RU2831840C2 - High-affinity anti-cd3 antibodies and methods for production and use thereof - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: invention relates to biochemistry, particularly to an anti-CD3 antibody or an antigen-binding fragment of an antibody. Also disclosed is a nucleic acid coding said antibody, an expression vector, a host cell for expressing said antibody, a pharmaceutical composition containing said antibody, an anti-CD3 chimeric antigen CAR receptor. Disclosed is a method of treating the disorder in a mammal using said antibody.

EFFECT: invention makes it possible to effectively treat disorders in a mammal using said antibody.

51 cl, 1 ex, 7 tbl

Description

This application claims the benefit of U.S. Provisional Patent Application No. 62/858,949, filed June 7, 2019, the contents of which are incorporated by reference in their entirety.

List of sequences

This application contains a sequence listing that was filed electronically in ASCII format and is hereby incorporated by reference in its entirety. The ASCII copy created on June 3, 2020 is named 1160430o001813.txt and is 92,322 bytes in size.

Field of technology to which the invention relates

The present invention relates, inter alia, to antibodies to cluster of differentiation 3 (CD3), including multispecific antibodies and functional fragments thereof, as well as to methods and reagents for their identification, isolation, production and use.

Prerequisites for the creation of an invention

Cell proliferative disorders such as cancer are characterized by the uncontrolled growth of subpopulations of cells. They are the leading cause of death in the developed world and the second leading cause of death in developing countries, with the total number of new cancer cases per year expected to rise to 23.6 million by 2030. The National Cancer Institute estimates that nearly 2 million new cases of cancer will be diagnosed in the United States and more than 600,000 Americans will die from cancer in 2018. Cancer care thus represents a significant and increasing burden on society.

The idea of harnessing the cytotoxic capacity of T cells to kill tumor cells by using bispecific antibodies that target CD3 emerged in the mid-1980s (Staerz et al. Nature 1985 314: 628-32). Many bispecific antibodies developed to date contain a first binding site specific for CD3 to recruit and activate T cells and a second binding site for a target antigen associated with a disease, such as an antigen produced by a tumor cell. CD3 bispecific antibodies activate the CD3 surface receptor on T cells by binding to their second target protein expressed on tumors, so that available T cells can bind to target-expressing cells via binding to the CD3 bispecific antibody, regardless of the peptide/MHC specificity of their T cell receptor. (See, e.g., Bassan, 2012, Blood 120:5094-95). Binding of T cells and tumor cells by CD3 bispecific antibodies can induce dramatic regression of advanced malignancies and, in some cases, lead to complete remission. Currently, more than 25 different CD3 bispecific antibodies are in clinical development for the treatment of hematological malignancies or solid cancers by targeting CD19, CD20, CD33 and CD123 or EpCAM, HER2, PSMA and CEA, respectively. (See, e.g., Liu et al. Front Immunol 2017 8:38).

Although bispecific antibodies have shown significant advantages over monospecific antibodies in the treatment and detection of cancer, the widespread commercial use of bispecific antibodies has been hampered by the lack of efficient/inexpensive production methods, the lack of stability of bispecific polypeptides, and the lack of a long half-life in humans. Over the past few decades, a vast number of methods have been developed for the production of bispecific monoclonal antibodies. However, many bispecific antibody candidates with exceptional selectivity and high potency for the target of interest often suffer from problems in subsequent development and in terms of clinical efficacy metrics, including polyspecific binding (or "polyspecificity"); off-target binding; non-specific binding; low expression levels or profiles in eukaryotic host cells such as mammalian host cells and yeast cells; unsatisfactory chemical and physical properties such as unsatisfactory storage stability (e.g. unsatisfactory/poor stability during shelf life), unsatisfactory (low) solubility, unsatisfactory (high) viscosity, tendency to aggregation, etc.; and unsatisfactory clinical and biophysical profiles such as unsatisfactory pharmacokinetic profiles, unsatisfactory pharmacodynamic profiles, rapid or unsatisfactory in vivo elimination rate, short half-life in the bloodstream, some of which lead to the discontinuation of their development.

There are specific methodologies and assays available to assess many of the above-mentioned developability characteristics for discovered antibodies in the context of subsequent development activities (“post-discovery antibodies”), such as CIC, SIC, BVP-ELISA, TMA, and other assays; however, such assays are generally not suitable for high-throughput formats in early antibody discovery platforms. Furthermore, milligram to gram-scale protein quantities are typically required to assess these properties, thus often effectively limiting the number of lead products that can pragmatically be considered for development, and thus reducing the likelihood of program success. As a result, significant resources are often wasted attempting to improve poorly performing lead candidates using the small number of fallback options available later in development.

Various anti-CD3 antibodies are known in the art, including monoclonal and bispecific antibody formats. See, for example, U.S. Patent Nos. 7,262,276; 7,635,472; 7,862,813; 9,587,021; and 10,174,124. However, many of these anti-CD3 antibodies suffer from developability issues such as those described above and/or have insufficient binding affinity for CD3 for a particular multispecific or chimeric antigen receptor (CAR) format, delivery approach, or the like. Accordingly, there is an unmet need in the art for bispecific antibodies to CD3 antibodies that exhibit the desired developability profiles and have high affinity for CD3 for use in the treatment of cancer.

Brief description of the invention

The present invention relates to CD3 antibodies and methods of using the same, wherein the antibodies are characterized by a particularly high affinity for CD3, e.g., a monovalent K _D of approximately 250 picomoles or less.

The present invention provides an antibody and/or antigen-binding fragment to CD3 comprising a complementarity determining region (CDR) in the variable region of a light chain, CDRL3, wherein CDRL3 comprises the amino acid sequence _X1QSYFRRT (SEQ ID NO: 1), wherein _X1 is K, A, T, or V. In some embodiments, _X1 is A, T, or V. In certain embodiments, CDRL3 comprises AQSYFRRT (SEQ ID NO: 2); TQSYFRRT (SEQ ID NO: 3); VQSYFRRT (SEQ ID NO: 4), or KQSYFRRT (SEQ ID NO: 5).

In some embodiments _{, the anti-CD3 antibody and/or antigen-binding fragment further comprises a complementarity determining region (CDR) in the variable region of the heavy chain, CDRH3, wherein CDRH3 comprises the amino acid sequence X3RDAYGX4YFYDV (} SEQ ID NO:6), wherein _X3 is A or V, and _X4 is R or Q. In certain embodiments, _X3 is A.

In other embodiments, the anti-CD3 antibody and/or antigen-binding fragment further comprises a complementarity determining region (CDR) in the variable region of the light chain, CDRL1, wherein CDRL1 comprises the amino acid sequence KSSQSLLNARTX ₅ KNYLA (SEQ ID NO: 7), wherein X ₅ is G, M, N, or R. In certain embodiments, X ₅ is G or R. In some embodiments, CDRL1 may comprise KSSQSLLNARTGKNYLA (SEQ ID NO: 8) or KSSQSLLNARTRKNYLA (SEQ ID NO: 9).

In some embodiments, the antibody and/or antigen-binding fragment to CD3 further comprises a complementarity determining region (CDR) in the variable region of the light chain, CDRL2, wherein CDRL2 comprises the amino acid sequence WASTRES (SEQ ID NO: 10) or WASTRSS (SEQ ID NO: 11).

In some embodiments, the anti-CD3 antibody and/or antigen-binding fragment further comprises a complementarity determining region (CDR) in the variable region of the heavy chain, CDRH1, wherein CDRH1 comprises the amino acid sequence FNX ₆ KDYYX ₇ H (SEQ ID NO: 12), wherein X ₆ is I, N, or V, and X ₇ is M or I. In certain embodiments, X ₆ is I, and/or X ₇ is M. In certain embodiments, CDRH1 comprises FNIKDYYMH (SEQ ID NO: 13).

In some embodiments, the anti-CD3 antibody and/or antigen-binding fragment further comprises a complementarity determining region (CDR) in the variable region of the heavy chain, CDRH2, wherein CDRH2 comprises the amino acid sequence WIDLX ₈ NANTVYDX ₉ KX ₁₀ QG (SEQ ID NO: 14), wherein X ₈ is E or N, X ₉ is A, H or T, and X ₁₀ is F or L. In certain embodiments, X ₈ is E and/or X ₉ is A and/or X ₁₀ is F. In certain embodiments, CDRH2 comprises WIDLENANTVYDAKFQG (SEQ ID NO: 15) or WIDLENANTIYDAKFQG (SEQ ID NO: 16).

In some embodiments, the present invention provides an anti-CD3 antibody and/or antigen-binding fragment thereof comprising one or more of CDRL1, CDRL2, and CDRL3. Such an antibody in some embodiments further comprises CDRH1, CDRH2, and CDRH3.

In some embodiments, the anti-CD3 antibodies and/or antigen-binding fragments thereof described herein have a binding affinity ( _KD ) of about 500 pM or less. In some embodiments, the anti-CD3 antibodies and/or antigen-binding fragments thereof have a binding affinity ( _KD ) of about 250 pM or less. In some embodiments, the anti-CD3 antibodies and/or antigen-binding fragments thereof have a binding affinity ( _KD ) of about 100 pM or less.

Moreover, the present invention generally relates to an antibody or antigen-binding fragment of an antibody to cluster of differentiation three ("CD3"), wherein said antibody or antigen-binding fragment of an antibody to CD3 comprises: a. a heavy chain variable region (V _H ) polypeptide comprising: i. V H CDR1 (CDRH1) with FNIKDYYMH (SEQ ID NO: 13); ii. V _H CDR2 (CDRH2) with WIDLENANTVYDAKFQG (SEQ ID NO: 15); iii. V _H CDR3 (CDRH3) with ARDAYGRYFYDV (SEQ ID NO: 103); and/or b. a light chain variable region (V _L ) polypeptide comprising: i. V _L CDR1 (CDRL1) with KSSQSLLNARTGKNYLA (SEQ ID NO: 8); ii. CDR2 V _L (CDRL2) with WASTRX ₁ S (SEQ ID NO: 133); and iii. CDR3 V _L (CDRL3) with X ₂ QSYFRRT (SEQ ID NO: 134); each of X ₁ and X ₂ is independently any amino acid. In some embodiments, X ₁ is E or S; and/or X ₂ is K or V. In some embodiments, said antibody or antigen-binding fragment of an antibody comprises CDRL2 of SEQ ID NO: 10 or SEQ ID NO: 11 and/or CDRL3 of SEQ ID NO: 5 or SEQ ID NO: 4. In some embodiments, said anti-CD3 antibody or antigen-binding fragment of an antibody is selected from the group consisting of: a. those comprising V _H chain CDR1 (CDRH1) with FNIKDYYMH (SEQ ID NO: 13), V _H chain CDR2 (CDRH2) with WIDLENANTVYDAKFQG (SEQ ID NO: 15) and V _H chain CDR3 (CDRH3) with ARDAYGRYFYDV (SEQ ID NO: 103) and/or V _L chain CDR1 (CDRL1) with KSSQSLLNARTGKNYLA (SEQ ID NO: 8), V _L chain CDR2 (CDRL2) with WASTRES (SEQ ID NO: 10) and V _L chain CDR3 (CDRL3) with KQSYFRRT (SEQ ID NO: 5); and b. those containing V _H chain CDR1 (CDRH1) with FNIKDYYMH (SEQ ID NO: 13), V _H chain CDR2 (CDRH2) with WIDLENANTVYDAKFQG (SEQ ID NO: 15) and V _H chain CDR3 (CDRH3) with ARDAYGRYFYDV (SEQ ID NO: 103) and/or V _L chain CDR1 (CDRL1) with KSSQSLLNARTGKNYLA (SEQ ID NO: 8), V _L chain CDR2 (CDRL2) with WASTRSS (SEQ ID NO: 11) and V _L chain CDR3 (CDRL3) with VQSYFRRT (SEQ ID NO: 4).

In some embodiments, said anti-CD3 antibody or antigen-binding fragment of an antibody comprises a V _L chain polypeptide comprising an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 18 or SEQ ID NO: 34. In some embodiments, said anti-CD3 antibody or antigen-binding fragment of an antibody comprises a V _L chain polypeptide comprising an amino acid sequence that has at least 95% sequence identity to SEQ ID NO: 18 or SEQ ID NO: 34. In some embodiments, said anti-CD3 antibody or antigen-binding fragment of an antibody comprises a V _L chain polypeptide comprising the amino acid sequence of SEQ ID NO: 18 or SEQ ID NO: 34. In some embodiments, said anti-CD3 antibody or antigen-binding fragment of an antibody comprises a V _{H chain polypeptide comprising an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 17. In some embodiments, said anti-CD3 antibody or antigen-binding fragment of an antibody comprises a V H} chain polypeptide comprising an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 17. the antigen-binding fragment of an anti-CD3 antibody comprises a V _H chain polypeptide comprising an amino acid sequence that has at least 95% sequence identity to SEQ ID NO: 17. In some embodiments, said anti-CD3 antibody or antigen-binding fragment of an antibody comprises a V _H chain comprising the amino acid sequence of SEQ ID NO: 17. In some embodiments, said anti-CD3 antibody or antigen-binding fragment of an antibody comprises a V _L chain polypeptide comprising an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 18 or SEQ ID NO: 34; and a V _H chain polypeptide comprising an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 17. In some embodiments, said anti-CD3 antibody or antigen-binding fragment of an antibody comprises a V _L chain polypeptide comprising an amino acid sequence that has at least 95% sequence identity to SEQ ID NO: 18 or SEQ ID NO: 34; and a V _H chain polypeptide comprising an amino acid sequence that has at least 95% sequence identity to SEQ ID NO: 17. In some embodiments, said anti-CD3 antibody or antigen-binding fragment of an antibody comprises a V _L chain polypeptide comprising the amino acid sequence of SEQ ID NO: 18 or SEQ ID NO: 34; and a V _H chain comprising the amino acid sequence of SEQ ID NO: 17. In some embodiments, said anti-CD3 antibody or antigen-binding fragment of an antibody comprises a V _L chain polypeptide comprising the amino acid sequence of SEQ ID NO: 18; and a V _H chain comprising the amino acid sequence of SEQ ID NO: 17. In some embodiments, said anti-CD3 antibody or antigen-binding fragment of an antibody comprises a V _L chain polypeptide comprising the amino acid sequence of SEQ ID NO: 34; and a V _H chain comprising the amino acid sequence of SEQ ID NO: 17.

In some embodiments, said anti-CD3 antibody or antigen-binding fragment of an antibody binds to CD3 with a dissociation constant ( _KD ) of about 500 pM or less, about 450 pM or less, about 400 pM or less, about 350 pM or less, about 300 pM or less, about 250 pM or less, about 200 pM or less, about 150 pM or less, or about 100 pM or less, wherein optionally said CD3 is human and/or cynomolgus monkey, wherein even more optionally said binding affinity is measured by surface plasmon resonance. In some embodiments, said _KD is a monovalent _KD and/or said _KD is measured using an scFv fragment of said anti-CD3 antibody or antibody fragment.

In some embodiments, the antibody or antigen-binding fragment of an antibody induces T cell activation or T cell cytolysis while exhibiting a reduced propensity to induce cytokine production to levels capable of inducing cytokine release syndrome. In some embodiments, the antibody or antigen-binding fragment of an antibody is a multispecific antibody. In some embodiments, the antibody or antigen-binding fragment of an antibody is a bispecific antibody. In some embodiments, the antibody or antigen-binding fragment of an antibody is an scFv. In some embodiments, the antibody or antigen-binding fragment of an antibody comprises at least a second antigen-binding domain that specifically binds to an oncological disease target antigen; an immuno-oncological disease target antigen; a neurodegenerative disease target antigen; an autoimmune disorder target antigen; an infectious disease target antigen; a metabolic disease target antigen; a cognitive impairment target antigen; blood-brain barrier target antigen or blood disease target antigen. In some embodiments, the antibody or antigen-binding fragment of an antibody comprises at least a second antigen-binding domain that specifically binds to an antigen selected from the group consisting of: 17-IA, 4-1BB, 4Dc, 6-keto-PGFla, 8-iso-PGF2a, 8-oxo-dG, adenosine receptor A1, A33, ACE, ACE-2, activin, activin A, activin AB, activin B, activin C, activin RIA, activin RIA ALK-2, activin RIB ALK-4, activin RIIA, activin RUB, ADAM, ADAM10, ADAM12, ADAM 15, ADAM 17/T ACE, ADAM8, ADAM9, ADAMTS, ADAMTS4, ADAMTS5, addressins, aFGF, ALCAM, ALK, ALK-1, ALK-7, alpha-1-antitrypsin, alpha-V/beta-1 antagonist, ANG, Ang, APAF-1, APE, APJ, APP, APRIL, AR, ARC, ART, artemin, anti-idiotype, ASPARTIC, atrial natriuretic factor, integrin av/b3, Ax1, b2M, B7-1, B7-2, B7-H, B-lymphocyte stimulator (BlyS), BACE, BACE-1, Bad, BAFF, BAFF-R, Bag-1, BAK, Bax, BCA-1, BCAM, Bel, BCMA, BDNF, b-ECGF, bFGF, BID, Bik, BFM, BLC, BL-CAM, BLK, BMP, BMP-2 BMP-2a, osteogenin BMP-3, BMP-4 BMP-2b, BMP-5, BMP-6 Vgr-1, BMP-7 (OP-1), BMP-8 (BMP-8a, OP-2), BMPR, BMPR-IA (ALK-3), BMPR-IB (ALK-6), BRK-2, RPK-1, BMPR-II (BRK-3), BMP, b-NGF, BOK, bombesin, bone neurotrophic factor, BPDE, BPDE-DNA, BTC, complement factor 3 (C3), C3a, C4, C5, C5a, CIO, CA125, CAD-8, calcitonin, cAMP, carcinoembryonic antigen (CEA), carcinoma-associated antigen, cathepsin A, cathepsin B, cathepsin C/DPPI, cathepsin D, cathepsin E, cathepsin H, cathepsin L, cathepsin O, cathepsin S, cathepsin V, cathepsin X/Z/P, CBL, CCI, CCK2, CCL, CCL1, CCL11, CCL12, CCL13, CCL 14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL2, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CCL3, CCL4, CCL5, CCL6, CCL7, CCL8, CCL9/10, CCR, CCR1, CCR10, CCR10, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CD1, CD2, CD4, CD5, CD6, CD7, CD8, CD10, CD11a, CD11b, CD11c, CD13, CD14, CD15, CD16, CD18, CD19, CD20, CD21, CD22, CD23, CD25, CD27L, CD28, CD29, CD30, CD30L, CD32, CD33 (p67 proteins), CD34, CD38, CD40, CD40L, CD44, CD45, CD46, CD49a, CD52, CD54, CD55, CD56, CD61, CD64, CD66e, CD74, CD80 (B7-1), CD89, CD95, CD123, CD137, CD138, CD140a, CD146, CD147, CD148, CD152, CD164, CEACAM5, CFTR, cGMP, CINC, Clostridium toxin botulinum, Clostridium perfringens toxin, CKb8-1, CLC, CMV, CMV UL, CNTF, CNTN-1, COX, C-Ret, CRG-2, CT-1, CTACK, CTGF, CTLA-4, CX3CL1, CX3CR1, CXCL, CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCR, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, cytokeratin tumor-associated antigen, DAN, DCC, DcR3, DC-SIGN, hemolysis stimulator, des(l-3)-IGF-I (IGF-1 brain), Dhh, digoxin, DNAM-1, Dnase, Dpp, DPPIV/CD26, Dtk, ECAD, EDA, EDA-A1, EDA-A2, EDAR, EGF, EGFR (ErbB-1), EMA, EMMPRIN, EN A, endothelin receptor, enkephalinase, eNOS, Eot, eotaxin l, EpCAM, ephrin B2/EphB4, EPO, ERCC, E-selectin, ET-1, factor Ila, factor VII, factor VIIIc, factor IX, fibroblast activation protein (FAP), Fas, FcRl, FEN-1, ferritin, FGF, FGF-19, FGF-2, FGF3, FGF-8, FGFR, FGFR-3, fibrin, FL, FLIP, Flt-3, Flt-4, follicle-stimulating hormone, fractalkine, FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, FZD10, G250, Gas 6, GCP-2, GCSF, GD2, GD3, GDF, GDF-1, GDF-3 (Vgr-2), GDF-5 (BMP-14, CDMP-1), GDF-6 (BMP-13, CDMP-2), GDF-7 (BMP-12, CDMP-3), GDF-8 (myostatin), GDF-9, GDF-15 (MIC-1), GDNF, GFAP, GFRa-1, GFR-alpha 1, GFR-alpha 2, GFR-alpha 3, GITR, glucagon, Glut 4, glycoprotein I1b/IIIa (GP I1b/IIIa), GM-CSF, gp130, gp72, GRO, somatotropin-releasing factor, hapten (NP-cap or NIP-cap), HB-EGF, HCC, HCMV envelope glycoprotein gB, HCMV envelope glycoprotein gH, HCMV UL, hematopoietic growth factor (HGF), Hep B gp120, heparanase, Her2, Her2/neu (ErbB-2), Her3 (ErbB-3), Her4 (ErbB-4), herpes simplex virus (HSV) glycoprotein gB, HSV glycoprotein gD, HGFA, high molecular weight melanoma-associated antigen (HMW-MAA), HIV gpl20, V3-loop of gp 120 HIV IIIB, HLA, HLA-DR, HM1.24, HMFG PEM, HRG, Hrk, human cardiac myosin, human cytomegalovirus (HCMV), human growth hormone (HGH), HVEM, 1-309, IAP, ICAM, ICAM-1, ICAM-3, ICE, ICOS, IFNg, Ig, IgA receptor, IgE, IGF, IGF-binding proteins, IGF-1R, IGFBP, IGF-I, IGF-II, IL, IL-1, IL-1R, IL-2, IL-2R, IL-4, IL-4R, IL-5, IL-5R, IL-6, IL-6R, IL-8, IL-9, IL-10, IL-12, IL-13, IL-15, IL-18, IL-18R, IL-23, alpha interferon (INF), INF-beta, INF-gamma, inhibin, iNOS, insulin A chain, insulin B chain, insulin-like growth factor 1, alpha-2 integrin, alpha-3 integrin, alpha-4 integrin, alpha-4/beta-l-integrin, alpha-4/beta-7 integrin, alpha-5 (alpha-V) integrin, alpha-5/beta-l-integrin, alpha-5/beta-3 integrin, alpha-6 integrin, beta-l-integrin, beta-2 integrin, gamma interferon, IP-10, 1-TAC, JE, kallikrein 2, kallikrein 5, kallikrein 6, kallikrein 11, kallikrein 12, kallikrein 14, kallikrein 15, kallikrein LI, kallikrein L2, kallikrein L3, kallikrein L4, KC, KDR, keratinocyte growth factor (KGF), laminin 5, LAMP, LAP, LAP (TGF-1), latent TGF-1, bpl latent TGF-1, LBP, LDGF, LECT2, Lefty, Lewis-Y antigen, Lewis-Y-related antigen, LFA-1, LFA-3, Lfo, LIF, LIGHT, lipoproteins, LIX, LKN, Lptn, L-selectin, LT-a, LT-b, LTB4, LTBP-1, pulmonary surfactant, luteinizing hormone, lymphotoxin beta receptor, Mac-1, MAdCAM, MAG, MAP2, MARC, MCAM, MCAM, MCK-2, MCP, M-CSF, MDC, Mer, metalloproteases, MGDF receptor, MGMT, MHC (HLA-DR), MIF, MIG, MIP, MIP-1alpha, MK, MMAC1, MMP, MMP-1, MMP-10, MMP-11, MMP-12, MMP-13, MMP-14, MMP-15, MMP-2, MMP-24, MMP-3, MMP-7, MMP-8, MMP-9, MPIF, Mpo, MSK, MSP, mucin (Mucl), MUC18, anti-Müllerian hormone, Mug, MuSK, NAIP, NAP, NCAD, N-cadherin, NCA 90, NCAM, NCAM, neprilysin, neurotrophin-3, -4, or -6, neuroturin, neuronal growth factor (NGF), NGFR, NGF-beta, nNOS, NO, NOS, Npn, NRG-3, NT, NTN, OB, OGG1, OPG, OPN, OSM, OX40L, OX40R, pl50, p95, PADPr, parathyroid hormone, PARC, PARP, PBR, PBSF, PCAD, P-cadherin, PCNA, PDGF, PDGF, PDK-1, PECAM, PEM, PF4, PGE, PGF, PGI2, PGJ2, PIN, PLA2, placental alkaline phosphatase (PLAP), PIGF, PLP, PP14, proinsulin, prorelaxin, C-protein, PS, PSA, PSCA, prostate-specific membrane antigen (PSMA), PTEN, PTHrp, Ptk, PTN, R51, RANK, RANKL, RANTES, relaxin A-chain, relaxin B-chain relaxin, renin, respiratory syncytial virus (RSV) F, RSV Fgp, Ret, rheumatoid factors, RLIP76, RPA2, RSK, S100, SCF/KL, SDF-1, SERINE, serum albumin, sFRP-3, Shh, SIGIRR, SK-1, SLAM, SLPI, SMAC, SMDF, SMOH, SOD, SPARC, Stat, STEAP, STEAP-II, TACE, TACI, TAG-72 (tumor-associated glycoprotein-72), TARC, TCA-3, T-cell receptors (e.g., T-cell receptor alpha/beta), TdT, TECK, TEM1, TEM5, TEM7, TEM8, TERT, testicular PLAP-like alkaline phosphatase, TfR, TGF, TGF-alpha, TGF-beta, polyspecific TGF-beta, RI TGF-beta (ALK-5), RII TGF-beta, R11b TGF-beta, RIII TGF-beta, TGF-beta 1, TGF-beta 2, TGF-beta 3, TGF-beta 4, TGF-beta 5, thrombin, Ck-1 thymus, thyroid stimulating hormone, Tie, TIMP, TIQ, tissue factor, TMEFF2, Tmpo, TMPRSS2, TNF, TNF-alpha, TNF-alpha/beta, TNF-beta 2, TNFc, TNF-RI, TNF-RII, TNFRSF10A (TRAIL Rl Apo-2, DR4), TNFRSFIOB (TRAIL R2 DR5, KILLER, TRICK-2A, TRICK-B), TNFRSF10C (TRAIL R3 DcRl, LIT, TRID), TNFRSF10D (TRAIL R4 DcR2, TRUNDD), TNFRSF11A (RANK ODF R, TRANCE R), TNFRSFllB (OPG OCIF, TR1), TNFRSF12 (TWEAK R FN14), TNFRSF13B (TACI), TNFRSF13C (BAFF R), TNFRSF14 (HVEM ATAR, HveA, LIGHT R, TR2), TNFRSF16 (NGFR p75NTR), TNFRSF17 (BCMA), TNFRSF 18 (GITR AITR), TNFRSF19 (TROY TAJ, TRADE), TNFRSF19L (RELT), TNFRSFIA (TNF RI CD120a, p55-60), TNFRSFIB (TNF RII CD120b, p75-80), TNFRSF26 (TNFRH3), TNFRSF3 (LTbR TNF RIII, TNFC R), TNFRSF4 (OX40 ACT35, TXGP1 R), TNFRSF 5 (CD40 p50), TNFRSF6 (Fas Apo-1, APT1, CD95), TNFRSF6B (DcR3 M68, TR6), TNFRSF7 (CD27), TNFRSF8 (CD30), TNFRSF9 (4-1BB CD137, ILA), TNFRSF21 (DR6), TNFRSF22 (DcTRAIL R2 TNFRH2), TNFRST23 (DcTRAIL Rl TNFRH1), TNFRSF25 (DR3 Apo-3, LARD, TR-3, TRAMP, WSL-1), TNFSF10 (ligand TRAIL Apo-2, TL2), TNFSF11 (ODF TRANCE/RANK ligand, OPG ligand), TNFSF12 (TWEAK Apo-3 ligand, DR3 ligand), TNFSF13 (APRIL TALL2), TNFSF13B (BAFF BLYS, TALL1, THANK, TNFSF20), TNFSF14 (LIGHT HVEM ligand, LTg), TNFSF15 (TLIA/VEGI), TNFSF18 (GITR ligand, AITR ligand, TL6), TNFSFIA (TNF-a conectin, DIF, TNFSF2), TNFSF1B (TNF-b LTa, TNFSF1), TNFSF3 (LTb TNFC, p33), TNFSF4 (gp34 ligand OX40, TXGP1), TNFSF5 (CD40 ligand CD154, gp39, HIGM1, IMD3, TRAP), TNFSF6 (Fas Ligand, Apo-1 Ligand, APT1 Ligand), TNFSF7 (CD27 CD70 Ligand), TNFSF8 (CD30 CD153 Ligand), TNFSF9 (4-1BB CD137 Ligand), TP-1, t-PA, Tpo, TRAIL, TRAIL R, TRAIL-R1, TRAIL-R2, TRANCE, transferrin receptor, TRF, Trk, TROP-2, TSG, TSLP, tumor-associated antigen CA 125, tumor-associated antigen characterized by the expression of carbohydrate antigen-related Lewis Y, TWEAK, TXB2, Ung, uPAR, uPAR-1, urokinase, VCAM, VCAM-1, VECAD, VE-cadherin, VE-cadherin-2, VEFGR-1 (flt-1), VEGF, VEGFR, VEGFR-3 (flt-4), VEGI, VFM, viral antigens, VLA, VLA-1, VLA-4, VNR integrin, von Willebrand factor, WIF-1, WNT1, WNT2, WNT2B/13, WNT3, WNT3A, WNT4, WNT5A, WNT5B, WNT6, WNT7A, WNT7B, WNT8A, WNT8B, WNT9A, WNT9A, WNT9B, WNT10A, WNT10B, WNT11, WNT16, XCL1, XCL2, XCR1, XCR1, XEDAR, XIAP, XPD, CTLA4 (cytotoxic T lymphocyte antigen 4), PD1 (programmed cell death protein 1), PD-L1 (programmed cell death ligand 1), LAG-3 (lymphocyte activating gene 3), TIM-3 (T-cell immunoglobulin and mucin domain-containing protein 3), hormone receptors and growth factors.

In some embodiments, the antibody or antigen-binding fragment of an antibody comprises at least a second antigen-binding domain that specifically binds to an antigen selected from the group consisting of: BCMA, CTLA4 (cytotoxic T lymphocyte antigen 4), PD1 (programmed cell death protein 1), PD-L1 (programmed cell death protein ligand 1), LAG-3 (lymphocyte activating gene 3), TIM-3, CD20, CD2, CD19, Her2, EGFR, EpCAM, FcyRIIIa (CD16), FcyRIIa (CD32a), FcyRIIb (CD32b), FcyRI (CD64), Toll-like receptors (TLRs), TLR4, TLR9, cytokines, IL-2, IL-5, IL-13, IL-6, IL-17, IL-12, IL-23, TNFa, TGFb, cytokine receptors, IL-2R, chemokines, chemokine receptors, growth factors, VEGF and HGF. In some embodiments, the antibody or antigen-binding fragment of an antibody is comprised in a chimeric antigen receptor (CAR), which optionally comprises at least one transmembrane domain and at least one intracellular domain from a T cell receptor, optionally a CD3ζ subunit, and at least one costimulatory domain. In some embodiments, the antibody or antigen-binding fragment of an antibody comprises scFv2-Fc2 and/or scFv-IgG. In some embodiments, the antibody or antigen-binding fragment of an antibody comprises an IgG constant domain. In some embodiments, the antibody or antigen-binding fragment of an antibody comprises at least a second antigen-binding domain that specifically binds to an antigen, wherein said antibody has a multispecific format selected from the group consisting of: Fab-Fc-scFv, "bottle opener", Mab-scFv, Mab-Fv, Dual scFv, core Fv, core scFv, core scFv with one arm, Fab-Fab, Fab-Fv, mAb-Fv, mAb-Fab, DART, BiTE, common light chain IgG, TandAb, Cross-Mab, SEED, BEAT, TrioMab and DuetMab. Moreover, the present invention generally relates to an isolated or recombinant nucleic acid sequence encoding an antibody or antigen-binding fragment of an antibody as described herein. In addition, the present invention generally relates to an expression vector comprising an isolated or recombinant nucleic acid sequence as described herein. In addition, the present invention generally relates to a host cell transfected, transformed or transduced with a nucleic acid sequence, optionally a mammalian or yeast cell, or a vector comprising said nucleic acid sequence as described herein.

Moreover, the present invention generally relates to a pharmaceutical composition comprising an antibody or antigen-binding fragment of an antibody as described herein; and a pharmaceutically acceptable carrier and/or excipient.

Furthermore, the present invention generally relates to a method of treating a disorder in a mammal in need of such treatment, wherein the disorder comprises a proliferative disorder, an oncological disorder, an immuno-oncological disorder, a neurological disorder, a neurodegenerative disorder, or an autoimmune disorder, comprising administering an effective amount of one or more antibodies or antibody fragments described herein, or a host cell that expresses one of said antibodies or antibody fragments described herein, optionally an immune cell, further optionally a T or NK cell. In some embodiments, the method further comprises administering to the mammal an additional therapeutic agent, optionally wherein the mammal is a human.

Moreover, the present invention generally relates to an antibody or antibody fragment to cluster of differentiation three ("CD3"), wherein said antibody or antigen-binding fragment of an antibody to CD3 comprises one or more CDRs of any one or more of Ab1-Ab50. Furthermore, the present invention generally relates to an antibody or antibody fragment to cluster of differentiation three ("CD3"), wherein said antibody or antigen-binding fragment of an antibody to CD3 comprises one or more V _H and/or V _L chains comprising an amino acid sequence selected from the sequences in Table 4.

Detailed description of the invention

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. As used herein, the term "about" when used with respect to a specific stated numerical value means that the stated value may differ from the stated value by no more than 1%. For example, as used herein, the expression "about 100" includes 99 and 101 and all values in between (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

It should be understood that aspects and embodiments of the present invention described herein include “comprising,” “consisting,” and “consisting essentially of” aspects and embodiments.

Provided herein are anti-CD3 antibodies and/or antigen-binding fragments thereof that have a high affinity (K _D ) for CD3. In some embodiments, the K _D of the anti-CD3 antibody is about 500 pM or less. In some embodiments, the K _D is about 250 pM or less. In some embodiments, the K _D is about 100 pM or less. In some embodiments, said anti-CD3 antibody or antigen-binding fragment of an antibody binds to CD3 with a dissociation constant ( _KD ) of about 500 pM or greater, about 500 pM or less, about 470 pM or less, about 450 pM or less, about 400 pM or less, about 350 pM or less, about 300 pM or less, about 250 pM or less, about 200 pM or less, about 150 pM or less, or about 100 pM or less, wherein optionally said CD3 is human and/or cynomolgus monkey, wherein even more optionally said binding affinity is measured by surface plasmon resonance. In some embodiments, said _KD is a monovalent _KD and/or said _KD is measured using an scFv fragment of said anti-CD3 antibody or antibody fragment. In some embodiments, the anti-CD3 antibodies and/or antigen-binding fragments thereof comprise certain CDR sequence motifs. In some embodiments, the CDR sequence motif comprises CDRL3 comprising SEQ ID NO: 1. In some embodiments, the anti-CD3 antibodies have favorable developability profiles.

Illustrative high-affinity CD3 antibodies and their antigen-binding fragments

“Cluster of Differentiation 3” or “CD3” refers generally to any native CD3 from any vertebrate, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise specified, including, for example, the CD3ε, CD3γ, CD3α, and CD3β chains. The term encompasses “full-length,” unprocessed CD3 (e.g., unprocessed or unmodified CD3ε or CD3γ), as well as any form of CD3 that results from processing within the cell. The term also encompasses naturally occurring variants of CD3, including, for example, splice variants or allelic variants. CD3 includes, for example, the human CD3ε protein (NCBI Reference Sequence No. NP-000724), which is 207 amino acids long, and the human CD3γ protein (NCBI Reference Sequence No. NP-000064), which is 182 amino acids long. The term also refers to either human or cynomolgus monkey CD3 epsilon protein, the amino acid sequence of which is SEQ ID NO: 131 and 132, respectively (Table 7). "CD3εN27" and "CD3εN13" refer to the 27 N-terminal amino acids and the 13 N-terminal amino acids of CD3, respectively, and they optionally contain chemical modifications or have conjugations made to them.

The term "anti-CD3 antibody" refers to an antibody or antigen-binding fragment thereof that is capable of binding to CD3, such as human CD3ε and/or CD3γ, such as human CD3ε and/or CD3γ, with sufficient affinity and/or specificity such that the antibody is useful as a diagnostic and/or therapeutic agent for targeting CD3. The anti-CD3 antibodies and antigen-binding fragments thereof described herein are characterized by a particularly high affinity for CD3.

The term "high affinity" refers to those antibodies that have a binding affinity for CD3 expressed as K _D . In some embodiments, the K _D is about 5 x 10 ^-10 M or less. In some embodiments, the K _D is about 2.5 x 10 ^-10 M or less. In some embodiments, the K _D is about 1 x 10 ^-11 M or less. In some embodiments, said antibody or antigen-binding fragment of an antibody to CD3 binds to CD3 with a dissociation constant (K _D ) of about 500 pM or greater, about 500 pM or less, about 470 pM or less, about 450 pM or less, about 400 pM or less, about 350 pM or less, about 300 pM or less, about 250 pM or less, about 200 pM or less, about 150 pM or less, or about 100 pM or less, wherein optionally said CD3 is human and/or cynomolgus monkey, wherein even more optionally said binding affinity is measured by surface plasmon resonance. In some embodiments, K _D is measured using surface plasmon resonance, such as BIACORE, by biolayer interferometry measurements using, for example, a FORTEBIO Octet HTX instrument (Pall Life Sciences), or by determining the affinity in solution using an ELISA. In some embodiments, K _D is measured using an anti-CD3 antibody scFv fragment. In some embodiments, monovalent K _D is measured.

In some embodiments, the anti-CD3 antibody binds to CD3 with a dissociation constant ( _KD ) of less than about 500 pM (5 x ^10-10 M). In some embodiments, the anti-CD3 antibody binds to CD3 with a dissociation constant ( _KD ) of less than about 250 pM. In some embodiments, the anti-CD3 antibody binds to CD3 with a dissociation constant ( _KD ) of about 100 pM. In certain embodiments, the anti-CD3 antibody binds to a CD3 epitope that is conserved between CD3 from different species, such as providing cross-reactivity between humans and cynomolgus monkeys.

K _D can be measured using assays well known to those skilled in the art, including but not limited to: biolayer interferometry (BLI), surface plasmon resonance (SPR), solution equilibrium kinetic exclusion assays, KinExA direct association assay, assays using FORTEBIO instruments and reagents such as Octet RED 384 and HTX BLI-based instruments, enzyme-linked immunosorbent assays (ELISA), or radioimmunoassay (RIA). K _D measurement can be performed using an intact anti-CD3 antibody or an antigen-binding fragment thereof, such as an scFv, using the BIACORE® Surface Plasmon Resonance Assay.

Although high-affinity CD3 binding molecules have been associated with the activation or promotion of cytokine release syndrome (CRS), the binding affinity of an antibody to CD3 is not the sole determinant of CRS. For example, a high-affinity CD3 antibody (with a K _D in nM in the ~low single-charge range) used in a bispecific format has successfully demonstrated potent T-cell cytotoxicity against tumor cells with limited cytokine (IFNγ, IL-6, TNFα) release (see, e.g., Mol Cancer Ther. 2016 Sep; 15(9); 2155-65 and the APTEVO AACR poster on preclinical studies of APOV436 presented April 16, 2018). Without being bound by theory, a high-affinity CD3 antibody may be particularly useful to provide specific targeting and minimal risk of CRS in multispecific antibodies with certain formats (e.g., scFv2-Fc2 or scFv-IgG or other similar polymeric formats) or chimeric antigen receptors with different components assembled in different orders.

The term cytokine release syndrome (or CRS) refers to a proinflammatory positive feedback loop between cytokines and immune cells that results in excessive or uncontrolled release of proinflammatory cytokines by cells of the immune system (see, e.g., Lee et al., Blood, Vol. 124, pages 188–195 (2014) and Tisoncik et al., Microbiol Mol Biol Rev, Vol. 76, pages 16–32 (2012). Once stimulated and activated, T cells release a range of cytokines to a level and extent that cause adverse biological/physiological effects of varying degrees and severity, including acute inflammation characterized by, e.g., erythema (redness), swelling or oedema, heat (elevated temperature), pain (pain), and dysfunction (loss of function). When localized to the skin or other tissue, Biological/physiological effects include increased blood flow allowing vascular leukocytes and plasma proteins to reach extravascular sites of injury, increased local temperature and pain, tissue edema and extravascular pressure, and decreased tissue perfusion. Other biological/physiological effects include organ system dysfunction such as cardiac dysfunction, adult respiratory distress syndrome, neurologic toxicity, renal and/or hepatic failure, and disseminated intravascular coagulation. Elevated levels of IFNγ, IL-6, TNFα, TGF-beta, IL-2, granulocyte macrophage colony-stimulating factor (GM-CSF), IL-10, IL-8, IL-5, and/or fractalkine are predictive of and/or causative of CRS or the propensity to cause CRS upon T cell stimulation.

In certain embodiments, the anti-CD3 antibodies and/or antigen-binding fragments thereof described herein are altered and/or modified to reduce the likelihood or severity of antibody-induced CRS. Non-limiting exemplary modifications can include silencing of Fc regions (e.g., complete removal of Fc or alteration of an Fc region to reduce or eliminate effector function) and/or masking (e.g., a polypeptide mask positioned such that it reduces or inhibits the ability of the antibody or antigen-binding fragment thereof to specifically bind CD3).

As used herein, the term "antibody" is used in its broadest sense and encompasses a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and/or antibody fragments (preferably those fragments that exhibit the desired antigen binding activity).

The term "monoclonal antibody" or "mAb" refers to an antibody obtained from a group of substantially homogeneous antibodies, i.e., the individual antibodies that comprise the group of antibodies that are identical and/or bind the same epitope except for possible antibody variants (e.g., containing mutations that occur naturally or that arise during the production of the monoclonal antibody preparation), such variants being typically present in small amounts. Unlike polyclonal antibody preparations, which typically contain different antibodies directed against different determinants (epitopes), each monoclonal antibody in a monoclonal antibody preparation is directed to a single determinant on an antigen.

With respect to multispecific antibodies, such antibodies comprise at least two different antigen-binding domains that recognize and specifically bind to at least two different antigens. With respect to bispecific antibodies, such antibodies comprise two different antigen-binding domains that recognize and specifically bind to at least two different antigens. A "distinct antigen" may refer to different and/or separate proteins, polypeptides, or molecules; and to different and/or separate epitopes, wherein such epitopes may be contained within a single protein, polypeptide, or other molecule.

The term "epitope" refers to an antigenic determinant that interacts with a specific antigen-binding site in the variable region of an antibody molecule, known as a paratope. A single antigen may have more than one epitope. Thus, different antibodies may bind to different regions of the antigen and may have different biological effects. The term "epitope" also refers to the site on an antigen to which B and/or T cells respond. It also refers to the region of an antigen that is bound by an antibody. Epitopes may be defined as structural or functional. Functional epitopes are generally a subset of structural epitopes and contain residues that directly contribute to the affinity of the interaction. Epitopes may also be conformational, that is, composed of non-linearly arranged amino acids. In certain embodiments, epitopes may include determinants that are chemically active surface groups of molecules, such as amino acids, sugar side chains, phosphoryl groups, or sulfonate groups, and in certain embodiments may have specific three-dimensional structural characteristics and/or specific charge characteristics.

In some cases, an antibody contains four polypeptide chains: two heavy (H) chains and two light (L) chains, linked by disulfide bonds. There are five main classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and some of these can be further divided into subclasses (isotypes), such as IgG ₁ , IgG ₂ , IgG ₃ , IgG ₄ , IgA ₁ , and IgA ₂ . The heavy chain constant domains that correspond to the different immunoglobulin classes are called α, δ, ε, γ, and μ, respectively.

In other cases, an antibody may instead contain multimers thereof (e.g., IgM) or antigen-binding fragments thereof. Each heavy chain consists of a heavy chain variable region ("VH") and a heavy chain constant region ("CH"), which consists of the _CH1 , _CH2 , and _CH3 domains. Each light chain consists of a light chain variable region ("VL") and a light chain constant region ("CL"). The V _H and V _L regions can be further divided into regions of hypervariability called complementarity determining regions (CDRs) interspersed with more conserved regions called framework regions (FRs). Each V _H and V _L consists of three CDRs and four FRs, arranged from amino terminus to carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In certain embodiments of the present invention, the FRs of the antibody (or antigen-binding fragment thereof) may be identical to human germline sequences or may be naturally or artificially modified. The amino acid consensus sequence may be determined based on an analysis of two or more adjacent CDRs. Accordingly, the CDRs in the heavy chain are designated as "CDRH1", "CDRH2" and "CDRH3", respectively, and the CDRs in the light chain are designated as "CDRL1", "CDRL2" and "CDRL3".

Unless otherwise specifically stated, the term "antibody" as used herein encompasses molecules comprising two immunoglobulin heavy chains and two immunoglobulin light chains (i.e., "whole antibody molecules" or "intact antibodies"), as well as antigen-binding fragments thereof.

The term "antigen-binding fragment" refers to the portion of an intact antibody that binds the antigen to which the intact antibody binds (in this case, CD3). The terms "full-length antibody," "intact antibody," and "whole antibody" or the like are used interchangeably herein to refer to an antibody having a structure that is substantially similar to the structure of a native antibody.

An antigen-binding fragment of an antibody includes any naturally occurring, enzymatically produced, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds an antigen to form a complex. Exemplary antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab') ₂ ; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv or VH or VL domains only), and multispecific antibodies formed from antibody fragments. In some embodiments, the antigen-binding fragments of the anti-CD3 antibodies described herein are scFv.

As with whole antibody molecules, antigen-binding fragments may be monospecific or multispecific (e.g., bispecific). A multispecific antigen-binding fragment of an antibody will typically comprise at least two distinct variable domains, wherein each variable domain is capable of specifically binding to a distinct antigen or to a distinct epitope on the same antigen. Various multispecific antibody formats may be used in the context of the anti-CD3 antibody antigen-binding fragment described herein. Non-limiting examples of multispecific and bispecific formats include, for example, Fab-Fc-scFv (“bottle opener”) (XENCOR), Mab-scFv (XENCOR), Mab-Fv (XENCOR), Dual scFv (XENCOR), Core Fv (XENCOR), Core scFv(XENCOR), Single-arm Core scFv (XENCOR), Fab-Fab (XENCOR), Fab-Fv (XENCOR), mAb-Fv (XENCOR), mAb-Fab (XENCOR), DART (MacroGenics), BiTE (Amgen/Micromet), KiTE, Universal Light Chain IgG (Genentech), TandAb (SFFIMED) Cross-Mab (Roche), SEED (EMD Serono), BEAT (Glenmark), TrioMab (Trion Pharma/Fresenius Biotech), DuetMab (MedImmune), and others, which are disclosed, for example, in WO 95/09917; WO 2008/119566; WO 2008/119567; WO2011/121110; WO 2010/037835; WO 2007/042261; WO 2007/110205; WO 2011/121110; WO 2012/055961; WO 2012/16067; WO 2016/086189; WO 2016/182751; WO 2015/006749; WO 2014/049003; WO 2013/177101; WO 2015/128509; US 7,951,917; US 2009/0252729; US 2014/0348839; US 7,183,076; Mazor et al., Mabs, Vol. 7, pages 377-389 (2015); Muda et al., Protein Engineering, Design, & Selection, Vol. 24, pages 447-454 (2011); and Del Bano et al., Antibodies, Vol. 5, pages 1-23 (2016). In some embodiments, the anti-CD3 scFv fragments described herein comprise one or more variable domains of a multispecific (e.g., bispecific) antibody.

In certain embodiments, the anti-CD3 antibodies and/or antigen-binding fragments thereof described herein are comprised in a multispecific antibody, in particular a bispecific antibody, that has binding specificity for a second antigen. Such a second antigen may be an entirely different target than the first target or a different epitope present on the same target. In some embodiments, the binding specificity is for two different epitopes of CD3 (e.g., CD3ε or CD3γ). In other embodiments, one of the binding specificities is for binding to CD3 (e.g., CD3ε or CD3γ) and the other is for binding to another biological molecule (e.g., a cell surface antigen, such as a tumor antigen).

Non-limiting examples of a second antigen against which a bispecific antibody comprising the anti-CD3 antibodies and/or antigen-binding fragments thereof described herein is active include targets selected from the group consisting of: 17-IA, 4-1BB, 4Dc, 6-keto-PGFla, 8-iso-PGF2a, 8-oxo-dG, adenosine receptor A1, A33, ACE, ACE-2, activin, activin A, activin AB, activin B, activin C, activin RIA, activin RIA ALK-2, activin RIB ALK-4, activin RIIA, activin RUB, ADAM, ADAM10, ADAM12, ADAM 15, ADAM17/TACE, ADAM8, ADAM9, ADAMTS, ADAMTS4, ADAMTS5, addressins, aFGF, ALCAM, ALK, ALK-1, ALK-7, alpha-1-antitrypsin, alpha-V/beta-1 antagonist, ANG, Ang, APAF-1, APE, APJ, APP, APRIL, AR, ARC, ART, artemin, anti-idiotype, ASPARTIC, atrial natriuretic factor, integrin av/b3, Axl, b2M, B7-1, B7-2, B7-H, B-lymphocyte stimulator (BlyS), BACE, BACE-1, Bad, BAFF, BAFF-R, Bag-1, BAK, Bax, BCA-1, BCAM, Bel, BCMA, BDNF, b-ECGF, bFGF, BID, Bik, BIM, BLC, BL-CAM, BLK, BMP, BMP-2 BMP-2a, osteogenin BMP-3, BMP-4 BMP-2b, BMP-5, BMP-6 Vgr-1, BMP-7 (OP-1), BMP-8 (BMP-8a, OP-2), BMPR, BMPR-IA (ALK-3), BMPR-IB (ALK-6), BRK-2, RPK-1, BMPR-II (BRK-3), BMP, b-NGF, BOK, bombesin, bone neurotrophic factor, BPDE, BPDE-DNA, BTC, complement factor 3 (C3), C3a, C4, C5, C5a, CIO, CA125, CAD-8, calcitonin, cAMP, carcinoembryonic antigen (CEA), carcinoma-associated antigen, cathepsin A, cathepsin B, cathepsin C/DPPI, cathepsin D, cathepsin E, cathepsin H, cathepsin L, cathepsin O, cathepsin S, cathepsin V, cathepsin X/Z/P, CBL, CCI, CCK2, CCL, CCL1, CCL11, CCL12, CCL13, CCL 14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL2, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CCL3, CCL4, CCL5, CCL6, CCL7, CCL8, CCL9/10, CCR, CCR1, CCR10, CCR10, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CD1, CD2, CD4, CD5, CD6, CD7, CD8, CD10, CD11a, CD11b, CD11c, CD13, CD14, CD15, CD16, CD18, CD19, CD20, CD21, CD22, CD23, CD25, CD27L, CD28, CD29, CD30, CD30L, CD32, CD33 (p67 proteins), CD34, CD38, CD40, CD40L, CD44, CD45, CD46, CD49a, CD52, CD54, CD55, CD56, CD61, CD64, CD66e, CD74, CD80 (B7-1), CD89, CD95, CD123, CD137, CD138, CD140a, CD146, CD147, CD148, CD152, CD164, CEACAM5, CFTR, cGMP, CINC, toxin Clostridium botulinum, Clostridium perfringens toxin, CKb8-1, CLC, CMV, CMV UL, CNTF, CNTN-1, COX, C-Ret, CRG-2, CT-1, CTACK, CTGF, CTLA-4, CX3CL1, CX3CR1, CXCL, CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCR, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, cytokeratin tumor-associated antigen, DAN, DCC, DcR3, DC-SIGN, hemolysis stimulator, des(l-3)-IGF-I (IGF-1 brain), Dhh, digoxin, DNAM-1, Dnase, Dpp, DPPIV/CD26, Dtk, ECAD, EDA, EDA-A1, EDA-A2, EDAR, EGF, EGFR (ErbB-1), EMA, EMMPRIN, EN A, endothelin receptor, enkephalinase, eNOS, Eot, eotaxin l, EpCAM, ephrin B2/EphB4, EPO, ERCC, E-selectin, ET-1, factor Ila, factor VII, factor VIIIc, factor IX, fibroblast activating protein (FAP), Fas, FcRl, FEN-1, ferritin, FGF, FGF-19, FGF-2, FGF3, FGF-8, FGFR, FGFR-3, fibrin, FL, FLIP, Flt-3, Flt-4, follicle-stimulating hormone, fractalkine, FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, FZD10, G250, Gas 6, GCP-2, GCSF, GD2, GD3, GDF, GDF-1, GDF-3 (Vgr-2), GDF-5 (BMP-14, CDMP-1), GDF-6 (BMP-13, CDMP-2), GDF-7 (BMP-12, CDMP-3), GDF-8 (myostatin), GDF-9, GDF- 15 (MIC-1), GDNF, GFAP, GFRa-1, GFR-alpha 1, GFR-alpha 2, GFR-alpha 3, GITR, glucagon, Glut 4, glycoprotein Ilb/IIIa (GP Ilb/IIIa), GM-CSF, gpl30, gp72, GRO, somatotropin-releasing factor, hapten (NP-cap or NIP-cap), HB-EGF, HCC, HCMV envelope glycoprotein gB, HCMV envelope glycoprotein gH, HCMV UL, hematopoietic growth factor (HGF), Hep B gpl20, heparanase, Her2, Her2/neu (ErbB-2), Her3 (ErbB-3), Her4 (ErbB-4), herpes simplex virus (HSV) glycoprotein gB, HSV glycoprotein gD, HGFA, high molecular weight melanoma-associated antigen (HMW-MAA), HIV gpl20, V3-loop of gp 120 HIV IIIB, HLA, HLA-DR, HM1.24, HMFG PEM, HRG, Hrk, human cardiac myosin, human cytomegalovirus (HCMV), human growth hormone (HGH), HVEM, 1-309, IAP, ICAM, ICAM-1, ICAM-3, ICE, ICOS, IFNg, Ig, IgA receptor, IgE, IGF, IGF-binding proteins, IGF-1R, IGFBP, IGF-I, IGF-II, IL, IL-1, IL-1R, IL-2, IL-2R, IL-4, IL-4R, IL-5, IL-5R, IL-6, IL-6R, IL-8, IL-9, IL-10, IL-12, IL-13, IL-15, IL-18, IL-18R, IL-23, alpha interferon (INF), INF-beta, INF-gamma, inhibin, iNOS, insulin A chain, insulin B chain, insulin-like growth factor 1, alpha-2 integrin, alpha-3 integrin, alpha-4 integrin, alpha-4/beta-1 integrin, alpha-4/beta-7 integrin, alpha-5 (alpha-V) integrin, alpha-5/beta-1 integrin, alpha-5/beta-3 integrin, alpha-6 integrin, beta-1 integrin, beta-2 integrin, interferon gamma, IP-10, 1-TAC, JE, kallikrein 2, kallikrein 5, kallikrein 6, kallikrein 11, kallikrein 12, kallikrein 14, kallikrein 15, kallikrein LI, kallikrein L2, kallikrein L3, kallikrein L4, KC, KDR, keratinocyte growth factor (KGF), laminin 5, LAMP, LAP, LAP (TGF-1), latent TGF-1, bpl latent TGF-1, LBP, LDGF, LECT2, Lefty, Lewis-Y antigen, Lewis-Y-related antigen, LFA-1, LFA-3, Lfo, LIF, LIGHT, lipoproteins, LIX, LKN, Lptn, L-selectin, LT-a, LT-b, LTB4, LTBP-1, pulmonary surfactant, luteinizing hormone, beta-lymphotoxin receptor, Mac-1, MAdCAM, MAG, MAP2, MARC, MCAM, MCAM, MCK-2, MCP, M-CSF, MDC, Mer, metalloproteases, MGDF receptor, MGMT, MHC (HLA-DR), MIF, MIG, MIP, MIP-1alpha, MK, MMAC1, MMP, MMP-1, MMP-10, MMP-11, MMP-12, MMP-13, MMP-14, MMP-15, MMP-2, MMP-24, MMP-3, MMP-7, MMP-8, MMP-9, MPIF, Mpo, MSK, MSP, mucin (Mucl), MUC18, anti-Müllerian hormone, Mug, MuSK, NAIP, NAP, NCAD, N-cadherin, NCA 90, NCAM, NCAM, neprilysin, neurotrophin-3, -4, or -6, neuroturin, neuronal growth factor (NGF), NGFR, NGF-beta, nNOS, NO, NOS, Npn, NRG-3, NT, NTN, OB, OGG1, OPG, OPN, OSM, OX40L, OX40R, pl50, p95, PADPr, parathyroid hormone, PARC, PARP, PBR, PBSF, PCAD, P-cadherin, PCNA, PDGF, PDGF, PDK-1, PECAM, PEM, PF4, PGE, PGF, PGI2, PGJ2, PIN, PLA2, placental alkaline phosphatase (PLAP), PIGF, PLP, PP14, proinsulin, prorelaxin, C-protein, PS, PSA, PSCA, prostate-specific membrane antigen (PSMA), PTEN, PTHrp, Ptk, PTN, R51, RANK, RANKL, RANTES, relaxin A-chain, relaxin B-chain relaxin, renin, respiratory syncytial virus (RSV) F, RSV Fgp, Ret, rheumatoid factors, RLIP76, RPA2, RSK, S100, SCF/KL, SDF-1, SERINE, serum albumin, sFRP-3, Shh, SIGIRR, SK-1, SLAM, SLPI, SMAC, SMDF, SMOH, SOD, SPARC, Stat, STEAP, STEAP-II, TACE, TACI, TAG-72 (tumor-associated glycoprotein-72), TARC, TCA-3, T-cell receptors (e.g., T-cell receptor alpha/beta), TdT, TECK, TEM1, TEM5, TEM7, TEM8, TERT, testicular PLAP-like alkaline phosphatase, TfR, TGF, TGF-alpha, TGF-beta, polyspecific TGF-beta, RI TGF-beta (ALK-5), RII TGF-beta, Rllb TGF-beta, RIII TGF-beta, TGF-beta l, TGF-beta 2, TGF-beta 3, TGF-beta 4, TGF-beta 5, thrombin, Ck-1 thymus, thyroid stimulating hormone, Tie, TIMP, TIQ, tissue factor, TMEFF2, Tmpo, TMPRSS2, TNF, TNF-alpha, TNF-alpha/beta, TNF-beta 2, TNFc, TNF-RI, TNF-RII, TNFRSF10A (TRAIL Rl Apo-2, DR4), TNFRSFIOB (TRAIL R2 DR5, KILLER, TRICK-2A, TRICK-B), TNFRSF10C (TRAIL R3 DcRl, LIT, TRID), TNFRSF10D (TRAIL R4 DcR2, TRUNDD), TNFRSF11A (RANK ODF R, TRANCE R), TNFRSFllB (OPG OCIF, TR1), TNFRSF12 (TWEAK R FN14), TNFRSF13B (TACI), TNFRSF13C (BAFF R), TNFRSF14 (HVEM ATAR, HveA, LIGHT R, TR2), TNFRSF16 (NGFR p75NTR), TNFRSF17 (BCMA), TNFRSF 18 (GITR AITR), TNFRSF19 (TROY TAJ, TRADE), TNFRSF19L (RELT), TNFRSFIA (TNF RI CD120a, p55-60), TNFRSFIB (TNF RII CD120b, p75-80), TNFRSF26 (TNFRH3), TNFRSF3 (LTbR TNF RIII, TNFC R), TNFRSF4 (OX40 ACT35, TXGP1 R), TNFRSF 5 (CD40 p50), TNFRSF6 (Fas Apo-1, APT1, CD95), TNFRSF6B (DcR3 M68, TR6), TNFRSF7 (CD27), TNFRSF8 (CD30), TNFRSF9 (4-1BB CD137, ILA), TNFRSF21 (DR6), TNFRSF22 (DcTRAIL R2 TNFRH2), TNFRST23 (DcTRAIL Rl TNFRH1), TNFRSF25 (DR3 Apo-3, LARD, TR-3, TRAMP, WSL-1), TNFSF10 (ligand TRAIL Apo-2, TL2), TNFSF11 (TRANCE/RANK ODF ligand, OPG ligand), TNFSF12 (TWEAK Apo-3 ligand, DR3 ligand), TNFSF13 (APRIL TALL2), TNFSF13B (BAFF BLYS, TALL1, THANK, TNFSF20), TNFSF14 (LIGHT HVEM ligand, LTg), TNFSF15 (TLIA/VEGI), TNFSF18 (GITR ligand, AITR ligand, TL6), TNFSFIA (TNF-a conectin, DIF, TNFSF2), TNFSF1B (TNF-b LTa, TNFSF1), TNFSF3 (LTb TNFC, p33), TNFSF4 (OX40 gp34 ligand, TXGP1), TNFSF5 (CD40 ligand CD154, gp39, HIGM1, IMD3, TRAP), TNFSF6 (Fas Ligand, Apo-1 Ligand, APT1 Ligand), TNFSF7 (CD27 CD70 Ligand), TNFSF8 (CD30 CD153 Ligand), TNFSF9 (4-1BB Ligand, CD137 Ligand), TP-1, t-PA, Tpo, TRAIL, TRAIL R, TRAIL-R1, TRAIL-R2, TRANCE, transferrin receptor, TRF, Trk, TROP-2, TSG, TSLP, tumor-associated antigen CA 125, tumor-associated antigen characterized by the expression of carbohydrate antigen-related Lewis Y, TWEAK, TXB2, Ung, uPAR, uPAR-1, urokinase, VCAM, VCAM-1, VECAD, VE-cadherin, VE-cadherin-2, VEFGR-1 (flt-1), VEGF, VEGFR, VEGFR-3 (flt-4), VEGI, VFM, viral antigens, VLA, VLA-1, VLA-4, VNR integrin, von Willebrand factor, WIF- 1, WNT1, WNT2, WNT2B/13, WNT3, WNT3A, WNT4, WNT5A, WNT5B, WNT6, WNT7A, WNT7B, WNT8A, WNT8B, WNT9A, WNT9A, WNT9B, WNT10A, WNT10B, WNT11, WNT16, XCL1, XCL2, XCR1, XCR1, XEDAR, XIAP, XPD, CTLA4 (cytotoxic T lymphocyte antigen 4), PD1 (programmed cell death protein 1), PD-L1 (programmed cell death ligand 1), LAG-3 (lymphocyte activating gene 3), TIM-3 (T-cell immunoglobulin and mucin domain-containing protein 3), hormone receptors and growth factors.

Multispecific agents comprising antibodies and antigen-binding fragments to CD3 disclosed herein can be prepared by a variety of techniques, including, but not limited to, recombinant co-expression of two immunoglobulin heavy chain-light chain pairs with different specificities (see Milstein and Cuello, Nature 305: 537 (1983)), WO 93/08829 and Traunecker et al., EMBO J. 10: 3655 (1991)), knob-in-hole construction (see, e.g., U.S. Patent No. 5,731,168); immunoglobulin crossover technology (also known as Fab domain exchange or CrossMab format) (see, e.g., WO 2009/080253; Schaefer et al., Proc. Natl. Acad. Sci. USA, 108:11187–11192 (2011)); construction of Fc heterodimeric antibody molecules using the electrostatic interaction effect (WO 2009/089004 A1); cross-linking of two or more antibodies or fragments (see, e.g., U.S. Patent No. 4,676,980 and Brennan et al., Science, 229: 81 (1985)); leucine zippers (see, e.g., Kostelny et al., J. Immunol, 148(5):1547–1553 (1992)); "diabody" technology (see, e.g., Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444–6448 (1993)); single-chain Fv (sFv) dimers (see, e.g., Gruber et al., J. ImmunoL, 152:5368 (1994)) and trispecific antibodies, as described in, e.g., Tutt et al. J. ImmunoL 147:60 (1991).

The present invention also provides for modification of the CD3 antibodies disclosed herein, wherein such modifications comprise one or more amino acid substitutions, insertions and/or deletions in the FR and/or CDR regions of the variable domains of the heavy and light chains. Once produced, such derivative antibodies and/or antigen-binding fragments can be tested for one or more desired properties, such as improved binding specificity, increased binding affinity, improved developability, etc.

In some embodiments, the anti-CD3 antibodies and/or antigen-binding fragments thereof comprise a heavy chain (HC) sequence, a light chain (LC) sequence, a CDRH3 sequence, a CDRH2 sequence, a CHRH1 sequence, a CDRL3 sequence, a CDRL2 sequence, a CDRL1 sequence, and/or a framework sequence. In some embodiments, the anti-CD3 antibodies and/or antigen-binding fragments thereof have an amino acid sequence identity to the corresponding anti-CD3 antibodies disclosed in Table 4 (Ab1-Ab50) of at least about 100%, at least about 99%, at least about 98%, at least about 97%, at least about 96%, at least about 95%, at least about 94%, at least about 93%, at least about 92%, at least about 91%, at least about 90%, at least about 89%, at least about 88%, at least about 87%, at least about 86%, at least about 85%, at least about 84%, at least about 83%, at least about 82%, at least about 80%, and/or all points in between. percent identity values. In some embodiments, percent identity is measured using any well-known sequence identity algorithm, such as FASTA, BLAST, or GAP.

In some embodiments, residue positions that are not identical differ by conservative amino acid substitutions. A "conservative amino acid substitution" is one in which an amino acid residue is replaced by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). In general, a conservative amino acid substitution will not substantially alter the functional properties of the protein. In cases where two or more amino acid sequences differ from each other by conservative substitutions, the percentage or degree of similarity may be adjusted upward to account for the conservative nature of the substitution. Means for making this adjustment are well known to those skilled in the art. (See, e.g., Pearson (1994) Methods Mol. Biol. 24: 307-331.) Examples of groups of amino acids that have side chains with similar chemical properties include 1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; 2) aliphatic hydroxyl side chains: serine and threonine; 3) amide-containing side chains: asparagine and glutamine; 4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; 5) basic side chains: lysine, arginine, and histidine; 6) acidic side chains: aspartate and glutamate; and 7) sulfur-containing side chains: cysteine and methionine. In some embodiments, the conservative amino acid substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamate-aspartate, and asparagine-glutamine. Alternatively, in some embodiments, a conservative substitution includes any change characterized by a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science 256: 1443 45. In some embodiments, a “moderately conservative” substitution includes any change characterized by a non-negative value in the PAM250 log-likelihood matrix.

It is also possible to replace one or more residues in a CDR or to delete one or more CDRs. The literature describes antibodies in which one or two CDRs can be transferred to alter binding. Padlan et al. (1995 FASEB J. 9:133–139) analyzed the contact sites between antibodies and their antigens from published crystal structures and concluded that only about one-fifth to one-third of the residues in a CDR actually contact their associated antigen. Padlan also found many antibodies in which one or two CDRs did not contain amino acids that made contact with antigen (see also Vajdos et al. 2002 J Mol Biol 320:415–428). CDR residues that do not contact the antigen may be identified based on previous studies (e.g., residues H60-H65 in CDRH2 are often not essential), based on regions of the Kabat CDRs that lie outside the Chothia CDRs, by molecular modeling, and/or empirically. If a CDR or residue(s) thereof is excluded, it is typically replaced by an amino acid occupying the corresponding position in another human antibody sequence or by a consensus sequence for such sequences. Positions to replace within the CDRs and amino acids to replace may also be selected empirically.

In certain embodiments, substitutions, insertions, or deletions may occur within one or more CDRs of the CD3 antibodies described herein, so long as such changes do not substantially reduce the ability of the antibody to bind its antigen. For example, conservative changes (e.g., conservative substitutions provided herein) may be made to the CDRs that do not substantially reduce binding affinity. Such changes may, for example, be located in the CDRs outside of residues that contact the antigen. In certain embodiments of the VH and VL sequence variants provided above, each CDR is unaltered or has no more than one, two, or three amino acid substitutions.

A useful method for identifying residues or sites on an antibody that can be targeted by mutagenesis is called "alanine scanning mutagenesis" as described in Cunningham and Wells (1989) Science 244:1081-1085. In this method, a residue or group of target residues (e.g., charged residues such as arg, asp, his, lys, and glu) is identified and replaced with a neutral or negatively charged amino acid (e.g., alanine or polyalanine) to determine whether it affects the interaction of the antibody with antigen. Additional substitutions can be introduced at amino acid positions that demonstrate functional sensitivity to the initial substitutions. Alternatively, or in addition, a crystal structure of the antigen-antibody complex is determined to identify contact points between antibody and antigen. Such contact residues and adjacent residues can serve as candidates for substitution, which are targeted or eliminated. Variants can be screened to determine whether they have the desired properties.

Amino acid sequence insertions include amino- and/or carboxyl-terminal fusions ranging in length from a single residue to polypeptides containing a hundred or more residues, as well as intrasequence insertions of one or more amino acid residues. Examples of terminal insertions include an antibody with an N-terminal methionyl residue. Other antibody insertion options include fusion of the N- or C-terminus of the antibody to an enzyme (e.g., in the case of ADEPT) or a polypeptide that increases the serum half-life of the antibody.

As described throughout this document, the CD3 antibodies and/or antigen-binding fragments thereof provided herein are characterized by favorable developability and are thus considered to be relatively developable.

The term "developmentally suitable" refers to the extent to which one or more polypeptides from a population of polypeptides possess desired characteristics such as, for example, desired expression, such as in mammalian cells; solubility; viscosity; aggregation; chemical and/or physical stability; desired "shelf life"; melting point; pharmacokinetic profiles; circulating half-life and clearance characteristics. Such characteristics may act as independent features, as combinations of subsets of such features, or collectively to determine the likelihood that such one or more polypeptides may be successfully developed as a therapeutic candidate and, ultimately, an approved drug. Accordingly, it is generally understood from the prior art that polypeptides with the desired developability characteristics have, for example, relatively high solubility, relatively low viscosity, relatively low aggregation susceptibility, relatively high chemical stability, relatively high physical stability, relatively long "shelf life", relatively high melting point, relatively long half-life in the bloodstream, relatively long elimination time, etc. Polypeptides with undesirable developability characteristics have, for example, relatively low solubility, relatively high viscosity, relatively high aggregation susceptibility, relatively unsatisfactory chemical stability, relatively unsatisfactory physical stability, relatively short "shelf life", relatively low melting point, relatively short half-life in the bloodstream, relatively short elimination time, etc.

Methods and assays that can be used to determine the extent to which polypeptides, such as anti-CD3 antibodies and/or antigen-binding fragments thereof, described herein exhibit the desired developability properties are available in the art and include, for example, PCR-based assays (WO 2014/179363 and Xu et al., Protein Eng Des Sol, Vol. 26, pages 663-670 (2013)); SMP and SCP assays and the like; cross-interaction chromatography (CIC); self-interaction chromatography (SIC); dynamic light scattering; size exclusion chromatography (SEC), dynamic light scattering spectroscopy (DLS); photon correlation spectroscopy; quasi-elastic light scattering, circular dichroism (CD), viscosity measurements; whole cell binding; tissue microarray techniques; BVP ELISA-based assays; AC-SINS assays (Liu et al; MAbs, Vol. 6, pages 483–492 (2014); differential scanning calorimetry, etc. (see, e.g., He et al., J. Pharm. Sci., Vol. 100(4), pp. 1330–1340 (2011); Wagner et al., Pharm. Develop. & Technol (published online 2012; hypertext transfer protocol: informahealthcare.com/doi/abs/10.3109/10837450.2011.649851); Hotzel et al., mAbs, Vol. 4(6), pages 753–7601 (2012); Weiqiang et al., J. Pharm. Sci., Vol. 101(5), pp. 1701-1720 (2012); Banks et al., J. Pharm. Sci., Vol. 101(8), pp. 2720-2732 (2012); Lie et al., J. Pharm. Sci., Vol. 94(9), pp. 1928-1948 (2005) and Payne et al., Biopolymers, Vol. 85(5), pp. 527-533 (2006)).

In some embodiments, antibodies that are identified as having reduced developability are considered to be such due to their interaction with a multispecificity reagent ("PSR") and are, in fact, referred to as "multispecific" polypeptides. Such multispecific antibodies may be referred to as relatively "non-developable" or considered to be relatively "undevelopable" antibodies.

"Developability profile" refers to an index that can be assigned to antibodies when assessing their developability. A developability profile is a score or measure by which the developability of CD3 antibodies can be assessed, compared, and/or ranked. Such developability profiles serve as an estimate of the degree of interaction between CD3 binding molecules and antibodies containing them. The degree of interaction can be assessed by any of a number of means available in the art that provide an output value that correlates with the strength or affinity of a polypeptide for the moiety to which it binds. Exemplary means include flow cytometry-based means such as FACS; ELISA; quantitative immune affinity assays or immunoprecipitation assays; mammalian two-hybrid assays or yeast two-hybrid assays, and the like. In the context of FACS, as demonstrated in the Examples section, the degree of interaction between polypeptides from a population and a PSR can be determined by obtaining the mean fluorescence intensity (MFI) for each detected polypeptide-PSR interaction and then ranking the MFI in either ascending or descending order, thereby ranking the polypeptides from the population according to the relative degree of interaction between each detected polypeptide and the PSR. Such ranking provides a ranking of the polypeptides from the population such that polypeptides that have improved developability and polypeptides that have decreased developability can be easily identified.

The developability profile may also take the form of a normalized score, such as by normalizing the developability of the CD3 antibodies described herein to the developability of a standard (or control) antibody, such as an anti-HEL antibody.

In certain embodiments, the CD3 binding domains and antibodies comprising them of the present invention may be further modified to contain additional non-proteinaceous moieties that are known in the art and readily available. Moieties suitable for derivatization of the antibody include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol/propylene glycol copolymers, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene/maleic anhydride copolymer, polyamino acids (or homopolymers or random copolymers), as well as dextran or poly(n-vinylpyrrolidone)polyethylene glycol, polypropylene glycol homopolymers, polypropylene oxide/ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerin), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol-propionaldehyde may have manufacturing advantages due to its stability in water. The polymer may have any molecular weight and may be branched or unbranched. The number of polymers attached to the antibody may vary, and if more than one polymer is attached, they may be the same or different molecules. Typically, the number and/or type of polymers used for derivatization may be determined based on factors including, but not limited to, the particular properties or functions of the antibody to be enhanced, whether the antibody derivative will be used in therapy under certain conditions, etc.

In certain embodiments, CD3 binding domains and antibodies comprising them of the present invention are provided that exhibit an improved developability profile. The developability profile of the CD3 antibodies is obtained by performing one or more of a PSR assay; an SCP assay; an AC-SINS assay; an ELISA; a DSF assay; a Tm assay; a HIC assay; a CIC assay, or a combination thereof.

In other embodiments, the anti-CD3 antibodies and/or antigen-binding fragments thereof described herein exhibit a multispecificity reagent score (PSR) of from about 0.0 to about 0.45; from about 0.0 to about 0.4; from about 0.0 to about 0.35; from about 0.0 to about 0.3; from about 0.0 to about 0.25; from about 0.0 to about 0.2; from about 0.0 to about 0.15; or from about 0.0 to about 0.1. A score of 0.0-0.1 means “clear PSR.” A score of 0.1 to 0.33 means “low PSR.” A score of 0.33 to 0.66 means “medium PSR.” A score of 0.66-1.00 means “high PSR.” A high PSR indicates low (or poor) developability. Generally, the lower the PSR, the more favorable the antibody's developability profile.

In other embodiments, the anti-CD3 antibodies and/or antigen-binding fragments thereof described herein exhibit a HIC of less than about 10.5 minutes (clear to low HIC); from about 10.5 minutes to 11.5 minutes (medium HIC), or greater than about 11.5 minutes (high HIC). Generally, the lower the HIC, the more favorable the antibody's developability profile.

In yet other embodiments, the anti-CD3 antibodies and/or antigen-binding fragments thereof described herein exhibit a SEC of less than about 95%, indicating that the antibody is monomeric, i.e., does not aggregate.

In yet other embodiments, the anti-CD3 antibodies and/or antigen-binding fragments thereof described herein exhibit a Tm of less than about 65°C.

In some embodiments, the anti-CD3 antibodies and/or antigen-binding fragments thereof described herein may be further modified to minimize effector function, such as Fc silencing.

"Effector function" refers to those biological activities that are associated with the Fc region of an antibody, which varies depending on the antibody isotype. Illustrative effector functions include: C1q binding and complement-dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., the B cell receptor); and B cell activation.

An "Fc region" is the C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region, including native sequence Fc regions and variant Fc regions. The Fc region of a human IgG heavy chain may extend from Cys226 or from Pro230 to the carboxy terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. Unless otherwise indicated herein, the numbering of amino acid residues in the Fc region or constant region follows the EU numbering system, also referred to as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991.

In certain embodiments, one or more amino acid modifications may be introduced into the Fc region of an anti-CD3 antibody of the present invention, thereby producing a variant Fc region (see, e.g., US 2012/0251531). The variant Fc region may comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) comprising an amino acid modification (e.g., a substitution) at one or more amino acid positions.

In certain embodiments, the present invention provides an anti-CD3 antibody variant that has some, but not all, effector functions, making it a suitable candidate for applications in which the in vivo half-life of the antibody is important, while some effector functions (such as the complement system and ADCC) are dispensable or detrimental. In vitro and/or in vivo cytotoxicity assays can be used to confirm the reduction/ablation of CDC and/or ADCC activity. For example, Fc receptor (FcR) binding assays can be used to ensure that the antibody does not exhibit binding to FcγR (and therefore likely lacks ADCC activity) but retains the ability to bind to FcRn. Primary cells that mediate ADCC (e.g., NK cells) express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991). Non-limiting examples of in vitro assays for assessing ADCC activity of a molecule of interest are described in U.S. Patent No. 5,500,362 (see, e.g., Hellstrom, I. et al. Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985); U.S. Patent No. 5,821,337 (see Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assays can be used (see, for example, the ACTI™ Non-Radioactive Flow Cytotoxicity Assay (Cell Technology, Inc., Mountain View, CA, USA) and the CytoTox 96® Non-Radioactive Cytotoxicity Assay (Promega, Madison, WI, USA)). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Alternatively, or in addition, the ADCC activity of the molecule of interest can be assessed in vivo, for example in an animal model such as that disclosed in Clynes et al. Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). C1q binding assays can also be performed to confirm that the antibody is unable to bind to C1q and therefore lacks CDC activity. See, for example, the C1q and C3c binding ELISA in WO 2006/029879 and WO 2005/100402. To assess complement activation, the CDC assay can be performed (see, for example, Gazzano-Santoro et al. J. Immunol. Methods 202:163 (1996); Cragg, M. S. et al. Blood. 101:1045-1052 (2003); and Cragg, M. S. and M. J. Glennie Blood. 103:2738-2743 (2004)). Binding to FcRn and determination of in vivo clearance/half-life can also be accomplished using methods known in the art (see, e.g., Petkova, S. B. et al. Int'l. Immunol. 18(12):1759-1769 (2006)).

In some embodiments, antibodies with reduced effector function include antibodies with a substitution of one or more residues 238, 265, 269, 270, 297, 327, and 329 of the Fc region (U.S. Pat. Nos. 6,737,056 and 8,219,149). In some embodiments, such Fc mutants include Fc mutants with substitutions at two or more amino acid positions 265, 269, 270, 297, and 327, including the so-called "DANA" Fc mutant with a substitution of residues 265 and 297 to alanine (U.S. Pat. Nos. 7,332,581 and 8,219,149).

In other embodiments, the anti-CD3 antibodies and/or antigen-binding fragments thereof described herein are further modified to include a masking agent, such as a polypeptide mask, attached via a cleavable linker.

In some embodiments, the anti-CD3 antibodies and/or antigen-binding fragments thereof described herein are conjugated to a therapeutic moiety, thereby producing an immunoconjugate. An "immunoconjugate" is an antibody conjugated to one or more heterologous molecules, such as, for example, an antibiotic, a second anti-CD3 antibody, a vaccine, or a toxoid, or any other therapeutic moiety.

In certain embodiments, the anti-CD3 antibodies and/or antigen-binding fragments thereof described herein are altered to increase or decrease the extent to which the antibody is glycosylated. Addition or removal of glycosylation sites in an anti-CD3 antibody of the present invention can be readily accomplished by altering the amino acid sequence so as to create or remove one or more glycosylation sites.

Moreover, the present invention generally relates to an anti-CD3 antibody or antibody fragment, wherein said anti-CD3 antibody or antigen-binding fragment comprises: a. a heavy chain variable region (VH) polypeptide comprising: i. CDR1 of V _H (CDRH1) with FNIKDYYMH (SEQ ID NO: 13); ii. CDR2 of V _H (CDRH2) with WIDLENANTVYDAKFQG (SEQ ID NO: 15); iii. CDR3 of V _H (CDRH3) with ARDAYGRYFYDV (SEQ ID NO: 103); and/or b. a light chain variable region (V _L ) polypeptide comprising: i. CDR1 of V _L (CDRL1) with KSSQSLLNARTGKNYLA (SEQ ID NO: 8); CDR2 V _L (CDRL2) with WASTRX ₁ S (SEQ ID NO: 133) and CDR3 V _L (CDRL3) with X ₂ QSYFRRT (SEQ ID NO: 134); wherein each of X ₁ and X ₂ is independently any amino acid. In some embodiments, X ₁ is E or S. In some embodiments, X ₂ is K or V. In some embodiments, said antibody or antigen-binding fragment of an antibody comprises CDRL2 of SEQ ID NO: 10 or SEQ ID NO: 11. In some embodiments, said antibody or antigen-binding fragment of an antibody comprises CDRL3 of SEQ ID NO: 5 or SEQ ID NO: 4.

In some embodiments, said antibody or antigen-binding fragment of an antibody comprises V _H chain CDR1 (CDRH1) with FNIKDYYMH (SEQ ID NO: 13), V _H chain CDR2 (CDRH2) with WIDLENANTVYDAKFQG (SEQ ID NO: 15) and V _H chain CDR3 (CDRH3) with ARDAYGRYFYDV (SEQ ID NO: 103) and/or V _L chain CDR1 (CDRL1) with KSSQSLLNARTGKNYLA (SEQ ID NO: 8), V _L chain CDR2 (CDRL2) with WASTRES (SEQ ID NO: 10) and V _L chain CDR3 (CDRL3) with KQSYFRRT (SEQ ID NO: 5). In some embodiments, said antibody or antigen-binding fragment of an antibody comprises V _H chain CDR1 (CDRH1) with FNIKDYYMH (SEQ ID NO: 13), V _H chain CDR2 (CDRH2) with WIDLENANTVYDAKFQG (SEQ ID NO: 15), and V _H chain CDR3 (CDRH3) with ARDAYGRYFYDV (SEQ ID NO: 103), and/or V _L chain CDR1 (CDRL1) with KSSQSLLNARTGKNYLA (SEQ ID NO: 8), V _L chain CDR2 (CDRL2) with WASTRSS (SEQ ID NO: 11), and V _L chain CDR3 (CDRL3) with VQSYFRRT (SEQ ID NO: 4). In some embodiments, the anti-CD3 antibody may comprise any one or more CDRs of an antibody referred to as Ab1 and/or any one or more CDRs of an antibody referred to as Ab13. In some embodiments, an anti-CD3 antibody may bind to the same or substantially similar CD3 epitope or epitopes as Ab1 and/or Ab13.

In some embodiments, said anti-CD3 antibody or antigen-binding fragment of an antibody comprises a V _L chain polypeptide comprising an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 18 or SEQ ID NO: 34. In some embodiments, said anti-CD3 antibody or antigen-binding fragment of an antibody comprises a V _L chain polypeptide comprising an amino acid sequence that has at least 95% sequence identity to SEQ ID NO: 18 or SEQ ID NO: 34. In some embodiments, said anti-CD3 antibody or antigen-binding fragment of an antibody comprises a V _L chain polypeptide comprising the amino acid sequence of SEQ ID NO: 18 or SEQ ID NO: 34. In some embodiments, said anti-CD3 antibody or antigen-binding fragment of an antibody comprises a V _{H chain polypeptide comprising an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 17. In some embodiments, said anti-CD3 antibody or antigen-binding fragment of an antibody comprises a V H} chain polypeptide comprising an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 17. the antigen-binding fragment of the anti-CD3 antibody comprises a V _H chain polypeptide comprising an amino acid sequence that has at least 95% sequence identity to SEQ ID NO: 17. In some embodiments, said antibody or antigen-binding fragment of the anti-CD3 antibody comprises a V _H chain comprising the amino acid sequence of SEQ ID NO: 17.

In some embodiments, said anti-CD3 antibody or antigen-binding fragment of an antibody comprises a V _L chain polypeptide comprising an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 18 or SEQ ID NO: 34; and a V _H chain polypeptide comprising an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 17. In some embodiments, said anti-CD3 antibody or antigen-binding fragment of an antibody comprises a V _L chain polypeptide comprising an amino acid sequence that has at least 95% sequence identity to SEQ ID NO: 18 or SEQ ID NO: 34; and a V _H chain polypeptide comprising an amino acid sequence that has at least 95% sequence identity to SEQ ID NO: 17. In some embodiments, said anti-CD3 antibody or antigen-binding fragment of an antibody comprises a V _L chain polypeptide comprising the amino acid sequence of SEQ ID NO: 18 or SEQ ID NO: 34; and a V _H chain comprising the amino acid sequence of SEQ ID NO: 17. In some embodiments, said anti-CD3 antibody or antigen-binding fragment of an antibody comprises a V _L chain polypeptide comprising the amino acid sequence of SEQ ID NO: 18; and a V _H chain comprising the amino acid sequence of SEQ ID NO: 17. In some embodiments, said anti-CD3 antibody or antigen-binding fragment of an antibody comprises a V _L chain polypeptide comprising the amino acid sequence of SEQ ID NO: 34; and a V _H chain comprising the amino acid sequence of SEQ ID NO: 17.

In some embodiments, said anti-CD3 antibody or antigen-binding fragment of an antibody binds to CD3 with a dissociation constant ( _KD ) of about 500 pM or less, about 470 pM or less, about 450 pM or less, about 400 pM or less, about 350 pM or less, about 300 pM or less, about 250 pM or less, about 200 pM or less, about 150 pM or less, or about 100 pM or less, wherein optionally said CD3 is human and/or cynomolgus monkey, wherein even more optionally said binding affinity is measured by surface plasmon resonance. In some embodiments, said _KD is a monovalent _KD and/or said _KD is measured using an scFv fragment of said anti-CD3 antibody or antibody fragment.

Moreover, the present invention generally relates to an antibody or antibody fragment to cluster of differentiation three ("CD3"), wherein said antibody or antigen-binding fragment of an anti-CD3 antibody comprises one or more CDRs of any one or more of Ab1-Ab50 and/or binds to the same epitope(s) as any one or more of Ab1-Ab50. Furthermore, the present invention generally relates to an antibody or antibody fragment to cluster of differentiation three ("CD3"), wherein said antibody or antigen-binding fragment of an anti-CD3 antibody comprises one or more V _H and/or V _L chains comprising an amino acid sequence selected from the sequences in Table 4.

Production of antibodies to CD3 and their antigen-binding fragments

Anti-CD3 antibodies and/or antigen-binding fragments thereof can be produced using recombinant techniques. For example, isolated nucleic acids encoding an anti-CD3 antibody described herein are provided. Such nucleic acids can encode an amino acid sequence comprising a VL and/or an amino acid sequence comprising a VH of an antibody (e.g., a light and/or heavy chain of an antibody). In a further embodiment, one or more vectors (e.g., expression vectors) comprising such nucleic acids are provided. In a further embodiment, a host cell comprising such nucleic acids is provided. In one such embodiment, the host cell comprises (e.g., has been transformed with): (1) a vector comprising a nucleic acid sequence that encodes an amino acid sequence providing a VL antibody and an amino acid sequence providing a VH antibody, or (2) a first vector comprising a nucleic acid that encodes an amino acid sequence providing a VL antibody and a second vector comprising a nucleic acid that encodes an amino acid sequence providing a VH antibody. In one embodiment, the host cell is eukaryotic, such as a Chinese hamster ovary (CHO) cell or a lymphoid cell (e.g., a Y0, NS0, Sp20 cell). In one embodiment, a method for producing an anti-CD3 antibody is provided, wherein the method comprises culturing a host cell comprising a nucleic acid encoding the above antibody under conditions suitable for expression of the antibody, and optionally recovering the antibody from the host cell (or a culture medium of the host cell).

The term "host cell" refers to cells into which an exogenous nucleic acid sequence has been introduced, including the progeny of such cells. Host cells include transformants and transformed cells, which include the primary transformed cell and the progeny derived from it, regardless of the number of passages.

In the case of recombinant production of an anti-CD3 antibody, nucleic acids encoding the antibody, such as those described above, are isolated and inserted into one or more vectors for subsequent cloning into and/or expression in a host cell. Such nucleic acids can be readily isolated and sequenced using conventional procedures (e.g., using oligonucleotide probes that are capable of specifically binding to genes encoding the heavy and light chains of the antibody).

Suitable host cells for cloning and/or expressing antibody-encoding vectors include prokaryotic or eukaryotic cells. For example, antibodies can be produced in bacteria, particularly where glycosylation and Fc effector function are not required. For expression of antibody fragments and polypeptides in bacteria, see, for example, U.S. Patent Nos. 5,648,237, 5,789,199, and 5,840,523. (See also Charlton, Methods in Molecular Biology, Vol. 248 (B.K.C. Lo, ed., Humana Press, Totowa, N.J., 2003), pp. 245-254, for a description of expression of antibody fragments in E. coli.) Following expression, the antibody can be isolated from the bacterial cell mass as a soluble fraction and can be further purified. In addition to prokaryotes, suitable hosts for cloning or expression of antibody-encoding vectors include eukaryotic microorganisms such as filamentous fungi or yeast, including fungi and yeast strains whose glycosylation pathways have been "humanized" to produce antibodies with partially or completely human glycosylation patterns. See, e.g., Gerngross, Nat. Biotech. 22:1409-1414 (2004) and Li et al., Nat. Biotech. 24:210-215 (2006); WO 2009/036379; WO 2010/105256; and WO 2012/009568. Plant cell cultures can also be used as hosts. See, e.g., U.S. Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (describing the PLANTIBODIES™ technology for producing antibodies in transgenic plants). Vertebrate cells can also be used as hosts. For example, mammalian cell lines that are adapted to grow in suspension can be used. Other examples of useful mammalian cell host cell lines include the SV40-transformed monkey kidney cell line CV1 (COS-7); the human embryonic kidney line (293 or 293 cells, described, e.g., in Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney (BHK) cells; mouse Sertoli cells (TM4 cells, described, for example, in Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical carcinoma cells (HELA); canine kidney cells (MDCK); buffalo rat liver cells (BRL 3A); human lung cells (W138); human liver cells (Hep G2); mouse mammary tumor (MMT 060562); TRI cells, as described in, e.g., Mather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982); MRC 5 cells; and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR-CHO cells (Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); and myeloma cell lines such as Y0, NS0, and Sp2/0. For a review of some mammalian host cell lines suitable for antibody production, see, e.g., Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (B.K.C. Lo, ed., Humana Press, Totowa, N.J.), pp. 255-268 (2003).

Anti-CD3 antibodies and/or antigen-binding fragments thereof can be identified, screened, selected or characterized for their physical/chemical properties and/or biological activities using various assays known in the art, such as ELISA, Western blotting, etc., or competitive binding assays can be used to identify an antibody that competes with an anti-CD3 antibody of the present invention for binding to CD3. In an exemplary competitive binding assay, immobilized CD3 is incubated in a solution containing a first labeled antibody that binds to CD3 and a second unlabeled antibody that is to be tested for its ability to compete with the first antibody for binding to CD3. The second antibody may be present in the hybridoma supernatant. As a control, immobilized CD3 is incubated in a solution containing the first labeled antibody but not the second unlabeled antibody. After incubation under conditions that permit binding of the primary antibody to CD3, excess unbound antibody is removed and the amount of label associated with immobilized CD3 is measured. If the amount of label associated with immobilized CD3 is significantly reduced in the test sample compared to the control sample, then this indicates that the secondary antibody is competing with the primary antibody for binding to CD3. See, e.g., Harlow and Lane (1988) Antibodies: A Laboratory Manual. Ch.14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.).

Anti-CD3 antibodies and/or antigen-binding fragments thereof that exhibit biological activity can be identified using standard approaches. Biological activity can include, for example, binding to CD3 on the surface of a T cell, either in vivo or in vitro or ex vivo. In the case of a multispecific anti-CD3 antibody (such as a bispecific antibody with one arm that binds to CD3 and the other arm that binds to another target, such as a cell surface antigen, such as a tumor antigen), biological activity can also include activation of effector cells (such as activation of CD8+ and/or CD4+ T cells), expansion of the effector cell population (i.e., an increase in the number of T cells), reduction of the population of target cells (i.e., a decrease in the population of cells expressing a second biological molecule on their cell surface), and/or killing of target cells.

Diagnostic and therapeutic applications of CD3 antibodies and their antigen-binding fragments

The antibodies and/or antigen-binding fragments to CD3 described herein can be used for diagnosis and/or detection. As used herein, the term "detection" includes quantitative or qualitative detection. In one embodiment, a method is provided for detecting the presence of CD3 in a biological sample. Such a method comprises (i) contacting the biological sample with an anti-CD3 antibody described herein under conditions that allow binding of the anti-CD3 antibody to CD3, and (ii) detecting whether a complex is formed between the anti-CD3 antibody and CD3. Such a method can be an in vitro or in vivo method. In certain embodiments, the biological sample comprises a cell or tissue(s).

In certain embodiments, labeled anti-CD3 antibodies are provided. The anti-CD3 antibodies and/or antigen-binding fragments thereof described herein include a label or moiety that is detected directly (e.g., fluorescent, chromophore, electron-dense, chemiluminescent, and radioactive labels) or indirectly (e.g., enzymes or ligands). Non-limiting exemplary labels include radioisotopes such as 32P, 14C, 125I, 3H, and 131I; fluorophores such as rare earth chelates or fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, luciferases such as firefly luciferase and bacterial luciferase (U.S. Patent No. 4,737,456), luciferin, 2,3-dihydrophthalazinediones, horseradish peroxidase (HRP), alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, saccharide oxidases such as glucose oxidase, galactose oxidase and glucose-6-phosphate dehydrogenase; heterocyclic oxidases such as uricase and xanthine oxidase coupled to an enzyme that uses hydrogen peroxide to oxidize a precursor dye such as HRP, lactoperoxidase, or microperoxidase; biotin/avidin; spin labels; bacteriophage-based labels; stable free radicals, etc.

The CD3 antibodies and/or antigen-binding fragments thereof described herein, as well as pharmaceutical compositions of such antibodies, can be used in therapeutic methods. In one embodiment, the CD3 antibodies and/or antigen-binding fragments thereof described herein, or pharmaceutical compositions comprising such antibodies, can be used to treat or slow the progression of a cell proliferative disorder or an autoimmune disorder.

"Disorder" refers to any condition or disease that would benefit from treatment, including without limitation chronic and acute disorders or diseases, including those pathological conditions that cause the disorder in question in a mammal.

The terms "cell proliferative disorder" and "proliferative disorder" refer to disorders that are associated with abnormal cell proliferation to some degree. Cell proliferative disorders include cancer, such as tumor.

As used in this document, the term "tumor" refers to the growth and proliferation of all neoplastic cells, whether malignant or benign, and to all precancerous and cancerous cells and tissues.

"Cancer" refers to a physiological condition in mammals characterized by unregulated cell growth. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or malignant neoplasms of lymphoid tissue; wherein more specific examples include squamous cell carcinoma (e.g. epithelial squamous cell carcinoma), lung cancer including small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma and squamous cell lung carcinoma, peritoneal cancer, hepatocellular carcinoma, gastric cancer or stomach cancer including gastrointestinal cancer and gastrointestinal stromal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, urinary tract cancer, hepatoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney cancer or renal cancer, prostate cancer, vulvar cancer, thyroid cancer, liver carcinoma, anal carcinoma, penile carcinoma, melanoma, superficial spreading melanoma, malignant melanoma lentigo, acral lentiginous melanoma types, nodular melanoma types, multiple myeloma, and B-cell lymphoma (including low-grade/follicular non-Hodgkin's lymphoma (NHL); small lymphocytic (SL) NHL; intermediate-grade/follicular NHL; intermediate-grade diffuse NHL; high-grade immunoblastic NHL; high-grade lymphoblastic NHL; high-grade small undissected NHL; generalized NHL; mantle cell lymphoma; AIDS-associated lymphoma; and Waldenstrom's macroglobulinemia); chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); hairy cell leukemia; chronic myeloid leukemia and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal vascular proliferation associated with phakomatoses, edema (such as associated with brain tumors), Meigs syndrome, brain cancer, and head and neck cancer and associated metastases. In certain embodiments, cancers that are treatable with the antibodies of the present invention include breast cancer, colorectal cancer, rectal cancer, non-small cell lung cancer, glioblastoma, non-Hodgkin's lymphoma (NHL), renal cell carcinoma, prostate cancer, liver cancer, pancreatic cancer, soft tissue sarcoma, Kaposi's sarcoma, carcinoid carcinoma, head and neck cancer, ovarian cancer, mesothelioma, and multiple myeloma. In some embodiments, the cancer is selected from small cell lung cancer, glioblastoma, neuroblastoma types, melanoma, breast carcinoma, gastric cancer, colorectal cancer (CRC), and hepatocellular carcinoma. In some embodiments, the cancer is selected from non-small cell lung cancer, colorectal cancer, glioblastoma, and breast carcinoma, including metastatic forms of these cancers. In other embodiments, the cancer is selected from the class of mature B-cell cancers except for Hodgkin's lymphoma, but including germinal center B-cell DLBCL (GCB), active B-cell DLBCL (ABC), follicular lymphoma (FL), mantle cell lymphoma (MCL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), marginal zone cell lymphoma (MZL), small lymphocytic leukemia (SLL), lymphoplasmacytic lymphoma (LL), Waldenstrom's macroglobulinemia (WM), central nervous system lymphoma (CNSL), Burkitt's lymphoma (BL), B-cell prolymphocytic leukemia, splenic marginal zone lymphoma, hairy cell leukemia, splenic lymphoma/leukemia, diffuse unclassified Splenic red pulp small B cell lymphoma, variant hairy cell leukemia, Waldenstrom's macroglobulinemia, heavy chain diseases, heavy chain disease, γ heavy chain disease, μ heavy chain disease, plasma cell myeloma, solitary plasmacytoma of bone, extraosseous plasmacytoma, extranodal mucosa-associated lymphoid tissue (MALT) lymphoma, nodular marginal zone cell lymphoma, nodular marginal zone cell lymphoma in children, follicular lymphoma in children, primary cutaneous follicular center cell lymphoma, large B cell lymphoma rich in T cells/histiocytes, primary CNS DLBCL, primary cutaneous DLBCL, leg type, EBV-positive DLBCL in the elderly, DLBCL associated with chronic inflammation, lymphomatoid granulomatosis, primary mediastinal (thymic) large B-cell lymphoma, intravascular large B-cell lymphoma, ALK-positive large B-cell lymphoma, plasmablastic lymphoma, large B-cell lymphoma arising in HHV8-associated multicentric Castleman disease, primary effusion lymphoma: B-cell lymphoma, unclassifiable, with features intermediate between diffuse large B-cell lymphoma and Burkitt lymphoma, and B-cell lymphoma, unclassifiable, with features intermediate between diffuse large B-cell lymphoma and classical Hodgkin lymphoma.

As used herein, "treatment" or "treat" or "treat" refers to a clinical intervention in an attempt to alter the natural history of a disease in the individual being treated, and may be administered either prophylactically or during a clinical trial. Desired treatment effects include, but are not limited to, preventing the onset or recurrence of the disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, reducing the severity or alleviating the disease state, and causing remission or improving prognosis.

As used in this document, the terms "prevent," "prevention," and "prevention" refer to the prevention or suppression of the development or occurrence of a disorder or disease.

As used herein, the terms “alleviation” and “relief” refer to a reduction or decrease in the severity of a condition or any of its symptoms.

In some embodiments, the antibodies of the present invention are used to slow the development of a disorder or disease or to slow the progression of a disorder or disease. As used herein, "slowing the progression" of a disorder or disease means delaying, inhibiting, reducing the rate of, slowing, stabilizing, and/or postponing the progression of a disease or disorder (e.g., a cell proliferative disorder, such as cancer). This slowing may vary in time depending on the history of the disease and/or the individual being treated.

An effective amount of such an antibody or composition can be administered to an individual suffering from cancer or arthritis, rheumatoid arthritis, colitis, inflammatory bowel disease, autoimmune diabetes type I, etc. An "effective amount" of an anti-CD3 antibody or composition (e.g., a pharmaceutical composition) comprising such an antibody disclosed herein is at least the minimum amount necessary to achieve the desired therapeutic or prophylactic result, such as a measurable improvement or prevention of a particular disorder, such as a cell proliferative disorder, such as cancer, preferably with minimal or no toxic or deleterious effects. An effective amount can vary depending on, among other things, the disease state, age, sex, and weight of the patient, as well as the ability of the antibody (or antigen-binding fragment thereof) to elicit the desired response in the individual and, in some cases, by co-administration of one or more additional therapeutic agents.

In some embodiments, the anti-CD3 antibodies and/or antigen-binding fragments thereof described herein can be used to enhance immune function in an individual suffering from a cellular proliferative disorder or an autoimmune disorder. Upon administration, such an antibody or composition can enhance immune function in an individual having a cellular proliferative disorder or an autoimmune disorder by activating effector cells (e.g., T cells, e.g., CD8+ and/or CD4+ T cells, including Tregs), expanding (increasing) the effector cell population, decreasing the target cell population (e.g., a cell expressing a second biological molecule recognized by an anti-CD3 antibody of the present invention, such as a bispecific antibody), and/or killing a target cell (e.g., a target tumor cell).

The CD3 antibodies and/or antigen-binding fragments thereof disclosed herein can be used to treat disorders, including but not limited to a proliferative disorder, an oncological disorder, an immuno-oncological disorder, a neurological disorder, a cognitive disorder, a neurodegenerative disorder, an autoimmune disorder. In one embodiment, an effective amount of such an anti-CD3 antibody can be administered alone or in combination with at least one additional agent to an individual suffering from such a disorder. Such an "individual" can be a mammal and, in particular, a human.

Non-limiting illustrative additional therapeutic agents include a chemotherapeutic agent, an antibody-drug conjugate (ADC), and/or a biological modifier. Chemotherapeutic agents may be selected from cyclophosphamide, doxorubicin, vincristine, and prednisolone (CHOP). The ADC may be selected from an anti-CD79b antibody drug conjugate (such as an anti-CD79b-MC-vc-PAB-MMAE antibody or an anti-CD79b antibody drug conjugate described in any of U.S. Patent No. 8,088,378 and/or U.S. Patent No. 2014/0030280, or polatuzumab-vedotin), an anti-CD19 antibody drug conjugate, an anti-CD22 antibody drug conjugate, an anti-CD45 antibody drug conjugate, and an anti-CD32 antibody drug conjugate. The biologic modifier may be selected from a BCL-2 inhibitor (such as GDC-0199/ABT-199), lenalidomide (Revlimid®), a PI3K delta inhibitor (such as idelalisib (Zydelig®)), a PD-1 axis binding antagonist, an agonist, such as an agonist antibody, directed against an activating costimulatory molecule, such as CD40, CD226, CD28, OX40 (e.g. AgonOX), GITR, CD137 (also known as TNFRSF9, 4-1 BB, or ILA), CD27 (e.g. CDX-1127), HVEM, or CD127, an antagonist, such as an antagonist antibody directed against an inhibitory costimulatory molecule, such as CTLA-4 (also known as CD152), PD-1, TIM-3, BTLA, VISTA, LAG-3, B7-H3, B7-H4, IDO (e.g., 1-methyl-D-tryptophan (also known as 1-D-MT)), TIGIT, MICA/B, GITR (e.g., TRX518) or arginase, ipilimumab (also known as MDX-010, MDX-101, or Yervoy®), tremelimumab (also known as ticilimumab or CP-675206), urelumab (also known as BMS-663513), MGA271, an antagonist directed against TGF-beta such as metelimumab (also known as CAT-192), frezolimumab (also known as GC1008), LY2157299k, and adoptive transfer of a T cell (e.g., a cytotoxic T cell, or CTL) expressing chimeric antigen receptor (CAR), such as adoptive transfer of a T cell containing a dominant negative TGF-beta receptor, such as dominant negative TGF-beta receptor type II.

The CD3 antibodies and/or antigen-binding fragments thereof disclosed herein can be used to enhance immune function in an individual, such as a human having such a disorder, an individual having such a disorder. In one embodiment, a method of enhancing immune function comprises administering to an individual an effective amount of an anti-CD3 antibody to activate effector cells (e.g., T cells, such as CD8+ and/or CD4+ T cells), increase (increase) the population of effector cells, decrease the population of target cells, and/or kill a target cell (e.g., a target tumor cell).

In a further aspect, pharmaceutical formulations comprising the antibodies and/or antigen-binding fragments to CD3 described herein are also provided, for example, for use in any of the above therapeutic methods. "Pharmaceutical formulation" refers to a preparation in such a form as to ensure the effectiveness of the biological activity of the active ingredient contained therein, such as the antibodies to CD3 described herein, and which preferably does not contain additional components that are unacceptably toxic to the subject to which the formulation is to be administered.

In one embodiment, the pharmaceutical formulation comprises any of the CD3 antibodies disclosed herein and a pharmaceutically acceptable carrier. "Pharmaceutically acceptable carrier" refers to an ingredient in the pharmaceutical formulation that is not an active ingredient and is non-toxic to a subject. A pharmaceutically acceptable carrier includes, but is not limited to, a buffer, an excipient, a stabilizer, or a preservative. In another embodiment, the pharmaceutical formulation comprises any of the CD3 antibodies provided herein and at least one additional therapeutic agent.

The antibodies of the present invention can be used either alone or in combination with other agents in therapy, for example, an anti-CD3 antibody and/or antigen-binding fragment thereof can be administered together with at least one additional therapeutic agent. In certain embodiments, the additional therapeutic agent is a chemotherapeutic agent, a growth inhibitory agent, a cytotoxic agent, an agent used in radiation therapy, an antiangiogenic agent, an apoptotic agent, an antitubulin agent, or another agent such as an epidermal growth factor receptor (EGFR) antagonist (e.g., a tyrosine kinase inhibitor), a HER1/EGFR inhibitor (e.g., erlotinib (Tarceva™)), a platelet-derived growth factor inhibitor (e.g., Gleevec™ (imatinib mesylate)), a COX-2 inhibitor (e.g., celecoxib), an interferon, a cytokine, an antibody other than an anti-CD3 antibody of the invention, such as an antibody that binds to one or more of the following targets: ErbB2, ErbB3, ErbB4, PDGFR-beta, BIyS, APRIL, BCMA VEGF, or receptor(s) VEGF, TRAIL/Apo2, PD-1, PD-L1, PD-L2 or other biologically active or organic chemical agent.

In some embodiments, the present invention provides a method wherein the additional therapeutic agent is a glucocorticoid. In one embodiment, the glucocorticoid is dexamethasone.

Such combination therapies noted above encompass combined administration (in which two or more therapeutic agents are included in the same or in separate formulations) and separate administration, in which case the administration of an antibody of the present invention may occur before, simultaneously with, and/or after the administration of the additional therapeutic agent or agents. In one embodiment, the administration of the anti-CD3 antibody and the administration of the additional therapeutic agent occur within about one month, or within about one, two, or three weeks, or within about one, two, three, four, five, or six days of each other. The anti-CD3 antibodies of the present invention (e.g., the bispecific anti-CD3 antibodies of the present invention that bind to CD3 and a second biological molecule, such as a cell surface antigen, such as a tumor antigen, such as the TDB antibody of the present invention or a variant thereof) can also be used in combination with radiation therapy.

The antibody of the present invention (and/or any additional therapeutic agent) can be administered by any suitable routes, including parenteral, intrapulmonary, intranasal, and, if desired for local treatment, intralesional administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. In some embodiments, the antibody is administered by subcutaneous administration. In some embodiments, an anti-CD3 antibody administered by subcutaneous injection exhibits a less toxic response in a patient than the same anti-CD3 antibody administered by intravenous injection. Dosing can be by any suitable route of administration, for example, by injections such as intravenous or subcutaneous injections, depending in part on whether the administration is short-term or chronic. This document includes various dosing regimens including, but not limited to, single or multiple doses at different time points, bolus administration, and pulse infusion.

The antibodies of the present invention will be formulated, dosed and administered in accordance with sound medical practice. Factors considered in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the agent, the route of administration, the schedule of administration and other factors known to practitioners. The antibody need not, but optionally can, be formulated with one or more agents currently used to prevent or treat the disorder in question. The effective amount of such other agents will depend on the amount of antibody present in the formulation, the type of disorder or treatment and the other factors discussed above. They are typically administered at the same doses and routes of administration as described herein, or at doses from about 1% to 99% of the doses described herein, or at any dose and by any route empirically/clinically determined to be appropriate.

In the case of preventing or treating a disease, the appropriate dose of the antibody of the present invention (used alone or in combination with one or more other additional therapeutic agents) will depend on the type of disease to be treated, the type of antibody, the severity and course of the disease, whether the antibody is administered for prophylactic or therapeutic purposes, previous therapy, the patient's clinical history and response to the antibody, and the discretion of the treating physician. The antibody is suitably administered to the patient at one time or over a series of treatments.

As a general suggestion, a therapeutically effective amount of an anti-CD3 antibody administered to a human will be in the range of about 0.01 to about 100 mg/kg of the patient's body weight, whether administered in one or more administrations. In some embodiments, for example, the antibody used is administered at a rate of about 0.01 to about 45 mg/kg, about 0.01 to about 40 mg/kg, about 0.01 to about 35 mg/kg, about 0.01 to about 30 mg/kg, about 0.01 to about 25 mg/kg, about 0.01 to about 20 mg/kg, about 0.01 to about 15 mg/kg, about 0.01 to about 10 mg/kg, about 0.01 to about 5 mg/kg, or about 0.01 to about 1 mg/kg daily. In one embodiment, the anti-CD3 antibody described herein is administered to a human at a dose of about 100 mg, about 200 mg, about 300 mg, about 400 mg, about 500 mg, about 600 mg, about 700 mg, about 800 mg, about 900 mg, about 1000 mg, about 1100 mg, about 1200 mg, about 1300 mg, or about 1400 mg per day 1 for 21-day cycles. The dose may be administered as a single dose or as multiple doses (e.g., 2 or 3 doses), such as infusions. If repeated administrations are administered over several days or more, depending on the condition, treatment will typically be maintained until the desired suppression of disease symptoms occurs. One exemplary dose of the antibody will be in the range of about 0.05 mg/kg to about 10 mg/kg. Thus, the patient may be administered one or more doses of approximately 0.5 mg/kg, 2.0 mg/kg, 4.0 mg/kg, or 10 mg/kg (or any combination thereof). Such doses may be administered intermittently, for example, every week or every three weeks (e.g., so that the patient receives from about two to about twenty, or, for example, about six doses of the CD3 antibody). An initial higher loading dose may be administered, followed by one or more lower doses. The progress of this therapy is readily monitored using conventional techniques and assays.

In some embodiments, the methods of the present invention may further comprise additional therapy. The additional therapy may be radiation therapy, surgery, chemotherapy, gene therapy, DNA therapy, viral therapy, RNA therapy, immunotherapy, bone marrow transplantation, nanotherapy, monoclonal antibody therapy, or a combination of the foregoing. The additional therapy may be in the form of adjuvant or neoadjuvant therapy. In some embodiments, the additional therapy is the administration of a small molecule enzyme inhibitor or an antimetastatic agent. In some embodiments, the additional therapy is the administration of side effect limiting agents (e.g., agents designed to reduce the incidence and/or severity of side effects of treatment, such as antiemetics, etc.). In some embodiments, the additional therapy is radiation therapy. In some embodiments, the additional therapy is surgery. In some embodiments, the additional therapy is a combination of radiation therapy and surgery. In some embodiments, the additional therapy is gamma irradiation. In some embodiments, the additional therapy may be a separate administration of one or more therapeutic agents described above.

In another aspect of the present invention, there is provided an article of manufacture comprising materials useful for treating, preventing and/or diagnosing the disorders described above. The article comprises a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV bags, etc. The containers may be formed from various materials, such as glass or plastic. The container contains a composition, which is itself therein or combined with another composition effective for treating, preventing and/or diagnosing the condition, and it may have a sterile entry port (for example, the container may be an IV bag or a vial having a stopper that is pierced with a hypodermic needle). At least one active agent in the composition is an antibody of the present invention. The label or package insert indicates that the composition is used to treat the selected condition. Moreover, the article of manufacture may comprise (a) a first container with a composition contained therein, wherein the composition comprises an antibody of the present invention; and (b) a second container with a composition contained therein, wherein the composition comprises an additional cytotoxic agent or another therapeutic agent. In this embodiment of the present invention, the article of manufacture may further comprise a package insert indicating that the compositions can be used to treat a particular condition. Alternatively or additionally, the article of manufacture may further comprise a second (or third) container containing a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate buffered saline, Ringer's solution and dextrose solution. It may further comprise other materials required from a commercial and user point of view, including other buffers, diluents, filters, needles and syringes.

Accordingly, it is also contemplated to manufacture and/or obtain a pharmaceutical composition comprising the CD3 antibodies and/or antigen-binding fragments disclosed herein. The composition can be used alone or in combination with other active agents for the treatment of a cell proliferative disorder (e.g., cancer) or an autoimmune disorder (e.g., arthritis, rheumatoid arthritis, colitis, inflammatory bowel disease, autoimmune diabetes type I, etc.).

In some embodiments, pharmaceutical compositions comprising the anti-CD3 antibodies and/or antigen-binding fragments thereof described herein are prepared, for example, by mixing such an antibody of the desired degree of purity with one or more optional pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)) in the form of lyophilized formulations or aqueous solutions, optionally formulated for modified (e.g., sustained) release. Exemplary lyophilized antibody formulations are described in U.S. Patent No. 6,267,958. Aqueous antibody formulations include those described in U.S. Patent No. 6,171,586 and WO2006/044908, the formulations described in the latter document comprising a histidine acetate buffer.

Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed and include, without limitation, buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl, or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol, and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates including glucose, mannose or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal-containing complexes (e.g. Zn-protein complexes) and/or nonionic surfactants such as polyethylene glycol (PEG). Herein, exemplary pharmaceutically acceptable carriers further include agents for dispersing drugs in the extracellular space, such as soluble neutral active hyaluronidase glycoproteins (sHASEGPs), for example human soluble PH-20 hyaluronidase glycoproteins such as rHuPH20 (HYLENEX®, Baxter International, Inc.). Certain exemplary sHASEGPs and methods of use involving rHuPH20 are described in U.S. Patent Application Publication Nos. 2005/0260186 and 2006/0104968.

Such compositions may contain more than one active ingredient if necessary for the particular indication to be treated, preferably ingredients with complementary activities that do not adversely affect each other and are present in amounts effective for the intended purpose. For example, it may further be necessary to provide an additional therapeutic agent (e.g., a chemotherapeutic agent, a cytotoxic agent, a growth inhibitory agent, and/or an antihormonal agent).

The active ingredients may be incorporated into microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, such as hydroxymethylcellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules, respectively, into colloidal drug delivery systems (e.g. liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or into macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

Examples

Table 1. Binding data for illustrative high-affinity CD3 antibodies in bispecific format

Antibody K _D Biacore biotinylated Fc to human CD3 εδ (M) monovalent Cell binding
Jurkat Human CD3+ Fold Over Background (FOB) Cell binding
Jurkat CD3+ cynomolgus macaque Fold over background (FOB) ADI-42594 4.7E-10 14 16 ADI-46357 2,5E-10 13 20 ADI-46359 2,2E-10 15 28 ADI-46366 2,3E-10 16 32 ADI-46378 1,2E-10 17 30 ADI-45959 1,2E-10 18 30 ADI-45975 2.1E-10 10 17

Table 2. Development profile of CD3 antibodies in bispecific format

Table 3. Thermal stability measurements for illustrative CD3 antibodies

ADI ID Tm DSF scFv-Fc (°C) ADI-42594 66.0 ADI-42596 66.0 ADI-46356 62.0 ADI-46357 66.5 ADI-46358 62.0 ADI-46359 62.5 ADI-46360 52.5 ADI-46361 58.5 ADI-46362 59.0 ADI-46363 58.5 ADI-46364 58.0 ADI-46365 58.5 ADI-46366 63.5 ADI-46367 66.5 ADI-46369 62.5 ADI-46370 62.0 ADI-46371 59.0 ADI-46372 59.0 ADI-46374 64.0 ADI-46375 58.5 ADI-46376 57.0 ADI-46378 58.5 ADI-46379 58.5 ADI-46381 56.5 ADI-26140 n.d.

Materials and methods

In addition to the description provided above, the following materials and methods were used in the examples.

FACS affinity pressure selection methods. Briefly, yeast cells (at least ~2 x 10 ⁷ cells/labeling parameter) were incubated with sufficient biotinylated antigen to provide a stoichiometric excess over the average IgG presentation. Albumin labeling parameters were 100-1 nM at equilibrium; labeling was typically carried out for 20 min to several hours at room temperature in FACS wash buffer (phosphate buffered saline (PBS)/0.1% bovine serum albumin (BSA)). Then, after washing three times with wash buffer, yeast were stained with secondary reagents, anti-human light chain antibody-FITC (LC-FITC) diluted 1:100 and either streptavidin-633 (SA-633) diluted 1:500 or extravidin-phycoerythrin (EA-PE) diluted 1:50, for 15 min at 4°C. After washing twice with ice-cold wash buffer, cell pellets were resuspended in wash buffer in a typical volume of at least 1 ml per 1 x 10 ⁷ yeast and transferred to sorting tubes sealed with a snap-on lid and strainer. Sorting was performed using a FACS ARIA sorter (BD Biosciences) and sorting gates were defined to select binding molecules. After the final round of sorting, yeast was plated and individual colonies were picked for characterization.

FACS thermal pressure selection methods. A stock antibody was diversified by reduced fidelity PCR to generate an optimization library in yeast. The yeast library was first subjected to a positive round of selection with antigen to select for cells expressing binding molecules using CD3εN27. The enriched population was then passed through a battery of thermal pressure parameters ranging from ~50°C to 65°C inclusive for 10 min, with room temperature as a control. Optimal pressure parameters were gated on LC presentation (anti-human lambda chain antibody conjugated to PE) and antigen binding (SA-APC), reflecting residual folded IgG competent to bind selection reagents. Sorted cells were pelleted and plasmids were extracted using a commercial yeast plasmid purification kit (Zymo Research), whereby the yeast cell walls were disrupted using zymolase and the DNA was subsequently purified using a DNA purification minicolumn. Plasmid DNA was then transformed into E. coli for amplification, followed by the isolation of plasmid DNA minipreps using an E. coli plasmid purification kit (Qiagen). Plasmid DNA was then prepared for transformation into the appropriate yeast strain for subsequent rounds of selection or sequencing and IgG production.

Optimization using reduced-fidelity PCR. Random diversity was introduced by reduced-fidelity PCR-based mutagenesis of the heavy chain (VH) and/or light chain (VL) using standard molecular biology techniques. Briefly, mutagenic nucleotide analogues dPTP and 8-oxo-dGTP were introduced into the VH and VL amplification process at a concentration of 1 μM to increase the base incorporation error rate to approximately 0.01 bp. The mutated PCR product was recombined in situ by homologous recombination with a linearized vector containing HC or LC constant region sequences. This typically yielded a library with a diversity of 1 x 10 ^7–8 . Affinity and expression pressure were used in combination by incubating the antigen-antibody-yeast cell complex at decreasing concentrations of antigen (equilibrium pressure) or competing with the starting Fab (equilibrium and kinetic pressure) various numbers of times to select the highest affinity antibodies in FACS over successive rounds of selection.

Oligonucleotide primer-based CDR H3 mutagenesis. Identified or previously optimized antibodies can be improved by further optimization by introducing diversity into the CDR H3 sequence. For this purpose, the light chain variable region of the starting antibody is PCR amplified and then introduced into a yeast strain containing an empty light chain vector using yeast homologous recombination. This represents the yeast strain with the starting light chain. The heavy chain of the starting antibody is used as starting material for PCR in combination with germline-specific primers, resulting in a PCR product that contains the heavy chain from framework 1 to framework 3. This amplification is performed using the mutagenic nucleotide 8-oxo-dGTP to provide additional low levels of mutagenesis in the amplified region of the heavy chain. To create engineered diversity in the CDR H3 region of the initial antibody, a CDR H3 oligonucleotide library was generated/ordered (i.e., from IDT). The oligonucleotide pool was amplified using primers containing 5' tails that allow for germline-specific recombination with the amplified region of FW1-FW3 and empty vector. For FW4, a universal 3' primer was used. Alternatively, mutagenic PCR that introduces 8-oxo-dGTP into the PCR reaction can be performed using germline-specific 5' primers, a universal 3' primer, and VH DNA. Once the starting materials of the LC strain, FW1-FW3 HC, and the diversified CDR H3-FW4 were obtained, a triple transformation was performed by introducing the two HC components along with an empty HC vector into the LC strain. The cells were then grown under selection pressure to ensure the presence of the HC and LC components.

Antibody production in yeast and purification. Yeast clones were grown to saturation and then induced for 48 h at 30°C with shaking. After induction, yeast cells were pelleted and supernatants were collected for purification. IgG was purified using a protein A column and eluted with acetic acid, pH 2.0. Fab fragments were generated by papain digestion and purified by passage through KappaSelect or CaptureSelect IgG-CH1 (GE Healthcare LifeSciences).

Antibody production in HEK and purification. Expression of IgG in mammalian cells was accomplished by subcloning the antibodies into a new expression vector, followed by transient transfection and expression in HEK293ADI1, a HEK293-derived monoclonal cell line (DSMZ) selected for its clumping-free growth, growth rate, and transfectability. Briefly, expression vectors containing the antibody of interest were transfected by complexing with transfection reagent, followed by exposure of HEK cells for one hour, followed by dilution with growth medium to a final density of 4 million cells/ml. Cells were then cultured for 7 days with fresh medium fed every 48 hours. After 7 days, the supernatant was collected by centrifugation and purified using protein A. Additional CHT column purification was used when necessary to achieve >95% monomers.

ForteBio KD measurements (biolayer interferometry; BLI). ForteBio affinity measurements were performed essentially as described previously (Estep, P., et al., High throughput solution-based measurement of antibody-antigen affinity and epitope binning. MAbs, 2013. 5(2): p. 270-8). Briefly, ForteBio affinity measurements were performed by running IgG loading onto AHQ sensors. Sensors were blank equilibrated with assay buffer for 30 min and then monitored running for 60 s to establish baseline values. IgG-loaded sensors were exposed to 100 nM antigen for 5 min and then transferred to assay buffer for 5 min to measure the off-rate. Kinetics were analyzed using a 1:1 binding model.

KD measurements using BiaCore (surface plasmon resonance; SPR). Biosensor analysis was performed at 25°C in HBS-EP buffer system (10 mM HEPES, pH 7.3, 150 mM NaCl, 3 mM EDTA, 0.05% P20 surfactant) using a Biacore 8K optical biosensor attached to a CM5 sensor chip (GE Healthcare, Marlborough, MA, USA). The sample was maintained at 10°C. Capture antibody, goat anti-human IgG (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA; 109-005-098), was immobilized (11,700 +/- 400 RU) on both flow cells of the sensor chip using standard amine immobilization chemistry. This surface type provides a format for repeatable capture of fresh analyte antigen after each regeneration step. Flow cell 2 was used to analyze the captured antigen (35.7 +/- 0.8 RU), while flow cell 1 was used as a reference flow cell. Fab was prepared in flow buffer at concentrations ranging from 100 to 0.412 nM (3-fold dilutions). Each Fab sample concentration was run as a single replicate. Two blank injections (buffer) were also run and used to estimate and subtract system artifacts. Association and dissociation phases for all Fab concentrations were monitored for 180 s each at a flow rate of 30 μl/min. The surface was regenerated with 10 mM glycine, pH 1.5, for 30 s at a flow rate of 30 μl/min. Data were aligned, double-standardized, and fitted using Biacore 8K estimation software, version 1.0.

Epitope-specific ligand sorting/blocking with Octet Red384. Epitope-specific ligand sorting/blocking was performed using a standard cross-blocking assay in a sandwich format. Control anti-target IgGs were loaded onto AHQ sensors and unoccupied Fc binding sites on the sensor were blocked with an irrelevant human IgG1 antibody. The sensors were then challenged with 100 nM target antigen followed by a second anti-target antibody or ligand. Data were analyzed using ForteBio 7.0 data analysis software. Additional binding by the second antibody or ligand after antigen association indicates an unoccupied epitope (non-competing molecule), while no binding indicates epitope blocking (competing molecule or blocking ligand).

Size exclusion chromatography. A TSKgel SuperSW mAb HTP column (22855) was used for rapid SEC analysis of mAbs produced in yeast and mammalian cells at a flow rate of 0.4 mL/min with a cycle time of 6 min/run. The mobile phase was 200 mM sodium phosphate and 250 mM sodium chloride.

Dynamic scanning fluorimetry (DSF). 10 µl 20x Sypro Orange was added to 20 µl of 0.2-1 mg/ml mAb or Fab solution. An RT-PCR device (BioRad CFX96 RT PCR) was used to linearly increase the temperature of the sample plate from 40 to 95°C in 0.5°C steps with equilibration for 2 min at each temperature. The negative first derivative of the input data was used to extract the Tm.

Preparation of PSR. The polyspecific reactivity reagent (PSR) was prepared as described, for example, in WO 2014/179363 and Xu et al., mAbs, 2013. Briefly, 2.5 liters of CHO-S cells were used as starting material. Cells were pelleted at 2400 x g for 5 min in 500 ml centrifuge bottles filled to 400 ml. Cell pellets were pooled and then resuspended in 25 ml buffer B and pelleted at 2400 x g for 3 min. The buffer was decanted and the wash was repeated one more time. Cell pellets were resuspended in 3x pellet volume of buffer B containing 1x protease inhibitors (Roche, Complete, EDTA-free) using a Polytron homogenizer while the cells were kept on ice. The homogenate was then centrifuged at 2,400 x g for 5 min and the supernatant was allowed to stand and further pelleted once (2,400 x g/5 min) to ensure removal of intact cells, cell debris, and nuclei; the resulting supernatant represents the total protein preparation. The supernatant was then transferred to two 45 ml Nalgene Oak Ridge centrifuge tubes and pelleted at 40,000 x g for 40 min at 4°C. The supernatants containing the separated cytosolic proteins (SCPs) were then transferred to fresh Oak Ridge tubes and further centrifuged once at 40,000 x g. In parallel, the pellets containing the membrane fraction (EMF) were held and centrifuged at 40,000 for 20 min to remove residual supernatant. The EMF pellets were then rinsed with buffer B. Then, 8 ml of buffer B was added to the membrane-containing pellets to loosen the pellets and they were transferred to a Dounce homogenizer. After homogenization of the pellets, they were transferred to a 50 ml conical tube and constituted the final EMF preparation.

One billion mammalian cells (e.g., CHO, ^HEK293 , Sf9) at ~ ^106-107 cells/ml were transferred from tissue culture medium to 4x 250 ml conical tubes and pelleted at 550 xg for 3 min. All subsequent steps were performed at 4°C or on ice with ice-cold buffers. Cells were washed with 100 ml PBSF (1x PBS + 1 mg/ml BSA) and pooled in one conical tube. After removal of the supernatant, the cell pellet was further resuspended in 30 ml buffer B (50 mM HEPES, 0.15 M NaCl, 2 mM _CaCl2 , 5 mM KCl, 5 mM _MgCl2 , 10% glycerol, pH 7.2) and pelleted at 550 xg for 3 min. The supernatant from buffer B was decanted and the cells were resuspended in 3x pellet volume of buffer B with 2.5x protease inhibitor (Roche, Complete, EDTA-free). From this point onwards, buffer B contained protease inhibitors. The cells were homogenized four times with 30 sec pulses (Polytron homogenizer, PT1200E) and the membrane fraction was pelleted at 40,000 xg for 1 hr at 4°C. The pellet was rinsed with 1 ml buffer B; the supernatant was allowed to stand and is the . The pellet was transferred to a Dounce homogenizer with 3 ml buffer B and resuspended by slowly raising and lowering the pestle for 30-35 strokes. The enriched membrane fraction (EMF) was transferred to a new collection tube, rinsing the pestle to collect all possible protein. The protein concentration of purified EMF was determined using the Dc Protein Assay Kit (BioRad). To solubilize EMF, it was transferred to solubilization buffer (50 mM HEPES, 0.15 M NaCl, 2 mM _CaCl2 , 5 mM KCl, 5 mM MgCl2, ₁ % n-dodecyl-b-D-maltopyranoside (DDM), 1x protease inhibitor, pH 7.2) to give a final concentration of 1 mg/mL. The mixture was vortexed overnight at 4°C, followed by centrifugation in a 50 mL Oak Ridge tube (Fisher Scientific, 050529-ID) at 40,000 xg for 1 h. The supernatant, which represents soluble membrane proteins (SMPs), was collected and the protein yield was quantified as described above.

For biotinylation, NHS-LC-biotin stock solution was prepared according to the manufacturer's protocol (Pierce, Thermo Fisher). Briefly, 20 μl biotin reagent was added for each 1 mg EMF sample and incubated at 4°C for 3 h with gentle agitation. The volume was adjusted to 25 ml with buffer B and transferred to an Oak Ridge centrifuge tube. Biotinylated EMF (b-EMF) was pelleted at 40,000 x g for 1 h and washed twice with 3 ml buffer C (buffer B without glycerol) without disturbing the pellet. The residual solution was discarded. The pellet was resuspended with a Dounce homogenizer in 3 ml buffer C as described previously. The resuspended pellet now represented biotinylated EMF (b-EMF). Solubilization was carried out as described above to obtain b-SMP.

PSR binding assays. Assays were performed essentially as described, for example, in Xu et al. To characterize the PSR profile of yeast-presented monoclonal antibodies, two million yeast presenting IgG were transferred to a 96-well assay plate and pelleted at 3000 x g for 3 min to remove the supernatant. The pellet was resuspended in 50 μl of a freshly prepared 1:10 dilution of b-PSR stock solutions and incubated on ice for 20 min. Cells were washed twice with 200 μl of cold PBSF and the pellet was resuspended in 50 μl of secondary labeling mixture (extravidin-R-PE, FITC-conjugated anti-human LC antibody, and propidium iodide). The mixture was incubated on ice for 20 min followed by two washes with 200 μl ice-cold PBSF. Cells were resuspended in 100 μl ice-cold PBSF and the plate was run on a FACSCanto (BD Biosciences) using the HTS sampler. Flow cytometric data were analyzed for mean fluorescence intensity in the R-PE channel and normalized to the appropriate controls to assess non-specific binding. Numerous methods for presenting or displaying antibodies or antibody fragments on the yeast surface have been previously described, all of which follow this protocol (Blaise et al., 2004, Boder and Wittrup, 1997, Kuroda and Ueda, 2011, Orcutt and Wittrup, 2010, Rakestraw et al., 2011, Sazinsky et al., 2008, Tasumi et al., 2009, Vasquez et al., 2009).

Kinetic analysis using ForteBio. FortBio Octet HTX devices were used in 12-channel mode (8 sensors per channel, 96 sensors per experiment) with one of the AHC, SA, or AHQ sensors. The device was controlled using the manufacturer's software (versions 8.2 and 9.0). Sample names and concentrations were entered on the plate data page, and proteins associated with the sensor were identified in the "information" column of the sensor data page. Data for kinetic experiments were collected at 90 or 180 s of baseline, 180 s of association phase, and 180 s of dissociation phase. Data from the sorting experiments were collected in 5 stages: 90 s of baseline 1, 90 s of sensor binding to the secondary binding molecule, 90 s of baseline 2, 180 s of association, and 180 s of dissociation in the well containing the secondary mAb. All files were saved to a shared network drive using a naming convention that identifies the experiment format.

HIC. IgG1 samples were buffer exchanged with 1 M ammonium sulfate and 0.1 M sodium phosphate at pH 6.5 using a 0.5 mL Zeba 40 kDa spin column (Thermo Pierce, Cat #87766). A salt gradient was run on a Dionex ProPac HIC-10 column from 1.8 M ammonium sulfate, 0.1 M sodium phosphate at pH 6.5 to the same parameters without ammonium sulfate. The gradient was run for 17 min at a flow rate of 0.75 mL/min. An acetonitrile wash step was added at the end of the run to remove any remaining protein, and the column was re-equilibrated with 7 column volumes before the next injection cycle. Peak retention times were monitored with A280 absorption and ammonium sulfate concentrations during elution were calculated based on the gradient and flow rate.

LCMS. mAb samples were recovered by DTT followed by downstream LCMS analysis to a Bruker maXis4G mass spectrometer coupled to an Agilent 1100 HPLC (Agilent). A POROS R2 10 μm (2.1 x 30 mm) reverse phase chromatography column was used to remove salt from the samples. Fast LC flow at 2 mL/min ensures separation between sample and salt, and sample elution and column regeneration were completed within a 2.1 min run. A T-junction was used to deliver only 0.15 mL/min of sample flow to the mass spectrometer for sample analysis. The Bruker Maxis 4G mass spectrometer was run in positive ion mode with detection in the 750-2500 m/z range. The remaining source parameters were set as follows: the capillary was set to 5500 V, the atomizer to 4.0 bar, the drying gas at a rate of 4.0 l/min, and the drying temperature was set to 200 °C.

MS spectra were analyzed using Bruker data analysis software version 4.1 and deconvolution was performed using maximum entropy deconvolution with a mass in the range of 20–30 kDa.

An unofficial sequence listing is presented in Table 4. The unofficial sequence listing shows the amino acid sequence of the variable region of the heavy chain ("HC") with the CDRs of each variable region of the heavy chain underlined, and the variable region of the light chain ("LC") with the CDRs of each variable region of the light chain underlined.

Table 4. Unofficial list of sequences

Table 5. Antibody VH sequence information

Table 6. Antibody VL sequence information

Table 7. Human and cynomolgus macaque CD3ε sequences

Name Subsequence SEQ ID NO: Hu CD3ε Fc QDGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILWQHNDKNIGGDEDDKNIGSDEDHLSLKEFSELEQSGYYVCYPRGSKPEDANFYLYLRARVCENCMEMDGGSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 131 Cy CD3ε Fc QDGNEEMGSITQTPYQVSISGTTVILTCSQHLGSEAQWQHNGKNKEDSGDRLFLPEFSEMEQSGYYVCYPRGSNPEDASHHLYLKARVCENCMEMDGGSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV HNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 132

Claims (123)

1. An anti-CD3 antibody or antigen-binding fragment of an antibody, which comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein said VH and VL comprise amino acid sequences according to: (i) SEQ ID NOS: 17 and 34 respectively; (ii) SEQ ID NOS: 22 and 23 respectively; (iii) SEQ ID NOS: 29 and 23, respectively; (iv) SEQ ID NOS: 49 and 23 respectively; (v) SEQ ID NOS: 58 and 23 respectively; (vi) SEQ ID NOS: 59 and 23 respectively; (vii) SEQ ID NOS: 70 and 23, respectively; (viii) SEQ ID NOS: 24 and 25 respectively; (ix) SEQ ID NOS: 17 and 47 respectively; (x) SEQ ID NOS: 17 and 25 respectively; (xi) SEQ ID NOS: 55 and 25 respectively; (xii) SEQ ID NOS: 20 and 21 respectively; (xiii) SEQ ID NOS: 54 and 36 respectively; (xiv) SEQ ID NOS: 31 and 36 respectively; (xv) SEQ ID NOS: 67 and 68 respectively; (xvi) SEQ ID NOS: 17 and 36 respectively; (xvii) SEQ ID NOS: 59 and 36 respectively; (xviii) SEQ ID NOS: 30 and 18 respectively; (xix) SEQ ID NOS: 31 and 18 respectively; or (xx) SEQ ID NOS: 50 and 18 respectively. 2. An anti-CD3 antibody or antigen-binding fragment according to claim 1, wherein VH and VL comprise amino acid sequences according to SEQ ID NOS: 17 and 34, respectively. 3. An anti-CD3 antibody or antigen-binding fragment according to claim 1 or 2, which is represented by scFv, Fv, Fab, Fab', Fab'-SH, F(ab') ₂ , diabody, scFv2-Fc2 or scFv-IgG. 4. An anti-CD3 antibody or antigen-binding fragment according to any one of claims 1-3, which comprises: (A) a heavy chain containing a VH and a heavy chain constant region (CH); and (B) light chain containing VL and light chain constant region (CL). 5. An anti-CD3 antibody or antigen-binding fragment according to claim 4, which is an immunoglobulin molecule comprising said two heavy chains and said two light chains linked together by disulfide bonds, or a multimer of said immunoglobulin molecule. 6. An anti-CD3 antibody or antigen-binding fragment according to claim 4 or 5, which is or is an IgG, IgA, IgD, IgE or IgM antibody. 7. An anti-CD3 antibody or antigen-binding fragment according to claim 4 or 5, which is or is an IgG1, IgG2, IgG3, IgG4, IgA1 or IgA2 antibody. 8. An anti-CD3 antibody or antigen-binding fragment according to any one of claims 1-7, which comprises an IgG constant domain. 9. An isolated or recombinant nucleic acid encoding an anti-CD3 antibody or antigen-binding fragment according to any one of claims 1-8. 10. An expression vector containing the nucleic acid of claim 9. 11. A host cell for expressing an anti-CD3 antibody or antigen-binding fragment according to any one of claims 1-8, which is transfected, transformed or transduced with a nucleic acid according to claim 9. 12. A host cell for expressing an anti-CD3 antibody or antigen-binding fragment according to any one of claims 1-8, which is transfected, transformed or transduced with an expression vector according to claim 10. 13. The host cell according to claim 11 or 12, which is a eukaryotic cell. 14. The host cell according to claim 11 or 12, which is a mammalian or yeast cell. 15. The host cell according to claim 11 or 12, which is a Chinese hamster ovary (CHO) cell, a human embryonic kidney (HEK) cell, or a lymphoid cell. 16. The host cell according to claim 11 or 12, which is a prokaryotic cell. 17. The host cell according to claim 11 or 12, which is a bacterial cell. 18. A pharmaceutical composition for enhancing immune function, comprising an anti-CD3 antibody or an antigen-binding fragment of an antibody according to any one of claims 1-8 and a pharmaceutically acceptable carrier and/or excipient. 19. An anti-CD3 chimeric antigen receptor (CAR) that comprises an anti-CD3 antigen-binding fragment of an antibody comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein said VH and VL comprise amino acid sequences according to: (i) SEQ ID NOS: 17 and 34 respectively; (ii) SEQ ID NOS: 22 and 23 respectively; (iii) SEQ ID NOS: 29 and 23, respectively; (iv) SEQ ID NOS: 49 and 23 respectively; (v) SEQ ID NOS: 58 and 23 respectively; (vi) SEQ ID NOS: 59 and 23 respectively; (vii) SEQ ID NOS: 70 and 23, respectively; (viii) SEQ ID NOS: 24 and 25 respectively; (ix) SEQ ID NOS: 17 and 47 respectively; (x) SEQ ID NOS: 17 and 25 respectively; (xi) SEQ ID NOS: 55 and 25 respectively; (xii) SEQ ID NOS: 20 and 21 respectively; (xiii) SEQ ID NOS: 54 and 36 respectively; (xiv) SEQ ID NOS: 31 and 36 respectively; (xv) SEQ ID NOS: 67 and 68 respectively; (xvi) SEQ ID NOS: 17 and 36 respectively; (xvii) SEQ ID NOS: 59 and 36 respectively; (xviii) SEQ ID NOS: 30 and 18 respectively; (xix) SEQ ID NOS: 31 and 18 respectively; or (xx) SEQ ID NOS: 50 and 18 respectively. 20. The anti-CD3 CAR of claim 19, comprising at least one transmembrane domain and at least one intracellular domain from a T cell receptor and at least one costimulatory domain. 21. The anti-CD3 CAR of claim 20, wherein at least one transmembrane domain is a CD3ζ subunit. 22. An isolated or recombinant nucleic acid encoding an anti-CD3 CAR according to any one of claims 19-21. 23. An expression vector containing the nucleic acid of claim 22. 24. A host cell for expressing an anti-CD3 CAR according to any one of claims 19-21, which is transfected, transformed or transduced with the nucleic acid according to claim 22. 25. A host cell for expressing an anti-CD3 CAR according to any one of claims 19-21, which is transfected, transformed or transduced with the expression vector according to claim 23. 26. The host cell according to claim 24 or 25, which is a mammalian or yeast cell. 27. The host cell according to claim 24 or 25, which is a lymphoid cell. 28. A pharmaceutical composition for enhancing immune function, comprising an anti-CD3 CAR according to any one of claims 19-21 and a pharmaceutically acceptable carrier and/or excipient. 29. A bispecific antibody or antibody fragment that specifically binds to CD3 and a second antigen that is a cell surface antigen, wherein the bispecific antibody or antibody fragment comprises: (A) the first antigen-binding domain, which specifically binds to CD3, and (B) a second antigen-binding domain that specifically binds a second antigen, and wherein the first antigen-binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein said VH and VL comprise amino acid sequences according to: (i) SEQ ID NOS: 17 and 34 respectively; (ii) SEQ ID NOS: 22 and 23 respectively; (iii) SEQ ID NOS: 29 and 23, respectively; (iv) SEQ ID NOS: 49 and 23 respectively; (v) SEQ ID NOS: 58 and 23 respectively; (vi) SEQ ID NOS: 59 and 23 respectively; (vii) SEQ ID NOS: 70 and 23, respectively; (viii) SEQ ID NOS: 24 and 25 respectively; (ix) SEQ ID NOS: 17 and 47 respectively; (x) SEQ ID NOS: 17 and 25 respectively; (xi) SEQ ID NOS: 55 and 25 respectively; (xii) SEQ ID NOS: 20 and 21 respectively; (xiii) SEQ ID NOS: 54 and 36 respectively; (xiv) SEQ ID NOS: 31 and 36 respectively; (xv) SEQ ID NOS: 67 and 68 respectively; (xvi) SEQ ID NOS: 17 and 36 respectively; (xvii) SEQ ID NOS: 59 and 36 respectively; (xviii) SEQ ID NOS: 30 and 18 respectively; (xix) SEQ ID NOS: 31 and 18 respectively; or (xx) SEQ ID NOS: 50 and 18 respectively. 30. The bispecific antibody or antigen-binding fragment of claim 29, wherein VH and VL of the first antigen-binding domain comprise amino acid sequences according to SEQ ID NOS: 17 and 34, respectively. 31. A bispecific antibody or antigen-binding fragment according to claim 29 or 30, which comprises: (I) scFv, Fv, Fab, Fab', Fab'-SH, F(ab') ₂ , diabody, scFv2-Fc2 or scFv-IgG; (II) (A) a heavy chain comprising VH and a heavy chain constant region (CH), and (B) a light chain comprising VL and a light chain constant region (CL); (III) an immunoglobulin molecule comprising said two heavy chains and said two light chains linked together by disulfide bonds, or a multimer of said immunoglobulin molecule; (IV) IgG, IgA, IgD, IgE or IgM antibody; and/or (V) IgG1, IgG2, IgG3, IgG4, IgA1 or IgA2 antibody. 32. A bispecific antibody or antigen-binding fragment according to any one of claims 29-31, wherein the second antigen-binding domain specifically binds to an oncological disease target antigen; an immuno-oncological disease target antigen; a neurodegenerative disease target antigen; an autoimmune disorder target antigen; an infectious disease target antigen; a metabolic disease target antigen; a cognitive impairment target antigen; a blood-brain barrier target antigen or a blood disease target antigen. 33. A bispecific antibody or antigen-binding fragment according to any one of claims 29-32, wherein the second antigen-binding domain specifically binds to an antigen selected from the group consisting of: 17-IA, 4-1BB, 4Dc, 6-keto-PGFla, 8-iso-PGF2a, 8-oxo-dG, adenosine receptor A1, A33, ACE, ACE-2, activin, activin A, activin AB, activin B, activin C, activin RIA, activin RIA ALK-2, activin RIB ALK-4, activin RIIA, activin RUB, ADAM, ADAM10, ADAM12, ADAM 15, ADAM 17/T ACE, ADAM8, ADAM9, ADAMTS, ADAMTS4, ADAMTS5, addressins, aFGF, ALCAM, ALK, ALK-1, ALK-7, alpha-1-antitrypsin, alpha-V/beta-1 antagonist, ANG, Ang, APAF-1, APE, APJ, APP, APRIL, AR, ARC, ART, artemin, anti-idiotype, ASPARTIC, atrial natriuretic factor, integrin av/b3, Axl, b2M, B7-1, B7-2, B7-H, B-lymphocyte stimulator (BlyS), BACE, BACE-1, Bad, BAFF, BAFF-R, Bag-1, BAK, Bax, BCA-1, BCAM, Bel, BCMA, BDNF, b-ECGF, bFGF, BID, Bik, BFM, BLC, BL-CAM, BLK, BMP, BMP-2 BMP-2a, osteogenin BMP-3, BMP-4 BMP-2b, BMP-5, BMP-6 Vgr-1, BMP-7 (OP-1), BMP-8 (BMP-8a, OP-2), BMPR, BMPR-IA (ALK-3), BMPR-IB (ALK-6), BRK-2, RPK-1, BMPR-II (BRK-3), BMP, b-NGF, BOK, bombesin, bone neurotrophic factor, BPDE, BPDE-DNA, BTC, complement factor 3 (C3), C3a, C4, C5, C5a, CIO, CA125, CAD-8, calcitonin, cAMP, carcinoembryonic antigen (CEA), carcinoma-associated antigen, cathepsin A, cathepsin B, cathepsin C/DPPI, cathepsin D, cathepsin E, cathepsin H, cathepsin L, cathepsin O, cathepsin S, cathepsin V, cathepsin X/Z/P, CBL, CCI, CCK2, CCL, CCL1, CCL11, CCL12, CCL13, CCL 14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL2, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CCL3, CCL4, CCL5, CCL6, CCL7, CCL8, CCL9/10, CCR, CCR1, CCR10, CCR10, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CD1, CD2, CD4, CD5, CD6, CD7, CD8, CD10, CDlla, CDllb, CDllc, CD13, CD14, CD15, CD16, CD18, CD19, CD20, CD21, CD22, CD23, CD25, CD27L, CD28, CD29, CD30, CD30L, CD32, CD33 (p67 proteins), CD34, CD38, CD40, CD40L, CD44, CD45, CD46, CD49a, CD52, CD54, CD55, CD56, CD61, CD64, CD66e, CD74, CD80 (B7-1), CD89, CD95, CD123, CD137, CD138, CD140a, CD146, CD147, CD148, CD152, CD164, CEACAM5, CFTR, cGMP, CINC, Clostridium botulinum toxin, Clostridium perfringens toxin, CKb8-1, CLC, CMV, CMV UL, CNTF, CNTN-1, COX, C-Ret, CRG-2, CT-1, CTACK, CTGF, CTLA-4, CX3CL1, CX3CR1, CXCL, CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCR, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, cytokeratin tumor-associated antigen, DAN, DCC, DcR3, DC-SIGN, hemolysis stimulator, des(l-3)-IGF-I (IGF-1 brain), Dhh, digoxin, DNAM-1, Dnase, Dpp, DPPIV/CD26, Dtk, ECAD, EDA, EDA-A1, EDA-A2, EDAR, EGF, EGFR (ErbB-1), EMA, EMMPRIN, EN A, endothelin receptor, enkephalinase, eNOS, Eot, eotaxin l, EpCAM, ephrin B2/EphB4, EPO, ERCC, E-selectin, ET-1, factor Ila, factor VII, factor VIIIc, factor IX, fibroblast activation protein (FAP), Fas, FcRl, FEN-1, ferritin, FGF, FGF-19, FGF-2, FGF3, FGF-8, FGFR, FGFR-3, fibrin, FL, FLIP, Flt-3, Flt-4, follicle-stimulating hormone, fractalkine, FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, FZD10, G250, Gas 6, GCP-2, GCSF, GD2, GD3, GDF, GDF-1, GDF-3 (Vgr-2), GDF-5 (BMP-14, CDMP-1), GDF-6 (BMP-13, CDMP-2), GDF-7 (BMP-12, CDMP-3), GDF-8 (myostatin), GDF-9, GDF-15 (MIC-1), GDNF, GFAP, GFRa-1, GFR-alpha 1, GFR-alpha 2, GFR-alpha 3, GITR, glucagon, Glut 4, glycoprotein Ilb/IIIa (GP Ilb/IIIa), GM-CSF, gpl30, gp72, GRO, somatotropin-releasing factor, hapten (NP-cap or NIP-cap), HB-EGF, HCC, HCMV envelope glycoprotein gB, HCMV envelope glycoprotein gH, HCMV UL, hematopoietic growth factor (HGF), Hep B gpl20, heparanase, Her2, Her2/neu (ErbB-2), Her3 (ErbB-3), Her4 (ErbB-4), herpes simplex virus (HSV) glycoprotein gB, HSV glycoprotein gD, HGFA, high molecular weight melanoma-associated antigen (HMW-MAA), HIV gpl20, V3-loop of gp 120 HIV IIIB, HLA, HLA-DR, HM1.24, HMFG PEM, HRG, Hrk, human cardiac myosin, human cytomegalovirus (HCMV), human growth hormone (HGH), HVEM, 1-309, IAP, ICAM, ICAM-1, ICAM-3, ICE, ICOS, IFNg, Ig, IgA receptor, IgE, IGF, IGF-binding proteins, IGF-1R, IGFBP, IGF-I, IGF-II, IL, IL-1, IL-1R, IL-2, IL-2R, IL-4, IL-4R, IL-5, IL-5R, IL-6, IL-6R, IL-8, IL-9, IL-10, IL-12, IL-13, IL-15, IL-18, IL-18R, IL-23, alpha interferon (INF), INF-beta, INF-gamma, inhibin, iNOS, insulin A chain, insulin B chain, insulin-like growth factor 1, alpha-2-integrin, alpha-3-integrin, alpha-4-integrin, alpha-4/beta-l-integrin, alpha-4/beta-7-integrin, alpha-5(alpha-V)-integrin, alpha-5/beta-l-integrin, alpha-5/beta-3-integrin, alpha-6-integrin, beta-l-integrin, beta-2-integrin, interferon-gamma, IP-10, 1-TAC, JE, kallikrein 2, kallikrein 5, kallikrein 6, kallikrein 11, kallikrein 12, kallikrein 14, kallikrein 15, kallikrein LI, kallikrein L2, kallikrein L3, kallikrein L4, KC, KDR, keratinocyte growth factor (KGF), laminin 5, LAMP, LAP, LAP (TGF-1), latent TGF-1, bpl latent TGF-1, LBP, LDGF, LECT2, Lefty, Lewis-Y antigen, Lewis-Y-related antigen, LFA-1, LFA-3, Lfo, LIF, LIGHT, lipoproteins, LIX, LKN, Lptn, L-selectin, LT-a, LT-b, LTB4, LTBP-1, pulmonary surfactant, luteinizing hormone, beta-lymphotoxin receptor, Mac-1, MAdCAM, MAG, MAP2, MARC, MCAM, MCAM, MCK-2, MCP, M-CSF, MDC, Mer, metalloprotease, MGDF receptor, MGMT, MHC (HLA-DR), MIF, MIG, MIP, MIP-1alpha, MK, MMAC1, MMP, MMP-1, MMP-10, MMP-11, MMP-12, MMP-13, MMP-14, MMP-15, MMP-2, MMP-24, MMP-3, MMP-7, MMP-8, MMP-9, MPIF, Mpo, MSK, MSP, mucin (Mucl), MUC18, anti-Müllerian hormone, Mug, MuSK, NAIP, NAP, NCAD, N-cadherin, NCA 90, NCAM, NCAM, neprilysin, neurotrophin-3, -4, or -6, neuroturin, neuronal growth factor (NGF), NGFR, NGF-beta, nNOS, NO, NOS, Npn, NRG-3, NT, NTN, OB, OGG1, OPG, OPN, OSM, OX40L, OX40R, pl50, p95, PADPr, parathyroid hormone, PARC, PARP, PBR, PBSF, PCAD, P-cadherin, PCNA, PDGF, PDGF, PDK-1, PECAM, PEM, PF4, PGE, PGF, PGI2, PGJ2, PIN, PLA2, placental alkaline phosphatase (PLAP), PIGF, PLP, PP14, proinsulin, prorelaxin, C-protein, PS, PSA, PSCA, prostate-specific membrane antigen (PSMA), PTEN, PTHrp, Ptk, PTN, R51, RANK, RANKL, RANTES, relaxin A-chain, relaxin B-chain, renin, respiratory syncytial virus (RSV) F, RSV Fgp, Ret, rheumatoid factors, RLIP76, RPA2, RSK, S100, SCF/KL, SDF-1, SERINE, serum albumin, sFRP-3, Shh, SIGIRR, SK-1, SLAM, SLPI, SMAC, SMDF, SMOH, SOD, SPARC, Stat, STEAP, STEAP-II, TACE, TACI, TAG-72 (tumor-associated glycoprotein-72), TARC, TCA-3, T-cell receptors (e.g., T-cell receptor alpha/beta), TdT, TECK, TEM1, TEM5, TEM7, TEM8, TERT, testicular PLAP-like alkaline phosphatase, TfR, TGF, TGF-alpha, TGF-beta, multispecific TGF-beta, RI TGF-beta (ALK-5), RII TGF-beta, Rllb TGF-beta, RIII TGF-beta, TGF-beta 1, TGF-beta 2, TGF-beta 3, TGF-beta 4, TGF-beta 5, thrombin, Ck-1 thymus, thyroid stimulating hormone, Tie, TIMP, TIQ, tissue factor, TMEFF2, Tmpo, TMPRSS2, TNF, TNF-alpha, TNF-alpha/beta, TNF-beta 2, TNFc, TNF-RI, TNF-RII, TNFRSF10A (TRAIL Rl Apo-2, DR4), TNFRSFIOB (TRAIL R2 DR5, KILLER, TRICK-2A, TRICK-B), TNFRSF10C (TRAIL R3 DcRl, LIT, TRID), TNFRSF10D (TRAIL R4 DcR2, TRUNDD), TNFRSF11A (RANK ODF R, TRANCE R), TNFRSFllB (OPG OCIF, TR1), TNFRSF12 (TWEAK R FN14), TNFRSF13B (TACI), TNFRSF13C (BAFF R), TNFRSF14 (HVEM ATAR, HveA, LIGHT R, TR2), TNFRSF16 (NGFR p75NTR), TNFRSF17 (BCMA), TNFRSF 18 (GITR AITR), TNFRSF19 (TROY TAJ, TRADE), TNFRSF19L (RELT), TNFRSFIA (TNF RI CD120a, p55-60), TNFRSFIB (TNF RII CD120b, p75-80), TNFRSF26 (TNFRH3), TNFRSF3 (LTbR TNF RIII, TNFC R), TNFRSF4 (OX40 ACT35, TXGP1 R), TNFRSF 5 (CD40 p50), TNFRSF6 (Fas Apo-1, APT1, CD95), TNFRSF6B (DcR3 M68, TR6), TNFRSF7 (CD27), TNFRSF8 (CD30), TNFRSF9 (4-1BB CD137, ILA), TNFRSF21 (DR6), TNFRSF22 (DcTRAIL R2 TNFRH2), TNFRST23 (DcTRAIL Rl TNFRH1), TNFRSF25 (DR3 Apo-3, LARD, TR-3, TRAMP, WSL-1), TNFSF10 (TRAIL Apo-2 ligand, TL2), TNFSF11 (ligand ODF TRANCE/RANK, ligand OPG), TNFSF12 (TWEAK Apo-3 ligand, DR3 ligand), TNFSF13 (APRIL TALL2), TNFSF13B (BAFF BLYS, TALL1, THANK, TNFSF20), TNFSF14 (LIGHT HVEM ligand, LTg), TNFSF15 (TLIA/VEGI), TNFSF18 (GITR ligand, AITR ligand, TL6), TNFSFIA (connectin TNF-a, DIF, TNFSF2), TNFSF1B (TNF-b LTa, TNFSF1), TNFSF3 (LTb TNFC, p33), TNFSF4 (gp34 ligand OX40, TXGP1), TNFSF5 (CD40 ligand CD154, gp39, HIGM1, IMD3, TRAP), TNFSF6 (Fas ligand, Apo-1 ligand, ligand APT1), TNFSF7 (CD27 CD70 ligand), TNFSF8 (CD30 CD153 ligand), TNFSF9 (4-1BB CD137 ligand), TP-1, t-PA, Tpo, TRAIL, TRAIL R, TRAIL-R1, TRAIL-R2, TRANCE, transferrin receptor, TRF, Trk, TROP-2, TSG, TSLP, tumor-associated antigen CA 125, tumor-associated antigen characterized by the expression of Lewis Y-related carbohydrate antigen, TWEAK, TXB2, Ung, uPAR, uPAR-1, urokinase, VCAM, VCAM-1, VECAD, VE-cadherin, VE-cadherin-2, VEFGR-1 (flt-1), VEGF, VEGFR, VEGFR-3 (flt-4), VEGI, VFM, viral antigens, VLA, VLA-1, VLA-4, VNR integrin, von Willebrand factor, WIF-1, WNT1, WNT2, WNT2B/13, WNT3, WNT3A, WNT4, WNT5A, WNT5B, WNT6, WNT7A, WNT7B, WNT8A, WNT8B, WNT9A, WNT9A, WNT9B, WNT10A, WNT10B, WNT11, WNT16, XCL1, XCL2, XCR1, XCR1, XEDAR, XIAP, XPD, CTLA4 (cytotoxic T lymphocyte antigen 4), PD1 (programmed cell death protein 1), PD-L1 (programmed cell death ligand 1), LAG-3 (lymphocyte activation gene 3), TIM-3 (T-cell receptor domain-containing protein 3), immunoglobulin and mucin), hormone receptors and growth factors. 34. A bispecific antibody or antigen-binding fragment according to any one of claims 29-33, wherein the second antigen-binding domain specifically binds to an antigen selected from the group consisting of: BCMA, CTLA4 (cytotoxic T lymphocyte antigen 4), PD1 (programmed cell death protein 1), PD-L1 (programmed cell death protein ligand 1), LAG-3 (lymphocyte activating gene 3), TIM-3, CD20, CD2, CD19, Her2, EGFR, EpCAM, FcyRIIIa (CD16), FcyRIIa (CD32a), FcyRIIb (CD32b), FcyRI (CD64), Toll-like receptors (TLR), TLR4, TLR9, cytokines, IL-2, IL-5, IL-13, IL-6, IL-17, IL-12, IL-23, TNFa, TGFb, receptors cytokines, IL-2R, chemokines, chemokine receptors, growth factors, VEGF and HGF. 35. A bispecific antibody or antigen-binding fragment according to any one of claims 29-34, which comprises an IgG constant domain. 36. The bispecific antibody or antigen-binding fragment of any one of claims 29-35, which has a multispecific format selected from the group consisting of: Fab-Fc-scFv, "bottle opener", Mab-scFv, Mab-Fv, Dual scFv, core Fv, core scFv, core scFv with one arm, Fab-Fab, Fab-Fv, mAb-Fv, mAb-Fab, DART, BiTE, IgG with a common light chain, TandAb, Cross-Mab, SEED, BEAT, TrioMab and DuetMab. 37. An isolated or recombinant nucleic acid encoding a bispecific antibody or antibody fragment according to any one of claims 29-36, wherein the nucleic acid comprises: (A) a nucleic acid sequence encoding the amino acid sequence of the first antigen-binding domain, and (B) a nucleic acid sequence encoding the amino acid sequence of the second antigen-binding domain. 38. An expression vector containing an isolated or recombinant nucleic acid according to claim 37. 39. A host cell for expressing a bispecific antibody or antibody fragment according to any one of claims 29-36, which is transfected, transformed or transduced with a nucleic acid according to claim 37. 40. A host cell for expressing a bispecific antibody or antibody fragment according to any one of claims 29-36, which is transfected, transformed or transduced with an expression vector according to claim 38. 41. The host cell according to claim 39 or 40, which is a eukaryotic cell. 42. The host cell according to claim 39 or 40, which is a mammalian or yeast cell. 43. The host cell according to claim 39 or 40, which is a Chinese hamster ovary (CHO) cell, a human embryonic kidney (HEK) cell, or a lymphoid cell. 44. The host cell according to claim 39 or 40, which is a prokaryotic cell. 45. The host cell according to claim 39 or 40, which is a bacterial cell. 46. A pharmaceutical composition for enhancing immune function, comprising a bispecific antibody or antibody fragment according to any one of paragraphs 29-36 and a pharmaceutically acceptable carrier and/or excipient. 47. A method of treating a disorder in a mammal in need of such treatment, wherein the disorder comprises a proliferative disorder, an oncological disorder, an immuno-oncological disorder, a neurological disorder, a neurodegenerative disorder or an autoimmune disorder, wherein the method comprises administering an effective amount of one or more anti-CD3 antibodies or antibody fragments according to any one of claims 1-8, one or more anti-CD3 CARs according to any one of claims 19-21, one or more bispecific antibodies or antibody fragments according to any one of claims 29-36, a host cell according to any one of claims 11-17, 24-27 or 39-45 or a pharmaceutical composition according to claim 46. 48. The method according to claim 47, wherein the host cell is an immune cell. 49. The method according to claim 48, wherein the immune cell is a T or NK cell. 50. The method according to any one of claims 47-49, wherein the method also comprises administering to the mammal an additional therapeutic agent. 51. The method according to any one of paragraphs 47-50, wherein the mammal is a human.