies (OKT3). Data shown are representative of n=2 donors and are representative of two independent experiments. [Figure 28F] Figures 28A-28L show cytokine or chemokine secretion in PBMCs activated with anti-TCRVb antibodies (molecule H), a control isotype (122), or bispecific anti-TCRVb antibodies (AH.1, BH.1) containing anti-CD3e antibodies (OKT3). Data shown are representative of n=2 donors and are representative of two independent experiments. [Figure 28G]Figures 28A-28L show cytokine or chemokine secretion in PBMCs activated with anti-TCRVb antibodies (molecule H), a control isotype (122), or bispecific anti-TCRVb antibodies (AH.1, BH.1) containing anti-CD3e antibodies (OKT3). Data shown are representative of n=2 donors and are representative of two independent experiments. [Figure 28H] Figures 28A-28L show cytokine or chemokine secretion in PBMCs activated with anti-TCRVb antibodies (molecule H), a control isotype (122), or bispecific anti-TCRVb antibodies (AH.1, BH.1) containing anti-CD3e antibodies (OKT3). Data shown are representative of n=2 donors and are representative of two independent experiments. [Figure 28I] Figures 28A-28L show cytokine or chemokine secretion in PBMCs activated with anti-TCRVb antibodies (molecule H), a control isotype (122), or bispecific anti-TCRVb antibodies (AH.1, BH.1) containing anti-CD3e antibodies (OKT3). Data shown are representative of n=2 donors and are representative of two independent experiments. [Figure 28J] Figures 28A-28L show cytokine or chemokine secretion in PBMCs activated with anti-TCRVb antibodies (molecule H), a control isotype (122), or bispecific anti-TCRVb antibodies (AH.1, BH.1) containing anti-CD3e antibodies (OKT3). Data shown are representative of n=2 donors and are representative of two independent experiments. [Figure 28K] Figures 28A-28L show cytokine or chemokine secretion in PBMCs activated with anti-TCRVb antibodies (molecule H), a control isotype (122), or bispecific anti-TCRVb antibodies (AH.1, BH.1) containing anti-CD3e antibodies (OKT3). Data shown are representative of n=2 donors and are representative of two independent experiments. [Figure 28L]Figures 28A-28L show cytokine or chemokine secretion in PBMCs activated with anti-TCRVb antibodies (molecule H), a control isotype (122), or bispecific anti-TCRVb antibodies (AH.1, BH.1) containing anti-CD3e antibodies (OKT3). Data shown are representative of n=2 donors and are representative of two independent experiments. [Figure 29]

[0175] Graph showing mean tumor volume in NOD / SCID / IL-2Rγ null (NSG) mice implanted with Raji-luc cells on days 10-28. Stars indicate PBMC implantation. Open triangles indicate antibody treatment with the indicated antibodies. [Figure 30A]

[0176] Figures 30A-30F are graphs showing cytokine secretion stimulated by anti-TRBC1 (antibody F) or anti-CD3 (OKT3) on days 2 and 5. Cytokines examined include IFNγ (Figure 30A), IL-2 (Figure 30B), IL-1β (Figure 30C), IL-6 (Figure 30D), IL-10 (Figure 30E), and TNFα (Figure 31F). [Figure 30B] Figures 30A-30F are graphs showing cytokine secretion stimulated by anti-TRBC1 (antibody F) or anti-CD3 (OKT3) on days 2 and 5. Cytokines examined include IFNγ (Figure 30A), IL-2 (Figure 30B), IL-1β (Figure 30C), IL-6 (Figure 30D), IL-10 (Figure 30E), and TNFα (Figure 31F). [Figure 30C] Figures 30A-30F are graphs showing cytokine secretion stimulated by anti-TRBC1 (antibody F) or anti-CD3 (OKT3) on days 2 and 5. Cytokines examined include IFNγ (Figure 30A), IL-2 (Figure 30B), IL-1β (Figure 30C), IL-6 (Figure 30D), IL-10 (Figure 30E), and TNFα (Figure 31F). [Figure 30D]Figures 30A-30F are graphs showing cytokine secretion stimulated by anti-TRBC1 (antibody F) or anti-CD3 (OKT3) on days 2 and 5. Cytokines examined include IFNγ (Figure 30A), IL-2 (Figure 30B), IL-1β (Figure 30C), IL-6 (Figure 30D), IL-10 (Figure 30E), and TNFα (Figure 31F). [Figure 30E] Figures 30A-30F are graphs showing cytokine secretion stimulated by anti-TRBC1 (antibody F) or anti-CD3 (OKT3) on days 2 and 5. Cytokines examined include IFNγ (Figure 30A), IL-2 (Figure 30B), IL-1β (Figure 30C), IL-6 (Figure 30D), IL-10 (Figure 30E), and TNFα (Figure 31F). [Figure 30F] Figures 30A-30F are graphs showing cytokine secretion stimulated by anti-TRBC1 (antibody F) or anti-CD3 (OKT3) on days 2 and 5. Cytokines examined include IFNγ (Figure 30A), IL-2 (Figure 30B), IL-1β (Figure 30C), IL-6 (Figure 30D), IL-10 (Figure 30E), and TNFα (Figure 31F). [Figure 31]

[0177] Figure 31 is a FACS plot showing the expansion of TCRvb 6-5+ T cells over 8 days using anti-TCRvb 6-5 v1. [Figure 32]

[0178] FIG. 32 is a bar graph showing the expansion of TCRvb 6-5+ CD4+ and TCRvb 6-5+ CD8+ T cells over 8 days using the anti-CD3ε antibody OKT3 (100 nM). [Figure 33]

[0179] Figure 36 is a bar graph showing the expansion of TCRvb 6-5+ CD4+ and TCRvb 6-5+ CD8+ T cells over 8 days using anti-TCRvb 6-5 v1 antibody (100 nM). [Figure 34]

[0180] FIG. 34 is a FACS plot showing the expansion of TCRvb 6-5+ T cells over 8 days using anti-TCRvb 6-5 v1 or anti-CD3ε antibody OKT3. [Figure 35A]

[0181] Figure 35A is a bar graph showing the percentage of TCRβV6-5+ T cells in PBMC cultures after 8 days of culture with the indicated antibodies. Data for 5 replicates are shown. [Figure 35B] Figure 35B is a bar graph showing the percentage of TCRβV6-5+ T cells in purified T cell cultures after 8 days of culture with the indicated antibodies. Data for 5 replicates are shown. [Figure 36A]

[0182] FIG. 36A is a bar graph showing the relative numbers of TCRβV 6-5+ T cells in PBMC cultures after 8 days of culture with the indicated antibodies. [Figure 36B] FIG. 36B is a bar graph showing the relative numbers of TCRβV 6-5+ T cells in PBMC cultures after 8 days of culture with the indicated antibodies. [Figure 37A]

[0183] FIG. 37A is a bar graph showing the relative numbers of TCRβV 6-5+ T cells in purified T cell cultures after 8 days of culture with the indicated antibodies. [Figure 37B] FIG. 37B is a bar graph showing the relative numbers of TCRβV 6-5+ T cells in purified T cell cultures after 8 days of culture with the indicated antibodies. [Figure 38]

[0184] FIG. 38 is a line graph showing total CD3+ T cell numbers (fold increase) after 8 days of T cell culture with either anti-CD3ε antibody OKT3 or anti-TCRvb 6-5 v1 antibody. [Figure 39]

[0185] Figure 39 is a series of line graphs showing target cell kinetics by TCRβV6-5 v1-activated T cells or anti-CD3ε (OKT3)-activated T cells. T cells from three different donors were utilized (donor 6769, donor 9880, donor 5411). [Figure 40A]

[0186] Figure 40A is a scatter plot showing the percent of T cell-mediated target cell lysis by TCRβV6-5 v1-activated T cells or anti-CD3ε (OKT3)-activated T cells without T cell preactivation. Data are presented on day 6 of co-culture between target and effector T cells. [Figure 40B] Figure 40B is a scatter plot showing the percent of T cell-mediated target cell lysis by TCRβV6-5 v1-activated T cells or anti-CD3ε (OKT3)-activated T cells with 4 days of T cell preactivation. Data are presented on day 2 of co-culture between target and effector T cells (day 4 after T cell preactivation). [Figure 41]

[0187] Figure 41 is a scatter plot showing the percent of T cell-mediated target cell lysis by TCRβV6-5 v1-activated T cells or anti-CD3ε (OKT3)-activated T cells with 4 days of T cell preactivation. Data are presented on day 2 of co-culture between target and effector T cells (day 4 after T cell preactivation). [Figure 42]

[0188] Figure 42 is a bar graph showing T cell-mediated target cell lysis by TCRβV6-5 v1-activated T cells or anti-CD3ε (OKT3)-activated T cells (100 nM of each antibody). Data include seven replicates for each experimental condition. [Figure 43]

[0189] Figure 43 is a series of FACS plots showing cell surface expression of CD3ε on CD4+ TCRβV6-5− or CD4+ TCRβV6-5+ T cells activated with either SP34-2 (anti-CD3ε antibody) or anti-TCRβV6-5 v1 (anti-TCRβV6-5 antibody) on days 0, 1, 2, 4, 6, or 8 after antibody activation. [Figure 44]

[0190] Figure 44 is a series of FACS plots showing cell surface expression of CD3ε on CD8+ TCRβV6-5− or CD8+ TCRβV6-5+ T cells activated with either SP34-2 (anti-CD3ε antibody) or anti-TCRβV6-5 v1 (anti-TCRβV6-5 antibody) on days 0, 1, 2, 4, 6, or 8 after antibody activation. [Figure 45]

[0191] Figure 45 is a series of FACS plots showing cell surface expression of TCRβV on CD4+ TCRβV 6-5- or CD4+ TCRβV 6-5+ T cells activated with either SP34-2 (anti-CD3ε antibody) or anti-TCRβV 6-5 v1 (anti-TCRβV 6-5 antibody) on days 0, 1, 2, 4, 6, or 8 after antibody activation. [Figure 46]

[0192] Figure 46 is a series of FACS plots showing cell surface expression of TCRβV on CD8+ TCRβV 6-5- or CD8+ TCRβV 6-5+ T cells activated with either SP34-2 (anti-CD3ε antibody) or anti-TCRβV 6-5 v1 (anti-TCRβV 6-5 antibody) on days 0, 1, 2, 4, 6, or 8 after antibody activation. [Figure 47A]

[0193] Figure 47A shows FACS plots of TCRβV 6-5+ cynomolgus T cell expansion 7 days after activation of cynomolgus PBMCs, either unstimulated (left) or stimulated with anti-TCRβV 6-5 v1 (right). PBMCs from donor DW8N (fresh PBMC sample, male, 8 years old, weighing 7.9 kg) were used. [Figure 47B] Figure 47B shows FACS plots of TCRβV 6-5+ cynomolgus T cell expansion after 7 days of activation of cynomolgus PBMCs, either unstimulated (left) or stimulated with anti-TCRβV 6-5 v1 (right). PBMCs from donor G709 (cryopreserved sample, male, 6 years old, weighing 4.7 kg) were used. [Figure 48]

[0194] Figure 48 shows FACS plots and corresponding microscopy images of TCRβV 6-5+ cynomolgus T cell expansion following activation of cryopreserved donor DW8N cynomolgus monkey PBMCs: unstimulated (left), stimulated with SP34-2 (anti-CD3ε antibody) (center), or stimulated with anti-TCRβV 6-5 v1 (right). The microscopy images show cell cluster formation (indicated by circles). [Figure 49]

[0195] FIG. 49 shows a diagram of a FACS plot showing FACS gating / staining of PBMCs prior to γδ T cell purification. [Figure 50]

[0196] FIG. 50 shows a diagram of a FACS plot showing FACS gating / staining of purified γδ T cell populations. [Figure 51]

[0197] FIG. 51 shows activation of purified γδ T cell populations with anti-CD3ε antibody (SP34-2) (left) or anti-TCRβV antibody (anti-TCRβV 6-5 v1) (right). [Figure 52A]

[0198] FIG. 55A shows IFNγ release from purified γδ T cell populations activated with anti-CD3ε antibody (SP34-2), anti-TCRβV antibody (anti-TCRβV 6-5 v1), or unstimulated. [Figure 52B] FIG. 52B shows the release of TNFα from purified γδ T cell populations activated with anti-CD3ε antibody (SP34-2), anti-TCRβV antibody (anti-TCRβV 6-5 v1), or unstimulated. [Figure 52C] FIG. 52C shows IL-2 release from purified γδ T cell populations activated with anti-CD3ε antibody (SP34-2), anti-TCRβV antibody (anti-TCRβV 6-5 v1), or unstimulated. [Figure 52D] FIG. 52D shows the release of IL-17A from purified γδ T cell populations activated with anti-CD3ε antibody (SP34-2), anti-TCRβV antibody (anti-TCRβV 6-5 v1), or unstimulated. [Figure 52E]FIG. 52E shows IL-1α release from purified γδ T cell populations activated with anti-CD3ε antibody (SP34-2), anti-TCRβV antibody (anti-TCRβV 6-5 v1), or unstimulated. [Figure 52F] FIG. 52F shows the release of IL-1β from purified γδ T cell populations activated with anti-CD3ε antibody (SP34-2), anti-TCRβV antibody (anti-TCRβV 6-5 v1), or unstimulated. [Figure 52G] FIG. 52G shows IL-6 release from purified γδ T cell populations activated with anti-CD3ε antibody (SP34-2), anti-TCRβV antibody (anti-TCRβV 6-5 v1), or unstimulated. [Figure 52H] FIG. 52H shows IL-10 release from purified γδ T cell populations activated with anti-CD3ε antibody (SP34-2), anti-TCRβV antibody (anti-TCRβV 6-5 v1), or unstimulated. [Figure 53]

[0199] Figure 53 shows the relative representation of all TCR alpha V-segments (TRAV gene cluster) and their variants (top), all TCR beta V-segment 6-5 variants (TRBV6-5 gene) (bottom left), and all TCR beta V-segments and variants excluding 6-5 (bottom right). [Figure 54A]

[0200] Figure 54A is a FACS plot showing phenotypic markers of CD4+ T cells expanded with an anti-TCRβV antibody (anti-TCRβV 6-5 v1). Defined phenotypes include TEMRA (top left), naive / TSCM (top right), TEM (bottom left), and TCM (bottom right). [Figure 54B] Figure 54B is a FACS plot showing phenotypic markers of CD4+ T cells expanded with anti-CD3ε antibody (OKT3). Defined phenotypes include TEMRA (top left), naive / TSCM (top right), TEM (bottom left), and TCM (bottom right). [Figure 55A]

[0201] Figure 55A is a FACS plot showing phenotypic markers of CD8+ T cells expanded with an anti-TCRβV antibody (anti-TCRβV 6-5 v1). Defined phenotypes include TEMRA (top left), naive / TSCM (top right), TEM (bottom left), and TCM (bottom right). [Figure 55B] Figure 55B is a FACS plot showing phenotypic markers of CD8+ T cells expanded with anti-CD3ε antibody (OKT3). Defined phenotypes include TEMRA (top left), naive / TSCM (top right), TEM (bottom left), and TCM (bottom right). [Figure 56A]

[0202] Figure 56A is a bar graph showing the percentage of PD1-expressing CD4+ T cells from T cell cultures activated with anti-TCRβV antibody (anti-TCRβV 6-5 v1), anti-CD3ε antibody (OKT3), or unstimulated. [Figure 56B] Figure 56B is a bar graph showing the percentage of PD1-expressing CD8+ T cells from T cell cultures activated with anti-TCRβV antibody (anti-TCRβV 6-5 v1), anti-CD3ε antibody (OKT3), or unstimulated. [Figure 57A]

[0203] Figure 57A is a bar graph showing Ki-67 expression by CD4+ T cells from T cell cultures activated with anti-TCRβV antibody (anti-TCRβV 6-5 v1), anti-CD3ε antibody (OKT3), or unstimulated. [Figure 57B] Figure 57B is a bar graph showing Ki-67 expression by CD8+ T cells from T cell cultures activated with anti-TCRβV antibody (anti-TCRβV 6-5 v1), anti-CD3ε antibody (OKT3), or unstimulated. [Figure 58A]

[0204] Figure 58A is a FACS plot showing the percentage of TEMRA-like CD8+ T cells activated using an anti-TCRβV antibody (anti-TCRβV 6-5 v1) that express CD57 (18.7%). [Figure 58B]Figure 58B is a FACS plot showing the percentage of TEM-like CD8+ T cells activated using anti-CD3ε antibody (OKT3) that express CD57 (46.8%) and the percentage of TCM-like CD8+ T cells activated using anti-CD3ε antibody (OKT3) that express CD57 (18.9%). [Figure 59]

[0205] Figure 59 shows a series of FACS plots showing CD27 expression by CD4+ (top) or CD8+ (bottom) T cells from T cell cultures activated with an anti-TCRβV antibody (anti-TCRβV 6-5 v1), an anti-CD3ε antibody (OKT3), or unstimulated. [Figure 60]

[0206] Figure 60 shows a series of FACS plots showing the expression of OX40, 41BB, and ICOS by CD4+ (top) or CD8+ (bottom) T cells from T cell cultures activated with an anti-TCRβV antibody (anti-TCRβV 6-5 v1), an anti-CD3ε antibody (OKT3), or unstimulated. [Figure 61]

[0207] Figure 61 shows a series of FACS plots showing the percentage of CD3+ (CD4 gated) TCRβV6-5+ T cells 1, 2, 3, 4, 5, 6, and 8 days after activation with BCMA and an anti-TCR Vβ antibody, anti-TCR Vβ 6-5 v1. [Figure 62A]

[0208] Figure 62A shows a series of FACS plots showing the percentage of CD4+ T cells expanded using isotype control (IgG1 N297A), anti-TCRβV (anti-TCR Vβ 6-5 v1), or anti-CD3ε (OKT3) antibodies at day 0 post activation. [Figure 62B] Figure 62B shows a series of FACS plots showing the percentage of CD4+ T cells expanded using isotype control (IgG1 N297A), anti-TCRβV (anti-TCR Vβ 6-5 v1), or anti-CD3ε (OKT3) antibodies at day 1 post-activation. [Figure 62C]Figure 62C shows a series of FACS plots showing the percentage of CD4+ T cells expanded using isotype control (IgG1 N297A), anti-TCRβV (anti-TCR Vβ 6-5 v1), or anti-CD3ε (OKT3) antibodies at day 2 post-activation. [Figure 62D] Figure 62D shows a series of FACS plots showing the percentage of CD4+ T cells expanded using isotype control (IgG1 N297A), anti-TCRβV (anti-TCR Vβ 6-5 v1), or anti-CD3ε (OKT3) antibodies at day 3 post-activation. [Figure 62E] Figure 62E shows a series of FACS plots showing the percentage of CD4+ T cells expanded using isotype control (IgG1 N297A), anti-TCRβV (anti-TCR Vβ 6-5 v1), or anti-CD3ε (OKT3) antibodies at day 4 post-activation. [Figure 62F] Figure 62F shows a series of FACS plots showing the percentage of CD4+ T cells expanded using isotype control (IgG1 N297A), anti-TCRβV (anti-TCR Vβ 6-5 v1), or anti-CD3ε (OKT3) antibodies at day 5 post-activation. [Figure 62G] Figure 62G shows a series of FACS plots showing the percentage of CD4+ T cells expanded using isotype control (IgG1 N297A), anti-TCRβV (anti-TCR Vβ 6-5 v1), or anti-CD3ε (OKT3) antibodies at day 6 post-activation. [Figure 62H] Figure 62H shows a series of FACS plots showing the percentage of CD4+ T cells expanded using isotype control (IgG1 N297A), anti-TCRβV (anti-TCR Vβ 6-5 v1), or anti-CD3ε (OKT3) antibodies at day 8 post-activation. [Figure 63A]

[0209] Figure 63A is a bar graph showing ATP production from glycolysis of T cell cultures activated with the indicated antibodies. [Figure 63B]Figure 63B is a bar graph showing ATP production from oxidative phosphorylation of T cell cultures activated with the indicated antibodies. [Figure 64]

[0210] Figure 64 is a line graph showing the oxygen consumption rate (OCR) of T cells activated with the indicated antibodies from about 0 to 75 minutes. [Figure 65A]

[0211] Figure 65A shows the oxygen consumption rate (OCR) of T cells activated with the indicated antibodies during basal respiration. [Figure 65B] Figure 65B shows the oxygen consumption rate (OCR) of T cells activated with the indicated antibodies during maximal respiration. [Figure 65C] Figure 65C shows the oxygen consumption rate (OCR) of T cells activated with the indicated antibodies during spare respiratory capacity. [Figure 65D] FIG. 65D is a line graph showing the areas of basal and maximum respiration shown in FIGS. 64A and 64B, respectively. [Figure 66A]

[0212] Figure 66A is a bar graph showing ATP production from glycolysis of T cell cultures activated with anti-TCRβV6-5 v1 and restimulated with the indicated antibodies. [Figure 66B] Figure 66B is a bar graph showing ATP production from oxidative phosphorylation of T cell cultures activated with anti-TCRβV6-5 v1 and restimulated with the indicated antibodies. [Figure 67A]

[0213] Figures 67A-67G are graphs showing the expression of IFNγ (Figure 67A), TNFα (Figure 67E), IL-1α (Figure 67B), IL-1β (Figure 67C), IL-6 (CRS- and neurotoxicity-associated cytokine) (Figure 67D), IL-10 (Figure 67F), and IL-17A (Figure 67G) using BHM1710 (anti-TCRVB), a reduced-affinity anti-CD3 antibody (TB), and SP34 anti-CD3e antibody. [Figure 67B]Figures 67A-67G are graphs showing the expression of IFNγ (Figure 67A), TNFα (Figure 67E), IL-1α (Figure 67B), IL-1β (Figure 67C), IL-6 (CRS- and neurotoxicity-associated cytokine) (Figure 67D), IL-10 (Figure 67F), and IL-17A (Figure 67G) using BHM1710 (anti-TCRVB), a reduced-affinity anti-CD3 antibody (TB), and SP34 anti-CD3e antibody. [Figure 67C] Figures 67A-67G are graphs showing the expression of IFNγ (Figure 67A), TNFα (Figure 67E), IL-1α (Figure 67B), IL-1β (Figure 67C), IL-6 (CRS- and neurotoxicity-associated cytokine) (Figure 67D), IL-10 (Figure 67F), and IL-17A (Figure 67G) using BHM1710 (anti-TCRVB), a reduced-affinity anti-CD3 antibody (TB), and SP34 anti-CD3e antibody. [Figure 67D] Figures 67A-67G are graphs showing the expression of IFNγ (Figure 67A), TNFα (Figure 67E), IL-1α (Figure 67B), IL-1β (Figure 67C), IL-6 (CRS- and neurotoxicity-associated cytokine) (Figure 67D), IL-10 (Figure 67F), and IL-17A (Figure 67G) using BHM1710 (anti-TCRVB), a reduced-affinity anti-CD3 antibody (TB), and SP34 anti-CD3e antibody. [Figure 67E] Figures 67A-67G are graphs showing the expression of IFNγ (Figure 67A), TNFα (Figure 67E), IL-1α (Figure 67B), IL-1β (Figure 67C), IL-6 (CRS- and neurotoxicity-associated cytokine) (Figure 67D), IL-10 (Figure 67F), and IL-17A (Figure 67G) using BHM1710 (anti-TCRVB), a reduced-affinity anti-CD3 antibody (TB), and SP34 anti-CD3e antibody. [Figure 67F] Figures 67A-67G are graphs showing the expression of IFNγ (Figure 67A), TNFα (Figure 67E), IL-1α (Figure 67B), IL-1β (Figure 67C), IL-6 (CRS- and neurotoxicity-associated cytokine) (Figure 67D), IL-10 (Figure 67F), and IL-17A (Figure 67G) using BHM1710 (anti-TCRVB), a reduced-affinity anti-CD3 antibody (TB), and SP34 anti-CD3e antibody. [Figure 67G]Figures 67A-67G are graphs showing the expression of IFNγ (Figure 67A), TNFα (Figure 67E), IL-1α (Figure 67B), IL-1β (Figure 67C), IL-6 (CRS- and neurotoxicity-associated cytokine) (Figure 67D), IL-10 (Figure 67F), and IL-17A (Figure 67G) using BHM1710 (anti-TCRVB), a reduced-affinity anti-CD3 antibody (TB), and SP34 anti-CD3e antibody. [Figure 68]

[0214] Figure 68 is a FACS plot showing the percentage of NK cells expanded from T cell cultures activated with the indicated antibodies. [Figure 69]

[0215] Figure 69 is a bar graph showing the number of NK cells expanded from T cell cultures activated with the indicated antibodies. [Figure 70]

[0216] Figure 70 shows a series of FACS plots showing NK cell proliferation induced by T cell cultures activated with the indicated antibodies. [Figure 71]

[0217] FIG. 71 is a diagram showing the assay described in the Examples for determining NK cell-mediated lysis of target K562 cells. [Figure 72]

[0218] FIG. 72 is a bar graph showing the percent target cell lysis mediated by NK cells activated by PBMCs activated with the indicated antibodies. [Figure 73]

[0219] Figure 73 shows a series of FACS plots showing the proliferation of NK cells from PBMC cultures activated / expanded with the indicated antibodies (isotype control or OKT3). PBMCs from three donors (D1, D2, and D3) were analyzed. [Figure 74]

[0220] Figure 74 shows a series of FACS plots showing proliferation of NK cells from PBMC cultures activated / expanded with the indicated antibodies (anti-TCRvβ 12-3 / 4 v1 or anti-TCRvβ 12-3 / 4 v2). PBMCs from three donors (D1, D2, and D3) were analyzed. [Figure 75]

[0221] Figure 75 shows a series of FACS plots showing the proliferation of NK cells from PBMC cultures activated / expanded with the indicated antibodies (anti-TCRvβ 12-3 / 4 v3 or SP34-2). PBMCs from three donors (D1, D2, and D3) were analyzed. [Figure 76]

[0222] Figure 76 is a bar graph showing the levels of IFNγ secretion by T cells activated / expanded with the indicated antibody (anti-TCRβV 6-5 v1, OKT3, or SP34) and cultured with the antibody for the indicated number of days (1, 3, or 5). [Figure 77]

[0223] Figure 77 is a bar graph showing the levels of IL-2 secretion by T cells activated / expanded with the indicated antibody (anti-TCRβV 6-5 v1, OKT3, or SP34) and cultured with the antibody for the indicated number of days (1, 3, or 5). [Figure 78]

[0224] Figure 78 is a bar graph showing the level of IL-15 secretion by T cells activated / expanded with the indicated antibody (anti-TCRβV 6-5 v1, OKT3, or SP34) and cultured with the antibody for the indicated number of days (1, 3, or 5). [Figure 79]

[0225] Figure 79 is a bar graph showing the levels of IL-1β secretion by T cells activated / expanded with the indicated antibody (anti-TCRβV 6-5 v1, OKT3, or SP34) and cultured with the antibody for the indicated number of days (1, 3, or 5). [Figure 80]

[0226] Figure 80 is a bar graph showing the levels of IL-6 secretion by T cells activated / expanded with the indicated antibody (anti-TCRβV 6-5 v1, OKT3, or SP34) and cultured with the antibody for the indicated number of days (1, 3, or 5). [Figure 81]

[0227] Figure 81 is a bar graph showing the level of IL-10 secretion by T cells activated / expanded with the indicated antibody (anti-TCRβV 6-5 v1, OKT3, or SP34) and cultured with the antibody for the indicated number of days (1, 3, or 5). [Figure 82]

[0228] Figure 82 is a bar graph showing the levels of the indicated cytokines secreted by T cells activated / expanded with the indicated antibodies (anti-TCRβV 6-5 v1 or SP34). Data include the use of 17 individual PBMC donors. [Figure 83A]

[0229] Figure 83A is a bar graph showing the levels of IFNγ secretion by T cells activated / expanded with the indicated antibody (anti-TCRβV 6-5 v1 or OKT3) and cultured with the antibody for the indicated number of days (1, 2, 3, 5, or 6). [Figure 83B] Figure 83B is a bar graph showing the levels of IL-1β secretion by T cells activated / expanded with the indicated antibody (anti-TCRβV 6-5 v1 or OKT3) and cultured with the antibody for the indicated number of days (1, 2, 3, 5, or 6). [Figure 83C] Figure 83C is a bar graph showing the levels of IL-4 secretion by T cells activated / expanded with the indicated antibody (anti-TCRβV 6-5 v1 or OKT3) and cultured with the antibody for the indicated number of days (1, 2, 3, 5, or 6). [Figure 83D] Figure 83D is a bar graph showing the levels of IL-6 secretion by T cells activated / expanded with the indicated antibody (anti-TCRβV 6-5 v1 or OKT3) and cultured with the antibody for the indicated number of days (1, 2, 3, 5, or 6). [Figure 83E] Figure 83E is a bar graph showing the level of IL-10 secretion by T cells activated / expanded with the indicated antibody (anti-TCRβV 6-5 v1 or OKT3) and cultured with the antibody for the indicated number of days (1, 2, 3, 5, or 6). [Figure 83F]Figure 83F is a bar graph showing the levels of TNFα secretion by T cells activated / expanded with the indicated antibody (anti-TCRβV 6-5 v1 or OKT3) and cultured with the antibody for the indicated number of days (1, 2, 3, 5, or 6). [Figure 83G] Figure 83G is a bar graph showing the levels of IL-2 secretion by T cells activated / expanded with the indicated antibody (anti-TCRβV 6-5 v1 or OKT3) and cultured with the antibody for the indicated number of days (1, 2, 3, 5, or 6). [Figure 84A]

[0230] Figure 84A is a bar graph showing the levels of IFNγ secretion by T cells activated / expanded with the indicated antibodies (anti-TCRβV 6-5 v1, anti-TCRβV 6-5 v1, OKT3, SP34-2, or isotype control) and cultured with the antibodies for the indicated number of days (1, 2, 3, 5, or 6). [Figure 84B] Figure 84B is a bar graph showing the levels of IL-1β secretion by T cells activated / expanded with the indicated antibodies (anti-TCRβV 6-5 v1, anti-TCRβV 6-5 v1, OKT3, SP34-2, or isotype control) and cultured with the antibodies for the indicated number of days (1, 2, 3, 5, or 6). [Figure 84C] Figure 84C is a bar graph showing the levels of IL-4 secretion by T cells activated / expanded with the indicated antibodies (anti-TCRβV 6-5 v1, anti-TCRβV 6-5 v1, OKT3, SP34-2, or isotype control) and cultured with the antibodies for the indicated number of days (1, 2, 3, 5, or 6). [Figure 84D] Figure 84D is a bar graph showing the levels of IL-6 secretion by T cells activated / expanded with the indicated antibodies (anti-TCRβV 6-5 v1, anti-TCRβV 6-5 v1, OKT3, SP34-2, or isotype control) and cultured with the antibodies for the indicated number of days (1, 2, 3, 5, or 6). [Figure 84E]Figure 84E is a bar graph showing the level of IL-10 secretion by T cells activated / expanded with the indicated antibodies (anti-TCRβV 6-5 v1, anti-TCRβV 6-5 v1, OKT3, SP34-2, or isotype control) and cultured with the antibodies for the indicated number of days (1, 2, 3, 5, or 6). [Figure 84F] Figure 84F is a bar graph showing the levels of TNFα secretion by T cells activated / expanded with the indicated antibodies (anti-TCRβV 6-5 v1, anti-TCRβV 6-5 v1, OKT3, SP34-2, or isotype control) and cultured with the antibodies for the indicated number of days (1, 2, 3, 5, or 6). [Figure 84G] Figure 84G is a bar graph showing the levels of IL-2 secretion by T cells activated / expanded with the indicated antibodies (anti-TCRβV 6-5 v1, anti-TCRβV 6-5 v1, OKT3, SP34-2, or isotype control) and cultured with the antibodies for the indicated number of days (1, 2, 3, 5, or 6). [Figure 85A]

[0231] Figure 85A is a bar graph showing the levels of IFNγ secretion by T cells activated / expanded with the indicated antibody (anti-TCRβV 6-5 v1, anti-TCRβV 6-5 v1, OKT3, or SP34-2) and cultured with the antibody for the indicated number of days (1, 2, 3, 4, 5, 6, or 8). [Figure 85B] Figure 85B is a bar graph showing the level of IL-1β secretion by T cells activated / expanded with the indicated antibody (anti-TCRβV 6-5 v1, anti-TCRβV 6-5 v1, OKT3, or SP34-2) and cultured with the antibody for the indicated number of days (1, 2, 3, 4, 5, 6, or 8). [Figure 85C] Figure 85C is a bar graph showing the levels of IL-4 secretion by T cells activated / expanded with the indicated antibody (anti-TCRβV 6-5 v1, anti-TCRβV 6-5 v1, OKT3, or SP34-2) and cultured with the antibody for the indicated number of days (1, 2, 3, 4, 5, 6, or 8). [Figure 85D] Figure 85D is a bar graph showing the levels of IL-6 secretion by T cells activated / expanded with the indicated antibody (anti-TCRβV 6-5 v1, anti-TCRβV 6-5 v1, OKT3, or SP34-2) and cultured with the antibody for the indicated number of days (1, 2, 3, 4, 5, 6, or 8). [Figure 85E] Figure 85E is a bar graph showing the level of IL-10 secretion by T cells activated / expanded with the indicated antibody (anti-TCRβV 6-5 v1, anti-TCRβV 6-5 v1, OKT3, or SP34-2) and cultured with the antibody for the indicated number of days (1, 2, 3, 4, 5, 6, or 8). [Figure 85F] Figure 85F is a bar graph showing the levels of TNFα secretion by T cells activated / expanded with the indicated antibody (anti-TCRβV 6-5 v1, anti-TCRβV 6-5 v1, OKT3, or SP34-2) and cultured with the antibody for the indicated number of days (1, 2, 3, 4, 5, 6, or 8). [Figure 85G] Figure 85G is a bar graph showing the levels of IL-2 secretion by T cells activated / expanded with the indicated antibody (anti-TCRβV 6-5 v1, anti-TCRβV 6-5 v1, OKT3, or SP34-2) and cultured with the antibody for the indicated number of days (1, 2, 3, 4, 5, 6, or 8). [Figure 86A]

[0232] Figure 86A is a bar graph showing the levels of IL-17A secretion by T cells activated / expanded with the indicated antibody (anti-TCRβV 6-5 v1, OKT3, or SP34-2) and cultured with the antibody for the indicated number of days (2, 5, or 7). [Figure 86B] Figure 86B is a bar graph showing the levels of IL-17A secretion by T cells activated / expanded with the indicated antibodies (anti-TCRβV 6-5 v1, OKT3, or SP34-2) and cultured with the antibodies for the indicated number of days (2, 5, or 8). [Figure 86C]Figure 86C is a bar graph showing the levels of IL-17A secretion by T cells activated / expanded with the indicated antibody (anti-TCRβV 6-5 v1, OKT3, or SP34-2) and cultured with the antibody for the indicated number of days (2, 5, or 7). [Figure 86D] Figure 86D is a bar graph showing the levels of IL-17A secretion by T cells activated / expanded with the indicated antibody (anti-TCRβV 6-5 v1 or SP34-2) and cultured with the antibody for the indicated number of days (1, 3, 5, or 7). [Figure 87A]

[0233] Figure 87A is a bar graph showing the levels of IFNγ secretion by T cells activated / expanded with the indicated antibodies (isotype control; anti-TCRβV 6-5 v1 with anti-BCMA antibody; anti-TCRβV 6-5 v1; anti-TCRβV 123 / 4 v1, or SP34-2) and cultured with the antibodies for the indicated number of days (1, 2, 3, 4, 5, 6, or 8). [Figure 87B] Figure 87B is a bar graph showing the levels of IL-1β secretion by T cells activated / expanded with the indicated antibodies (isotype control; anti-TCRβV 6-5 v1 with anti-BCMA antibody; anti-TCRβV 6-5 v1; anti-TCRβV 123 / 4 v1, or SP34-2) and cultured with the antibodies for the indicated number of days (1, 2, 3, 4, 5, 6, or 8). [Figure 87C] Figure 87C is a bar graph showing the levels of IL-4 secreted by T cells activated / expanded with the indicated antibodies (isotype control; anti-TCRβV 6-5 v1 with anti-BCMA antibody; anti-TCRβV 6-5 v1; anti-TCRβV 123 / 4 v1, or SP34-2) and cultured with the antibodies for the indicated number of days (1, 2, 3, 4, 5, 6, or 8). [Figure 87D]Figure 87D is a bar graph showing the levels of IL-6 secreted by T cells activated / expanded with the indicated antibodies (isotype control; anti-TCRβV 6-5 v1 with anti-BCMA antibody; anti-TCRβV 6-5 v1; anti-TCRβV 123 / 4 v1, or SP34-2) and cultured with the antibodies for the indicated number of days (1, 2, 3, 4, 5, 6, or 8). [Figure 87E] Figure 87E is a bar graph showing the level of IL-10 secretion by T cells activated / expanded with the indicated antibodies (isotype control; anti-TCRβV 6-5 v1 with anti-BCMA antibody; anti-TCRβV 6-5 v1; anti-TCRβV 123 / 4 v1, or SP34-2) and cultured with the antibodies for the indicated number of days (1, 2, 3, 4, 5, 6, or 8). [Figure 87F] Figure 87F is a bar graph showing the levels of TNFα secretion by T cells activated / expanded with the indicated antibodies (isotype control; anti-TCRβV 6-5 v1 with anti-BCMA antibody; anti-TCRβV 6-5 v1; anti-TCRβV 123 / 4 v1, or SP34-2) and cultured with the antibodies for the indicated number of days (1, 2, 3, 4, 5, 6, or 8). [Figure 87G] Figure 87G is a bar graph showing the levels of IL-2 secreted by T cells activated / expanded with the indicated antibodies (isotype control; anti-TCRβV 6-5 v1 with anti-BCMA antibody; anti-TCRβV 6-5 v1; anti-TCRβV 123 / 4 v1, or SP34-2) and cultured with the antibodies for the indicated number of days (1, 2, 3, 4, 5, 6, or 8). [Figure 87H] Figure 87H is a bar graph showing the levels of IL-12p70 secretion by T cells activated / expanded with the indicated antibodies (isotype control; anti-TCRβV 6-5 v1 with anti-BCMA antibody; anti-TCRβV 6-5 v1; anti-TCRβV 123 / 4 v1, or SP34-2) and cultured with the antibodies for the indicated number of days (1, 2, 3, 4, 5, 6, or 8). [Figure 87I]Figure 87I is a bar graph showing the level of IL-13 secretion by T cells activated / expanded with the indicated antibodies (isotype control; anti-TCRβV 6-5 v1 with anti-BCMA antibody; anti-TCRβV 6-5 v1; anti-TCRβV 123 / 4 v1, or SP34-2) and cultured with the antibodies for the indicated number of days (1, 2, 3, 4, 5, 6, or 8). [Figure 87J] Figure 87J is a bar graph showing the levels of IL-8 secretion by T cells activated / expanded with the indicated antibodies (isotype control; anti-TCRβV 6-5 v1 with anti-BCMA antibody; anti-TCRβV 6-5 v1; anti-TCRβV 123 / 4 v1, or SP34-2) and cultured with the antibodies for the indicated number of days (1, 2, 3, 4, 5, 6, or 8). [Figure 87K] Figure 87K is a bar graph showing the level of exotaxin secretion by T cells activated / expanded with the indicated antibodies (isotype control; anti-TCRβV 6-5 v1 with anti-BCMA antibody; anti-TCRβV 6-5 v1; anti-TCRβV 123 / 4 v1, or SP34-2) and cultured with the antibodies for the indicated number of days (1, 2, 3, 4, 5, 6, or 8). [Figure 87L] Figure 87L is a bar graph showing the levels of exotoxin-3 secretion by T cells activated / expanded with the indicated antibody (isotype control; anti-TCRβV 6-5 v1 with anti-BCMA antibody; anti-TCRβV 6-5 v1; anti-TCRβV 123 / 4 v1, or SP34-2) and cultured with the antibody for the indicated number of days (1, 2, 3, 4, 5, 6, or 8). [Figure 87M] Figure 87M is a bar graph showing the levels of IL-8 secretion by T cells activated / expanded with the indicated antibodies (isotype control; anti-TCRβV 6-5 v1 with anti-BCMA antibody; anti-TCRβV 6-5 v1; anti-TCRβV 123 / 4 v1, or SP34-2) and cultured with the antibodies for the indicated number of days (1, 2, 3, 4, 5, 6, or 8). [Figure 87N]Figure 87N is a bar graph showing the level of IP-10 secretion by T cells activated / expanded with the indicated antibodies (isotype control; anti-TCRβV 6-5 v1 with anti-BCMA antibody; anti-TCRβV 6-5 v1; anti-TCRβV 123 / 4 v1, or SP34-2) and cultured with the antibodies for the indicated number of days (1, 2, 3, 4, 5, 6, or 8). [Figure 87O] Figure 87O is a bar graph showing the levels of MCP-1 secretion by T cells activated / expanded with the indicated antibodies (isotype control; anti-TCRβV 6-5 v1 with anti-BCMA antibody; anti-TCRβV 6-5 v1; anti-TCRβV 123 / 4 v1, or SP34-2) and cultured with the antibodies for the indicated number of days (1, 2, 3, 4, 5, 6, or 8). [Figure 87P] Figure 87P is a bar graph showing the levels of MCP-4 secretion by T cells activated / expanded with the indicated antibodies (isotype control; anti-TCRβV 6-5 v1 with anti-BCMA antibody; anti-TCRβV 6-5 v1; anti-TCRβV 123 / 4 v1, or SP34-2) and cultured with the antibodies for the indicated number of days (1, 2, 3, 4, 5, 6, or 8). [Figure 87Q] Figure 87Q is a bar graph showing the level of MDC secretion by T cells activated / expanded with the indicated antibodies (isotype control; anti-TCRβV 6-5 v1 with anti-BCMA antibody; anti-TCRβV 6-5 v1; anti-TCRβV 123 / 4 v1, or SP34-2) and cultured with the antibodies for the indicated number of days (1, 2, 3, 4, 5, 6, or 8). [Figure 87R] Figure 87R is a bar graph showing the levels of MIP-1a secretion by T cells activated / expanded with the indicated antibodies (isotype control; anti-TCRβV 6-5 v1 with anti-BCMA antibody; anti-TCRβV 6-5 v1; anti-TCRβV 123 / 4 v1, or SP34-2) and cultured with the antibodies for the indicated number of days (1, 2, 3, 4, 5, 6, or 8). [Figure 87S]Figure 87S is a bar graph showing the levels of MIP-1b secretion by T cells activated / expanded with the indicated antibodies (isotype control; anti-TCRβV 6-5 v1 with anti-BCMA antibody; anti-TCRβV 6-5 v1; anti-TCRβV 123 / 4 v1, or SP34-2) and cultured with the antibodies for the indicated number of days (1, 2, 3, 4, 5, 6, or 8). [Figure 87T] Figure 87T is a bar graph showing the level of TARC secretion by T cells activated / expanded with the indicated antibodies (isotype control; anti-TCRβV 6-5 v1 with anti-BCMA antibody; anti-TCRβV 6-5 v1; anti-TCRβV 123 / 4 v1, or SP34-2) and cultured with the antibodies for the indicated number of days (1, 2, 3, 4, 5, 6, or 8). [Figure 87U] Figure 87U is a bar graph showing the levels of GMCSF secretion by T cells activated / expanded with the indicated antibodies (isotype control; anti-TCRβV 6-5 v1 with anti-BCMA antibody; anti-TCRβV 6-5 v1; anti-TCRβV 123 / 4 v1, or SP34-2) and cultured with the antibodies for the indicated number of days (1, 2, 3, 4, 5, 6, or 8). [Figure 87V] Figure 87V is a bar graph showing the levels of IL-12-23p40 secretion by T cells activated / expanded with the indicated antibody (isotype control; anti-TCRβV 6-5 v1 with anti-BCMA antibody; anti-TCRβV 6-5 v1; anti-TCRβV 123 / 4 v1, or SP34-2) and cultured with the antibody for the indicated number of days (1, 2, 3, 4, 5, 6, or 8). [Figure 87W] Figure 87W is a bar graph showing the level of IL-15 secretion by T cells activated / expanded with the indicated antibody (isotype control; anti-TCRβV 6-5 v1 with anti-BCMA antibody; anti-TCRβV 6-5 v1; anti-TCRβV 123 / 4 v1, or SP34-2) and cultured with the antibody for the indicated number of days (1, 2, 3, 4, 5, 6, or 8). [Figure 87X]Figure 87X is a bar graph showing the level of IL-16 secretion by T cells activated / expanded with the indicated antibody (isotype control; anti-TCRβV 6-5 v1 with anti-BCMA antibody; anti-TCRβV 6-5 v1; anti-TCRβV 123 / 4 v1, or SP34-2) and cultured with the antibody for the indicated number of days (1, 2, 3, 4, 5, 6, or 8). [Figure 87Y] Figure 87Y is a bar graph showing the levels of IL-17a secretion by T cells activated / expanded with the indicated antibodies (isotype control; anti-TCRβV 6-5 v1 with anti-BCMA antibody; anti-TCRβV 6-5 v1; anti-TCRβV 123 / 4 v1, or SP34-2) and cultured with the antibodies for the indicated number of days (1, 2, 3, 4, 5, 6, or 8). [Figure 87Z] Figure 87Z is a bar graph showing the level of IL-1a secretion by T cells activated / expanded with the indicated antibody (isotype control; anti-TCRβV 6-5 v1 with anti-BCMA antibody; anti-TCRβV 6-5 v1; anti-TCRβV 123 / 4 v1, or SP34-2) and cultured with the antibody for the indicated number of days (1, 2, 3, 4, 5, 6, or 8). [Figure 87AA] Figure 87AA is a bar graph showing the levels of IL-5 secretion by T cells activated / expanded with the indicated antibodies (isotype control; anti-TCRβV 6-5 v1 with anti-BCMA antibody; anti-TCRβV 6-5 v1; anti-TCRβV 123 / 4 v1, or SP34-2) and cultured with the antibodies for the indicated number of days (1, 2, 3, 4, 5, 6, or 8). [Figure 87BB] Figure 87BB is a bar graph showing the levels of IL-7 secretion by T cells activated / expanded with the indicated antibodies (isotype control; anti-TCRβV 6-5 v1 with anti-BCMA antibody; anti-TCRβV 6-5 v1; anti-TCRβV 123 / 4 v1, or SP34-2) and cultured with the antibodies for the indicated number of days (1, 2, 3, 4, 5, 6, or 8). [Figure 87CC]Figure 87CC is a bar graph showing the levels of TNF-B secretion by T cells activated / expanded with the indicated antibodies (isotype control; anti-TCRβV 6-5 v1 with anti-BCMA antibody; anti-TCRβV 6-5 v1; anti-TCRβV 123 / 4 v1, or SP34-2) and cultured with the antibodies for the indicated number of days (1, 2, 3, 4, 5, 6, or 8). [Figure 87DD] Figure 87DD is a bar graph showing the level of VEGF secretion by T cells activated / expanded with the indicated antibodies (isotype control; anti-TCRβV 6-5 v1 with anti-BCMA antibody; anti-TCRβV 6-5 v1; anti-TCRβV 123 / 4 v1, or SP34-2) and cultured with the antibodies for the indicated number of days (1, 2, 3, 4, 5, 6, or 8). [Figure 88]

[0234] Figure 88 shows a graphical representation of the sequence relationships between different TCRVB clonotype subfamilies. [Figure 89A]

[0235] Figure 89A is a bar graph showing the percentage of cytokine release from PBMCs activated / expanded for 8 days with the indicated antibodies (anti-TCRβV 12-3 / 4 v1 or SP34-2). [Figure 89B] Figure 89B is a bar graph showing the percentage of cytokine release from PBMCs activated / expanded for 8 days with the indicated antibodies (anti-TCRβV5 or SP34-2). [Figure 89C] Figure 89C is a bar graph showing the percentage of cytokine release from PBMCs activated / expanded for 8 days with the indicated antibodies (anti-TCRβV 10 or SP34-2). [Figure 90A]

[0236] Figure 90A is a bar graph showing the level of IFNγ secretion by T cells activated / expanded with the indicated antibodies for the indicated number of days (3 or 6). [Figure 90B] FIG. 90B is a bar graph showing the level of IL-10 secretion by T cells activated / expanded with the indicated antibodies for the indicated number of days (3 or 6). [Figure 90C] Figure 90C is a bar graph showing the level of IL-17A secretion by T cells activated / expanded with the indicated antibodies for the indicated number of days (3 or 6). [Figure 90D] FIG. 90D is a bar graph showing the level of IL-1α secretion by T cells activated / expanded with the indicated antibodies for the indicated number of days (3 or 6). [Figure 90E] FIG. 90E is a bar graph showing the level of IL-1β secretion by T cells activated / expanded with the indicated antibodies for the indicated number of days (3 or 6). [Figure 90F] Figure 90F is a bar graph showing the level of IL-6 secretion by T cells activated / expanded with the indicated antibodies for the indicated number of days (3 or 6). [Figure 90G] Figure 90G is a bar graph showing the level of TNFα secretion by T cells activated / expanded with the indicated antibodies for the indicated number of days (3 or 6). [Figure 90H] FIG. 90H is a bar graph showing the level of IL-2 secretion by T cells activated / expanded with the indicated antibodies for the indicated number of days (3 or 6). [Figure 91]

[0237] Figure 91 is a bar graph summarizing data from FACS analysis of PBMCs activated / expanded for 6 days using the indicated anti-TCRVβ antibodies. [Figure 92A]

[0238] Figure 92A is a bar graph showing the level of IFNγ secretion by T cells activated / expanded with the indicated antibodies for the indicated number of days (1, 3, 5, or 7). [Figure 92B] Figure 92B is a bar graph showing the level of IL-10 secretion by T cells activated / expanded with the indicated antibodies for the indicated number of days (1, 3, 5, or 7). [Figure 92C] Figure 92C is a bar graph showing the level of IL-17A secretion by T cells activated / expanded with the indicated antibodies for the indicated number of days (1, 3, 5, or 7). [Figure 92D] Figure 92D is a bar graph showing the level of IL-1α secretion by T cells activated / expanded with the indicated antibodies for the indicated number of days (1, 3, 5, or 7). [Figure 92E] Figure 92E is a bar graph showing the level of IL-1β secretion by T cells activated / expanded with the indicated antibodies for the indicated number of days (1, 3, 5, or 7). [Figure 92F] Figure 92F is a bar graph showing the level of IL-6 secretion by T cells activated / expanded with the indicated antibodies for the indicated number of days (1, 3, 5, or 7). [Figure 92G] Figure 92G is a bar graph showing the level of IL-4 secretion by T cells activated / expanded with the indicated antibodies for the indicated number of days (1, 3, 5, or 7). [Figure 92H] Figure 92H is a bar graph showing the level of IL-2 secretion by T cells activated / expanded with the indicated antibodies for the indicated number of days (1, 3, 5, or 7). [Figure 93]

[0239] Figure 93 is a bar graph summarizing data from FACS analysis of PBMCs activated / expanded for 7 days using the indicated anti-TCRVβ antibodies. [Figure 94A]

[0240] Figure 94A is a bar graph showing the level of IFNγ secretion by T cells activated / expanded with the indicated antibodies for the indicated number of days (3 or 6). [Figure 94B] Figure 94B is a bar graph showing the level of IL-10 secretion by T cells activated / expanded with the indicated antibodies for the indicated number of days (3 or 6). [Figure 94C] Figure 94C is a bar graph showing the level of IL-17A secretion by T cells activated / expanded with the indicated antibodies for the indicated number of days (3 or 6). [Figure 94D]Figure 94D is a bar graph showing the level of IL-1α secretion by T cells activated / expanded with the indicated antibodies for the indicated number of days (3 or 6). [Figure 94E] Figure 94E is a bar graph showing the level of IL-1β secretion by T cells activated / expanded with the indicated antibodies for the indicated number of days (3 or 6). [Figure 94F] Figure 94F is a bar graph showing the level of IL-6 secretion by T cells activated / expanded with the indicated antibodies for the indicated number of days (3 or 6). [Figure 94G] Figure 94G is a bar graph showing the level of IL-4 secretion by T cells activated / expanded with the indicated antibodies for the indicated number of days (3 or 6). [Figure 94H] Figure 94H is a bar graph showing the level of TNFα secretion by T cells activated / expanded with the indicated antibodies for the indicated number of days (3 or 6). [Figure 94I] Figure 94I is a bar graph showing the level of IL-2 secretion by T cells activated / expanded with the indicated antibodies for the indicated number of days (3 or 6). [Figure 95A]

[0241] Figure 95A is a bar graph showing the level of IFN-γ secretion by T cells activated / expanded with the indicated antibodies (anti-TCRβV 6-5 v1 (plate coated), anti-CD3ε (plate coated), anti-TCRβV 6-5 v1 (in solution), or anti-CD3ε (in solution)) and cultured with the antibodies for the indicated number of days (1, 3, 5, or 7). [Figure 95B] Figure 95B is a bar graph showing the level of IFN-γ secretion by T cells activated / expanded with the indicated antibodies (anti-TCRβV 6-5 v1 (plate coated), anti-CD3ε (plate coated), anti-TCRβV 6-5 v1 (in solution), or anti-CD3ε (in solution)) and cultured with the antibodies for the indicated number of days (1, 3, 5, or 7). [Figure 95C]Figure 95C is a bar graph showing the level of IL-1b secretion by T cells activated / expanded with the indicated antibodies (anti-TCRβV 6-5 v1 (plate coated), anti-CD3ε (plate coated), anti-TCRβV 6-5 v1 (in solution), or anti-CD3ε (in solution)) and cultured with the antibodies for the indicated number of days (1, 3, 5, or 7). [Figure 95D] Figure 95D is a bar graph showing the level of IL-6 secretion by T cells activated / expanded with the indicated antibodies (anti-TCRβV 6-5 v1 (plate coated), anti-CD3ε (plate coated), anti-TCRβV 6-5 v1 (in solution), or anti-CD3ε (in solution)) and cultured with the antibodies for the indicated number of days (1, 3, 5, or 7). [Figure 95E] Figure 95E is a bar graph showing the level of IL-10 secretion by T cells activated / expanded with the indicated antibodies (anti-TCRβV 6-5 v1 (plate coated), anti-CD3ε (plate coated), anti-TCRβV 6-5 v1 (in solution), or anti-CD3ε (in solution)) and cultured with the antibodies for the indicated number of days (1, 3, 5, or 7). [Figure 95F] Figure 95F is a bar graph showing the level of IL-15 secretion by T cells activated / expanded with the indicated antibodies (anti-TCRβV 6-5 v1 (plate coated), anti-CD3ε (plate coated), anti-TCRβV 6-5 v1 (in solution), or anti-CD3ε (in solution)) and cultured with the antibodies for the indicated number of days (1, 3, 5, or 7). [Figure 95G] Figure 95G is a bar graph showing the level of IL-17A secretion by T cells activated / expanded with the indicated antibodies (anti-TCRβV 6-5 v1 (plate coated), anti-CD3ε (plate coated), anti-TCRβV 6-5 v1 (in solution), or anti-CD3ε (in solution)) and cultured with the antibodies for the indicated number of days (1, 3, 5, or 7). [Figure 95H]Figure 95H is a bar graph showing the level of IL-1a secretion by T cells activated / expanded with the indicated antibodies (anti-TCRβV 6-5 v1 (plate coated), anti-CD3ε (plate coated), anti-TCRβV 6-5 v1 (in solution), or anti-CD3ε (in solution)) and cultured with the antibodies for the indicated number of days (1, 3, 5, or 7). [Figure 95I] Figure 95I is a bar graph showing the level of IL-1b secretion by T cells activated / expanded with the indicated antibodies (anti-TCRβV 6-5 v1 (plate coated), anti-CD3ε (plate coated), anti-TCRβV 6-5 v1 (in solution), or anti-CD3ε (in solution)) and cultured with the antibodies for the indicated number of days (1, 3, 5, or 7). [Figure 95J] Figure 95J is a bar graph showing the level of IL-2 secretion by T cells activated / expanded with the indicated antibodies (anti-TCRβV 6-5 v1 (plate coated), anti-CD3ε (plate coated), anti-TCRβV 6-5 v1 (in solution), or anti-CD3ε (in solution)) and cultured with the antibodies for the indicated number of days (1, 3, 5, or 7). [Figure 95K] Figure 95K is a bar graph showing the level of IL-4 secretion by T cells activated / expanded with the indicated antibodies (anti-TCRβV 6-5 v1 (plate coated), anti-CD3ε (plate coated), anti-TCRβV 6-5 v1 (in solution), or anti-CD3ε (in solution)) and cultured with the antibodies for the indicated number of days (1, 3, 5, or 7). [Figure 95L] Figure 95L is a bar graph showing the levels of TNF-α secretion by T cells activated / expanded with the indicated antibodies (anti-TCRβV 6-5 v1 (plate coated), anti-CD3ε (plate coated), anti-TCRβV 6-5 v1 (in solution), or anti-CD3ε (in solution)) and cultured with the antibodies for the indicated number of days (1, 3, 5, or 7). [Figure 96]

[0242] Figure 96 is a FACS plot showing the ability of MH3-2 to bind to PBMC from one of two donors when the PBMC were either pre-incubated with TM23 or not (MH3-2 alone). [Figure 97]

[0243] Figure 97 is a FACS plot showing the ability of MH3-2 to bind to PBMC from one of two donors when the PBMC were pre-incubated with TM23 or not (MH3-2 alone). [Figure 98A]

[0244] Figure 98A is a bar graph showing the polyfunctional strength index (PSI) of PBMC CD4+ T cells, CD4+ T cells expanded with anti-CD3 antibody (CD3-expanded T cells), and CD4+ T cells expanded with anti-TCRVβ 6-5 antibody (drug-expanded T cells). The effector mediators are granzyme B, IFNγ, MIP-1α, perforin, TNFα, and TNFβ. The stimulatory mediator is IL-5. The chemoattractant mediator is MIP-1b. [Figure 98B] Figure 98B is a bar graph showing the polyfunctional strength index (PSI) of PBMC CD8+ T cells, CD8+ T cells expanded with anti-CD3 antibody (CD3-expanded T cells), and CD8+ T cells expanded with anti-TCRVβ 6-5 antibody (drug-expanded T cells). Effector mediators are granzyme B, IFNγ, MIP-1α, perforin, and TNFβ. Chemoattractant mediators are MIP-1b and RANTES. [Figure 99]

[0245] Figure 99 is a diagram of the experimental design for the pharmacokinetic (PK) profile and dosing strategy of the multifunctional polypeptide molecules described herein. [Figure 100-1]

[0246] Figure 100 shows Table 9, which shows an alignment of TCRBV amino acid sequences (SEQ ID NOS: 3457-3516, 3669-3673, 3522, 3674-3675, 3525, 3676-3687, 3538, 3688-3698, 3550-3639, and 3699-3790, respectively, in order of appearance). The alignment of TCRBV amino acid sequences in Table 9 highlights the diversity of TCR sequences. In particular, TCRBV sequences from different subfamilies are significantly different from each other. [Figure 100-2] Same as above. [Figure 100-3] Same as above. [Figure 101]

[0247] FIG. 101 shows an alignment of affinity-matured humanized antibody AH VL sequences (SEQ ID NOs: 3377 to 3389, respectively, in order of appearance). [Figure 102-1]

[0248] Figure 102 shows an alignment of affinity-matured humanized antibody AH VH sequences (SEQ ID NOs: 3390 to 3436, respectively, in order of appearance). [Figure 102-2] Same as above. [Figure 102-3] Same as above. [Figure 103A]

[0249] Figure 103A shows an exemplary embodiment (e.g., BKM0186) of a multifunctional molecule comprising a TCRβV-binding moiety and a cytokine polypeptide (e.g., IL2 or IL2-C125A) described herein. Figure 103B shows an exemplary embodiment of a multifunctional molecule comprising a TCRβV-binding moiety and a cytokine polypeptide described herein. Figures 103C, 103D, 103E, and 103F show exemplary embodiments of a multifunctional molecule comprising a first TCRβV-binding moiety, a second TCRβV-binding moiety, and two cytokine polypeptides described herein. In some embodiments, the cytokine polypeptide comprises IL-2 or a functional fragment or variant thereof, IL2-C125A or a functional fragment or variant thereof, IL-15 or a functional fragment or variant thereof, IL-7 or a functional fragment or variant thereof, IL-12 or a functional fragment or variant thereof, or IL-21 or a functional fragment or variant thereof. In embodiments, the cytokine polypeptide further comprises a cytokine receptor. In some embodiments, the cytokine polypeptide comprises IL-15 linked to IL-15Ra. In some embodiments, the cytokine polypeptide comprises IL-15 linked to an IL-15Ra sushi domain. In some embodiments, the cytokine polypeptide comprises a cytokine dimer. In some embodiments, the cytokine polypeptide comprises an IL-12 beta subunit linked to an IL-12 alpha subunit. [Figure 103B]Figure 103A shows an exemplary embodiment (e.g., BKM0186) of a multifunctional molecule comprising a TCRβV-binding moiety and a cytokine polypeptide (e.g., IL2 or IL2-C125A) described herein. Figure 103B shows an exemplary embodiment of a multifunctional molecule comprising a TCRβV-binding moiety and a cytokine polypeptide described herein. Figures 103C, 103D, 103E, and 103F show exemplary embodiments of a multifunctional molecule comprising a first TCRβV-binding moiety, a second TCRβV-binding moiety, and two cytokine polypeptides described herein. In some embodiments, the cytokine polypeptide comprises IL-2 or a functional fragment or variant thereof, IL2-C125A or a functional fragment or variant thereof, IL-15 or a functional fragment or variant thereof, IL-7 or a functional fragment or variant thereof, IL-12 or a functional fragment or variant thereof, or IL-21 or a functional fragment or variant thereof. In embodiments, the cytokine polypeptide further comprises a cytokine receptor. In some embodiments, the cytokine polypeptide comprises IL-15 linked to IL-15Ra. In some embodiments, the cytokine polypeptide comprises IL-15 linked to an IL-15Ra sushi domain. In some embodiments, the cytokine polypeptide comprises a cytokine dimer. In some embodiments, the cytokine polypeptide comprises an IL-12 beta subunit linked to an IL-12 alpha subunit. [Figure 103C]Figure 103A shows an exemplary embodiment (e.g., BKM0186) of a multifunctional molecule comprising a TCRβV-binding moiety and a cytokine polypeptide (e.g., IL2 or IL2-C125A) described herein. Figure 103B shows an exemplary embodiment of a multifunctional molecule comprising a TCRβV-binding moiety and a cytokine polypeptide described herein. Figures 103C, 103D, 103E, and 103F show exemplary embodiments of a multifunctional molecule comprising a first TCRβV-binding moiety, a second TCRβV-binding moiety, and two cytokine polypeptides described herein. In some embodiments, the cytokine polypeptide comprises IL-2 or a functional fragment or variant thereof, IL2-C125A or a functional fragment or variant thereof, IL-15 or a functional fragment or variant thereof, IL-7 or a functional fragment or variant thereof, IL-12 or a functional fragment or variant thereof, or IL-21 or a functional fragment or variant thereof. In embodiments, the cytokine polypeptide further comprises a cytokine receptor. In some embodiments, the cytokine polypeptide comprises IL-15 linked to IL-15Ra. In some embodiments, the cytokine polypeptide comprises IL-15 linked to an IL-15Ra sushi domain. In some embodiments, the cytokine polypeptide comprises a cytokine dimer. In some embodiments, the cytokine polypeptide comprises an IL-12 beta subunit linked to an IL-12 alpha subunit. [Figure 103D]Figure 103A shows an exemplary embodiment (e.g., BKM0186) of a multifunctional molecule comprising a TCRβV-binding moiety and a cytokine polypeptide (e.g., IL2 or IL2-C125A) described herein. Figure 103B shows an exemplary embodiment of a multifunctional molecule comprising a TCRβV-binding moiety and a cytokine polypeptide described herein. Figures 103C, 103D, 103E, and 103F show exemplary embodiments of a multifunctional molecule comprising a first TCRβV-binding moiety, a second TCRβV-binding moiety, and two cytokine polypeptides described herein. In some embodiments, the cytokine polypeptide comprises IL-2 or a functional fragment or variant thereof, IL2-C125A or a functional fragment or variant thereof, IL-15 or a functional fragment or variant thereof, IL-7 or a functional fragment or variant thereof, IL-12 or a functional fragment or variant thereof, or IL-21 or a functional fragment or variant thereof. In embodiments, the cytokine polypeptide further comprises a cytokine receptor. In some embodiments, the cytokine polypeptide comprises IL-15 linked to IL-15Ra. In some embodiments, the cytokine polypeptide comprises IL-15 linked to an IL-15Ra sushi domain. In some embodiments, the cytokine polypeptide comprises a cytokine dimer. In some embodiments, the cytokine polypeptide comprises an IL-12 beta subunit linked to an IL-12 alpha subunit. [Figure 103E]Figure 103A shows an exemplary embodiment (e.g., BKM0186) of a multifunctional molecule comprising a TCRβV-binding moiety and a cytokine polypeptide (e.g., IL2 or IL2-C125A) described herein. Figure 103B shows an exemplary embodiment of a multifunctional molecule comprising a TCRβV-binding moiety and a cytokine polypeptide described herein. Figures 103C, 103D, 103E, and 103F show exemplary embodiments of a multifunctional molecule comprising a first TCRβV-binding moiety, a second TCRβV-binding moiety, and two cytokine polypeptides described herein. In some embodiments, the cytokine polypeptide comprises IL-2 or a functional fragment or variant thereof, IL2-C125A or a functional fragment or variant thereof, IL-15 or a functional fragment or variant thereof, IL-7 or a functional fragment or variant thereof, IL-12 or a functional fragment or variant thereof, or IL-21 or a functional fragment or variant thereof. In embodiments, the cytokine polypeptide further comprises a cytokine receptor. In some embodiments, the cytokine polypeptide comprises IL-15 linked to IL-15Ra. In some embodiments, the cytokine polypeptide comprises IL-15 linked to an IL-15Ra sushi domain. In some embodiments, the cytokine polypeptide comprises a cytokine dimer. In some embodiments, the cytokine polypeptide comprises an IL-12 beta subunit linked to an IL-12 alpha subunit. [Figure 103F]Figure 103A shows an exemplary embodiment (e.g., BKM0186) of a multifunctional molecule comprising a TCRβV-binding moiety and a cytokine polypeptide (e.g., IL2 or IL2-C125A) described herein. Figure 103B shows an exemplary embodiment of a multifunctional molecule comprising a TCRβV-binding moiety and a cytokine polypeptide described herein. Figures 103C, 103D, 103E, and 103F show exemplary embodiments of a multifunctional molecule comprising a first TCRβV-binding moiety, a second TCRβV-binding moiety, and two cytokine polypeptides described herein. In some embodiments, the cytokine polypeptide comprises IL-2 or a functional fragment or variant thereof, IL2-C125A or a functional fragment or variant thereof, IL-15 or a functional fragment or variant thereof, IL-7 or a functional fragment or variant thereof, IL-12 or a functional fragment or variant thereof, or IL-21 or a functional fragment or variant thereof. In embodiments, the cytokine polypeptide further comprises a cytokine receptor. In some embodiments, the cytokine polypeptide comprises IL-15 linked to IL-15Ra. In some embodiments, the cytokine polypeptide comprises IL-15 linked to an IL-15Ra sushi domain. In some embodiments, the cytokine polypeptide comprises a cytokine dimer. In some embodiments, the cytokine polypeptide comprises an IL-12 beta subunit linked to an IL-12 alpha subunit. [Figure 104]

[0250] Figure 104 shows a FACS plot showing the binding of BKM0186 to different immune cell populations in human PBMCs. [Figure 105]

[0251] Figure 105 shows the binding of BKM0186 to pure human T cells expressing either Vβ6 or CD25 (IL-2Rα) or both. [Figure 106]

[0252] Figure 106 shows the in vitro concentration-effect relationship for BKM0186-mediated in vitro expansion of Vβ6 and activated (CD25) Vβ6 T cells from human PBMCs on day 5 as a % of total cytotoxic T cell (CD8) and helper T cell (CD4) populations. Left graph: cytotoxic T lymphocytes; right graph: helper T lymphocytes. [Figure 107]

[0253] Figure 107 shows in vitro TCR sequencing. PBMCs were incubated with 100 nM BKM0186 for 5 days, and T cells were sequenced for the TCR β chain V (TRBV) gene. Compared to unstimulated T cells (gray), BKM0186 selectively expanded T cells harboring TRBV6-1, TRBV6-2, TRBV6-3, TRBV6-5, and TRBV10-3. [Figure 108A]

[0254] Figures 108A and 108B show a series of graphs (Figure 108A) and a series of FACS plots (Figure 108B) depicting activation of CD4+ and CD8+ T cells as assessed by CD25 expression following stimulation with BKM0186, RSV-IL2, and anti-TCRVβ6 control in solution. [Figure 108B] Figures 108A and 108B show a series of graphs (Figure 108A) and a series of FACS plots (Figure 108B) depicting activation of CD4+ and CD8+ T cells as assessed by CD25 expression following stimulation with BKM0186, RSV-IL2, and anti-TCRVβ6 control in solution. [Figure 109]

[0255] Figure 109 shows a series of FACS plots demonstrating memory T cell differentiation mediated by BKM0186 compared to unstimulated and control RSV-IL2 and anti-TCRVβ6. The upper left quadrant represents central memory (CM), the lower left quadrant represents effector memory (EM), the upper right quadrant represents naive (N), and the lower right quadrant represents effector memory RA (TEMRA). [Figure 110]

[0256] Figure 110 shows the in vitro concentration-effect relationship for BKM0186-induced cytokine release from human PBMCs on day 4 using the MSD V-plex human cytokine panel. [Figure 111]

[0257] Figure 111 shows the BKM0186-mediated killing of human tumor organoids generated from primary patient-derived tissues from colorectal and NSCLC cancer patients.Vertical bars represent the percentage of organoid area that is reduced after BKM0186 and autologous TIL incubation of organoids compared to isotype control. [Figure 112]

[0258] Figure 112 shows tumor growth curves of EMT6 tumor-bearing mice treated with mBKM0186. Studies were conducted in randomized mice with tumor volumes of 80-150 mm. For all models except MC38, mice were dosed at weekly doses of 0.5-1.5 mg / kg for 3 weeks, and survival was determined based on an endpoint of a tumor volume of 2000 mm. [Figure 113]

[0259] Figure 113 shows tumor growth curves for mice treated with mBKM0186. Studies were conducted in randomized mice with tumor volumes of 80-150 mm3. For all models except MC38, mice were dosed at weekly doses of 1-1.5 mg / kg for 4 weeks, and survival was determined based on an endpoint of a tumor volume of 2000 mm3. For MC38, mice received a first dose of 3 mg / kg, followed by 1 mg / kg for three subsequent weekly (QW) doses. [Figure 114]

[0260] Figure 114 shows Kaplan-Meier survival curves for treated mice. Studies were conducted in randomized mice with tumor volumes of 80-150 mm3. For all models except MC38, mice were dosed at weekly doses of 1-1.5 mg / kg for 4 weeks, and survival was determined based on an endpoint of a tumor volume of 2000 mm3. For MC38, mice received a first dose of 3 mg / kg, followed by 1 mg / kg for three subsequent weekly (QW) doses. [Figure 115]

[0261] Figure 115 shows the experimental design for the tumor re-challenge study. Cured EMT6 tumor-bearing mice were re-challenged with EMT6 tumor cells in one flank and with CT26 tumor cells in the other flank and monitored for tumor growth for 28 days. [Figure 116]

[0262] Figure 116 shows the results of a tumor re-challenge study. EMT6 tumors were rejected, but CT26 tumors grew, suggesting the establishment of a memory response against EMT6 tumors likely mediated through mBKM0186 treatment. [Figure 117]

[0263] Figure 117 shows immune profiling of T cells in blood and tumor tissues at day 14 after dosing with mBKM0186. [Figure 118]

[0264] Figure 118 shows tumor growth curves of EMT6 tumors after weekly (QW) treatment of mice bearing 150 mm tumors with 1 mg / kg mBKM0186, with and without depletion of Vβ-specific T cells. Filled triangles indicate dosing intervals for the depleting antibody, and open triangles indicate dosing intervals for mBKM0186. [Figure 119A]

[0265] Figures 119A and 119B show the pharmacokinetic profiles of BKM0186 (Figure 119A) and BKM0281 (Figure 119B) administered as a single dose IV in cynomolgus monkeys. [Figure 119B] Figures 119A and 119B show the pharmacokinetic profiles of BKM0186 (Figure 119A) and BKM0281 (Figure 119B) administered as a single dose IV in cynomolgus monkeys. [Figure 120A]

[0266] Figure 120A shows T cell expansion after a single IV dose of BKM0186. [Figure 120B] Figure 120B shows T cell expansion after a single IV dose of BKM0281, n=3 monkeys, vehicle control in n=1 monkey. [Figure 121]

[0267] Figure 121 shows serum soluble CD25 levels in monkeys administered a single IV dose of BKM0186. Mean values, n=2-3 monkeys / group. [Figure 122A]

[0268] Figure 122A shows serum IL-6 levels in monkeys administered a single IV dose of BKM0186. Figure 122B shows serum IL-6 levels in monkeys administered a single IV dose of BKM0281. Mean values, n=2-3 monkeys / group. [Figure 122B] Figure 122A shows serum IL-6 levels in monkeys administered a single IV dose of BKM0186. Figure 122B shows serum IL-6 levels in monkeys administered a single IV dose of BKM0281. Mean values, n=2-3 monkeys / group. [Figure 123A]

[0269] Figure 123A shows serum IFN-γ levels in monkeys administered a single IV dose of BKM0186. Figure 123B shows IFN-γ levels in monkeys administered a single IV dose of BKM0281. Mean values, n=2-3 monkeys / group. Mean values, n=2-3 monkeys / group. [Figure 123B] Figure 123A shows serum IFN-γ levels in monkeys administered a single IV dose of BKM0186. Figure 123B shows IFN-γ levels in monkeys administered a single IV dose of BKM0281. Mean values, n=2-3 monkeys / group. Mean values, n=2-3 monkeys / group. [Figure 124]

[0270] FIG. 124 shows the in vitro concentration-effect relationship for bispecific-mediated in vitro expansion of Vβ6 T cells. [Figure 125A]

[0271] Figures 125A and 125B show exemplary embodiments of multifunctional molecules comprising a TCRβV-binding portion described herein and a cytokine molecule (e.g., wild-type human IL2 or IL15-IL15R sushi fusion). Figures 125C-125T show exemplary embodiments of multifunctional molecules, e.g., comprising a tumor-associated antigen (TAA)-binding portion described herein, a TCRβV-binding portion, and at least one cytokine molecule, or a functional fragment or functional variant thereof. In some embodiments, the tumor-associated antigen (TAA)-binding portion and / or the TCRβV-binding portion is an antibody, antigen-binding fragment thereof, or antibody fragment. In some embodiments, the antigen-binding fragment or antibody fragment comprises a Fab, Fab', F(ab'), F(ab), variable fragment (Fv), domain antibody (dAb), or single-chain variable fragment (scFv). In some embodiments, the at least one cytokine molecule, or functional fragment or functional variant thereof, is selected from the group consisting of interleukin-2 (IL-2) or a functional fragment or functional variant thereof, interleukin-7 (IL-7) or a functional fragment or functional variant thereof, interleukin-12 (IL-12) or a functional fragment or functional variant thereof, interleukin-15 (IL-15) or a functional fragment or functional variant thereof, interleukin-18 (IL-18) or a functional fragment or functional variant thereof, interleukin-21 (IL-21) or a functional fragment or functional variant thereof, or interferon gamma or a functional fragment or functional variant thereof, or a combination thereof. In some embodiments, an exemplary embodiment of a multifunctional molecule, e.g., a multifunctional molecule comprising a tumor-associated antigen (TAA)-binding portion, a TCRβV-binding portion, and at least one cytokine molecule, or a functional fragment or functional variant thereof, described herein, comprises a dimerization module comprising an Fc region comprising an N297A mutation.Figures 125C-125H show exemplary embodiments of multifunctional molecules, e.g., multifunctional molecules comprising a tumor-associated antigen (TAA)-binding portion described herein, a TCRβV-binding portion, and at least one cytokine molecule, or a functional fragment or variant thereof, where the multifunctional molecules comprise an exemplary dimerization module, e.g., an Fc region comprising a knobs-in-holes mutation and a disulfide bridge. Figures 125I-125N show exemplary embodiments of multifunctional molecules, e.g., multifunctional molecules comprising a tumor-associated antigen (TAA)-binding portion described herein, a TCRβV-binding portion, and at least one cytokine molecule, or a functional fragment or variant thereof, where the multifunctional molecules comprise an exemplary dimerization module, e.g., an Fc region comprising a disulfide bridge but not a knobs-in-holes mutation. Figures 125O-125T show exemplary embodiments of multifunctional molecules, e.g., multifunctional molecules comprising a tumor-associated antigen (TAA)-binding portion described herein, a TCRβV-binding portion, and at least one cytokine molecule, or a functional fragment or variant thereof, where the multifunctional molecule comprises an exemplary dimerization module, e.g., an Fc region that does not comprise a disulfide bridge or a knob-in-hole mutation. Figures 125C-125E, 125I-125K, and 125O-135Q show exemplary embodiments of multifunctional molecules, e.g., multifunctional molecules comprising a tumor-associated antigen (TAA)-binding portion described herein, a TCRβV-binding portion, and at least one cytokine molecule, or a functional fragment or variant thereof, where the TCRβV-binding portion comprises an scFv. Figures 125F-125H, 125L-125N, and 125R-125T show exemplary embodiments of multifunctional molecules, e.g., multifunctional molecules comprising a tumor-associated antigen (TAA) binding portion described herein, a TCRβV binding portion, and at least one cytokine molecule or functional fragment or functional variant thereof, wherein the TCRβV binding portion comprises a Fab.Figure 125U shows an exemplary embodiment of a multifunctional molecule, e.g., a multifunctional molecule comprising a tumor-associated antigen (TAA) binding portion described herein, a TCRβV binding portion, and at least one cytokine molecule or a functional fragment or functional variant thereof, wherein the multifunctional molecule comprises two TAA binding portions. In some embodiments, the at least one cytokine molecule or functional fragment or variant thereof is not interleukin-2 (IL-2) or a functional fragment or variant thereof, denoted as "IL2," but is interleukin-7 (IL-7) or a functional fragment or variant thereof, interleukin-12 (IL-12) or a functional fragment or variant thereof, interleukin-15 (IL-15) or a functional fragment or variant thereof, interleukin-18 (IL-18) or a functional fragment or variant thereof, interleukin-21 (IL-21) or a functional fragment or variant thereof, or interferon gamma or a functional fragment or variant thereof, or a combination thereof. [Figure 125B]Figures 125A and 125B show exemplary embodiments of multifunctional molecules comprising a TCRβV-binding portion described herein and a cytokine molecule (e.g., wild-type human IL2 or IL15-IL15R sushi fusion). Figures 125C-125T show exemplary embodiments of multifunctional molecules, e.g., comprising a tumor-associated antigen (TAA)-binding portion described herein, a TCRβV-binding portion, and at least one cytokine molecule, or a functional fragment or functional variant thereof. In some embodiments, the tumor-associated antigen (TAA)-binding portion and / or the TCRβV-binding portion is an antibody, antigen-binding fragment thereof, or antibody fragment. In some embodiments, the antigen-binding fragment or antibody fragment comprises a Fab, Fab', F(ab'), F(ab), variable fragment (Fv), domain antibody (dAb), or single-chain variable fragment (scFv). In some embodiments, the at least one cytokine molecule, or functional fragment or functional variant thereof, is selected from the group consisting of interleukin-2 (IL-2) or a functional fragment or functional variant thereof, interleukin-7 (IL-7) or a functional fragment or functional variant thereof, interleukin-12 (IL-12) or a functional fragment or functional variant thereof, interleukin-15 (IL-15) or a functional fragment or functional variant thereof, interleukin-18 (IL-18) or a functional fragment or functional variant thereof, interleukin-21 (IL-21) or a functional fragment or functional variant thereof, or interferon gamma or a functional fragment or functional variant thereof, or a combination thereof. In some embodiments, an exemplary embodiment of a multifunctional molecule, e.g., a multifunctional molecule comprising a tumor-associated antigen (TAA)-binding portion, a TCRβV-binding portion, and at least one cytokine molecule, or a functional fragment or functional variant thereof, described herein, comprises a dimerization module comprising an Fc region comprising an N297A mutation.Figures 125C-125H show exemplary embodiments of multifunctional molecules, e.g., multifunctional molecules comprising a tumor-associated antigen (TAA)-binding portion described herein, a TCRβV-binding portion, and at least one cytokine molecule, or a functional fragment or variant thereof, where the multifunctional molecules comprise an exemplary dimerization module, e.g., an Fc region comprising a knobs-in-holes mutation and a disulfide bridge. Figures 125I-125N show exemplary embodiments of multifunctional molecules, e.g., multifunctional molecules comprising a tumor-associated antigen (TAA)-binding portion described herein, a TCRβV-binding portion, and at least one cytokine molecule, or a functional fragment or variant thereof, where the multifunctional molecules comprise an exemplary dimerization module, e.g., an Fc region comprising a disulfide bridge but not a knobs-in-holes mutation. Figures 125O-125T show exemplary embodiments of multifunctional molecules, e.g., multifunctional molecules comprising a tumor-associated antigen (TAA)-binding portion described herein, a TCRβV-binding portion, and at least one cytokine molecule, or a functional fragment or variant thereof, where the multifunctional molecule comprises an exemplary dimerization module, e.g., an Fc region that does not comprise a disulfide bridge or a knob-in-hole mutation. Figures 125C-125E, 125I-125K, and 125O-135Q show exemplary embodiments of multifunctional molecules, e.g., multifunctional molecules comprising a tumor-associated antigen (TAA)-binding portion described herein, a TCRβV-binding portion, and at least one cytokine molecule, or a functional fragment or variant thereof, where the TCRβV-binding portion comprises an scFv. Figures 125F-125H, 125L-125N, and 125R-125T show exemplary embodiments of multifunctional molecules, e.g., multifunctional molecules comprising a tumor-associated antigen (TAA) binding portion described herein, a TCRβV binding portion, and at least one cytokine molecule or functional fragment or functional variant thereof, wherein the TCRβV binding portion comprises a Fab.Figure 125U shows an exemplary embodiment of a multifunctional molecule, e.g., a multifunctional molecule comprising a tumor-associated antigen (TAA) binding portion described herein, a TCRβV binding portion, and at least one cytokine molecule or a functional fragment or functional variant thereof, wherein the multifunctional molecule comprises two TAA binding portions. In some embodiments, the at least one cytokine molecule or functional fragment or variant thereof is not interleukin-2 (IL-2) or a functional fragment or variant thereof, denoted as "IL2," but is interleukin-7 (IL-7) or a functional fragment or variant thereof, interleukin-12 (IL-12) or a functional fragment or variant thereof, interleukin-15 (IL-15) or a functional fragment or variant thereof, interleukin-18 (IL-18) or a functional fragment or variant thereof, interleukin-21 (IL-21) or a functional fragment or variant thereof, or interferon gamma or a functional fragment or variant thereof, or a combination thereof. [Fig. 125C-125D]Figures 125A and 125B show exemplary embodiments of multifunctional molecules comprising a TCRβV-binding portion described herein and a cytokine molecule (e.g., wild-type human IL2 or IL15-IL15R sushi fusion). Figures 125C-125T show exemplary embodiments of multifunctional molecules, e.g., comprising a tumor-associated antigen (TAA)-binding portion described herein, a TCRβV-binding portion, and at least one cytokine molecule, or a functional fragment or functional variant thereof. In some embodiments, the tumor-associated antigen (TAA)-binding portion and / or the TCRβV-binding portion is an antibody, antigen-binding fragment thereof, or antibody fragment. In some embodiments, the antigen-binding fragment or antibody fragment comprises a Fab, Fab', F(ab'), F(ab), variable fragment (Fv), domain antibody (dAb), or single-chain variable fragment (scFv). In some embodiments, the at least one cytokine molecule, or functional fragment or functional variant thereof, is selected from the group consisting of interleukin-2 (IL-2) or a functional fragment or functional variant thereof, interleukin-7 (IL-7) or a functional fragment or functional variant thereof, interleukin-12 (IL-12) or a functional fragment or functional variant thereof, interleukin-15 (IL-15) or a functional fragment or functional variant thereof, interleukin-18 (IL-18) or a functional fragment or functional variant thereof, interleukin-21 (IL-21) or a functional fragment or functional variant thereof, or interferon gamma or a functional fragment or functional variant thereof, or a combination thereof. In some embodiments, an exemplary embodiment of a multifunctional molecule, e.g., a multifunctional molecule comprising a tumor-associated antigen (TAA)-binding portion, a TCRβV-binding portion, and at least one cytokine molecule, or a functional fragment or functional variant thereof, described herein, comprises a dimerization module comprising an Fc region comprising an N297A mutation.Figures 125C-125H show exemplary embodiments of multifunctional molecules, e.g., multifunctional molecules comprising a tumor-associated antigen (TAA)-binding portion described herein, a TCRβV-binding portion, and at least one cytokine molecule, or a functional fragment or variant thereof, where the multifunctional molecules comprise an exemplary dimerization module, e.g., an Fc region comprising a knobs-in-holes mutation and a disulfide bridge. Figures 125I-125N show exemplary embodiments of multifunctional molecules, e.g., multifunctional molecules comprising a tumor-associated antigen (TAA)-binding portion described herein, a TCRβV-binding portion, and at least one cytokine molecule, or a functional fragment or variant thereof, where the multifunctional molecules comprise an exemplary dimerization module, e.g., an Fc region comprising a disulfide bridge but not a knobs-in-holes mutation. Figures 125O-125T show exemplary embodiments of multifunctional molecules, e.g., multifunctional molecules comprising a tumor-associated antigen (TAA)-binding portion described herein, a TCRβV-binding portion, and at least one cytokine molecule, or a functional fragment or variant thereof, where the multifunctional molecule comprises an exemplary dimerization module, e.g., an Fc region that does not comprise a disulfide bridge or a knob-in-hole mutation. Figures 125C-125E, 125I-125K, and 125O-135Q show exemplary embodiments of multifunctional molecules, e.g., multifunctional molecules comprising a tumor-associated antigen (TAA)-binding portion described herein, a TCRβV-binding portion, and at least one cytokine molecule, or a functional fragment or variant thereof, where the TCRβV-binding portion comprises an scFv. Figures 125F-125H, 125L-125N, and 125R-125T show exemplary embodiments of multifunctional molecules, e.g., multifunctional molecules comprising a tumor-associated antigen (TAA) binding portion described herein, a TCRβV binding portion, and at least one cytokine molecule or functional fragment or functional variant thereof, wherein the TCRβV binding portion comprises a Fab.Figure 125U shows an exemplary embodiment of a multifunctional molecule, e.g., a multifunctional molecule comprising a tumor-associated antigen (TAA) binding portion described herein, a TCRβV binding portion, and at least one cytokine molecule or a functional fragment or functional variant thereof, wherein the multifunctional molecule comprises two TAA binding portions. In some embodiments, the at least one cytokine molecule or functional fragment or variant thereof is not interleukin-2 (IL-2) or a functional fragment or variant thereof, denoted as "IL2," but is interleukin-7 (IL-7) or a functional fragment or variant thereof, interleukin-12 (IL-12) or a functional fragment or variant thereof, interleukin-15 (IL-15) or a functional fragment or variant thereof, interleukin-18 (IL-18) or a functional fragment or variant thereof, interleukin-21 (IL-21) or a functional fragment or variant thereof, or interferon gamma or a functional fragment or variant thereof, or a combination thereof. [Figure 125E-125H]Figures 125A and 125B show exemplary embodiments of multifunctional molecules comprising a TCRβV-binding portion described herein and a cytokine molecule (e.g., wild-type human IL2 or IL15-IL15R sushi fusion). Figures 125C-125T show exemplary embodiments of multifunctional molecules, e.g., comprising a tumor-associated antigen (TAA)-binding portion described herein, a TCRβV-binding portion, and at least one cytokine molecule, or a functional fragment or functional variant thereof. In some embodiments, the tumor-associated antigen (TAA)-binding portion and / or the TCRβV-binding portion is an antibody, antigen-binding fragment thereof, or antibody fragment. In some embodiments, the antigen-binding fragment or antibody fragment comprises a Fab, Fab', F(ab'), F(ab), variable fragment (Fv), domain antibody (dAb), or single-chain variable fragment (scFv). In some embodiments, the at least one cytokine molecule, or functional fragment or functional variant thereof, is selected from the group consisting of interleukin-2 (IL-2) or a functional fragment or functional variant thereof, interleukin-7 (IL-7) or a functional fragment or functional variant thereof, interleukin-12 (IL-12) or a functional fragment or functional variant thereof, interleukin-15 (IL-15) or a functional fragment or functional variant thereof, interleukin-18 (IL-18) or a functional fragment or functional variant thereof, interleukin-21 (IL-21) or a functional fragment or functional variant thereof, or interferon gamma or a functional fragment or functional variant thereof, or a combination thereof. In some embodiments, an exemplary embodiment of a multifunctional molecule, e.g., a multifunctional molecule comprising a tumor-associated antigen (TAA)-binding portion, a TCRβV-binding portion, and at least one cytokine molecule, or a functional fragment or functional variant thereof, described herein, comprises a dimerization module comprising an Fc region comprising an N297A mutation.Figures 125C-125H show exemplary embodiments of multifunctional molecules, e.g., multifunctional molecules comprising a tumor-associated antigen (TAA)-binding portion described herein, a TCRβV-binding portion, and at least one cytokine molecule, or a functional fragment or variant thereof, where the multifunctional molecules comprise an exemplary dimerization module, e.g., an Fc region comprising a knobs-in-holes mutation and a disulfide bridge. Figures 125I-125N show exemplary embodiments of multifunctional molecules, e.g., multifunctional molecules comprising a tumor-associated antigen (TAA)-binding portion described herein, a TCRβV-binding portion, and at least one cytokine molecule, or a functional fragment or variant thereof, where the multifunctional molecules comprise an exemplary dimerization module, e.g., an Fc region comprising a disulfide bridge but not a knobs-in-holes mutation. Figures 125O-125T show exemplary embodiments of multifunctional molecules, e.g., multifunctional molecules comprising a tumor-associated antigen (TAA)-binding portion described herein, a TCRβV-binding portion, and at least one cytokine molecule, or a functional fragment or variant thereof, where the multifunctional molecule comprises an exemplary dimerization module, e.g., an Fc region that does not comprise a disulfide bridge or a knob-in-hole mutation. Figures 125C-125E, 125I-125K, and 125O-135Q show exemplary embodiments of multifunctional molecules, e.g., multifunctional molecules comprising a tumor-associated antigen (TAA)-binding portion described herein, a TCRβV-binding portion, and at least one cytokine molecule, or a functional fragment or variant thereof, where the TCRβV-binding portion comprises an scFv. Figures 125F-125H, 125L-125N, and 125R-125T show exemplary embodiments of multifunctional molecules, e.g., multifunctional molecules comprising a tumor-associated antigen (TAA) binding portion described herein, a TCRβV binding portion, and at least one cytokine molecule or functional fragment or functional variant thereof, wherein the TCRβV binding portion comprises a Fab.Figure 125U shows an exemplary embodiment of a multifunctional molecule, e.g., a multifunctional molecule comprising a tumor-associated antigen (TAA) binding portion described herein, a TCRβV binding portion, and at least one cytokine molecule or a functional fragment or functional variant thereof, wherein the multifunctional molecule comprises two TAA binding portions. In some embodiments, the at least one cytokine molecule or functional fragment or variant thereof is not interleukin-2 (IL-2) or a functional fragment or variant thereof, denoted as "IL2," but is interleukin-7 (IL-7) or a functional fragment or variant thereof, interleukin-12 (IL-12) or a functional fragment or variant thereof, interleukin-15 (IL-15) or a functional fragment or variant thereof, interleukin-18 (IL-18) or a functional fragment or variant thereof, interleukin-21 (IL-21) or a functional fragment or variant thereof, or interferon gamma or a functional fragment or variant thereof, or a combination thereof. [Figure 125I-125L]Figures 125A and 125B show exemplary embodiments of multifunctional molecules comprising a TCRβV-binding portion described herein and a cytokine molecule (e.g., wild-type human IL2 or IL15-IL15R sushi fusion). Figures 125C-125T show exemplary embodiments of multifunctional molecules, e.g., comprising a tumor-associated antigen (TAA)-binding portion described herein, a TCRβV-binding portion, and at least one cytokine molecule, or a functional fragment or functional variant thereof. In some embodiments, the tumor-associated antigen (TAA)-binding portion and / or the TCRβV-binding portion is an antibody, antigen-binding fragment thereof, or antibody fragment. In some embodiments, the antigen-binding fragment or antibody fragment comprises a Fab, Fab', F(ab'), F(ab), variable fragment (Fv), domain antibody (dAb), or single-chain variable fragment (scFv). In some embodiments, the at least one cytokine molecule, or functional fragment or functional variant thereof, is selected from the group consisting of interleukin-2 (IL-2) or a functional fragment or functional variant thereof, interleukin-7 (IL-7) or a functional fragment or functional variant thereof, interleukin-12 (IL-12) or a functional fragment or functional variant thereof, interleukin-15 (IL-15) or a functional fragment or functional variant thereof, interleukin-18 (IL-18) or a functional fragment or functional variant thereof, interleukin-21 (IL-21) or a functional fragment or functional variant thereof, or interferon gamma or a functional fragment or functional variant thereof, or a combination thereof. In some embodiments, an exemplary embodiment of a multifunctional molecule, e.g., a multifunctional molecule comprising a tumor-associated antigen (TAA)-binding portion, a TCRβV-binding portion, and at least one cytokine molecule, or a functional fragment or functional variant thereof, described herein, comprises a dimerization module comprising an Fc region comprising an N297A mutation.Figures 125C-125H show exemplary embodiments of multifunctional molecules, e.g., multifunctional molecules comprising a tumor-associated antigen (TAA)-binding portion described herein, a TCRβV-binding portion, and at least one cytokine molecule, or a functional fragment or variant thereof, where the multifunctional molecules comprise an exemplary dimerization module, e.g., an Fc region comprising a knobs-in-holes mutation and a disulfide bridge. Figures 125I-125N show exemplary embodiments of multifunctional molecules, e.g., multifunctional molecules comprising a tumor-associated antigen (TAA)-binding portion described herein, a TCRβV-binding portion, and at least one cytokine molecule, or a functional fragment or variant thereof, where the multifunctional molecules comprise an exemplary dimerization module, e.g., an Fc region comprising a disulfide bridge but not a knobs-in-holes mutation. Figures 125O-125T show exemplary embodiments of multifunctional molecules, e.g., multifunctional molecules comprising a tumor-associated antigen (TAA)-binding portion described herein, a TCRβV-binding portion, and at least one cytokine molecule, or a functional fragment or variant thereof, where the multifunctional molecule comprises an exemplary dimerization module, e.g., an Fc region that does not comprise a disulfide bridge or a knob-in-hole mutation. Figures 125C-125E, 125I-125K, and 125O-135Q show exemplary embodiments of multifunctional molecules, e.g., multifunctional molecules comprising a tumor-associated antigen (TAA)-binding portion described herein, a TCRβV-binding portion, and at least one cytokine molecule, or a functional fragment or variant thereof, where the TCRβV-binding portion comprises an scFv. Figures 125F-125H, 125L-125N, and 125R-125T show exemplary embodiments of multifunctional molecules, e.g., multifunctional molecules comprising a tumor-associated antigen (TAA) binding portion described herein, a TCRβV binding portion, and at least one cytokine molecule or functional fragment or functional variant thereof, wherein the TCRβV binding portion comprises a Fab.Figure 125U shows an exemplary embodiment of a multifunctional molecule, e.g., a multifunctional molecule comprising a tumor-associated antigen (TAA) binding portion described herein, a TCRβV binding portion, and at least one cytokine molecule or a functional fragment or functional variant thereof, wherein the multifunctional molecule comprises two TAA binding portions. In some embodiments, the at least one cytokine molecule or functional fragment or variant thereof is not interleukin-2 (IL-2) or a functional fragment or variant thereof, denoted as "IL2," but is interleukin-7 (IL-7) or a functional fragment or variant thereof, interleukin-12 (IL-12) or a functional fragment or variant thereof, interleukin-15 (IL-15) or a functional fragment or variant thereof, interleukin-18 (IL-18) or a functional fragment or variant thereof, interleukin-21 (IL-21) or a functional fragment or variant thereof, or interferon gamma or a functional fragment or variant thereof, or a combination thereof. [Fig. 125M-125P]Figures 125A and 125B show exemplary embodiments of multifunctional molecules comprising a TCRβV-binding portion described herein and a cytokine molecule (e.g., wild-type human IL2 or IL15-IL15R sushi fusion). Figures 125C-125T show exemplary embodiments of multifunctional molecules, e.g., comprising a tumor-associated antigen (TAA)-binding portion described herein, a TCRβV-binding portion, and at least one cytokine molecule, or a functional fragment or functional variant thereof. In some embodiments, the tumor-associated antigen (TAA)-binding portion and / or the TCRβV-binding portion is an antibody, antigen-binding fragment thereof, or antibody fragment. In some embodiments, the antigen-binding fragment or antibody fragment comprises a Fab, Fab', F(ab'), F(ab), variable fragment (Fv), domain antibody (dAb), or single-chain variable fragment (scFv). In some embodiments, the at least one cytokine molecule, or functional fragment or functional variant thereof, is selected from the group consisting of interleukin-2 (IL-2) or a functional fragment or functional variant thereof, interleukin-7 (IL-7) or a functional fragment or functional variant thereof, interleukin-12 (IL-12) or a functional fragment or functional variant thereof, interleukin-15 (IL-15) or a functional fragment or functional variant thereof, interleukin-18 (IL-18) or a functional fragment or functional variant thereof, interleukin-21 (IL-21) or a functional fragment or functional variant thereof, or interferon gamma or a functional fragment or functional variant thereof, or a combination thereof. In some embodiments, an exemplary embodiment of a multifunctional molecule, e.g., a multifunctional molecule comprising a tumor-associated antigen (TAA)-binding portion, a TCRβV-binding portion, and at least one cytokine molecule, or a functional fragment or functional variant thereof, described herein, comprises a dimerization module comprising an Fc region comprising an N297A mutation.Figures 125C-125H show exemplary embodiments of multifunctional molecules, e.g., multifunctional molecules comprising a tumor-associated antigen (TAA)-binding portion described herein, a TCRβV-binding portion, and at least one cytokine molecule, or a functional fragment or variant thereof, where the multifunctional molecules comprise an exemplary dimerization module, e.g., an Fc region comprising a knobs-in-holes mutation and a disulfide bridge. Figures 125I-125N show exemplary embodiments of multifunctional molecules, e.g., multifunctional molecules comprising a tumor-associated antigen (TAA)-binding portion described herein, a TCRβV-binding portion, and at least one cytokine molecule, or a functional fragment or variant thereof, where the multifunctional molecules comprise an exemplary dimerization module, e.g., an Fc region comprising a disulfide bridge but not a knobs-in-holes mutation. Figures 125O-125T show exemplary embodiments of multifunctional molecules, e.g., multifunctional molecules comprising a tumor-associated antigen (TAA)-binding portion described herein, a TCRβV-binding portion, and at least one cytokine molecule, or a functional fragment or variant thereof, where the multifunctional molecule comprises an exemplary dimerization module, e.g., an Fc region that does not comprise a disulfide bridge or a knob-in-hole mutation. Figures 125C-125E, 125I-125K, and 125O-135Q show exemplary embodiments of multifunctional molecules, e.g., multifunctional molecules comprising a tumor-associated antigen (TAA)-binding portion described herein, a TCRβV-binding portion, and at least one cytokine molecule, or a functional fragment or variant thereof, where the TCRβV-binding portion comprises an scFv. Figures 125F-125H, 125L-125N, and 125R-125T show exemplary embodiments of multifunctional molecules, e.g., multifunctional molecules comprising a tumor-associated antigen (TAA) binding portion described herein, a TCRβV binding portion, and at least one cytokine molecule or functional fragment or functional variant thereof, wherein the TCRβV binding portion comprises a Fab.Figure 125U shows an exemplary embodiment of a multifunctional molecule, e.g., a multifunctional molecule comprising a tumor-associated antigen (TAA) binding portion described herein, a TCRβV binding portion, and at least one cytokine molecule or a functional fragment or functional variant thereof, wherein the multifunctional molecule comprises two TAA binding portions. In some embodiments, the at least one cytokine molecule or functional fragment or variant thereof is not interleukin-2 (IL-2) or a functional fragment or variant thereof, denoted as "IL2," but is interleukin-7 (IL-7) or a functional fragment or variant thereof, interleukin-12 (IL-12) or a functional fragment or variant thereof, interleukin-15 (IL-15) or a functional fragment or variant thereof, interleukin-18 (IL-18) or a functional fragment or variant thereof, interleukin-21 (IL-21) or a functional fragment or variant thereof, or interferon gamma or a functional fragment or variant thereof, or a combination thereof. [Figure 125Q-125T]Figures 125A and 125B show exemplary embodiments of multifunctional molecules comprising a TCRβV-binding portion described herein and a cytokine molecule (e.g., wild-type human IL2 or IL15-IL15R sushi fusion). Figures 125C-125T show exemplary embodiments of multifunctional molecules, e.g., comprising a tumor-associated antigen (TAA)-binding portion described herein, a TCRβV-binding portion, and at least one cytokine molecule, or a functional fragment or functional variant thereof. In some embodiments, the tumor-associated antigen (TAA)-binding portion and / or the TCRβV-binding portion is an antibody, antigen-binding fragment thereof, or antibody fragment. In some embodiments, the antigen-binding fragment or antibody fragment comprises a Fab, Fab', F(ab'), F(ab), variable fragment (Fv), domain antibody (dAb), or single-chain variable fragment (scFv). In some embodiments, the at least one cytokine molecule, or functional fragment or functional variant thereof, is selected from the group consisting of interleukin-2 (IL-2) or a functional fragment or functional variant thereof, interleukin-7 (IL-7) or a functional fragment or functional variant thereof, interleukin-12 (IL-12) or a functional fragment or functional variant thereof, interleukin-15 (IL-15) or a functional fragment or functional variant thereof, interleukin-18 (IL-18) or a functional fragment or functional variant thereof, interleukin-21 (IL-21) or a functional fragment or functional variant thereof, or interferon gamma or a functional fragment or functional variant thereof, or a combination thereof. In some embodiments, an exemplary embodiment of a multifunctional molecule, e.g., a multifunctional molecule comprising a tumor-associated antigen (TAA)-binding portion, a TCRβV-binding portion, and at least one cytokine molecule, or a functional fragment or functional variant thereof, described herein, comprises a dimerization module comprising an Fc region comprising an N297A mutation.Figures 125C-125H show exemplary embodiments of multifunctional molecules, e.g., multifunctional molecules comprising a tumor-associated antigen (TAA)-binding portion described herein, a TCRβV-binding portion, and at least one cytokine molecule, or a functional fragment or variant thereof, where the multifunctional molecules comprise an exemplary dimerization module, e.g., an Fc region comprising a knobs-in-holes mutation and a disulfide bridge. Figures 125I-125N show exemplary embodiments of multifunctional molecules, e.g., multifunctional molecules comprising a tumor-associated antigen (TAA)-binding portion described herein, a TCRβV-binding portion, and at least one cytokine molecule, or a functional fragment or variant thereof, where the multifunctional molecules comprise an exemplary dimerization module, e.g., an Fc region comprising a disulfide bridge but not a knobs-in-holes mutation. Figures 125O-125T show exemplary embodiments of multifunctional molecules, e.g., multifunctional molecules comprising a tumor-associated antigen (TAA)-binding portion described herein, a TCRβV-binding portion, and at least one cytokine molecule, or a functional fragment or variant thereof, where the multifunctional molecule comprises an exemplary dimerization module, e.g., an Fc region that does not comprise a disulfide bridge or a knob-in-hole mutation. Figures 125C-125E, 125I-125K, and 125O-135Q show exemplary embodiments of multifunctional molecules, e.g., multifunctional molecules comprising a tumor-associated antigen (TAA)-binding portion described herein, a TCRβV-binding portion, and at least one cytokine molecule, or a functional fragment or variant thereof, where the TCRβV-binding portion comprises an scFv. Figures 125F-125H, 125L-125N, and 125R-125T show exemplary embodiments of multifunctional molecules, e.g., multifunctional molecules comprising a tumor-associated antigen (TAA) binding portion described herein, a TCRβV binding portion, and at least one cytokine molecule or functional fragment or functional variant thereof, wherein the TCRβV binding portion comprises a Fab.Figure 125U shows an exemplary embodiment of a multifunctional molecule, e.g., a multifunctional molecule comprising a tumor-associated antigen (TAA) binding portion described herein, a TCRβV binding portion, and at least one cytokine molecule or a functional fragment or functional variant thereof, wherein the multifunctional molecule comprises two TAA binding portions. In some embodiments, the at least one cytokine molecule or functional fragment or variant thereof is not interleukin-2 (IL-2) or a functional fragment or variant thereof, denoted as "IL2," but is interleukin-7 (IL-7) or a functional fragment or variant thereof, interleukin-12 (IL-12) or a functional fragment or variant thereof, interleukin-15 (IL-15) or a functional fragment or variant thereof, interleukin-18 (IL-18) or a functional fragment or variant thereof, interleukin-21 (IL-21) or a functional fragment or variant thereof, or interferon gamma or a functional fragment or variant thereof, or a combination thereof. [Figure 125U]Figures 125A and 125B show exemplary embodiments of multifunctional molecules comprising a TCRβV-binding portion described herein and a cytokine molecule (e.g., wild-type human IL2 or IL15-IL15R sushi fusion). Figures 125C-125T show exemplary embodiments of multifunctional molecules, e.g., comprising a tumor-associated antigen (TAA)-binding portion described herein, a TCRβV-binding portion, and at least one cytokine molecule, or a functional fragment or functional variant thereof. In some embodiments, the tumor-associated antigen (TAA)-binding portion and / or the TCRβV-binding portion is an antibody, antigen-binding fragment thereof, or antibody fragment. In some embodiments, the antigen-binding fragment or antibody fragment comprises a Fab, Fab', F(ab'), F(ab), variable fragment (Fv), domain antibody (dAb), or single-chain variable fragment (scFv). In some embodiments, the at least one cytokine molecule, or functional fragment or functional variant thereof, is selected from the group consisting of interleukin-2 (IL-2) or a functional fragment or functional variant thereof, interleukin-7 (IL-7) or a functional fragment or functional variant thereof, interleukin-12 (IL-12) or a functional fragment or functional variant thereof, interleukin-15 (IL-15) or a functional fragment or functional variant thereof, interleukin-18 (IL-18) or a functional fragment or functional variant thereof, interleukin-21 (IL-21) or a functional fragment or functional variant thereof, or interferon gamma or a functional fragment or functional variant thereof, or a combination thereof. In some embodiments, an exemplary embodiment of a multifunctional molecule, e.g., a multifunctional molecule comprising a tumor-associated antigen (TAA)-binding portion, a TCRβV-binding portion, and at least one cytokine molecule, or a functional fragment or functional variant thereof, described herein, comprises a dimerization module comprising an Fc region comprising an N297A mutation.Figures 125C-125H show exemplary embodiments of multifunctional molecules, e.g., multifunctional molecules comprising a tumor-associated antigen (TAA)-binding portion described herein, a TCRβV-binding portion, and at least one cytokine molecule, or a functional fragment or variant thereof, where the multifunctional molecules comprise an exemplary dimerization module, e.g., an Fc region comprising a knobs-in-holes mutation and a disulfide bridge. Figures 125I-125N show exemplary embodiments of multifunctional molecules, e.g., multifunctional molecules comprising a tumor-associated antigen (TAA)-binding portion described herein, a TCRβV-binding portion, and at least one cytokine molecule, or a functional fragment or variant thereof, where the multifunctional molecules comprise an exemplary dimerization module, e.g., an Fc region comprising a disulfide bridge but not a knobs-in-holes mutation. Figures 125O-125T show exemplary embodiments of multifunctional molecules, e.g., multifunctional molecules comprising a tumor-associated antigen (TAA)-binding portion described herein, a TCRβV-binding portion, and at least one cytokine molecule, or a functional fragment or variant thereof, where the multifunctional molecule comprises an exemplary dimerization module, e.g., an Fc region that does not comprise a disulfide bridge or a knob-in-hole mutation. Figures 125C-125E, 125I-125K, and 125O-135Q show exemplary embodiments of multifunctional molecules, e.g., multifunctional molecules comprising a tumor-associated antigen (TAA)-binding portion described herein, a TCRβV-binding portion, and at least one cytokine molecule, or a functional fragment or variant thereof, where the TCRβV-binding portion comprises an scFv. Figures 125F-125H, 125L-125N, and 125R-125T show exemplary embodiments of multifunctional molecules, e.g., multifunctional molecules comprising a tumor-associated antigen (TAA) binding portion described herein, a TCRβV binding portion, and at least one cytokine molecule or functional fragment or functional variant thereof, wherein the TCRβV binding portion comprises a Fab.Figure 125U shows an exemplary embodiment of a multifunctional molecule, e.g., a multifunctional molecule comprising a tumor-associated antigen (TAA) binding portion described herein, a TCRβV binding portion, and at least one cytokine molecule or a functional fragment or functional variant thereof, wherein the multifunctional molecule comprises two TAA binding portions. In some embodiments, the at least one cytokine molecule or functional fragment or variant thereof is not interleukin-2 (IL-2) or a functional fragment or variant thereof, denoted as "IL2," but is interleukin-7 (IL-7) or a functional fragment or variant thereof, interleukin-12 (IL-12) or a functional fragment or variant thereof, interleukin-15 (IL-15) or a functional fragment or variant thereof, interleukin-18 (IL-18) or a functional fragment or variant thereof, interleukin-21 (IL-21) or a functional fragment or variant thereof, or interferon gamma or a functional fragment or variant thereof, or a combination thereof. [Figure 126A]

[0272] Figures 126A, 126B, and 126C show that an anti-CD20 TriSTAR molecule, an exemplary embodiment of a multifunctional molecule provided herein, induces highly potent Vβ-specific expansion, CD8+ T cell activation, and B cell killing in vitro. Figure 126A shows in vitro murine Vβ T cell expansion, Figure 126B shows in vitro murine CD8+ T cell activation and expansion, and Figure 126C shows in vitro murine B cell depletion. [Figure 126B] Figures 126A, 126B, and 126C show that an anti-CD20 TriSTAR molecule, an exemplary embodiment of a multifunctional molecule provided herein, induces highly potent Vβ-specific expansion, CD8+ T cell activation, and B cell killing in vitro. Figure 126A shows in vitro murine Vβ T cell expansion, Figure 126B shows in vitro murine CD8+ T cell activation and expansion, and Figure 126C shows in vitro murine B cell depletion. [Figure 126C] Figures 126A, 126B, and 126C show that an anti-CD20 TriSTAR molecule, an exemplary embodiment of a multifunctional molecule provided herein, induces highly potent Vβ-specific expansion, CD8+ T cell activation, and B cell killing in vitro. Figure 126A shows in vitro murine Vβ T cell expansion, Figure 126B shows in vitro murine CD8+ T cell activation and expansion, and Figure 126C shows in vitro murine B cell depletion. [Figure 127]

[0273] Figure 127 shows that an exemplary embodiment of a multifunctional molecule described herein, the anti-CD20 TriSTAR T cell engager (TCE), also exhibits potent B cell depletion in vivo upon a single IP dose of 3 mg / kg. In the same study, an anti-CD3 / CD20 bispecific antibody was also tested and shown to have comparable efficacy in vivo. [Figure 128A]

[0274] Figures 128A and 128B show in vitro expansion using anti-MSLN TriSTAR (BLM0155), an exemplary embodiment of a multifunctional molecule provided herein, and CD3 TCE (BMM0388). Both TriSTAR and CD3 engagers expanded in the presence of plate-bound MSLN. Figure 128A shows CD25+ % and Figure 128B shows EC50 and Emax (%). [Figure 128B] Same as above. [Figure 129A]

[0275] Figures 129A and 129B show in vitro expansion using an anti-MSLN Vb bispecific TCE (BLM0158, 1x1 format; BMM0274, 2x2 format), an exemplary embodiment of a multifunctional molecule provided herein, and a CD3 TCE. Both Vb and CD3 engagers expanded in the presence of plate-bound MSLN. The CD3 TCE expanded much more. Figure 129A shows the CD25+ % and Figure 129B shows the EC50 and Emax (%). [Figure 129B] Same as above. [Figure 130A]

[0276] Figures 130A and 130B show in vitro cytotoxicity by anti-MSLN TriSTAR (BLM0155, 1x1x1 TriSTAR; BMM0462, 2x1x1 TriSTAR format), an exemplary embodiment of a multifunctional molecule provided herein, and CD3 TCE (BMM0388). Figure 130A shows % cytotoxicity, and Figure 130B shows EC50 and Emax (%). [Figure 130B] Same as above. [Figure 131A]

[0277] Figures 131A and 131B show in vitro cytotoxicity by anti-MSLN Vb bispecific TCEs (BLM0158, 1x1 format; BMM0274, 2x2 format), which are exemplary embodiments of the multifunctional molecules provided herein, and CD3 TCEs. Figure 131A shows % cytotoxicity, and Figure 131B shows EC50 and Emax (%). [Figure 131B] Same as above. [Figure 132A]

[0278] Figures 132A and 132B show in vitro expansion using exemplary embodiments of the multifunctional molecules provided herein, anti-MSLN TriSTAR mouse surrogates (BMM0449, 1x1x1 TriSTAR format; BMM0456, 2x1x1 TriSTAR format), and CD3 TCE (BMM0456, 1x1 CD3 TCE; BMM0469, 2x1 CD3 TCE). All TCE constructs showed robust expansion in the presence of plate-bound MSLN. Figure 132A shows CD25+ % and Figure 132B shows EC50 and Emax (%). [Figure 132B] Same as above. [Figure 133A-133B]

[0279] Figures 133A-133D show in vitro expansion using exemplary embodiments of the multifunctional molecules provided herein, anti-MSLN Vb TCE mouse surrogates (BMM0446, 1x1 format; BMM0439, 2x2 format), and CD3 TCE (BMM0456). All constructs showed expansion in the presence of plate-bound MSLN. Figures 133A and 133C show CD25+ % and Figures 133B and 133D show EC50 and Emax (%). [Fig. 133C-133D] Same as above. [Figure 134]

[0280] Figure 134 shows in vitro cytotoxicity of EMT6 cells by anti-MSLN TriSTAR mouse surrogate (BMM0449) and CD3 TCE (BMM0456). [Figure 135A]

[0281] Figure 135A and Figure 135B show the in vitro cytotoxicity of exemplary embodiments of the multifunctional molecules provided herein, EMT6 cell anti-MSLN Vb TCE mouse surrogates (BMM0446, 1x1 format; BMM0439, 2x2 format), and CD3 TCE. Figure 135A shows % cytotoxicity, and Figure 135B shows EC50 and Emax (%). [Figure 135B] Same as above. [Figure 136A]

[0282] Figures 136A and 136B show that an exemplary embodiment of the multifunctional molecule provided herein, an anti-MSLN TriSTAR mouse surrogate (BMM0449), exhibits potent anti-tumor activity compared to αCD3 TCE (BMM0456) in an EMT6 syngeneic mouse model. Tumor-bearing mice were given the test substance at a weekly dose of 3 mg / kg for 4 weeks. Figure 136A shows a scheme of the experimental design, and Figure 136B shows the average tumor volume. [Figure 136B] Same as above. [Figure 137]

[0283] Figure 137 shows PK data for an anti-MSLN TriSTAR mouse surrogate (BMM0449), an exemplary embodiment of a multifunctional molecule provided herein. [Figure 138]

[0284] Figure 138 shows that anti-gp75Vβ bispecific TCE mouse surrogates (BNM0104, 1x1 format and BNM0869, 2x2 format), exemplary embodiments of multifunctional molecules provided herein, showed limited activity in solid (B16F10) syngeneic tumors. Limited anti-tumor activity of 1x1 and 2x2Vβxgp75 TCE, exemplary embodiments of multifunctional molecules provided herein, in the B16F10 solid tumor model. [Figure 139]

[0285] Figure 139 shows that exemplary embodiments of multifunctional molecules provided herein, anti-gp75 Vβ mouse surrogate TCEs (BNM0104, 1x1 format and BNM0869, 2x2 format), are highly active when combined with an exemplary multifunctional molecule comprising an anti-Vβ binder and IL-2 (BKM0307). The 1x1 and 2x2 Vβxgp75 T cell engagers are active in the B16F10 tumor model when administered with or after an exemplary multifunctional molecule comprising an anti-Vβ binder and IL-2. [Figure 140]

[0286] Figure 140 shows that an exemplary embodiment of a multifunctional molecule provided herein, the anti-gp75 TriSTAR TCE mouse surrogate (BNM0094), promotes superior anti-tumor activity compared to anti-gp75 / αCD3 TCE (BNM0092) in the B16F10 model. Significant anti-tumor activity was observed in refractory B16 mice with TriSTAR TCE. [Figure 141A]

[0287] Figures 141A-C show the pharmacokinetics of the TriSTAR construct (BNM0094), an exemplary embodiment of a multifunctional molecule provided herein. Figure 141A shows the results for BNM0094, an exemplary embodiment of a multifunctional molecule provided herein, and Figure 141B shows the results for the CD3xgp75 bispecific molecule BNM0092. Figure 141C shows the experimental design. [Figure 141B] Same as above. [Figure 141C] Same as above. [Figure 142A]

[0288] Figures 142A-142C show that TriSTAR, an exemplary embodiment of a multifunctional molecule provided herein, promotes greater expansion of cytotoxic CD8 T cells in the tumor microenvironment in B16F10 tumors. Figure 142A shows the CD8% of CD45 cells, Figure 142B shows the CD8% of CD25 cells, and Figure 142C shows the CD8% of granzyme B cells. [Figure 142B] Same as above. [Figure 142C] Same as above. [Figure 143A]

[0289] Figures 143A and 143B show Vβ13+ T cell expansion in the tumor microenvironment (TME) by Tri-STAR, an exemplary embodiment of a multifunctional molecule provided herein. Figure 143A shows CD8 Vb cells / mg tumor, and Figure 143B shows CD8% Vb subset frequency. [Figure 143B] Same as above. DETAILED DESCRIPTION OF THE INVENTION

[0117] Detailed Description definition

[0290] Certain details are described herein to provide a thorough understanding of various embodiments. However, those skilled in the art will understand that the present disclosure may be practiced without these details. In other instances, well-known structures are not shown or described in detail to avoid unnecessarily obscuring the description of the embodiments.

[0118]

[0291] Unless the context otherwise requires, throughout this specification and the claims that follow, the terms "comprise" and variations thereof, such as "comprises" and "comprising," are to be interpreted in their open, inclusive sense, i.e., "including, but not limited to." Additionally, the headings provided herein are for convenience only and do not interpret the scope or meaning of the claimed disclosure.

[0119]

[0292] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. The use of the terms "a" or "an," when used with the term "comprising" herein, can mean "one," but is also consistent with the meaning of "one or more," "at least one," and "one or more than one."

[0120]

[0293] It should also be noted that the term "or" is generally used in its sense including "and / or" unless the context clearly indicates otherwise.

[0294] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, suitable methods and materials are described below.

[0121]

[0295] When referring to a measurable value, such as an amount, duration, or the like, the term "about" is meant to encompass a variation of ±20% from the particular value, or in some instances ±10%, or in some instances ±5%, or in some instances ±1%, or in some instances ±0.1%, where such variation is appropriate for practicing the disclosed methods. As used herein, "about" and "approximately" generally refer to an acceptable degree of error for the measured quantity given the nature or precision of the measurement. Exemplary degrees of error are within 20 percent (%), typically within 10%, and more typically within 5% of a given range of values.

[0122]

[0296] The term "obtain" or "obtaining," as used herein, refers to gaining possession of a physical entity (e.g., a sample, a polypeptide, a nucleic acid, or a sequence) or a value, e.g., a numerical value, by "directly obtaining" or "indirectly obtaining" the physical entity or value. "Directly obtaining" means performing a process (e.g., performing a synthetic or analytical method) to obtain the physical entity or value. "Indirectly obtaining" refers to receiving the physical entity or value from another party or source (e.g., a third party that directly obtained the physical entity or value). Directly obtaining a physical entity includes performing a process that involves a physical change of a physical substance, e.g., a starting material. Directly obtaining a value includes performing a process that involves a physical change of a sample or another substance, e.g., performing an analytical process that involves a physical change of a substance, e.g., a sample.

[0123]

[0297] As used herein, "antibody molecule" refers to a protein, e.g., an immunoglobulin chain, or fragment thereof, that contains at least one immunoglobulin variable domain structure and / or sequence. Antibody molecules encompass antibodies (e.g., full-length antibodies) and antibody fragments. In some embodiments, an antibody molecule comprises an antigen-binding or functional fragment of a full-length antibody or a full-length immunoglobulin chain. For example, a full-length antibody is an immunoglobulin (Ig) molecule (e.g., an IgG antibody) that occurs naturally or is formed by the recombinant process of normal immunoglobulin gene fragments. In embodiments, an antibody molecule refers to an immunologically active antigen-binding portion of an immunoglobulin molecule, e.g., an antibody fragment. An antibody fragment, e.g., a functional fragment, is a portion of an antibody, e.g., a Fab, Fab', F(ab')2, F(ab)2, variable fragment (Fv), domain antibody (dAb), or single-chain variable fragment (scFv). A functional antibody fragment binds to the same antigen recognized by an intact (e.g., full-length) antibody. The term "antibody fragment" or "functional fragment" also includes isolated fragments consisting of the variable regions, such as an "Fv" fragment consisting of the variable regions of the heavy and light chains, or a recombinant single-chain polypeptide molecule in which the variable regions of the light and heavy chains are linked by a peptide linker ("scFv protein"). In some embodiments, an antibody fragment does not include a portion of an antibody that does not have antigen-binding activity, such as an Fc fragment or a single amino acid residue. Exemplary antibody molecules include full-length antibodies and antibody fragments, such as dAb (domain antibodies), single-chain, Fab, Fab', and F(ab')2 fragments, and single-chain variable fragments (scFv). In some embodiments, the antibody molecule is an antibody mimetic. In some embodiments, the antibody molecule is or includes an antibody-like framework or scaffold, such as fibronectin, ankyrin repeats (e.g., designed ankyrin repeat proteins (DARPins)), avimers, affibody affinity ligands, anticalins, or affilin molecules.

[0124]

[0298] The term "human-like antibody molecule," as used herein, refers to a humanized antibody molecule, a human antibody molecule, or an antibody molecule having at least 95% sequence identity to a non-murine germline framework region, e.g., FR1, FR2, FR3, and / or FR4. In some embodiments, a human-like antibody molecule comprises a human germline framework region, e.g., a framework region having at least 95% sequence identity to FR1, FR2, FR3, and / or FR4 of a human germline framework region. In some embodiments, a human-like antibody molecule is a recombinant antibody. In some embodiments, a human-like antibody molecule is a humanized antibody molecule. In some embodiments, a human-like antibody molecule is a human antibody molecule. In some embodiments, a human-like antibody molecule is a phage-displayed or yeast-displayed antibody molecule. In some embodiments, a human-like antibody molecule is a chimeric antibody molecule. In some embodiments, a human-like antibody molecule is a CDR-grafted antibody molecule.

[0125]

[0299] As used herein, "immunoglobulin variable domain sequence" refers to an amino acid sequence capable of forming the structure of an immunoglobulin variable domain. For example, the sequence may include all or part of the amino acid sequence of a naturally occurring variable domain. For example, the sequence may or may not include one, two, or more N-terminal or C-terminal amino acids, or may include other modifications compatible with forming a protein structure.

[0126]

[0300] In embodiments, the antibody molecule is monospecific, e.g., comprises binding specificity for a single epitope. In some embodiments, the antibody molecule is multispecific, e.g., comprises multiple immunoglobulin variable domain sequences, wherein a first immunoglobulin variable domain sequence has binding specificity for a first epitope and a second immunoglobulin variable domain sequence has binding specificity for a second epitope. In some embodiments, the antibody molecule is a bispecific antibody molecule. As used herein, a "bispecific antibody molecule" refers to an antibody molecule that has specificity for more than one (e.g., two, three, four, or more) epitopes and / or antigens.

[0127]

[0301] As used herein, "antigen" (Ag) refers to a molecule capable of eliciting an immune response, e.g., involving activation of certain immune cells and / or antibody production. Any macromolecule, including almost any protein or peptide, can be an antigen. Antigens can also be derived from genomic recombinants or DNA. For example, any DNA containing a nucleotide sequence or partial nucleotide sequence encoding a protein capable of eliciting an immune response encodes an "antigen." In embodiments, an antigen need not be encoded solely by the full-length nucleotide sequence of a gene, nor need it be encoded by a gene at all. In embodiments, antigens can be synthesized or derived from a biological sample, e.g., a tissue sample, a tumor sample, cells, or a fluid containing other biological components. As used herein, "tumor antigen" or, synonymously, "cancer antigen" includes any molecule present on or associated with a cancer, e.g., a cancer cell, or the tumor microenvironment, capable of eliciting an immune response. As used herein, "immune cell antigen" includes any molecule present on or associated with an immune cell, capable of eliciting an immune response.

[0128]

[0302] The "antigen-binding site" or "binding site" of an antibody molecule refers to the portion of an antibody molecule, e.g., an immunoglobulin (Ig) molecule, that participates in antigen binding. In embodiments, the antigen-binding site is formed by amino acid residues from the variable regions (V) of the heavy (H) and light (L) chains. Three highly divergent stretches within the variable regions of the heavy and light chains, called hypervariable regions, are located between more conserved adjacent regions called "framework regions" (FR). FRs are amino acid sequences naturally found between and adjacent to the hypervariable regions in immunoglobulins. In embodiments, in an antibody molecule, the three hypervariable regions of the light chain and the three hypervariable regions of the heavy chain are positioned relative to each other in three-dimensional space to form an antigen-binding surface complementary to the three-dimensional surface of a bound antigen. The three hypervariable regions of each of the heavy and light chains are referred to as "complementarity-determining regions" or "CDRs." Framework regions and CDRs are defined and described, for example, in Kabat, E. A. et al. (1991) Sequences of Proteins of Immunological Interest, 5th ed., USDapartment of Health and Human Services, NIH Publication No. 91-3242, and Chothia, C. et al. (1987) J. Mol. Biol. 196:901-917. Each variable chain (e.g., variable heavy chain and variable light chain) typically consists of three CDRs and four FRs, arranged from amino terminus to carboxy terminus in the following amino acid order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.

[0129]

[0303] As used herein, "immune cell" refers to any of a variety of cells that function in the immune system, e.g., defend against infection and foreign agents. In embodiments, the term includes leukocytes, e.g., neutrophils, eosinophils, basophils, lymphocytes, and monocytes. Innate leukocytes include phagocytes (e.g., macrophages, neutrophils, and dendritic cells), mast cells, eosinophils, basophils, and natural killer cells. Innate leukocytes identify and eliminate pathogens by attacking larger pathogens through contact or by engulfing and killing microorganisms, and are mediators of the activation of the adaptive immune response. Cells of the adaptive immune system are a specialized type of leukocyte called lymphocytes. B cells and T cells are important types of lymphocytes and are derived from hematopoietic stem cells in the bone marrow. B cells are involved in the humoral immune response, and T cells are involved in the cellular immune response. The term "immune cell" includes immune effector cells.

[0130]

[0304] The term "immune effector cell," as used herein, refers to a cell that is involved in an immune response, e.g., promoting an immune effector response. Examples of immune effector cells include, but are not limited to, T cells, e.g., alpha / beta T cells and gamma / delta T cells, B cells, natural killer (NK) cells, natural killer T cells, and mast cells.

[0131]

[0305] The term "effector function" or "effector response" refers to a specialized function of a cell. The effector function of a T cell can be, for example, cytolytic activity or helper activity, including secretion of cytokines.

[0132]

[0306] The terms "polypeptide," "peptide," and "protein" (when single-chain) are used interchangeably herein to refer to polymers of amino acids of any length. The polymers can be linear or branched, can contain modified amino acids, and can be interrupted by non-amino acids. These terms also encompass amino acid polymers that have been modified; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation, such as conjugation with a labeling component. Polypeptides can be isolated from natural sources, produced by recombinant techniques from eukaryotic or prokaryotic hosts, or can be the product of synthetic techniques.

[0133]

[0307] The terms "nucleic acid," "nucleic acid sequence," "nucleotide sequence," or "polynucleotide sequence," and "polynucleotide" are used interchangeably. They refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or their analogs. A polynucleotide can be either single-stranded or double-stranded, and if single-stranded, can be the coding strand or the non-coding (antisense) strand. A polynucleotide can comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. The sequence of nucleotides can be interrupted by non-nucleotide components. A polynucleotide can be further modified after polymerization, for example, by conjugation with a labeling component. A nucleic acid can be a recombinant polynucleotide, or a polynucleotide of genomic, cDNA, semi-synthetic, or synthetic origin that does not occur in nature or is linked to another polynucleotide in a non-naturally occurring sequence.

[0134]

[0308] The term "isolated," as used herein, refers to material that is removed from its original or natural environment (e.g., the natural environment if it occurs in nature). For example, a naturally occurring polynucleotide or polypeptide present in a living animal is not isolated, but an identical polynucleotide or polypeptide that has been separated by human intervention from some or all of the coexisting materials in the natural system is isolated. Such a polynucleotide may be part of a vector, and / or such a polynucleotide or polypeptide may be part of a composition, and such a vector or composition may still be isolated in that it is not part of the environment in which it is found in nature. An isolated polynucleotide (ribonucleic acid (RNA), deoxyribonucleic acid (DNA)) or polypeptide does not contain the genes / nucleic acids or sequences / amino acids that flank it in its naturally occurring state.

[0135]

[0309] The compositions and methods of the present invention encompass polypeptides and nucleic acids having a specific sequence or a sequence substantially identical or similar thereto, for example, a sequence that is at least 80%, 85%, 90%, 95% identical, or more identical to the specific sequence. In the context of amino acid sequences, the term "substantially identical" is used herein to refer to a first amino acid sequence that contains a sufficient or minimal number of amino acid residues that are i) identical to a second amino acid sequence, or ii) are conservative substitutions for aligned amino acid residues in a second amino acid sequence, such that the first and second amino acid sequences can have a common structural domain and / or a common functional activity. For example, an amino acid sequence containing a common structural domain that has at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to a reference sequence, such as the sequences provided herein. The term "substantially identical" as used herein in the context of nucleotide sequences refers to a first nucleic acid sequence that contains a sufficient number or a minimum number of nucleotides identical to aligned nucleotides in a second nucleic acid sequence such that the first and second nucleotide sequences encode polypeptides having a common functional activity, or encode a common polypeptide structural domain or common polypeptide functional activity. For example, a nucleotide sequence having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to a reference sequence, such as a sequence provided herein.

[0136]

[0310] The term "variant" refers to a polypeptide having substantially the same amino acid sequence as a reference amino acid sequence or encoded by a substantially identical nucleotide sequence. In some embodiments, the variant is a functional variant. In some embodiments, the TCRβV variant can bind to TCRα and form a TCRα:β complex.

[0137]

[0311] The term "functional variant" refers to a polypeptide having an amino acid sequence substantially identical to a reference amino acid sequence, or a polypeptide encoded by a substantially identical nucleotide sequence, which may have one or more activations of the reference amino acid sequence.

[0138]

[0312] Calculation of homology or sequence identity (these terms are used interchangeably herein) between sequences is performed as follows: To determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., for optimal alignment, gaps can be introduced into one or both of the first and second amino acid or nucleic acid sequences, and non-homologous sequences can be ignored for comparison purposes). In a preferred embodiment, the length of the reference sequence aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%, 60%, and even more preferably at least 70%, 80%, 90%, or 100% of the length of the reference sequence. Next, the amino acid residues or nucleotides at corresponding amino acid or nucleotide positions are compared. If a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, the molecules are identical at that position (as used herein, amino acid or nucleic acid "identity" is equivalent to amino acid or nucleic acid "homology").

[0139]

[0313] The percent identity between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps and the length of each gap that need to be introduced for optimal alignment of the two sequences.Comparing sequences and determining the percent identity between two sequences can be achieved using a mathematical algorithm.In a preferred embodiment, the percent identity between two amino acid sequences is determined using the Needleman and Wunsch ((1970) J. Mol. Biol. 48:444-453) algorithm, which is incorporated into the GAP program in the GCG software package (available at http: / / www.gcg.com), using either a Blossum62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4, and a length weight of 1, 2, 3, 4, 5, or 6. In yet another preferred embodiment, percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (available at http: / / www.gcg.com), using the NWSgapdna.CMP matrix and gap weights of 40, 50, 60, 70, or 80 and length weights of 1, 2, 3, 4, 5, or 6. A particularly preferred set of parameters (and those to be used unless otherwise specified) is a Blossum62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5.

[0140]

[0314] Percent identity between two amino acid or nucleotide sequences can be determined using the algorithm of E. Meyers and W. Miller ((1989) CABIOS 4:11-17) as incorporated into the ALIGN program (version 2.0), using a PAM120 residue weighting table, a gap length penalty of 12, and a gap penalty of 4. The nucleic acid and protein sequences described herein can be used as "query sequences" to conduct searches against public databases, e.g., to identify other family members or related sequences. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul et al. (1990) J. Mol. Biol. 215:403-10. BLAST nucleotide searches can be performed using the NBLAST program, score=100, word length=12, to obtain nucleotide sequences homologous to the nucleic acid molecules of the invention. BLAST protein searches can be performed with the XBLAST program, score=50, wordlength=3 to obtain amino acid sequences homologous to the protein molecules of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used.

[0141]

[0315] It is understood that the molecules of the present invention may have additional conservative or non-essential amino acid substitutions which do not substantially affect their function.

[0316] The term "amino acid" is intended to encompass all molecules, whether natural or synthetic, that contain both amino and acid functionalities and can be included in naturally occurring amino acid polymers. Exemplary amino acids include naturally occurring amino acids; their analogs, derivatives and homologs; amino acid analogs with variant side chains; and all stereoisomers of any of the foregoing. As used herein, the term "amino acid" includes both D or L optical isomers and peptidomimetics.

[0142]

[0317] A "conservative amino acid substitution" is one in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[0143]

[0318] As used herein, the term "molecule," as used in, e.g., antibody molecules, cytokine molecules, receptor molecules, includes full-length naturally occurring molecules as well as variants, e.g., functional variants (e.g., truncations, fragments, mutations (e.g., substantially similar sequences) or derivatized forms thereof), so long as at least one function and / or activity of the unmodified (e.g., naturally occurring) molecule remains.

[0144]

[0319] As used herein, the term "mutation" refers to an alteration in the nucleotide sequence of the genome of an organism, virus, or extrachromosomal DNA. In some embodiments, a mutation may be a large-scale mutation, such as an amplification (or gene duplication) or repetition of a chromosomal segment, a deletion of a large chromosomal region, a chromosomal rearrangement (e.g., a chromosomal translocation, a chromosomal inversion, a non-homologous chromosomal crossover, and an interstitial deletion), and a loss of heterozygosity. In some embodiments, a mutation may be a small-scale mutation, such as an insertion, deletion, or substitution mutation. As used herein, the term "substitution mutation" refers to a transition in which a single nucleotide is exchanged for another nucleotide.

[0145]

[0320] "Interleukin-2," also known as IL2, IL-2, IL 2, TCGF, ​​lymphokine, and interleukin 2, as referred to herein, includes any recombinant or naturally occurring form of IL-2, or a variant or homolog thereof, that has or maintains IL-2 activity (e.g., at least 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity). In some aspects, the variant or homolog has at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity over the entire sequence or a portion of the sequence (e.g., a 50, 100, 150, or 200 contiguous amino acid portion) compared to naturally occurring IL-2. In some embodiments, the IL-2 is substantially identical to the protein identified by UniProt reference number P60568, or a variant or homolog having substantial identity thereto. Anti-TCRβV antibody Human T cell receptor (TCR) complex

[0321] TCRs are typically disulfide-linked, membrane-anchored heterodimeric proteins consisting of highly variable alpha (α) and beta (β) chains expressed as part of a complex with an invariant CD3 chain molecule. The TCR on αβ T cells is formed by a heterodimer of one alpha chain and one beta chain. Each alpha or beta chain consists of a constant domain and a highly variable domain classified as an immunoglobulin superfamily (IgSF) fold. TCRβ V chains can be further classified into 30 subfamilies (TRBV1-30). Despite their high structural and functional homology, amino acid sequence homology within TRBV genes is very low. Only four of the approximately 95 amino acids are identical, while 10 additional amino acids are conserved across all subfamilies (see Table 9 for an alignment of TCRβ amino acid sequences). Nevertheless, TCRs formed between highly divergent alpha and beta chains show significant structural homology (Figures 25A and 25B) and elicit similar functions, e.g., T cell activation.

[0146]

[0322] T cell receptors (TCRs) can be found on the surface of T cells. TCRs recognize antigens, e.g., peptides, bound to major histocompatibility complex (MHC) molecules, presented on the surface of cells, e.g., antigen-presenting cells. TCRs are heterodimeric molecules and can include an alpha chain, a beta chain, a gamma chain, or a delta chain. TCRs containing an alpha chain and a beta chain are also called TCRαβ. The TCRβ chain consists of the following regions (also known as segments): variable (V), diverse (D), joining (J), and constant (C) (see Mayer G. and Nyland J. (2010) Chapter 10: Major Histocompatibility Complex and T-cell Receptors—Role in Immune Responses. In: Microbiology and Immunology online, University of South Carolina School of Medicine). The TCRα chain consists of the V, J, and C regions. Rearrangement of the T cell receptor (TCR) by somatic recombination of the V (variable), D (diverse), J (joining), and C (constant) regions is a critical event in the development and maturation of T cells. TCR gene rearrangement occurs in the thymus.

[0147]

[0323] A TCR may comprise a receptor complex known as a TCR complex, which comprises a TCR heterodimer comprising an alpha chain and a beta chain, and a dimeric signaling molecule, e.g., a CD3 co-receptor, e.g., CD3δ / ε and / or CD3γ / ε.

[0148]

[0324] As used herein, the term "T cell receptor beta variable chain" or "TCRβV" refers to the extracellular region of the T cell receptor beta chain that contains the antigen recognition domain of the T cell receptor. The term TCRβV includes isoforms, mammalian, e.g., human TCRβV, human species homologs, and analogs that share at least one epitope with the TCRβV. Human TCRβVs include, but are not limited to, TCRβ V6 subfamily, TCRβ V10 subfamily, TCRβ V12 subfamily, TCRβ V5 subfamily, TCRβ V7 subfamily, TCRβ V11 subfamily, TCRβ V14 subfamily, TCRβ V16 subfamily, TCRβ V18 subfamily, TCRβ V9 subfamily, TCRβ V13 subfamily, TCRβ V4 subfamily, TCRβ V3 subfamily, TCRβ V2 subfamily, TCRβ V15 subfamily, TCRβ V30 subfamily, TCRβ V19 subfamily, TCRβ V27 subfamily, TCRβ V28 subfamily, TCRβ V24 subfamily, TCRβ V20 subfamily, TCRβ V25 subfamily, TCRβ V29 subfamily, TCRβ V1 subfamily, TCRβ V1 subfamily, TCRβ V17 subfamily, TCRβ V21 subfamily, TCRβ In some embodiments, the TCRβ V6 subfamily includes gene families including the TCRβ V6-4 subfamily, the TCRβ V23 subfamily, or the TCRβ V26 subfamily, as well as subfamilies including members of said subfamilies and variants thereof (e.g., structural or functional variants thereof). * 01. TCRβ V6-4 * 02. TCRβ V6-9 * 01. TCRβ V6-8 * 01. TCRβ V6-5 * 01. TCRβ V6-6 * 02. TCRβ V6-6 * 01. TCRβ V6-2 * 01. TCRβ V6-3 * 01 or TCRβ V6-1 *In some embodiments, the TCRβV comprises TCRβV6-5. * 01, or variants thereof, e.g., variants having 85%, 90%, 95%, 99% or more identity to the naturally occurring sequence. * 01 is also known as TRBV65; TCRBV6S5; TCRBV13S1, or TCRβ V13.1. * 01, for example, human TCRβV6-5 * The amino acid sequence of TCRβV6-5 is known in the art and is provided, for example, by IMGT ID L36092. * 01 is encoded by the nucleic acid sequence of SEQ ID NO: 43, or a sequence having 85%, 90%, 95%, 99% or more identity thereto. * 01 comprises the amino acid sequence of SEQ ID NO: 44, or a sequence having 85%, 90%, 95%, 99% or more identity thereto.

[0149] SEQ ID NO: 43 ATGAGCATCGGCCTCCTGTGCTGTGCAGCCTTGTCTCTCCTGGGCAGGTCCAGTGAATGCTGGTGTCACTCAGACCCCAAAATTCCAGGTCCTGAAGACAGGACAGAGCATGACACTGCAGTGTGCCCAGGATATGAACCATGAATACATGTCCTGGTATCGACAAGACC CAGGCATGGGGCTGAGGCTGATTCATTACTCAGTTGGTGCTGGTATCACTGACCAAGGAGAAGTCCCCAATGGCTACAATGTCTCCAGATCAACCACAGAGGATTTCCCGCTCAGGCTGCTGTCGGCTGCTCCCTCCCAGACATCTGTGTACTTCTGTGCCAGCAGTTACTC.

[0150] SEQ ID NO: 44 MSIGLCCAALSLLWAGPVNAGVTQTPKFQVLKTGQSMTLQCAQDMNHEYMSWYRQDPGMGLRLIHY-SVGAGITDQGEVPNGYNVSRSTTEDFPLRLLSAAPSQTSVYFCASSY.

[0151] TCR beta V (TCRβV)

[0325] Diversity in the immune system allows for defense against a vast array of pathogens. Because the size of the germline genome is limited, diversity is achieved not only through the process of V(D)J recombination, but also through deletion of nucleotide junctions (the junctions between the VD and DJ segments) and pseudorandom, non-templated addition of nucleotides. TCR beta genes undergo genetic rearrangement to generate diversity.

[0152]

[0326] The TCR V beta repertoire varies between individuals and populations due to, for example, seven frequent inactivating polymorphisms in functional gene segments and large insertion / deletion-associated polymorphisms encompassing two V beta gene segments.

[0153]

[0327] Provided herein, for example, are, inter alia, antibody molecules and fragments thereof that bind to, e.g., specifically bind to, e.g., a human TCR beta V chain (TCRβV), e.g., a TCRβV gene family (also referred to as a group), e.g., a TCRβV subfamily (also referred to as a subgroup), as described herein. TCR beta V families and subfamilies are known in the art and are described, for example, in Yassai et al. (2009) Immunogenetics 61(7):493-502; Wei S. and Concannon P. (1994) Human Immunology 41(3):201-206. The antibodies described herein can be recombinant antibodies, e.g., recombinant non-murine antibodies, e.g., recombinant human or humanized antibodies.

[0154]

[0328] The terms TCRBV, TCRVB, TRBV, TCRβV, TCRVβ or TRβV are used interchangeably herein and refer to a TCR beta V chain, e.g., as described herein.

[0155]

[0329] In some embodiments, provided herein are anti-TCRβ antibody molecules that bind to a human TCRβV, e.g., a TCRβV family, e.g., a gene family, or variant thereof. In some embodiments, the TCRβV gene family includes one or more subfamilies, e.g., as described herein, e.g., as described in Figure 4, Table 8A, or 8B. In some embodiments, the TCRβ V gene family is selected from the group consisting of TCRβ V6 subfamily, TCRβ V10 subfamily, TCRβ V12 subfamily, TCRβ V5 subfamily, TCRβ V7 subfamily, TCRβ V11 subfamily, TCRβ V14 subfamily, TCRβ V16 subfamily, TCRβ V18 subfamily, TCRβ V9 subfamily, TCRβ V13 subfamily, TCRβ V4 subfamily, TCRβ V3 subfamily, TCRβ V2 subfamily, TCRβ V15 subfamily, TCRβ V30 subfamily, TCRβ V19 subfamily, TCRβ V27 subfamily, TCRβ V28 subfamily, TCRβ V24 subfamily, TCRβ V20 subfamily, TCRβ V25 subfamily, TCRβ V29 subfamily, TCRβ V1 subfamily, TCRβ V1 subfamily, TCRβ V17 subfamily, TCRβ V21 subfamily, TCRβ V23 subfamily, or TCRβ V26 subfamily.

[0156]

[0330] In some embodiments, the TCRβ V6 subfamily is also known as TCRβ V13.1. In some embodiments, the TCRβ V6 subfamily is also known as TCRβ V6-4 * 01. TCRβ V6-4 * 02. TCRβ V6-9 * 01. TCRβ V6-8 * 01. TCRβ V6-5* 01. TCRβ V6-6 * 02. TCRβ V6-6 * 01. TCRβ V6-2 * 01. TCRβ V6-3 * 01 or TCRβ V6-1 * 01, or a variant thereof. In some embodiments, the TCRβ V6 comprises TCRβ V6-4 * 01, or a variant thereof. In some embodiments, the TCRβ V6 comprises TCRβ V6-4 * 02, or a variant thereof. In some embodiments, the TCRβ V6 comprises TCRβ V6-9 * 01, or a variant thereof. In some embodiments, the TCRβ V6 comprises TCRβ V6-8 * 01, or a variant thereof. In some embodiments, the TCRβ V6 comprises TCRβ V6-5 * 01, or a variant thereof. In some embodiments, the TCRβ V6 comprises TCRβ V6-6 * 02, or a variant thereof. In some embodiments, the TCRβ V6 comprises TCRβ V6-6 * 01, or a variant thereof. In some embodiments, the TCRβ V6 comprises TCRβV6-2 * 01, or a variant thereof. In some embodiments, the TCRβ V6 comprises TCRβ V6-3 * 01, or a variant thereof. In some embodiments, the TCRβ V6 comprises TCRβ V6-1 * 01, or any variant thereof.

[0157]

[0331] In some embodiments, TCRβ V6 is TCRβ V6-5 * 01, or a variant thereof. In some embodiments, TCRβ V6, e.g., TCRβ V6-5 * 01 is recognized, e.g., bound, by SEQ ID NO: 1 and / or SEQ ID NO: 2. In some embodiments, TCRβ V6, e.g., TCRβ V6-5 *01 is recognized, e.g., bound by, SEQ ID NO: 9 and / or SEQ ID NO: 10. In some embodiments, TCRβ V6 is recognized, e.g., bound by, SEQ ID NO: 9 and / or SEQ ID NO: 11.

[0158]

[0332] In some embodiments, the TCRβ V10 subfamily is also known as TCRβ V12. In some embodiments, the TCRβ V10 subfamily is also known as TCRβ V10-1 * 01. TCRβ V10-1 * 02. TCRβ V10-3 * 01 or TCRβ V10-2 * 01, or any variant thereof.

[0159]

[0333] In some embodiments, the TCRβ V12 subfamily is also known as TCRβ V8.1. In some embodiments, the TCRβ V12 subfamily is also known as TCRβ V12-4 * 01. TCRβ V12-3 * 01, or TCRβ V12-5 * 01, or variants thereof. In some embodiments, TCR β V12 is recognized by, e.g., bound by, SEQ ID NO: 15 and / or SEQ ID NO: 16. In some embodiments, TCR β V12 is recognized by, e.g., bound by, any one of SEQ ID NOs: 23-25 ​​and / or any one of SEQ ID NOs: 26-30.

[0160]

[0334] In some embodiments, the TCRβ V5 subfamily is TCRβ V5-5 * 01. TCRβ V5-6 * 01. TCRβ V5-4 * 01. TCRβ V5-8 * 01. TCRβ V5-1 * 01, or a variant thereof.

[0161]

[0335] In some embodiments, the TCRβ V7 subfamily is TCRβ V7-7 *01. TCRβ V7-6 * 01. TCRβ V7-8 * 02. TCRβ V7-4 * 01. TCRβ V7-2 * 02. TCRβ V7-2 * 03. TCRβ V7-2 * 01. TCRβ V7-3 * 01. TCRβ V7-9 * 03, or TCRβ V7-9 * 01, or any variant thereof.

[0162]

[0336] In some embodiments, the TCRβ V11 subfamily is TCRβ V11-1 * 01. TCRβ V11-2 * 01 or TCRβ V11-3 * 01, or a variant thereof. In some embodiments, the TCRβ V14 subfamily includes TCRβ V14 * 01, or a variant thereof. In some embodiments, the TCRβ V16 subfamily comprises TCRβ V16 * 01, or a variant thereof. In some embodiments, the TCRβ V18 subfamily comprises TCRβ V18 * 01, or a variant thereof. In some embodiments, the TCRβ V9 subfamily includes TCRβ V9 * 01 or TCRβ V9 * 02, or a variant thereof. In some embodiments, the TCRβ V13 subfamily comprises TCRβ V13 * 01, or a variant thereof. In some embodiments, the TCRβ V4 subfamily includes TCRβ V4-2 * 01. TCRβ V4-3 * 01, or TCRβ V4-1 * In some embodiments, the TCRβ V3 subfamily includes TCRβ V3-1, TCRβ V3-2, TCRβ V3-3, TCRβ V3-4, TCRβ V3-5, TCRβ V3-6, TCRβ V3-7, TCRβ V3-8, TCRβ V3-9, TCRβ V3-10, TCRβ V3-11, TCRβ V3-12, TCRβ V3-13, TCRβ V3-14, TCRβ V3-15, TCRβ V3-16, TCRβ V3-17, TCRβ V3-18, TCRβ V3-19 ... * 01, or a variant thereof. In some embodiments, the TCRβ V2 subfamily includes TCRβ V2 *01, or a variant thereof. In some embodiments, the TCRβ V15 subfamily includes TCRβ V15 * 01, or a variant thereof. In some embodiments, the TCRβ V30 subfamily includes TCRβ V30 * 01, or TCRβ V30 * 02, or a variant thereof. In some embodiments, the TCRβ V19 subfamily comprises TCRβ V19 * 01, or TCRβ V19 * 02, or a variant thereof. In some embodiments, the TCRβ V27 subfamily comprises TCRβ V27 * 01, or a variant thereof. In some embodiments, the TCRβ V28 subfamily comprises TCRβ V28 * In some embodiments, the TCRβ V24 subfamily includes TCRβ V24-1, or a variant thereof. * 01, or a variant thereof. In some embodiments, the TCRβ V20 subfamily includes TCRβ V20-1 * 01, or TCRβ V20-1 * In some embodiments, the TCRβ V25 subfamily includes TCRβ V25-1, TCRβ V25-2, or a variant thereof. * 01, or a variant thereof. In some embodiments, the TCRβ V29 subfamily includes TCRβ V29-1 * 01, or any variant thereof.

[0163]

[0337] Exemplary amino acid sequences of TCRβV subfamily members can be found on the ImMunoGeneTics Information System website: http: / / www.imgt.org / , or similar resources.

[0164] Anti-TCRβV antibody

[0338] Current bispecific constructs designed to redirect T cells to promote tumor cell lysis for cancer immunotherapy typically utilize antibody fragments (e.g., Fab, scFv, VH, single-domain antibodies) derived from monoclonal antibodies (mAbs) directed against the CD3e subunit of the T cell receptor (TCR). However, this approach has limitations that may prevent the full realization of the therapeutic potential of such bispecific constructs. Previous studies have shown that even low "activating" doses of anti-CD3e mAbs can cause long-term T cell dysfunction and exert immunosuppressive effects. Furthermore, anti-CD3e mAbs are associated with side effects due to massive T cell activation. Large numbers of activated T cells secrete substantial amounts of cytokines, the most important of which is interferon gamma (IFNγ). This excess amount of IFNγ then activates macrophages, which then overproduce inflammatory cytokines such as IL-1 beta, IL-6, IL-10, and TNF-alpha, causing a "cytokine storm" known as cytokine release syndrome (CRS) (Shimabukuro-Vornhagen et al., J Immunother Cancer. 2018 Jun. 15;6(1):56, incorporated herein by reference in its entirety). Thus, there is a need to develop antibodies that can bind to and activate only a subset of effector T cells, for example, to reduce CRS and / or neurotoxicity (NT).

[0165]

[0339] Described herein are molecules and methods for targeting the TCRβV chain of a TCR. Without being bound by theory, such molecules can bind to, activate, and / or expand only a subset of T cells, avoiding or reducing CRS and / or NT, and minimizing the potential immunosuppressive effects of anti-CD3 mAbs.

[0166]

[0340] Described herein is a class of antibodies, i.e., the anti-TCRβV antibody molecules described herein, that recognize structurally conserved but sequence-variable regions, e.g., domains, on the TCRβV protein (as represented by the circled sections in Figure 25A) and have similar functions (e.g., T cell activation and similar cytokine profiles as described herein), despite having low sequence similarity (e.g., low sequence identity among different antibody molecules that recognize different TCRβV subfamilies). Thus, the anti-TCRβV antibody molecules described herein share a structure-function relationship.

[0167]

[0341] Without being bound by theory, in some embodiments, the anti-TCRβV antibody molecules described herein bind to an outward-facing epitope of the TCRβV protein when complexed with the TCR alpha protein, for example, as shown by the circled region in Figure 25A. In some embodiments, the anti-TCRβV antibody molecules described herein recognize (e.g., bind to) a domain (e.g., epitope) on the TCRβV protein that is (1) structurally conserved among different TCRβV subfamilies; and (2) has minimal sequence identity among different TCRβV subfamilies. As shown in Table 9, TCRβV proteins from different TCRβV subfamilies share minimal sequence similarity. However, as shown in Figures 25A-25B, TCRβV proteins with minimal sequence similarity share similar 3D conformations and structures.

[0168]

[0342] The alignment of TCRBV amino acid sequences in Table 9 highlights the diversity of TCR sequences. In particular, TRBV sequences from different subfamilies are quite different from each other.

[0343] In some embodiments, the anti-TCRβV antibody molecules described herein do not recognize, e.g., do not bind to, the interface of the TCRβV:TCR alpha complex. In some embodiments, the anti-TCRβV antibody molecules described herein do not recognize, e.g., do not bind to, the constant region of the TCRβV protein. An exemplary antibody that binds to the constant region of the TCRβV region is JOVI.1, described by Viney et al. (Hybridoma. 1992 Dec;11(6):701-13). In some embodiments, the anti-TCRβV antibody molecules described herein do not recognize, e.g., do not bind to, one or more (e.g., all) of the complementarity determining regions (e.g., CDR1, CDR2, and / or CDR3) of the TCRβV protein.

[0169]

[0344] In particular, provided herein are antibody molecules directed against the variable chain of the beta subunit of the TCR (TCRβV), which bind to and, e.g., activate, a subset of T cells. The anti-TCRβV antibody molecules described herein result in reduced or no production of CRS-associated cytokines, such as IL-6, IL-1beta, IL-10, and TNF-alpha; and enhanced and / or delayed production of IL-2 and IFNγ. In some embodiments, the anti-TCRβV antibodies described herein have a cytokine profile, e.g., as described herein, that differs from the cytokine profile of T cell engagers that bind to receptors or molecules other than the TCRβV region ("non-TCRβV-binding T cell engagers"). In some embodiments, non-TCRβV-binding T cell engagers include antibodies that bind to CD3 molecules (e.g., CD3 epsilon (CD3e) molecules) or TCR alpha (TCRα) molecules. In some embodiments, the non-TCRβV-binding T cell engager is the OKT3 antibody or the SP34-2 antibody.

[0170]

[0345] In some embodiments, the anti-TCRβV antibodies described herein result in the expansion of TCRβV+ T cells, e.g., a subset of memory effector T cells known as TEMRA. Without being bound by theory, in some embodiments, TEMRA cells are believed to be capable of promoting tumor cell lysis but not CRS. Accordingly, methods for producing the anti-TCRβV antibody molecules and uses thereof are provided herein. Also described herein are multispecific, e.g., bispecific, molecules comprising the anti-TCRβV antibody molecules. In some embodiments, compositions comprising the anti-TCRβV antibody molecules of the present disclosure can be used, for example, to (1) activate and redirect T cells to promote tumor cell lysis for cancer immunotherapy; and / or (2) expand TCRβV+ T cells. In some embodiments, compositions comprising the anti-TCRβV antibody molecules described herein limit the adverse side effects of CRS and / or NT, e.g., CRS and / or NT associated with anti-CD3e targeting.

[0171]

[0346] In some embodiments, the anti-TCRβV antibody molecule is selected from the group consisting of TRBV2, TRBV3-1, TRBV4-1, TRBV4-2, TRBV4-3, TRBV5-1, TRBV5-4, TRBV5-5, TRBV5-6, TRBV5-8, TRBV6-1, TRBV6-2, TRBV6-3, TRBV6-4, TRBV6-5, TRBV6-6, TRBV6-8, TRBV6-9, TRBV7-2, TRBV7-3, TRBV7-4, TRBV7-6, TRBV7-7, TRBV7-8, and binds to one or more of TRBV7-9, TRBV9, TRBV10-1, TRBV10-2, TRBV10-3, TRBV11-1, TRBV11-2, TRBV11-3, TRBV12-3, TRBV12-4, TRBV12-5, TRBV13, TRBV14, TRBV15, TRBV16, TRBV18, TRBV19, TRBV20-1, TRBV24-1, TRBV25-1, TRBV27, TRBV28, TRBV29-1 and TRBV30. In some embodiments, the anti-TCRβV antibody molecule binds to one or more of TRBV6-1, TRBV6-2, TRBV6-3, TRBV6-4, TRBV6-5, TRBV6-6, TRBV6-8 and TRBV6-9. In some embodiments, the anti-TCRβV antibody molecule is anti-TRBV2, anti-TRBV3-1, anti-TRBV4-1, anti-TRBV4-2, anti-TRBV4-3, anti-TRBV5-1, anti-TRBV5-4, anti-TRBV5-5, anti-TRBV5-6, anti-TRBV5-8, anti-TRBV 6-1, anti-TRBV6-2, anti-TRBV6-3, anti-TRBV6-4, anti-TRBV6-5, anti-TRBV6-6, anti-TRBV6-8, anti-TRBV6-9, anti-TRBV7-2, anti-TRBV7-3, anti-TRBV7-4, anti-TRBV7-6, anti-TRBV7-7, anti-TR BV7-8, anti-TRBV7-9, anti-TRBV9, anti-TRBV10-1, anti-TRBV10-2, anti-TRBV10-3, anti-TRBV11-1, anti-TRBV11-2, anti-TRBV11-3, anti-TRBV12-3, anti-TRBV12-4, anti-TRBV12-5, anti-TRBV13, anti-TRBV14, anti-TRBV15, anti-TRBV16, anti-TRBV18, anti-TRBV19, anti-TRBV20-1, anti-TRBV24-1, anti-TRBV25-1, anti-TRBV27, anti-TRBV28, anti-TRBV29-1, or anti-TRBV30.Exemplary anti-TCRβV antibody molecules and the corresponding TCRβV subfamilies recognized by said anti-TCRβV antibody molecules are disclosed in Table 10A.

[0172]

[0347] In some embodiments, the anti-TCRβV antibody molecule is selected from the group consisting of TRBV2, TRBV3-1, TRBV4-1, TRBV4-2, TRBV4-3, TRBV5-1, TRBV5-4, TRBV5-5, TRBV5-6, TRBV5-8, TRBV6-1, TRBV6-2, TRBV6-3, TRBV6-4, TRBV6-5, TRBV6-6, TRBV6-8, TRBV6-9, TRBV7-2, TRBV7-3, TRBV7-4, TRBV7-6, TRBV7-7, TRBV 7-8, TRBV7-9, TRBV9, TRBV10-1, TRBV10-2, TRBV10-3, TRBV11-1, TRBV11-2, TRBV11-3, TRBV12-3, TRBV12-4, TRBV12-5, TRBV13, TRBV14, TRBV15, TRBV16, TRBV18, TRBV19, TRBV20-1, TRBV24-1, TRBV25-1, TRBV27, TRBV28, TRBV29-1, or TRBV30. In some embodiments, the anti-TCRβV antibody molecule specifically binds to TRBV6-1. In some embodiments, the anti-TCRβV antibody molecule specifically binds to TRBV6-2. In some embodiments, the anti-TCRβV antibody molecule specifically binds to TRBV6-3. In some embodiments, the anti-TCRβV antibody molecule specifically binds to TRBV6-4. In some embodiments, the anti-TCRβV antibody molecule specifically binds to TRBV6-5. In some embodiments, the anti-TCRβV antibody molecule specifically binds to TRBV6-6. In some embodiments, the anti-TCRβV antibody molecule specifically binds to TRBV6-8. In some embodiments, the anti-TCRβV antibody molecule specifically binds to TRBV6-9.

[0173]

[0348] In some embodiments, the anti-TCRβ antibody molecule does not bind to TCRβ V12 or binds to TCRβ V12 with an affinity and / or binding specificity that is lower (e.g., about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or about 2-, 5-, or 10-fold lower) than the affinity and / or binding specificity of the 16G8 murine antibody or a humanized version thereof described in U.S. Pat. No. 5,861,155.

[0174]

[0349] In some embodiments, the anti-TCRβ antibody molecule binds to TCRβ V12 with an affinity and / or binding specificity that is higher (e.g., about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or about 2, 5, or 10 times higher) than the affinity and / or binding specificity of the 16G8 murine antibody or a humanized version thereof described in U.S. Pat. No. 5,861,155.

[0175]

[0350] In some embodiments, the anti-TCRβ antibody molecule binds to a TCRβ V region other than TCRβ V12 (e.g., a TCRβ V region described herein, e.g., the TCRβ V6 subfamily (e.g., TCRβ V6-5*01)) with an affinity and / or binding specificity that is greater (e.g., about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or about 2-, 5-, or 10-fold greater) than the affinity and / or binding specificity of the 16G8 murine antibody or a humanized version thereof described in U.S. Pat. No. 5,861,155.

[0176]

[0351] In some embodiments, the anti-TCRβV antibody molecule does not comprise the CDRs of the Antibody B murine antibody.

[0352] In some embodiments, the anti-TCRβ antibody molecule does not bind to TCRβ V5-5*01 or TCRβ V5-1*01, or binds to TCRβ V5-5*01 or TCRβ V5-1*01 with an affinity and / or binding specificity that is lower (e.g., about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or about 2-, 5-, or 10-fold lower) than the affinity and / or binding specificity of the TM23 murine antibody or a humanized version thereof described in U.S. Pat. No. 5,861,155.

[0177]

[0353] In some embodiments, the anti-TCRβ antibody molecule binds to TCRβ V5-5*01 or TCRβ V5-1*01 with an affinity and / or binding specificity that is higher (e.g., about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or about 2, 5, or 10 times higher) than the affinity and / or binding specificity of the TM23 murine antibody or humanized version thereof described in U.S. Pat. No. 5,861,155.

[0178]

[0354] In some embodiments, the anti-TCRβ antibody molecule binds to a TCRβ V region other than TCRβ V5-5*01 or TCRβ V5-1*01 (e.g., a TCRβ V region described herein, e.g., the TCRβ V6 subfamily (e.g., TCRβ V6-5*01)) with an affinity and / or binding specificity that is greater (e.g., about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or about 2-, 5-, or 10-fold greater) than the affinity and / or binding specificity of the TM23 murine antibody or a humanized version thereof described in U.S. Pat. No. 5,861,155.

[0179]

[0355] In some embodiments, the anti-TCRβV antibody molecule does not comprise the CDRs of the TM23 murine antibody.

[0356] In some embodiments, the light or heavy chain variable framework (e.g., a region encompassing at least FR1, FR2, FR3, and optionally FR4) of an anti-TCRβ antibody molecule, e.g., an anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule, contains (a) at least 80%, 85%, 87% of the amino acid residues from a human light or heavy chain variable framework, e.g., from a human mature antibody, a human germline sequence, or a human consensus sequence. (b) a light or heavy chain variable framework that comprises 90%, 92%, 93%, 95%, 97%, 98%, or 100% of the amino acid residues from a human light or heavy chain variable framework, e.g., 20% to 80%, 40% to 60%, 60% to 90%, or 70% to 95% of the light or heavy chain variable framework residues from a human mature antibody, a human germline sequence, or a human consensus sequence; (c) a non-human framework (e.g., a rodent framework); or (d) a non-human framework that has been modified, e.g., deimmunized or partially humanized, e.g., to remove antigenic or cytotoxic determinants. In some embodiments, the light or heavy chain variable framework region (particularly FR1, FR2 and / or FR3) comprises a light or heavy chain variable framework sequence that is at least 70, 75, 80, 85, 87, 88, 90, 92, 94, 95, 96, 97, 98, 99% identical or identical to the framework of a VL or VH segment of a human germline gene.

[0180]

[0357] In some embodiments, an anti-TCR β antibody molecule, e.g., an anti-TCR β V6 (e.g., anti-TCR β V6-5*01) antibody molecule, comprises a heavy chain variable domain having at least one, two, three, four, five, six, seven, ten, fifteen, twenty or more changes, e.g., amino acid substitutions or deletions, from the amino acid sequence of the FR region in the entire variable region from any one of the amino acid sequences AH.1 to AH.85, e.g., AH.1, AH.2 or AH.68, e.g., as shown in FIG. 2A, or SEQ ID NO:9.

[0181]

[0358] Alternatively, or in combination with the heavy chain substitutions described herein, an anti-TCRβ antibody molecule, e.g., an anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule, comprises a light chain variable domain having at least one, two, three, four, five, six, seven, ten, fifteen, twenty or more amino acid changes, e.g., amino acid substitutions or deletions, from the amino acid sequence of the FR region in the entire variable region from any one of the amino acid sequences AH.1 to AH.85, e.g., AH.1, AH.2 or AH.68, e.g., as shown in Figure 2B, or SEQ ID NO: 10 or SEQ ID NO: 11.

[0182]

[0359] In some embodiments, the anti-TCRβ antibody molecule, e.g., the anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule, comprises one, two, three, or four heavy chain framework regions shown in Figure 2A, or a sequence substantially identical thereto.

[0183]

[0360] In some embodiments, the anti-TCRβ antibody molecule, e.g., the anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule, comprises one, two, three, or four light chain framework regions shown in Figure 2B, or a sequence substantially identical thereto.

[0184]

[0361] In some embodiments, the anti-TCRβ V antibody molecule, eg, the anti-TCRβ V6 (eg, anti-TCRβ V6-5*01) antibody molecule, comprises a light chain framework region 1 of AH.1 or AH.2, eg, as shown in FIG. 2B.

[0185]

[0362] In some embodiments, the anti-TCRβ V antibody molecule, eg, anti-TCRβ V6 (eg, anti-TCRβ V6-5*01) antibody molecule, comprises light chain framework region 2 of AH.1 or AH.2, eg, as shown in FIG. 2B.

[0186]

[0363] In some embodiments, the anti-TCRβ V antibody molecule, eg, the anti-TCRβ V6 (eg, anti-TCRβ V6-5*01) antibody molecule, comprises light chain framework region 3 of AH.1 or AH.2, eg, as shown in FIG. 2B.

[0187]

[0364] In some embodiments, the anti-TCRβ V antibody molecule, eg, the anti-TCRβ V6 (eg, anti-TCRβ V6-5*01) antibody molecule, comprises light chain framework region 4 of AH.1 or AH.2, eg, as shown in FIG. 2B.

[0188]

[0365] In some embodiments, the anti-TCRβ V antibody molecule, e.g., anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule, comprises a light chain variable domain comprising a framework region, e.g., framework region 1 (FR1), that comprises an alteration, e.g., a substitution (e.g., a conservative substitution), e.g., at position 10 according to Kabat numbering. In some embodiments, FR1 comprises a phenylalanine, e.g., a serine to phenylalanine substitution, at position 10. In some embodiments, the substitution is relative to a human germline light chain framework region sequence.

[0189]

[0366] In some embodiments, an anti-TCRβ V antibody molecule, e.g., an anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule, comprises a light chain variable domain comprising a framework region, e.g., framework region 2 (FR2), that comprises an alteration, e.g., a substitution (e.g., a conservative substitution), at a position described herein according to Kabat numbering. In some embodiments, FR2 comprises a histidine at position 36 according to Kabat numbering, e.g., a substitution at position 36, e.g., a tyrosine to histidine substitution. In some embodiments, FR2 comprises an alanine at position 46 according to Kabat numbering, e.g., a substitution at position 46, e.g., an arginine to alanine substitution. In some embodiments, the substitution is relative to a human germline light chain framework region sequence.

[0190]

[0367] In some embodiments, an anti-TCRβ V antibody molecule, e.g., an anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule, comprises a light chain variable domain comprising a framework region, e.g., framework region 3 (FR3), that comprises an alteration, e.g., a substitution (e.g., a conservative substitution), at a position described herein according to Kabat numbering. In some embodiments, FR3 comprises a phenylalanine at position 87 according to Kabat numbering, e.g., a substitution at position 87, e.g., a tyrosine to phenylalanine substitution. In some embodiments, the substitution is relative to a human germline light chain framework region sequence.

[0191]

[0368] In some embodiments, an anti-TCRβ V antibody molecule, e.g., an anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule, comprises a light chain variable domain comprising: (a) framework region 1 (FR1) comprising a phenylalanine at position 10 according to Kabat numbering, e.g., a substitution at position 10, e.g., a serine to phenylalanine substitution; (b) framework region 2 (FR2) comprising a histidine at position 36 according to Kabat numbering, e.g., a substitution at position 36, e.g., a tyrosine to histidine substitution, and an alanine at position 46 according to Kabat numbering, e.g., a substitution at position 46, e.g., an arginine to alanine substitution; and (c) framework region 3 (FR3) comprising a phenylalanine at position 87 according to Kabat numbering, e.g., a substitution at position 87, e.g., a tyrosine to phenylalanine substitution, e.g., as set forth in the amino acid sequence of SEQ ID NO: 10. In some embodiments, the substitution is relative to a human germline light chain framework region sequence.

[0192]

[0369] In some embodiments, an anti-TCRβ V antibody molecule, e.g., an anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule, comprises a light chain variable domain comprising: (a) framework region 2 (FR2) comprising a histidine at position 36 according to Kabat numbering, e.g., a substitution at position 36, e.g., a tyrosine to histidine substitution, and an alanine at position 46 according to Kabat numbering, e.g., a substitution at position 46, e.g., an arginine to alanine substitution, and (b) framework region 3 (FR3) comprising a phenylalanine at position 87 according to Kabat numbering, e.g., a substitution at position 87, e.g., a tyrosine to phenylalanine substitution, e.g., as set forth in the amino acid sequence of SEQ ID NO: 11. In some embodiments, the substitution is relative to a human germline light chain framework region sequence.

[0193]

[0370] In some embodiments, an anti-TCRβ V antibody molecule, e.g., an anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule, comprises a light chain variable domain comprising: (a) framework region 1 (FR1) comprising an alteration, e.g., a substitution (e.g., a conservative substitution), at one or more (e.g., all) positions described herein according to Kabat numbering; (b) framework region 2 (FR2) comprising an alteration, e.g., a substitution (e.g., a conservative substitution), at one or more (e.g., all) positions described herein according to Kabat numbering; and (c) framework region 3 (FR3) comprising an alteration, e.g., a substitution (e.g., a conservative substitution), at one or more (e.g., all) positions described herein according to Kabat numbering. In some embodiments, the substitutions are relative to a human germline light chain framework region sequence.

[0194]

[0371] In some embodiments, an anti-TCRβV antibody molecule, e.g., an anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule, comprises heavy chain framework region 1 of AH.1 or AH.2, e.g., as shown in Figure 2A. In some embodiments, an anti-TCRβV antibody molecule, e.g., an anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule, comprises heavy chain framework region 2 of AH.1 or AH.2, e.g., as shown in Figure 2A. In some embodiments, an anti-TCRβV antibody molecule, e.g., an anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule, comprises heavy chain framework region 3 of AH.1 or AH.2, e.g., as shown in Figure 2A. In some embodiments, an anti-TCRβ V antibody molecule, e.g., an anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule, comprises heavy chain framework region 4 of AH.1 or AH.2, e.g., as shown in Figure 2A.

[0195]

[0372] In some embodiments, an anti-TCRβ V antibody molecule, e.g., an anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule, comprises a heavy chain variable domain comprising a framework region, e.g., framework region 3 (FR3), that comprises an alteration, e.g., a substitution (e.g., a conservative substitution), at a position described herein according to Kabat numbering. In some embodiments, FR3 comprises a threonine at position 73 according to Kabat numbering, e.g., a substitution at position 73, e.g., a glutamic acid to threonine substitution. In some embodiments, FR3 comprises a glycine at position 94 according to Kabat numbering, e.g., a substitution at position 94, e.g., an arginine to glycine substitution. In some embodiments, the substitution is relative to a human germline heavy chain framework region sequence.

[0196]

[0373] In some embodiments, an anti-TCRβ antibody molecule, e.g., an anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule, comprises a heavy chain variable domain comprising framework region 3 (FR3) comprising a threonine at position 73 according to Kabat numbering, e.g., a substitution at position 73, e.g., a glutamic acid to threonine substitution, and a glycine at position 94 according to Kabat numbering, e.g., a substitution at position 94, e.g., an arginine to glycine substitution, e.g., as shown in the amino acid sequence of SEQ ID NO: 10.

[0197]

[0374] In some embodiments, an anti-TCRβ antibody molecule, e.g., an anti-TCRβ V6 (e.g., an anti-TCRβ V6-5*01) antibody molecule, comprises heavy chain framework regions 1-4 of AH.1 or AH.2, e.g., SEQ ID NO: 9, or as depicted in Figures 2A and 2B. In some embodiments, an anti-TCRβ antibody molecule, e.g., an anti-TCRβ V6 (e.g., an anti-TCRβ V6-5*01) antibody molecule, comprises light chain framework regions 1-4 of AH.1, e.g., SEQ ID NO: 10, or as depicted in Figures 2A and 2B. In some embodiments, an anti-TCRβ antibody molecule, e.g., an anti-TCRβ V6 (e.g., an anti-TCRβ V6-5*01) antibody molecule, comprises light chain framework regions 1-4 of AH.2, e.g., SEQ ID NO: 11, or as depicted in Figures 2A and 2B. In some embodiments, an anti-TCRβ antibody molecule, e.g., an anti-TCRβ V6 (e.g., an anti-TCRβ V6-5*01) antibody molecule, comprises heavy chain framework regions 1-4 of AH.1, e.g., SEQ ID NO: 9, and light chain framework regions 1-4 of AH.1, e.g., SEQ ID NO: 10, or as depicted in Figures 2A and 2B. In some embodiments, an anti-TCRβ antibody molecule, e.g., an anti-TCRβ V6 (e.g., an anti-TCRβ V6-5*01) antibody molecule, comprises heavy chain framework regions 1-4 of AH.2, e.g., SEQ ID NO: 9, and light chain framework regions 1-4 of AH.2, e.g., SEQ ID NO: 11, or as depicted in Figures 2A and 2B.

[0198]

[0375] In some embodiments, the heavy or light chain variable domain, or both, of an anti-TCRβ antibody molecule, e.g., an anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule, comprises an amino acid sequence that is substantially identical to an amino acid described herein, e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical to a variable region of an antibody described herein, e.g., an antibody selected from any one of AH.1-AH.85, e.g., AH.1, AH.2, or AH.68, or described in Table 1 or encoded by a nucleotide sequence in Table 1, or that differs by at least 1 or 5 residues, and fewer than 40, 30, 20, or 10 residues, from a variable region of an antibody described herein.

[0199]

[0376] In some embodiments, the anti-TCRβ antibody molecule, e.g., anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule, comprises at least one, two, three, or four antigen-binding regions, e.g., variable regions, having an amino acid sequence set forth in Table 1, or a sequence substantially identical thereto (e.g., at least about 85%, 90%, 95%, 99% or more identical thereto, or a sequence that differs by 1, 2, 5, 10, or 15 or fewer amino acid residues from a sequence set forth in Table 1). In another embodiment, the anti-TCRβ antibody molecule, e.g., anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecules comprise a VH and / or VL domain encoded by a nucleic acid having a nucleotide sequence set forth in Table 1, or a sequence substantially identical thereto (e.g., at least about 85%, 90%, 95%, 99% or more identical thereto, or a sequence that differs by no more than 3, 6, 15, 30, or 45 nucleotides from a sequence set forth in Table 1).

[0200]

[0377] In some embodiments, an anti-TCRβ V antibody molecule, e.g., an anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule, comprises a VH domain comprising the amino acid sequence of SEQ ID NO:9, an amino acid sequence at least about 85%, 90%, 95%, 99% or more identical to the amino acid sequence of SEQ ID NO:9, or an amino acid sequence that differs by no more than 1, 2, 5, 10, or 15 amino acid residues from the amino acid sequence of SEQ ID NO:9, and / or a VL domain comprising the amino acid sequence of SEQ ID NO:10, an amino acid sequence at least about 85%, 90%, 95%, 99% or more identical to the amino acid sequence of SEQ ID NO:10, or an amino acid sequence that differs by no more than 1, 2, 5, 10, or 15 amino acid residues from the amino acid sequence of SEQ ID NO:10.

[0201]

[0378] In some embodiments, an anti-TCRβ V antibody molecule, e.g., an anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule, comprises a VH domain comprising the amino acid sequence of SEQ ID NO:9, an amino acid sequence at least about 85%, 90%, 95%, 99% or more identical to the amino acid sequence of SEQ ID NO:9, or an amino acid sequence that differs by no more than 1, 2, 5, 10, or 15 amino acid residues from the amino acid sequence of SEQ ID NO:9, and / or a VL domain comprising the amino acid sequence of SEQ ID NO:11, an amino acid sequence at least about 85%, 90%, 95%, 99% or more identical to the amino acid sequence of SEQ ID NO:11, or an amino acid sequence that differs by no more than 1, 2, 5, 10, or 15 amino acid residues from the amino acid sequence of SEQ ID NO:11.

[0202]

[0379] In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule, is a full-length antibody or a fragment thereof (e.g., Fab, F(ab')2, Fv, single-domain antibody, or single-chain Fv fragment (scFv)). In embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule, is a monoclonal antibody or an antibody having a single specificity. In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule, may also be a humanized, chimeric, camelid, shark, or in vitro-generated antibody molecule. In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule, is a humanized antibody molecule. The heavy and light chains of an anti-TCRβ antibody molecule, e.g., an anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule, can be full length (e.g., the antibody can comprise at least one, and preferably two, complete heavy chains, and at least one, and preferably two, complete light chains), or can comprise an antigen-binding fragment (e.g., a Fab, F(ab'), Fv, a single-chain Fv fragment, a single-domain antibody, a diabody (dAb), a bivalent antibody, or a bispecific antibody or fragment thereof, a single-domain variant thereof, or a camelid antibody).

[0203]

[0380] In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule, is in the form of a multispecific molecule, e.g., a bispecific molecule, e.g., as described herein.

[0204]

[0381] In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule, has a heavy chain constant region (Fc) selected from, e.g., the heavy chain constant regions of IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE. In some embodiments, the Fc region is selected from the heavy chain constant regions of IgG1, IgG2, IgG3, and IgG4. In some embodiments, the Fc region is selected from the heavy chain constant region of IgG1 or IgG2 (e.g., human IgG1 or IgG2). In some embodiments, the heavy chain constant region is human IgG1. In some embodiments, the Fc region comprises an Fc region variant, e.g., as described herein.

[0205]

[0382] In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule, has a light chain constant region chosen from, e.g., a kappa or lambda light chain constant region, preferably a kappa (e.g., human kappa) light chain constant region. In some embodiments, the constant region is altered, e.g., mutated, to modify the properties of the anti-TCRβV antibody molecule, e.g., anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule (e.g., to increase or decrease one or more of Fc receptor binding, antibody glycosylation, the number of cysteine ​​residues, effector cell function, or complement function). For example, the constant region has been mutated at positions 296 (M to Y), 298 (S to T), 300 (T to E), 477 (H to K) and 478 (N to F) to alter Fc receptor binding, e.g., compared to human IgG1 (e.g., the mutated positions correspond to positions 132 (M to Y), 134 (S to T), 136 (T to E), 313 (H to K) and 314 (N to F) of SEQ ID NO: 212 or 214, or positions 135 (M to Y), 137 (S to T), 139 (T to E), 316 (H to K) and 317 (N to F) of SEQ ID NO: 215, 216, 217 or 218).

[0206]

[0383] Antibody AH.1 comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 3278 and a light chain comprising the amino acid sequence of SEQ ID NO: 72. Antibody AH.2 comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 3278 and a light chain comprising the amino acid sequence of SEQ ID NO: 3279. Antibody AH.68 comprises the amino acid sequence of SEQ ID NO: 1337, or a sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity thereto. Antibody AH.69 comprises the amino acid sequence of SEQ ID NO: 1500, or a sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity thereto.

[0207]

[0384] Additional exemplary humanized anti-TCRB V6 antibodies are provided in Table 1. In some embodiments, the anti-TCRβ V6 is Antibody A provided in Table 1, e.g., humanized Antibody A (Antibody AH). In some embodiments, the anti-TCRβ V antibody comprises one or more (e.g., all three) of the LC CDR1, LC CDR2, and LC CDR3 provided in Table 1; and / or one or more (e.g., all three) of the HC CDR1, HC CDR2, and HC CDR3 provided in Table 1, or a sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity thereto. In some embodiments, Antibody A comprises a variable heavy chain (VH) and / or variable light chain (VL) provided in Table 1, or a sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity thereto.

[0208]

[0385] In some embodiments, the anti-TCR β V antibody molecule, e.g., anti-TCR β V6 (e.g., anti-TCR β V6-5*01) antibody molecule is selected from the group consisting of AH.1, AH.2, AH.3, AH.4, AH.5, AH.6, AH.7, AH.8, AH.9, AH.10, AH.11, AH.12, AH.13, AH.14, AH.15, AH.16, AH.17, AH.18, AH.19, AH.20, AH.21, AH.22, AH.23, AH.24, AH.25, AH.26, AH.27, AH.28, AH.29, AH.30, AH.31, AH.32, AH.33, AH.34, AH.35, AH.36, AH.37, AH.38, AH.39, AH.40, AH.41, AH.42, AH.43, AH.44, AH.45, AH.46, AH.47, AH.48, AH.49, AH.50, AH.51, AH.52, AH.53, AH.54, AH.55, AH.56, AH.57, AH.58, AH.59, AH.60, AH.61, AH.62, AH.63, AH.64, AH.65, AH.66, AH.67, AH.68, AH.69, AH.70, AH.71, AH.72, AH.73, AH.74, AH.75, AH.76, AH.77, A 4, AH.25, AH.26, AH.27, AH.28, AH.29, AH.30, AH.31, AH.32, AH.33, AH.34, AH.35, AH.36 , AH.37, AH.38, AH.39, AH.40, AH.1, AH.42, AH.43, AH.44, AH.45, AH.46, AH.47, AH.48, AH .49, AH.50, AH.51, AH.52, AH.53, AH.54, AH.55, AH.56, AH.57, AH.58, AH.59, AH.60, AH. 61, AH.62, AH.63, AH.64, AH.65, AH.66, AH.67, AH.68, AH.69, AH.70, AH.71, AH.72, AH.73 , AH.74, AH.75, AH.76, AH.77, AH.78, AH.79, AH.80, AH.81, AH.82, AH.83, AH.84, or AH.85, or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity thereto.

[0209]

[0386] In some embodiments, the anti-TCR β V antibody molecule, e.g., anti-TCR β V6 (e.g., anti-TCR β V6-5*01) antibody molecule is selected from the group consisting of AH.1, AH.2, AH.3, AH.4, AH.5, AH.6, AH.7, AH.8, AH.9, AH.10, AH.11, AH.12, AH.13, AH.14, AH.15, AH.16, AH.17, AH.18, AH.19, AH.20, AH.21, AH.22, AH.23, AH.24, AH.25, AH.26, AH.27, AH.28, AH.29, AH.30, AH.31, AH.32, AH.33, AH.34, AH.35, AH.36, AH.37, AH.38, AH.39, AH.40, AH.41, AH.42, AH.43, AH.44, AH.45, AH.46, AH.47, AH.48, AH.49, AH.50, AH.51, AH.52, AH.53, AH.54, AH.55, AH.56, AH.57, AH.58, AH.59, AH.60, AH.61, AH.62, AH.63, AH.64, AH.65, AH.66, AH.67, AH.68, AH.69, AH.70, AH.71, AH.72, AH.73, AH.74, AH.75, AH.76, AH.77, A 4, AH.25, AH.26, AH.27, AH.28, AH.29, AH.30, AH.31, AH.32, AH.33, AH.34, AH.35, AH.36 , AH.37, AH.38, AH.39, AH.40, AH.1, AH.42, AH.43, AH.44, AH.45, AH.46, AH.47, AH.48, AH .49, AH.50, AH.51, AH.52, AH.53, AH.54, AH.55, AH.56, AH.57, AH.58, AH.59, AH.60, AH. 61, AH.62, AH.63, AH.64, AH.65, AH.66, AH.67, AH.68, AH.69, AH.70, AH.71, AH.72, AH.73 , AH.74, AH.75, AH.76, AH.77, AH.78, AH.79, AH.80, AH.81, AH.82, AH.83, AH.84, or AH.85, or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity thereto.

[0210]

[0387] One of the preferred strains is the TCRβV strain and is TCRβ V6(TCRβ). V6-5*01) Remove AH.1, AH.2, AH.3, AH.4, AH.5, AH.6, AH.7, AH .8、AH.9、AH.10、AH.11、AH.12、AH.13、AH.14、AH.15、AH.16、AH .17、AH.18、AH.19、AH.20、AH.21、AH.22、AH.23、AH.24、AH.25 AH.26, AH.27, AH.28, AH.29, AH.30, AH.31, AH.32, AH.33, AH. 34 AH.35 AH.36 AH.37 AH.38 AH.39 AH.40 AH.1 AH.42A H.43, AH.44, AH.45, AH.46, AH.47, AH.48, AH.49, AH.50, AH AH.52, AH.53, AH.54, AH.55, AH.56, AH.57, AH.58, AH.59, AH. 60, AH.61, AH.62, AH.63, AH.64, AH.65, AH.66, AH.67, AH.68, A H.69, AH.70, AH.71, AH.72, AH.73, AH.74, AH.75, AH.76, AH 7, AH.78, AH.79, AH.80, AH.81, AH.82, AH.83, AH.84, AH The VH of 5 is still 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% There are several different types of AH.1, AH.2, AH.3, AH.4, AH.5, AH.6, AH .7、AH.8、AH.9、AH.10、AH.11、AH.12、AH.13、AH.14、AH.15、AH .16、AH.17、AH.18、AH.19、AH.20、AH.21、AH.22、AH.23、AH.24、 AH.25, AH.26, AH.27, AH.28, AH.29, AH.30, AH.31, AH.32, AH 3, AH.34, AH.35, AH.36, AH.37, AH.38, AH.39, AH.40, AH.1, AH.42, AH.43, AH.44, AH.45, AH.46, AH.47, AH.48, AH.49, AH.50, AH.51, AH.52, AH.53, AH.54, AH.55, AH .56, AH.57, AH.58, AH.59, AH.60, AH.61, AH.62, AH.63, AH.64, AH.65, AH.66, AH.67, AH.68, AH.69, AH AH.70, AH.71, AH.72, AH.73, AH.74, AH.75, AH.76, AH.77, AH.78, AH.79, AH.80, AH.81, AH.82, AH.83, AH.84, or AH.85, or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity thereto.

[0211]

[0388] Exemplary anti-TCRβV antibody molecules and the corresponding TCRβV subfamilies recognized by said anti-TCRβV antibody molecules are disclosed in Table 10A.

[0389] Various TCRβV subfamilies and / or subfamily members may be expressed at different levels in individuals, e.g., healthy individuals, as disclosed in Kitaura K. et al., (2016), BMC Immunology vol 17: 38 (the entire contents of which are incorporated herein by reference). For example, TCRβ V6-5 is expressed in approximately 3-6% of healthy donors.

[0212]

[0390] The expression of various TCRβ subfamilies and / or subfamily members may also vary in cancer cells. For example, TCRβV is present in approximately 3-6% of tumor-infiltrating T cells regardless of tumor type (see Li B. et al., Nature Genetics, 2016, vol:48(7):725-32, the entire contents of which are incorporated herein by reference). Li et al. also disclose that TCRβV6-5 is frequently present in tumor cells.

[0213] Anti-TCRβV6 antibody

[0391] In one embodiment, a human TCRβ V6, e.g., TCRβ V6-4 * 01. TCRβ V6-4 * 02. TCRβ V6-9 * 01. TCRβ V6-8 * 01. TCRβ V6-5 * 01. TCRβ V6-6 * 02. TCRβ V6-6 * 01. TCRβ V6-2 * 01. TCRβ V6-3 * 01 or TCRβ V6-1 * Provided herein are anti-TCRβV antibody molecules that bind to the TCRβ V6 subfamily, including TCRβ V6-5. In some embodiments, the TCRβ V6 subfamily includes TCRβ V6-5. * 01 or a variant thereof. In some embodiments, the TCRβ V6 comprises TCRβ V6-4 * 01, or a variant thereof. In some embodiments, the TCRβ V6 comprises TCRβ V6-4 * 02, or a variant thereof. In some embodiments, the TCRβ V6 comprises TCRβ V6-9 * 01, or a variant thereof. In some embodiments, the TCRβ V6 comprises TCRβ V6-8 * 01, or a variant thereof. In some embodiments, the TCRβ V6 comprises TCRβ V6-5 * 01, or a variant thereof. In some embodiments, the TCRβ V6 comprises TCRβ V6-6 * 02, or a variant thereof. In some embodiments, the TCRβ V6 comprises TCRβ V6-6 * 01, or a variant thereof. In some embodiments, the TCRβ V6 comprises TCRβ V6-2 * 01, or a variant thereof. In some embodiments, the TCRβ V6 comprises TCRβ V6-3 * 01, or a variant thereof. In some embodiments, the TCRβ V6 comprises TCRβ V6-1 * 01, or any variant thereof.

[0214]

[0392] In some embodiments, TCRβ V6-5 * 01 is encoded by the nucleic acid sequence of SEQ ID NO: 43, or a sequence having 85%, 90%, 95%, 99% or higher identity thereto. * 01 comprises the amino acid sequence of SEQ ID NO: 44, or an amino acid sequence having 85%, 90%, 95%, 99% or more identity thereto.

[0215]

[0393] In some embodiments, an anti-TCRβ antibody molecule, such as anti-TCRβ V6 (e.g., anti-TCRβ V6-5 * 01) The antibody molecule is a non-murine antibody molecule, e.g., a human or humanized antibody molecule. In some embodiments, the anti-TCRβV antibody molecule, e.g., the anti-TCRβ V6 (e.g., the anti-TCRβ V6-5*01) antibody molecule, is a human antibody molecule. In some embodiments, the anti-TCRβV antibody molecule, e.g., the anti-TCRβ V6 (e.g., the anti-TCRβ V6-5*01) antibody molecule, is a humanized antibody molecule.

[0216]

[0394] In some embodiments, an anti-TCRβ antibody molecule, such as anti-TCRβ V6 (e.g., anti-TCRβ V6-5 * 01) The antibody molecule is isolated or recombinant.

[0217]

[0395] In some embodiments, an anti-TCRβ antibody molecule, such as anti-TCRβ V6 (e.g., anti-TCRβ V6-5 * 01) The antibody molecule comprises at least one antigen-binding region, e.g., a variable region or antigen-binding fragment thereof, from an antibody described herein, e.g., an antibody selected from any one of AH.1-AH.85, e.g., AH.1, AH.2, or AH.68, or an antibody described in Table 1, or encoded by a nucleotide sequence in Table 1, or a sequence substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical) to any of the foregoing sequences.

[0218]

[0396] In some embodiments, the anti-TCRβ antibody molecule, e.g., anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule, comprises at least one, two, three, or four variable regions from an antibody described herein, e.g., an antibody selected from any one of AH.1 through A-H85, e.g., AH.1, AH.2, or AH.68, or an antibody described in Table 1, or encoded by a nucleotide sequence in Table 1, or a sequence substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical) to any of the foregoing sequences.

[0219]

[0397] In some embodiments, the anti-TCRβ antibody molecule, e.g., anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule, comprises at least one or two heavy chain variable regions from an antibody described herein, e.g., an antibody selected from any one of AH.1-AH.85, e.g., AH.1, AH.2, or AH.68, or an antibody molecule described in Table 1, or encoded by a nucleotide sequence in Table 1, or a sequence substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical) to any of the foregoing sequences.

[0220]

[0398] In some embodiments, the anti-TCRβV antibody molecule comprises a heavy chain variable region (VH) having the consensus sequence of SEQ ID NO: 231 or 3290.

[0399] SEQ ID NO: 231 - Consensus VH QVQLVQSGAEVKKPGSSVKVSCKASGH / T / G / YD / T / SFH / R / D / K / TL / D / K / T / NW / F / T / I / Y / GYIHWVRQAPGQGLEWMGR / WV / I / F F / S / YA / PGSGN / ST / V / Y / IK / RYNEKFKGRVTITADTSTSTAYMELSSLRSEDTAVYYCAG / VSY / IYSY / AD / GVLDYWGQGTTVTVSS.

[0221]

[0400] SEQ ID NO: 3290 - Consensus VH QVQLVQSGAEVKKPGSSVKVSCKASGX1X2FX3X4X5YIHWVRQAPGQGLEWMGX6X7X8X9GSGX 10 X 11 X 12 YNEKFKGRVTITADTSTSTAYMELSSLRSEDTAVYYCAX 13 SX 14 YSX 15 X 16 VLDYWGQGTTVTVSS (X1 is H or T or G or Y, X2 is D or T or S, X3 is H or R or D or K or T, X4 is L or D or K or T or N, X5 is W or F or T or I or Y or G, X6 is R or W, X7 is V or I or F, X8 is F or S or Y, X9 is A or P, X10 is N or S, X11 is T or V or Y or I, X12 is K or R, X13 is G or V, X14 is Y or I, X15 is Y or A, and X16 is D or G).

[0222]

[0401] In some embodiments, the anti-TCRβ antibody molecule, e.g., anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule, comprises at least one or two light chain variable regions from an antibody described herein, e.g., an antibody selected from any one of AH.1-AH.85, e.g., AH.1, AH.2, or AH.68, or an antibody described in Table 1, or encoded by a nucleotide sequence in Table 1, or a sequence substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical) to any of the foregoing sequences.

[0223]

[0402] In some embodiments, the anti-TCRβV antibody molecule comprises a light chain variable region (VL) having the consensus sequence of SEQ ID NO: 230 or 3289.

[0403] SEQ ID NO: 230 - Consensus VL DIQMTQSPSFLSASVGDRVTITCKASQNVG / E / A / DN / DR / KVAWY / HQQKPGKAPKALIYSSSHRYK / SGVPSRFSGSGSGTEFTLTISSLQPEDFATYFCQQFKSYPLTFGQGTKLEIK

[0404] SEQ ID NO: 3289 - Consensus VL DIQMTQSPSFLSASVGDRVTITCKASQNVX1X2X3VAWX4QQKPGKAPKALIYSSSHRYX5GVPSRFSGSGSGTEFTLTISSLQPEDFATYFCQQFKSYPLTFGQGTKLEIK X1 is G, E, A or D; X2 is N or D; X3 is R or K; X4 is Y or H; and X5 is K or S.

[0224]

[0405] In some embodiments, an anti-TCRβ antibody molecule, such as anti-TCRβ V6 (e.g., anti-TCRβ V6-5 * 01) antibody molecule comprises a heavy chain constant region of IgG4, e.g., human IgG4. In yet another embodiment, an anti-TCRβ V antibody molecule, e.g., anti-TCRβ V6 (e.g., anti-TCRβ V6-5 * 01) The antibody molecule comprises a heavy chain constant region of IgG1, e.g., human IgG1. In some embodiments, the heavy chain constant region comprises an amino acid sequence set forth in Table 3, or a sequence substantially identical thereto (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical).

[0225]

[0406] In some embodiments, an anti-TCRβ antibody molecule, such as anti-TCRβ V6 (e.g., anti-TCRβ V6-5 *01) The antibody molecule comprises a kappa light chain constant region, e.g., a human kappa light chain constant region. In some embodiments, the light chain constant region comprises an amino acid sequence set forth in Table 3, 21, or 22, or a sequence substantially identical thereto (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical).

[0226]

[0407] In some embodiments, the anti-TCRβ antibody molecule, e.g., anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule, comprises at least one, two, or three complementarity determining regions (CDRs) from the heavy chain variable region (VH) of an antibody described herein, e.g., an antibody selected from any one of AH.1-AH.85, e.g., AH.1, AH.2, or AH.68, or an antibody described in Table 1, or encoded by a nucleotide sequence in Table 1, or a sequence substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical) to any of the foregoing sequences.

[0227]

[0408] In some embodiments, an anti-TCRβ V antibody molecule, e.g., an anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule, comprises at least one, two, or three CDRs (or collectively all of the CDRs) from a heavy chain variable region comprising an amino acid sequence shown in Table 1 or encoded by a nucleotide sequence shown in Table 1. In some embodiments, one or more of the CDRs (or collectively all of the CDRs) have one, two, three, four, five, six, or more changes, e.g., amino acid substitutions or deletions, compared to the amino acid sequence shown in Table 1 or encoded by the nucleotide sequence shown in Table 1.

[0228]

[0409] In some embodiments, the anti-TCRβ antibody molecule, e.g., anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule, comprises at least one, two, or three complementarity determining regions (CDRs) from a light chain variable region of an antibody described herein, e.g., an antibody selected from any one of AH.1-AH.85, e.g., AH.1, AH.2, or AH.68, or an antibody described in Table 1, or encoded by a nucleotide sequence in Table 1, or a sequence substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical) to any of the foregoing sequences.

[0229]

[0410] In some embodiments, an anti-TCRβ V antibody molecule, e.g., an anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule, comprises at least one, two, or three CDRs (or collectively all of the CDRs) from a light chain variable region comprising an amino acid sequence shown in Table 1 or encoded by a nucleotide sequence shown in Table 1. In some embodiments, one or more of the CDRs (or collectively all of the CDRs) have one, two, three, four, five, six, or more changes, e.g., amino acid substitutions or deletions, compared to the amino acid sequence shown in Table 1 or encoded by the nucleotide sequence shown in Table 1.

[0230]

[0411] In some embodiments, an anti-TCRβ V antibody molecule, e.g., an anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule, comprises at least one, two, three, four, five, or six CDRs (or collectively all of the CDRs) from heavy and light chain variable regions comprising the amino acid sequences shown in Table 1 or encoded by the nucleotide sequences shown in Table 1. In some embodiments, one or more of the CDRs (or collectively all of the CDRs) have one, two, three, four, five, six, o...

Claims

1. (a) a tumor-associated antigen-binding portion; (b) at least one cytokine molecule or a functional fragment or functional variant thereof; and (c) a TCRβV-binding portion covalently linked to at least one cytokine molecule or a functional fragment or functional variant thereof. A multifunctional molecule comprising:

2. the multifunctional molecule comprises a first polypeptide chain comprising a first portion of a dimerization module and a second polypeptide chain comprising a second portion of a dimerization module; the first polypeptide chain and the second polypeptide chain are non-contiguous; 2. The multifunctional molecule of claim 1, wherein the tumor-associated antigen binding portion is linked to a first portion of the dimerization module, and at least one cytokine molecule or a functional fragment or functional variant thereof is linked to the first portion of the dimerization module, the second portion of the dimerization module, or a combination thereof.

3. (i) the tumor-associated antigen binding portion is linked to the N-terminus of the first portion of the dimerization module, and at least one cytokine molecule, or a functional fragment or variant thereof, is linked to the C-terminus of the first portion of the dimerization module, the N-terminus of the second portion of the dimerization module, the C-terminus of the second portion of the dimerization module, or any combination thereof; or (ii) the tumor-associated antigen binding portion is linked to the C-terminus of the first portion of the dimerization module, and at least one cytokine molecule, or a functional fragment or functional variant thereof, is linked to the N-terminus of the first portion of the dimerization module, the N-terminus of the second portion of the dimerization module, the C-terminus of the second portion of the dimerization module, or any combination thereof.

4. A multifunctional molecule described in any one of claims 2 to 3, wherein the TCRβV binding portion and at least one cytokine molecule or a functional fragment or functional variant thereof are present in a single continuous polypeptide chain of the first polypeptide chain or the second polypeptide chain.

5. The multifunctional molecule of any one of claims 1 to 4, wherein the tumor-associated antigen-binding portion, the TCRβV-binding portion, or a combination thereof comprises an antibody or an antigen-binding fragment thereof, and the antigen-binding fragment comprises any one selected from the group consisting of Fab, F(ab')2, Fv, single-chain Fv (scFv), single-domain antibody, diabody (dAb), camel antibody, and any combination thereof.

6. The multifunctional molecule of claim 1 , wherein the TCRβV-binding portion comprises a heavy chain variable domain (VH) and a light chain variable domain (VL), or a single domain antibody.

7. A multifunctional molecule described in any one of claims 2 to 5, wherein the TCRβV binding portion comprises a first portion of the TCRβV binding portion, and the multifunctional molecule further comprises a third polypeptide chain comprising a second portion of the TCRβV binding portion, and the third polypeptide chain is non-contiguous with the first polypeptide chain and the second polypeptide chain.

8. The multifunctional molecule of claim 7, wherein the first portion of the TCRβV binding portion comprises the VH of the TCRβV binding portion and the second portion of the TCRβV binding portion comprises the VL of the TCRβV binding portion, or the first portion of the TCRβV binding portion comprises the VL of the TCRβV binding portion and the second portion of the TCRβV binding portion comprises the VH of the TCRβV binding portion.

9. The multifunctional molecule of claim 1 , wherein the tumor-associated antigen-binding portion comprises a VH and a VL, or a single domain antibody.

10. 9. The multifunctional molecule of any one of claims 2 to 8, wherein the tumor-associated antigen binding portion comprises a first portion of the tumor-associated antigen binding portion, and the multifunctional molecule further comprises a fourth polypeptide chain comprising a second portion of the tumor-associated antigen binding portion, wherein the fourth polypeptide chain is non-contiguous with the first polypeptide chain, the second polypeptide chain, and the third polypeptide chain.

11. The multifunctional molecule of claim 10, wherein the first portion of the tumor-associated antigen binding moiety comprises the VH of the tumor-associated antigen binding moiety and the second portion of the tumor-associated antigen binding moiety comprises the VL of the tumor-associated antigen binding moiety, or the first portion of the tumor-associated antigen binding moiety comprises the VL of the tumor-associated antigen binding moiety and the second portion of the tumor-associated antigen binding moiety comprises the VH of the tumor-associated antigen binding moiety.

12. 12. The multifunctional molecule of claim 1, wherein the first portion of the dimerization module and the second portion of the dimerization module are dimerized.

13. (i) the tumor-associated antigen-binding portion further comprises a heavy chain constant domain 1 (CH1) linked to the VH of the tumor-associated antigen-binding portion; (ii) the TCRβV-binding portion further comprises a heavy chain constant domain 1 (CH1) linked to the VH of the TCRβV-binding portion; or (iii) A multifunctional molecule according to any one of claims 1 to 12, which is a combination thereof.

14. (i) the tumor-associated antigen-binding moiety further comprises a light chain constant domain (CL) linked to the VL of the tumor-associated antigen-binding moiety; (ii) the TCRβV-binding portion further comprises a light chain constant domain (CL) linked to the VL of the TCRβV-binding portion; or (iii) A multifunctional molecule according to any one of claims 1 to 13, which is a combination thereof.

15. (i) whether the CL linked to the VL of the tumor-associated antigen-binding moiety comprises a kappa chain constant domain or a lambda chain constant domain; (ii) the C L linked to the V L of the TCRβV-binding portion comprises a kappa chain constant domain or a lambda chain constant domain; or (iii) The multifunctional molecule of claim 14, which is a combination thereof.

16. 16. The multifunctional molecule of claim 15, wherein the kappa chain constant domain or the lambda chain constant domain comprises any one of the light chain constant region sequences listed in Tables 3, 21, or 22.

17. (i) a linker between the first portion of the dimerization module and the tumor-associated antigen-binding portion or the first portion of the tumor-associated antigen; (ii) a linker between at least one cytokine molecule, or a functional fragment or functional variant thereof, and a first portion of the dimerization module, a linker between at least one cytokine molecule, or a functional fragment or functional variant thereof, and a second portion of the dimerization module, or a combination thereof; (iii) a linker between at least one cytokine molecule, or a functional fragment or functional variant thereof, and the TCRβV-binding portion or the first portion of the TCRβV-binding portion; (iv) a linker between the VH and the VL of the tumor-associated antigen-binding portion; (v) a linker between the VH and the VL of the TCRβV-binding portion; (vi) a linker between the CH1 and the VH of the tumor-associated antigen-binding portion; (vii) a linker between the CH1 and the VH of the TCRβV-binding portion; (viii) a linker between the CL and the VL of the tumor-associated antigen-binding moiety; (ix) a linker between the C and the V of the TCRβV-binding portion; or (x) any combination of these 17. The multifunctional molecule of claim 1, further comprising:

18. 18. The multifunctional molecule of claim 17, wherein the linker is selected from the group consisting of a cleavable linker, a non-cleavable linker, a peptide linker, a flexible linker, a rigid linker, a helical linker, and a non-helical linker.

19. 19. The multifunctional molecule of claim 18, wherein the linker is a peptide linker comprising the sequence of SEQ ID NO: 3308 or SEQ ID NO: 3643.

20. 20. The multifunctional molecule of any one of claims 1 to 19, which is an isolated multifunctional molecule.

21. 21. The multifunctional molecule of any one of claims 1 to 20, wherein the tumor-associated antigen-binding portion, the TCRβV-binding portion, or a combination thereof comprises a Fab or an scFv.

22. 22. The multifunctional molecule of any one of claims 1 to 21, wherein the at least one cytokine molecule or functional fragment or functional variant thereof is selected from the group consisting of interleukin-2 (IL-2) or a functional fragment or functional variant thereof, interleukin-7 (IL-7) or a functional fragment or functional variant thereof, interleukin-12 (IL-12) or a functional fragment or functional variant thereof, interleukin-15 (IL-15) or a functional fragment or functional variant thereof, interleukin-18 (IL-18) or a functional fragment or functional variant thereof, interleukin-21 (IL-21) or a functional fragment or functional variant thereof, or interferon gamma or a functional fragment or functional variant thereof, or any combination thereof.

23. 22. The multifunctional molecule of any one of claims 1 to 21, wherein the at least one cytokine molecule or functional fragment or functional variant thereof comprises interleukin-2 (IL-2) or a functional fragment or functional variant thereof.

24. 24. The multifunctional molecule of claim 23, wherein at least one cytokine molecule or functional fragment or variant thereof is an IL-2 variant comprising a substitution mutation.

25. 25. The multifunctional molecule of claim 24, wherein at least one cytokine molecule or functional fragment or variant thereof is an IL-2 variant comprising a C125A mutation.

26. 26. The multifunctional molecule of any one of claims 23 to 25, wherein at least one cytokine molecule or functional fragment or functional variant thereof comprises a sequence having at least 75% sequence identity to the sequence of SEQ ID NO: 2270 or the sequence of SEQ ID NO: 2191.

27. 26. The multifunctional molecule of any one of claims 23 to 25, wherein at least one cytokine molecule or functional fragment or functional variant thereof comprises the sequence of SEQ ID NO: 2270 or the sequence of SEQ ID NO: 2191.

28. 28. The multifunctional molecule of any one of claims 1 to 27, wherein a first portion of the dimerization module comprises a first immunoglobulin constant region (Fc region) and a second portion of the dimerization module comprises a second Fc region.

29. The multifunctional molecule of claim 28, wherein the first Fc region, the second Fc region, or a combination thereof is selected from the group consisting of an IgG1 Fc region or fragment thereof, an IgG2 Fc region or fragment thereof, an IgG3 Fc region or fragment thereof, an IgGA1 Fc region or fragment thereof, an IgG2 Fc region or fragment thereof, an IgG4 Fc region or fragment thereof, an IgJ Fc region or fragment thereof, an IgM Fc region or fragment thereof, an IgD Fc region or fragment thereof, and an IgE Fc region or fragment thereof.

30. 30. The multifunctional molecule of claim 29, wherein the first Fc region, the second Fc region, or a combination thereof is selected from the group consisting of a human IgG1 Fc region or fragment thereof, a human IgG2 Fc region or fragment thereof, and a human IgG4 Fc region or fragment thereof.

31. 31. The multifunctional molecule of any one of claims 28 to 30, wherein the first Fc region, the second Fc region, or a combination thereof comprises an Fc interface having one or more of paired holes and protrusions, electrostatic interactions, or strand exchange, and wherein dimerization of the first Fc region and the second Fc region is enhanced as indicated by a higher ratio of heteromultimer:homomultimer forms compared to dimerization of an Fc region having an unengineered interface.

32. 32. The multifunctional molecule of claim 31 , wherein the first Fc region, the second Fc region, or a combination thereof comprises an amino acid substitution listed in Table 14.

33. 33. The multifunctional molecule of claim 32, wherein the first Fc region, the second Fc region, or a combination thereof comprises an Asn297Ala (N297A) mutation or a Leu234Ala / Leu235Ala (LALA) mutation.

34. The multifunctional molecule of claim 31 , wherein the first Fc region, the second Fc region, or a combination thereof comprises a sequence having at least 75% sequence identity to the sequence of SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 3645, SEQ ID NO: 3646, SEQ ID NO: 3647, SEQ ID NO: 3648, SEQ ID NO: 3649, SEQ ID NO: 3792, or SEQ ID NO: 3794.

35. The multifunctional molecule of claim 31 , wherein the first Fc region, the second Fc region, or a combination thereof comprises the sequence of SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 3645, SEQ ID NO: 3646, SEQ ID NO: 3647, SEQ ID NO: 3648, SEQ ID NO: 3649, SEQ ID NO: 3792, or SEQ ID NO: 3794.

36. The multifunctional molecule of claim 31, wherein the first Fc region comprises a sequence having at least 75% sequence identity to the sequence of SEQ ID NO: 3649 or SEQ ID NO: 3792, and the second Fc region comprises a sequence having at least 75% sequence identity to the sequence of SEQ ID NO: 3648 or SEQ ID NO: 3794, or the first Fc region comprises a sequence having at least 75% sequence identity to the sequence of SEQ ID NO: 3648 or SEQ ID NO: 3794, and the second Fc region comprises a sequence having at least 75% sequence identity to the sequence of SEQ ID NO: 3648 or SEQ ID NO: 3794.

37. The multifunctional molecule of claim 31 , wherein the first Fc region comprises a sequence having the sequence of SEQ ID NO: 3649 or SEQ ID NO: 3792, and the second Fc region comprises a sequence of SEQ ID NO: 3648 or SEQ ID NO: 3794, or wherein the first Fc region comprises a sequence of SEQ ID NO: 3648 or SEQ ID NO: 3794, and the second Fc region comprises a sequence of SEQ ID NO: 3648 or SEQ ID NO: 3794.

38. 38. The multifunctional molecule of any one of claims 1 to 37, wherein the TCRβV binding portion binds to one or more TCRβV subfamilies selected from the group consisting of TCRβ V2 subfamily, TCRβ V3 subfamily, TCRβ V4 subfamily, TCRβ V5 subfamily, TCRβ V6 subfamily, TCRβ V9 subfamily, TCRβ V10 subfamily, TCRβ V11 subfamily, TCRβ V12 subfamily, TCRβ V13 subfamily, TCRβ V16 subfamily, TCRβ V19, TCRβ V20 subfamily, TCRβ V21 subfamily, TCRβ V23 subfamily, TCRβ V27 subfamily, and TCRβ V28 subfamily.

39. The TCRβV binding portion is (i) the TCRβ V2 subfamily, which includes TCRβ V2*01; (ii) the TCRβ V3 subfamily, including TCRβ V3-1*01; (iii) the TCRβ V4 subfamily, including one or more selected from TCRβ V4-1, TCRβ V4-2, and TCRβ V4-3; (iv) the TCRβ V5 subfamily, including one or more selected from TCRβ V5-6*01, TCRβ V5-4*01, TCRβ V5-1*01, and TCRβ V5-8*01; (v) the TCRβ V6 subfamily, including one or more selected from TCRβ V6-4*01, TCRβ V6-4*02, TCRβ V6-9*01, TCRβ V6-8*01, TCRβ V6-5*01, TCRβ V6-6*02, TCRβ V6-6*01, TCRβ V6-2*01, TCRβ V6-3*01, and TCRβ V6-1*01; (vi) the TCRβ V10 subfamily, including one or more selected from TCRβ V10-1*01, TCRβ V10-1*02, TCRβ V10-3*01, and TCRβ V10-2*01; (vii) the TCRβ V11 subfamily, including TCRβ V11-2; (viii) the TCRβ V12 subfamily, including one or more selected from TCRβ V12-4*01, TCRβ V12-3*01, and TCRβ V12-5*01; (ix) the TCRβ V13 subfamily, including TCRβ V13*01; (x) the TCRβ V16 subfamily, including TCRβ V16*01; (xi) the TCRβ V19 subfamily, including one or more selected from TCRβ V19*01 and TCRβ V19*02; or (xii) TCRβ V20 subfamily including TCRβ V20-1*01 or TCRβ V20-1*02 39. The multifunctional molecule of any one of claims 1 to 38, which binds to one or more of the TCRβV subfamilies selected from the group consisting of:

40. 39. The multifunctional molecule of any one of claims 1 to 38, wherein the TCRβV-binding portion binds to the TCRβ V6 subfamily or the TCRβ V20 subfamily.

41. The TCRβV binding portion is (i) a VH comprising a combination of HC CDR1, HC CDR2, and HC CDR3 listed in Table 1, 2, 10B, 11, 12, 13, 21, or 22; (ii) a VL comprising a combination of LC CDR1, LC CDR2, and LC CDR3 listed in Table 1, 2, 10B, 11, 12, 13, 21, or 22; or (iii) combinations thereof 41. The multifunctional molecule of any one of claims 1 to 40, comprising:

42. The TCRβV binding portion is (i) a VH comprising a HC CDR1, a HC CDR2, and a HC CDR3 of any one of the amino acid sequences of a heavy chain variable domain listed in Table 1, 2, 10B, 11, 12, 13, 21, or 22; (ii) a VL comprising the LC CDR1, LC CDR2, and LC CDR3 of any one of the amino acid sequences of a light chain variable domain listed in Table 1, 2, 10B, 11, 12, 13, 21, or 22; or (iii) combinations thereof 42. The multifunctional molecule of any one of claims 1 to 41, comprising:

43. The TCRβV binding portion is (i) a VH comprising a sequence having at least 70% sequence identity to any one of the amino acid sequences of a heavy chain variable domain listed in Table 1, 2, 10B, 11, 12, 13, 21, or 22; (ii) a VL comprising a sequence having at least 70% sequence identity to any one of the amino acid sequences of a light chain variable domain listed in Table 1, 2, 10B, 11, 12, 13, 21, or 22; (iii) combinations thereof 43. The multifunctional molecule of any one of claims 1 to 42, comprising:

44. The TCRβV binding portion is (i) a VH comprising any one of the amino acid sequences of a heavy chain variable domain listed in Table 1, 2, 10B, 11, 12, 13, 21, or 22; (ii) a VL comprising any one of the amino acid sequences of a light chain variable domain listed in Table 1, 2, 10B, 11, 12, 13, 21, or 22; (iii) combinations thereof 44. The multifunctional molecule of any one of claims 1 to 43, comprising:

45. 45. The multifunctional molecule of any one of claims 2 to 44, wherein the second polypeptide chain comprises a sequence having at least 70% sequence identity to the sequence of SEQ ID NO: 1346, a sequence having at least 70% sequence identity to the sequence of SEQ ID NO: 1349, a sequence having at least 70% sequence identity to the sequence of SEQ ID NO: 2270, and a sequence having at least 70% sequence identity to the sequence of SEQ ID NO: 3648.

46. 46. ​​The multifunctional molecule of any one of claims 2 to 45, wherein the second polypeptide chain comprises the sequence of SEQ ID NO: 1346, the sequence of SEQ ID NO: 1349, the sequence of SEQ ID NO: 2270, and the sequence of SEQ ID NO: 3648.

47. 47. The multifunctional molecule of claim 45 or 46, wherein the second polypeptide chain further comprises the sequence of SEQ ID NO: 3801, the sequence of SEQ ID NO: 3309, the sequence of SEQ ID NO: 3308, or any combination thereof.

48. The multifunctional molecule of any one of claims 2 to 45, wherein the second polypeptide chain comprises a sequence having at least 70% sequence identity to the sequence of SEQ ID NO: 1346, wherein the sequence of SEQ ID NO: 1346 is operably linked to a sequence having at least 70% sequence identity to the sequence of SEQ ID NO: 1349, wherein the sequence having at least 70% sequence identity to the sequence of SEQ ID NO: 1349 is operably linked to a sequence having at least 70% sequence identity to the sequence of SEQ ID NO: 2270, wherein the sequence having at least 70% sequence identity to the sequence of SEQ ID NO: 2270 is operably linked to a sequence having at least 70% sequence identity to the sequence of SEQ ID NO: 3648.

49. 49. The multifunctional molecule of claim 48, wherein a sequence having at least 70% sequence identity to the sequence of SEQ ID NO: 1346 is operably linked to a sequence having at least 70% sequence identity to the sequence of SEQ ID NO: 1349 via the sequence of SEQ ID NO: 3801.

50. A multifunctional molecule as described in claim 48 or 49, wherein a sequence having at least 70% sequence identity to the sequence of SEQ ID NO: 1349 is operably linked to a sequence having at least 70% sequence identity to the sequence of SEQ ID NO: 2270 via the sequence of SEQ ID NO: 3309.

51. A multifunctional molecule described in any one of claims 48 to 50, wherein a sequence having at least 70% sequence identity to the sequence of SEQ ID NO: 2270 is operably linked to a sequence having at least 70% sequence identity to the sequence of SEQ ID NO: 3648 via the sequence of SEQ ID NO: 3308.

52. The multifunctional molecule of any one of claims 2 to 45 and 48, wherein the second polypeptide chain comprises the sequence of SEQ ID NO: 1346, which is operably linked to the sequence of SEQ ID NO: 1349, which is operably linked to the sequence of SEQ ID NO: 2270, which is operably linked to the sequence of SEQ ID NO: 3648.

53. 53. The multifunctional molecule of claim 52, wherein the sequence of SEQ ID NO: 1346 is operably linked to the sequence of SEQ ID NO: 1349 via the sequence of SEQ ID NO: 3801.

54. 54. The multifunctional molecule of claim 52 or 53, wherein the sequence of SEQ ID NO: 1349 is operably linked to the sequence of SEQ ID NO: 2270 via the sequence of SEQ ID NO: 3309.

55. 55. A multifunctional molecule described in any one of claims 52 to 54, wherein the sequence of SEQ ID NO: 2270 is operably linked to the sequence of SEQ ID NO: 3648 via the sequence of SEQ ID NO: 3308.

56. 56. The multifunctional molecule of any one of claims 2 to 55, wherein the second polypeptide chain comprises a sequence having at least 70% sequence identity to the sequence of SEQ ID NO: 3800.

57. 57. The multifunctional molecule of any one of claims 2 to 56, wherein the second polypeptide chain comprises the sequence of SEQ ID NO: 3800.

58. 58. The multifunctional molecule of any one of claims 1 to 57, wherein the multifunctional molecule comprises a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain comprises a tumor-associated antigen binding moiety linked to an Fc region, wherein the tumor-associated antigen binding moiety is an scFv that binds to the tumor-associated antigen, and wherein the second polypeptide chain comprises the sequence of SEQ ID NO: 3800.

59. 59. The multifunctional molecule of any one of claims 1 to 58, wherein the multifunctional molecule comprises a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain comprises a tumor-associated antigen binding moiety linked to a sequence having at least 75% sequence identity to the sequence of SEQ ID NO: 3649 or SEQ ID NO: 3792, wherein the tumor-associated antigen binding moiety is an scFv that binds to the tumor-associated antigen, and wherein the second polypeptide chain comprises the sequence of SEQ ID NO: 3800.

60. 60. The multifunctional molecule of any one of claims 1 to 59, wherein the multifunctional molecule comprises a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain comprises a tumor-associated antigen binding moiety linked to the sequence of SEQ ID NO: 3649 or SEQ ID NO: 3792, wherein the tumor-associated antigen binding moiety is an scFv that binds to the tumor-associated antigen, and wherein the second polypeptide chain comprises the sequence of SEQ ID NO: 3800.

61. 58. The multifunctional molecule of any one of claims 1 to 57, wherein the multifunctional molecule comprises a first polypeptide chain, a second polypeptide chain, and a third polypeptide chain, wherein the first polypeptide chain comprises a first portion of a tumor-associated antigen binding moiety linked to an Fc region, the tumor-associated antigen binding moiety being a Fab that binds to the tumor-associated antigen; the second polypeptide chain comprises the sequence of SEQ ID NO: 3800; and the third polypeptide chain comprises a second portion of the tumor-associated antigen binding moiety.

62. 62. The multifunctional molecule of any one of claims 1 to 57 and 61, wherein the multifunctional molecule comprises a first polypeptide chain, a second polypeptide chain, and a third polypeptide chain, wherein the first polypeptide chain comprises a first portion of a tumor-associated antigen binding moiety linked to a sequence having at least 75% sequence identity to the sequence of SEQ ID NO: 3649 or SEQ ID NO: 3792, wherein the tumor-associated antigen binding moiety is a Fab that binds to the tumor-associated antigen; the second polypeptide chain comprises the sequence of SEQ ID NO: 3800; and the third polypeptide chain comprises a second portion of the tumor-associated antigen binding moiety.

63. 63. The multifunctional molecule of any one of claims 1 to 57, 61, and 62, wherein the multifunctional molecule comprises a first polypeptide chain, a second polypeptide chain, and a third polypeptide chain, wherein the first polypeptide chain comprises a first portion of a tumor-associated antigen binding moiety linked to the sequence of SEQ ID NO: 3649 or SEQ ID NO: 3792, wherein the tumor-associated antigen binding moiety is a Fab that binds to the tumor-associated antigen; the second polypeptide chain comprises the sequence of SEQ ID NO: 3800; and the third polypeptide chain comprises a second portion of the tumor-associated antigen binding moiety.

64. The tumor-associated antigen binding portion may be CD19, CD123, CD22, CD30, CD171, CS-1, C-type lectin-like molecule-1, CD33, epidermal growth factor receptor variant III (EGFRvIII), ganglioside G2 (GD2), ganglioside GD3, TNF receptor family member B-cell maturation (BCMA), Tn antigen (TnAg) or (GalNAcα-Ser / Thr)), prostate-specific membrane antigen (PSMA), receptor tyrosine kinase-like orphan receptor 1 (ROR1), Fms-like tyrosine kinase 3 (FLT3), tumor-associated glycoprotein 72 (TAG72), CD38, CD44v6, carcinoembryonic antigen (CEA), epithelial cell adhesion molecule (EPCAM), B7H3 (CD276), KIT (CD117), interleukin-13 receptor subunit alpha-2, mesothelin, interleukin-11 receptor alpha (IL-11Ra), prostate stem cell antigen (PSCA), protease serine 21, vascular endothelial growth factor receptor 2 (VEGFR2), Lewis (Y) antigen, CD24, platelet-derived growth factor receptor beta (PDGFR-beta), stage-specific embryonic antigen-4 (SSEA-4), CD20, folate receptor alpha, receptor tyrosine-protein kinase ERBB2 (Her2 / neu), cell surface-associated mucin 1 (MUC1), epidermal growth factor receptor (EGFR), neural cell adhesion molecule (NCAM), prostase, prostatic acid phosphatase (PAP), mutant elongation factor 2 (ELF2M) ), ephrinB2, fibroblast activation protein alpha (FAP), insulin-like growth factor 1 receptor (IGF-I receptor), carbonic anhydrase IX (CAIX), proteasome (prosome, macropein) subunit, beta type 9 (LMP2), glycoprotein 100 (gp100 / pmel17), oncogene fusion protein (bcr-abl) consisting of breakpoint cluster region (BCR) and Abelson murine leukemia viral oncogene homolog 1 (Abl), tyrosinase, ephrin type A receptor 2 (EphA2 ), fucosyl GM1, sialyl Lewis adhesion molecule (sLe), ganglioside GM3, transglutaminase 5 (TGS5), high molecular weight melanoma-associated antigen (HMWMAA), o-acetyl-GD2 ganglioside (OAcGD2), folate receptor beta, tumor endothelial marker 1 (TEM1 / CD248), tumor endothelial marker 7-related (TEM7R), claudin 6 (CLDN6), thyroid-stimulating hormone receptor (TSHR), G protein-coupled receptor class C group 5, member D (GPRC5D), chromosome X open reading frame61 (CXORF61), CD97, CD179a, anaplastic lymphoma kinase (ALK), polysialic acid, placenta-specific 1 (PLAC1), hexasaccharide moiety of globoH glycoceramide (GloboH), mammary differentiation antigen (NY-BR-1), uroplakin 2 (UPK2), hepatitis A virus cell receptor 1 (HAVCR1), adrenoceptor beta 3 (ADRB3), pannexin 3 (PANX3), G protein-coupled receptor 20 (GPR20), lymphocyte antigen 6 complex, locus K9 (LY6K), olfactory receptor 5 1E2 (OR51E2), TCR gamma alternate reading frame protein (TARP), Wilms tumor protein (WT1), cancer / testis antigen 1 (NY-ESO-1 / LAGE-1), cancer / testis antigen 2 (LAGE-1a), melanoma-associated antigen 1 (MAGE-A1), ETS translocation variant gene 6 located on chromosome 12p (ETV6-AML), sperm protein 17 (SPA17), X antigen family, member 1A (XAGE1), angiopoietin-binding cell surface receptor 2 (Tie 2), melanoma cancer testis antigen-1 (MAD-CT-1), melanoma cancer testis antigen-2 (MAD-CT-2), Fos-related antigen 1, tumor protein p53 (p53), p53 mutant, prostein, survivin, telomerase, prostate cancer tumor antigen-1, melanoma antigen 1 recognized by T cells, rat sarcoma (Ras) mutant, human telomerase reverse transcriptase (hTERT), sarcoma translocation breakpoint, melanoma inhibitor of apoptosis (ML-IAP), ER G (transmembrane protease, serine 2 (TMPRSS2) ETS fusion gene), N-acetylglucosaminyl-transferase V (NA17), paired box protein Pax-3 (PAX3), androgen receptor, cyclin B1, v-myc avian myelocytomatosis viral oncogene neuroblastoma-derived homolog (MYCN), Ras homolog family member C (RhoC), tyrosinase-related protein 2 (TRP-2), cytochrome P4501B1 (CYP1B1), CCCTC-binding factor (zinc finger protein)-like, squamous cell carcinoma antigen 3 recognized by T cells (SART3), paired box protein Pax-5 (PAX5), proacrosin-binding protein sp32 (OY-TES1), lymphocyte-specific protein tyrosine kinase (LCK), A-kinase anchor protein 4 (AKAP-4), synovial sarcoma X breakpoint 2 (SSX2), receptor for advanced glycation end products (RAGE-1), renal ubiquitous 1 (RU1), renal ubiquitous 2 (RU2), legumain, human papillomavirus E6 (HPV E6), human papillomavirus E7 (HPV E7), intestinal carboxylesterase, mutant heat shock protein 70-2 (muthsp70-2), CD79a, CD79b, CD72, leukocyte-associated immunoglobulin-like receptor 1 (LAIR1), Fc fragment of IgA receptor (FCAR or CD89), leukocyte immunoglobulin-like receptor subfamily A member 2 (LILRA2), CD300 molecule-like family member f (CD300LF), C-type lectin domain family 12 member A (CLEC12A), bone marrow stromal cell antigen 2 (BST2), EGF-like molecule Glucocorticoid-containing mucin-like hormone receptor-like 2 (EMR2), lymphocyte antigen 75 (LY75), glypican-3 (GPC3), Fc receptor-like 5 (FCRL5), FcRH5, PDL1, CD47, prostate-specific membrane antigen (PMSA), prostate-specific antigen (PSA), Ron kinase, c-Met, immature laminin receptor, TAG-72, BING-4, calcium-activated chloride channel 2, cyclin-B1, 9D7, Ep-CAM, EphA3 , SAP-1, PRAME, SSX-2, Melan-A / MART-1, TRP1 / gp75, MC1R, β-catenin, BRCA1 / 2, CDK4, CML66, fibronectin, Ras, TGF-B receptor, AFP, ETA, MAGE, CA-125, BAGE, GAGE, CDC27, α-actinin-4, gangliosides, MART-2, MUC2, MUM1, MUM2, MUM3, NA88-1, NPM, OA1, OGT, R 64. The multifunctional molecule of any one of claims 1 to 63, which binds to a cancer antigen selected from the group consisting of CC, RU11, RU12, SAGE, TRG, TSTA, L1-CAM, gpA33, GM2, VEGFR, integrin, carbohydrate, TRAILR1, TRAILR2, RANKL, TGF-beta, hyaluronic acid, collagen, tenascin-C, tenascin-W, and immunoglobulin lambda-like polypeptide 1 (IGLL1).

65. 65. The multifunctional molecule of any one of claims 1 to 64, wherein the tumor-associated antigen-binding portion binds to a cancer antigen selected from the group consisting of CD20, MSLN, gp75 (Tryp1), or any combination thereof.

66. 66. The multifunctional molecule of any one of claims 1 to 65, wherein the multifunctional molecule is a polypeptide molecule.

67. 67. The multifunctional molecule of any one of claims 1 to 66, wherein the tumor-associated antigen binding portion comprises a VH comprising HC CDR1, HC CDR2 and HC CDR3 comprising the sequences of SEQ ID NO: 524, SEQ ID NO: 525 and SEQ ID NO: 526, respectively, or a VH comprising HC CDR1, HC CDR2 and HC CDR3 comprising the sequences of SEQ ID NO: 538, SEQ ID NO: 539 and SEQ ID NO: 540, respectively.

68. 68. The multifunctional molecule of any one of claims 1 to 67, wherein the tumor-associated antigen binding portion comprises a VL comprising LC CDR1, LC CDR2 and LC CDR3 comprising the sequences of SEQ ID NO: 1168, SEQ ID NO: 1295, and SEQ ID NO: 528, respectively, or a VL comprising LC CDR1, LC CDR2 and LC CDR3 comprising the sequences of SEQ ID NO: 562, SEQ ID NO: 563, and SEQ ID NO: 564, respectively.

69. 69. The multifunctional molecule of any one of claims 1 to 68, wherein the tumor-associated antigen binding portion comprises a VH comprising HC CDR1, HC CDR2 and HC CDR3 comprising the sequences of SEQ ID NO: 524, SEQ ID NO: 525 and SEQ ID NO: 526, respectively, and a VL comprising LC CDR1, LC CDR2 and LC CDR3 comprising the sequences of SEQ ID NO: 1168, SEQ ID NO: 1295 and SEQ ID NO: 528, respectively.

70. 69. The multifunctional molecule of any one of claims 1 to 68, wherein the tumor-associated antigen binding portion comprises a VH comprising HC CDR1, HC CDR2 and HC CDR3 comprising the sequences of SEQ ID NO: 538, SEQ ID NO: 539 and SEQ ID NO: 540, respectively, and a VL comprising LC CDR1, LC CDR2 and LC CDR3 comprising the sequences of SEQ ID NO: 562, SEQ ID NO: 563 and SEQ ID NO: 564, respectively.

71. 71. The multifunctional molecule of any one of claims 1 to 70, wherein the tumor-associated antigen binding portion comprises a VH comprising a sequence having at least 70% sequence identity to the sequence of SEQ ID NO: 523 or the sequence of SEQ ID NO:

537.

72. 72. A multifunctional molecule according to any one of claims 1 to 71, wherein the tumor-associated antigen binding portion comprises a VL comprising a sequence having at least 70% sequence identity to the sequence of SEQ ID NO: 527 or the sequence of SEQ ID NO:

561.

73. 73. The multifunctional molecule of any one of claims 1 to 72, wherein the tumor-associated antigen binding portion comprises a VH comprising the sequence of SEQ ID NO: 523 or the sequence of SEQ ID NO:

537.

74. 74. A multifunctional molecule according to any one of claims 1 to 73, wherein the tumor-associated antigen binding portion comprises a VL comprising the sequence of SEQ ID NO: 527 or the sequence of SEQ ID NO:

561.

75. 75. The multifunctional molecule of any one of claims 1 to 74, wherein the tumor-associated antigen binding portion comprises a VH comprising a sequence having at least 70% sequence identity to the sequence of SEQ ID NO: 523, and a VL comprising a sequence having at least 70% sequence identity to the sequence of SEQ ID NO:

527.

76. 76. The multifunctional molecule of any one of claims 1 to 75, wherein the tumor-associated antigen binding portion comprises a VH comprising the sequence of SEQ ID NO: 523 and a VL comprising the sequence of SEQ ID NO:

527.

77. 75. The multifunctional molecule of any one of claims 1 to 74, wherein the tumor-associated antigen binding portion comprises a VH comprising a sequence having at least 70% sequence identity to the sequence of SEQ ID NO: 537, and a VL comprising a sequence having at least 70% sequence identity to the sequence of SEQ ID NO:

561.

78. 80. The multifunctional molecule of any one of claims 1 to 74 and 77, wherein the tumor-associated antigen binding portion comprises a VH comprising the sequence of SEQ ID NO: 537 and a VL comprising the sequence of SEQ ID NO:

561.

79. 67. The multifunctional molecule of any one of claims 1 to 66, wherein the tumor-associated antigen binding portion comprises a VH comprising HC CDR1, HC CDR2 and HC CDR3 comprising the sequences of SEQ ID NO: 581, SEQ ID NO: 582 and SEQ ID NO: 583, respectively.

80. 80. The multifunctional molecule of any one of claims 1 to 66 and 79, wherein the tumor-associated antigen binding portion comprises a VL comprising LC CDR1, LC CDR2 and LC CDR3 comprising the sequences of SEQ ID NO: 586, SEQ ID NO: 587 and SEQ ID NO: 588, respectively.

81. 81. The multifunctional molecule of any one of claims 1 to 66, 79, and 80, wherein the tumor-associated antigen binding portion comprises a VH comprising HC CDR1, HC CDR2, and HC CDR3 comprising the sequences of SEQ ID NO: 581, SEQ ID NO: 582, and SEQ ID NO: 583, respectively, and a VL comprising LC CDR1, LC CDR2, and LC CDR3 comprising the sequences of SEQ ID NO: 586, SEQ ID NO: 587, and SEQ ID NO: 588, respectively.

82. 82. The multifunctional molecule of any one of claims 1 to 66 and 79 to 81, wherein the tumor-associated antigen binding portion comprises a VH comprising a sequence having at least 70% sequence identity to the sequence of SEQ ID NO:

580.

83. A multifunctional molecule described in any one of claims 1 to 66 and 79 to 82, wherein the tumor-associated antigen binding portion comprises a VL comprising a sequence having at least 70% sequence identity to the sequence of SEQ ID NO:

585.

84. 84. The multifunctional molecule of any one of claims 1 to 66 and 79 to 83, wherein the tumor-associated antigen binding portion comprises a VH comprising the sequence of SEQ ID NO:

580.

85. A multifunctional molecule described in any one of claims 1 to 66 and 79 to 84, wherein the tumor-associated antigen binding portion comprises a VL comprising the sequence of SEQ ID NO:

585.

86. 86. The multifunctional molecule of any one of claims 1 to 66 and 79 to 85, wherein the tumor-associated antigen binding portion comprises a VH comprising a sequence having at least 70% sequence identity to the sequence of SEQ ID NO: 580, and a VL comprising a sequence having at least 70% sequence identity to the sequence of SEQ ID NO:

585.

87. 87. The multifunctional molecule of any one of claims 1 to 66 and 79 to 86, wherein the tumor-associated antigen binding portion comprises a VH comprising the sequence of SEQ ID NO: 580 and a VL comprising the sequence of SEQ ID NO:

585.

88. 88. The multifunctional molecule of any one of claims 1 to 87, wherein the TCRβV binding portion comprises a VH comprising HC CDR1, HC CDR2 and HC CDR3 comprising the sequences of SEQ ID NO: 3650, SEQ ID NO: 3651, and SEQ ID NO: 5, respectively, or a VH comprising HC CDR1, HC CDR2 and HC CDR3 comprising the sequences of SEQ ID NO: 542, SEQ ID NO: 543, and SEQ ID NO: 544, respectively.

89. 89. The multifunctional molecule of any one of claims 1 to 88, wherein the TCRβV binding portion comprises a VL comprising LC CDR1, LC CDR2 and LC CDR3 comprising the sequences of SEQ ID NO: 3655, SEQ ID NO: 3653, and SEQ ID NO: 8, respectively, or a VL comprising LC CDR1, LC CDR2 and LC CDR3 comprising the sequences of SEQ ID NO: 545, SEQ ID NO: 546, and SEQ ID NO: 547, respectively.

90. 90. The multifunctional molecule of any one of claims 1 to 89, wherein the TCRβV binding portion comprises a VH comprising HC CDR1, HC CDR2 and HC CDR3 comprising the sequences of SEQ ID NO: 3650, SEQ ID NO: 3651, and SEQ ID NO: 5, respectively, and a VL comprising LC CDR1, LC CDR2 and LC CDR3 comprising the sequences of SEQ ID NO: 3655, SEQ ID NO: 3653, and SEQ ID NO: 8, respectively.

91. 90. The multifunctional molecule of any one of claims 1 to 89, wherein the TCRβV binding portion comprises a VH comprising HC CDR1, HC CDR2 and HC CDR3 comprising the sequences of SEQ ID NO: 542, SEQ ID NO: 543, and SEQ ID NO: 544, respectively, and a VL comprising LC CDR1, LC CDR2 and LC CDR3 comprising the sequences of SEQ ID NO: 545, SEQ ID NO: 546, and SEQ ID NO: 547, respectively.

92. 92. A multifunctional molecule according to any one of claims 1 to 91, wherein the TCRβV binding portion comprises a VH comprising a sequence having at least 70% sequence identity to the sequence of SEQ ID NO: 1346 or the sequence of SEQ ID NO:

541.

93. 93. A multifunctional molecule according to any one of claims 1 to 92, wherein the TCRβV binding portion comprises a VL comprising a sequence having at least 70% sequence identity to the sequence of SEQ ID NO: 1349 or the sequence of SEQ ID NO: 3527.

94. 94. The multifunctional molecule of any one of claims 1 to 93, wherein the TCRβV binding portion comprises a VH comprising the sequence of SEQ ID NO: 1346 or the sequence of SEQ ID NO:

541.

95. 95. A multifunctional molecule according to any one of claims 1 to 94, wherein the TCRβV binding portion comprises a VL comprising the sequence of SEQ ID NO: 1349 or the sequence of SEQ ID NO: 3527.

96. 96. A multifunctional molecule according to any one of claims 1 to 95, wherein the TCRβV binding portion comprises a VH comprising a sequence having at least 70% sequence identity to the sequence of SEQ ID NO: 1346, and a VL comprising a sequence having at least 70% sequence identity to the sequence of SEQ ID NO: 1349.

97. 97. The multifunctional molecule of any one of claims 1 to 96, wherein the TCRβV binding portion comprises a VH comprising the sequence of SEQ ID NO: 1346 and a VL comprising the sequence of SEQ ID NO: 1349.

98. 96. A multifunctional molecule according to any one of claims 1 to 95, wherein the TCRβV binding portion comprises a VH comprising a sequence having at least 70% sequence identity to the sequence of SEQ ID NO: 541, and a VL comprising a sequence having at least 70% sequence identity to the sequence of SEQ ID NO: 3527.

99. 99. The multifunctional molecule of any one of claims 1 to 95 and 98, wherein the TCRβV binding portion comprises a VH comprising the sequence of SEQ ID NO: 541 and a VL comprising the sequence of SEQ ID NO: 3527.

100. A multifunctional molecule described in any one of claims 1 to 99, wherein the TCRβV binding portion comprises an scFv comprising the sequence of SEQ ID NO: 1346 operably linked to the sequence of SEQ ID NO: 1349 via a linker comprising the sequence of SEQ ID NO: 3801.

101. A multifunctional molecule described in any one of claims 1 to 99, wherein the TCRβV binding portion comprises an scFv comprising the sequence of SEQ ID NO: 541 operably linked to the sequence of SEQ ID NO: 3527 via a linker comprising the sequence of SEQ ID NO: 3801.

102. 102. The multifunctional molecule of any one of claims 1 to 101, wherein the TCRβV binding portion comprises an scFv comprising a sequence having at least 70% sequence identity to the sequence of SEQ ID NO: 1331 or the sequence of SEQ ID NO: 1376.

103. 103. The multifunctional molecule of any one of claims 1 to 102, wherein the TCRβV binding portion comprises an scFv comprising the sequence of SEQ ID NO: 1331 or the sequence of SEQ ID NO: 1376.

104. the multifunctional molecule comprises a first polypeptide chain, a second polypeptide chain, and a third polypeptide chain; (i) the first polypeptide chain comprises the sequence of SEQ ID NO: 523 and the sequence of SEQ ID NO: 3649; (ii) the second polypeptide chain comprises the sequence of SEQ ID NO: 1331, the sequence of SEQ ID NO: 2270, and the sequence of SEQ ID NO: 3648; (iii) The multifunctional molecule of any one of claims 1 to 103, wherein the third polypeptide chain comprises the sequence of SEQ ID NO: 527 and the sequence of SEQ ID NO: 3644.

105. the multifunctional molecule comprises a first polypeptide chain, a second polypeptide chain, and a third polypeptide chain; (i) the first polypeptide chain comprises the sequence of SEQ ID NO: 523 operably linked to the sequence of SEQ ID NO: 3649; (ii) the second polypeptide chain comprises the sequence of SEQ ID NO: 1331 operably linked to the sequence of SEQ ID NO: 2270, which is operably linked to the sequence of SEQ ID NO: 3648; (iii) The multifunctional molecule of any one of claims 1 to 104, wherein the third polypeptide chain comprises the sequence of SEQ ID NO: 527 operably linked to the sequence of SEQ ID NO: 3644.

106. the multifunctional molecule comprises a first polypeptide chain, a second polypeptide chain, and a third polypeptide chain; (i) the first polypeptide chain comprises a sequence having at least 70% sequence identity to the sequence of SEQ ID NO: 501; (ii) the second polypeptide chain comprises a sequence having at least 70% sequence identity to the sequence of SEQ ID NO: 3800; (iii) The multifunctional molecule of any one of claims 1 to 105, wherein the third polypeptide chain comprises a sequence having at least 70% sequence identity to the sequence of SEQ ID NO:

502.

107. the multifunctional molecule comprises a first polypeptide chain, a second polypeptide chain, and a third polypeptide chain; (i) the first polypeptide chain comprises the sequence of SEQ ID NO: 501; (ii) the second polypeptide chain comprises the sequence of SEQ ID NO: 3800; (iii) The multifunctional molecule of any one of claims 1 to 106, wherein the third polypeptide chain comprises the sequence of SEQ ID NO:

502.

108. the multifunctional molecule comprises a first polypeptide chain, a second polypeptide chain, and a third polypeptide chain; (i) the first polypeptide chain comprises a first sequence of SEQ ID NO:523, a second sequence of SEQ ID NO:523, and a sequence of SEQ ID NO:3649; (ii) the second polypeptide chain comprises the sequence of SEQ ID NO: 1331, the sequence of SEQ ID NO: 2270, and the sequence of SEQ ID NO: 3648; (iii) The multifunctional molecule of any one of claims 1 to 103, wherein the third polypeptide chain comprises the sequence of SEQ ID NO: 527 and the sequence of SEQ ID NO: 3644.

109. the multifunctional molecule comprises a first polypeptide chain, a second polypeptide chain, and a third polypeptide chain; (i) the first polypeptide chain comprises a first sequence of SEQ ID NO: 523 operably linked to a second sequence of SEQ ID NO: 523 operably linked to a sequence of SEQ ID NO: 3649; (ii) the second polypeptide chain comprises the sequence of SEQ ID NO: 1331 operably linked to the sequence of SEQ ID NO: 2270, which is operably linked to the sequence of SEQ ID NO: 3648; (iii) The multifunctional molecule of any one of claims 1 to 103 and 108, wherein the third polypeptide chain comprises the sequence of SEQ ID NO: 527 operably linked to the sequence of SEQ ID NO: 3644.

110. the multifunctional molecule comprises a first polypeptide chain, a second polypeptide chain, and a third polypeptide chain; (i) the first polypeptide chain comprises a sequence having at least 70% sequence identity to the sequence of SEQ ID NO: 590; (ii) the second polypeptide chain comprises a sequence having at least 70% sequence identity to the sequence of SEQ ID NO: 3800; (iii) The multifunctional molecule of any one of claims 1 to 103, 108, and 109, wherein the third polypeptide chain comprises a sequence having at least 70% sequence identity to the sequence of SEQ ID NO:

502.

111. the multifunctional molecule comprises a first polypeptide chain, a second polypeptide chain, and a third polypeptide chain; (i) the first polypeptide chain comprises the sequence of SEQ ID NO: 590; (ii) the second polypeptide chain comprises the sequence of SEQ ID NO: 3800; (iii) The multifunctional molecule of any one of claims 1 to 103 and 108 to 110, wherein the third polypeptide chain comprises the sequence of SEQ ID NO:

502.

112. the multifunctional molecule comprises a first polypeptide chain, a second polypeptide chain, and a third polypeptide chain; (i) the first polypeptide chain comprises the sequence of SEQ ID NO: 537 and the sequence of SEQ ID NO: 559; (ii) the second polypeptide chain comprises the sequence of SEQ ID NO: 1376, the sequence of SEQ ID NO: 2191, and the sequence of SEQ ID NO: 3533; (iii) The multifunctional molecule of any one of claims 1 to 103, wherein the third polypeptide chain comprises the sequence of SEQ ID NO: 561 and the sequence of SEQ ID NO:

558.

113. the multifunctional molecule comprises a first polypeptide chain, a second polypeptide chain, and a third polypeptide chain; (i) the first polypeptide chain comprises the sequence of SEQ ID NO: 537 operably linked to the sequence of SEQ ID NO: 559; (ii) the second polypeptide chain comprises the sequence of SEQ ID NO: 1376 operably linked to the sequence of SEQ ID NO: 2191, which is operably linked to the sequence of SEQ ID NO: 3533; (iii) The multifunctional molecule of any one of claims 1 to 103 and 112, wherein the third polypeptide chain comprises the sequence of SEQ ID NO: 561 operably linked to the sequence of SEQ ID NO:

558.

114. the multifunctional molecule comprises a first polypeptide chain, a second polypeptide chain, and a third polypeptide chain; (i) the first polypeptide chain comprises a sequence having at least 70% sequence identity to the sequence of SEQ ID NO: 509; (ii) the second polypeptide chain comprises a sequence having at least 70% sequence identity to the sequence of SEQ ID NO: 511; (iii) The multifunctional molecule of any one of claims 1 to 103, 112, and 113, wherein the third polypeptide chain comprises a sequence having at least 70% sequence identity to the sequence of SEQ ID NO:

510.

115. the multifunctional molecule comprises a first polypeptide chain, a second polypeptide chain, and a third polypeptide chain; (i) the first polypeptide chain comprises the sequence of SEQ ID NO: 509; (ii) the second polypeptide chain comprises the sequence of SEQ ID NO: 511; (iii) A multifunctional molecule described in any one of claims 1 to 103 and 112 to 114, wherein the third polypeptide chain comprises the sequence of SEQ ID NO:

510.

116. the multifunctional molecule comprises a first polypeptide chain, a second polypeptide chain, and a third polypeptide chain; (i) the first polypeptide chain comprises a first sequence of SEQ ID NO:537, a second sequence of SEQ ID NO:537, and a sequence of SEQ ID NO:559; (ii) the second polypeptide chain comprises the sequence of SEQ ID NO: 1376, the sequence of SEQ ID NO: 2191, and the sequence of SEQ ID NO: 3533; (iii) The multifunctional molecule of any one of claims 1 to 103, wherein the third polypeptide chain comprises the sequence of SEQ ID NO: 561 and the sequence of SEQ ID NO:

558.

117. the multifunctional molecule comprises a first polypeptide chain, a second polypeptide chain, and a third polypeptide chain; (i) the first polypeptide chain comprises a first sequence of SEQ ID NO: 537 operably linked to a second sequence of SEQ ID NO: 537 operably linked to a sequence of SEQ ID NO: 559; (ii) the second polypeptide chain comprises the sequence of SEQ ID NO: 1376 operably linked to the sequence of SEQ ID NO: 2191, which is operably linked to the sequence of SEQ ID NO: 3533; (iii) The multifunctional molecule of any one of claims 1 to 103 and 116, wherein the third polypeptide chain comprises the sequence of SEQ ID NO: 561 operably linked to the sequence of SEQ ID NO:

558.

118. the multifunctional molecule comprises a first polypeptide chain, a second polypeptide chain, and a third polypeptide chain; (i) the first polypeptide chain comprises a sequence having at least 70% sequence identity to the sequence of SEQ ID NO: 512; (ii) the second polypeptide chain comprises a sequence having at least 70% sequence identity to the sequence of SEQ ID NO: 511; (iii) The multifunctional molecule of any one of claims 1 to 103, 116, and 117, wherein the third polypeptide chain comprises a sequence having at least 70% sequence identity to the sequence of SEQ ID NO:

510.

119. the multifunctional molecule comprises a first polypeptide chain, a second polypeptide chain, and a third polypeptide chain; (i) the first polypeptide chain comprises the sequence of SEQ ID NO: 512; (ii) the second polypeptide chain comprises the sequence of SEQ ID NO: 511; (iii) A multifunctional molecule described in any one of claims 1 to 103 and 116 to 118, wherein the third polypeptide chain comprises the sequence of SEQ ID NO:

510.

120. the multifunctional molecule comprises a first polypeptide chain, a second polypeptide chain, and a third polypeptide chain; (i) the first polypeptide chain comprises the sequence of SEQ ID NO: 580 and the sequence of SEQ ID NO: 584; (ii) the second polypeptide chain comprises the sequence of SEQ ID NO: 1376, the sequence of SEQ ID NO: 2191, and the sequence of SEQ ID NO: 3533; (iii) The multifunctional molecule of any one of claims 1 to 103, wherein the third polypeptide chain comprises the sequence of SEQ ID NO: 585 and the sequence of SEQ ID NO: 3528.

121. the multifunctional molecule comprises a first polypeptide chain, a second polypeptide chain, and a third polypeptide chain; (i) the first polypeptide chain comprises the sequence of SEQ ID NO: 580 operably linked to the sequence of SEQ ID NO: 584; (ii) the second polypeptide chain comprises the sequence of SEQ ID NO: 1376 operably linked to the sequence of SEQ ID NO: 2191, which is operably linked to the sequence of SEQ ID NO: 3533; (iii) The multifunctional molecule of any one of claims 1 to 103 and 120, wherein the third polypeptide chain comprises the sequence of SEQ ID NO: 585 operably linked to the sequence of SEQ ID NO: 3528.

122. the multifunctional molecule comprises a first polypeptide chain, a second polypeptide chain, and a third polypeptide chain; (i) the first polypeptide chain comprises a sequence having at least 70% sequence identity to the sequence of SEQ ID NO: 519; (ii) the second polypeptide chain comprises a sequence having at least 70% sequence identity to the sequence of SEQ ID NO: 511; (iii) The multifunctional molecule of any one of claims 1 to 103, 120, and 121, wherein the third polypeptide chain comprises a sequence having at least 70% sequence identity to the sequence of SEQ ID NO:

520.

123. the multifunctional molecule comprises a first polypeptide chain, a second polypeptide chain, and a third polypeptide chain; (i) the first polypeptide chain comprises the sequence of SEQ ID NO: 519; (ii) the second polypeptide chain comprises the sequence of SEQ ID NO: 511; (iii) A multifunctional molecule described in any one of claims 1 to 103 and 120 to 122, wherein the third polypeptide chain comprises the sequence of SEQ ID NO:

520.

124. 124. The multifunctional molecule of any one of claims 1 to 123, wherein the multifunctional molecule is a multispecific molecule.

125. 125. A polynucleotide comprising a sequence encoding the multifunctional molecule of any one of claims 1 to 124.

126. 126. The polynucleotide of claim 125, which is an isolated nucleic acid molecule.

127. 127. A vector comprising one or more of the polynucleotides of claim 125 or 126.

128. A cell comprising the polynucleotide of claim 125 or 126, or the vector of claim 127.

129. A pharmaceutical composition comprising a multifunctional molecule described in any one of claims 1 to 124, a polynucleotide described in claim 125 or 126, a vector described in claim 127, or a cell described in claim 128, and a pharmaceutically acceptable carrier, excipient, or diluent.

130. 128. A method of treating a condition or disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a multifunctional molecule of any one of claims 1 to 124, a polynucleotide of claim 125 or 126, a vector of claim 127, a cell of claim 128, a pharmaceutical composition of claim 129, or any combination thereof; The method, wherein the administering step is effective to treat a condition or disease in the subject.

131. 131. The method of claim 130, wherein the condition or disease is cancer.

132. 132. The method of claim 131, wherein the cancer is a solid tumor, a blood cancer, a metastatic cancer, a soft tissue tumor, or any combination thereof.

133. 133. The method of claim 132, wherein the cancer is a solid tumor, and the solid tumor is selected from the group consisting of melanoma, pancreatic cancer, breast cancer, colorectal cancer, lung cancer, skin cancer, ovarian cancer, liver cancer, and any combination thereof.

134. 133. The method of claim 132, wherein the cancer is a blood cancer, and the blood cancer is selected from the group consisting of Hodgkin's lymphoma, non-Hodgkin's lymphoma, acute myeloid leukemia (AML), chronic myeloid leukemia, myelodysplastic syndrome, multiple myeloma, T-cell lymphoma, acute lymphocytic leukemia, and any combination thereof.

135. 135. The method of claim 134, wherein the non-Hodgkin's lymphoma is selected from the group consisting of B-cell lymphoma, diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, chronic lymphocytic leukemia (B-CLL), mantle cell lymphoma, marginal zone B-cell lymphoma, Burkitt lymphoma, lymphoplasmacytic lymphoma, hairy cell leukemia, and any combination thereof.

136. 135. The method of claim 134, wherein the T-cell lymphoma is peripheral T-cell lymphoma.

137. 137. The method of any one of claims 131 to 136, wherein the cancer is characterized by a cancer antigen present on the cancer.

138. 138. The method of claim 137, wherein the cancer antigen is a tumor antigen, a stromal antigen, or a blood antigen.

139. Cancer antigens include CD19, CD123, CD22, CD30, CD171, CS-1, C-type lectin-like molecule-1, CD33, epidermal growth factor receptor variant III (EGFRvIII), ganglioside G2 (GD2), ganglioside GD3, TNF receptor family member B cell maturation (BCMA), and Tn antigen (TnAg) or (GalNAcα-Ser / Thr)), prostate-specific membrane antigen (PSMA), receptor tyrosine kinase-like orphan receptor 1 (ROR1), Fms-like tyrosine kinase 3 (FLT3), tumor-associated glycoprotein 72 (TAG72), CD38, CD44v6, carcinoembryonic antigen (CEA), epithelial cell adhesion molecule (EPCAM), B7H3 (CD276), KIT (CD117), interleukin-13 receptor subunit alpha-2, mesothelin, interleukin-11 receptor alpha (IL-11Ra), prostate stem cell antigen (PS CA), protease serine 21, vascular endothelial growth factor receptor 2 (VEGFR2), Lewis (Y) antigen, CD24, platelet-derived growth factor receptor beta (PDGFR-beta), stage-specific embryonic antigen-4 (SSEA-4), CD20, folate receptor alpha, receptor tyrosine-protein kinase ERBB2 (Her2 / neu), cell surface-associated mucin 1 (MUC1), epidermal growth factor receptor (EGFR), neural cell adhesion molecule (NCAM), prostase, prostatic acid phosphatase (PAP), mutant elongation factor 2 ( ELF2M), ephrinB2, fibroblast activation protein alpha (FAP), insulin-like growth factor 1 receptor (IGF-I receptor), carbonic anhydrase IX (CAIX), proteasome (prosome, macropein) subunit, beta type 9 (LMP2), glycoprotein 100 (gp100 / pmel17), oncogene fusion protein (bcr-abl) consisting of breakpoint cluster region (BCR) and Abelson murine leukemia viral oncogene homolog 1 (Abl), tyrosinase, ephrin type A receptor 2 (Eph A2), fucosyl GM1, sialyl Lewis adhesion molecule (sLe), ganglioside GM3, transglutaminase 5 (TGS5), high molecular weight melanoma-associated antigen (HMWMAA), o-acetyl-GD2 ganglioside (OAcGD2), folate receptor beta, tumor endothelial marker 1 (TEM1 / CD248), tumor endothelial marker 7-related (TEM7R), claudin 6 (CLDN6), thyroid-stimulating hormone receptor (TSHR), G protein-coupled receptor class C group 5, member D (GPRC5D), chromosome X open reading frame61 (CXORF61), CD97, CD179a, anaplastic lymphoma kinase (ALK), polysialic acid, placenta-specific 1 (PLAC1), hexasaccharide moiety of globoH glycoceramide (GloboH), mammary differentiation antigen (NY-BR-1), uroplakin 2 (UPK2), hepatitis A virus cell receptor 1 (HAVCR1), adrenoceptor beta 3 (ADRB3), pannexin 3 (PANX3), G protein-coupled receptor 20 (GPR20), lymphocyte antigen 6 complex, locus K9 (LY6K), olfactory receptor 5 1E2 (OR51E2), TCR gamma alternate reading frame protein (TARP), Wilms tumor protein (WT1), cancer / testis antigen 1 (NY-ESO-1 / LAGE-1), cancer / testis antigen 2 (LAGE-1a), melanoma-associated antigen 1 (MAGE-A1), ETS translocation variant gene 6 located on chromosome 12p (ETV6-AML), sperm protein 17 (SPA17), X antigen family, member 1A (XAGE1), angiopoietin-binding cell surface receptor 2 (Tie 2), melanoma cancer testis antigen-1 (MAD-CT-1), melanoma cancer testis antigen-2 (MAD-CT-2), Fos-related antigen 1, tumor protein p53 (p53), p53 mutant, prostein, survivin, telomerase, prostate cancer tumor antigen-1, melanoma antigen 1 recognized by T cells, rat sarcoma (Ras) mutant, human telomerase reverse transcriptase (hTERT), sarcoma translocation breakpoint, melanoma inhibitor of apoptosis (ML-IAP), ER G (transmembrane protease, serine 2 (TMPRSS2) ETS fusion gene), N-acetylglucosaminyl-transferase V (NA17), paired box protein Pax-3 (PAX3), androgen receptor, cyclin B1, v-myc avian myelocytomatosis viral oncogene neuroblastoma-derived homolog (MYCN), Ras homolog family member C (RhoC), tyrosinase-related protein 2 (TRP-2), cytochrome P4501B1 (CYP1B1), CCCTC-binding factor (zinc finger protein)-like, squamous cell carcinoma antigen 3 recognized by T cells (SART3), paired box protein Pax-5 (PAX5), proacrosin-binding protein sp32 (OY-TES1), lymphocyte-specific protein tyrosine kinase (LCK), A-kinase anchor protein 4 (AKAP-4), synovial sarcoma X breakpoint 2 (SSX2), receptor for advanced glycation end products (RAGE-1), renal ubiquitous 1 (RU1), renal ubiquitous 2 (RU2), legumain, human papillomavirus E6 (HPV E6), human papillomavirus E7 (HPV E7), intestinal carboxylesterase, mutant heat shock protein 70-2 (muthsp70-2), CD79a, CD79b, CD72, leukocyte-associated immunoglobulin-like receptor 1 (LAIR1), Fc fragment of IgA receptor (FCAR or CD89), leukocyte immunoglobulin-like receptor subfamily A member 2 (LILRA2), CD300 molecule-like family member f (CD300LF), C-type lectin domain family 12 member A (CLEC12A), bone marrow stromal cell antigen 2 (BST2), EGF-like module E-containing mucin-like hormone receptor-like 2 (EMR2), lymphocyte antigen 75 (LY75), glypican-3 (GPC3), Fc receptor-like 5 (FCRL5), FcRH5, PDL1, CD47, prostate-specific membrane antigen (PMSA), prostate-specific antigen (PSA), Ron kinase, c-Met, immature laminin receptor, TAG-72, BING-4, calcium-activated chloride channel 2, cyclin-B1, 9D7, Ep-CAM, EphA3, SAP-1, PRAME, SSX-2, Melan-A / MART-1, TRP1 / gp75, MC1R, β-catenin, BRCA1 / 2, CDK4, CML66, fibronectin, Ras, TGF-B receptor, AFP, ETA, MAGE, CA-125, BAGE, GAGE, CDC27, α-actinin-4, ganglioside, MART-2, MUC2, MUM1, MUM2, MUM3, NA88-1, NPM, OA1, OGT, RC 139. The method of any one of claims 137 to 138, wherein the target polypeptide is selected from the group consisting of C, RU11, RU12, SAGE, TRG, TSTA, L1-CAM, gpA33, GM2, VEGFR, integrins, carbohydrates, TRAILR1, TRAILR2, RANKL, TGF-beta, hyaluronic acid, collagen, tenascin-C, tenascin-W, and immunoglobulin lambda-like polypeptide 1 (IGLL1).

140. 140. The method of any one of claims 130 to 139, further comprising administering a second therapeutic agent or treatment to the subject.

141. 141. The method of claim 140, wherein the second therapeutic agent or treatment comprises a chemotherapeutic agent, a biological agent, hormone therapy, radiation, or surgery.

142. The method of any one of claims 140 to 141, wherein a second therapeutic agent or treatment is administered in combination, sequentially, simultaneously, or in parallel with a multifunctional molecule of any one of claims 1 to 124, a polynucleotide of claim 125 or 126, a vector of claim 127, a cell of claim 128, or a pharmaceutical composition of claim 129.

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