High affinity anti-CD3 antibodies and methods for producing and using same

Abstract

Antibodies and antigen-binding fragments thereof having high affinity for CD3 and desirable developability profiles are provided, as are methods for making and using the same.

Description

[Technical Field]

[0001] Related Applications This application claims priority to U.S. Provisional Patent Application No. 62 / 858,949, filed June 7, 2019, the contents of which are incorporated by reference in their entirety.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy was created on June 3, 2020, is named 1160430o001813.txt, and is 92,322 bytes in size.

[0003] In particular, the present invention relates to anti-Cluster of Differentiation 3 (CD3) antibodies, including multispecific antibodies, and functional fragments thereof, as well as methods and reagents for their identification, isolation, preparation and use. [Background technology]

[0004] Cell proliferative disorders, such as cancer, are characterized by the uncontrolled proliferation of cell subpopulations. Cell proliferative disorders are the leading cause of death in developed countries and the second leading cause of death in developing countries. The total number of newly diagnosed cases of cancer each year is predicted to reach 23.6 million by 2030. The National Cancer Institute predicts that approximately 2 million new cases of cancer will be diagnosed in the United States in 2018, and that more than 600,000 Americans will die from cancer. Therefore, cancer treatment represents a significant and ever-increasing societal burden.

[0005] The idea of ​​harnessing the cytotoxic ability of T cells to kill tumor cells through the use of CD3-targeting bispecific antibodies dates back to 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 for T cell recruitment and activation, and a second binding site for a target disease-associated antigen, such as an antigen produced by tumor cells. By binding to its second target protein expressed on tumors, CD3 bispecific antibodies trigger the CD3 surface receptor on T cells, allowing competent T cells to bind to target-expressing cells via cross-linking by the CD3 bispecific antibody, regardless of the peptide / MHC specificity of their T cell receptors. (See, e.g., Bassan, 2012, Blood 120:5094-95). Using CD3 bispecific antibodies to cross-link T cells and tumor cells can induce dramatic regression of advanced malignancies, even resulting in complete remission in some cases. More than 25 different CD3 bispecific antibodies are currently 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).

[0006] While bispecific antibodies have shown significant advantages over monospecific antibodies for cancer treatment and detection, their widespread commercial application has been hindered by the lack of efficient / low-cost production methods, the lack of stability of bispecific polypeptides, and their short half-life in humans. Many methods for generating bispecific monoclonal antibodies have been developed over the past few decades. However, many bispecific antibody candidates with excellent selectivity and high potency for their target of interest often have challenges in downstream development and clinical efficacy, including: multispecific binding (or "multispecificity"), off-target binding, non-specific binding, poor expression levels or profiles in eukaryotic host cells, e.g., mammalian host cells or yeast cells, poor chemical and physical properties, e.g., poor / low "shelf-life" stability, poor (low) solubility, poor (high) viscosity, tendency to aggregate, and poor clinical and biophysical profiles, e.g., poor pharmacokinetic profile, poor pharmacological profile, fast or poor in vivo clearance rate, short circulatory half-life, some of which cause them to be discontinued in development.

[0007] Specific techniques and assays exist for evaluating many of the aforementioned developability characteristics of discovered antibodies in the context of downstream development activities ("post-development antibodies"), e.g., CIC, SIC, BVP-ELISA, TMA, and other assays. However, such assays are often incompatible with the high-throughput formats of early-stage antibody discovery platforms. Furthermore, these characterizations often require milligram- to gram-scale quantities of protein, which often imposes a practical limit on the number of leads that can be considered for development. This, in turn, reduces the likelihood of program success. As a result, significant resources are often expended attempting to qualify poorly characterized lead candidates, with few backups available in later development stages.

[0008] Various CD3 antibodies, including monoclonal antibodies and bispecific antibodies, are known in the art. 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 CD3 antibodies have development challenges, such as those outlined above, and / or do not have sufficiently high binding affinity for CD3 for certain multispecific or chimeric antigen receptor (CAR) formats or delivery methods. Therefore, there is an unmet need in the art for anti-CD3 bispecific antibodies that exhibit a desirable developability profile and have high affinity for CD3 for use in cancer therapy. Summary of the Invention

[0009] The present disclosure relates to anti-CD3 antibodies and methods of use thereof, which have a particularly high affinity for CD3, e.g., a monovalent K of about 250 picomolar or less. D It has an affinity for

[0010] The present disclosure provides anti-CD3 antibodies and / or antigen-binding fragments comprising a complementarity-determining region (CDR) in CDRL3 of a light chain variable region, wherein the CDRL3 comprises the amino acid sequence of X1QSYFRRT (SEQ ID NO: 1), where 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).

[0011] In some embodiments, the anti-CD3 antibody and / or antigen-binding fragment further comprises a complementarity-determining region (CDR) in CDRH3 of the heavy chain variable region, wherein the CDRH3 comprises the amino acid sequence X3R1GX4YFYDV (SEQ ID NO: 6), where X3 is A or V and X4 is R or Q. In certain embodiments, X3 is A.

[0012] In some embodiments, the anti-CD3 antibody and / or antigen-binding fragment further comprises a complementarity determining region (CDR) in CDRL1 of the light chain variable region, wherein CDRL1 comprises the amino acid sequence KSSQSLLNARTX5KNYLA (SEQ ID NO: 7), where X5 is G, M, N, or R. In certain embodiments, X5 is G or R. In some embodiments, CDRL1 may comprise KSSQSLLNARTGKNYLA (SEQ ID NO: 8) or KSSQSLLNARTRKNYLA (SEQ ID NO: 9).

[0013] In some embodiments, the anti-CD3 antibody and / or antigen-binding fragment further comprises a complementarity-determining region (CDR) in CDRL2 of the light chain variable region, wherein CDRL2 comprises the amino acid sequence WASTRES (SEQ ID NO: 10) or WASTRSS (SEQ ID NO: 11).

[0014] In some embodiments, the anti-CD3 antibody and / or antigen-binding fragment further comprises a complementarity determining region (CDR) in CDRH1 of the heavy chain variable region, wherein the CDRH1 comprises the amino acid sequence FNX6KDYYX7H (SEQ ID NO: 12), where X6 is I, N, or V and X7 is M or I. In certain embodiments, X6 is I and / or X7 is M. In certain embodiments, the CDRH1 comprises FNIKDYYMH (SEQ ID NO: 13).

[0015] In some embodiments, the anti-CD3 antibody and / or antigen-binding fragment further comprises a complementarity determining region (CDR) in CDRH2 of the heavy chain variable region, said CDRH2 comprising the amino acid sequence WIDLX8NANTVYDX9KX 10 QG (SEQ ID NO: 14), wherein X8 is E or N, X9 is A, H or T, and X 10 is F or L. In some embodiments, X is E and / or X is A and / or X 10 is F. In one embodiment, CDRH2 comprises WIDLENANTVYDAKFQG (SEQ ID NO: 15) or WIDLENANTIYDAKFQG (SEQ ID NO: 16).

[0016] In some embodiments, the present disclosure provides anti-CD3 antibodies and / or antigen-binding fragments thereof comprising one or more of CDRL1, CDRL2, and CDRL3. Such antibodies, in some embodiments, further comprise CDRH1, CDRH2, and CDRH3.

[0017] In some embodiments, the anti-CD3 antibodies and / or antigen-binding fragments thereof described herein have a binding affinity (K D In some embodiments, the anti-CD3 antibody and / or antigen-binding fragment thereof has a binding affinity (K D In some embodiments, the anti-CD3 antibody and / or antigen-binding fragment thereof has a binding affinity (K D ) is provided.

[0018] The present disclosure further relates generally to an anti-cluster of differentiation 3 (CD3) antibody or antigen-binding antibody fragment thereof, the anti-CD3 antibody or antigen-binding antibody fragment thereof comprising: a. a variable heavy chain (V) comprising: H ) Polypeptides: i. VH CDR1 (CDRH1) of FNIKDYYMH (SEQ ID NO: 13), ii. VH CDR1 (CDRH2) of WIDLENANTVYDAKFQG (SEQ ID NO: 15) H CDR2 (CDRH2), iii.ARGRYFYDV (SEQ ID NO: 103) H a. a CDR3 (CDRH3), and / or b. a variable light chain (V L ) Polypeptide: i.KSSQSLLNARTGKNYLA (SEQ ID NO: 8) V L CDR1 (CDRL1), ii. V of WASTRX1S (SEQ ID NO: 133) L CDR2 (CDRL2), and iii.V of X2QSYFRRT (SEQ ID NO: 134) LIn CDR3 (CDRL3), X1 and X2 are each independently any amino acid. In some embodiments, X1 is E or S, and / or X2 is K or V. In some embodiments, the antibody or antigen-binding antibody fragment comprises a CDRL2 of SEQ ID NO: 10 or SEQ ID NO: 11, and / or a CDRL3 of SEQ ID NO: 5 or SEQ ID NO: 4. In some embodiments, the anti-CD3 ... H Chain CDR1 (CDRH1), V of WIDLENANTVYDAKFQG (SEQ ID NO: 15) H Chain CDR2 (CDRH2), and V of AR-GRYFYDV (SEQ ID NO: 103) H Chain CDR3 (CDRH3), and / or V of KSSQSLLNARTGKNYLA (SEQ ID NO: 8) L Chain CDR1 (CDRL1), V of WASTRES (SEQ ID NO: 10) L Chain CDR2 (CDRL2), and V of KQSYFRRT (SEQ ID NO: 5) L a.V.FNIKDYYMH (SEQ ID NO: 13) H Chain CDR1 (CDRH1), V of WIDLENANTVYDAKFQG (SEQ ID NO: 15) H Chain CDR2 (CDRH2), and V of AR-GRYFYDV (SEQ ID NO: 103) H Chain CDR3 (CDRH3), and / or V of KSSQSLLNARTGKNYLA (SEQ ID NO: 8) L Chain CDR1 (CDRL1), V of WASTRSS (SEQ ID NO: 11) L V of VQSYFRRT (SEQ ID NO: 4) L Those containing the CDR3 (CDRL3).

[0019] In some embodiments, the anti-CD3 antibody or antigen-binding antibody fragment comprises a V sequence having an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 18 or SEQ ID NO: 34. LIn some embodiments, the anti-CD3 antibody or antigen-binding antibody fragment comprises a V chain polypeptide comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 18 or SEQ ID NO: 34. L In some embodiments, the anti-CD3 antibody or antigen-binding antibody fragment comprises a V chain polypeptide comprising the amino acid sequence of SEQ ID NO: 18 or SEQ ID NO: 34. L In some embodiments, the anti-CD3 antibody or antigen-binding antibody fragment comprises a V chain polypeptide comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 17. H In some embodiments, the anti-CD3 antibody or antigen-binding antibody fragment comprises a V chain polypeptide comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 17. H In some embodiments, the anti-CD3 antibody or antigen-binding antibody fragment comprises a V chain polypeptide comprising the amino acid sequence of SEQ ID NO: 17. H In some embodiments, the anti-CD3 antibody or antigen-binding antibody fragment comprises a V chain comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 18 or SEQ ID NO: 34. L a V chain polypeptide, and an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 17 H In some embodiments, the anti-CD3 antibody or antigen-binding antibody fragment comprises a V chain polypeptide comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 18 or SEQ ID NO: 34. L a V chain polypeptide, and an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 17 H In some embodiments, the anti-CD3 antibody or antigen-binding antibody fragment comprises a V chain polypeptide comprising the amino acid sequence of SEQ ID NO: 18 or SEQ ID NO: 34. L a V chain polypeptide comprising the amino acid sequence of SEQ ID NO: 17; H In some embodiments, the anti-CD3 antibody or antigen-binding antibody fragment comprises a V chain comprising the amino acid sequence of SEQ ID NO: 18. L a V chain polypeptide comprising the amino acid sequence of SEQ ID NO: 17; HIn some embodiments, the anti-CD3 antibody or antigen-binding antibody fragment comprises a V chain comprising the amino acid sequence of SEQ ID NO: 34. L a V chain polypeptide comprising the amino acid sequence of SEQ ID NO: 17; H Contains chains.

[0020] In some embodiments, the anti-CD3 antibody or antigen-binding antibody fragment has a dissociation constant (K) 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. D ) binds to CD3, optionally wherein the CD3 is human and / or cynomolgus monkey CD3, and further optionally wherein the binding affinity is measured by surface plasmon resonance. D is a monovalent K D and / or in this case, the K D is measured using the scFv fragment of the anti-CD3 antibody or antibody fragment.

[0021] In some embodiments, the antibody or antigen-binding antibody fragment exhibits a reduced tendency to elicit cytokine production to levels capable of inducing cytokine release syndrome while eliciting T cell activation or T cell killing. In some embodiments, the antibody or antigen-binding antibody fragment comprises a multispecific antibody. In some embodiments, the antibody or antigen-binding antibody fragment comprises a bispecific antibody. In some embodiments, the antibody or antigen-binding antibody fragment comprises an scFv. In some embodiments, the antibody or antigen-binding antibody fragment comprises at least a second antigen-binding domain that specifically binds to a tumor target, an immune-oncology target, a neurodegenerative disease target, an autoimmune disorder target, an infectious disease target, a metabolic disease target, a cognitive disorder target, a blood-brain barrier target, or a hematological disorder target. In some embodiments, the antibody or antigen-binding antibody fragment 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-PGF1a, 8-iso-PGF2a, 8-oxo-dG, Al adenosine receptor, 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, ADAM15, ADAM17 / TACE, ADAM8, ADAM9, ADAMTS, ADAMTS4, ADAMTS5, addressin, aFGF, ALCAM, ALK, ALK-1, ALK-7, alpha-l-antitrypsin, alpha-V / beta-1 antagonist, ANG, Ang, APAF-1, APE, APJ, APP, APRIL, AR, ARC, ART, Artemin, anti-Id, asparagine, atrial natriuretic factor, av / b3 integrin, Axl, b2M, B7-1, B7-2, B7-H, B-lymphocyte stimulatory factor (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, BMP-3, osteogenin, 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), BMPs, β-NGF, BOK, Bone-derived neurotrophic factor, BPDE, BPDE-DNA, BTC, Complement Factor 3 (C3), C3a, C4, C5, C5a, CIO, CA125, CAD-8, Carboxytonin, cAMP, Carcinoma-fetal antigen (CEA), Tumor-related antigen, Catechin A, Catechin B , カテプシンC / DPPI, カテプシンD, カテプシンE, カテプシンH, カテプシンL, カテプシンO, カテプシンS, カテプシンV, カテプシンX / Z / P, CBL, CCI, CCK2, CCL, CCL1, CCL11, CCL12, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL2, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CCL3, CCL4, CCL5, CCL 6. 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(p6 7-tanpakine), 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, botzinus toxin, velshene toxin, CKb8-l, 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, D cR3, DC-SIGN, decay accelerating factor, des(l-3)-IGF-I (brain IGF-1), 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, Gas6, 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-alpha1, GFR-alpha2, GFR-alpha3, GITR, glucagon, Glut4, glycoprotein Ilb / IIIa (GPI) Ilb / IIIa), GM-CSF, gpl30, gp72, GRO, growth hormone-releasing factor, hapten (NP-cap or NIP-cap), HB-EGF, HCC, HCMV gB envelope glycoprotein, HCMV gH envelope glycoprotein, HCMV UL, hemopoietic growth factor (HGF), Hep B gpl20, heparanase, Her2, Her2 / neu (ErbB-2),Her3 (ErbB-3), Her4 (ErbB-4), herpes simplex virus (HSV) gB glycoprotein, HSV gD glycoprotein, HGFA, high molecular weight melanoma-associated antigen (HMW-MAA), HIV gpl20, HIV IIIB gp 120 V3 loop, 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 protein, 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, interferon (INF)-alpha, INF-beta, INF- Gamma, inhibin, iNOS, insulin A-chain, insulin B-chain, insulin-like growth factor 1, integrin 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, 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, latent TGF-1bpl, LBP, LDGF, LECT2, Lefty, Lewis-Y antigen, Lewis-Y-related antigen, LFA-1, LFA-3, Lfo, LIF, LIGHT, lipoprotein, LIX, LKN, Lptn, L-selectin, LT-a,LT-b, LTB4, LTBP-1, pulmonary surfactant, progesterone, lymphotoxin beta receptor, Mac-1, MAdCAM, MAG, MAP2, MARC, MCAM, MCAM, MCK-2, MCP, M-CSF, MDC, Mer, metalloproteinase, MGDF receptor, MGMT, MHC (HLA-DR), MIF, MIG, MIP, MIP-1-alpha, MK, MMAC1, MMP, MMP-1, MMP-1 0, 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, Müllerian inhibitory factor, Mug, MuSK, NAIP, NAP, NCAD, N-cadherin, NCA90, NCAM, NCAM, neprilysin, neurotrophin-3, -4, or - 6, Neurturin, Nerve 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, PDK-1, PECAM, PEM, PF4, PGE, PGF, PGI2, PGJ2, PIN, PLA2, placental alkaline phosphatase (PLAP), PIGF, PLP, PP14, proinsulin, prorelaxin, protein C, 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 factor, 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 receptor (e.g., T cell receptor alpha / beta), TdT, TECK, TEM1, TEM5, TEM7TEM8, TERT, testicular PLAP-like alkaline phosphatase, TfR, TGF, TGF-alpha, TGF-beta, TGF-beta pan-specific, TGF-beta RI (ALK-5), TGF-beta RII, TGF-beta Rllb, TGF-beta, RIII, TGF-linkl, TGF-link2, TGF-link3, T GF-factor 4 and TGF-factor 5 activate Ck-1 Tie, TIMP, TIQ, TMEFF2, Tmpo, TMPRSS2, TNF, TNF-protein, TNF-protease Liquids, TNF-link 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(WHEM ATAR、HveA、LIGHT R、TR2)、TNFRSF16(NGFR p75NTR), TNFRSF17(BCMA), TNFRSF 18 (GITR AITR), TNFRSF19 (TROY CROWN), 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(TRAILApo-2 ligand, TL2), TNFSF11 (TRANCE / RANK ligand ODF, 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-α Conectin, DIF, TNFSF2), TNFSF1B (TNF-β LTa, TNFSF1), TNFSF3 (LTb TNFC, p33), TNFSF4 (OX40 ligand gp34, TXGP1), TNFSF5 (CD40 ligand) CD154, gp39, HIGM1, IMD3, TRAP), TNFSF6 (Fas ligand, Apo-1 ligand, APT1 ligand), TNFSF7 (CD27 ligand, CD70), TNFSF8 (CD30 ligand, CD153), TNFSF9 (4-1BB ligand, CD137 ligand), TP-1, t-PA, Tpo, TRAIL, TRAIL R, TRAIL-R1, TRAIL-R2, TRANCE, transfer receptor receptor), TRF, Trk, TROP-2, TSG, TSLP, tumor-associated antigen CA125, tumor-associated antigen expressing Lewis Y-related glycoprotein, 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 antigen, 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, WN T9A, WNT9B, WNT10A, WNT10B, WNT11, WNT16, XCL1, XCL2, XCR1, XCR1, XEDAR, XIAP, XPD, CTLA4(cytotoxic Tocyte lymphantigen-4), PD1 (programmed cell death protein 1), PD-L1 (programmed cell death ligand 1), LAG-3 (lymphocyte activation gene-3), TIM-3 (T cell immunoglobulin and mucin protein-3), hormone receptors, and growth factors.

[0022] In some embodiments, the antibody or antigen-binding antibody fragment 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 ligand 1), LAG-3 (lymphocyte activation 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 antibody fragment is a chimeric antigen receptor (CAR). chimeric antigen receptor ), wherein the CAR optionally comprises at least one transmembrane domain and at least one intracellular domain derived from a T cell receptor, and optionally comprises a CD3ζ subunit and at least one costimulatory domain. In some embodiments, the antibody or antigen-binding antibody fragment comprises scFv2-Fc2 and / or scFv-IgG. In some embodiments, the antibody or antigen-binding antibody fragment comprises an IgG constant domain. In some embodiments, the k. antibody or antigen-binding antibody fragment comprises at least a second antigen-binding domain that specifically binds to an antigen, and the antibody is comprised of a Fab-Fc-scFv( Bottle opener type ) The present disclosure also relates to a multispecific format selected from the group consisting of: Mab-scFv, Mab-Fv, Dual scFv, Central Fv, Central scFv, One-arm Central scFv, Fab-Fab, Fab-Fv, mAb-Fv, mAb-Fab, DART, BiTE, Common Light Chain-IgG, TanDab, Cross-Mab, SEED, BEAT, TrioMab, and DuetMab. Furthermore, the present disclosure generally relates to isolated or recombinant nucleic acid sequences encoding the antibodies or antigen-binding antibody fragments described herein. Furthermore, the present disclosure generally relates to expression vectors comprising the isolated or recombinant nucleic acid sequences described herein. Furthermore, the present disclosure generally relates to host cells transfected, transformed, or transduced with the nucleic acid sequences, and optionally to mammalian or yeast cells or vectors containing the nucleic acid sequences described herein.

[0023] The present disclosure further relates generally to pharmaceutical compositions comprising an antibody or antigen-binding antibody fragment described herein and a pharmaceutically acceptable carrier and / or excipient.

[0024] The present disclosure further relates generally to methods of treating a disorder in a mammal in need of such treatment, including a proliferative disorder, an oncological disorder, an immune-oncological disorder, a neurological disorder, a neurodegenerative disorder, or an autoimmune disorder, the method comprising administering an effective amount of one or more antibodies or antibody fragments described herein, or a host cell, optionally an immune cell, and optionally a T cell or an NK cell, expressing one of the antibodies or antibody fragments described herein. In some embodiments, the method further comprises administering an additional therapeutic agent to the mammal, optionally in which case the mammal is a human.

[0025] The present disclosure further relates generally to anti-cluster of differentiation 3 (CD3) antibodies or antibody fragments, wherein the anti-CD3 antibodies or antigen-binding antibody fragments comprise one or more CDRs of any one or more of Ab1-Ab50. The present disclosure further relates generally to anti-cluster of differentiation 3 (CD3) antibodies or antibody fragments, wherein the anti-CD3 antibodies or antigen-binding antibody fragments comprise a V CDR comprising an amino acid sequence selected from the sequences set forth in Table 4. H Chain and / or V L Contains one or more chains. DETAILED DESCRIPTION OF THE INVENTION

[0026] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs.As used herein, the term "about" when used in connection with a specific recited numerical value means that the value can vary by 1% or less from the recited value.For example, as used herein, the expression "about 100" includes 99 and 101, and all values ​​therebetween (for example, 99.1, 99.2, 99.3, 99.4, etc.).

[0027] It will be understood that aspects and embodiments of the invention described herein include "comprising," "consisting of," and "consisting essentially of" aspects and embodiments.

[0028] As used herein, a protein with high affinity for CD3 (K D In some embodiments, an anti-CD3 antibody and / or antigen-binding fragment thereof is provided, having a K D is about 500 pM or less. D is about 250 pM or less. DIn some embodiments, the anti-CD3 antibody or antigen-binding antibody fragment has a dissociation constant (K) of about 500 pM or more, 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. D ) binds to CD3, optionally wherein the CD3 is human and / or cynomolgus monkey CD3, and further optionally wherein the binding affinity is measured by surface plasmon resonance. D is a monovalent K D and / or in this case, the K D is measured using an scFv fragment of the anti-CD3 antibody or antibody fragment. In some embodiments, the anti-CD3 antibody and / or antigen-binding fragment thereof comprises a particular CDR sequence motif. In some embodiments, the CDR sequence motif comprises CDRL3 comprising SEQ ID NO: 1. In some embodiments, the anti-CD3 antibody has a favorable developability profile.

[0029] Examples of anti-affinity anti-CD3 antibodies and their antigen-binding fragments "Cluster of Differentiation 3" or "CD3" generally refers to any native CD3 from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), and includes, for example, the CD3ε chain, the CD3γ chain, the CD3α chain, and the CD3β chain, unless otherwise indicated. 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 in cells. The term also encompasses naturally occurring variants of CD3, such as splice variants or allelic variants. CD3 includes, for example, the human CD3ε protein (NCBI Reference SEQ ID NO: NP _ 000724), which is a 207 amino acid long protein, and human CD3γ protein (NCBI Reference SEQ ID NO: NP_ 000064), which is 182 amino acids in length. The term also refers to human or cynomolgus monkey CD3ε proteins, the amino acid sequences of which are SEQ ID NOs: 131 and 132, respectively (Table 7). "CD3εN27" and "CD3εN13" refer to the N-terminal 27 amino acids and the N-terminal 13 amino acids of CD3, respectively, including any chemical modifications or linkages thereto.

[0030] "Anti-CD3 antibody" refers to an antibody or antigen-binding fragment thereof capable of binding to CD3, e.g., CD3ε and / or CD3γ, e.g., human CD3ε and / or CD3γ, with sufficient affinity and / or specificity such that the antibody is useful as a diagnostic and / or therapeutic agent in targeting CD3. The anti-CD3 antibodies and antigen-binding fragments thereof described herein have particularly high affinity for CD3.

[0031] The term "high affinity" refers to an antibody that has a binding affinity for CD3, D In some embodiments, K D is approximately 5x10 -10 In some embodiments, K D is approximately 2.5x10 -10 In some embodiments, K D is approximately 1 x 10 -11 In some embodiments, the anti-CD3 antibody or antigen-binding antibody fragment has a dissociation constant (K) of about 500 pM or more, 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. D ) binds to CD3, optionally wherein the CD3 is human and / or cynomolgus monkey CD3, and further optionally wherein the binding affinity is measured by surface plasmon resonance. Dis measured by surface plasmon resonance, e.g., BIACORE, biolayer interferometry using, e.g., a FORTEBIO Octet HTX instrument (Pall Life Sciences), or solution-affinity ELISA. D is measured using an scFv fragment of an anti-CD3 antibody. In some embodiments, a monovalent K D is measured.

[0032] In some embodiments, the anti-CD3 antibody is at a concentration of about 500 pM (5×10 -10 The dissociation constant (K) is less than M D In some embodiments, the anti-CD3 antibody binds to CD3 with a dissociation constant (K) of less than about 250 pM. D In some embodiments, the anti-CD3 antibody binds to CD3 with a dissociation constant (K) of about 100 pM. D In certain embodiments, the anti-CD3 antibody binds to an epitope of CD3 that is conserved among CD3 from various species, such as the cross-reactive species of human and cynomolgus monkey.

[0033] K D K can be measured using assays known to those skilled in the art, including, but not limited to, biolayer interferometry (BLI), surface plasmon resonance (SPR), solution equilibrium based kinetic exclusion assays, KinExA direct association assays using ForteBio instruments and reagents, such as Octet RED 384 and HTX BLI-based instruments, enzyme-linked immunosorbent assays (ELISAs), and radioimmunoassays (RIAs). D Measurements may be performed using a BIACORE® surface plasmon resonance assay with an intact anti-CD3 antibody or an antigen-binding fragment thereof, such as an scFv.

[0034] While high affinity CD3 binders have been associated with inducing or causing cytokine release syndrome (CRS) in patients, the binding affinity of an antibody to CD3 is not the sole determinant of CRS. For example, high affinity anti-CD3 antibodies (K D Low single-digit nM concentrations have demonstrated potent T cell cytotoxicity against target 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 on April 16, 2018). Without being bound by theory, high-affinity anti-CD3 antibodies may be particularly beneficial for providing specific targeting and minimizing CRS risk in certain formats of multispecific antibodies (e.g., scFv2-Fc2 or scFv-IgG, or other similar multimeric formats) or chimeric antigen receptors with various components assembled in different orders.

[0035] The term "cytokine release syndrome" (or CRS) refers to a pro-inflammatory positive feedback loop between cytokines and immune cells, resulting in the excessive and uncontrolled release of pro-inflammatory cytokines by cells within the immune system (see, for example, Lee et al., Blood, Vol. 124, pages 188-195 (2014) and Tisoncik et al., Microbiol Mol Biol Rev, Vol. 76, pages 16-32 (2012)). Upon stimulation and activation, T cells release a range of cytokines at levels and to an extent that produce adverse biological / physiological effects of varying degrees and severity. Such effects include, for example, acute inflammation characterized by redness, swelling or edema, fever, pain, and loss of function. When localized to the skin or other tissues, biological / physiological effects include increased blood flow, allowing vascular leukocytes and plasma proteins to reach extravascular sites of injury, increased local temperature, and the development of pain, tissue edema and extravascular pressure, and decreased tissue perfusion. Other biological / physiological effects include organ and systemic dysfunction, such as heart failure, adult respiratory distress syndrome, neurotoxicity, renal and / or hepatic failure, and disseminated intravascular coagulation. Elevated levels of IFNγ, IL-6, TNFα, TGFβ, IL-2, granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-10, IL-8, IL-5, and / or fractalkine have been implicated as indicators and / or causes of CRS or a propensity for T cell stimulation to induce CRS.

[0036] In certain embodiments, the anti-CD3 antibodies and / or antigen-binding fragments thereof described herein are dumbed down and / or modified to reduce the likelihood or severity of antibody-induced CRS. Non-limiting examples of modifications include silent Fc regions (e.g., removing the Fc entirely or modifying the Fc region to reduce or eliminate effector function) and / or masking (e.g., a polypeptide mask positioned to reduce or inhibit the ability of the antibody or antigen-binding fragment thereof to specifically bind to CD3).

[0037] The term "antibody" is used herein in the 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 fragments that exhibit the desired antigen-binding activity).

[0038] A "monoclonal antibody" or "mAb" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical and / or bind to the same epitope, excluding potential variant antibodies (e.g., containing natural mutations or arising during generation of the monoclonal antibody preparation). Such variants are generally present in small amounts. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen.

[0039] For multispecific antibodies, such antibodies contain at least two different antigen-binding domains that recognize and specifically bind to at least two different antigens. For bispecific antibodies, such antibodies contain two different antigen-binding domains that recognize and specifically bind to at least two different antigens. "Different antigens" can refer to different and / or distinct proteins, polypeptides, or molecules, and can also refer to different and / or distinct epitopes. The epitopes may be contained within a single protein, polypeptide, or other molecule.

[0040] 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 the paratope. A single antigen may have multiple epitopes. Thus, different antibodies may bind to different regions of an antigen and have different biological effects. The term "epitope" also refers to the site on an antigen to which B cells and / or T cells respond. It also refers to the region of an antigen that is bound by an antibody. Epitopes may be defined structurally or functionally. 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 amino acids in a nonlinear configuration. In certain embodiments, epitopes may include determinants that are chemically active surface groupings of molecules, such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups. In certain embodiments, epitopes may have specific three-dimensional structural characteristics and / or specific charge characteristics.

[0041] In some instances, antibodies comprise four polypeptide chains: two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, some of which may be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy-chain constant domains that correspond to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0042] In other examples, the antibody may instead include a multimeric form thereof (e.g., IgM) or an antigen-binding fragment thereof. Each heavy chain is composed of a heavy chain variable region (VH) and a heavy chain constant region, the heavy chain constant region comprising domains C, D, E, F, G, H, I, G, I, M ... M, I, M, M, I, M, M, M, M, M, M, M, M H 1. C H 2 and C H Each light chain is composed of a light chain variable region (VL) and a light chain constant region (CL). H Area and V L The regions can be further divided into more conserved regions termed framework regions (FR) and intervening regions of hypervariability termed complementarity determining regions (CDR). H and V L is composed of three CDRs and four FRs arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In certain embodiments of the invention, the FRs of an antibody (or antigen-binding fragment thereof) may be identical to human germline sequences, or may be naturally occurring or artificially modified. A consensus sequence of amino acids may be defined based on the alignment and analysis of two or more CDRs. Thus, the heavy chain CDRs are designated "CHRH1," "CDRH2," and "CDRH3," respectively, and the light chain CDRs are designated "CDRL1," "CDRL2," and "CDRL3," respectively.

[0043] Unless specifically indicated otherwise, the term "antibody," as used herein, includes molecules comprising two heavy immunoglobulin chains and two light immunoglobulin chains (i.e., "intact antibody molecules" or "intact antibodies"), as well as antigen-binding fragments thereof.

[0044] An "antigen-binding fragment" refers to a portion of an intact antibody that binds to the antigen to which the intact antibody binds (in this case, CD3). The terms "full length antibody," "intact antibody," and "whole antibody" are used interchangeably herein and refer to an antibody having substantially the same structure as a native antibody.

[0045] Antigen-binding fragments of antibodies include any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds to 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 only the VH or VL domain), and multispecific antibodies formed from antibody fragments. In some embodiments, the antigen-binding fragment of an anti-CD3 antibody described herein is an scFv.

[0046] As with intact antibody molecules, antigen-binding fragments may be monospecific or multispecific (e.g., bispecific). Multispecific antigen-binding fragments of antibodies may contain at least two different variable domains, in which case each variable domain can specifically bind to a distinct antigen or to different epitopes on the same antigen. In the case of the antigen-binding fragments of anti-CD3 antibodies described herein, a variety of multispecific antibody formats can be used.Non-limiting examples of multispecific and bispecific formats include Fab-Fc-scFv (bottle opener type) (XENCOR), Mab-scFv (XENCOR), Mab-Fv (XENCOR), dual scFv (XENCOR), central Fv (XENCOR), central scFv (XENCOR), one-arm central scFv (XENCOR), Fab-Fab (XENCOR), Fab-Fv (XENCOR), mAb-Fv (XENCOR), mAb-Fab (XENCOR), DART (MACROGENICS), BiTE (AMGEN / MICROMET), KiTE, common light chain-IgG (Genentech), TandAb (SFIMED), Cross-Mab (ROCHE), SEED (EMD Serono), BEAT (Glenmark), TrioMab (Trion Pharma / Fresenius Biotech), DuetMab (Medimune), and the like, for example, (WO95 / 09917; WO2008 / 119566; WO2008 / 119567; WO2011 / 121110; WO2010 / 037835; WO2007 / 042261; WO2007 / 110205; WO2011 / 121110; WO2012 / 055961;WO2012 / 16067;WO2016 / 086189;WO2016 / 182751;WO2015 / 006749;WO2014 / 049003;WO201 3 / 177101;WO2015 / 128509;US7,951,917;US2009 / 0252729;US2014 / 0348839;US7,183,076;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.

[0047] In certain embodiments, the anti-CD3 antibodies and / or antigen-binding fragments thereof described herein are contained in multispecific antibodies, particularly bispecific antibodies, having binding specificity for a second antigen. Such a second antigen may be an entirely different target from the first target, or may be a different epitope on the same target. In some embodiments, the binding specificities are for two different epitopes of CD3 (e.g., CD3ε or CD3γ). In other embodiments, one of the binding specificities is for CD3 (e.g., CD3ε or CD3γ), and the other is for a different biological molecule (e.g., a cell surface antigen, e.g., a tumor antigen).

[0048] Non-limiting examples of second antigens to which bispecific antibodies, including anti-CD3 antibodies and / or antigen-binding fragments thereof, described herein are directed include targets selected from the group consisting of: 17-IA, 4-1BB, 4Dc, 6-keto-PGF1a, 8-iso-PGF2a, 8-oxo-dG, Al adenosine receptor, 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, ADAM15, ADAM17 / TACE, ADAM8, ADAM9, ADAMTS, ADAMTS4, ADAMTS5, addressin, aFGF, ALCAM, ALK, ALK-1, ALK-7, alpha-l-antitrypsin, alpha-V / beta-1 antagonist, ANG, Ang, APAF-1, APE, APJ, APP, APRIL, AR, ARC, ART, artemin, anti-Id, asparagine, atrial natriuretic factor, av / b3 Integrin, Axl, b2M, B7-1, B7-2, B7-H, B-lymphocyte stimulatory factor (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, BMP-3 osteogenin, BMP-4 BMP-2b, BMP-5, BMP-6Vgr-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), BMPs, b-NGF, BOK, bombesin, bone-derived neurotrophic factor, BPDE, BPDE-DNA, BTC, complement factor 3 (C3), C3a, C4, C5, C5a, CIO, CA125, CAD-8, calcitonin, cAMP, carcinoembryonic antigen (CEA), tumor-associated antigen, cathepsin A, catecholamines 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, CCLl, CCLll, CCL12, CCL13, CCL14, CCL15, CCL16, CCLl7, 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, C D10, CDlla, CDllb, CDllc, CD13, CD14, CD15, CD16, CD18, CD19, CD20, CD21, CD22, CD23, CD25, CD27L, CD28, CD29, CD30, CD30L, CD32, CD33( p67 protein), 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, botulinum toxin, Clostridium perfringens toxin, CKb8-l, CLC, CMV, CMVUL, CNTF, CNTN-1, COX, C-Ret, CRG-2, CT-1, CTACK, CTGF, CTLA-4, CX3CL1, CX3CR1, CXCL, CXCLl, CXCL2, CXCL3, CXCL 4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCR, CXCR1, CXCR 2, CXCR3, CXCR4, CXCR5, CXCR6, cytokeratin tumor-associated antigen, DAN, DCC, DcR3, DC-SIGN, decay-accelerating factor, des(l-3)-IGF-I (brain IGF-1), 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, Gas6, 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, GDNF, GFAP, GFRa-1, GFR-alpha1, GFR-alpha2, GFR-alpha3, GITR, glucagon, Glut4, glycoprotein Ilb / IIIa (GPIlb / IIIa), GM-CSF, gpl30, gp72, GRO, growth hormone-releasing factor, hapten (NP-cap or NIP-cap), HB-EGF, HCC, HCMV gBEnvelope glycoprotein, HCMV gH envelope glycoprotein, HCMV UL, hemopoietic growth factor (HGF), Hep B gpl20, heparanase, Her2, Her2 / neu (ErbB-2), Her3 (ErbB-3), Her4 (ErbB-4), herpes simplex virus (HSV) gB glycoprotein, HSV gD glycoprotein, HGFA, high molecular weight melanoma-associated antigen (HMW-MAA), HIV gpl20, HIV IIIB gp 120 V3 loop, HLA, HLA-DR, HM1.24, HMFGPEM, 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 protein, 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, interferon (INF)-alpha, INF-beta, INF-gamma, inhibin, iNOS, insulin A-chain, insulin B-chain, insulin-like growth factor 1, Integrin 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, interferon gamma, IP-10, l-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, latent TGF-1bpl, LBP, LDGF, LECT2, Lefty, Lewis-Y antigen, Lewis-Y related antigen, LFA-1, LFA-3, Lfo, LIF, LIGHT, lipoprotein, LIX, LKN, Lptn, L-selectin, LT-a, LT-b, LTB4, LTBP-1, pulmonary surfactant, progesterone, lymphotoxin beta receptor, Mac-1, MAdCAM, MAG, MAP2, MARC, MCAM, MCAM, MCK-2, MCP, M-CSF, MDC, Mer, metalloprotein ase, MGDF receptor, MGMT, MHC (HLA-DR), MIF, MIG, MIP, MIP-1-alpha, 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, Müllerian inhibitory factor, Mug, MuSK, NAIP, NAP, NCAD, N-Ca cadherin, NCA90, NCAM, NCAM, neprilysin, neurotrophin-3, -4, or -6, neurturin, nerve 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, PDK -1, PECAM, PEM, PF4, PGE, PGF, PGI2, PGJ2, PIN, PLA2, placental alkaline phosphatase (PLAP), P1GF, PLP, PP14, proinsulin, prorelaxin, protein C, 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, RSVFgp, Ret, rheumatoid factor, 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 receptor (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, TGF-beta pan-specific (Pan Specific), TGF-betaRI (ALK-5), TGF-betaRII, TGF-betaRllb, TGF-betaRIII, TGF-beta1, TGF-beta2, TGF-beta3, TGF-beta4, TGF-beta5, thrombin, thymic Ck-1, thyroid-stimulating hormone, Tie, TIMP, TIQ, tissue factor, TMEFF2, Tmpo, TMPRSS2, TNF, TNF-alpha, TNF-alpha beta, TNF-beta2, 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)、TNFRSF18(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), TNFRSF5(CD40 p50), TNFRSF6(Fas Apo-1, APT1, CD95), TNFRSF6B(DcR3 M68, TR6), TNFRSF7(CD27), TNFRSF8(CD30), TNFRSF9(4-lBB). 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 polymer TL2) TNFSF11(TRANCE / RANK dimer ODF) TNFSF12(TWEAK). Apo-3リンド, DR3リンド, TNFSF13(APRIL TALL2), TNFSF13B(BAFF LIGHT, TALL1, THANK, TNFSF20), TNFSF14(LIGHT HVEM ligand LTg) TNFSF15(TL1A / VEGI) TNFSF18(GITR ligand AITR ligand TL6) TNFSFIA(TNF-a Connectin, DIF, TNFSF2, TNFSF1B (TNF-b LTa, TNFSF1), TNFSF3 (LTb TNFC, p33), TNFSF4 (OX40-linked gp34, TXGP1), and TNFSF5 (CD40-linked).CD154, gp39, HIGM1, IMD3, TRAP), TNFSF6 (Fas ligand, Apo-1 ligand, APT1 ligand), TNFSF7 (CD27 ligand, CD70), TNFSF8 (CD30 ligand, CD153), TNFSF9 (4-lBB ligand, CD137 ligand), TP-1, t-PA, Tpo, TRAIL, TRAIL R, TRAIL-R1, TRAIL-R2, TRANCE, transfer receptor, TRF, Trk, TROP-2, TSG, TSLP, tumor-associated antigen CA 125, Lewis Y-related glycoprotein-expressing tumor-associated 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, VIM, viral antigen, VLA, VLA-1, VLA-4, VNR integrin, von V. Revland 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 immunoglobulin and mucin protein-3), hormone receptors, and growth factors.

[0049] Multispecific agents, including anti-CD3 antibodies and antigen-binding fragments disclosed herein, can be engineered using techniques such as, but not limited to, recombinant co-expression of two immunoglobulin heavy chain-light chains with different specificities (see, e.g., Milstein and Cuello, Nature 305:537 (1983)), WO 93 / 08829, and Traunecker et al., EMBO J. 10:3655 (1991)), "knobs-in-holes" engineering (see, e.g., U.S. Pat. No. 5,731,168), immunoglobulin crossover (also known as Fab domain swapping or CrossMab format) technology (see, e.g., WO 2009 / 080253; Schaefer et al., EMBO J. 10:3655 (1991)), and other techniques. al., Proc. Natl. Acad. Sci. USA, 108:11187-11192 (2011)), engineering electrostatic steering effects on antibody Fc heterodimeric molecules (WO2009 / 089004A1), cross-linking of two or more antibodies or fragments (see, e.g., U.S. Pat. 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, 148(5):1547-1553 (1992)), and the like. al., J. ImmunoL, 152:5368 (1994)), and trispecific antibodies as described, for example, in Tutt et al. J. ImmunoL 147:60 (1991).

[0050] The present disclosure also contemplates modifications of the anti-CD3 antibodies disclosed herein, including substitutions, insertions, and / or deletions of one or more amino acids in the FR and / or CDR regions of the heavy and light chain variable domains. Once obtained, such derived antibodies and / or antigen-binding fragments may be tested for one or more desirable properties, such as improved binding specificity, increased binding affinity, improved developability, etc.

[0051] 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 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 percentages of identity therebetween, of amino acid sequence identity to the corresponding sequences of the anti-CD3 antibodies disclosed in Table 4 (Ab1-Ab50). In some embodiments, percent identity is measured by any known algorithm for sequence identity, such as, for example, FASTA, BLAST, or GAP.

[0052] 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 with another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). Generally, conservative amino acid substitutions do not substantially change the functional properties of a protein. When two or more amino acid sequences differ from each other by conservative substitutions, the percent or degree of similarity may be adjusted upward to correct for the conservative nature of the substitution. Means for making this adjustment are known to those of skill in the art. (See, e.g., Pearson (1994) Methods Mol. Biol. 24:307-331.) Examples of groups of amino acids with side chains having 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: aspartic acid and glutamic acid, and 7) sulfur-containing side chains: cysteine ​​and methionine. In some embodiments, conservative amino acid substitution groups are valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid-aspartic acid, and asparagine-glutamine. Alternatively, in some embodiments, a conservative substitution includes any change that has 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 that has a non-negative value in the PAM250 log-likelihood matrix.

[0053] Substitution of one or more CDR residues or omission of one or more CDRs is also possible. The scientific literature reports antibodies in which one or two CDRs can be deleted to alter binding. Padlan et al. (1995 FASEB J. 9:133-139) analyzed the contact regions between antibodies and their antigens based on published crystal structures and concluded that only approximately one-fifth to one-third of the CDR residues actually contact their associated antigen. Padlan also found that in many antibodies, one or two CDRs do not contain any amino acids that contact the antigen (see also Vajdos et al. 2002 J Mol Biol 320:415-428). CDR residues that do not contact the antigen can be identified by molecular modeling and / or empirically based on previous studies from regions of the Kabat CDRs outside the Cho Chia CDRs (e.g., residues H60-H65 of CDRH2 are often not required). When a CDR or residue thereof is omitted, it is usually replaced with an amino acid at the corresponding position in another human antibody sequence or a consensus of such sequences. The position of substitution within the CDR, and the amino acid to be substituted, can also be selected empirically.

[0054] In certain embodiments, substitutions, insertions, or deletions may be present in one or more CDRs of the anti-CD3 antibodies described herein, so long as such changes do not substantially reduce the ability of the antibody to bind to its antigen. For example, conservative changes (e.g., conservative substitutions provided herein) that do not substantially reduce binding affinity may be made in a CDR. Such changes may, for example, be outside the antigen-contacting residues in the CDR. In certain embodiments of the variant VH and VL sequences provided above, each CDR contains no alterations or no more than one, two, or three amino acid substitutions.

[0055] A useful method for identifying antibody residues or regions that can be targeted for mutagenesis is called "alanine scanning mutagenesis," 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 neutral or negatively charged amino acids (e.g., alanine or polyalanine), and it is determined whether the antibody-antigen interaction is affected. Further substitutions can be introduced at amino acid positions that show functional sensitivity to the initial substitution. Alternatively, or in addition, crystal structures of antigen-antibody complexes can be used to identify contact points between the antibody and antigen. Such contact residues and adjacent residues can be targeted or excluded from substitution candidates. Variants can be screened to determine whether they contain desirable properties.

[0056] Amino acid sequence insertions include amino- and / or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing several hundred or more residues, as well as intersequence insertions of single or multiple amino acid residues. An example of a terminal insertion is an antibody with an N-terminal methionyl residue. Other insertional variants of antibody molecules include the fusion to the antibody N- or C-terminus to an enzyme (e.g., an enzyme for ADEPT) or a polypeptide that increases the serum half-life of the antibody.

[0057] As described throughout, the anti-CD3 antibodies and / or antigen-binding fragments thereof provided herein have favorable developability and are therefore relatively amenable to development.

[0058] The term "developable" refers to the extent to which one or more polypeptides in a plurality of polypeptides possess desirable properties, such as, for example, desirable expression in mammalian cells, solubility, viscosity, aggregation, chemical and / or physical stability, a desirable shelf life, melting point, pharmacokinetic profile, circulating half-life, and clearance characteristics. Such properties may be treated individually, as a combination of a subset of such properties, or collectively, as indicators of the likelihood that the one or more polypeptides will be successfully developed as therapeutic candidates and ultimately become approved drugs. Thus, as understood in the art, polypeptides having desirable developability properties generally possess, for example, relatively high solubility, relatively low viscosity, relatively low tendency to aggregate, relatively high chemical stability, relatively high physical stability, a relatively long shelf life, a relatively high melting point, a relatively long circulating half-life, a relatively long clearance time, etc. Polypeptides with undesirable developability properties possess, for example, relatively low solubility, relatively high viscosity, relatively high tendency to aggregate, relatively poor chemical stability, relatively poor physical stability, relatively short shelf life, relatively low melting point, relatively short circulatory half-life, relatively long clearance time, etc.

[0059] Methods and assays that can be employed to confirm the extent to which a polypeptide, such as, for example, an anti-CD3 antibody and / or antigen-binding fragment thereof described herein, possesses desirable developability properties are available in the art, such as, for example, PSR assays (WO2014 / 179363, and Xu et al., Protein Eng Des Sol, Vol. 26, pages 663-670 (2013)), SMP assays and SCP assays, cross-interaction chromatography (CIC), self-interaction chromatography (SIC), dynamic light scattering, size-exclusion chromatography (SEC), dynamic light scattering (DLS) spectroscopy, photon correlation spectroscopy, quasi-elastic light scattering, circular dichroism (CD), viscosity measurements, whole cell binding, tissue microarray methodology, BVP ELISA assays, AC-SINS assays (Liu et al; MAbs, Vol. 6, 483-492 (2014)); differential scanning calorimetry, and the like (e.g., He et al. al.,J.Pharm.Sci.,Vol.100(4),pp.1330-1340(2011);Wagner et al.,Pharm.Develop.& Technol(Posted online in 2012;hyper-text transfer protocol:informahealthcare.com / doi / abs / 10.3109 / 10837450.2011.649851);Hotzel et al.,mAbs,Vol.4(6),753-7601(2012);Weiqiang et al. 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. al., J. Pharm. Sci., Vol. 94(9), pp. 1928-1948 (2005); and Payne et al., Biopolymers, Vol. 85(5), pp. 527-533 (2006).

[0060] In some embodiments, antibodies identified as having low developability are so detected by their interaction with a multispecific reagent (PSR) and are therefore referred to as "multispecific" polypeptides. Such multispecific antibodies may also be referred to as relatively "undevelopable" or relatively "non-developable."

[0061] A "developability profile" refers to an index that can be assigned to an antibody when assessing its developability. A developability profile is a scale or metric by which the developability of anti-CD3 antibodies can be evaluated, compared, and / or ranked. Such a developability profile serves as a measure of the degree of interaction between a CD3-binding substance and an antibody comprising the same. The degree of interaction may be assessed by any number of means available in the art that provide an output value that correlates with the strength or affinity of a polypeptide for a conjugated moiety. Exemplary means include, for example, flow cytometry such as FACS, ELISA, quantitative immunoaffinity assays, immunoprecipitation assays, mammalian two-hybrid assays, or yeast two-hybrid assays. In the case of FACS, as shown in the examples, the degree of interaction between a polypeptide in a plurality of polypeptides and PSR may be ascertained by generating a mean fluorescence intensity (MFI) for each detected polypeptide-PSR interaction, then ordering the MFIs in either ascending or descending order, thereby ranking the polypeptides in the plurality of polypeptides according to the relative degree of interaction between each detected polypeptide and PSR. By performing such a ranking on a plurality of polypeptides, polypeptides with high developability can be easily identified, and polypeptides with low developability can also be easily identified.

[0062] The developability profile may also take the form of a normalized score, for example, by normalizing the developability of an anti-CD3 antibody described herein to the developability of a standard (or control) antibody, such as an anti-HEL antibody.

[0063] In certain embodiments, the CD3-binding domains of the present invention and antibodies comprising the same may be further modified to contain additional nonproteinaceous moieties known and readily available in the art. Suitable moieties for derivatization of antibodies include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (either homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone), polyethylene glycol, polypropylene glycol homopolymer, polypropylene oxide / ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and combinations thereof. Polyethylene glycol propionaldehyde may be advantageous in manufacturing due to its stability in water. The polymer may be of any molecular weight and may be branched or unbranched. The number of polymers attached to an antibody can vary, and when multiple polymers are attached, they can be the same molecule or different molecules. Generally, the number and / or type of polymers used for derivatization can be determined depending on considerations including, but not limited to, the particular property or function of the antibody to be improved, whether the antibody derivative will be used therapeutically under defined conditions, etc.

[0064] In one embodiment, the CD3 binding domains of the present invention and antibodies comprising the same are provided with enhanced developability profiles, which are obtained by performing one or more of a PSR assay, a SCP assay, an AC-SINS assay, an ELISA, a DSF assay, a Tm assay, an HIC assay, a CIC assay, or a combination thereof.

[0065] In other embodiments, the anti-CD3 antibodies and / or antigen-binding fragments thereof described herein exhibit a poly-specificity reagent (PSR) score of about 0.0 to about 0.45, about 0.0 to about 0.4, about 0.0 to about 0.35, about 0.0 to about 0.3, about 0.0 to about 0.25, about 0.0 to about 0.2, about 0.0 to about 0.15, or about 0.0 to about 0.1. A score of 0.0 to 0.1 is a "clean PSR." A score of 0.1 to 0.33 is a "low PSR." A score of 0.33 to 0.66 is a "medium PSR." A score of 0.66 to 1.00 is a "high PSR." A high PSR score indicates reduced (or poor) developability. Generally, the lower the PSR score, the more favorable the antibody's developability.

[0066] In still other embodiments, the anti-CD3 antibodies and / or antigen-binding fragments thereof described herein exhibit an HIC score of less than about 10.5 minutes (clean to low HIC score), between about 10.5 minutes and 11.5 minutes (medium HIC score), or greater than about 11.5 minutes (high HIC score). Generally, the lower the HIC score, the more favorable the antibody's developability.

[0067] In still other embodiments, the anti-CD3 antibodies and / or antigen-binding fragments thereof described herein exhibit an SEC score of less than about 95%, indicating that the antibody is monomeric, i.e., not aggregated.

[0068] In still other embodiments, the anti-CD3 antibodies and / or antigen-binding fragments thereof described herein exhibit a Tm of less than about 65°C.

[0069] In some embodiments, the anti-CD3 antibodies and / or antigen-binding fragments thereof described herein may be further modified to minimize effector function, e.g., a silenced Fc.

[0070] "Effector function" refers to a biological activity attributable to the Fc region of an antibody and varies depending on the antibody isotype. Exemplary effector functions include C1q binding, complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, down-regulation of cell surface receptors (e.g., B cell receptors), and B cell activation.

[0071] The "Fc region" is the C-terminal region of an immunoglobulin heavy chain containing 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 can range from Cys226 or from Pro230 to the carboxyl terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may be present or absent. Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region follows the EU numbering system, also known as the EU index. This system is described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed., Public Health Service, National Institutes of Health, Bethesda, Md., 1991.

[0072] In certain embodiments, one or more amino acid modifications may be introduced into the Fc region of an anti-CD3 antibody of the disclosure, thereby generating an Fc region variant (see, e.g., US2012 / 0251531). The Fc region variant may comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) containing an amino acid modification (e.g., substitution) at one or more amino acid positions.

[0073] In certain embodiments, the present disclosure contemplates anti-CD3 antibody variants that retain some, but not all, effector functions, making them desirable candidates for applications in which in vivo antibody half-life is important but certain effector functions (e.g., complement and ADCC) are unnecessary or deleterious. In vitro and / or in vivo cytotoxicity assays can be performed to confirm the reduction / loss of CDC and / or ADCC activity. For example, Fc receptor (FcR) binding assays can be performed to confirm that the antibody lacks FcγR binding (and thus likely lacks ADCC activity) but retains FcRn binding ability. Primary cells that mediate ADCC (e.g., NK cells) express only FcγIII, whereas 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. Pat. No. 5,500,362 (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. Pat. No. 5,821,337 (see Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assay methods may be employed (see, e.g., ACTI™ Non-Radioactive Cytotoxicity Assay for Flow Cytometry (Cell Technology, Inc., Mountain View, CA); and CytoTox 96® Non-Radioactive Cytotoxicity Assay (Promega, Madison, WI)). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer cells (NK).Alternatively, or in addition, the ADCC activity of the molecule of interest may be assessed in vivo in an animal model, such as that disclosed in Clynes et al. Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). A C1q binding assay may 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 ELISAs in WO2006 / 029879 and WO2005 / 100402. To assess complement activation, a CDC assay may be performed (see, e.g., Gazzano-Santoro et al. J. ImmunoL Methods 202:163 (1996); Cragg, MS et al. Blood. 101:1045-1052 (2003); and Cragg, MS and MJ Glennie Blood. 103:2738-2743 (2004)). FcRn binding and in vivo clearance / half-life determinations can also be performed using methods known in the art (see, e.g., Petkova, SB et al. Int'l. ImmunoL 18(12):1759-1769 (2006)).

[0074] In some embodiments, antibodies with reduced effector function include antibodies with substitutions at one or more of residues 238, 265, 269, 270, 297, 327, and 329 in the Fc region (U.S. Patent Nos. 6,737,056 and 8,219,149). In some embodiments, Fc variants include Fc variants with substitutions at two or more of amino acid positions 265, 269, 270, 297, and 327, including the so-called "DANA" Fc variant in which residues 265 and 297 are substituted with alanine (U.S. Patent Nos. 7,332,581 and 8,219,149).

[0075] 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.

[0076] In some embodiments, the anti-CD3 antibodies and / or antigen-binding fragments thereof described herein are conjugated to a therapeutic moiety, thereby forming 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.

[0077] In certain embodiments, the anti-CD3 antibodies and / or antigen-binding fragments thereof described herein are modified to increase or decrease the extent to which the antibodies are glycosylated. Adding or deleting glycosylation sites from an anti-CD3 antibody of the present disclosure can be readily accomplished by altering the amino acid sequence to create or remove one or more glycosylation sites.

[0078] The present disclosure further relates generally to an anti-CD3 antibody or antibody fragment, the anti-CD3 antibody or antigen-binding antibody fragment comprising: a. a variable heavy chain (VH) polypeptide comprising: i. a VH polypeptide of FNIKDYYMH (SEQ ID NO: 13); H CDR1 (CDRH1), ii. V of WIDLENANTVYDAKFQG (SEQ ID NO: 15) H CDR2 (CDRH2), iii.ARGRYFYDV (SEQ ID NO: 103) H a. a CDR3 (CDRH3), and / or b. a variable light chain (V L ) Polypeptide: i.KSSQSLLNARTGKNYLA (SEQ ID NO: 8) V L CDR1 (CDRL1), V of WASTRX1S (SEQ ID NO: 133) L CDR2 (CDRL2), and V of X2QSYFRRT (SEQ ID NO: 134) LCDR3 (CDRL3), wherein X1 and X2 are each independently any amino acid. In some embodiments, X1 is E or S. In some embodiments, X2 is K or V. In some embodiments, the antibody or antigen-binding antibody fragment comprises a CDRL2 of SEQ ID NO: 10 or SEQ ID NO: 11. In some embodiments, the antibody or antigen-binding antibody fragment comprises a CDRL3 of SEQ ID NO: 5 or SEQ ID NO: 4.

[0079] In some embodiments, the antibody or antigen-binding antibody fragment has a V H Chain CDR1 (CDRH1), V of WIDLENANTVYDAKFQG (SEQ ID NO: 15) H Chain CDR2 (CDRH2), and V of AR-GRYFYDV (SEQ ID NO: 103) H Chain CDR3 (CDRH3), and / or V of KSSQSLLNARTGKNYLA (SEQ ID NO: 8) L Chain CDR1 (CDRL1), V of WASTRES (SEQ ID NO: 10) L Chain CDR2 (CDRL2), and V of KQSYFRRT (SEQ ID NO: 5) L In some embodiments, the antibody or antigen-binding antibody fragment comprises a V CDR3 (CDRL3) of FNIKDYYMH (SEQ ID NO: 13). H Chain CDR1 (CDRH1), V of WIDLENANTVYDAKFQG (SEQ ID NO: 15) H Chain CDR2 (CDRH2), and V of AR-GRYFYDV (SEQ ID NO: 103) H Chain CDR3 (CDRH3), and / or V of KSSQSLLNARTGKNYLA (SEQ ID NO: 8) L Chain CDR1 (CDRL1), V of WASTRSS (SEQ ID NO: 11) L V of VQSYFRRT (SEQ ID NO: 4) Land those comprising the CDR3 (CDRL3) of the antibody designated Ab1. In some embodiments, an anti-CD3 antibody may comprise any one or more CDRs of the antibody designated Ab1 and / or any one or more CDRs of the antibody designated Ab13. In some embodiments, an anti-CD3 antibody may bind to the same or a substantially similar epitope of CD3 as Ab1 and / or Ab13.

[0080] In some embodiments, the anti-CD3 antibody or antigen-binding antibody fragment comprises a V sequence having an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 18 or SEQ ID NO: 34. L In some embodiments, the anti-CD3 antibody or antigen-binding antibody fragment comprises a V chain polypeptide comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 18 or SEQ ID NO: 34. L In some embodiments, the anti-CD3 antibody or antigen-binding antibody fragment comprises a V chain polypeptide comprising the amino acid sequence of SEQ ID NO: 18 or SEQ ID NO: 34. L In some embodiments, the anti-CD3 antibody or antigen-binding antibody fragment comprises a V chain polypeptide comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 17. H In some embodiments, the anti-CD3 antibody or antigen-binding antibody fragment comprises a V chain polypeptide comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 17. H In some embodiments, the anti-CD3 antibody or antigen-binding antibody fragment comprises a V chain polypeptide comprising the amino acid sequence of SEQ ID NO: 17. H Contains chains.

[0081] In some embodiments, the anti-CD3 antibody or antigen-binding antibody fragment comprises a V sequence having an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 18 or SEQ ID NO: 34. L a V chain polypeptide, and an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 17 HIn some embodiments, the anti-CD3 antibody or antigen-binding antibody fragment comprises a V chain polypeptide comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 18 or SEQ ID NO: 34. L a V chain polypeptide, and an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 17 H In some embodiments, the anti-CD3 antibody or antigen-binding antibody fragment comprises a V chain polypeptide comprising the amino acid sequence of SEQ ID NO: 18 or SEQ ID NO: 34. L a V chain polypeptide comprising the amino acid sequence of SEQ ID NO: 17; H In some embodiments, the anti-CD3 antibody or antigen-binding antibody fragment comprises a V chain comprising the amino acid sequence of SEQ ID NO: 18. L a V chain polypeptide comprising the amino acid sequence of SEQ ID NO: 17; H In some embodiments, the anti-CD3 antibody or antigen-binding antibody fragment comprises a V chain comprising the amino acid sequence of SEQ ID NO: 34. L a V chain polypeptide comprising the amino acid sequence of SEQ ID NO: 17; H Contains chains.

[0082] In some embodiments, the anti-CD3 antibody or antigen-binding antibody fragment has a dissociation constant (K) 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. D ) binds to CD3, optionally wherein the CD3 is human and / or cynomolgus monkey CD3, and further optionally wherein the binding affinity is measured by surface plasmon resonance. D is a monovalent K D and / or in this case, the K D is measured using the scFv fragment of the anti-CD3 antibody or antibody fragment.

[0083] The present disclosure further relates generally to anti-cluster of differentiation 3 (CD3) antibodies or antibody fragments, which comprise one or more CDRs of any one or more of Ab1-Ab50 and / or bind to the same epitope as any one or more of Ab1-Ab50. The present disclosure further relates generally to anti-cluster of differentiation 3 (CD3) antibodies or antibody fragments, which comprise a V sequence comprising an amino acid sequence selected from the sequences set forth in Table 4. H Chain and / or V L Contains one or more chains.

[0084] Preparation of anti-CD3 antibodies and their antigen-binding fragments Anti-CD3 antibodies and / or antigen-binding fragments thereof may be produced using recombinant methods. For example, isolated nucleic acids encoding the anti-CD3 antibodies described herein are provided. The nucleic acids may encode an amino acid sequence comprising the antibody VL and / or an amino acid sequence comprising the antibody VH (e.g., the antibody light chain and / or heavy chain). In further embodiments, one or more vectors (e.g., expression vectors) comprising such nucleic acids are provided. In further embodiments, host cells comprising such nucleic acids are provided. In one such embodiment, the host cell comprises (e.g., is transformed with) (1) a vector comprising a nucleic acid encoding an amino acid sequence comprising the antibody VL and an amino acid sequence comprising the antibody VH, or (2) a first vector comprising a nucleic acid encoding an amino acid sequence comprising the antibody VL and a second vector comprising a nucleic acid encoding an amino acid sequence comprising the antibody VH. In one embodiment, the host cell is a eukaryotic cell, such as a Chinese hamster ovary (CHO) cell or a lymphoid cell (e.g., a Y0, NS0, or Sp20 cell). In one embodiment, a method of producing an anti-CD3 antibody is provided, the method comprising culturing a host cell containing nucleic acid encoding the antibody, as described above, under conditions suitable for expression of the antibody, and optionally recovering the antibody from the host cell (or host cell culture medium).

[0085] The term "host cell" refers to a cell into which exogenous nucleic acid has been introduced, including the progeny of such a cell. Host cells include transformants and transformed cells, which include the primary transformed cell and its progeny without regard to the number of transfers.

[0086] For recombinant production of an anti-CD3 antibody, nucleic acid encoding the antibody is isolated, e.g., as described above, and inserted into one or more vectors for further cloning and / or expression in host cells. The nucleic acid can be readily isolated and sequenced using conventional methods (e.g., by using oligonucleotide probes capable of binding specifically to genes encoding the antibody heavy and light chains).

[0087] Suitable host cells for cloning or expressing antibody-encoding vectors include prokaryotic or eukaryotic cells. For example, antibodies can be produced in bacteria, particularly if glycosylation and Fc effector functions are not required. For the 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. (Furthermore, see Charlton, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ, 2003), pp. 245-254, which describes the expression of antibody fragments in E. coli.) After expression, the antibody can be isolated in a soluble fraction from the bacterial cell paste and further purified. In addition to prokaryotes, eukaryotes such as filamentous fungi or yeast, including fungal and yeast strains whose glycosylation pathways have been "humanized" to produce antibodies with partially or fully human glycosylation patterns, are also suitable cloning or expression hosts for antibody-encoding vectors. See, for example, Gerngross, Nat. Biotech. 22:1409-1414 (2004), and Li et al., Nat. Biotech. 24:210-215 (2006); WO2009 / 036379; WO2010 / 105256; and WO2012 / 009568. Plant cell cultures can also be used as hosts. See, for example, U.S. Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (which describe the PL Antibody™ technology for producing antibodies in transgenic plants). Vertebrate cells can also be used as hosts. For example, mammalian trunk cell lines adapted to grow in suspension may be useful.Other examples of useful mammalian host cell lines include SV40 (COS-7) transformed monkey kidney CV1 cells, human embryonic kidney cell lines (e.g., 293 or 293 cells described in Graham et al., J. Gen Virol. 36:59 (1977)), baby hamster kidney cells (BHK), mouse Sertoli cells (e.g., TM4 cells described 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), e.g., Mather et al., Annals TRI cells, MRC 5 cells, and FS4 cells, described in NYAcad. Sci. 383:44-68 (1982). 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 specific mammalian host cell lines suitable for antibody production, see, for example, Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ), pp. 255-268 (2003).

[0088] Anti-CD3 antibodies and / or antigen-binding fragments thereof may be identified, screened, selected, or characterized for their physical / chemical properties and / or biological activity using various assays known in the art, such as ELISA and Western blotting. Alternatively, a competitive assay may be used to identify antibodies that compete with the anti-CD3 antibodies of the present invention for binding to CD3. In an example of a competitive assay, immobilized CD3 is incubated in a solution containing a first labeled antibody that binds to CD3 and a second unlabeled antibody that is tested for its ability to compete with the first antibody for binding to CD3. The second antibody may be present in 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 allow the first antibody to bind to CD3, excess unbound antibody is removed, and the amount of label bound to the immobilized CD3 is measured. A significant decrease in the amount of label bound to immobilized CD3 in the test sample compared to the control sample indicates that the first and second antibodies compete for binding to CD3 (see, e.g., Harlow and Lane (1988) Antibodies: A Laboratory Manual, Ch. 14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY)).

[0089] Biologically active anti-CD3 antibodies and / or antigen-binding fragments thereof may be identified using standard methods. Biological activity may include, for example, binding to CD3 on the surface of T cells, either in vivo, in vitro, or ex vivo. In the case of multispecific anti-CD3 antibodies (e.g., bispecific antibodies having one arm that binds to CD3 and another arm that binds to a different target, such as a tumor antigen or a cell surface antigen), biological activity may also include effector cell activation (e.g., activation of CD8+ T cells and / or CD4+ T cells), expansion of the effector cell population (i.e., an increase in the number of T cells), depletion of the target cell population (i.e., a decrease in the population of cells expressing a second biological molecule on their surface), and / or killing of target cells.

[0090] Diagnostic and therapeutic uses of anti-CD3 antibodies and antigen-binding fragments thereof The anti-CD3 antibodies and / or antigen-binding fragments described herein may be used for diagnosis and / or detection. As used herein, "detection" encompasses quantitative or qualitative detection. In one embodiment, a method for detecting the presence of CD3 in a biological sample is provided. The method includes (i) contacting the biological sample with an anti-CD3 antibody described herein under conditions that allow the anti-CD3 antibody to bind to CD3, and (ii) detecting whether a complex is formed between the anti-CD3 antibody and CD3. The method may be an in vitro method or an in vivo method. In one embodiment, the biological sample comprises cells or tissue.

[0091] In some embodiments, a labeled anti-CD3 antibody is provided. The anti-CD3 antibodies and / or antigen-binding fragments thereof described herein include labels or moieties that are detectable directly (e.g., fluorescent labels, dye labels, electron-dense labels, chemiluminescent labels, and radioactive labels) or indirectly (e.g., enzymes or ligands). Non-limiting examples of 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. Pat. No. 4,737,456); luciferin; 2,3-dihydrophthalazinedione; horseradish peroxidase (HRP); alkaline phosphatase; β-galactosidase; and the like. Examples of suitable oxidases include oxidases, glucoamylases, lysozymes, monosaccharide oxidases such as glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase, heterocyclic oxidases such as uricase and xanthine oxidase coupled with enzymes that use hydrogen peroxide to oxidize dye precursors such as HRP, lactoperoxidase, or microperoxidase, biotin / avidin, spin labels, bacteriophage labels, stable free radicals, and the like.

[0092] The CD3 antibodies and / or antigen-binding fragments thereof described herein, and pharmaceutical compositions of such antibodies, may be used in therapeutic methods. In one embodiment, the anti-CD3 antibodies and / or antigen-binding fragments thereof described herein, or pharmaceutical compositions comprising such antibodies, may be used to treat or delay the progression of a cell proliferative disorder or an autoimmune disorder.

[0093] "Disorder" refers to any condition or disease that would benefit from treatment, including, but not limited to, chronic and acute disorders or diseases, including pathological conditions that predispose the mammal to the disorder in question.

[0094] The terms "cell proliferative disorder" and "proliferative disorder" refer to disorders associated with some degree of abnormal cell proliferation. Cell proliferative disorders include cancers, e.g., tumors.

[0095] As used herein, the term "tumor" refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues.

[0096] "Cancer" refers to a physiological condition in mammals characterized by uncontrolled cell growth. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies, and more specific examples include squamous cell carcinoma (e.g., squamous cell carcinoma), lung cancer including small cell lung cancer, non-small cell lung cancer, lung adenoma and lung squamous cell carcinoma, cancer of the peritoneum, hepatocellular carcinoma, gastric cancer including gastrointestinal cancer and gastrointestinal stromal cancer (gastric cancer or gastric carcinoma), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, cancer of the urinary tract, liver cancer (hepatoma), breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial cancer or uterine cancer, salivary gland cancer, kidney cancer (renal cancer or renal cancer). Cancer), prostate cancer, vulvar cancer, thyroid cancer, liver cancer, anal cancer, penile cancer, melanoma, superficial spreading melanoma, lentigo maligna melanoma, acral lentigo melanoma, nodular melanoma, multiple myeloma and B-cell lymphoma (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 noncleaved cell NHL, Cancers suitable for treatment with the antibodies of the present disclosure 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, melanoma, breast cancer, gastric cancer, colorectal cancer (CRC), and hepatocellular carcinoma, although in some embodiments, the cancer is selected from non-small cell lung cancer, colorectal cancer, glioblastoma, and breast cancer, including metastatic forms of those cancers.In other embodiments, the cancer excludes Hodgkin's lymphoma, but is selected from the group consisting of germinal center B-cell-like (GCB) DLBCL, activated B-cell-like (ABC) DLBCL, follicular lymphoma (FL), mantle cell lymphoma (MCL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), marginal zone lymphoma (MZL), small lymphocytic leukemia (SLL), lymphoplasmacytic lymphoma (LL), Waldenstrom's macroglobulinemia (WM), and leukemia-associated lymphoma (LEM). Central nervous system lymphoma (CNSL), Burkitt lymphoma (BL), B-cell prolymphocytic leukemia, splenic marginal zone lymphoma, hairy cell leukemia, splenic lymphoma / leukemia, unclassifiable, splenic diffuse red pulp small B-cell lymphoma, hairy cell leukemia variant, Waldenstrom's macroglobulinemia, heavy chain disease, alpha heavy chain disease, gamma heavy chain disease, mucous membrane-associated lymphoid tissue disease, plasma cell myeloma, isolated plasmacytoma of bone, extraskeletal plasmacytoma, extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue Zone lymphoma (MALT lymphoma), nodal marginal zone lymphoma, pediatric nodal marginal zone lymphoma, pediatric follicular lymphoma, primary cutaneous follicle-centered lymphoma, T-cell / histiocytic-rich large B-cell lymphoma, primary DLBCL of the CNS, primary cutaneous DLBCL, foot 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, AL Selected from K-positive large B-cell lymphoma, plasmablastic lymphoma, large B-cell lymphoma arising in HHV8-associated multicentric Castleman disease, a class of mature B-cell carcinomas including 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.

[0097] As used herein, "treatment" or "treat" or "treating" refers to clinical intervention in an attempt to alter the natural course of the individual being treated, and can be performed either for preventative purposes or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, prevention of disease onset or recurrence, alleviation of symptoms, reduction of any direct or indirect pathological consequences of the disease, prevention of metastasis, reduction in the rate of disease progression, improvement or palliation of the disease state, and remission or improved prognosis.

[0098] As used herein, the terms "prevent," "preventing," and "prevention" refer to the prevention or inhibition of the development or occurrence of a disorder or disease.

[0099] As used herein, the terms "amelioration" and "alleviation" refer to a decrease or lessening in the severity of a condition or any of its symptoms.

[0100] In some embodiments, the antibodies of the invention are used to delay the onset of a disorder or disease or to slow the progression of a disorder or disease. As used herein, "delaying the progression" of a disorder or disease means postponing, preventing, slowing, inhibiting, stabilizing, and / or postponing the onset of the disorder or disease (e.g., a cell proliferative disorder, e.g., cancer). Delay can be for varying lengths of time, depending on the history of the disease and / or the individual being treated.

[0101] An effective amount of the antibody or composition may be administered to an individual suffering from cancer, or arthritis, rheumatoid arthritis, colitis, inflammatory bowel disease, autoimmune type 1 diabetes, etc. For example, an "effective amount" of an anti-CD3 antibody disclosed herein, or a composition (e.g., a pharmaceutical composition) comprising the antibody, is at least the minimum amount required to achieve a desired therapeutic or prophylactic result, such as a measurable improvement or prevention of a particular disorder, e.g., a cell proliferative disorder such as cancer, preferably without or with minimal toxic or adverse consequences. The effective amount may vary depending, inter alia, on the patient's disease state, age, sex, and weight, and the ability of the antibody (or antigen-binding fragment thereof) to elicit a desired response in the individual, and in some cases, by co-administration with one or more additional therapeutic agents.

[0102] In some embodiments, the anti-CD3 antibodies and / or antigen-binding fragments thereof described herein may be used to enhance immune function in individuals with cell proliferative or autoimmune disorders. Following administration, the antibodies or compositions may enhance immune function in individuals with cell proliferative or autoimmune disorders by activating effector cells (e.g., T cells, such as CD8+ and / or CD4+ T cells, including Tregs), expanding (increasing) the effector cell population, depleting the target cell population (e.g., cells expressing a second biological molecule recognized by an anti-CD3 antibody, such as a bispecific antibody of the invention), and / or killing target cells (e.g., target tumor cells).

[0103] The anti-CD3 antibodies and / or antigen-binding fragments thereof disclosed herein can be used to treat disorders including, but not limited to, proliferative disorders, oncological disorders, immuno-oncological disorders, neurological disorders, cognitive disorders, neurodegenerative disorders, and autoimmune disorders. In one embodiment, an effective amount of the anti-CD3 antibodies, alone or in combination with at least one additional agent, can be administered to an individual with such a disorder. Such an "individual" can be a mammal, particularly a human.

[0104] Non-limiting examples of additional therapeutic agents include chemotherapeutic agents, antibody-drug conjugates (ADCs), and / or biological modifiers. The chemotherapeutic agent may be selected from cyclophosphamide, doxorubicin, vincristine, and prednisolone (CHOP). The ADC may be selected from an anti-CD79b antibody-drug conjugate (e.g., CD79b-MC-vc-PAB-MMAE or an anti-CD79b antibody-drug conjugate described in any one of U.S. Patent No. 8,088,378 and / or US 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.Biological modifiers include BCL-2 inhibitors (e.g., GDC-0199 / ABT-199), lenalidomide (Revlimid®), PI3K-delta inhibitors (e.g., idelalisib (Zydelig®)), PD-1 axis binding antagonists, such as CD40, CD226, CD28, OX40 (e.g., AgonOX), GITR, CD137 (TNFRSF9, 4-1 agonists, such as agonistic antibodies directed against activating costimulatory molecules such as CD27 (also known as BB or ILA), CD27 (e.g., CDX-1127), HVEM or CD127; antagonists, such as antagonist antibodies directed against inhibitory costimulatory molecules 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-675,206, urelumab (also known as BMS-663513), MGA271, antagonists directed against TGF beta, such as, for example, metelimumab (also known as CAT-192), fresolimumab (also known as GC1008), LY2157299k, and adoptive transfer of T cells (e.g., cytotoxic T cells or CTLs) expressing a chimeric antigen receptor (CAR), for example, adoptive transfer of T cells comprising a dominant negative TGF beta receptor, such as a dominant negative TGF beta type II receptor.

[0105] The anti-CD3 antibodies and / or antigen-binding fragments thereof disclosed herein can be used to enhance immune function in individuals, e.g., humans, with such disorders. In one embodiment, a method of enhancing immune function comprises administering to the individual an amount of an anti-CD3 antibody effective to activate effector cells (e.g., T cells, e.g., CD8+ T cells and / or CD4+ T cells), expand (increase) the effector cell population, deplete the target cell population, and / or kill target cells (e.g., target tumor cells).

[0106] In a further aspect, pharmaceutical formulations are also provided that include the anti-CD3 antibodies and / or antigen-binding fragments described herein, e.g., for use in any of the above-described methods of treatment. A "pharmaceutical formulation" refers to a preparation in a form that allows the biological activity of an active ingredient contained therein, such as an anti-CD3 antibody described herein, to be effective, and preferably does not contain additional ingredients that are unacceptably toxic to the subject to which the formulation is administered.

[0107] In one embodiment, a pharmaceutical formulation comprises any of the anti-CD3 antibodies disclosed herein and a pharmaceutically acceptable carrier. A "pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical formulation, other than the active ingredient, that is not toxic to a subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives. In another embodiment, a pharmaceutical formulation comprises any of the anti-CD3 antibodies provided herein and at least one additional therapeutic agent.

[0108] The antibodies of the present disclosure may be used alone or in combination with other agents in a treatment, for example, an anti-CD3 antibody and / or antigen-binding fragment thereof may be co-administered 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 anti-angiogenic agent, an apoptotic agent, an anti-tubulin agent, or other 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: ErbB3, ErbB4, PDGFR-beta, BIyS, APRIL, BCMA VEGF or VEGF receptor, TRAIL / Apo2, PD-1, PD-L1, PD-L2, or other bioactive or organic chemical agent.

[0109] In some embodiments, the present disclosure provides methods wherein the additional therapeutic agent is a glucocorticoid. In one embodiment, the glucocorticoid is dexamethasone.

[0110] Such combination therapy encompasses mixed administration (two or more therapeutic agents in the same formulation or in separate formulations) and separate administration, where administration of an antibody of the present disclosure can occur before, simultaneously with, and / or after administration of the additional therapeutic agent. In one embodiment, administration of an anti-CD3 antibody and administration of an 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. Anti-CD3 antibodies of the present disclosure (e.g., bispecific anti-CD3 antibodies of the present invention that bind to CD3 and a second biological molecule, e.g., a cell surface antigen such as a tumor antigen, e.g., a TDB antibody or variant thereof of the present invention) can also be used in combination with radiation therapy.

[0111] The disclosed antibodies (and / or any additional therapeutic agents) can be administered by any suitable means, including parenteral, intrapulmonary, and intranasal administration, or, if localized treatment is desired, by intralesional administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. In some embodiments, the antibody is administered subcutaneously. In some embodiments, an anti-CD3 antibody administered by subcutaneous injection exhibits a lower toxic response in patients than the same anti-CD3 antibody administered by intravenous injection. Dosing can be by any suitable route, e.g., by injection, e.g., intravenous or subcutaneous injection, depending in part on whether the administration is temporary or chronic. Various dosing schedules are contemplated herein, including, but not limited to, a single dose or multiple doses over various time points, a bolus dose, and pulse infusion.

[0112] The antibodies of the present disclosure are formulated, dosed, and administered in a manner consistent with sound medical practice. Factors to consider 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 method of administration, the administration schedule, and other factors known to medical professionals. Although not required, the antibodies may optionally 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 other factors discussed above. These will generally be used at the same doses and by the same routes of administration as those described herein, or at approximately 1-99% of the doses described herein, or at any dose and by any route empirically / clinically determined to be appropriate.

[0113] The appropriate dose of the disclosed antibodies (whether used alone or in combination with one or more other additional therapeutic agents) for the prevention or treatment of disease will depend on the type of disease being treated, the type of antibody, the severity and course of the disease, whether the antibody is being administered for prophylactic or therapeutic purposes, previous treatments, the patient's medical history and responsiveness to the antibody, and the discretion of the attending physician. The antibody is suitably administered to the patient at one time or over a series of treatments.

[0114] As a general rule, a therapeutically effective amount of an anti-CD3 antibody administered to a human, whether administered as a single dose or multiple doses, will be in the range of about 0.01 to about 100 mg / kg of patient body weight. In some embodiments, the antibody used is administered daily at, for example, 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. In one embodiment, an 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 on day 1 of a 21-day cycle. The dose may be administered as a single dose or multiple doses (e.g., two or three doses), e.g., by infusion. When administered repeatedly over several days or longer, treatment is generally sustained until a desired suppression of disease symptoms occurs, depending on the condition. One exemplary dose of the antibody ranges from about 0.05 mg / kg to about 10 mg / kg. Thus, one or more doses of about 0.5 mg / kg, 2.0 mg / kg, 4.0 mg / kg, or 10 mg / kg (or any combination thereof) may be administered to the patient. Such doses may be administered intermittently, for example, weekly or every three weeks (e.g., the patient receives about two to about twenty, or, for example, about six, doses of anti-CD3 antibody). An initial higher loading dose may be administered, followed by one or more lower loading doses. The progress of this therapy is easily monitored by conventional techniques and assays.

[0115] In some embodiments, the disclosed methods may further comprise an additional therapy. The additional therapy may be radiation therapy, surgery, chemotherapy, gene therapy, DNA therapy, viral therapy, RNA therapy, immunotherapy, bone marrow transplant, nanotherapy, monoclonal antibody therapy, or a combination of the foregoing. The additional therapy may be in the form of adjuvant therapy or neoadjuvant therapy. In some embodiments, the additional therapy is administration of a small molecule enzyme inhibitor or an anti-metastatic agent. In some embodiments, the additional therapy is administration of a side effect-limiting agent (e.g., an agent intended to reduce the occurrence and / or severity of side effects of the therapy, such as an antiemetic). 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 the separate administration of one or more of the aforementioned therapeutic agents.

[0116] Another aspect of the present disclosure provides an article of manufacture containing materials useful for the treatment, prevention, and / or diagnosis of the aforementioned disorders. The article of manufacture includes a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, etc. The container may be formed from a variety of materials, such as glass or plastic. The container may hold the composition alone or in combination with another composition effective for the treatment, prevention, and / or diagnosis of a condition, and may have a sterile access port (e.g., the container may be an intravenous solution bag or a vial with a stopper pierceable by a hypodermic injection 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 for treating the selected condition. Additionally, the article of manufacture may include (a) a first container containing a composition contained therein, the composition comprising an antibody of the present invention, and (b) a second container containing a composition contained therein, the composition comprising an additional cytotoxic agent or another therapeutic agent. The article of manufacture in this embodiment of the invention may further include a package insert indicating that the composition can be used to treat a particular condition. Alternatively, or in addition, the article of manufacture may further include 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 include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

[0117] Accordingly, the manufacture and / or preparation of pharmaceutical compositions comprising the anti-CD3 antibodies and / or antigen-binding fragments disclosed herein are also contemplated. The compositions may be used alone or in combination with other active agents to treat cell proliferative disorders (e.g., cancer) or autoimmune disorders (e.g., arthritis, rheumatoid arthritis, colitis, inflammatory bowel disease, autoimmune type 1 diabetes, etc.).

[0118] In some embodiments, pharmaceutical compositions comprising the anti-CD3 antibodies and / or antigen-binding fragments thereof described herein are prepared, for example, by combining the antibodies having the desired purity with one or more pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)) in the form of a lyophilized formulation or an aqueous solution, optionally prepared for modified (e.g., sustained) release. Exemplary lyophilized antibody formulations are described in U.S. Pat. No. 6,267,958. Aqueous antibody formulations include those described in U.S. Pat. No. 6,171,586 and WO 2006 / 044908, the latter of which contains a histidine-acetate buffer.

[0119] Pharmaceutically acceptable carriers are generally non-toxic to recipients at the dosages and concentrations employed and include, but are not limited to, buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl alcohol or benzyl alcohol, alkyl parabens such as methyl paraben or propyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol); low molecular weight (less than about 10 residues) polypeptides; Examples of pharmaceutically acceptable carriers include 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 dextrin, chelating agents such as EDTA, sugars such as sucrose, mannitol, trehalose, or sorbitol, salt-forming counterions such as sodium, metal complexes (e.g., Zn-protein complexes), and / or non-ionic surfactants such as polyethylene glycol (PEG). Pharmaceutically acceptable carriers herein also include interstitial drug dispersion agents, such as soluble neutral-active hyaluronidase glycoprotein (sHASEGP), for example, human soluble PH-20 hyaluronidase glycoprotein, for example, rHuPH20 (HYLENEX®, Baxter International, Inc.). Certain exemplary sHASEGPs, including rHuPH20, and methods of using same are described in U.S. Patent Application Publications 2005 / 0260186 and 2006 / 0104968.

[0120] Such formulations may contain more than one active ingredient, if necessary for the particular indication being treated, preferably ingredients with complementary activities that do not adversely affect each other, and that are present in amounts that are effective for the purpose intended. For example, it may be desirable to further provide additional therapeutic agents (e.g., chemotherapeutic agents, cytotoxic agents, growth inhibitory agents, and / or antihormonal agents, etc.).

[0121] The active ingredient may be encapsulated in microcapsules prepared by droplet formation techniques or by interfacial polymerization, such as hydroxymethylcellulose or gelatin microcapsules, and poly(methyl methacrylate) microcapsules, in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules), or in the form of macroemulsions, respectively. Such techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980). [Example]

[0122] [Table 1]

[0123] [Table 2]

[0124] [Table 3]

[0125] [Table 4]

[0126] [Table 5]

[0127] material and method In addition to the above description, the following materials and methods were employed in this example.

[0128] FACS affinity pressure selection method. Briefly, yeast cells (at least approximately 2 x 10 7 Cells (labeling condition) were incubated with biotinylated antigen in sufficient stoichiometric excess relative to the average IgG presentation. Antigen labeling conditions were 100–1 nM under equilibration conditions, typically performed for 20 minutes to several hours at room temperature in FACS wash buffer (phosphate-buffered saline (PBS) / 0.1% bovine serum albumin (BSA)). After three washes with wash buffer, yeast were stained for 15 minutes at 4°C with a 1:100 dilution of the secondary reagent anti-human light chain FITC conjugate (LC-FITC) and either a 1:500 dilution of streptavidin-633 (SA-633) or a 1:50 dilution of extraavidin-phycoerythrin (EA-PA). After two washes with ice-cold wash buffer, the cell pellet was stained with a 1x10 IgG antibody. 7 Yeast were resuspended in at least 1 mL of wash buffer per yeast and transferred to a strainer-capped sort tube. Sorting was performed using a FACS ARIA sorter (BD Biosciences) to determine sort gating and select binders. After the final round of sorting, yeast were plated and individual colonies were picked and characterized.

[0129] FACS heat-stress selection method. Parent antibodies were diversified by error-prone PCR to derive an optimized library in yeast. This yeast library first underwent a positive antigen selection round and was selected for binder expression using CD3εN27. The enriched population was then subjected to a series of heat-stress conditions ranging from approximately 50°C to a maximum of 65°C for 10 minutes, with room temperature as a control. Optimal pressure conditions were gated on LC presentation (anti-human lambda PE) and antigen binding (SA-APC), reflecting residual folded IgG capable of binding to the selection reagent. Sorted cells were pelleted, and plasmids were extracted using a commercially available yeast plasmid purification kit (Zymo Research). In this kit, the yeast cell wall is disrupted with Zymolase, and DNA is subsequently purified using DNA minicolumns. Plasmid DNA was then transformed into E. coli for amplification, and plasmid DNA was isolated by miniprep using an E. coli plasmid purification kit (Qiagen). Plasmid DNA is then prepared for transformation into an appropriate yeast strain followed by cycle selection or sequence analysis and IgG production.

[0130] Optimization of error-prone PCR. Stochastic diversity was introduced by error-prone PCR-based mutagenesis of the heavy chain (VH) and / or light chain (VL) using standard molecular biology techniques. Briefly, the mutagenic nucleotide analogs dPTP and 8-oxo-dGTP were incorporated into the VH and VL amplification process at a concentration of 1 μM to increase the frequency of base misincorporation by approximately 0.01 bp. The mutated PCR products were recombined in situ by homologous recombination with linearized vectors containing the HC or LC constant region sequences. Typically, this resulted in 1x10 7-8 A diverse library of 1000 nucleotides was obtained. The antigen-antibody-yeast complex was incubated for varying periods of time with decreasing concentrations of antigen (equilibrium pressure) or parental Fab competitor (equilibrium and dynamic pressure), applying joint pressure on affinity and expression, and the highest affinity antibodies were selected on FACS during successive selection rounds.

[0131] Oligonucleotide-Based CDR H3 Mutagenesis. Discovered or previously optimized antibodies can be further optimized by diversifying the CDR H3 sequence. To do so, the light chain variable region of the starting antibody is PCR-amplified and then inserted into a yeast strain containing a light chain empty vector using yeast homologous recombination. This becomes the parent light chain yeast strain. The heavy chain of the starting antibody is used as PCR input in combination with germline-specific primers, which generate a PCR product containing frameworks 1 through 3 of the heavy chain. This amplification is performed using the mutagenic nucleotide 8-oxo-dGTP, resulting in additional low-level mutations in the amplified heavy chain region. To generate engineered diversity in the CDR H3 region of the starting antibody, a library of CDR H3 oligos is generated / ordered (i.e., from IDT). The oligo pool is amplified using a primer containing a 5' tail that allows germline-specific recombination with the amplified FW1-FW3 region and an empty plasmid. A universal 3' primer is used for FW4. Alternatively, mutagenic PCR can be performed to incorporate 8-oxo-dGTP into the PCR reaction product using a germline-specific 5' primer, a universal 3' primer, and VH DNA. Once the LC strain, HC FW1-FW3, and diversified CDR H3-FW4 inputs are generated, a three-piece transformation is performed by introducing the two HC components into the LC strain along with the HC empty vector. Cells are then grown under selective pressure to confirm the presence of the HC and LC components.

[0132] Yeast production and purification of antibodies. Yeast clones were grown to saturation and then induced for 48 hours at 30°C with shaking. After induction, yeast cells were pelleted and the supernatant was collected for purification. IgG was purified using a Protein A column and eluted with acetic acid at pH 2.0. Fab fragments were generated by papain digestion and purified with KappaSelect or CaptureSelect IgG-CH1 (GE Healthcare LifeSciences).

[0133] HEK production and purification of antibodies. Mammalian expression of IgG was achieved by subcloning the antibody into a new expression vector, followed by transient transfection and expression in HEK293ADI1. A monoclonal cell line derived from HEK293 (DSMZ) was selected for its aggregation-free growth, growth rate, and transfectability. Briefly, the expression vector containing the antibody of interest was transfected by complexation with a transfection reagent, followed by exposure to HEK cells for 1 hour, followed by dilution of the culture medium to a final density of 4 million cells per mL. The cells were then cultured for 7 days, with fresh feed medium changes every 48 hours. After 7 days, the supernatant was collected after centrifugation and purified using Protein A. Where necessary, CHT column purification was added to achieve >95% monomericity.

[0134] ForteBio KD Measurement (BioLayer Interferometry; BLI). ForteBio affinity measurements were generally performed as previously reported (Estep, P., et al., High throughput solution-based measurement of antibody-antigen affinity and epitope binning. MAbs, 2013.5(2):pp.270-8). Briefly, ForteBio affinity measurements were performed by online loading of IgG onto the AHQ sensor. The sensor was equilibrated offline in assay buffer for 30 minutes and then monitored online for 60 seconds to establish a baseline. The IgG-loaded sensors were exposed to 100 nM antigen for 5 minutes, after which they were transferred to assay buffer for 5 minutes and dissociation rate measurements were performed. A 1:1 binding model was used to analyze the reaction kinetics.

[0135] BiaCore KD measurements (surface plasmon resonance; SPR). Biosensor analysis was performed at 25°C using a Biacore 8K optical biosensor (GE Healthcare, Marlborough, MA) equipped with a CM5 sensor chip in an HBS-EP buffer system (10 mM HEPES pH 7.3, 150 mM NaCl, 3 mM EDTA, 0.05% Surfactant P20). The sample solution was maintained at 10°C. Goat anti-human IgG capture antibodies (Jackson Immunoresearch Laboratories, Inc., West Grove, PA; 109-005-098) were immobilized to both flow cells of the sensor chip using standard amine coupling chemistry (11,700 + / - 400 RU). This type of surface provided a novel format for highly reproducible analytical antigen capture after each replication step. Flow cell 2 was used to analyze the captured antigen (35.7 + / - 0.8 RU). Flow cell 1 was used as the reference flow cell. Fab concentrations ranging from 100 to 0.412 nM (3-fold dilutions) were prepared in running buffer. Each Fab sample concentration was run as a single duplicate. Two blank (buffer) injections were also performed and used to evaluate and subtract system artifacts. The association and dissociation phases of all Fab concentrations were monitored for 180 seconds at a flow rate of 30 μL / min, respectively. The surface was regenerated with 10 mM glycine, pH 1.5, for 30 seconds at a flow rate of 30 μL / min. Data were aligned, double-referenced, and fitted using Biacore 8K Evaluation Software version 1.0.

[0136] Octet Red 384 Epitope Binning / Ligand Blocking. Epitope binning / ligand blocking was performed using a standard sandwich-format cross-blocking assay. A control anti-target IgG was loaded onto the AHQ sensor, and the available Fc binding sites on the sensor were blocked with an irrelevant human IgG1 antibody. The sensor was then exposed to 100 nM of target antigen, followed by a second anti-target antibody or ligand. Data were processed using ForteBio's Data Analysis Software 7.0. Additional binding of the second antibody or ligand after antigen association indicates an available epitope (non-competitor), whereas a lack of binding indicates a blocked epitope (competitor or ligand blocking).

[0137] Size-exclusion chromatography. For rapid SEC analysis of yeast- and mammalian-produced mAbs, a TSKgel SuperSW mAb HTP column (22855) was used at 0.4 mL / min with a cycle time of 6 min / run. 200 mM sodium phosphate and 250 mM sodium chloride were used as the mobile phase.

[0138] Dynamic Scanning Fluorimetry (DSF). 10 μL of 20x Sypo Orange was added to 20 μL of 0.2–1 mg / mL mAb or Fab solution. Using an RT-PCR instrument (BioRad CFX96 RT PCR), the temperature of the sample plate was increased from 40°C to 95°C in 0.5°C increments, with equilibration at each temperature for 2 minutes. Tm was estimated using the negative of the first derivative of the raw data.

[0139] PSR Preparation. The polyspecific reaction reagent (PSR) was prepared as described, for example, in WO2014 / 179363 and Xu et al., Mabs, 2013. Briefly, 2.5 liters of CHO-S cells were used as starting material. In a 500 mL centrifuge bottle filled to 400 mL, the cells were pelleted at 2,400 x g for 5 minutes. The cell pellets were combined and then resuspended in 25 mL of Buffer B and pelleted at 2,400 x g for 3 minutes. The buffer was decanted, and the wash was repeated once more. The cell pellets were resuspended in 3x the pellet volume of Buffer B containing 1x protease inhibitor (Roche, Complete, EDTA-free) using a Polytron homogenizer while keeping the cells on ice. The homogenate was then centrifuged at 2,400 × g for 5 minutes, and the supernatant was retained and pelleted once more (2,400 × g / 5 minutes) to ensure removal of unbroken cells, cell debris, and nuclei. The resulting supernatant constituted the total protein preparation. The supernatant was then transferred to two Nalgene Oak Ridge 45 mL centrifuge tubes and pelleted at 40,000 × g for 40 minutes at 4°C. The supernatant containing the separated cytosolic proteins (SCP) was then transferred to a clean Oak Ridge tube and centrifuged once more at 40,000 × g. In parallel, the pellet containing the membrane fraction (EMF) was retained and centrifuged at 40,000 × g for 20 minutes to remove any remaining supernatant. The EMF pellet was then rinsed with Buffer B. 8 mL of Buffer B was then added to the membrane pellet, which was then scraped and transferred to a Dounce homogenizer. After the pellets were homogenized, they were transferred to a 50 mL conical tube to represent the final EMF preparation.

[0140] about 10 6 ~10 7One billion cells / mL of mammalian cells (e.g., CHO, HEK293, Sf9, etc.) were transferred from tissue culture medium into four 250 mL conical tubes and pelleted at 550 x g for 3 minutes. All subsequent steps were performed at 4 °C or on ice using ice-cold buffers. The cells were washed with 100 mL of PBSF (1x PBS + 1 mg / mL BSA) and combined in one conical tube. After removing the supernatant, the cell pellet was resuspended in 30 mL of buffer B (50 mM HEPES, 0.15 M NaCl, 2 mM CaCl, 5 mM KCl, 5 mM HCl). The cells were resuspended in 10% MgCl2, 10% glycerol, pH 7.2, and pelleted at 550 x g for 3 min. The supernatant in Buffer B was decanted, and the cells were resuspended in 3x the pellet volume of Buffer B supplemented with 2.5x protease inhibitors (Roche, cOmplete, EDTA-free). From this point on, protease inhibitors were included in Buffer B. The cells were homogenized (Polyton homogenizer, PT1200E) with four 30-second pulses, and the membrane fraction was pelleted at 40,000 x g for 1 h at 4°C. The pellet was collected in 1 mL of PBS. The pellet was transferred to a Dounce homogenizer with 3 mL of buffer B and resuspended by slowly moving the pestle up and down 30-35 times. The enriched membrane fraction (EMF) was transferred to a new collection tube, and the pestle was rinsed to collect all potential proteins. The protein concentration of the purified EMF was determined using a Dc-protein assay kit (BioRad). To solubilize the EMF, solubilization buffer (50 mM NaCl) was added. The mixture was transferred to a final concentration of 1 mg / mL in a 50 mL Oak Ridge tube (Fisher Scientific, 050529-ID) containing 1% n-dodecyl-bD-maltopyranoside (DDM), 1x protease inhibitors, pH 7.2. The mixture was rotated overnight at 4°C and then centrifuged at 40,000 x g for 1 hour in a 50 mL Oak Ridge tube (Fisher Scientific, 050529-ID). The supernatant, which contains soluble membrane proteins (SMPs), was collected and protein production was quantified as described above.

[0141] For biotinylation, prepare NHS-LC-biotin stock solution according to the manufacturer's protocol (Pierce, Thermo Fisher). Briefly, 20 μl of biotin reagent was added to every 1 mg of EMF sample and incubated at 4°C for 3 hours with gentle agitation. Adjust the volume to 25 mL with Buffer B and transfer to an Oak Ridge centrifuge tube. Precipitate biotinylated EMF (b-EMF) at 40,000 × g for 1 hour and rinse twice with 3 mL of Buffer C (Buffer B minus glycerol) without disturbing the pellet. Remove residual solution. Resuspend the pellet in 3 mL of Buffer C using a Dounce homogenizer as described above. The resuspended pellet is now biotinylated EMF (b-EMF). It was solubilized as described above for preparing b-SMP.

[0142] PSR binding analysis. Assays were generally performed as described, for example, in Xu et al. To characterize the PSR profile of monoclonal antibodies displayed on yeast, 2 million IgG-displaying yeast cells were transferred to a 96-well assay plate, pelleted at 3000 x g for 3 minutes, and the supernatant was removed. The pellet was resuspended in 50 μl of a 1:10 dilution of freshly prepared stock b-PSR and incubated on ice for 20 minutes. The cells were washed twice with 200 μl of ice-cold PBSF and resuspended in 50 μl of secondary labeling mix (Extravidin-R-PE, anti-human LC-FITC, and propidium iodide). This mix was incubated on ice for 20 minutes, followed by two washes with 200 μl of ice-cold PBSF. The cells were resuspended in 100 μl of ice-cold PBSF and plated on a FACSCanto (BD Biosciences) using an HTS sample injector. Flow cytometry data were analyzed for mean fluorescence intensity in the R-PE channel and normalized to appropriate controls for assessment of nonspecific binding. Many methods for displaying or presenting antibodies or antibody fragments on yeast surfaces have been previously reported, all of which are compatible with 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).

[0143] ForteBio Kinetics. A ForteBio Octet HTX instrument was used in 12-channel mode (8 sensors per channel, 96 sensors per experiment) with either AHC, SA, or AHQ sensors. The instrument was operated with manufacturer-supplied software (versions 8.2 and 9.0). Sample names and concentrations were entered on the plate data page, and protein bound to the sensor was identified in the "Information" column on the sensor data page. Kinetic experiments were collected with either a 90- or 180-second baseline, a 180-second association phase, and a 180-second dissociation phase. Binning experiments were collected in five steps: a 90-second baseline 1, a 90-second sensor binding check with the secondary binder, a 90-second baseline 2, 180 seconds of binding, and a 180-second dissociation in wells containing the secondary mAb. All files were saved to a shared network drive using a naming convention that identifies the experiment type.

[0144] HIC. The IgG1 sample was buffer exchanged into 1 M ammonium sulfate and 0.1 M sodium phosphate at pH 6.5 using a Zeba 40 kDa 0.5 mL spin column (Thermo Pierce, catalog number 87766). A salt gradient was established on a Dionex ProPac HIC-10 column from 1.8 M ammonium sulfate and 0.1 M sodium phosphate at pH 6.5 to the same conditions 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 for over 7 column volumes before the next injection cycle. Peak retention times were monitored by absorbance at A280, and the ammonium sulfate concentration at elution was calculated based on the gradient and flow rate.

[0145] LCMS. The mAb sample was reduced with DTT and then subjected to mid-down LCMS analysis on a Bruker maXis 4G mass spectrometer coupled to an Agilent 1100 HPLC (Agilent). A POROS R2 10 μm (2.1 x 30 mm) reversed-phase column was used to remove salts from the sample. A fast LC flow of 2 mL / min allowed for separation of the sample and salt, allowing sample elution and column regeneration to be completed within a 2.1-minute cycle. A T-junction was used to deliver a sample flow of only 0.15 mL / min to the mass spectrometer for sample analysis. The Bruker maXis 4G mass spectrometer was operated 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 at 5500 V, the nebulizer at 4.0 bar, the drying gas at 4.0 L / min, and the drying temperature at 200 °C.

[0146] MS spectra were analyzed using Bruker Data Analysis version 4.1, and deconvolution was performed using maximum entropy deconvolution in the mass range of 20–30 kDa.

[0147] A concise sequence listing is provided below in Table 4. The concise sequence listing provides the amino acid sequence of the heavy chain variable region (HC), with each of the heavy chain variable region CDRs underlined. The amino acid sequence of the light chain variable region (LC) is also provided, with each of the light chain variable region CDRs underlined.

[0148] [Table 6]

[0149] [Table 7]

[0150] [Table 8]

[0151] Table 9

[0152] Table 10

[0153] Table 11

[0154] Table 12

[0155] Table 13

[0156] Table 14

[0157] Table 15

[0158] Table 16

[0159] Table 17

[0160] Table 18

[0161] Table 19

[0162] Table 20

[0163] Table 21

[0164] Table 22

[0165] Table 23

[0166] Table 24

[0167] Table 25

[0168] Table 26

[0169] Table 27

[0170] Table 28

[0171] Table 29

[0172] Table 30

[0173] Table 31

[0174] Table 32

[0175] Table 33 Embodiments of the present invention Embodiments of the present invention are further described in the following sections: [Section 1] An anti-cluster of differentiation 3 (CD3) antibody or antigen-binding antibody fragment, the anti-CD3 antibody or antigen-binding antibody fragment comprising: a. i.V of FNIKDYYMH (SEQ ID NO: 13) H CDR1 (CDRH1), ii. V of WIDLENANTVYDAKFQG (SEQ ID NO: 15) H CDR2 (CDRH2), iii. V of ARDAYGRYFYDV (SEQ ID NO: 103) H Variable weight (V) containing CDR3 (CDRH3) H ) chain polypeptides, and / or b. i.KSSQSLLNARTGKNYLA (SEQ ID NO: 8) L CDR1 (CDRL1), ii.WASTRX 1 S (SEQ ID NO: 133) V L CDR2 (CDRL2), and iii.X 2 QSYFRRT (SEQ ID NO: 134) V L Variable light chain (V) containing CDR3 (CDRL3) L ) chain polypeptide, X 1 and X 2 and each independently represents any amino acid. [Section 2] aX 1 is E or S, and / or bX 2 is K or V. [Section 3] The antibody or antigen-binding antibody fragment comprises: a. CDRL2 of SEQ ID NO: 10 or SEQ ID NO: 11, and / or b. The anti-CD3 antibody or antigen-binding antibody fragment of paragraph 1 or 2, which comprises CDRL3 of SEQ ID NO:5 or SEQ ID NO:4. [Section 4] The anti-CD3 antibody or antigen-binding antibody fragment a. V of FNIKDYYMH (SEQ ID NO: 13) H Chain CDR1 (CDRH1), V of WIDLENANTVYDAKFQG (SEQ ID NO: 15) H Chain CDR2 (CDRH2), and V of ARDAYGRYFYDV (SEQ ID NO: 103) H Chain CDR3 (CDRH3), and / or V of KSSQSLLNARTGKNYLA (SEQ ID NO: 8) L Chain CDR1 (CDRL1), V of WASTRES (SEQ ID NO: 10) L Chain CDR2 (CDRL2), and V of KQSYFRRT (SEQ ID NO: 5) L those comprising the CDR3 (CDRL3) of the amino acid sequence ... b. V of FNIKDYYMH (SEQ ID NO: 13) H Chain CDR1 (CDRH1), V of WIDLENANTVYDAKFQG (SEQ ID NO: 15) H Chain CDR2 (CDRH2), and V of ARDAYGRYFYDV (SEQ ID NO: 103) H Chain CDR3 (CDRH3), and / or V of KSSQSLLNARTGKNYLA (SEQ ID NO: 8) L Chain CDR1 (CDRL1), V of WASTRSS (SEQ ID NO: 11) L V of VQSYFRRT (SEQ ID NO: 4) L 4. The anti-CD3 antibody or antigen-binding antibody fragment according to any one of items 1 to 3, wherein the anti-CD3 antibody or antigen-binding antibody fragment comprises a CDR3 (CDRL3) of the nucleotide sequence of the target gene. [Section 5] The anti-CD3 antibody or antigen-binding antibody fragment a. V comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 18 or SEQ ID NO: 34 L chain polypeptide, b. V comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 18 or SEQ ID NO: 34 L chain polypeptide, c. V comprising the amino acid sequence of SEQ ID NO: 18 or SEQ ID NO: 34 L chain polypeptide, d. V comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 17 H chain polypeptide, e. V comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 17 H chain polypeptides, and / or f. V comprising the amino acid sequence of SEQ ID NO: 17 H 5. The anti-CD3 antibody or antigen-binding antibody fragment according to any one of items 1 to 4, comprising a chain. [Section 6] The anti-CD3 antibody or antigen-binding antibody fragment a. V comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 18 or SEQ ID NO: 34 L a V chain polypeptide, and an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 17 H chain polypeptide, b. V comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 18 or SEQ ID NO: 34 L a V chain polypeptide, and an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 17 H chain polypeptide, c. V comprising the amino acid sequence of SEQ ID NO: 18 or SEQ ID NO: 34 L a V chain polypeptide comprising the amino acid sequence of SEQ ID NO: 17; H chain, d. V comprising the amino acid sequence of SEQ ID NO: 18 L a V chain polypeptide comprising the amino acid sequence of SEQ ID NO: 17; H chains, and / or e. V comprising the amino acid sequence of SEQ ID NO: 34 L a V chain polypeptide comprising the amino acid sequence of SEQ ID NO: 17; H 6. The anti-CD3 antibody or antigen-binding antibody fragment according to any one of items 1 to 5, comprising a chain. [Section 7] The anti-CD3 antibody or antigen-binding antibody fragment has a dissociation constant (K D 7. The anti-CD3 antibody or antigen-binding antibody fragment of any one of items 1 to 6 above, wherein the anti-CD3 antibody or antigen-binding antibody fragment binds to CD3 via a nucleotide sequence (SEQ ID NO: 1), optionally wherein the CD3 is human and / or cynomolgus monkey CD3, and optionally wherein the binding affinity is measured by surface plasmon resonance. [Section 8] The above K D is a monovalent K D and / or said K D 8. The anti-CD3 antibody or antigen-binding antibody fragment according to Item 7, wherein the antibody binding activity is measured using an scFv fragment of the anti-CD3 antibody or antibody fragment. [Section 9] a. the antibody or antigen-binding antibody fragment, while eliciting T cell activation or T cell killing, exhibits a reduced tendency to elicit cytokine production to levels capable of inducing cytokine release syndrome; b. the antibody or antigen-binding antibody fragment comprises a multispecific antibody; c. the antibody or antigen-binding antibody fragment comprises a bispecific antibody; d. the antibody or antigen-binding antibody fragment comprises an scFv; e. the antibody or antigen-binding antibody fragment comprises at least a second antigen-binding domain that specifically binds to a tumor target, an immune-oncology target, a neurodegenerative disease target, an autoimmune disorder target, an infectious disease target, a metabolic disease target, a cognitive disorder target, a blood-brain barrier target, or a hematological disorder target; f. The antibody or antigen-binding antibody fragment is selected from the group consisting of 17-IA, 4-1BB, 4Dc, 6-keto-PGF1a, 8-iso-PGF2a, 8-oxo-dG, Al adenosine receptor, 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, ADAM15, and ADAM17 / T. ACE, ADAM8, ADAM9, ADAMTS, ADAMTS4, ADAMTS5, addressin, aFGF, ALCAM, ALK, ALK-1, ALK-7, alpha-l-antitrypsin, alpha-V / beta-1 antagonist, ANG, Ang, APAF-1, APE, APJ, APP, APRIL, AR, ARC, ART, artemin, anti-Id, ASPARTIC, atrial natriuretic factor, av / b3 integrin, Axl, b2M, B7-1, B7-2, B7-H, B-lymphocyte stimulatory factor (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, BMP-3 osteogenin, BMP-4 BMP-2b, BMP-5, BMP-6Vgr-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), BMPs, b-NGF, BOK, bombesin, bone-derived neurotrophic factor, BPDE, BPDE-DNA, BTC, complement factor 3 (C3), C3a, C4, C5, C5a, CIO, CA125, CAD-8, calcitonin, cAMP, carcinoembryonic antigen (CEA), tumor-associated antigen, cathepsin A, catecholamines 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, CCL14, 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, C D10, CDlla, CDllb, CDllc, CD13, CD14, CD15, CD16, CD18, CD19, CD20, CD21, CD22, CD23, CD25, CD27L, CD28, CD29, CD30, CD30L, CD32, CD33( p67 protein), 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, botulinum toxin, Clostridium perfringens toxin, CKb8-l, CLC, CMV, CMVUL, CNTF, CNTN-1, COX, C-Ret, CRG-2, CT-1, CTACK, CTGF, CTLA-4, CX3CL1, CX3CR1, CXCL, CXCL1, CXCL2, CXCL3, CXCL 4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCR, CXCR1, CXCR 2, CXCR3, CXCR4, CXCR5, CXCR6, cytokeratin tumor-associated antigen, DAN, DCC, DcR3, DC-SIGN, decay-accelerating factor, des(l-3)-IGF-I (brain IGF-1), 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-alpha1, GFR-alpha2, GFR-alpha3, GITR, glucagon, Glut 4, glycoprotein Ilb / IIIa (GP Ilb / IIIa), GM-CSF, gpl30, gp72, GRO, growth hormone-releasing factor, hapten (NP-cap or NIP-cap), HB-EGF, HCC, HCMV gBEnvelope glycoprotein, HCMV gH envelope glycoprotein, HCMV UL, hemopoietic growth factor (HGF), Hep B gpl20, heparanase, Her2, Her2 / neu (ErbB-2), Her3 (ErbB-3), Her4 (ErbB-4), herpes simplex virus (HSV) gB glycoprotein, HSV gD glycoprotein, HGFA, high molecular weight melanoma-associated antigen (HMW-MAA), HIV gpl20, HIV IIIB gp 120 V3 loop, 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 protein, 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, interferon (INF)-alpha, INF-beta, INF-gamma, inhibin, iNOS, insulin A-chain, insulin B-chain, insulin Integrin-like growth factor 1, integrin 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, interferon gamma, IP-10, l-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, latent TGF-1 bpl, LBP, LDGF, LECT2, Lefty, Lewis-Y antigen, Lewis-Y related antigen, LFA-1, LFA-3, Lfo, LIF, LIGHT, lipoprotein, LIX, LKN, Lptn, L-selectin, LT-a, LT-b, LTB4, LTBP-1, pulmonary surfactant, progesterone, lymphotoxin beta receptor, Mac-1, MAdCAM, MAG, MAP2, MARC, MCAM, MCAM, MCK-2, MCP, M-CSF, MDC, Mer, metalloproteases enzyme, MGDF receptor, MGMT, MHC (HLA-DR), MIF, MIG, MIP, MIP-1-alpha, 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 (Muc1), MUC18, Müllerian inhibitory factor, Mug, MuSK, NAIP, NAP, NCAD, N-cadherin, NCA90, NCAM, NCAM, neprilysin, neurotrophin-3, -4, or -6, neurturin, nerve growth factor (NGF), NGFR, NGF-beta, nNOS, NO, NOS, Npn, NRG-3, N T, 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, protein C, 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 factor, 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 receptor (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, TGF-beta pan-specific (PanSpecific) TGF-link RI(ALK-5), TGF-link RII, TGF-link Rl lb, TGF-link RIII, TGF-link l, TGF-link 2, TGF-link 3, TGF-lin 4, TGF-factor 5, glycoprotein Ck-1, activator of Tie, TIMP, and TI Q, TMEFF2, Tmpo, TMPRSS2, TNF, TNF-protein, TNF-protein Liquids, TNF-link 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(WHEM ATAR、HveA、LIGHT R、TR2)、TNFRSF16(NGFR p75NTR), TNFRSF17(BCMA), TNFRSF 18 (GITR AITR), TNFRSF19 (TROY CROWN), 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 (TRANCE / RANK ligand ODF, 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 ligand) gp34, TXGP1), TNFSF5 (CD40 ligand CD154, gp39, HIGM1, IMD3, TRAP), TNFSF6 (Fas ligand Apo-1 ligand, APT1 ligand), TNFSF7 (CD27 ligand CD70), TNFSF8 (CD30 ligand CD153), TNFSF9 (4-1BB ligand CD137 ligand), TP-1, t-PA, Tpo, TRAIL, TRAIL R, TRAIL-R1, TRAIL-R2, TRANCE, transfer receptor, TRF, Trk, TROP-2, TSG, TSLP, tumor-associated antigen CA125, Lewis Y-related glycoprotein-expressing tumor-associated 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 antigen, VLA, VLA-1, VLA-4, VNR integrin, von V. at least a second antigen-binding domain that specifically binds to an antigen selected from the group consisting of lebrand 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 immunoglobulin and mucin protein-3), hormone receptors, and growth factors; g. the antibody or antigen-binding antibody fragment comprises at least a second antigen-binding domain that specifically binds to an antigen selected from the group consisting of BCMA, cytotoxic T lymphocyte antigen-4 (CTLA4), programmed cell death protein 1 (PD1), programmed cell death ligand 1 (PD-L1), lymphocyte activation gene-3 (LAG-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; h. the antibody or antigen-binding antibody fragment is contained within a chimeric antigen receptor (CAR), the CAR optionally comprising at least one transmembrane domain and at least one intracellular domain derived from a T cell receptor, optionally comprising a CD3 zeta subunit, and at least one costimulatory domain; i. the antibody or antigen-binding antibody fragment comprises scFv2-Fc2 and / or scFv-IgG; j. the antibody or antigen-binding antibody fragment comprises an IgG constant domain, and / or k. The anti-CD3 antibody or antigen-binding antibody fragment of any one of items 1 to 8, wherein the antibody or antigen-binding antibody fragment comprises at least a second antigen-binding domain that specifically binds to an antigen, and the antibody or antigen-binding antibody fragment is in a multispecific format selected from the group consisting of Fab-Fc-scFv (bottle opener type), Mab-scFv, Mab-Fv, dual scFv, central Fv, central scFv, one-arm central scFv, Fab-Fab, Fab-Fv, mAb-Fv, mAb-Fab, DART, BiTE, common light chain-IgG, TandAb, Cross-Mab, SEED, BEAT, TrioMab, and DuetMab. [Section 10] An isolated or recombinant nucleic acid sequence encoding the antibody or antigen-binding antibody fragment according to any one of items 1 to 9 above. [Section 11] An expression vector containing the isolated or recombinant nucleic acid sequence according to item 10 above. [Section 12] A host cell transfected, transformed or transduced with a nucleic acid sequence according to paragraph 10 above, optionally a mammalian or yeast cell containing said nucleic acid sequence, or a vector. [Section 13] A pharmaceutical composition comprising the antibody or antigen-binding antibody fragment according to any one of items 1 to 12 above, and a pharmaceutically acceptable carrier and / or excipient. [Section 14] A method for treating a disorder in a mammal in need of such treatment, wherein the disorder comprises a proliferative disorder, an oncological disorder, an immune-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 in any one of paragraphs 1 to 9 above, or a host cell, optionally an immune cell, and optionally a T cell or an NK cell, expressing one of the antibodies or antibody fragments described in paragraph 12 above. [Section 15] 15. The method of claim 14, wherein the method further comprises administering an additional therapeutic agent to the mammal, and optionally the mammal is a human. [Section 16] An anti-cluster of differentiation 3 (CD3) antibody or antibody fragment, the anti-CD3 antibody or antigen-binding antibody fragment comprising: aX 1 QSYFRRT (SEQ ID NO: 1) V L Variable light chain (V) containing CDR3 (CDRL3) L ) chain polypeptide, optionally containing X 1 is K, A, T, or V, bX 3 RDAYGX4YFYDV (SEQ ID NO: 6) H Variable weight (V) containing CDR3 (CDRH3) H ) chain polypeptide, optionally containing X 3 is A or V, and X 4 is R or Q, c.KSSQSLLNARTX 5 KNYLA (SEQ ID NO: 7) V L Variable light chain (V) containing CDR1 (CDRL1) L ) chain polypeptide, optionally containing X 5 is G, M, N, or R, d. V of WASTRES (SEQ ID NO: 10) or WASTRSS (SEQ ID NO: 11) L Variable light chain (V) containing CDR2 (CDRL2) L ) chain polypeptide, e.FNX 6 KDYYX 7 H (SEQ ID NO: 12) V H Variable weight (V) containing CDR1 (CDRH1) H ) chain polypeptide, 6 is I, N or V, and X 7 is M or I, and / or f.WIDLX 8 NANTVYDX 9 KX 10 QG (SEQ ID NO: 14) V H Variable weight (V) containing CDR2 (CDRH2)H ) chain polypeptide, 8 is E or N, and X 9 is A, H, or T, and X 10 is F or L, including anti-CD3 antibodies or antibody fragments. [Section 17] aX 1 is A, T, or V, b. the CDRL3 comprises the amino acid sequence of AQSYFRRT (SEQ ID NO:2), TQSYFRRT (SEQ ID NO:3), VQSYFRRT (SEQ ID NO:4), or KQSYFRRT (SEQ ID NO:5); cX 3 is A, dX 5 is G or R, e. said CDRL1 comprises the amino acid sequence of KSSQSLLNARTGKNYLA (SEQ ID NO: 8) or KSSQSLLNARTRKNYLA (SEQ ID NO: 9); f. said CDRL2 comprises the amino acid sequence of WASTRES (SEQ ID NO: 10); g. said CDRL2 comprises the amino acid sequence of WASTRSS (SEQ ID NO: 11); hX 6 is I, iX 7 is M, j. said CDRH1 comprises the amino acid sequence of FNIKDYYMH (SEQ ID NO: 13); kX 8 is E, l.X 9 is A, mX 10 is F, n. said CDRH2 comprises the amino acid sequence of WIDLENANTVYDAKFQG (SEQ ID NO: 15), and / or o. The anti-CD3 antibody or antigen-binding antibody fragment of paragraph 16, wherein the CDRH2 comprises the amino acid sequence WIDLENANTIYDAKFQG (SEQ ID NO: 16). [Section 18] a. The anti-CD3 antibody or antigen-binding antibody fragment has a dissociation constant (K) of about 500 pM or more, 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 D ), wherein optionally said CD3 is human and / or cynomolgus monkey CD3, and further optionally said binding affinity is measured by surface plasmon resonance; b. the antibody or antigen-binding antibody fragment, while eliciting T cell activation or T cell killing, exhibits a reduced tendency to elicit cytokine production to levels capable of inducing cytokine release syndrome; c. the antibody or antigen-binding antibody fragment comprises a multispecific antibody; d. the antibody or antigen-binding antibody fragment comprises a bispecific antibody; e. the antibody or antigen-binding antibody fragment comprises an scFv; f. the antibody or antigen-binding antibody fragment comprises at least a second antigen-binding domain that specifically binds to a tumor target, an immune-oncology target, a neurodegenerative disease target, an autoimmune disorder target, an infectious disease target, a metabolic disease target, a cognitive disorder target, a blood-brain barrier target, or a hematological disease target; g. The antibody or antigen-binding antibody fragment is selected from the group consisting of 17-IA, 4-1BB, 4Dc, 6-keto-PGF1, 8-iso-PGF2a, 8-oxo-dG, Al adenosine receptor, A33, ACE, ACE-2, activin, activin A, activin AB, activin B, activin C, activin RIA, activin RIA ALK-2, and activin RIB. ALK-4, activin RIIA, activin RUB, ADAM, ADAM10, ADAM12, ADAM15, ADAM17 / TACE, ADAM8, ADAM9, ADAMTS, ADAMTS4, ADAMTS5, addressin, aFGF, ALCAM, ALK, ALK-1, ALK-7, alpha-l-antitrypsin, alpha-V / beta-1 antagonist, ANG, Ang, APAF-1, APE, APJ, APP, APRIL, AR, ARC, ART, artemin, anti-Id, ASPARTIC, atrial natriuretic factor, av / b3 Integrin, Axl, b2M, B7-1, B7-2, B7-H, B-lymphocyte stimulatory factor (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, BMP-3 osteogenin, BMP-4 BMP-2b, BMP-5, BMP-6Vgr-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), BMPs, b-NGF, BOK, bombesin, bone-derived neurotrophic factor, BPDE, BPDE-DNA, BTC, complement factor 3 (C3), C3a, C4, C5, C5a, CIO, CA125, CAD-8, calcitonin, cAMP, carcinoembryonic antigen (CEA), tumor-associated antigen, cathepsin A, catecholamines 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, CCL14, 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, C D10, CDlla, CDllb, CDllc, CD13, CD14, CD15, CD16, CD18, CD19, CD20, CD21, CD22, CD23, CD25, CD27L, CD28, CD29, CD30, CD30L, CD32, CD33( p67 protein), 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, botulinum toxin, Clostridium perfringens toxin, CKb8-l, CLC, CMV, CMVUL, CNTF, CNTN-1, COX, C-Ret, CRG-2, CT-1, CTACK, CTGF, CTLA-4, CX3CL1, CX3CR1, CXCL, CXCL1, CXCL2, CXCL3, CXCL 4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCR, CXCR1, CXCR 2, CXCR3, CXCR4, CXCR5, CXCR6, cytokeratin tumor-associated antigen, DAN, DCC, DcR3, DC-SIGN, decay-accelerating factor, des(l-3)-IGF-I (brain IGF-1), 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-alpha1, GFR-alpha2, GFR-alpha3, GITR, glucagon, Glut 4, glycoprotein Ilb / IIIa (GP Ilb / IIIa), GM-CSF, gpl30, gp72, GRO, growth hormone-releasing factor, hapten (NP-cap or NIP-cap), HB-EGF, HCC, HCMV gBEnvelope glycoprotein, HCMV gH envelope glycoprotein, HCMV UL, hemopoietic growth factor (HGF), Hep B gpl20, heparanase, Her2, Her2 / neu (ErbB-2), Her3 (ErbB-3), Her4 (ErbB-4), herpes simplex virus (HSV) gB glycoprotein, HSV gD glycoprotein, HGFA, high molecular weight melanoma-associated antigen (HMW-MAA), HIV gpl20, HIV IIIB gp 120 V3 loop, 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 protein, 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, interferon (INF)-alpha, INF-beta, INF-gamma, inhibin, iNOS, insulin A-chain, insulin B-chain, insulin Integrin-like growth factor 1, integrin 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, interferon gamma, IP-10, l-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, latent TGF-1 bpl, LBP, LDGF, LECT2, Lefty, Lewis-Y antigen, Lewis-Y related antigen, LFA-1, LFA-3, Lfo, LIF, LIGHT, lipoprotein, LIX, LKN, Lptn, L-selectin, LT-a, LT-b, LTB4, LTBP-1, pulmonary surfactant, progesterone, lymphotoxin beta receptor, Mac-1, MAdCAM, MAG, MAP2, MARC, MCAM, MCAM, MCK-2, MCP, M-CSF, MDC, Mer, metalloprotein Ze, MGDF receptor, MGMT, MHC (HLA-DR), MIF, MIG, MIP, MIP-1-alpha, 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, Müllerian inhibitory factor, Mug, MuSK, NAIP, NAP, NCAD, N-cadherin, NCA90, NCAM, NCAM, neprilysin, neurotrophin-3, -4, or -6, neurturin, nerve growth factor (NGF), NGFR, NGF-beta, nNOS, NO, NOS, Npn, NRG-3, N T, 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, protein C, 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 factor, 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 receptor (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, TGF-beta pan-specific (PanSpecific) TGF-link RI(ALK-5), TGF-link RII, TGF-link Rl lb, TGF-link RIII, TGF-link l, TGF-link 2, TGF-link 3, TGF-lin 4, TGF-factor 5, glycoprotein Ck-1, activator of Tie, TIMP, and TI Q, TMEFF2, Tmpo, TMPRSS2, TNF, TNF-protein, TNF-protein Liquids, TNF-link 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(WHEM ATAR、HveA、LIGHT R、TR2)、TNFRSF16(NGFR p75NTR), TNFRSF17(BCMA), TNFRSF 18 (GITR AITR), TNFRSF19 (TROY CROWN), 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 (TRANCE / RANK ligand ODF, 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 ligand) gp34, TXGP1), TNFSF5 (CD40 ligand CD154, gp39, HIGM1, IMD3, TRAP), TNFSF6 (Fas ligand Apo-1 ligand, APT1 ligand), TNFSF7 (CD27 ligand CD70), TNFSF8 (CD30 ligand CD153), TNFSF9 (4-1BB ligand CD137 ligand), TP-1, t-PA, Tpo, TRAIL, TRAIL R, TRAIL-R1, TRAIL-R2, TRANCE, transfer receptor, TRF, Trk, TROP-2, TSG, TSLP, tumor-associated antigen CA125, Lewis Y-related glycoprotein-expressing tumor-associated 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 antigen, VLA, VLA-1, VLA-4, VNR integrin, von V. at least a second antigen-binding domain that specifically binds to an antigen selected from the group consisting of lebrand 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 attenuator-4), PD1 (programmed cell death protein 1), PD-L1 (programmed cell death ligand 1), LAG-3 (lymphocyte activation gene-3), TIM-3 (T cell immunoglobulin and mucin protein-3), a hormone receptor, and a growth factor; h. the antibody or antigen-binding antibody fragment comprises at least a second antigen-binding domain that specifically binds to an antigen selected from the group consisting of BCMA, cytotoxic T lymphocyte antigen-4 (CTLA4), programmed cell death protein 1 (PD1), programmed cell death ligand 1 (PD-L1), lymphocyte activation gene-3 (LAG-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; i. the antibody or antigen-binding antibody fragment is contained within a chimeric antigen receptor (CAR), the CAR optionally comprising at least one transmembrane domain and at least one intracellular domain derived from a T cell receptor, optionally comprising a CD3 zeta subunit, and at least one costimulatory domain; j. the antibody or antigen-binding antibody fragment comprises scFv2-Fc2 and / or scFv-IgG; k. the antibody or antigen-binding antibody fragment comprises an IgG constant domain; and / or l. The anti-CD3 antibody or antigen-binding antibody fragment of paragraph 16 or 17, wherein the antibody or antigen-binding antibody fragment comprises at least a second antigen-binding domain that specifically binds to an antigen, and the antibody is in a multispecific format selected from the group consisting of Fab-Fc-scFv (bottle opener), Mab-scFv, Mab-Fv, dual scFv, central Fv, central scFv, one-arm central scFv, Fab-Fab, Fab-Fv, mAb-Fv, mAb-Fab, DART, BiTE, common light chain-IgG, TandAb, Cross-Mab, SEED, BEAT, TrioMab, and DuetMab. [Section 19] An anti-cluster of differentiation 3 (CD3) antibody or antibody fragment, wherein the anti-CD3 antibody or antigen-binding antibody fragment comprises one or more CDRs of any one or more of Ab1-Ab50. [Section 20] An anti-cluster of differentiation 3 (CD3) antibody or antibody fragment, wherein the anti-CD3 antibody or antigen-binding antibody fragment comprises an amino acid sequence selected from the sequences set forth in Table 4. H Chain and / or V L An anti-CD3 antibody or antibody fragment comprising one or more chains. [Section 21] 21. An isolated or recombinant nucleic acid sequence encoding the antibody or antigen-binding antibody fragment according to any one of items 16 to 20 above. [Section 22] 22. An expression vector containing the isolated or recombinant nucleic acid sequence according to 21 above. [Section 23] 22. A host cell transfected, transformed or transduced with a nucleic acid sequence according to paragraph 21 above, optionally a mammalian or yeast cell containing said nucleic acid sequence, or a vector. [Section 24] A pharmaceutical composition comprising the antibody or antigen-binding antibody fragment according to any one of items 16 to 20 above, and a pharmaceutically acceptable carrier and / or excipient. A method for treating a disorder in a mammal in need of such treatment, wherein the disorder comprises a proliferative disorder, an oncological disorder, an immune-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 according to any one of items 16 to 20 above, or a host cell expressing one of the antibodies or antibody fragments according to item 23 above, optionally an immune cell, and optionally a T cell or an NK cell.

Claims

1. An anti-cluster of differentiation 3 (CD3) antibody or antigen-binding antibody fragment, comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH and the VL comprise the amino acid sequences of SEQ ID NOs: 17 and 34, respectively.

2. scFv, Fv, Fab, Fab', Fab'-SH, F(ab') ２ 2. The anti-CD3 antibody or antigen-binding antibody fragment of claim 1, comprising a diabody, scFv2-Fc2, or scFv-IgG.

3. (A) a heavy chain comprising the VH and heavy chain constant region (CH); and (B) a light chain comprising the VL and light chain constant region (CL).

3. The anti-CD3 antibody or antigen-binding antibody fragment of claim 1 or 2, comprising:

4. 4. The anti-CD3 antibody or antigen-binding antibody fragment of claim 3, comprising an immunoglobulin molecule comprising two of the heavy chains and two of the light chains interconnected by disulfide bonds, or a multimeric form of the immunoglobulin molecule.

5. 5. The anti-CD3 antibody or antigen-binding antibody fragment of claim 3 or 4, which is or comprises an IgG, IgA, IgD, IgE, or IgM antibody.

6. 5. The anti-CD3 antibody or antigen-binding antibody fragment of claim 3 or 4, which is or comprises an IgG1, IgG2, IgG3, IgG4, IgA1 or IgA2 antibody.

7. The anti-CD3 antibody or antigen-binding antibody fragment of any one of claims 1 to 6, which is comprised in a chimeric antigen receptor (CAR).

8. The anti-CD3 antibody or antigen-binding antibody fragment of claim 7, wherein the CAR comprises at least one transmembrane domain, and at least one intracellular domain derived from a T cell receptor, and at least one costimulatory domain.

9. The anti-CD3 antibody or antigen-binding antibody fragment of claim 8, wherein the at least one intracellular domain is derived from the CD3ζ subunit.

10. 10. The anti-CD3 antibody or antigen-binding antibody fragment of any one of claims 1 to 9, comprising an IgG constant domain.

11. 11. The anti-CD3 antibody or antigen-binding antibody fragment of any one of claims 1 to 10, which is or comprises a multispecific antibody.

12. 12. The anti-CD3 antibody or antigen-binding antibody fragment of any one of claims 1 to 11, which is or comprises a bispecific antibody.

13. 13. The anti-CD3 antibody or antigen-binding antibody fragment of any one of claims 1 to 12, comprising at least a second antigen-binding domain that specifically binds to a tumor target, an immune-tumor target, a neurodegenerative disease target, an autoimmune disorder target, an infectious disease target, a metabolic disease target, a cognitive disorder target, a blood-brain barrier target, or a hematological disorder target.

14. 17-IA, 4-1BB, 4Dc, 6-keto-PGF1a, 8-iso-PGF2a, 8-oxo-dG, Al adenosine receptor, 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, ADAM15, ADAM17 / T ACE, ADAM8, ADAM9, ADAMTS, ADAMTS4, ADAMTS5, addressin, aFGF, ALCAM, ALK, ALK-1, ALK-7, alpha-l-antitrypsin, alpha-V / beta-1 antagonist, ANG, Ang, APAF-1, APE, APJ, APP, APRIL, AR, ARC, ART, artemin, anti-Id, ASPARTIC, atrial natriuretic factor, av / b3 Integrin, Axl, b2M, B7-1, B7-2, B7-H, B-lymphocyte stimulatory factor (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, BMP-3 osteogenin, BMP-4 BMP-2b, BMP-5, BMP-6Vgr-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), BMPs, b-NGF, BOK, bombesin, bone-derived neurotrophic factor, BPDE, BPDE-DNA, BTC, complement factor 3 (C3), C3a, C4, C5, C5a, CIO, CA125, CAD-8, calcitonin, cAMP, carcinoembryonic antigen (CEA), tumor-associated antigen, cathepsin A, catecholamines 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, CCL14, 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, C D10, CDlla, CDllb, CDllc, CD13, CD14, CD15, CD16, CD18, CD19, CD20, CD21, CD22, CD23, CD25, CD27L, CD28, CD29, CD30, CD30L, CD32, CD33 ( p67 protein), 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, botulinum toxin, Clostridium perfringens toxin, CKb8-1, CLC, CMV, CMVUL, CNTF, CNTN-1, COX, C-Ret, CRG-2, CT-1, CTACK, CTGF, CTLA-4, CX3CL1, CX3CR1, CXCL, CXCL1, CXCL2, CXCL3, CXCL 4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCR, CXCR1, CXCR 2, CXCR3, CXCR4, CXCR5, CXCR6, cytokeratin tumor-associated antigen, DAN, DCC, DcR3, DC-SIGN, decay-accelerating factor, des(1-3)-IGF-I (brain IGF-1), 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 I, 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, GFRα-1, GFR-alpha1, GFR-alpha2, GFR-alpha3, GITR, glucagon, Glut 4, glycoprotein Ilb / IIIa (GP Ilb / IIIa), GM-CSF, gpl30, gp72, GRO, growth hormone-releasing factor, hapten (NP-cap or NIP-cap), HB-EGF, HCC, HCMV gBEnvelope glycoprotein (HCMV) gH envelope glycoprotein, HCMV UL, hemopoietic growth factor (HGF), Hep B gpl20, heparanase, Her2, Her2 / neu (ErbB-2), Her3 (ErbB-3), Her4 (ErbB-4), herpes simplex virus (HSV) gB glycoprotein, HSV gD glycoprotein, HGFA, high molecular weight melanoma-associated antigen (HMW-MAA), HIV gpl20, HIV IIIB gp 120 V3 Loop, 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 protein, 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, interferon (INF)-alpha, INF-beta, INF-gamma, inhibin, iNOS, insulin A-chain, insulin B-chain, insulin Integrin-like growth factor 1, integrin 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, 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, latent TGF-1 bpl, LBP, LDGF, LECT2, Lefty, Lewis-Y antigen, Lewis-Y related antigen, LFA-1, LFA-3, Lfo, LIF, LIGHT, lipoprotein, LIX, LKN, Lptn, L-selectin, LT-a, LT-b, LTB4, LTBP-1, pulmonary surfactant, progesterone, lymphotoxin beta receptor, Mac-1, MAdCAM, MAG, MAP2, MARC, MCAM, MCK-2, MCP, M-CSF, MDC, Mer, metalloprin Protease, MGDF receptor, MGMT, MHC (HLA-DR), MIF, MIG, MIP, MIP-1-alpha, 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, Müllerian inhibitory factor, Mug, MuSK, NAIP, NAP, NCAD , N-cadherin, NCA90, NCAM, NCAM, neprilysin, neurotrophin-3, -4, or -6, neurturin, nerve growth factor (NGF), NGFR, NGF-beta, nNOS, NO, NOS, Npn, NRG-3, NT, NTN, OB, OGG1, OPG, OPN, OSM, OX40L, OX40R, p150, p95, PADPr, parathyroid hormone, PARC, PARP, PBR, PBSF, PCAD, P-cadherin, PCNA, PDGF, PDG F, PDK-1, PECAM, PEM, PF4, PGE, PGF, PGI2, PGJ2, PIN, PLA2, placental alkaline phosphatase (PLAP), PIGF, PLP, PP14, proinsulin, prorelaxin, protein C, 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, RSVFgp, Ret, rheumatoid factor, 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 receptor, T cell receptor alpha / beta, TdT, TECK, TEM1, TEM5, TEM7, TEM8, TERT, testicular PLAP-like alkaline phosphatase, TfR, TGF, TGF-alpha, TGF-beta, TGF-beta pan-specific (Pan Specific), TGF-betaRI (ALK-5), TGF-betaRII, TGF-betaRIIb, TGF-betaRIII, TGF-beta1, TGF-beta2, TGF-beta3, TGF-beta4, TGF-beta5, thrombin, thymic Ck-1, thyroid stimulating hormone, Tie, TIMP, TIQ, tissue factor, TMEFF2, Tmpo, TMPRSS2, TNF, TNF-alpha, TNF-alpha beta, TNF-beta2, TNFc, TNF-RI, TNF-RII, TNFRSF10A (TRAIL R1), Apo-2, DR4, TNFRSF10B (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 H14) 、SHOCKS H13(SYS)、SHOCKS H130 S)、SHASHS14(SHAS SHAS、SHYS、CHAS S、SY2)、SYSYS16(SYS) 07SYS、SYSYS17(SYSYS)、SYSYS 18(SYS) SHYSY、SYSYS10(SYS3 WHY、WHYWH、WHYWHYWHYWHYWHYWHYWHYWHYWHY 100000000000000000000000000000000000000000000000000000000000 H1200、F75806、SHOSH2 6(SYS3)、SYSYS3(SYS THIS SUCH、SHIS S6、SHOSCHS4(SYS)0 SIS33、SHIS1 S) 、WHYS 5(SH40 50)、SWHYS6 DA11、HAR1、DAY5)、DYSCHYS(DYS3 H68、S6)、S6S67(S027)、 FASHION(SHR30)、SHASHES(400) HR137、HARSH、SHRYSY21 DASH) 、SHASH22(DASH) 2 SHY2)、SYSYS23(SYSYS) SHYS1)、SHYSYS25(GY3 FASH3、SHASH、SHASH、SHASH 、SHAKE1)、SHASE100(SHARE). DAY2リガンド、LOY2)、SHAS11(SHAS SHASE RHリガンドDAY 、DAYリガンド)、DAYS12(SIR). DA3リガンド、DAY3リガンド)、DAYDAY13(SYS) LOVE2)、SIGNIFICANCE130 LOVE SY、 LOVE1 LOVE LOVE LOVE LOVE LOVE LOVE LIKEリガンド、THE)、SHASE15(30). SUCH SUCH 18(SUSHリガンドSUBJECTリガンド、CHASE) FASHION、DYS、SYSYS)、SYSYSYS LIKE、SYSYS1)、SYSYS(LOVE THIS、D33) THIS 4(S40リガンドW34、SH1)、SHOSIS5(CHA40リガンドH154、N39、NH10、13 、WATCHES、DAYSJS(THEリガンドDA11リガンド 、DA1リガンド) DASH 7(KS27リガンドCD70), TNFSF8 (CD30 ligand CD153), TNFSF9 (4-1BB ligand CD137 ligand), TP-1, t-PA, Tpo, TRAIL, TRAIL R, TRAIL-R1, TRAIL-R2, TRANCE, transfer receptor, TRF, Trk, TROP-2, TSG, TSLP, tumor-associated antigen CA 125, tumor-associated antigens expressing Lewis Y-related carbohydrates, 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 V Revland factor, WIF-1, WNT1, WNT2, WNT2B / 13, WNT3, WNT3A, WNT4, WNT5A, WNT5B, WNT6, WNT7A, WNT7B, WNT8A, WNT8B, WN T9A, WNT9A, WNT9B, WNT10A, WNT10B, WNT11, WNT16, XCL1, XCL2, XCR1, XCR1, XEDAR, XIAP, XPD, CTLA4 (cytotoxic The anti-CD3 antibody or antigen-binding antibody fragment of any one of claims 1 to 13, comprising at least a second antigen-binding domain that specifically binds to an antigen selected from the group consisting of 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 immunoglobulin and mucin protein-3), a hormone receptor, and a growth factor.

15. BCMA, CTLA4 (cytotoxic lymphocyte antigen-4), PD1 (programmed cell death protein 1), PD-L1 (programmed cell death ligand) 1), LAG-3 (lymphocyte activation 15. The anti-CD3 antibody or antigen-binding antibody fragment of any one of claims 1 to 14, comprising at least a second antigen-binding domain that specifically binds to an antigen selected from the group consisting of: FcγRIIIa (CD16), FcγRIIa (CD32a), FcγRIIb (CD32b), FcγRI (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.

16. 16. The anti-CD3 antibody or antigen-binding antibody fragment of any one of claims 1 to 15, comprising a multispecific format selected from the group consisting of Fab-Fc-scFv (bottle opener type), Mab-scFv, Mab-Fv, dual scFv, central Fv, central scFv, one-arm central scFv, Fab-Fab, Fab-Fv, mAb-Fv, mAb-Fab, DART, BiTE, common light chain-IgG, TandAb, Cross-Mab, SEED, BEAT, TrioMab, and DuetMab.

17. An isolated or recombinant nucleic acid encoding the anti-CD3 antibody or antigen-binding antibody fragment of any one of claims 1 to 16.

18. 18. An expression vector containing the isolated or recombinant nucleic acid of claim 17.

19. 18. A host cell transfected, transformed or transduced with the nucleic acid of claim 17 or a vector containing said nucleic acid.

20. 20. The host cell of claim 19, which is a eukaryotic cell.

21. 20. The host cell of claim 19, which is a mammalian cell or a yeast cell.

22. 20. The host cell of claim 19, which is a Chinese hamster ovary (CHO) cell, a human embryonic kidney (HEK) cell, or a lymphoid cell.

23. 20. The host cell of claim 19, which is a prokaryotic cell.

24. 20. The host cell of claim 19, which is a bacterial cell.

25. A pharmaceutical composition comprising the anti-CD3 antibody or antigen-binding antibody fragment of any one of claims 1 to 16, and a pharmaceutically acceptable carrier and / or excipient.

26. 26. A pharmaceutical composition for treating a disorder in a mammal in need of such treatment, comprising the anti-CD3 antibody or antigen-binding antibody fragment of any one of claims 1 to 16, or the host cell of any one of claims 19 to 24 expressing said anti-CD3 antibody or antigen-binding antibody fragment, wherein said disorder comprises a proliferative disorder, an oncological disorder, an immune-oncological disorder, a neurological disorder, a neurodegenerative disorder, or an autoimmune disorder.

27. 27. The pharmaceutical composition of claim 26, wherein the host cell comprises an immune cell.

28. 28. The pharmaceutical composition of claim 26 or 27, wherein the host cell comprises a T cell or an NK cell.

29. The pharmaceutical composition of any one of claims 26 to 28, further comprising or administered in combination with an additional therapeutic agent.

30. The pharmaceutical composition according to any one of claims 26 to 29, wherein the mammal is a human.

31. 26. Use of the anti-CD3 antibody or antigen-binding antibody fragment of any one of claims 1 to 16, or the host cell of any one of claims 19 to 24 expressing said anti-CD3 antibody or antigen-binding antibody fragment, in the manufacture of a medicament for 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.

32. 32. The use of claim 31 , wherein the host cell comprises an immune cell.

33. 33. The use of claim 31 or 32, wherein the host cell comprises a T cell or an NK cell.

34. The use according to any one of claims 31 to 33, wherein the treatment further comprises the use of an additional therapeutic agent.

35. The use according to any one of claims 31 to 34, wherein the mammal is a human.

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