PH-dependent anti-CD3 antibodies and methods related thereto

Abstract

The present disclosure provides anti-CD3 antibodies and antigen binding fragments as well as multispecific antibodies and antibody fragments thereof. The disclosure also provides nucleic acids and vectors encoding such antibodies or antibody fragments as well as cells comprising such nucleic acids. Also provided are pharmaceutical compositions, in vivo methods, screening methods, and methods of preparation related to the anti-CD3 antibodies and antigen binding fragments.

Description

[0001] Cross - Reference to Related Applications

[0002] This application claims priority to U.S. Provisional Application No. 63 / 417,118, entitled "PH - DEPENDENT ANTI - CD3 ANTIBODIES AND METHODS RELATING THERETO", filed on October 18, 2022, the content of which is incorporated herein by reference in its entirety.

[0003] Reference to Electronic Sequence Listing

[0004] The content of the electronic sequence listing (1160430o003813.xml; size: 508,346 bytes; creation date: October 11, 2023) is incorporated herein by reference in its entirety. Background of the Invention

[0005] Cell - proliferative disorders such as cancer are characterized by the uncontrolled growth of subsets of cells. They are the leading cause of death in developed countries and the second leading cause of death in developing countries, and the total number of new cancer cases per year is expected to increase to 23.6 million by 2030. The National Cancer Institute estimates that nearly 2 million new cancer cases will be diagnosed in the United States and that more than 600,000 Americans will die of cancer in 2020. Thus, cancer care represents a significant and growing social burden.

[0006] The idea of using the cytotoxic capabilities of T cells to kill tumor cells by using 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 directed against a disease-related antigen targeted, such as an antigen produced by tumor cells. CD3 bispecific antibodies bind both to the CD3 surface receptor on T cells and to their second target antigen, such as a protein expressed on a tumor, such that available T cells can bind to target-expressing cells via the bridging of the CD3 bispecific antibody, regardless of the peptide / MHC specificity of their T cell receptor. (See, e.g., Bassan, 2012, Blood 120:5094-95). Bridging T cells and tumor cells using CD3 bispecific antibodies can induce significant regression of advanced malignancies and, in some cases, can result in complete remission. Currently, over 25 different CD3 bispecific antibodies are in clinical development for the treatment of hematological malignancies or solid cancers by targeting CD19, CD20, CD33, and CD123, or EpCAM, HER2, PSMA, and CEA, respectively. (See, e.g., Liu et al., Front Immunol 2017 8:38).

[0007] Multiple anti-CD3 antibodies are known in the art, including in monoclonal antibody and bispecific antibody forms. See, e.g., U.S. Patent Nos. 7,262,276; 7,635,472; 7,862,813; 9,587,021; and 10,174,124. However, many of these anti-CD3 antibodies trigger the overproduction of cytokines, which often leads to cytokine release syndrome (CRS). Because the anti-CD3 binding region of a bispecific antibody engages all T cells, a subset of CD4 T cells with high cytokine production is recruited. Accordingly, there is an unmet need for an anti-CD3 antibody that exhibits a desired CRS risk profile and is safe and effective in specifically binding to CD3 expressed on T cells, activating T cells, and (re)directing the activated T cells to kill target cells and in so acting while reducing the risk of triggering CRS.

[0008] One way to develop a CD3-binding region that exhibits the desired CRS risk profile is to engineer a CD3-binding region with pH-dependent antigen binding. Incorporating histidine and / or other ionizable residues into the binding interface of antibodies and other proteins has previously been used to engineer pH-dependent antigen binding (see, e.g., Igawa et al., Nature Biotechnology 28:1203-1207 (2010)). Protonation of histidine side chains in the binding interface can alter electrostatic interactions and / or induce conformational changes that result in pH-dependent differences in binding affinity (Gera et al., PLOS ONE 7(11) e48928. doi:10.1371 / journal.pone.0048928 (2012)). Recognizing that the pH of human blood ranges from approximately 7.6 - 7.8, while tumor cells have an extracellular pH of approximately 6.3 - 6.5, at least in part due to the accumulation of metabolic acids that cannot be efficiently cleared due to poor tumor vasculature formation, the applicant previously engineered pH-dependent CD3-binding antibodies that have preferential CD3 binding at low (lower) pH values, which promotes binding and activity in and around the tumor microenvironment (WO2020247932A1). Without being bound by theory, it is believed that a CD3-binding region engineered to preferentially bind CD3 at a lower pH (e.g., pH ~6) can generate selective and persistent cytotoxic activity at or around the tumor site, thereby potentially reducing or eliminating off-target effects as well as improving the half-life and enabling reduced dosing. SUMMARY OF THE INVENTION

[0009] The present disclosure relates to: improved pH-dependent anti-CD3 antibodies and antigen-binding fragments thereof, e.g., those that bind not only to human CD3 but also to cynomolgus monkey CD3 and / or provide improved binding to CD3 and / or CD3-expressing cells at pH 6.0; and methods of using or generating the same.

[0010] In one aspect, the present disclosure provides pH-dependent anti-CD3 antibodies and antigen-binding fragments.

[0011] In some embodiments, the anti-CD3 antibody or antigen-binding fragment thereof may comprise: (A) a heavy chain variable domain (VH) polypeptide, the polypeptide comprising: (a) a VH complementarity determining region 1 (CDR-H1), the CDR-H1 comprising the amino acid sequence of: (i) the CDR-H1 contained in ADI-74968, ADI-74967, ADI-74966, ADI-74965, ADI-79842, ADI-79843, ADI-79848, ADI-79844, ADI-79845, ADI-79846 or ADI-79847; and / or (ii) FNIKDYYMH (SEQ ID NO: 12, 612, 512, 412, 312, 712, 812, 912, 1012, 1112, 1212 or 1312); (b) a VH complementarity determining region 2 (CDR-H2), the CDR-H2 comprising the amino acid sequence of: (i) the CDR-H2 contained in ADI-74968, ADI-74967, ADI-74966, ADI-74965, ADI-79842, ADI-79843, ADI-79848, ADI-79844, ADI-79845, ADI-79846 or ADI-79847; and / or (ii) WIDLENANTIYDAKFQG (SEQ ID NO: 14, 614, 514, 414, 314, 714, 814, 914, 1014, 1114, 1214 or 1314); and / or (c) a VH complementarity determining region 3 (CDR-H3), the CDR-H3 comprising the amino acid sequence of: (i) the CDR-H3 contained in ADI-74968, ADI-74967, ADI-74966, ADI-74965, ADI-79842, ADI-79843, ADI-79848, ADI-79844, ADI-79845, ADI-79846 or ADI-79847; (ii) ARDX2Y X3RYFYDV (SEQ ID NO: 16), wherein X2 is A or H and X3 is H or G; and / or (iii) ARDAYHRYFYDV (SEQ ID NO: 616, 316 or 816), ARDHYHRYFYDV (SEQ ID NO: 516, 416 or 1316), ARDHYGRYFYDV (SEQ ID NO: 716 or 1216) or ARDAYGRYFYDV (SEQ ID NO: 916, 1016 or 1116);and / or (B) a light chain variable domain (VL) polypeptide, said VL polypeptide comprising: (a) VL complementarity determining region 1 (CDR-L1), said CDR-L1 comprising the amino acid sequence of: (i) the CDR-L1 contained in ADI-74968, ADI-74967, ADI-74966, ADI-74965, ADI-79842, ADI-79843, ADI-79848, ADI-79844, ADI-79845, ADI-79846 or ADI-79847; (ii) KSSQSLLNARTGX5NYLA (SEQ ID NO: 22), wherein X5 is H or K; and / or (iii) KSSQSLLNARTGHNYLA (SEQ ID NO: 622, 422, 322, 922, 1222 or 1322) or KSSQSLLNARTGKNYLA (SEQ ID NO: 522, 722, 822, 1022 or 1122); (b) VL complementarity determining region 2 (CDR-L2), said CDR-L2 comprising the amino acid sequence of: (i) the CDR-L2 contained in ADI-74968, ADI-74967, ADI-74966, ADI-74965, ADI-79842, ADI-79843, ADI-79848, ADI-79844, ADI-79845, ADI-79846 or ADI-79847; and / or (ii) WASTRES (SEQ ID NO: 24, 624, 524, 424, 324, 724, 824, 924, 1024, 1124, 1224 or 1324); and / or (c) VL complementarity determining region 3 (CDR-L3), said CDR-L3 comprising the amino acid sequence of: (i) the CDR-L3 contained in ADI-74968, ADI-74967, ADI-74966, ADI-74965, ADI-79842, ADI-79843, ADI-79848, ADI-79844, ADI-79845, ADI-79846 or ADI-79847; (ii) KQSX6SX7RT (SEQ ID NO: 26), wherein X6 is Y or H and X7 is H or R; and / or (iii) KQSYSHRT (SEQ ID NO: 626, 426 or 1126), KQSHSHRT (SEQ ID NO: 526, 326 or 1226), KQSHSRRT (SEQ ID NO: 1026 or 1326) or KQSYSRRT (SEQ ID NO: 726, 826 or 926), or the anti-CD3 antibody or antigen-binding fragment may comprise a combination of one or more of the foregoing CDRs.;

[0012] In some cases, the anti-CD3 antibody and / or antigen-binding fragment does not comprise: (i) at least one of CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 contained in ADI-48587; or (ii) at least one of CDR-H1 comprising FNIKDYYMH (SEQ ID NO: 12 or 212), CDR-H2 comprising WIDLENANTIYDAKFQG (SEQ ID NO: 14 or 214), CDR-H3 comprising ARDHYHRYFYDV (SEQ ID NO: 216), CDR-L1 comprising KSSQSLLNARTGHNYLA (SEQ ID NO: 222), CDR-L2 comprising WASTRES (SEQ ID NO: 24 or 224), and CDR-L3 comprising KQSHSHRT (SEQ ID NO: 226).

[0013] In certain cases, the anti-CD3 antibody and / or antigen-binding fragment does not comprise: (i) at least one of CDR-H3, CDR-L1, and CDR-L3 contained in ADI-48587; or (ii) at least one of CDR-H3 comprising ARDHYHRYFYDV (SEQ ID NO: 216), CDR-L1 comprising KSSQSLLNARTGHNYLA (SEQ ID NO: 222), and CDR-L3 comprising KQSHSHRT (SEQ ID NO: 226).

[0014] In some cases, the anti-CD3 antibody and / or antigen-binding fragment does not comprise: (i) at least one of CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 contained in ADI-26906; or (ii) at least one of CDR-H1 comprising FNIKDYYMH (SEQ ID NO: 12 or 112), CDR-H2 comprising WIDLENANTIYDAKFQG (SEQ ID NO: 14 or 114), CDR-H3 comprising ARDAYGRYFYDV (SEQ ID NO: 116), CDR-L1 comprising KSSQSLLNARTGKNYLA (SEQ ID NO: 122), CDR-L2 comprising WASTRES (SEQ ID NO: 24 or 124), and CDR-L3 comprising KQSYSRRT (SEQ ID NO: 126).

[0015] In certain cases, the anti-CD3 antibody and / or antigen-binding fragment does not comprise: (i) the CDR-H3, CDR-L1, and CDR-L3 contained in ADI-26906; or (ii) a CDR-H3 comprising ARDAYGRYFYDV (SEQ ID NO: 116), a CDR-L1 comprising KSSQSLLNARTGKNYLA (SEQ ID NO: 122), and a CDR-L3 comprising KQSYSRRT (SEQ ID NO: 126).

[0016] In certain embodiments, the anti-CD3 antibody or antigen-binding fragment may comprise: (A) a VH polypeptide, the VH polypeptide comprising: (a) CDR-H1, the CDR-H1 comprising the amino acid sequence of: (i) the CDR-H1 contained in ADI-74968, ADI-74967, ADI-74966, or ADI-74965; and / or (ii) FNIKDYYMH (SEQ ID NO: 12, 612, 512, 412, or 312); (b) CDR-H2, the CDR-H2 comprising the amino acid sequence of: (i) the CDR-H2 contained in ADI-74968, ADI-74967, ADI-74966, or ADI-74965; and / or (ii) WIDLENANTIYDAKFQG (SEQ ID NO: 14, 614, 514, 414, or 314); and / or (c) CDR-H3, the CDR-H3 comprising the amino acid sequence of: (i) the CDR-H3 contained in ADI-74968, ADI-74967, ADI-74966, or ADI-74965; (ii) ARDX2Y X3RYFYDV (SEQ ID NO: 16), wherein X2 is A or H and X3 is H; and / or (iii) ARDAYHRYFYDV (SEQ ID NO: 616 or 316) or ARDHYHRYFYDV (SEQ ID NO: 516 or 416); and / or (B) a VL polypeptide, the VL polypeptide comprising: (a) CDR-L1, the CDR-L1 comprising the amino acid sequence of: (i) the CDR-L1 contained in ADI-74968, ADI-74967, ADI-74966, or ADI-74965; (ii) KSSQSLLNARTGX5NYLA (SEQ ID NO: 22), wherein X5 is H or K; and / or (iii) KSSQSLLNARTGHNYLA (SEQ ID NO: 622, 422, or 322) or KSSQSLLNARTGKNYLA (SEQ ID NO: 522); (b) CDR-L2, the CDR-L2 comprising the amino acid sequence of: (i) the CDR-L2 contained in ADI-74968, ADI-74967, ADI-74966, or ADI-74965; and / or (ii) WASTRES (SEQ ID NO: 24, 624, 524, 424, or 324); and / or (c) CDR-L3, the CDR-L3 comprising the amino acid sequence of: (i) the CDR-L3 contained in ADI-74968, ADI-74967, ADI-74966, or ADI-74965;(ii) KQSX6SX7RT (SEQ ID NO: 26), where X6 is Y or H and X7 is H; and / or (iii) KQSYSHRT (SEQ ID NO: 626 or 426) or KQSHSHRT (SEQ ID NO: 526 or 326).;

[0017] In certain embodiments, the anti-CD3 antibody or antigen-binding fragment may comprise: (A) a VH polypeptide, the VH polypeptide comprising: (a) CDR-H1, the CDR-H1 comprising the amino acid sequence of: (i) the CDR-H1 contained in ADI-74968; and / or (ii) FNIKDYYMH (SEQ ID NO: 12 or 612); (b) CDR-H2, the CDR-H2 comprising the amino acid sequence of: (i) the CDR-H2 contained in ADI-74968; and / or (ii) WIDLENANTIYDAKFQG (SEQ ID NO: 14 or 614); and / or (c) CDR-H3, the CDR-H3 comprising the amino acid sequence of: (i) the CDR-H3 contained in ADI-74968; and / or (ii) ARDAYHRYFYDV (SEQ ID NO: 616); and / or (B) a VL polypeptide, the VL polypeptide comprising: (a) CDR-L1, the CDR-L1 comprising the amino acid sequence of: (i) the CDR-L1 contained in ADI-74968; (ii) KSSQSLLNARTGHNYLA (SEQ ID NO: 622); (b) CDR-L2, the CDR-L2 comprising the amino acid sequence of: (i) the CDR-L2 contained in ADI-74968; and / or (ii) WASTRES (SEQ ID NO: 24 or 624); and / or (c) CDR-L3, the CDR-L3 comprising the amino acid sequence of: (i) the CDR-L3 contained in ADI-74968; (ii) KQSYSHRT (SEQ ID NO: 626).

[0018] In certain embodiments, the anti-CD3 antibody or antigen-binding fragment may comprise: (A) a VH polypeptide comprising: (a) CDR-H1 having the amino acid sequence of: (i) the CDR-H1 contained in ADI-74967; and / or (ii) FNIKDYYMH (SEQ ID NO: 12 or 512); (b) CDR-H2 having the amino acid sequence of: (i) the CDR-H2 contained in ADI-74967; and / or (ii) WIDLENANTIYDAKFQG (SEQ ID NO: 14 or 514); and / or (c) CDR-H3 having the amino acid sequence of: (i) the CDR-H3 contained in ADI-74967; (ii) ARDHYHRYFYDV (SEQ ID NO: 516); and / or (B) a VL polypeptide comprising: (a) CDR-L1 having the amino acid sequence of: (i) the CDR-L1 contained in ADI-74967; (ii) KSSQSLLNARTGKNYLA (SEQ ID NO: 522); (b) CDR-L2 having the amino acid sequence of: (i) the CDR-L2 contained in ADI-74967; and / or (ii) WASTRES (SEQ ID NO: 24, 624, 524); and / or (c) CDR-L3 having the amino acid sequence of: (i) the CDR-L3 contained in ADI-74967; (ii) KQSHSHRT (SEQ ID NO: 526).

[0019] In some cases, the anti-CD3 antibody or antigen-binding fragment according to any one of the above embodiments may comprise: (A) a VH polypeptide comprising the CDR-H1, the CDR-H2, and the CDR-H3; and / or (B) a VL polypeptide comprising the CDR-L1, the CDR-L2, and the CDR-L3.

[0020] In certain cases, the anti-CD3 antibody or antigen-binding fragment according to any one of the above embodiments may comprise: (A) a VH polypeptide comprising the CDR-H1, the CDR-H2, and the CDR-H3; and (B) a VL polypeptide comprising the CDR-L1, the CDR-L2, and the CDR-L3.

[0021] In certain embodiments, the anti-CD3 antibody or antigen-binding fragment may comprise: (i) the CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 contained in ADI-74968; or (ii) CDR-H1 comprising FNIKDYYMH (SEQ ID NO: 12 or 612), CDR-H2 comprising WIDLENANTIYDAKFQG (SEQ ID NO: 14 or 614), CDR-H3 comprising ARDAYHRYFYDV (SEQ ID NO: 616), CDR-L1 comprising KSSQSLLNARTGHNYLA (SEQ ID NO: 622), CDR-L2 comprising WASTRES (SEQ ID NO: 24 or 624), and CDR-L3 comprising KQSYSHRT (SEQ ID NO: 626).

[0022] In certain embodiments, the anti-CD3 antibody or antigen-binding fragment may comprise: (i) the CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 contained in ADI-74967; or (ii) CDR-H1 comprising FNIKDYYMH (SEQ ID NO: 12 or 512), CDR-H2 comprising WIDLENANTIYDAKFQG (SEQ ID NO: 14 or 514), CDR-H3 comprising ARDHYHRYFYDV (SEQ ID NO: 516), CDR-L1 comprising KSSQSLLNARTGKNYLA (SEQ ID NO: 522), CDR-L2 comprising WASTRES (SEQ ID NO: 24 or 524), and CDR-L3 comprising KQSHSHRT (SEQ ID NO: 526).

[0023] In certain embodiments, the anti-CD3 antibody or antigen-binding fragment may comprise: (i) the CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 contained in ADI-74966; or (ii) CDR-H1 comprising FNIKDYYMH (SEQ ID NO: 12 or 412), CDR-H2 comprising WIDLENANTIYDAKFQG (SEQ ID NO: 14 or 414), CDR-H3 comprising ARDHYHRYFYDV (SEQ ID NO: 416), CDR-L1 comprising KSSQSLLNARTGHNYLA (SEQ ID NO: 422), CDR-L2 comprising WASTRES (SEQ ID NO: 24 or 424), and CDR-L3 comprising KQSYSHRT (SEQ ID NO: 426).

[0024] In certain embodiments, the anti-CD3 antibody or antigen-binding fragment may comprise: (i) the CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 contained in ADI-74965; or (ii) CDR-H1 comprising FNIKDYYMH (SEQ ID NO: 12 or 312), CDR-H2 comprising WIDLENANTIYDAKFQG (SEQ ID NO: 14 or 314), CDR-H3 comprising ARDAYHRYFYDV (SEQ ID NO: 316), CDR-L1 comprising KSSQSLLNARTGHNYLA (SEQ ID NO: 322), CDR-L2 comprising WASTRES (SEQ ID NO: 24 or 324), and CDR-L3 comprising KQSHSHRT (SEQ ID NO: 326).

[0025] In certain embodiments, the anti-CD3 antibody or antigen-binding fragment may comprise: (i) the CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 contained in ADI-79842; or (ii) CDR-H1 comprising FNIKDYYMH (SEQ ID NO: 12 or 712), CDR-H2 comprising WIDLENANTIYDAKFQG (SEQ ID NO: 14 or 714), CDR-H3 comprising ARDHYGRYFYDV (SEQ ID NO: 716), CDR-L1 comprising KSSQSLLNARTGKNYLA (SEQ ID NO: 722), CDR-L2 comprising WASTRES (SEQ ID NO: 24 or 724), and CDR-L3 comprising KQSYSRRT (SEQ ID NO: 726).

[0026] In certain embodiments, the anti-CD3 antibody or antigen-binding fragment may comprise: (i) the CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 contained in ADI-79843; or (ii) CDR-H1 comprising FNIKDYYMH (SEQ ID NO: 12 or 812), CDR-H2 comprising WIDLENANTIYDAKFQG (SEQ ID NO: 14 or 814), CDR-H3 comprising ARDAYHRYFYDV (SEQ ID NO: 816), CDR-L1 comprising KSSQSLLNARTGKNYLA (SEQ ID NO: 822), CDR-L2 comprising WASTRES (SEQ ID NO: 24 or 824), and CDR-L3 comprising KQSYSRRT (SEQ ID NO: 826).

[0027] In certain embodiments, the anti-CD3 antibody or antigen-binding fragment may comprise: (i) the CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 contained in ADI-79848; or (ii) CDR-H1 comprising FNIKDYYMH (SEQ ID NO: 12 or 912), CDR-H2 comprising WIDLENANTIYDAKFQG (SEQ ID NO: 14 or 914), CDR-H3 comprising ARDAYGRYFYDV (SEQ ID NO: 916), CDR-L1 comprising KSSQSLLNARTGHNYLA (SEQ ID NO: 922), CDR-L2 comprising WASTRES (SEQ ID NO: 24 or 924), and CDR-L3 comprising KQSYSRRT (SEQ ID NO: 926).

[0028] In certain embodiments, the anti-CD3 antibody or antigen-binding fragment may comprise: (i) the CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 contained in ADI-79844; or (ii) CDR-H1 comprising FNIKDYYMH (SEQ ID NO: 12 or 1012), CDR-H2 comprising WIDLENANTIYDAKFQG (SEQ ID NO: 14 or 1014), CDR-H3 comprising ARDAYGRYFYDV (SEQ ID NO: 1016), CDR-L1 comprising KSSQSLLNARTGKNYLA (SEQ ID NO: 1022), CDR-L2 comprising WASTRES (SEQ ID NO: 24 or 1024), and CDR-L3 comprising KQSHSRRT (SEQ ID NO: 1026).

[0029] In certain embodiments, the anti-CD3 antibody or antigen-binding fragment may comprise: (i) the CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 contained in ADI-79845; or (ii) a CDR-H1 comprising FNIKDYYMH (SEQ ID NO: 12 or 1112), a CDR-H2 comprising WIDLENANTIYDAKFQG (SEQ ID NO: 14 or 1114), a CDR-H3 comprising ARDAYGRYFYDV (SEQ ID NO: 1116), a CDR-L1 comprising KSSQSLLNARTGKNYLA (SEQ ID NO: 1122), a CDR-L2 comprising WASTRES (SEQ ID NO: 24 or 1124), and a CDR-L3 comprising KQSYSHRT (SEQ ID NO: 1126).

[0030] In certain embodiments, the anti-CD3 antibody or antigen-binding fragment may comprise: (i) the CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 contained in ADI-79846; or (ii) a CDR-H1 comprising FNIKDYYMH (SEQ ID NO: 12 or 1212), a CDR-H2 comprising WIDLENANTIYDAKFQG (SEQ ID NO: 14 or 1214), a CDR-H3 comprising ARDHYGRYFYDV (SEQ ID NO: 1216), a CDR-L1 comprising KSSQSLLNARTGHNYLA (SEQ ID NO: 1222), a CDR-L2 comprising WASTRES (SEQ ID NO: 24 or 1224), and a CDR-L3 comprising KQSHSHRT (SEQ ID NO: 1226).

[0031] In certain embodiments, the anti-CD3 antibody or antigen-binding fragment may comprise: (i) the CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 contained in ADI-79846; or (ii) CDR-H1 comprising FNIKDYYMH (SEQ ID NO: 12 or 1312), CDR-H2 comprising WIDLENANTIYDAKFQG (SEQ ID NO: 14 or 1314), CDR-H3 comprising ARDHYHRYFYDV (SEQ ID NO: 1316), CDR-L1 comprising KSSQSLLNARTGHNYLA (SEQ ID NO: 1322), CDR-L2 comprising WASTRES (SEQ ID NO: 24 or 1324), and CDR-L3 comprising KQSHSRRT (SEQ ID NO: 1326).

[0032] In some cases, in an anti-CD3 antibody or antigen-binding fragment according to any of the above embodiments: (A) the VH polypeptide comprises: (a) a VH framework region 1 (FR-H1), the FR-H1 comprising the amino acid sequence of: (i) the FR-H1 contained in ADI-74968, ADI-74967, ADI-74966, ADI-74965, ADI-79842, ADI-79843, ADI-79848, ADI-79844, ADI-79845, ADI-79846 or ADI-79847; and / or (ii) QVQLVQSGAEVKKPGASVKVSCKASG (SEQ ID NO: 11, 611, 511, 411, 311, 711, 811, 911, 1011, 1111, 1211 or 1311); (b) a VH framework region 2 (FR-H2), the FR-H2 comprising the amino acid sequence of: (i) the FR-H2 contained in ADI-74968, ADI-74967, ADI-74966, ADI-74965, ADI-79842, ADI-79843, ADI-79848, ADI-79844, ADI-79845, ADI-79846 or ADI-79847; and / or (ii) WVRQAPGQRLEWMG (SEQ ID NO: 13, 613, 513, 413, 313, 713, 813, 913, 1013, 1113, 1213 or 1313); (c) a VH framework region 3 (FR-H3), the FR-H3 comprising the amino acid sequence of: (i) the FR-H3 contained in ADI-74968, ADI-74967, ADI-74966, ADI-74965, ADI-79842, ADI-79843, ADI-79848, ADI-79844, ADI-79845, ADI-79846 or ADI-79847; (ii) RVTITRDTSASTAYMX1LSSLRSEDTAVYYC (SEQ ID NO: 15), where X1 is E or G; and / or (iii) RVTITRDTSASTAYMELSSLRSEDTAVYYC (SEQ ID NO: 615, 515, 415, 315, 715, 815, 915, 1015, 1115 or 1315) or RVTITRDTSASTAYMGLSSLRSEDTAVYYC (SEQ ID NO: 1215);and / or (d) VH framework region 4 (FR-H4), wherein the FR-H4 comprises the amino acid sequence of: (i) the FR-H4 contained in ADI-74968, ADI-74967, ADI-74966, ADI-74965, ADI-79842, ADI-79843, ADI-79848, ADI-79844, ADI-79845, ADI-79846 or ADI-79847; and / or (ii) WGQGTLVTVSS (SEQ ID NO: 17, 617, 517, 417, 317, 717, 817, 917, 1017, 1117, 1217 or 1317); and / or (B) the VL polypeptide comprises: (a) VL framework region 1 (FR-L1), wherein the FR-L1 comprises the amino acid sequence of: (i) the FR-L1 contained in ADI-74968, ADI-74967, ADI-74966, ADI-74965, ADI-79842, ADI-79843, ADI-79848, ADI-79844, ADI-79845, ADI-79846 or ADI-79847; (ii) X4IVMTQSPDSLAVSLGERATINC (SEQ ID NO: 21), wherein X4 is D or G; and / or (iii) DIVMTQSPDSLAVSLGERATINC (SEQ ID NO: 621, 421, 321, 721, 821, 921, 1021, 1121, 1221 or 1321) or GIVMTQSPDSLAVSLGERATINC (SEQ ID NO: 521); (b) VL framework region 2 (FR-L2), wherein the FR-L2 comprises the amino acid sequence of: (i) the FR-L2 contained in ADI-74968, ADI-74967, ADI-74966, ADI-74965, ADI-79842, ADI-79843, ADI-79848, ADI-79844, ADI-79845, ADI-79846 or ADI-79847; and / or (ii) WYQQKPGQPPKLLIY (SEQ ID NO: 23, 623, 523, 423, 323, 723, 823, 923, 1023, 1123, 1223 or 1323); (c) VL framework region 3 (FR-L3), wherein the FR-L3 comprises the amino acid sequence of: (i) the FR-L3 contained in ADI-74968, ADI-74967, ADI-74966, ADI-74965, ADI-79842, ADI-79843, ADI-79848, ADI-79844, ADI-79845, ADI-79846 or ADI-79847;and / or (ii) GVPDRFSGSGSGTDFTLTISSLQAEDVAVYYC (SEQ ID NO: 25, 625, 525, 425, 325, 725, 825, 925, 1025, 1125, 1225 or 1325); and / or (d) VL framework region 4 (FR-L4), wherein the FR-L4 comprises the amino acid sequence of: (i) the FR-L4 contained in ADI-74968, ADI-74967, ADI-74966, ADI-74965, ADI-79842, ADI-79843, ADI-79848, ADI-79844, ADI-79845, ADI-79846 or ADI-79847; and / or (iii) FGGGTKVEIK (SEQ ID NO: 27, 627, 527, 427, 327, 727, 827, 927, 1027, 1127, 1227 or 1327), or the anti-CD3 antibody or antigen-binding fragment may comprise a VH and / or VL comprising any combination of the aforementioned VH and VL framework regions.;

[0033] In certain cases, the anti-CD3 antibody or antigen-binding fragment according to any one of the above embodiments may comprise: (i) the FR-H1, FR-H2, FR-H3, FLR-H4, FR-L1, FR-L2, FR-L3, and FR-L4 contained in ADI-74968, ADI-74966, ADI-74965, ADI-79842, ADI-79843, ADI-79848, ADI-79844, ADI-79845, or ADI-79847; or (ii) FR-H1 comprising QVQLVQSGAEVKKPGASVKVSCKASG (SEQ ID NO: 11, 611, 411, 311, 711, 811, 911, 1011, 1111, or 1311), FR-H2 comprising WVRQAPGQRLEWMG (SEQ ID NO: 13, 613, 413, 313, 713, 813, 913, 1013, 1113, or 1313), FR-H3 comprising RVTITRDTSASTAYMELSSLRSEDTAVYYC (SEQ ID NO: 615, 415, 315, 715, 815, 915, 1015, 1115, or 1315), FR-H4 comprising WGQGTLVTVSS (SEQ ID NO: 17, 617, 417, 317, 717, 817, 917, 1017, 1117, or 1317), FR-L1 comprising DIVMTQSPDSLAVSLGERATINC (SEQ ID NO: 621, 421, 321, 721, 821, 921, 1021, 1121, or 1321), FR-L2 comprising WYQQKPGQPPKLLIY (SEQ ID NO: 23, 623, 423, 323, 723, 823, 923, 1023, 1123, or 1323), FR-L3 comprising GVPDRFSGSGSGTDFTLTISSLQAEDVAVYYC (SEQ ID NO: 25, 625, 425, 325, 725, 825, 925, 1025, 1125, or 1325), and FR-L4 comprising FGGGTKVEIK (SEQ ID NO: 27, 627, 427, 327, 727, 827, 927, 1027, 1127, or 1327).

[0034] In some cases, the anti-CD3 antibody or antigen-binding fragment according to any of the above embodiments may comprise: (i) the FR-H1, FR-H2, FR-H3, FLR-H4, FR-L1, FR-L2, FR-L3, and FR-L4 contained in ADI-74967; or (ii) an FR-H1 comprising QVQLVQSGAEVKKPGASVKVSCKASG (SEQ ID NO: 11 or 511), an FR-H2 comprising WVRQAPGQRLEWMG (SEQ ID NO: 13 or 513), an FR-H3 comprising RVTITRDTSASTAYMELSSLRSEDTAVYYC (SEQ ID NO: 515), an FR-H4 comprising WGQGTLVTVSS (SEQ ID NO: 17 or 517), an FR-L1 comprising GIVMTQSPDSLAVSLGERATINC (SEQ ID NO: 521), an FR-L2 comprising WYQQKPGQPPKLLIY (SEQ ID NO: 23 or 523), an FR-L3 comprising GVPDRFSGSGSGTDFTLTISSLQAEDVAVYYC (SEQ ID NO: 25 or 525), and an FR-L4 comprising FGGGTKVEIK (SEQ ID NO: 27 or 527).

[0035] In some cases, the anti-CD3 antibody or antigen-binding fragment according to any one of the above embodiments may comprise: (i) the FR-H1, FR-H2, FR-H3, FLR-H4, FR-L1, FR-L2, FR-L3, and FR-L4 contained in ADI-74968, ADI-74966, ADI-74965, ADI-79842, ADI-79843, ADI-79848, ADI-79844, ADI-79845, or ADI-79847; or (ii) an FR-H1 comprising QVQLVQSGAEVKKPGASVKVSCKASG (SEQ ID NO: 11 or 1211), an FR-H2 comprising WVRQAPGQRLEWMG (SEQ ID NO: 13 or 1213), an FR-H3 comprising RVTITRDTSASTAYMGLSSLRSEDTAVYYC (SEQ ID NO: 1215), an FR-H4 comprising WGQGTLVTVSS (SEQ ID NO: 17 or 1217), an FR-L1 comprising DIVMTQSPDSLAVSLGERATINC (SEQ ID NO: 1221), an FR-L2 comprising WYQQKPGQPPKLLIY (SEQ ID NO: 1223), an FR-L3 comprising GVPDRFSGSGSGTDFTLTISSLQAEDVAVYYC (SEQ ID NO: 1225), and an FR-L4 comprising FGGGTKVEIK (SEQ ID NO: 1227).

[0036] In certain embodiments, the anti-CD3 antibody or antigen-binding fragment may comprise: (i) the FR-H1, CDR-H1, FR-H2, CDR-H2, FR-H3, CDR-H3, FR-H4, FR-L1, CDR-L1, FR-L2, CDR-L2, FR-L3, CDR-L3, and FR-L4 contained in ADI-74968; or

[0037] (ii) an FR-H1, CDR-H1, FR-H2, CDR-H2, FR-H3, CDR-H3, FR-H4, FR-L1, CDR-L1, FR-L2, CDR-L2, FR-L3, CDR-L3, and FR-L4 comprising SEQ ID NOs: 611, 612, 613, 614, 615, 616, 617, 621, 622, 623, 624, 625, 626, and 627, respectively.

[0038] In certain embodiments, the anti-CD3 antibody or antigen-binding fragment may comprise: (i) the FR-H1, CDR-H1, FR-H2, CDR-H2, FR-H3, CDR-H3, FR-H4, FR-L1, CDR-L1, FR-L2, CDR-L2, FR-L3, CDR-L3, and FR-L4 contained in ADI-74967; or (ii) FR-H1, CDR-H1, FR-H2, CDR-H2, FR-H3, CDR-H3, FR-H4, FR-L1, CDR-L1, FR-L2, CDR-L2, FR-L3, CDR-L3, and FR-L4 respectively comprising SEQ ID NOs: 511, 512, 513, 514, 515, 516, 517, 521, 522, 523, 524, 525, 526, and 527.

[0039] In certain embodiments, the anti-CD3 antibody or antigen-binding fragment may comprise: (i) the FR-H1, CDR-H1, FR-H2, CDR-H2, FR-H3, CDR-H3, FR-H4, FR-L1, CDR-L1, FR-L2, CDR-L2, FR-L3, CDR-L3, and FR-L4 contained in ADI-74966; or (ii) FR-H1, CDR-H1, FR-H2, CDR-H2, FR-H3, CDR-H3, FR-H4, FR-L1, CDR-L1, FR-L2, CDR-L2, FR-L3, CDR-L3, and FR-L4 respectively comprising SEQ ID NOs: 411, 412, 413, 414, 415, 416, 417, 421, 422, 423, 424, 425, 426, and 427.

[0040] In certain embodiments, the anti-CD3 antibody or antigen-binding fragment may comprise: (i) the FR-H1, CDR-H1, FR-H2, CDR-H2, FR-H3, CDR-H3, FR-H4, FR-L1, CDR-L1, FR-L2, CDR-L2, FR-L3, CDR-L3, and FR-L4 contained in ADI-74965; or (ii) FR-H1, CDR-H1, FR-H2, CDR-H2, FR-H3, CDR-H3, FR-H4, FR-L1, CDR-L1, FR-L2, CDR-L2, FR-L3, CDR-L3, and FR-L4 respectively comprising SEQ ID NOs: 311, 312, 313, 314, 315, 316, 317, 321, 322, 323, 324, 325, 326, and 327.

[0041] In certain embodiments, the anti-CD3 antibody or antigen-binding fragment may comprise: (i) the FR-H1, CDR-H1, FR-H2, CDR-H2, FR-H3, CDR-H3, FR-H4, FR-L1, CDR-L1, FR-L2, CDR-L2, FR-L3, CDR-L3, and FR-L4 contained in ADI-79842; or (ii) FR-H1, CDR-H1, FR-H2, CDR-H2, FR-H3, CDR-H3, FR-H4, FR-L1, CDR-L1, FR-L2, CDR-L2, FR-L3, CDR-L3, and FR-L4 comprising SEQ ID NOs: 711, 712, 713, 714, 715, 716, 717, 721, 722, 723, 724, 725, 726, and 727, respectively.

[0042] In certain embodiments, the anti-CD3 antibody or antigen-binding fragment may comprise: (i) the FR-H1, CDR-H1, FR-H2, cDR-H2, FR-H3, cDR-H3, FR-H4, FR-L1, CDR-L1, FR-L2, CDR-L2, FR-L3, CDR-L3, and FR-L4 contained in ADI-79843; or (ii) FR-H1, CDR-H1, FR-H2, CDR-H2, FR-H3, CDR-H3, FR-H4, FR-L1, CDR-L1, FR-L2, CDR-L2, FR-L3, CDR-L3, and FR-L4 comprising SEQ ID NOs: 811, 812, 813, 814, 815, 816, 817, 821, 822, 823, 824, 825, 826, and 827, respectively.

[0043] In certain embodiments, the anti-CD3 antibody or antigen-binding fragment may comprise: (i) the FR-H1, CDR-H1, FR-H2, CDR-H2, FR-H3, CDR-H3, FR-H4, FR-L1, CDR-L1, FR-L2, CDR-L2, FR-L3, CDR-L3, and FR-L4 contained in ADI-79848; or (ii) FR-H1, CDR-H1, FR-H2, CDR-H2, FR-H3, CDR-H3, FR-H4, FR-L1, CDR-L1, FR-L2, CDR-L2, FR-L3, CDR-L3, and FR-L4 comprising SEQ ID NOs: 911, 912, 913, 914, 915, 916, 917, 921, 922, 923, 924, 925, 926, and 927, respectively.

[0044] In certain embodiments, the anti-CD3 antibody or antigen-binding fragment may comprise: (i) the FR-H1, CDR-H1, FR-H2, CDR-H2, FR-H3, CDR-H3, FR-H4, FR-L1, CDR-L1, FR-L2, CDR-L2, FR-L3, CDR-L3, and FR-L4 contained in ADI-79844; or (ii) FR-H1, CDR-H1, FR-H2, CDR-H2, FR-H3, CDR-H3, FR-H4, FR-L1, CDR-L1, FR-L2, CDR-L2, FR-L3, CDR-L3, and FR-L4 comprising SEQ ID NOs: 1011, 1012, 1013, 1014, 1015, 1016, 1017, 1021, 1022, 1023, 1024, 1025, 1026, and 1027, respectively.

[0045] In certain embodiments, the anti-CD3 antibody or antigen-binding fragment may comprise: (i) the FR-H1, CDR-H1, FR-H2, CDR-H2, FR-H3, CDR-H3, FR-H4, FR-L1, CDR-L1, FR-L2, CDR-L2, FR-L3, CDR-L3, and FR-L4 contained in ADI-79845; or (ii) FR-H1, CDR-H1, FR-H2, CDR-H2, FR-H3, CDR-H3, FR-H4, FR-L1, CDR-L1, FR-L2, CDR-L2, FR-L3, CDR-L3, and FR-L4 comprising SEQ ID NOs: 1111, 1112, 1113, 1114, 1115, 1116, 1117, 1121, 1122, 1123, 1124, 1125, 1126, and 1127, respectively.

[0046] In certain embodiments, the anti-CD3 antibody or antigen-binding fragment may comprise: (i) the FR-H1, CDR-H1, FR-H2, CDR-H2, FR-H3, CDR-H3, FR-H4, FR-L1, CDR-L1, FR-L2, CDR-L2, FR-L3, CDR-L3, and FR-L4 contained in ADI-79846; or (ii) FR-H1, CDR-H1, FR-H2, CDR-H2, FR-H3, CDR-H3, FR-H4, FR-L1, CDR-L1, FR-L2, CDR-L2, FR-L3, CDR-L3, and FR-L4 comprising SEQ ID NOs: 1211, 1212, 1213, 1214, 1215, 1216, 1217, 1221, 1222, 1223, 1224, 1225, 1226, and 1227, respectively.

[0047] In certain embodiments, the anti-CD3 antibody or antigen-binding fragment may comprise: (i) the FR-H1, CDR-H1, FR-H2, CDR-H2, FR-H3, CDR-H3, FR-H4, FR-L1, CDR-L1, FR-L2, CDR-L2, FR-L3, CDR-L3, and FR-L4 contained in ADI-79847; or (ii) FR-H1, CDR-H1, FR-H2, CDR-H2, FR-H3, CDR-H3, FR-H4, FR-L1, CDR-L1, FR-L2, CDR-L2, FR-L3, CDR-L3, and FR-L4 comprising SEQ ID NOs: 1311, 1312, 1313, 1314, 1315, 1316, 1317, 1321, 1322, 1323, 1324, 1325, 1326, and 1327, respectively.

[0048] In some embodiments, the anti-CD3 antibody or antigen-binding fragment may comprise: (A) a VH polypeptide comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 610, 510, 410, 310, 710, 810, 910, 1010, 1110, 1210, or 1310; and / or (B) a VL polypeptide comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 620, 520, 420, 320, 720, 820, 920, 1020, 1120, 1220, or 1320.

[0049] In certain embodiments, the anti-CD3 antibody or antigen-binding fragment may comprise: (A) a VH polypeptide comprising (i) the CDR-H1, CDR-H2, and CFR-H3 sequences of SEQ ID NOs: 612, 614, and 616, respectively, and (ii) an amino acid sequence that is at least 90% identical to SEQ ID NO: 610; and (B) a VL polypeptide comprising (i) the CDR-L1, CDR-L2, and CFR-L3 sequences of SEQ ID NOs: 622, 624, and 626, respectively; and (ii) an amino acid sequence that is at least 90% identical to SEQ ID NO: 620.

[0050] In certain embodiments, the anti-CD3 antibody or antigen-binding fragment may comprise: (A) a VH polypeptide comprising (i) the CDR-H1, CDR-H2, and CDR-H3 sequences of SEQ ID NOs: 512, 514, and 516, respectively, and (ii) an amino acid sequence that is at least 90% identical to SEQ ID NO: 510; and (B) a VL polypeptide comprising (i) the CDR-L1, CDR-L2, and CDR-L3 sequences of SEQ ID NOs: 522, 524, and 526, respectively; and (ii) an amino acid sequence that is at least 90% identical to SEQ ID NO: 520.

[0051] In certain embodiments, the anti-CD3 antibody or antigen-binding fragment may comprise a VH polypeptide and a VL polypeptide, the VH polypeptide and the VL polypeptide comprising the amino acid sequences of SEQ ID NOs: 610 and 620, respectively.

[0052] In certain embodiments, the anti-CD3 antibody or antigen-binding fragment may comprise a VH polypeptide and a VL polypeptide, the VH polypeptide and the VL polypeptide comprising the amino acid sequences of SEQ ID NOs: 510 and 520, respectively.

[0053] In certain embodiments, the anti-CD3 antibody or antigen-binding fragment may comprise a VH polypeptide and a VL polypeptide, the VH polypeptide and the VL polypeptide comprising the amino acid sequences of SEQ ID NOs: 410 and 420, respectively.

[0054] In certain embodiments, the anti-CD3 antibody or antigen-binding fragment may comprise a VH polypeptide and a VL polypeptide, the VH polypeptide and the VL polypeptide comprising the amino acid sequences of SEQ ID NOs: 310 and 320, respectively.

[0055] In certain embodiments, the anti-CD3 antibody or antigen-binding fragment may comprise a VH polypeptide and a VL polypeptide, the VH polypeptide and the VL polypeptide comprising the amino acid sequences of SEQ ID NOs: 710 and 720, respectively.

[0056] In certain embodiments, the anti-CD3 antibody or antigen-binding fragment may comprise a VH polypeptide and a VL polypeptide, the VH polypeptide and the VL polypeptide comprising the amino acid sequences of SEQ ID NOs: 810 and 820, respectively.

[0057] In certain embodiments, the anti-CD3 antibody or antigen-binding fragment may comprise a VH polypeptide and a VL polypeptide, the VH polypeptide and the VL polypeptide comprising the amino acid sequences of SEQ ID NOs: 910 and 920, respectively.

[0058] In certain embodiments, the anti-CD3 antibody or antigen-binding fragment may comprise a VH polypeptide and a VL polypeptide, wherein the VH polypeptide and the VL polypeptide comprise the amino acid sequences of SEQ ID NO: 1010 and 1020, respectively.

[0059] In certain embodiments, the anti-CD3 antibody or antigen-binding fragment may comprise a VH polypeptide and a VL polypeptide, wherein the VH polypeptide and the VL polypeptide comprise the amino acid sequences of SEQ ID NO: 1110 and 1120, respectively.

[0060] In certain embodiments, the anti-CD3 antibody or antigen-binding fragment may comprise a VH polypeptide and a VL polypeptide, wherein the VH polypeptide and the VL polypeptide comprise the amino acid sequences of SEQ ID NO: 1210 and 1220, respectively.

[0061] In certain embodiments, the anti-CD3 antibody or antigen-binding fragment may comprise a VH polypeptide and a VL polypeptide, wherein the VH polypeptide and the VL polypeptide comprise the amino acid sequences of SEQ ID NO: 1310 and 1320, respectively.

[0062] In some embodiments, the anti-CD3 antibody or antigen-binding fragment may comprise an antibody constant region, a CH1 domain, a hinge, a CH2 domain, and / or a CH3 domain. In some cases, the antibody constant region, CH1 domain, hinge, CH2 domain, and / or CH3 domain may individually belong to or be derived from IgG or human IgG. In certain cases, the antibody constant region, CH1 domain, hinge, CH2 domain, and / or CH3 domain may individually belong to or be derived from human IgG1, IgG4, IgG2, or IgG3.

[0063] In some embodiments, the anti-CD3 antibody or antigen-binding fragment may comprise a crystallizable fragment (Fc) region.

[0064] In certain embodiments, the Fc region may belong to human IgG1. In some cases, the Fc region may comprise one or more of the following amino acid modifications: N297A, N297Q, D265A, L234A, L235A, C226S, C229S, P238S, E233P, L234V, G236 - deletion, P238A, A327Q, A327G, P329A, K322A, L234F, L235E, P331S, T394D, A330L, P331S, F243L, R292P, Y300L, V305I, P396L, S239D, I332E, S298A, E333A, K334A, L234Y, L235Q, G236W, S239M, H268D, D270E, K326D, A330M, K334E, G236A, K326W, S239D, E333S, S267E, H268F, S324T, E345R, E430G, S440Y M428L, N434S, L328F, M252Y, S254T, T256E or any combination thereof according to EU numbering.

[0065] In certain embodiments, the Fc region may belong to human IgG4. In some cases, the Fc region may comprise one or more of the following amino acid modifications: E233P, F234V, L235A, G237A, E318A, S228P, L236E, S241P, L248E, T394D, M252Y, S254T, T256E, N297A, N297Q or any combination thereof according to EU numbering.

[0066] In certain embodiments, the Fc region may belong to human IgG2. In some cases, the Fc region may comprise one or more of the following amino acid modifications: P238S, V234A, G237A, H268A, H268Q, H268E, V309L, N297A, N297Q, A330S, P331S, C232S, C233S, M252Y, S254T, T256E or any combination thereof according to EU numbering.

[0067] In certain embodiments, the Fc region may belong to human IgG3. In some cases, the Fc region may comprise E235Y according to EU numbering.

[0068] In some embodiments, the anti - CD3 antibody or antigen - binding fragment may comprise IgG, IgA, IgE, IgD or IgM. In some cases, the IgG may be IgG1, IgG4, IgG2 or IgG3.

[0069] In some embodiments, the anti-CD3 antibody or antigen-binding fragment may comprise an antibody fragment selected from: antigen-binding fragment (Fab); Fab2; Fab3; Fab' fragment; F(ab')2; variable fragment (Fv); single-chain Fv (scFv) fragment; bispecific antibody; trispecific antibody; minibody; scFv-Fc; scFv2-Fc2; scFv-IgG; monovalent IgG (or semi-IgG); and / or chimeric antigen receptor (CAR), the CAR comprising an antigen-binding region comprising the VH polypeptide and / or the VL polypeptide, a transmembrane domain, and at least one intracellular signaling domain (optionally derived from a T cell receptor, further optionally CD3ζ).

[0070] In some embodiments, the anti-CD3 antibody or antigen-binding fragment may comprise or be comprised in a multispecific antibody or antibody fragment, the multispecific antibody or antibody fragment having at least (a) a first antigen-binding region specific for CD3, the first antigen-binding region comprising the VH polypeptide and / or the VL polypeptide, and (b) a second antigen-binding region.

[0071] In certain embodiments, the second antigen-binding region may be specific for: an oncology target, a target molecule expressed on a cancer cell, an immuno-oncology target, a target molecule expressed on an immune cell, an autoimmune disorder target (optionally, an autoreactive immune molecule or a target molecule expressed on an immune cell expressing an autoreactive immune molecule), an inflammatory disease target (optionally, an inflammatory cytokine or chemokine or its receptor), a neurodegenerative disease target, an infectious disease target (optionally, a target molecule of a virus, bacterium, or fungus), a target molecule expressed on an infected cell (optionally, infected with a virus, bacterium, or fungus), a metabolic disease target, a cognitive disorder target, a blood-brain barrier target, or a blood disease target.

[0072] In certain embodiments, the second antigen-binding region can be specific for a second antigen selected from: 17-IA, 4-1BB, 4Dc, 6-keto-PGF1a, 8-iso-PGF2a, 8-oxo-dG, A1 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, ADAM 15, ADAM 17 / TACE, ADAM8, ADAM9, ADAMTS, ADAMTS4, ADAMTS5, addressin, aFGF, ALCAM, ALK, ALK-1, ALK-7, α-1-antitrypsin, α-V / β-1 antagonist, ANG, Ang, APAF-1, APE, APJ, APP, APRIL, AR, ARC, ART, amphiregulin, anti-Id, ASPARTIC, atrial natriuretic factor, av / b3 integrin, Axl, b2M, B7-1, B7-2, B7-H, B-lymphocyte stimulator (BlyS), BACE, BACE-1, Bad, BAFF, BAFF-R, Bag-1, BAK, Bax, BCA-1, BCAM, Bel, BCMA, BDNF, b-ECGF, bFGF, BID, Bik, BFM, BLC, BL-CAM, BLK, BMP, BMP-2, BMP-2a, 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, b-NGF, BOK, bombesin, bone-derived neurotrophic factor, BPDE, BPDE-DNA, BTC, complement factor 3 (C3), C3a, C4, C5, C5a, C10, CA125, CAD-8, calcitonin, cAMP, carcinoembryonic antigen (CEA), carcinoma-associated antigen, cathepsin A, cathepsin B, cathepsin C / DPPI, cathepsin D, cathepsin E, cathepsin H, cathepsin L, cathepsin O, cathepsin S, cathepsin V, cathepsin X / Z / P, CBL, CCI, CCK2, CCL, CCL1, CCL11, CCL12, CCL13, CCL 14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL2, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CCL3, CCL4, CCL5, CCL6, CCL7, CCL8, CCL9 / 10, CCR, CCR1, CCR10, CCR10, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CD1, CD2, CD4, CD5, CD6, CD7, CD8, CD10, CD11a, CD11b, CD11c, CD13, CD14, CD15, CD16, CD18, CD19, CD20, CD21, CD22, CD23, CD25, CD27L, CD28, CD29, CD30, CD30L, CD32, CD33 (p67 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, Clostridium botulinum toxin, Clostridium perfringens toxin, CKb8-1, CLC, CMV, CMV UL, CNTF, CNTN-1, COX, C-Ret, CRG-2, CT-1, CTACK, CTGF, CTLA-4, CX3CL1, CX3CR1, CXCL, CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCR, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, Cytokeratin tumor-associated antigen, DAN, DCC, DcR3, DC-SIGN, 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, ENA, Endothelin receptor, Neprilysin, eNOS, Eot, Eotaxin-1, EpCAM, Ephrin B2 / EphB4, EPO, ERCC, E-selectin, ET-1, Factor Ila, Factor VII, Factor VIIIc, Factor IX, Fibroblast activation protein (FAP), Fas, FcR1, 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-α1, GFR-α2, GFR-α3, GITR, Glucagon, Glut 4, Glycoprotein IIb / IIIa (GPIIb / IIIa), GM-CSF, gp130, gp72, GRO, Growth hormone releasing factor, Hapten (NP-cap or NIP-cap), HB-EGF, HCC, HCMV gB envelope glycoprotein, HCMV) gH envelope glycoprotein, HCMVUL, Hematopoietic growth factor (HGF), Hep B gp120, 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 gp120, HIVIIIB gp120 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) - α, INF - β, INF - γ, inhibin, iNOS, insulin A chain, insulin B chain, insulin - like growth factor 1, integrin α2, integrin α3, integrin α4, integrin α4 / β1, integrin α4 / β7, integrin α5(αV), integrin α5 / β1, integrin α5 / p3, integrin α6, integrin β1, integrin β2, interferon γ, 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 bp1, 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, luteinizing hormone, lymphotoxin β 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 - α, 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 duct inhibitor, Mug, MuSK, NAIP, NAP, NCAD, N - cadherin, NCA90, NCAM, NCAM, Neprilysin, Neurotrophin-3, -4 or -6, Neuroserpin, Nerve Growth Factor (NGF), NGFR, NGF-β, 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, 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 α / β), TdT, TECK, TEM1, TEM5, TEM7, TEM8, TERT, Testicular PLAP-Like Alkaline Phosphatase, TfR, TGF, TGF-α, TGF-β, TGF-β Pan-Specific, TGF-β RI (ALK-5), TGF-β RII, TGF-β RIIb, TGF-β RIII, TGF-β1, TGF-β2, TGF-β3, TGF-β4, TGF-β5, Thrombin, Thymic Ck-1, Thyroid-Stimulating Hormone, Tie, TIMP, TIQ, Tissue Factor, TMEFF2, Tmpo, TMPRSS2, TNF, TNF-α, TNF-αβ, TNF-β2, TNFc, TNF-RI, TNF-RII, TNFRSF10A (TRAIL R1, Apo-2, DR4), TNFRSF10B (TRAIL R2 DR5, KILLER, TRICK-2A, TRICK-B), TNFRSF10C (TRAILR3, DcR1, LIT, TRID), TNFRSF10D (TRAIL R4DcR2, TRUNDD, TNFRSF11A (RANK, ODF, R, TRANCER), TNFRSFIIB (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 - 1BB, CD137, ILA), TNFRSF21 (DR6), TNFRSF22 (DcTRAIL, R2, TNFRH2), TNFRST23 (DcTRAIL, R1, 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), TNFSF1A (TNF - a, titin, DIF, TNFSF2), TNFSF1B (TNF - b, LTa, TNFSF1), TNFSF3 (LTbTNFC, 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 CA 125, Lewis Y-related carbohydrate 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 Willebrand’s 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-3), and hormone receptors and growth factors.

[0073] In certain embodiments, the second antigen-binding region can be specific for 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, FcγRIIIa (CD16), FcγRIIa (CD32a), FcγRIIb (CD32b), FcγRI (CD64), Toll-like receptor (TLR), 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.

[0074] In certain embodiments, the multispecific antibody or antibody fragment can be bispecific. In certain embodiments, the multispecific antibody or antibody fragment can further comprise a third antigen-binding region. In certain embodiments, the multispecific antibody or antibody fragment can be trispecific.

[0075] In certain embodiments, the multispecific antibody or antibody fragment can comprise a multispecific format selected from the group consisting of Fab-Fc-scFv, scFv2-Fc2, scFv-IgG, "bottle-opener", Mab-scFv, Mab-Fv, bis-scFv, central Fv, central scFv, single-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.

[0076] In certain embodiments, the multispecific antibody or antibody fragment can comprise at least one CLκ-preferred variant CH1 domain, optionally the CLκ-preferred variant CH1 domain described in WO2021067404.

[0077] In certain embodiments, the multispecific antibody or antibody fragment can comprise at least one CLλ-preferred variant CH1 domain, optionally the CLλ-preferred variant CH1 domain described in WO2021067404.

[0078] In certain embodiments, the multispecific antibody or antibody fragment can comprise at least one pair of variant CH1 domains and variant CL domains that preferentially pair with each other, optionally the pair described in WO2022150787.

[0079] In certain embodiments, the multispecific antibody or antibody fragment may comprise at least a pair of variant CH3 domains that preferentially pair with each other and another variant CH3 domain, optionally the pair described in WO2022150785.

[0080] In some embodiments, the anti-CD3 antibody or antigen-binding fragment may bind to CD3 with greater binding affinity or avidity at acidic pH, optionally about pH 6.0, than at physiological pH, optionally about pH 7.4. In certain embodiments, the binding affinity or avidity is measured by the following methods: (1) surface plasmon resonance (SPR), optionally using system (GE healthcare or ); (2) biolayer interferometry (BLI), optionally using system; (3) enzyme-linked immunosorbent assay (ELISA); and / or (4) radioimmunoassay (RIA). In certain embodiments, CD3 may be: (1) human CD3, optionally CD3εδ; and / or (2) non-human primate, optionally monkey, further optionally cynomolgus monkey CD3, optionally CD3εδ. In certain embodiments, based on the equilibrium dissociation constant (Kd) value, the binding to CD3 at acidic pH, optionally about pH 6.0, may be at least ×1.2, at least ×1.5, at least ×2, at least ×5, at least ×10, at least ×20, at least ×50, at least ×100, at least ×10 3 、at least ×10 4 、at least ×10 5 、at least ×10 6 、at least ×10 7 、at least ×10 8 or at least ×10 9 greater than at physiological pH, optionally about pH 7.4. In certain embodiments, the anti-CD3 antibody or antigen-binding fragment may not bind to CD3 at physiological pH, optionally about pH 7.4.

[0081] In some embodiments, the anti-CD3 antibody or antigen-binding fragment may bind to human CD3 (optionally CD3εδ) at acidic pH (optionally about pH 6.0).

[0082] In certain embodiments, the anti-CD3 antibody or antigen-binding fragment may have a binding affinity for human CD3 (optionally CD3εδ) at acidic pH (optionally about pH 6.0) lower than 1.57×10 8 (M), lower than 1.0×10 8 (M), lower than 9.0×10 9 (M), lower than 8.0×10 9 (M), lower than 7.0×109 (M), less than 6.0×10 9 (M), less than 5.0×10 9 (M), less than 4.0×10 9 (M), less than 3.0×10 9 (M), less than 2.0×10 9 (M), less than 1.0×10 9 (M), less than 9.0×10 10 (M) or less than 8.0×10 10 (M) has a Kd value that binds to human CD3 (optionally CD3εδ). In some cases, the Kd value can be measured via SPR, optionally using system.

[0083] In certain embodiments, the anti-CD3 antibody or antigen-binding fragment can be at 1.56×10 8 (M) to 7.0×10 10 (M) between, 1.0×10 8 (M) to 7.0×10 10 (M) between, 1.0×10 8 (M) to 1.0×10 9 (M) between, 2.0×10 8 (M) to 1.0×10 9 (M) between, or 5.0×10 8 (M) to 1.0×10 9 (M) between and has a Kd value that binds to human CD3 (optionally CD3εδ). In some cases, the Kd value can be measured via SPR, optionally using system.

[0084] In certain embodiments, the anti-CD3 antibody or antigen-binding fragment can be at less than 9.33×10 9 (M), less than 9.0×10 9 (M), less than 8.0×10 9 (M), less than 7.0×10 9 (M), less than 6.0×10 9 (M), less than 5.0×10 9 (M), less than 4.0×10 9 (M), less than 3.0×10 9 (M) or less than 2.0×10 9 (M) has a Kd value that binds to human CD3 (optionally CD3cδ). In some cases, the Kd value can be measured via BLI, optionally using system.

[0085] In certain embodiments, the anti-CD3 antibody or antigen-binding fragment can be soluble in water at an acidic pH (optionally about pH 6.0) at 9.32×10 9 (M) to 1.0×10 9 (M) between 9.0×10 9 (M) to 1.0×10 9 (M) between 8.0×10 9 (M) to 3.0×10 9 (M) between 7.0×10 9 (M) to 4.0×10 9 Between or 6.0×10 9 (M) to 5.0×10 9 (M) with a Kd value between 1 and 20 μM and binds to human CD3 (optionally CD3εδ). In some cases, the Kd value can be measured via BLI, optionally using system.

[0086] In some embodiments, the anti-CD3 antibody or antigen-binding fragment can bind to cynomolgus monkey CD3 (optionally CD3εδ) at acidic pH (optionally about pH 6.0).

[0087] In certain embodiments, the anti-CD3 antibody or antigen-binding fragment can be soluble in water at an acidic pH (optionally about pH 6.0) at a concentration of less than 2.0×10 8 (M), less than 1.0×10 8 (M), less than 9.0×10 9 (M), less than 8.0×10 9 (M), less than 7.0×10 9 (M), less than 6.0×10 9 (M), less than 5.0×10 9 (M), less than 4.0×10 9 (M), less than 3.0×10 9 (M) or less than 2.0×10 9 (M) binds to cynomolgus monkey CD3 (optionally CD3εδ). In some cases, the Kd value can be measured via BLI, further optionally using system.

[0088] In certain embodiments, the anti-CD3 antibody or antigen-binding fragment can be quenched at 2.0×10 8 (M) to 1.0×10 9 (M) between 1.0×10 8 (M) to 1.0×10 9(M), between 9.0×10 9 (M) and 2.0×10 9 (M), or between 8.0×10 9 (M) and 5.0×10 9 The Kd value between binds to cynomolgus monkey CD3 (optionally CD3εδ). In some cases, the Kd value can be measured via BLI, further optionally using system.

[0089] In some embodiments, the anti-CD3 antibody or antigen-binding fragment can bind to cells expressing CD3 with greater binding at an acidic pH (optionally about pH 6.0) than at physiological pH (optionally about pH 7.4).

[0090] In certain embodiments, the cell binding can be measured via flow cytometry, further optionally based on the median fluorescence intensity (MFI). In certain embodiments, the cells can express human CD3, optionally CD3εδ. In certain embodiments, the cells can express non-human primate (optionally monkey, further optionally cynomolgus monkey) CD3 (optionally CD3εδ). In certain embodiments, the cells can be primary cells. In certain embodiments, the cells can be cells of a cell line. In certain embodiments, the cells can be human cells. In certain embodiments, the cells can be human T cells. In certain embodiments, the cells can be Jurkat cells. In certain embodiments, the cells can be non-human primate (optionally monkey, further optionally cynomolgus monkey) cells. In certain embodiments, the cells can be HSC-F cells.

[0091] In certain embodiments, based on the NCB value calculated using the MFI value when measured via flow cytometry, the binding to cells expressing CD3 is at least ×1.2, at least ×1.5, at least ×2, at least ×5, at least ×10, at least ×20, at least ×50, at least ×100, at least ×10 3 at least ×10 4 at least ×10 5 at least ×10 6 at least ×10 7 at least ×10 8 or at least ×10 9。In some cases, the NCB value is calculated as {(Sample MFI)-(Secondary only MFI)} / (Secondary only MFI), in other words, {MFI of (“incubated with test antibody and then with secondary antibody”)-(MFI of incubated without primary antibody and then with secondary antibody)} / (MFI of incubated without primary antibody and then with secondary antibody).

[0092] In certain embodiments, binding to CD3-expressing cells may not bind to CD3-expressing cells at physiological pH (optionally about pH 7.4).

[0093] In some embodiments, the anti-CD3 antibody or antigen-binding fragment may trigger activation of the cytotoxic function of the cell and / or enhance the cytotoxic function of the cell when binding to CD3 on the cell.

[0094] In certain embodiments, T cell activation or initiation of T cell killing may occur, while showing a reduced tendency to trigger cytokine production to levels capable of inducing cytokine release syndrome (CRS). In certain embodiments, when binding to CD3 on a cell (optionally a T cell), the anti-CD3 antibody or antigen-binding fragment may not trigger cytokine production at levels capable of inducing CRS when binding to CD3.

[0095] In certain embodiments, the anti-CD3 antibody or antigen-binding fragment may comprise or be comprised in a multispecific antibody or antibody fragment having at least (a) a first antigen-binding region specific for CD3, the first antigen-binding region comprising the VH polypeptide and / or the VL polypeptide, and (b) a second antigen-binding region specific for a second antigen; and when binding to (i) CD3 on a first cell (optionally a T cell) and (ii) the second antigen expressed on a second cell, the first cell may exhibit cytotoxicity towards the second cell.

[0096] In another aspect, the present disclosure provides a nucleic acid (e.g., an isolated or recombinant nucleic acid, one or more nucleic acids, such as one nucleic acid, a combination of two or more nucleic acids, etc.) encoding any one of the anti-CD3 antibodies and antigen-binding fragments described herein. Such nucleic acids may include DNA such as cDNA or RNA such as mRNA, for example for delivery to a cell and expression of the anti-CD3 antibody or antigen-binding fragment.

[0097] In some embodiments, a nucleic acid according to the present disclosure may comprise: (A) a nucleic acid sequence encoding a VH polypeptide that is at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 650, 550, 450, 350, 750, 850, 950, 1050, 1150, 1250 or 1350, or an RNA (e.g., mRNA) version of any of the foregoing; and / or (B) a nucleic acid sequence encoding a VL polypeptide that is at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 660, 560, 460, 360, 760, 860, 960, 1060, 1160, 1260 or 1360, or an RNA (e.g., mRNA) version of any of the foregoing.

[0098] In certain embodiments, the nucleic acid may comprise the nucleic acid sequence encoding the VH polypeptide and the nucleic acid sequence encoding the VL polypeptide of SEQ ID NO: 650 and 660, respectively.

[0099] In certain embodiments, the nucleic acid may comprise the nucleic acid sequence encoding the VH polypeptide and the nucleic acid sequence encoding the VL polypeptide of SEQ ID NO: 550 and 560, respectively.

[0100] In certain embodiments, the nucleic acid may comprise the nucleic acid sequence encoding the VH polypeptide and the nucleic acid sequence encoding the VL polypeptide of SEQ ID NO: 450 and 460, respectively.

[0101] In certain embodiments, the recombinant nucleic acid may comprise the nucleic acid sequence encoding the VH polypeptide and the nucleic acid sequence encoding the VL polypeptide of SEQ ID NO: 350 and 360, respectively.

[0102] In certain embodiments, the nucleic acid may comprise the nucleic acid sequence encoding the VH polypeptide and the nucleic acid sequence encoding the VL polypeptide of SEQ ID NO: 750 and 760, respectively.

[0103] In certain embodiments, the nucleic acid may comprise the nucleic acid sequence encoding the VH polypeptide and the nucleic acid sequence encoding the VL polypeptide of SEQ ID NO: 850 and 860, respectively.

[0104] In certain embodiments, the nucleic acid may comprise the nucleic acid sequence encoding the VH polypeptide and the nucleic acid sequence encoding the VL polypeptide of SEQ ID NO: 950 and 960, respectively.

[0105] In certain embodiments, the nucleic acids may comprise, respectively, a nucleic acid sequence encoding a VH polypeptide of SEQ ID NO: 1050 and a nucleic acid sequence encoding a VL polypeptide of SEQ ID NO: 1060.

[0106] In certain embodiments, the nucleic acids may comprise, respectively, a nucleic acid sequence encoding a VH polypeptide of SEQ ID NO: 1150 and a nucleic acid sequence encoding a VL polypeptide of SEQ ID NO: 1160.

[0107] In certain embodiments, the nucleic acids may comprise, respectively, a nucleic acid sequence encoding a VH polypeptide of SEQ ID NO: 1250 and a nucleic acid sequence encoding a VL polypeptide of SEQ ID NO: 1260.

[0108] In certain embodiments, the nucleic acids may comprise, respectively, a nucleic acid sequence encoding a VH polypeptide of SEQ ID NO: 1350 and a nucleic acid sequence encoding a VL polypeptide of SEQ ID NO: 1360.

[0109] In some cases, the nucleic acid may comprise RNA (e.g., mRNA), which may comprise an RNA sequence encoding a VH polypeptide and an RNA sequence encoding a VL polypeptide corresponding to any one of the foregoing nucleic acid sequences.

[0110] In another aspect, the present disclosure provides a vector (e.g., one or more vectors, such as a single vector, a combination of two or more vectors, etc.) encoding any one of the anti-CD3 antibodies and antigen-binding fragments described herein.

[0111] In some embodiments, the vector may comprise any one of the nucleic acids described herein.

[0112] In some embodiments, the vector may be an expression vector.

[0113] In some embodiments, the vector may comprise a plasmid, a viral vector (optionally an adenovirus, a lentivirus or a retrovirus), a lipid-based vector, a self-replicating RNA vector, a virus-like particle, a polymer-based vector and / or a nanoparticle, optionally a lipid-based nanoparticle.

[0114] In another aspect, the present disclosure provides an isolated or recombinant cell that comprises any one of the nucleic acids described herein and / or any one of the vectors described herein, is transfected with any one of the nucleic acids described herein and / or any one of the vectors described herein, is transformed with any one of the nucleic acids described herein and / or any one of the vectors described herein, or is transduced with any one of the nucleic acids described herein and / or any one of the vectors described herein.

[0115] In some embodiments, the isolated or recombinant cell can be mammalian. In certain embodiments, the isolated or recombinant cell can be human, non-human primate, monkey, rabbit, rodent, hamster, rat, or mouse. In some embodiments, the isolated or recombinant cell can be non-mammalian, optionally plant, bacterium, fungus, yeast, protozoan, or insect. In some embodiments, the isolated or recombinant cell can be an immune cell or a hybridoma.

[0116] In another aspect, the present disclosure provides pharmaceutical compositions.

[0117] In some embodiments, a pharmaceutical composition according to the present disclosure can comprise: (A) any one of the anti-CD3 antibodies and antigen-binding fragments described herein; and (B) a pharmaceutically acceptable carrier and / or excipient. In some embodiments, a pharmaceutical composition according to the present disclosure can comprise: (A) any one of the nucleic acids described herein; and (B) a pharmaceutically acceptable carrier and / or excipient. In some embodiments, a pharmaceutical composition according to the present disclosure can comprise: (A) any one of the vectors described herein; and (B) a pharmaceutically acceptable carrier and / or excipient. In some embodiments, a pharmaceutical composition according to the present disclosure can comprise: (A) any one of the isolated or recombinant cells described herein; and (B) a pharmaceutically acceptable carrier and / or excipient.

[0118] In another aspect, the present disclosure provides methods of treating a subject in need of such treatment and methods of treating or preventing a disease, disorder, or condition in a subject.

[0119] In some embodiments, the method can comprise administering to the subject an effective amount of any one of the anti-CD3 antibodies and antigen-binding fragments described herein. In some embodiments, the method can comprise administering to the subject an effective amount of any one of the nucleic acids described herein. In some embodiments, the method can comprise administering to the subject an effective amount of any one of the vectors described herein. In some embodiments, the method can comprise administering to the subject an effective amount of any one of the isolated or recombinant cells described herein. In some embodiments, the method can comprise administering to the subject an effective amount of any one of the pharmaceutical compositions described herein.

[0120] In another aspect, the present disclosure provides methods of inducing cytotoxicity to cells expressing a target molecule of interest.

[0121] In some embodiments, the method may include administering to a subject an effective amount of any of the multispecific antibodies or antibody fragments described herein, or an anti-CD3 antibody and antigen-binding fragment contained in a multispecific antibody or antibody fragment. In some embodiments, the method may include administering to a subject an effective amount of a nucleic acid encoding such an anti-CD3 antibody or antigen-binding fragment. In some embodiments, the method may include administering to a subject an effective amount of a vector comprising such a nucleic acid and / or encoding such an anti-CD3 antibody or antigen-binding fragment. In some embodiments, the method may include administering to a subject an effective amount of isolated or recombinant cells that contain such a nucleic acid or such a vector, are transfected with such a nucleic acid or such a vector, are transformed with such a nucleic acid or such a vector, or are transduced with such a nucleic acid or such a vector. In some embodiments, the method may include administering to a subject an effective amount of a pharmaceutical composition comprising (A) any of the multispecific antibodies or antibody fragments described herein, or an anti-CD3 antibody and antigen-binding fragment contained in a multispecific antibody or antibody fragment, a nucleic acid encoding such an anti-CD3 antibody or antigen-binding fragment, a vector comprising such a nucleic acid, and / or isolated or recombinant cells that contain such a nucleic acid or such a vector, are transfected with such a nucleic acid or such a vector, are transformed with such a nucleic acid or such a vector, or are transduced with such a nucleic acid or such a vector; and / or (B) a pharmaceutically acceptable carrier and / or excipient.

[0122] In any of the above method embodiments, in some cases, the subject may be a mammal, optionally a human, non-human primate, monkey, horse, cow, sheep, goat, pig, dog, cat, rabbit, rodent, hamster, rat, or mouse. In certain embodiments, the subject may be a non-mammalian vertebrate, optionally a bird, fish, amphibian, or reptile.

[0123] In any of the above method embodiments, in some cases, the method may further include administering to the subject an additional agent, optionally an adjuvant or therapeutic agent.

[0124] In any of the above method embodiments, in some cases, the subject may have or be at risk of having a disease, disorder, or condition.

[0125] In any of the above method embodiments, in some cases, the disease, disorder, or condition includes a cancer or neoplastic condition, an autoimmune disease, a neurodegenerative disease, an infectious disease, an inflammatory disease, or other disease.

[0126] In certain embodiments, the cancer can be a solid cancer, optionally selected from one or more of the following: mesothelioma, malignant pleural mesothelioma, non-small cell lung cancer, small cell lung cancer, squamous cell lung cancer, large cell lung cancer, pancreatic cancer, pancreatic ductal adenocarcinoma, esophageal adenocarcinoma, breast cancer, glioblastoma, ovarian cancer, colorectal cancer, prostate cancer, cervical cancer, skin cancer, melanoma, kidney cancer, liver cancer, brain cancer, thymoma, sarcoma, carcinoma, uterine cancer, kidney cancer, gastrointestinal cancer, urothelial cancer, pharyngeal cancer, head and neck cancer, rectal cancer, esophageal cancer, or bladder cancer or metastases thereof.

[0127] In certain embodiments, the cancer can be a liquid cancer, optionally selected from: chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), multiple myeloma, acute lymphoblastic leukemia (ALL), Hodgkin lymphoma, B-cell acute lymphoblastic leukemia (BALL), T-cell acute lymphoblastic leukemia (TALL), small lymphocytic leukemia (SLL), B-cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt lymphoma, diffuse large B-cell lymphoma (DLBCL), DLBCL associated with chronic inflammation, chronic myeloid leukemia, myeloproliferative neoplasm, follicular lymphoma, pediatric follicular lymphoma, hairy cell leukemia, small cell or large cell follicular lymphoma, malignant lymphoproliferative condition, MALT lymphoma (mucosa-associated lymphoid tissue extranodal marginal zone lymphoma), marginal zone lymphoma, myelodysplasia, myelodysplastic syndrome, non-Hodgkin lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenström macroglobulinemia, splenic marginal zone lymphoma, splenic lymphoma / leukemia, splenic diffuse red pulp small B-cell lymphoma, hairy cell leukemia variant, lymphoplasmacytic lymphoma, heavy chain disease, plasma cell myeloma, solitary plasmacytoma of bone, extramedullary plasmacytoma, nodal marginal zone lymphoma, pediatric nodal marginal zone lymphoma, primary cutaneous follicle center lymphoma, lymphomatoid granulomatosis, primary mediastinal (thymic) large B-cell lymphoma, intravascular large B-cell lymphoma, ALK+ large B-cell lymphoma, large B-cell lymphoma arising from HHV8-associated multicentric Castleman disease, primary effusion lymphoma, B-cell lymphoma, acute myeloid leukemia (AML), or lymphoma not otherwise specified.

[0128] In certain embodiments, the autoimmune or inflammatory disease can be psoriasis, rheumatoid arthritis, autoimmune arthritis, type I diabetes, sarcoidosis, systemic lupus erythematosus, myasthenia gravis, multiple sclerosis, scleroderma, inflammatory bowel disease, Crohn's disease, ulcerative colitis, Guillain-Barre syndrome, chronic inflammatory demyelinating polyneuropathy, pemphigus vulgaris, Sjogren syndrome, Addison disease, Behcet disease, Schmidt syndrome, celiac disease, dermatomyositis, autoimmune vitiligo, Graves′ disease, Hashimoto thyroiditis, Kawasaki disease, pernicious anemia, autoimmune vasculitis or fibrosis.

[0129] In certain embodiments, the neurodegenerative disease can be Alzheimer's disease, Huntington's disease, Parkinson's disease, amyotrophic lateral sclerosis, Friedreich ataxia, Lewy body disease, spinal muscular atrophy, motor neuron disease, multiple sclerosis, Batten disease, Creutzfeldt-Jakob disease.

[0130] In certain embodiments, the infectious disease can be a viral disease, a bacterial disease, a fungal disease, a yeast disease, a protozoal disease, a prion disease, or a parasitic disease, optionally wherein (1) the viral disease is human immunodeficiency virus (HIV), hepatitis virus (optionally hepatitis A, B, or C virus), human papillomavirus (HPV), herpes simplex virus (HSV) (optionally HSV-1 or HSV-2), enterovirus, human cytomegalovirus, adenovirus, rhinovirus, poxvirus, influenza virus, coronavirus (optionally MERS-CoV, SARS-CoV, or SARS-CoV-2 or common human coronaviruses), norovirus, West Nile virus, Zika virus, poliovirus, Ebola virus, or dengue virus (DENV) infection, (2) the bacterial disease is Salmonella, Escherichia coli, Mycobacterium tuberculosis, methicillin-resistant Staphylococcus aureus (MRSA), Clostridioides difficile, Streptococcus pneumoniae, Klebsiella pneumoniae, Pseudomonas aeruginosa, Helicobacter pylori, Neisseria gonorrhoeae, Vibrio vulnificus, and / or (3) the fungal disease is aspergillosis, Candida spp., Candida auris, Cryptococcus neoformans, Pneumocystis jirovecii, Mucorales, Talaromyces spp., tinea, Blastomyces spp., Coccidioides spp., Cryptococcus gattii, Histoplasma spp., Paracoccidioides spp., or Sporothrix spp. infection.

[0131] In another aspect, the present disclosure provides methods of preparing the anti-CD3 antibodies or antigen-binding fragments described herein.

[0132] In some embodiments, the method can include (a) culturing a cell comprising a nucleic acid encoding an anti-CD3 antibody or antigen-binding fragment under conditions that permit expression of the antibody or antigen-binding fragment, and (b) harvesting and purifying the antibody or antigen-binding fragment from the cell culture from (a).

[0133] In another aspect, the present disclosure provides methods of preparing an isolated or recombinant cell or a population of such cells according to the present disclosure.

[0134] In some embodiments, the method can include introducing (i) a nucleic acid encoding an anti-CD3 antibody or antigen-binding fragment according to the present disclosure and / or (ii) a vector encoding an anti-CD3 antibody or antigen-binding fragment according to the present disclosure or comprising such a nucleic acid into one or more cells.

[0135] In certain embodiments, the introducing can be performed in vitro, ex vivo, or in vivo.

[0136] Any one of the anti-CD3 antibodies and antigen-binding fragments according to the present disclosure, any one of the nucleic acids according to the present disclosure, any one of the vectors according to the present disclosure, any one of the isolated or recombinant cells or such cell populations according to the present disclosure, and / or any one of the pharmaceutical compositions according to the present disclosure can be used in medicine or for preparing a medicament for use in medicine.

[0137] Any one of the anti-CD3 antibodies and antigen-binding fragments according to the present disclosure, any one of the nucleic acids according to the present disclosure, any one of the vectors according to the present disclosure, any one of the isolated or recombinant cells or such cell populations according to the present disclosure, and / or any one of the pharmaceutical compositions according to the present disclosure can be used for treating a disease, disorder or condition, optionally any one of the diseases, disorders or conditions described herein.

[0138] The present disclosure also encompasses the use of any one of the anti-CD3 antibodies and antigen-binding fragments according to the present disclosure, any one of the nucleic acids according to the present disclosure, any one of the vectors according to the present disclosure, any one of the isolated or recombinant cells or a population of such cells according to the present disclosure, and / or any one of the pharmaceutical compositions according to the present disclosure for preparing a medicament for treating a disease, disorder or condition, optionally any one of the diseases, disorders or conditions described herein. Detailed Description

[0139] Definition

[0140] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0141] It should be understood that the terms used in the description are for the purpose of describing particular versions or embodiments only and are not intended to limit the scope of the invention, which is limited only by the appended claims.

[0142] All references cited herein, including patent documents and non-patent documents, are incorporated herein by reference in their entirety. Nothing herein shall be construed as an admission that the present invention is not entitled to antedate such disclosure by virtue of a prior invention.

[0143] Unless the context clearly dictates otherwise, as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural referents. Thus, reference to “a cell” refers to one or more cells known to those skilled in the art and their equivalents, and so forth.

[0144] As used herein, the term "about" when used in reference to a particular recited value means that the value can vary from the recited value by no more than 5%. For example, as used herein, the expression "about 100" includes 95 and 105 and all values in between (e.g., 96, 97, 98, 99, 99.1, 99.2, 99.3, 99.4, 100, 101, 102, 103, 104, etc.).

[0145] It is to be understood that the aspects and embodiments of the present disclosure described herein include "comprising" aspects and embodiments, "consisting of" aspects and embodiments, and "consisting essentially of" aspects and embodiments. The conjunctive terms "comprising", "including", "having", "containing", "involving", "consisting of", etc. are to be understood as open-ended, i.e., not excluding additional unrecited elements. Thus, such conjunctive terms encompass open-ended embodiments as well as, depending on the circumstances, closed and semi-closed embodiments (i.e., embodiments described, respectively, with "consisting of" and "consisting essentially of"). Only the conjunctive terms "consisting of" and "consisting essentially of" should be closed or semi-closed conjunctive terms, respectively.

[0146] The term "antibody" is used herein in its broadest sense and encompasses various antibody structures, including but not limited to intact antibodies and antibody fragments (preferably those fragments that exhibit the desired antigen-binding activity (i.e., antigen-binding fragments)), multispecific (e.g., bispecific, trispecific, etc.) antibodies and antibody fragments, monoclonal antibodies, polyclonal antibodies, etc.

[0147] The terms "intact antibody" and "whole antibody" etc. are used interchangeably herein and refer to an antibody having a structure that is substantially similar to a native antibody. In some cases, an antibody comprises heavy (H) chains and light (L) chains interconnected by disulfide bonds. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these classes can be further subdivided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to the different classes of immunoglobulins are designated α, δ, ε, γ, and μ, respectively. For example, an intact IgG (or IgD or IgE) antibody comprises two immunoglobulin heavy chains and two immunoglobulin light chains. Thus, in some cases, an antibody according to the present disclosure can comprise two pairs of heavy and light chains interconnected by disulfide bonds or an antigen-binding fragment thereof. Some intact antibodies comprise multiple units, each unit comprising two pairs of heavy and light chains interconnected by disulfide bonds. For example, intact IgA comprises two units, and intact IgM comprises five units. Thus, in other cases, an antibody according to the present disclosure can alternatively comprise multiple (e.g., two, three, four, five, etc.) units, each unit comprising two pairs of heavy and light chains interconnected by disulfide bonds, or an antigen-binding fragment thereof.

[0148] Each heavy chain comprises: a heavy chain variable domain (VH); and a heavy chain constant region (CH), typically comprising domains CH1, CH2, and CH3. Each light chain comprises: a light chain variable domain (VL); and a light chain constant region (CL). Typically (with some exceptions such as nanobodies, camelid heavy chain antibodies, IgNAR, etc.), one VH and one VL can form an antigen-binding region. The VH and VL can be further subdivided into hypervariable regions called complementarity-determining regions (CDRs), which are interspersed with more conserved regions called framework regions (FRs). Each VH and VL polypeptide is composed of three CDRs and four FRs arranged in the following order from the amino terminus to the carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The CDRs in the heavy chain are named "CDR-H1", "CDR-H2", and "CDR-H3", respectively, and the CDRs in the light chain are named "CDR-L1", "CDR-L2", and "CDR-L3". The FRs in the heavy chain are named "FR-H1", "FR-H2", "FR-3", and "FR-H4", respectively, and the FRs in the light chain are named "FR-L1", "FR-L2", "FR-L3", and "FR-L4". In certain embodiments of the present disclosure, the FRs of the antibody (or its antigen-binding fragment) can be identical to human germline-encoded sequences (e.g., the heavy chain FR sequence encoded by the VH1-03 germline and / or the light chain FR sequence encoded by the VK4-01 germline), or can be natural or artificially modified. Amino acid consensus sequences can be defined based on the juxtaposed analysis of two or more CDRs.

[0149] The numbering of amino acid residues in the antibody variable domain and / or constant domain can be performed by any suitable numbering scheme, method, and definition, e.g., based on a numbering scheme such as EU numbering (as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)), IMGT numbering, Kabat numbering, Chothia numbering, Martin numbering, Gelfand numbering, or Honneger numbering); or structurally (see, e.g., the NCBI online tool, IgBlast; Dondelinger et al., Front Immunol. October 16, 2018; 9:2278).

[0150] According to IMGT (the international ImMunoGeneTics information system for immunoglobulins or antibodies, T cell receptors, MH, immunoglobulin superfamily IgSF and MhSF), the CH1 domain, hinge region, CH2 domain and CH3 domain correspond to amino acid positions 118 - 215, 216 - 230, 231 - 340 and 341 - 446 (EU numbering), respectively. The terms "CH1 domain", "hinge", "CH2 domain" and "CH3" are used herein broadly to encompass any naturally occurring, corresponding heavy chain constant domain and / or region allotype and variants thereof, which may include fewer or more amino acids (e.g., the CH1 domain may include a portion of the hinge region) and / or amino acid modifications.

[0151] Exemplary CH1 domains of human IgG1 may comprise the amino acid sequences of SEQ ID NO: 41 or 42; an exemplary hinge of human IgG1 may include the amino acid sequence of SEQ ID NO: 51; and the CH2 domain of human IgG1 may include the amino acid sequence of SEQ ID NO: 61. Exemplary CH3 domains of human IgG1 may include the amino acid sequences of SEQ ID NO: 71, 72, 73 or 74, and a C-terminal K may be added to any of such CH3 sequences. Any variant of such exemplary sequences may be used in combination with the anti-CD3 variable sequences described herein.

[0152] The "Fc region" is the C-terminal region of an immunoglobulin heavy chain containing at least a portion of the constant region, including the native sequence Fc region and variant Fc regions. The Fc region of the human IgG heavy chain may extend from Cys226 or from Pro230 to the carboxyl terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also referred to as the EU index, as described below: Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991.

[0153] The expression "effector function" of an antibody refers to the biological activities attributable to the Fc region of the antibody, which vary with the antibody isotype. Exemplary effector functions include: complement (e.g., C1q) binding and complement-dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptors); and B cell activation.

[0154] There are two main light chain isotypes, kappa (κ) and lambda (λ), and the corresponding light chain constant domains are called the kappa CL domain (CLκ domain) and the lambda CL domain (CLλ domain), respectively.

[0155] According to IMGT, the CLκ domain is amino acid positions 108 - 214 (EU numbering). An exemplary CLκ domain of human IgG can include the amino acid sequence of SEQ ID NO: 81. According to IMGT, the CLλ domain is amino acid positions 107 - 215 (EU numbering). An exemplary CLλ domain of human IgG can include the amino acid sequence of SEQ ID NO: 82.

[0156] The terms "CLκ domain" and "CLλ domain" are used herein broadly to encompass any naturally occurring, corresponding light chain constant domain and / or regional allotype and variants thereof which may include fewer or more amino acids and / or amino acid modifications.

[0157] The various standard sequences (corresponding to different allotypes) of the constant domains of human IgG1, IgG2, IgG3, and IgG4 are known in the art and can be found, for example, in Vidarsson et al., Frontiers in Immunology, October 20, 2014; 5:520 and U.S. Patent No. 9,150,663, the disclosures of which are hereby incorporated by reference in their entirety herein. Similarly, these reference sequences are intended to be exemplary as the applicant intends for the human IgG1, IgG2, IgG3, and IgG4 sequences to encompass any naturally occurring human IgG1, IgG2, IgG3, and IgG4 allotypes.

[0158] "Antigen-binding fragment" or "antigen-binding antibody fragment" refers to a part of a whole antibody or a combination of parts derived from one or more whole antibodies, where the whole antibody binds to an antigen (in this case, CD3). The antigen-binding fragment of an antibody comprises any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex. Exemplary antigen-binding fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g., single-chain variable fragments (scFv), half-antibodies, nanobodies, or VH or VL alone); and multispecific antibodies formed from antibody fragments. In some embodiments, the antigen-binding fragment of the anti-CD3 antibody described herein is scFv. The term "half-molecule" or "half-antibody", which may be referred to as "half-IgG", "half-IgE", or "half-IgD" when referring to IgG, IgE, or IgD, respectively, is a collection consisting of one heavy chain and one light chain of a reference antibody.

[0159] "Antigen-binding region" refers to a part of an antibody or antigen-binding fragment that is specific for an antigen.

[0160] With respect to multispecific antibodies (e.g., bispecific, trispecific, tetra-specific, etc.), such antibodies comprise at least two different antigen-binding regions that recognize and specifically bind to at least two different antigens or epitopes. The at least two epitopes may or may not be within the same antigen. A "bispecific antibody" is a type of multispecific antibody and comprises two different antigen-binding regions that recognize and specifically bind to two different antigens or two epitopes. Bispecific antibodies can target, for example, two different surface receptors on the same or different (e.g., immune cells and cancer cells) cells.

[0161] The expression "different antigens" can refer to different and / or distinct proteins, polypeptides, or molecules; and different and / or distinct epitopes that may be contained within a protein, a polypeptide, or a molecule.

[0162] The term "epitope" refers to an antigenic determinant that interacts with the specific antigen-binding site in the variable region of an antibody molecule, called a paratope. A single antigen can have more than one epitope. Thus, different antibodies can bind to different regions on an antigen and can 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 can be defined as structural or functional. Functional epitopes are generally a subset of structural epitopes and have those residues that directly contribute to the interaction affinity. Epitopes can also be conformational, i.e., composed of non-linear amino acids. In certain embodiments, an epitope can contain determinants that are chemical reactive surface groupings as molecules, such as amino acids, sugar side chains, phosphoryl or sulfonyl groups, and in certain embodiments, can have specific three-dimensional structural characteristics and / or specific charge characteristics.

[0163] "Monoclonal antibody" or "mAb" refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies comprising the population are identical and / or bind the same epitope, except for possible variant antibodies (e.g., containing naturally occurring mutations or those arising during the production of the monoclonal antibody preparation) (such variants generally being present in minor amounts). As compared to polyclonal antibody preparations, which typically contain different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single

[0164] "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 non-human primates) and rodents (e.g., mice and rats), unless otherwise specified, including, for example, CD3ε, CD3γ, CD3α, and CD3β chains. The term encompasses "full-length", untreated CD3 (e.g., untreated or unmodified CD3ε or CD3γ), as well as any form of CD3 produced by processing in a cell. The term also encompasses naturally occurring variants of CD3, including, for example, splice variants or allelic variants. CD3 includes, for example, the human CD3ε protein of 207 amino acids in length (NCBI RefSeq number NP_000724) and the human CD3γ protein of 182 amino acids in length (NCBI RefSeq number NP_000064). The term also refers to the human or cynomolgus monkey CD3ε proteins, SEQ ID NO: 91 and 92, respectively (Table U). "CD3εN27" and "CD3εN13" refer to the N-terminal 27 amino acids and N-terminal 13 amino acids of CD3, respectively, and optionally contain chemical modifications or conjugations thereto.

[0165] "Anti-CD3 antibody" refers to an antibody or an antigen-binding fragment thereof that can bind to CD3, such as CD3ε and / or CD3γ, such as a human CD3ε and / or CD3γ having sufficient affinity and / or specificity such that the antibody can be used as a diagnostic agent and / or therapeutic agent targeting CD3. In some embodiments, the anti-CD3 antibody binds to CD3 with a dissociation constant (K -9 ) of about 100×10 -9 M or less, about 50×10 -9 M or less, about 25×10 -9 M or less, about 20×10 -9 M or less, or about 10×10 D M or less. In some embodiments, the anti-CD3 antibody binds to CD3 with a dissociation constant (K -9 ) of about 5×10 D M or less. In some embodiments, the anti-CD3 antibody binds to CD3 with a dissociation constant (K -9 ) of about 2.5×10 D M or less. In some embodiments, the anti-CD3 antibody binds to CD3 with a dissociation constant (K -10 ) of about 1×10 D M or less. In some embodiments, K is measured by SPR (e.g., ), BLI measurement using, for example, a FORTEBIO D HTX instrument (Pall Life Sciences), or solution affinity ELISA. In some embodiments, KD is measured using the scFv fragment of the anti-CD3 antibody. In some embodiments, the monovalent KD is measured. In some embodiments, the anti-CD3 antibody binds to an epitope of CD3 that is conserved in CD3 that is cross-reactive among different species (e.g., human and cynomolgus monkey).

[0166] "Immunoconjugate" is an antibody conjugated to one or more moieties such as a polymer, a label, an additional agent (e.g., an antibiotic, a second anti-CD3 antibody, a vaccine, or a toxoid), or any other therapeutic moiety or any other agent described herein.

[0167] The term "developable" or "developability" refers to the extent to which one or more of a plurality of polypeptides have desired properties such as, but not limited to: desired binding specificity, e.g., binding to a cognate antigen with a desired affinity and not significantly binding to a non-cognate antigen; desired expression, e.g., in mammalian cells; solubility; viscosity; aggregation; chemical and / or physical stability; required shelf life; melting temperature; toxicity; pharmacokinetic profile; circulating half-life; and clearance characteristics. Such characteristics can serve as markers independently; as a combination of subsets of such markers; or collectively, such that one or more of such polypeptides may be successfully developed as a therapeutic candidate and ultimately become an approved drug. Generally, polypeptides having desired developability properties have one or more of the following: relatively high solubility, relatively low viscosity, relatively low tendency to aggregate, relatively high chemical stability, relatively high physical stability, relatively long shelf life, relatively high melting temperature, relatively long circulating half-life, relatively slow clearance rate, and the like. In contrast, polypeptides having undesired developability properties generally have one or more of the following: 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 temperature, relatively short circulating half-life, relatively fast clearance rate, etc.

[0168] Methods and assays that can be used to determine the extent to which a polypeptide, such as an anti-CD3 antibody and / or antigen-binding fragment thereof as described herein, has desired developability characteristics are available in the art and can include, but are not limited to: cytokine release assays; multispecific reagent (PSR) assays (WO 2014 / 179363 and Xu et al., Protein Eng Des Sel, Vol. 26, pp. 663-670 (2013)); cross-interaction chromatography (CIC); self-interaction chromatography (SIC); hydrophobic interaction chromatography (HIC); 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 methods; ELISA assays, such as the BVP ELISA assay; AC-SINS assays (Liu et al.; MAbs, Vol. 6, pp. 483-492 (2014); melting temperature (Tm) assays; differential scanning calorimetry or differential scanning fluorimetry (DSF); and the like (see, for example, He et al., J. Pharm. Sci, Vol. 100(4), pp. 1330-1340 (2011); Wagner et al., Pharm. Develop. & Technol (published online in 2012; Hypertext Transfer Protocol: informahealthcare.com / doi / abs / 10.3109 / 10837450.2011.649851); Hotzel et al., MAbs, Vol. 4(6), pp. 753-7601 (2012); Weiqiang et al., J. Pharm. Sci., Vol. 101(5), pp. 1701-1720 (2012); Banks et al., J. Pharm. Sci., Vol. 101(8), pp. 2720-2732 (2012); Lie et al., J. Pharm. Sci., Vol. 94(9), pp. 1928-1948 (2005); and Payne et al., Biopolymers, Vol. 85(5), pp. 527-533 (2006)).

[0169] "Developability profile" refers to an index that can be assigned to an antibody when assessing the developability of the antibody. The developability profile is a measure 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 of a CD3 binder and an antibody comprising the same. The degree of interaction can be evaluated by any number of means available in the art, which provide an output value related to the strength or affinity of the polypeptide for the moiety to which it binds. Exemplary methods include flow cytometry methods such as fluorescence-activated cell sorting (FACS); enzyme-linked immunosorbent assay (ELISA); quantitative immunoaflfinity assay or immunoprecipitation assay; mammalian two-hybrid or yeast two-hybrid assay, etc. In the context of FACS, as demonstrated in the examples, the degree of interaction between a polypeptide and PSR among the plurality of polypeptides can be determined by generating the mean fluorescence intensity for each detected polypeptide-PSR interaction and then ranking the mean fluorescence intensities in ascending or descending order, thereby ranking the polypeptides among the plurality of polypeptides according to the relative degree of interaction between each detected polypeptide and PSR. Such ranking provides a ranking of the polypeptides among the plurality of polypeptides, such that it is easy to identify those polypeptides with enhanced developability, as well as those polypeptides with reduced developability.

[0170] The developability profile can also take the form of a normalized score, for example, by normalizing the developability of the anti-CD3 antibodies described herein to the developability of a standard (or control) antibody (e.g., anti-HEL antibody).

[0171] The term "cytokine release syndrome" (or "CRS") refers to a pro-inflammatory positive feedback loop between cytokines and immune cells that results in the excessive or uncontrolled release of pro-inflammatory cytokines within the immune system (see, e.g., Lee et al., Blood, vol. 124, pp. 188-195 (2014) and Tisoncik et al., Microbiol Mol Biol Rev, vol. 76, pp. 16-32 (2012)). Upon stimulation and activation, T cells release a range of cytokines, the levels and extent of which can have adverse bio / physiological effects or effects of varying degrees and severities, including, for example, acute inflammation characterized by inflammation (redness), swelling or edema, burning (heat), pain (pain), and "functio laesa" (loss of function). When localized in the skin or other tissues, the bio / physiological effects include increased blood flow, enabling vascular leukocytes and plasma proteins to reach the extravascular site of injury, increased local temperature, and the production of pain, tissue edema, and extravascular pressure, as well as reduced tissue perfusion. Other bio / physiological effects include organ and system dysfunction, such as cardiac dysfunction, adult respiratory distress syndrome, neurotoxicity, renal and / or liver 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 chemokines are associated with the prediction and / or etiology of CRS, or with the propensity to trigger CRS upon T cell stimulation.

[0172] The term "pH-dependent" antibody refers to an antibody having a modified amino acid sequence that permits preferential or selective antigen binding at a particular pH. For example, an antibody can be engineered (e.g., by modifying the amino acid sequence) to effect pH-dependent binding. pH-dependent binding refers to the preference of an antibody to bind an antigen at a given pH (or given pH range) as compared to different pHs (or pH ranges). In one embodiment, a pH-dependent antibody preferentially or selectively binds an antigen at an acidic pH (e.g., a pH around 6) as compared to physiological pH (e.g., a pH around 7 or 7.4). Antibody sequences can be modified, for example, by substituting one or more ionizable amino acid residues such as histidine, lysine, arginine, aspartic acid, and glutamic acid. Ionizable residues can be substituted into the CDRs and / or FRs. In some embodiments, 1-10 substitutions can be present in each variant VH or VK. In some embodiments, 1-6 substitutions can be present in each variant VH or VK. Although the pH range of human blood is about 7.6-7.8, the extracellular pH of tumor cells is about 6.3-6.5, at least in part due to the accumulation of metabolic acids that cannot be efficiently cleared due to poor tumor vascularization. Thus, antibodies having an antigen binding preference at a pH lower than physiological pH (e.g., pH ~6) are thought to provide selective and sustained cytotoxic activity at or around the tumor site, potentially reducing or eliminating off-target effects and minimizing the risk of CRS, as well as improving half-life and dosing. Thus, in certain embodiments, the anti-CD3 antibodies and / or antigen-binding fragments described herein can have a reduced likelihood of causing potential CRS or can reduce the severity of potential CRS.

[0173] The tendency of an antibody to bind to multiple targets is referred to as "multispecificity", which, together with target-specific therapeutic antibodies, can be associated with negative clinical outcomes. The anti-CD3 antibodies and antigen-binding fragments of the present disclosure can exhibit reduced multispecificity [e.g., as evaluated by interaction with a multispecificity reagent (PSR)]. Antibodies with reduced multispecificity can be engineered from starting antibodies and antibody fragments by substituting various variable domain amino acid residues with residues having charged side chains. Residues can be replaced with amino acid residues having negatively charged side chains, such as Asp and Glu residues. The residues selected for substitution can be selected from those predicted not to specifically interact with the CD3 amino acid residues to which the anti-CD3 antibodies and antigen-binding fragments bind. Antibodies having a high interaction with a PSR can be referred to as "multispecific" antibodies and can be referred to as relatively "undevelopable" or relatively "non-developable".

[0174] "Conservative amino acid substitution" is an amino acid substitution in which an amino acid residue is replaced by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). Generally, conservative amino acid substitutions will essentially not alter the functional properties of a protein. In cases where two or more amino acid sequences differ from each other by conservative substitutions, the percentage or degree of similarity can be up-regulated to correct for the conservative nature of the substitutions. Means for making such adjustments are well 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 having side chains with similar chemical properties include 1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; 2) aliphatic-hydroxy 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, the group of conservative amino acid substitutions is: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid-aspartic acid, and asparagine-glutamine. Alternatively, in some embodiments, conservative substitutions include any change having a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science 256: 1443-45. In some embodiments, "moderately conservative" substitutions include any change having a non-negative value in the PAM250 log-likelihood matrix.

[0175] The term "nucleic acid" or "polynucleotide" refers to RNA or DNA that is linear or branched, single-stranded or double-stranded, or a hybrid thereof. The term also encompasses RNA / DNA hybrids. Non-limiting examples of polynucleotides are: genes or gene fragments, exons, introns, mRNA, tRNA, rRNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. Nucleic acids can contain modified nucleotides such as methylated nucleotides and nucleotide analogs, uracil, other sugars and linking groups such as fluororibose and thiolate, and nucleotide branches. The sequence of nucleotides can be further modified after polymerization, such as by conjugation with a labeling component. Other types of modifications included in this definition are capping, replacement of one or more naturally occurring nucleotides with analogs, and introduction of means for attaching the polynucleotide to proteins, metal ions, labeling components, other polynucleotides, or solid supports. Polynucleotides can be obtained by chemical synthesis, recombinantly, or from microorganisms.

[0176] A "vector" is a compound or composition of matter that contains an isolated nucleic acid and can be used to deliver the isolated nucleic acid into the interior of a cell. Many vectors are known in the art, including but not limited to linear polynucleotides, polynucleotides associated with ions or amphiphilic compounds, plasmids, viruses, and virus-like particles (VLPs). Thus, the term "vector" includes autonomously replicating plasmids, self-replicating RNAs, or viral particles. The term should also be interpreted to include non-plasmid and non-viral compounds that facilitate the transfer of nucleic acids into cells, such as, for example, polylysine compounds, liposomes, and the like. Examples of viral vectors include but are not limited to adenoviral vectors, adeno-associated viral vectors, retroviral vectors, lentiviral vectors, and the like.

[0177] The term "host cell" refers to a cell that has been introduced with an exogenous nucleic acid sequence, including progeny of such a cell. Host cells include transformants and transformed cells, which include the primary transformed cell and progeny derived therefrom, regardless of the number of passages.

[0178] A "pharmaceutical formulation" refers to a preparation in a form that permits the biological activity of the active ingredient (such as the anti-CD3 antibody described herein) contained therein to be effective and preferably does not contain additional components that are unacceptable toxic to the subject to which the formulation will be administered.

[0179] A "pharmaceutically acceptable carrier" refers to the components of a pharmaceutical formulation other than the active ingredient, which are non-toxic to the subject. Pharmaceutically acceptable carriers include but are not limited to buffers, excipients, stabilizers, or preservatives. In another embodiment, the pharmaceutical formulation contains any of the anti-CD3 antibodies provided herein and at least one additional therapeutic agent.

[0180] An "effective amount" of an anti-CD3 antibody or a composition (e.g., a pharmaceutical composition) comprising such an antibody as disclosed herein is at least a minimal amount required to achieve a desired therapeutic or prophylactic outcome (e.g., a measurable improvement or prevention of a particular disorder (e.g., a proliferative disorder, such as cancer), preferably with minimal or no toxicity or adverse effects). The effective amount can vary depending in particular on the disease state, age, sex, and weight of the patient, as well as the ability of the antibody (or its antigen-binding fragment) to elicit a desired response in the individual and in some cases due to the co-administration of one or more additional therapeutic agents.

[0181] "Disorder" means any condition or disease that would benefit from treatment, including but not limited to chronic and acute conditions or diseases, including those pathological conditions that render a mammal susceptible to the disorder in question.

[0182] The terms "proliferative disorder" and "proliferative disease" refer to disorders associated with some degree of abnormal cell proliferation. Proliferative disorders include cancer, such as tumors.

[0183] As used herein, "tumor" refers to all tumor cell growth and proliferation, whether malignant or benign, as well as all pre-cancerous and cancerous cells and tissues.

[0184] "Cancer" refers to a physiological condition in mammals characterized by unregulated cell growth. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies; more specific examples thereof include squamous cell carcinoma (e.g., epithelial squamous cell carcinoma), lung cancer (including small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma), peritoneal cancer, hepatocellular carcinoma, gastric cancer (gastric or stomach cancer) (including gastrointestinal cancer and gastrointestinal stromal cancer), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, urethral cancer, hepatoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial cancer or uterine cancer, salivary gland cancer, kidney cancer (kidney or renal cancer), prostate cancer, vulvar cancer, thyroid cancer, liver cancer, anal cancer, penile cancer, melanoma, superficial spreading melanoma, lentigo maligna melanoma, acral lentiginous melanoma, nodular melanoma, multiple myeloma, and B cell lymphoma (including low grade / follicular non-Hodgkin lymphoma (NHL); small lymphocytic (SL) NHL; intermediate / follicular NHL; intermediate diffuse NHL; high grade immunoblastic NHL; high grade lymphocytic NHL; high grade small non-cleaved cell NHL; bulky lesion NHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenström's macroglobulinemia); chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); hairy cell leukemia; chronic myelogenous leukemia; and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal vascular proliferation associated with phakomatosis, edema (such as edema associated with brain tumors), Meigs' syndrome, brain, and head and neck cancer and related metastases. In certain embodiments, 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 lymphoma (NHL), renal cell carcinoma, prostate cancer, liver cancer, pancreatic cancer, soft tissue sarcoma, Kaposi's sarcoma, carcinoid, 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. However, 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 is selected from a class of mature B cell cancers, which does not include Hodgkin lymphoma, but includes 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), Waldenström macroglobulinemia (WM), 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, Waldenström macroglobulinemia, heavy chain disease, alpha heavy chain disease, gamma heavy chain disease, mu heavy chain disease, plasma cell myeloma, solitary plasmacytoma of bone, extramedullary plasmacytoma, extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue (MALT lymphoma), nodal marginal zone lymphoma, pediatric nodal marginal zone lymphoma, pediatric follicular lymphoma, primary cutaneous follicle center lymphoma, T cell / histiocyte-rich large B cell lymphoma, primary DLBCL of the CNS, primary cutaneous DLBCL (leg type), EBV-positive DLBCL in the elderly, DLBCL associated with chronic inflammation, lymphomatoid granulomatosis, primary mediastinal (thymic) large B cell lymphoma, intravascular large B cell lymphoma, ALK-positive large B cell lymphoma, plasmablastic lymphoma, large B cell lymphoma arising in HHV8-related multicentric Castleman disease, primary effusion lymphoma; unclassifiable B cell lymphoma with intermediate features between diffuse large B cell lymphoma and Burkitt lymphoma, and unclassifiable B cell lymphoma with intermediate features between diffuse large B cell lymphoma and classical Hodgkin lymphoma.

[0185] As used herein, "treatment" or "treat" or "treating" refers to a clinical intervention that attempts to alter the natural course of the individual being treated and can be used to prevent or during the clinical pathology. Desired effects of treatment include, but are not limited to, preventing the occurrence or recurrence of disease, alleviating symptoms, reducing and / or reversing any direct or indirect pathological consequences of the disease, preventing metastasis (e.g., in cancer), delaying the progression of the disease / condition / disorder, reducing the rate of disease progression, improving or alleviating the disease state, and alleviating or improving the prognosis.

[0186] As used herein, the terms "prevent", "preventing", and "prevention" refer to preventing or inhibiting the development or onset of a disorder or disease.

[0187] As used herein, the terms "ameliorate" and "alleviate" refer to a decrease or reduction in the severity of a condition or any of its symptoms.

[0188] As used herein, "delayed progression" of a disorder or disease means delaying, impeding, slowing, halting, stabilizing, and / or postponing the development of a disease or disorder (e.g., a cell proliferative disorder such as cancer). The delay can have different time lengths depending on the disease being treated and / or the individual's history.

[0189] As used herein, "detect" encompasses quantitative or qualitative detection.

[0190] Variable region sequences of anti-CD3 antibodies

[0191] Provided herein are anti-CD3 antibodies and antigen-binding fragments that exhibit pH-dependent binding and optionally a favorable developability profile.

[0192] In some embodiments, the anti-CD3 antibody or antigen-binding fragment may comprise one or more CDR sequences that (i) are contained in any of antibody designations A003 - A013, (ii) are contained in the variable domain sequences shown in Appendix Table A, and / or (iii) are encoded by the variable domain-encoding nucleic acid sequences contained in any of antibody designations A003 - A013 and / or shown in Appendix Table B. In some embodiments, the anti-CD3 antibody or antigen-binding fragment may comprise one or more CDR sequences shown in Appendix Tables D, F, H, K, M, and O.

[0193] In some embodiments, the anti-CD3 antibody or antigen-binding fragment may comprise the CDR-H3, CDR-L1, and CDR-L3 sequences of any of antibody designations A003 - A013. In certain embodiments, the anti-CD3 antibody or antigen-binding fragment may comprise the CDR-H3, CDR-L1, and CDR-L3 sequences of antibody designation 6 or 5. In specific embodiments, the anti-CD3 antibody or antigen-binding fragment may comprise the CDR-H3, CDR-L1, and CDR-L3 sequences of antibody designation 6.

[0194] In some embodiments, the anti-CD3 antibody or antigen-binding fragment may comprise the CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 sequences of any one of antibody numbers A003 - A013. In certain embodiments, the anti-CD3 antibody or antigen-binding fragment may comprise the CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 sequences of antibody number 6 or 5. In specific embodiments, the anti-CD3 antibody or antigen-binding fragment may comprise the CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 sequences of antibody number 6.

[0195] The anti-CD3 antibodies and antigen-binding fragments according to the present disclosure may comprise any suitable FR sequences.

[0196] In some embodiments, the anti-CD3 antibody or antigen-binding fragment may comprise one or more heavy-chain FR sequences encoded in the germline VH1-03 allele or another allele, and / or one or more heavy-chain FR sequences that contain one, two, three, four, five, six, or more amino acid differences (substitutions, insertions, or deletions) relative to such germline-encoded sequences.

[0197] In some embodiments, the anti-CD3 antibody or antigen-binding fragment may comprise one or more light-chain FR sequences encoded in the germline VK4-01 allele or another allele, and / or one or more light-chain FR sequences that contain one, two, three, four, five, six, or more amino acid differences (substitutions, insertions, or deletions) relative to the germline-encoded sequences.

[0198] In some embodiments, the anti-CD3 antibody or antigen-binding fragment may comprise one or more FR sequences that (i) are comprised in any one of antibody numbers A003 - A013, (ii) are comprised in the variable domain sequences shown in Appendix Table A, and / or (iii) are encoded in the variable domain-encoding nucleic acid sequences of any one of antibody numbers A003 - 013 and / or shown in Appendix Table B. In some embodiments, the anti-CD3 antibody or antigen-binding fragment may comprise one or more FR sequences shown in Appendix Tables C, E, G, I, J, L, N, and P.

[0199] In some embodiments, the anti-CD3 antibody or antigen-binding fragment may comprise the FR-H1, FR-H2, FR-H3, FR-H4, FR-L1, FR-L2, FR-L3, and FR-L4 sequences of any one of antibody numbers A003 - A013. In certain embodiments, the anti-CD3 antibody or antigen-binding fragment may comprise the FR-H1, FR-H2, FR-H3, FR-H4, FR-L1, FR-L2, FR-L3, and FR-L4 sequences of antibody number 6 or 5. In specific embodiments, the anti-CD3 antibody or antigen-binding fragment may comprise the FR-H1, FR-H2, FR-H3, FR-H4, FR-L1, FR-L2, FR-L3, and FR-L4 sequences of antibody number 6.

[0200] In some embodiments, the anti-CD3 antibody or antigen-binding fragment may comprise a VH polypeptide that comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the following sequences: (i) the VH polypeptide sequence of any one of antibody numbers A003 - A013; (ii) the VH polypeptide sequence encoded by the sequence encoding VH of any one of antibody numbers A003 - A013; (iii) any one of the VH polypeptide sequences shown in Appendix Table A; and / or (iv) the VH polypeptide sequence encoded by the sequence encoding VH shown in Appendix Table B.

[0201] In some embodiments, the anti-CD3 antibody or antigen-binding fragment may comprise a VL polypeptide that comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the following sequences: (i) the VL polypeptide sequence of any one of antibody numbers A003 - A013; (ii) the VL polypeptide sequence encoded by the sequence encoding VL of any one of antibody numbers A003 - A013; (iii) any one of the VL polypeptide sequences shown in Appendix Table A; and / or (iv) the VL polypeptide sequence encoded by the sequence encoding VL shown in Appendix Table B.

[0202] In some embodiments, the percent identity is measured by any well-known algorithm for sequence identity such as FASTA, BLAST, or GAP.

[0203] In certain embodiments, the anti-CD3 antibody or antigen-binding fragment may comprise: (A) a VH polypeptide comprising (i) CDR-H1, CDR-H2, and CDR-H3 sequences of SEQ ID NOs: 612, 614, and 616, respectively; and (ii) an amino acid sequence that is at least 90% identical to SEQ ID NO: 610; and (B) a VL polypeptide comprising (i) CDR-L1, CDR-L2, and CDR-L3 sequences of SEQ ID NOs: 622, 624, and 626, respectively; and (ii) an amino acid sequence that is at least 90% identical to SEQ ID NO: 620.

[0204] In certain embodiments, the anti-CD3 antibody or antigen-binding fragment may comprise: (A) a VH polypeptide comprising (i) CDR-H1, CDR-H2, and CDR-H3 sequences of SEQ ID NOs: 512, 514, and 516, respectively; and (ii) an amino acid sequence that is at least 90% identical to SEQ ID NO: 510; and (B) a VL polypeptide comprising (i) CDR-L1, CDR-L2, and CDR-L3 sequences of SEQ ID NOs: 522, 524, and 526, respectively; and (ii) an amino acid sequence that is at least 90% identical to SEQ ID NO: 520.

[0205] In some embodiments, the anti-CD3 antibody or antigen-binding fragment may comprise a VH polypeptide and a VL polypeptide, the VH polypeptide and VL polypeptide comprising any one of the VH and VL amino acid sequence combinations shown in Appendix Table A.

[0206] In certain embodiments, the anti-CD3 antibody or antigen-binding fragment may comprise: (A) a VH polypeptide comprising SEQ ID NO: 610; and (B) a VL polypeptide comprising SEQ ID NO: 620.

[0207] In certain embodiments, the anti-CD3 antibody or antigen-binding fragment may comprise: (A) a VH polypeptide comprising SEQ ID NO: 510; and (B) a VL polypeptide comprising SEQ ID NO: 520.

[0208] Antibody constant domains and regions

[0209] The anti-CD3 antibodies and antigen-binding fragments according to the present disclosure may or may not include one or more immunoglobulin (Ig) constant domains (e.g., CH1, CH2, and / or CH3), one or more Fc regions (e.g., hinge, a portion of CH2 and CH3), and / or one or more constant regions (e.g., one or more sets of CH1, hinge, CH2, and CH3) or one or more portions thereof (e.g., one or more sets of a portion of CH1 and hinge). Such constant domains, Fc regions, and / or their constant regions or portions may be or may be derived from any Ig isotype (e.g., human IgG, IgA, IgE, IgM, or IgD) and subclass (e.g., human IgG1, IgG2, IgG3, or IgG4) and their variants, and may optionally include any of the modifications described herein.

[0210] In some embodiments, one or more amino acid modifications may be introduced into the Fc region of the anti-CD3 antibodies of the present disclosure, thereby generating Fc region variants (see, e.g., US 2012 / 0251531). The Fc region variants may include a human Fc region sequence (e.g., human IgG1, IgG2, IgG3, or IgG4 Fc region) that includes amino acid modifications (e.g., substitutions) at one or more amino acid positions.

[0211] In certain embodiments, the present disclosure contemplates anti-CD3 antibody variants that possess some but not all effector functions, making them desirable candidates for applications where in vivo antibody half-life is important, but certain effector functions (such as complement and ADCC) are unnecessary or detrimental. In vitro and / or in vivo cytotoxicity assays can be performed to confirm the reduction / depletion of CDC and / or ADCC activity. For example, Fc receptor (FcR) binding assays can be performed to ensure that the antibody lacks FcγR binding (and thus may lack ADCC activity), but retains the ability to bind FcRn. Primary cells used to mediate ADCC (e.g., NK cells) express only FcγRIII, whereas monocytes express FcγRI, FcγRII, and FcTRIII. The expression of FcRs 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 evaluating the ADCC activity of a molecule of interest are described in U.S. Patent No. 5,500,362 (see, e.g., Hellstrom, I. et al., Proc. Nat′l Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I et al., Proc. Nat′l Acad. Sci. USA 82:1499-1502 (1985); U.S. Patent No. 5,821,337 (see Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assay methods can be employed (see, e.g., the ACTITM non-radioactive cytotoxicity assay for flow cytometry (Cell Technology, Inc. Mountain View, Calif.); and CYTOTOX Non-radioactive cytotoxicity assays (Promega, Madison, Wis.). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Alternatively or additionally, the ADCC activity of the molecule of interest can be evaluated in vivo, e.g., 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 can also be performed to confirm that the antibody does not bind C1q and thus lacks CDC activity. See, e.g., the C1q and C3c binding ELISAs in WO 2006 / 029879 and WO 2005 / 100402. To assess complement activation, a CDC assay can be performed (see, e.g., Gazzano - Santoro et al., J. Immunol Methods 202:163 (1996); Cragg, M.S. et al., Blood. 101:1045 - 1052 (2003); and Cragg, M.S. and M.J. Glennie Blood. 103:2738 - 2743 (2004)). FcRn binding and in vivo clearance / half - life determination can also be performed using methods known in the art (see, e.g., Petkova, S.B. et al., Int′l. Immunol 18(12):1759 - 1769 (2006)).

[0212] In certain embodiments, antibodies with reduced effector function include those in which one or more of the Fc region residues 238, 265, 269, 270, 297, 327, and 329 are substituted (U.S. Patent Nos. 6,737,056 and 8,219,149). In some embodiments, the Fc mutants include Fc mutants having substitutions at two or more amino acid positions among amino acid positions 265, 269, 270, 297, and 327, including the so - called "DANA" Fc mutant in which residues 265 and 297 are substituted with alanine (U.S. Patent Nos. 7,332,581 and 8,219,149).

[0213] In some embodiments, the anti - CD3 antibodies and / or antigen - binding fragments as described herein can include a silent Fc region (e.g., complete removal of the Fc or modification of the Fc region to reduce or eliminate effector function), and / or can include a masking agent (e.g., a polypeptide mask that is positioned (e.g., attached via a cleavable linker) such that it reduces or inhibits the ability of the antibody or antigen - binding fragment to induce effector function - inducing molecules such as complement (e.g., C1q)).

[0214] Antigen-binding fragments and multispecific antibodies

[0215] The anti-CD3 antigen-binding fragment according to the present disclosure can be in any suitable format, including but not limited to any of the formats described herein (e.g., Fab, scFv, etc.), provided that such modifications retain pH-dependent CD3 binding and do not substantially reduce the ability of the antibody to bind to CD3.

[0216] In certain embodiments, the anti-CD3 antibody and / or its antigen-binding fragment as described herein can comprise or be comprised in a multispecific antibody or antibody fragment, which can comprise at least two different variable regions, wherein each variable region is capable of specifically binding to a separate antigen or different epitopes on the same antigen, particularly, a bispecific antibody having binding specificity for a second antigen. In some embodiments, the binding specificities are directed against two different epitopes of CD3 (e.g., CD3ε or CD3γ). In other embodiments, one of the binding specificities is directed against CD3 (e.g., CD3ε or CD3γ), while the other is directed against a different biomolecule (e.g., a cell surface antigen, such as a tumor antigen).

[0217] Multispecific antibodies comprising at least one anti-CD3 antibody and / or antigen-binding fragment disclosed herein can be prepared according to a variety of techniques, including but not limited to recombinant co-expression of two immunoglobulin heavy-light chain pairs with different specificities (see Milstein and Cuello, Nature 305:537 (1983)); WO 93 / 08829 and Traunecker et al., EMBO J. 10:3655 (1991)); "knobs-into-holes" engineering (see, e.g., U.S. Patent No. 5,731,168); immunoglobulin crossover techniques (also known as Fab domain exchange or CrossMab format) (see, e.g., WO2009 / 080253; Schaefer et al., Proc. Natl. Acad. Sci. USA, 108:11187-11192 (2011)); engineered electrostatic steering effects for preparing antibody Fc-heterodimeric molecules (WO 2009 / 089004A1); crosslinking two or more antibodies or fragments (see, e.g., U.S. Patent No. 4,676,980; Brennan et al., Science, 229:81 (1985)); leucine zippers (see, e.g., Kostelny et al., J; Immunol, 148(5):1547-1553 (1992)); "diabody" technology (see, e.g., Hollinger et al Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)); single-chain Fv (sFv) dimers (see, e.g., Gruber et al., J. Immunol, 152:5368 (1994)); and trispecific antibodies, such as in, e.g., Tutt et al J. Immunol 147:60 (1991).

[0218] A variety of multispecific antibody formats can be used in the context of the antigen-binding fragments of the anti-CD3 antibodies described herein. Non-limiting examples of multispecific and bispecific formats include, for example, Fab-Fc-scFv ("opener") (Xencor), Mab-scFv (Xencor), Mab-Fv (Xencor), bis-scFv (Xencor), central Fv (Xencor), central scFv (Xencor), single-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 (Sffimed), Cross-Mab (Roche), SEED (EMD Serono), BEAT (Glenmark), TrioMab (Trion Pharma / Fresenius Biotech), DuetMab (MedImmune), and others, such as those disclosed in (WO2021067404; WO2022150787; WO2022150785; WO 95 / 09917; WO 2008 / 119566; WO2008 / 119567; WO2011 / 121110; WO 2010 / 037835; WO 2007 / 042261; WO 2007 / 110205; WO2011 / 121110; WO 2012 / 055961; WO 2012 / 16067; WO 2016 / 086189; WO 2016 / 182751; WO2015 / 006749; WO 2014 / 049003; WO 2013 / 177101; WO 2015 / 128509; US 7,951,917; US2009 / 0252729; US2014 / 0348839; US 7,183,076; Mazor et al., Mabs, Vol. 7, pp. 377-389 (2015); Muda et al., Protein Engineering, Design, & Selection, Vol. 24, pp. 447-454 (2011); and De1Bano et al., Antibodies, Vol. 5, pp. 1-23 (2016). In some embodiments, the anti-CD3 scFv fragments described herein may comprise one or more variable regions of a multispecific (e.g., bispecific) antibody.

[0219] In some embodiments, the multispecific antibody or antibody fragment may include one or more engineered variant constant domains that facilitate efficient polypeptide heterodimerization for bispecific antibody formation (e.g., a first heavy chain and a second heavy chain different from the first heavy chain).

[0220] In certain embodiments, the multispecific antibody or antibody fragment may include at least one CLκ-preferred variant CH1 domain and / or a CLλ-preferred variant CH1 domain. The CLκ-preferred variant CH1 domain preferentially pairs with a CLκ domain rather than a non-CLκ domain (such as a CLλ domain). The CLλ-preferred variant CH1 domain preferentially pairs with a CLλ domain rather than a non-CLλ domain (such as a CLκ domain). In certain embodiments, such CLκ-preferred variant CH1 domains and / or CLκ-preferred variant CH1 domains may be selected from the domains described in WO2021067404.

[0221] In certain embodiments, the multispecific antibody or antibody fragment may include at least one pair of CH1 domains and CL domains that preferably pair with each other. In the preferentially paired pair of CH1 and CL domains: the CH1 domain preferably pairs with a CL domain rather than with another given CL domain, such as a wild-type CL domain; and / or the CL domain preferably pairs with a CH1 domain rather than with another given CH1 domain name, such as a wild-type CH1 domain region. One or both of the CH1 domain and the CL domain may be variant domains. In certain embodiments, such preferentially paired pairs of CH1 and CL domains may be selected from the pairs described in WO2022150787.

[0222] In certain embodiments, the multispecific antibody or antibody fragment may include at least one pair of a first CH3 domain and a second CH3 domain different from the first CH3 that preferentially pair with each other (i.e., form a heterodimer). In such preferentially paired pairs, the first CH3 domain preferably pairs with the second CH3 domain rather than with another first CH3 domain; and / or the second CH3 domain preferably pairs with the first CH3 domain rather than with another second CH3 domain. One or both of the CH3 domains may be variant domains. In certain embodiments, such preferentially paired pairs of the first and second CH3 domains may be selected from the pairs described in WO 2022150785.

[0223] Exemplary technical advantages

[0224] Exemplary technical advantages provided in accordance with some embodiments of the present disclosure are listed below. It should be noted that the technical advantages of such embodiments may not be limited to the advantages specifically listed below. It should also be noted that embodiments other than those specifically mentioned below may also have one or more of the advantages listed below.

[0225] Binding to cynomolgus CD3

[0226] As shown in Table 2B, while the parental pH-dependent antibody of antibody number 2 does not bind to cynomolgus CD3, the novel pH-dependent antibodies of antibody numbers 3-10 and 12 bind to cynomolgus CD3.

[0227] Due to their similarity to humans, non-human primates such as cynomolgus monkeys are typically ideal species for establishing the pharmacokinetics of antibodies and collecting efficacy data of antibodies in preclinical studies. Without wishing to be bound by theory, the ability to bind to cynomolgus CD3 is a technical advantage because it allows for various in vitro studies using cynomolgus CD3, which will assist in designing such preclinical studies in cynomolgus monkeys.

[0228] In some embodiments, the anti-CD3 antibody or antigen-binding fragment according to the present disclosure may comprise one or more CDRs of any one of antibody numbers 3-10 and 12. In certain embodiments, the anti-CD3 antibody or antigen-binding fragment according to the present disclosure may comprise a combination of six CDRs of any one of antibody numbers 3-10 and 12. In specific embodiments, the anti-CD3 antibody or antigen-binding fragment according to the present disclosure may comprise a combination of six CDRs of antibody number 6 or 5.

[0229] Improved binding to human CD3 at acidic pH

[0230] As shown in Table 2A, when measured via BLI using the ForteBio system, the novel pH-dependent antibodies of antibody numbers 3-12 bind to human CD3 with higher affinity at pH 6.0 than the parental pH-dependent antibody of antibody number 2.

[0231] As mentioned above, tumor cells typically have an extracellular pH of approximately 6.3-6.5, which is at least partially due to the accumulation of metabolic acids that cannot be effectively cleared due to poor tumor vascularization. Therefore, without wishing to be bound by theory, antibodies with higher affinity at acidic pH such as pH 6.0 will exhibit more potent effects (e.g., higher cytotoxic activity) at or around the tumor site. Additionally, without wishing to be bound by theory, this will allow for the use of reduced doses and, therefore, toxicity will be minimized.

[0232] In some embodiments, the anti-CD3 antibody or antigen-binding fragment according to the present disclosure may comprise one or more CDRs of any one of antibody numbers 3-12. In certain embodiments, the anti-CD3 antibody or antigen-binding fragment according to the present disclosure may comprise a combination of six CDRs of any one of antibody numbers 3-12. In specific embodiments, the anti-CD3 antibody or antigen-binding fragment according to the present disclosure may comprise a combination of six CDRs of antibody number 6 or 5.

[0233] Improved binding to cells expressing human CD3 at acidic pH

[0234] As shown in Table 3, based on the NCB values obtained from flow cytometry analysis, the novel pH-dependent antibodies of antibody numbers 3 and 5-11 bind more to cells expressing human CD3 (CD3+ Jurkat cells) at pH 6.0 than the parental pH-dependent antibody of antibody number 2.

[0235] As described above, tumor cells typically have an extracellular pH of about 6.3-6.5, which is lower than the physiological pH (around pH 7.4). Thus, without wishing to be bound by theory, antibodies that bind better to cells at acidic pH such as pH 6.0 will exhibit more potent effects (e.g., higher cytotoxic activity) at or around the tumor site. For example, such antibodies may provide better binding to T cells while also binding to tumor cells, whereby the T cells can exert a more potent cytotoxic effect on the tumor cells. Additionally, without wishing to be bound by theory, this will allow for the use of reduced doses and, thus, toxicity will be minimized.

[0236] In some embodiments, the anti-CD3 antibody or antigen-binding fragment according to the present disclosure may comprise one or more CDRs of any one of antibody numbers 3-12. In certain embodiments, the anti-CD3 antibody or antigen-binding fragment according to the present disclosure may comprise a combination of six CDRs of any one of antibody numbers 3-12. In specific embodiments, the anti-CD3 antibody or antigen-binding fragment according to the present disclosure may comprise a combination of six CDRs of antibody number 6 or 5.

[0237] Improved binding to cells expressing cynomolgus monkey CD3 at acidic pH

[0238] As shown in Table 3, based on the NCB values obtained from flow cytometry analysis, the novel pH-dependent antibodies of antibody numbers 3 and 5-11 bind more to cells expressing cynomolgus monkey CD3 (HSC-F cells) at pH 6.0 than the parental pH-dependent antibody of antibody number 2.

[0239] As described above, due to their similarity to humans, non-human primates such as cynomolgus monkeys are typically ideal species for establishing the pharmacokinetics of antibodies and collecting efficacy data of antibodies in preclinical studies. Without wishing to be bound by theory, improved binding to cells expressing cynomolgus monkey CD3 is a technical advantage as it increases the chances of success in such preclinical studies, which is crucial for advancing an antibody into the clinical stage.

[0240] In some embodiments, an anti-CD3 antibody or antigen-binding fragment according to the present disclosure may comprise one or more CDRs of any one of antibody Nos. 3 and 5-11. In certain embodiments, an anti-CD3 antibody or antigen-binding fragment according to the present disclosure may comprise a combination of six CDRs of any one of antibody Nos. 3 and 5-11. In specific embodiments, an anti-CD3 antibody or antigen-binding fragment according to the present disclosure may comprise a combination of six CDRs of antibody No. 6 or 5.

[0241] Further modifications

[0242] Sequence modifications

[0243] The present disclosure also encompasses modifications of the anti-CD3 antibodies disclosed herein, such as one or more amino acid modifications (substitutions, insertions, or deletions) in the VH and / or VL polypeptides and / or constant region sequences relative to the corresponding sequences of any of the antibodies described above. Once obtained, one or more desired properties of such derived antibodies and / or antigen-binding fragments can be tested, such as improved binding specificity, increased binding affinity, improved pH-dependent antigen binding, improved developability, etc.

[0244] In some embodiments, such modifications may be in the FR regions and / or CDR regions.

[0245] In some embodiments, one or more substitutions, insertions, or deletions may occur within one or more CDRs of the anti-CD3 antibodies described herein, provided that such modifications retain pH sensitivity and do not substantially reduce the ability of the antibody to bind its antigen. For example, conservative changes (e.g., conservative amino acid substitutions as provided herein) that do not substantially reduce binding affinity may be made in the CDRs. Such changes may be located, for example, outside of the antigen contact residues in the CDRs. In certain embodiments, each of the six CDRs may contain no more than one, two, or three amino acid substitutions. In certain embodiments, CDR-H1, CDR-H2, and / or CDR-L2 may contain no more than one, two, or three amino acid substitutions relative to any of antibody numbering 3-13, preferably relative to antibody number 6 or 5. In a particular embodiment, CDR-H3, CDR-L1, and CDR-L3 may not contain any substitutions, insertions, or deletions relative to CDR-H3, CDR-L1, and CDR-L3 of any of antibody numbering 3-13, preferably antibody number 6 or 5.

[0246] A useful method for identifying residues or regions of an antibody that may be targeted for mutagenesis is called "alanine scanning mutagenesis," as described by Cunningham and Wells (1989) Science, 244:1081-1085. In this method, a residue or a set of target residues (e.g., charged residues such as arg, asp, his, lys, and glu) is identified and replaced with a neutral or negatively charged amino acid (e.g., alanine or polyalanine) to determine whether the interaction of the antibody with the antigen is affected. Additional substitutions may be introduced at the amino acid position to demonstrate functional sensitivity to the initial substitution. Alternatively or additionally, the crystal structure of the antigen-antibody complex may be used to identify the contact points between the antibody and the antigen. Such contact residues and adjacent residues may be targeted or eliminated as substitution candidates. Variants may be screened to determine whether they possess the desired properties.

[0247] Amino acid sequence insertions include amino- and / or carboxyl-terminal fusions ranging in length from one residue to polypeptides of one hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include antibodies having an N-terminal methionyl residue. Other insertion variants of antibody molecules include enzymes (e.g., for ADEPT) or polypeptides fused to the N-terminus or C-terminus of the antibody to increase the serum half-life of the antibody.

[0248] In addition to substituting one or more CDR residues, one or more CDRs may also be omitted. Antibodies have been described in the scientific literature in which one or two CDRs have been assigned 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 about one-fifth to one-third of the CDR residues actually contact their associated antigen. Padlan also found many antibodies in which one or two CDRs had no amino acids contacting 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 from regions of the Kabat CDRs that lie outside the Chothia CDRs, based on prior studies (e.g., residues H60-H65 in CDRH2 are often not required). If a CDR or its residues are omitted, they are generally replaced by the amino acids occupying the corresponding positions in another human antibody sequence or the consensus sequence of such sequences. Positions within the CDRs for substitution and the amino acids to be substituted can also be selected empirically.

[0249] Modifications via conjugation

[0250] In some embodiments, an anti-CD3 antibody and / or its antigen-binding fragment as described herein is conjugated to a moiety or agent to form an immunoconjugate.

[0251] In certain embodiments, an anti-CD3 antibody according to the present disclosure can be further modified to contain additional non-protein moieties known and readily available in the art. Moieties suitable for antibody derivatization include but are not limited to water-soluble polymers. Non-limiting examples of water-soluble polymers include but are not limited to polyethylene glycol (PEG), copolymers of ethylene glycol / propanediol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymer, polyamino acids (homopolymers or random copolymers), and dextran or poly(vinylpyrrolidone) polyethylene glycol, polyethylene glycol homopolymers, polypropylene oxide / ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde has advantages in manufacture due to its stability in water. The polymer can have any molecular weight and can be branched or unbranched. The number of polymers attached to the antibody can vary, and if more than one polymer is attached, they can be the same or different molecules. In general, the number and / or type of polymer used for derivatization can be determined based on considerations including but not limited to the specific properties or functions of the antibody to be improved, whether the antibody derivative is to be used in therapy under defined conditions, etc.

[0252] In certain embodiments, labeled anti-CD3 antibodies are provided. The anti-CD3 antibodies and / or antigen-binding fragments thereof as described herein may include labels or moieties that are detected directly (such as fluorescent, chromogenic, electrochemiluminescent, chemiluminescent, and radioactive labels) or indirectly (such as enzymes or ligands). Non-limiting exemplary labels include radioisotopes such as 32P, 14C, 125I, 3H, and 131I; fluorophores such as rare earth chelates or fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, luciferases (e.g., firefly luciferase and bacterial luciferase (U.S. Patent No. 4,737,456)), luciferin, 2,3-dihydrophthalazinedione, horseradish peroxidase (HRP), alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, glucose oxidases (e.g., glucose oxidase, galactose oxidase), and glucose-6-phosphate dehydrogenase; heterocyclic oxidases (such as uricase and xanthine oxidase) enzyme-coupled to oxidize dye precursors such as HRP, lactoperoxidase, or microperoxidase using hydrogen peroxide; biotin / avidin; spin labels; bacteriophage tags; stable free radicals; and the like.

[0253] In certain embodiments, the anti-CD3 antibodies or antigen-binding fragments of the present disclosure can be antibody-drug conjugates (ADCs), can comprise an ADC or can be comprised in an ADC. The ADC can comprise: (a) any anti-CD3 antibody or antigen-binding fragment described herein; and (b) a drug conjugated thereto. In certain embodiments, the drug can be, for example but not limited to, an anti-cancer drug, an anti-proliferative drug, a cytotoxic drug, an anti-angiogenic drug, an apoptotic drug, an immune-stimulating drug, an anti-microbial drug, an antibiotic drug, an anti-viral drug, an anti-inflammatory drug, an enzyme, a hormone, a toxin, a radioisotope, a compound, a small molecule, a small molecule inhibitor, a protein, a peptide, a vector, a plasmid, a viral replicon, a viral particle, a nanoparticle, a DNA molecule, an RNA molecule, siRNA, shRNA, microRNA, an oligonucleotide or an imaging drug. In some cases, the drug can be selected from: doxorubicin, daunorubicin, cucurbitacin, chaetomin, chaetomugilin, chlamydocin, calicheamicin, nemorubicin, cryptophyscin, mensacarcin, ansamitocin, mitomycin C, geldanamycin, mechercharmycin, papuamycin, crocetin, okilactomycin, oligomycin, actinomycin, sandramycin, ecteinascidin, polyketomycin, hydroxycamptothecin, thiocolchicine, methotrexate, triptolide, taltobulin, lactacystin, dolastatin, auristatin, monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), telomestatin, tubastatin A, combretastatin, maytansine, MMAD, MMAF, DM1, DM4, DTT, 16GMB APA GA, 17DMAP GA, JW 55, pyrrolobenzodiazepine SN-38, Ro53335, puwainaphycin, diplomycin, bafilomycin, taxane, tubulysin, ferulol, lusiol A, fumagillin, hygrolidin, glucosinolate, amanitin, anthramycin, emberomycin, phalloidin-like peptide, phalloidin, phytosphingosine, sphingosinolate, poronetin, podophyllotoxin, gramicidin A, sanguinarine, sinalbin, herboxidiene, microcolin B, microcystin, muscotoxin A, oscillatoxin, tripolin A, myoalbumin, mytoxin B, nocuolin A, pseudolaric acid B, pseudoneurotoxin A, cyclopamine, curvulin, colchicine, afidomycin, englerin, cordycepin, apoptin, epothilone A, limaquinone, isatropolone, isofistularin, quinadolide, ixabepilone, aeroplysinin, pyocyanin, agrochelin, epothilone, and derivatives of any of the foregoing.

[0254] Glycosylation

[0255] In some embodiments, the anti-CD3 antibodies and / or antigen-binding fragments thereof described herein are modified to increase or decrease the degree to which the antibody is glycosylated. Glycosylation sites can be added or removed from the anti-CD3 antibodies of the present disclosure by modifying the amino acid sequence such that one or more glycosylation sites are created or removed. In certain embodiments, the addition or deletion of glycosylation sites is not limited to the constant region of the anti-CD3 antibody or antigen-binding fragment.

[0256] Antibody characterization and further screening

[0257] In some embodiments, the anti-CD3 antibodies and / or antigen-binding fragments described herein can be characterized for their physical / chemical properties and / or biological activities by various assays known in the art. In some embodiments, variants of any of the anti-CD3 antibodies and antigen-binding fragments described herein can be identified, screened, selected, or characterized for their physical / chemical properties and / or biological activities based on various assays known in the art. In some embodiments, multispecific antibodies or antibody fragments comprising the anti-CD3 antibodies or antigen-binding fragments described herein can be designed and screened, selected, or characterized for their physical / chemical properties and / or biological activities based on various assays known in the art.

[0258] In certain embodiments, such assays can include SPR, BLI, ELISA, Western blotting, flow cytometry, and the like.

[0259] In certain embodiments, such assays can include competitive assays, which can be used in some cases to identify antibodies that compete with a given anti-CD3 antibody of the disclosure for binding to CD3. In an exemplary competitive assay, immobilized CD3 is incubated in a solution comprising a first labeled antibody that binds CD3 and a second unlabeled antibody whose ability to compete with the first antibody for binding to CD3 is being tested. The second antibody can be present in a hybridoma supernatant. As a control, immobilized CD3 is incubated in a solution comprising the first labeled antibody instead of the second unlabeled antibody. After incubation under conditions that permit the first antibody to bind to CD3, the excess unbound antibody is removed, and the amount of label associated with the immobilized CD3 is measured. If the amount of label associated with the immobilized CD3 in the test sample is substantially reduced relative to the control sample, it indicates that the second antibody competes with the first antibody for binding to CD3. See, e.g., Harlow and Lane (1988) Antibodies: A Laboratory Manual. Chapter 14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.).

[0260] Standard methods can be used to identify anti-CD3 antibodies and / or their antigen-binding fragments that possess biological activity. Biological activity can include, for example, in vivo, in vitro, or ex vivo binding to CD3 on the surface of T cells. In the case of multispecific anti-CD3 antibodies (such as bispecific antibodies having one arm that binds CD3 and another arm that binds a different target, e.g., a cell surface antigen, such as a tumor antigen), biological activity can also include effector cell activation (such as activation of CD8+ and / or CD4+ T cells), effector cell population expansion (i.e., an increase in T cell count), target cell population reduction (i.e., a reduction in the population of cells that express the second biomolecule on their cell surface), and / or target cell killing.

[0261] Evaluation Based on Target Binding

[0262] In some embodiments, an anti-CD3 antibody or antigen-binding fragment as described herein can exhibit a binding level to CD3 within a preferred range at an approximate pH of interest.

[0263] In certain embodiments, antigen binding can be determined based on the K D value obtained by surface plasmon resonance (SPR), optionally using a system. In certain embodiments, the K D can be determined by SPR as described in the examples.

[0264] In certain embodiments, the anti-CD3 antibody or antigen-binding fragment as described herein may have a K value between about 0.5 nM and about 50 nM for human CD3 at about pH 6.0. D In certain embodiments, the K for human CD3 measured by SPR at pH 6.0 D may be between about 0.5 nM and about 20 nM, between about 0.5 nM and about 20 nM, between about 1 nM and about 10 nM, or between about 5 nM and about 10 nM. In certain embodiments, the K for human CD3 measured by SPR at about pH 7.4 for the anti-CD3 antibody or antigen-binding fragment as described herein D value may be about 1 nM or higher. In certain embodiments, the K for human CD3 measured by SPR at pH 7.4 D may be about 10 nM or higher, or 100 nM or higher, 1000 nM or higher, or 10000 nM or higher. In certain specific embodiments, no binding is observed at pH 7.4.

[0265] In certain embodiments, antigen binding may be determined based on the K value obtained by Biolayer Interferometry (BLI), optionally using D a system. In certain embodiments, the K may be determined by BLI as described in the examples . D

[0266] In certain embodiments, the K for human CD3 measured by BLI at about pH 6.0 for the anti-CD3 antibody or antigen-binding fragment as described herein D value may be between about 0.5 nM and about 20 nM. In certain embodiments, the K for human CD3 measured by BLI at pH 6.0 D may be between about 1 nM and about 10 nM or between about 5 nM and about 7 nM. In certain embodiments, the K for human CD3 measured by BLI at about pH 7.4 for the anti-CD3 antibody or antigen-binding fragment as described herein D value may be between about 5 nM or higher. In certain embodiments, the K for human CD3 measured by BLI at pH 7.4 D may be about 10 nM or higher.

[0267] In certain embodiments, the K for non-human primate (e.g., cynomolgus monkey or rhesus monkey) CD3 measured by BLI at about pH 6.0 for the anti-CD3 antibody or antigen-binding fragment as described herein DThe value can be between about 0.5 nM and about 20 nM. In certain embodiments, the K for non - human primate CD3 measured by BLI at pH 6.0 D can be between about 1 nM and about 10 nM or between about 5 nM and about 8 nM. In certain embodiments, the K for non - human primate CD3 of an anti - CD3 antibody or antigen - binding fragment as described herein measured by BLI at about pH 7.4 D value can be between about 5 nM or higher. In certain embodiments, the K for non - human primate CD3 measured by BLI at pH 7.4 D can be about 20 nM or higher.

[0268] Without wishing to be bound by theory, antibodies with higher affinity (i.e., low K D value) can induce more potent cytokine release upon antigen binding and / or activation of CD3 - expressing cells upon antigen binding, and in some cases, the potent cytokine release and / or activation can help reduce the dose (e.g., effective amount) of the anti - CD3 antibody or antigen - binding fragment required for treatment.

[0269] In some embodiments, an anti - CD3 antibody or antigen - binding fragment as described herein can exhibit higher binding to CD3 - expressing cells at an approximate pH of interest.

[0270] In certain embodiments, pH - dependent binding to CD3 - expressing cells can be determined by fluorescence - activated cell sorting (FACS), optionally based on median fluorescence intensity (MFI) values and / or normalized cell binding (NCB) values calculated using MFI values. In certain embodiments, MFI and / or NCB values can be determined as described in the examples, where cells are incubated with the test antibody and then with a secondary antibody that binds to the test antibody (negative control, including incubation without the test antibody and then with the secondary antibody). In some cases, the NCB value is calculated as {(sample MFI)-(secondary only MFI)} / (secondary only MFI).

[0271] In certain embodiments, the NCB value of the binding of an anti - CD3 antibody or antigen - binding fragment as described to human CD3 - expressing cells at about pH 6.0 can be at least ×1.2 greater than the NCB value of the binding to human CD3 - expressing cells at about pH 7.4. In certain embodiments, the NCB value at about pH 6.0 can be at least ×1.3, at least ×1.4, at least ×1.5, at least ×2, at least ×3, at least ×4, at least ×5, at least ×10, at least ×20, at least ×50, at least ×100, at least ×10 3 、at least ×10 4, at least ×10 5 , at least ×10 6 , at least ×10 7 , at least ×10 8 or at least ×10 9 . In certain cases, the NCB value at about pH 6.0 can be at least ×3, at least ×4, or at least ×5 greater than the NCB value at about pH 7.4. Optionally, the cells expressing human CD3 can be Jurkat cells expressing CD3.

[0272] In some embodiments, the anti-CD3 antibodies or antigen-binding fragments as described herein can exhibit improved binding to cells expressing CD3 (e.g., cells expressing human CD3) at acidic pH (e.g., about pH 6.0) relative to known anti-CD3 antibodies.

[0273] In certain embodiments, improved binding to cells expressing CD3 can be determined by FACS, optionally based on the MFI value and / or the NCB value calculated using the MFI value. In certain embodiments, the MFI and / or NCB values can be determined as described in the examples. In some cases, the NCB value can be calculated as described above.

[0274] Without wishing to be bound by theory, antibodies that have higher or improved binding (i.e., higher NCB value) to cells expressing human CD3 (e.g., T cells) at acidic pH (e.g., about pH 6.0) can induce more potent cytokine release and / or activation of cells expressing CD3 upon antigen binding in an acidic environment, and in some cases, the potent cytokine release and / or activation can contribute to reducing the dose (e.g., effective amount) of the anti-CD3 antibody or antigen-binding fragment required for treatment.

[0275] Evaluation based on other developability parameters

[0276] In some embodiments, anti-CD3 antibodies and / or antigen-binding fragments, including the multispecific antibodies and their variants provided herein (e.g., multispecific antibodies designed to incorporate a second specificity of interest), can be identified based on developability, screened based on developability, selected based on developability, or characterized for developability.

[0277] In certain embodiments, the anti-CD3 antibody or antigen-binding fragment can exhibit a favorable developability profile. The developability profile of the anti-CD3 antibody can be obtained by performing one or more of the following: PSR assay; SCP assay; AC-SINS; ELISA; DSF assay; Tm assay; HIC assay; CIC assay; or a combination thereof.

[0278] In some embodiments, an anti-CD3 antibody or antigen-binding fragment as described herein may exhibit reduced multispecificity (a tendency to bind to multiple molecules or epitopes). In certain embodiments, the multispecificity may be determined based on the Multispecificity Reagent (PSR) score obtained by PSR assay. In some embodiments, the PSR score may be determined as described in the examples. In some embodiments, an anti-CD3 antibody or antigen-binding fragment as described herein shows a PSR score between about 0.0 and about 0.45. In some embodiments, the PSR score is between about 0.0 and about 0.4. In some embodiments, the PSR is between about 0.0 and about 0.35. In some embodiments, the PSR is between about 0.0 and about 0.3. In some embodiments, the PSR is between about 0.0 and about 0.25. In some embodiments, the PSR is between about 0.0 and about 0.2. In some embodiments, the PSR is between about 0.0 and about 0.15. In some embodiments, the PSR is between about 0.0 and about 0.1. In some embodiments, a score of 0.0 - 0.1 is "no PSR". In some embodiments, a score of 0.1 to 0.33 is "low PSR". In some embodiments, a score of 0.33 to 0.66 is "medium PSR". In some embodiments, a score of 0.66 - 1.00 is "high PSR". In some embodiments, a high PSR score indicates reduced (or poor) developability. Generally, the lower the PSR score, the more favorable the developability of the antibody.

[0279] In some embodiments, an anti-CD3 antibody or antigen-binding fragment as described herein may exhibit reduced hydrophobicity. In certain embodiments, the hydrophobicity may be determined based on the retention time observed during HIC. In certain embodiments, HIC may be performed and the retention time obtained as described in the examples. In some embodiments, an anti-CD3 antibody or its antigen-binding fragment as described herein shows an HIC score of less than about 10.5 minutes (no to low HIC score). In some embodiments, the HIC score is between about 10.5 minutes and 11.5 minutes (medium HIC score). In some embodiments, the HIC score is greater than about 11.5 minutes (high HIC score). Generally, the lower the HIC score, the more favorable the developability of the antibody.

[0280] In some embodiments, an anti-CD3 antibody or antigen-binding fragment as described herein may exhibit a tendency of reduced self-interaction. In certain embodiments, the tendency of self-interaction can be determined based on the Δλmax value observed during affinity capture self-interaction nanoparticle spectroscopy (AC-SINS). In certain embodiments, AC-SINS can be performed and the Δλmax value can be obtained as described in the examples. In some embodiments, an anti-CD3 antibody or its antigen-binding fragment as described herein may exhibit a Δλmax between about 0.0 nm and about 15.0 nm. In some embodiments, the Δλmax can be between about 0.0 nm and about 10.0 nm. In some embodiments, the Δλmax can be between about 0.0 nm and about 7.5 nm. In some embodiments, the Δλmax can be between about 0.0 nm and about 5.0 nm. In some embodiments, the Δλmax can be between about 0.0 nm and about 3.0 nm. In some embodiments, the Δλmax can be between about 0.0 nm and about 2.0 nm. In some embodiments, the Δλmax can be between about 0.0 nm and about 1.0 nm. In some embodiments, 0.0 nm ≤ Δλmax < 5.0 nm is considered "low self-interaction". In some embodiments, 5.0 nm ≤ Δλmax < 20.0 nm is considered "moderate self-interaction". In some embodiments, 10.0 nm ≤ Δλmax is considered "high self-interaction". Generally, the lower the tendency of self-interaction, the more favorable the developability of the antibody.

[0281] In some embodiments, an anti-CD3 antibody or antigen-binding fragment as described herein may exhibit reduced viscosity. In some embodiments, the viscosity can be determined based on the diffusion interaction parameter (kD) value observed during dynamic light scattering (DLS). In some embodiments, DLS can be performed and the kD value can be obtained as described in the examples, for example, using 10 mM histidine buffer (pH of about 6). In some embodiments, an anti-CD3 antibody or antigen-binding fragment as described herein may exhibit a kD of about 5 mL / g or higher. In some embodiments, the kD can be about 10 mL / g or higher. In some embodiments, the kD can be about 15 mL / g or higher. In some embodiments, the kD can be about 20 mL / g or higher. In some embodiments, the kD can be about 25 mL / g or higher. In some embodiments, Δλmax < 20 mL / g can be considered associated with high viscosity or high opalescence. Generally, the lower the viscosity, the more favorable the developability of the antibody.

[0282] In some embodiments, an anti-CD3 antibody or antigen-binding fragment as described herein may exhibit a reduced likelihood of heavy-light chain mispairing (including pairing failure). In some embodiments, heavy-light chain mispairing can be determined based on the presence of heavy chain peaks (indicating heavy chains that have not successfully paired with light chains) and / or light chain peaks (indicating light chains that have not successfully paired with heavy chains) observed during liquid chromatography-mass spectrometry (LC-MS). In some embodiments, LC-MS can be performed as described in the examples. In some embodiments, an anti-CD3 antibody or antigen-binding fragment as described herein may show no heavy chain peaks or light chain peaks. Generally, the smaller the heavy chain peaks and light chain peaks, the more favorable the developability of the antibody.

[0283] In some embodiments, an anti-CD3 antibody or antigen-binding fragment as described herein may exhibit a reduced tendency to aggregate. In some embodiments, the tendency to aggregate can be determined based on the monomer % value observed during size exclusion chromatography (SEC) (i.e., the % of antibody species (e.g., IgG or Fab) that are present in their full size without aggregation or multimerization in the protein from antibody production and optionally purification). In some embodiments, SEC can be performed as described in the examples. In some embodiments, an anti-CD3 antibody or antigen-binding fragment as described herein may show a monomer % of about 95% or higher in SEC, indicating that the antibody is present substantially in monomeric form, i.e., not aggregated. In some embodiments, the monomer % can be about 97% or higher. In some embodiments, the monomer % can be about 98% or higher. In some embodiments, the monomer % can be about 99% or higher. In some embodiments, the monomer % can be about 99.5% or higher. Generally, the higher the monomer % value, the more favorable the developability of the antibody.

[0284] In some embodiments, the anti-CD3 antibodies or antigen-binding fragments as described herein may exhibit improved tolerance to acidic environments or acidic stress, such as a pH of about 6 or lower, about 5 or lower, about 4 or lower, or about 3.5 or lower. In some embodiments, the tolerance to low pH can be determined based on the tendency to aggregate upon exposure to low pH. In some embodiments, the tendency to aggregate at low pH can be determined based on the monomer % value (e.g., monomeric IgG or monomeric Fab) observed during SEC after exposure to low pH. In some embodiments, this SEC for low pH tolerance testing can be performed as described in the examples. In some embodiments, the anti-CD3 antibodies or antigen-binding fragments as described herein may exhibit a monomer % of about 95% or higher in SEC after exposure to low pH, indicating that the antibody is substantially in monomeric form, i.e., not aggregated. In some embodiments, the monomer % can be about 96% or higher. In some embodiments, the monomer % can be about 97% or higher. In some embodiments, the monomer % can be about 98% or higher. In some embodiments, the monomer % can be about 99% or higher. Generally, the higher the monomer % value after exposure to low pH, the higher the tolerance to acidic stress, i.e., the more favorable the developability of the antibody. Without wishing to be bound by theory, increasing the tolerance to acidic stress may help provide a longer shelf life and / or improve in vivo stability (e.g., in an acidic cancer microenvironment).

[0285] In some embodiments, the anti-CD3 antibodies or antigen-binding fragments as described herein may exhibit improved stability. In certain embodiments, stability can be evaluated based on the Tm value. In some cases, the anti-CD3 antibodies or antigen-binding fragments as described herein may exhibit a Tm of about 65°C or higher. In some embodiments, DSF or any other suitable method that can be performed as described in the examples can be used to determine the Tm. Generally, the higher the Tm, the more stable the antibody, i.e., the more favorable the developability.

[0286] Nucleic acids, vectors, and cells

[0287] The present disclosure also encompasses nucleic acids (one nucleic acid or a combination of two (or more) nucleic acids) encoding the VH and VL of any of the anti-CD3 antibodies and antigen-binding fragments described herein. Exemplary nucleic acids include, but are not limited to, DNA and RNA.

[0288] In some embodiments, a nucleic acid molecule can encode (i) an amino acid sequence comprising the VH of an antibody and (ii) an amino acid sequence comprising the VL of an antibody. In certain embodiments, (i) and (ii) can be encoded on the same strand of the nucleic acid molecule. In some cases, (i) and (ii) can be encoded under a single promoter. In certain cases, (i) and (ii) can be encoded in the same orientation (in some cases, (i) and (ii) can be transcribed into a single transcript, and in some cases, (i) and (ii) can be transcribed into two separate transcripts). In certain cases, (i) and (ii) can be encoded in the opposite orientation. In some cases, (i) and (ii) can be encoded under separate promoters. In certain embodiments, (i) and (ii) can be encoded on different strands of the nucleic acid molecule.

[0289] In some embodiments, a nucleic acid encoding an antibody can comprise: (i) a first nucleic acid that encodes an amino acid sequence comprising VH; and (ii) a second nucleic acid that encodes an amino acid sequence comprising VL.

[0290] In certain embodiments, the nucleic acid can comprise the sequence encoding VH of SEQ ID NO: 650 and the sequence encoding VL of SEQ ID NO: 660. In certain embodiments, the nucleic acid can comprise the sequence encoding VH of SEQ ID NO: 550 and the sequence encoding VL of SEQ ID NO: 560. In some cases, the nucleic acid can comprise RNA, which can comprise an RNA version of such sequences.

[0291] The present disclosure also encompasses vectors (a single vector or a combination of two (or more) vectors, e.g., expression vectors) encoding VH and VL of any of the anti-CD3 antibodies and antigen-binding fragments described herein.

[0292] In some embodiments, a vector can comprise a nucleic acid encoding both (i) an amino acid sequence comprising the VH of an antibody and (ii) an amino acid sequence comprising the VL of an antibody. In certain embodiments, (i) and (ii) can be encoded on the same strand of the nucleic acid molecule. In some cases, (i) and (ii) can be encoded under a single promoter. In certain cases, (i) and (ii) can be encoded in the same orientation (in some cases, (i) and (ii) can be transcribed into a single transcript, and in some cases, (i) and (ii) can be transcribed into two separate transcripts). In certain cases, (i) and (ii) can be encoded in the opposite orientation. In some cases, (i) and (ii) can be encoded under separate promoters. In certain embodiments, (i) and (ii) can be encoded on different strands of the nucleic acid.

[0293] In some embodiments, the vector may comprise: (i) a first vector comprising a nucleic acid encoding an amino acid sequence comprising VH; and (ii) a second vector comprising a nucleic acid encoding an amino acid sequence comprising VL.

[0294] In certain embodiments, the vector may comprise a nucleic acid comprising the sequence encoding VH of SEQ ID NO: 650 and a nucleic acid comprising the sequence encoding VL of SEQ ID NO: 660. In certain embodiments, the vector may comprise a nucleic acid comprising the sequence encoding VH of SEQ ID NO: 550 and a nucleic acid comprising the sequence encoding VL of SEQ ID NO: 560.

[0295] The present disclosure also provides an isolated, recombinant, and / or host cell comprising any one of the anti-CD3 antibodies and antigen-binding fragments described above, comprising any one of the nucleic acids described above, and / or comprising any one of the vectors described above, transfected with any one of the vectors described above, transduced with any one of the vectors described above, or transformed with any one of the vectors described above.

[0296] In some embodiments, the cell comprises the following, has been transfected with the following, has been transduced with the following, and / or has been transformed with the following: (1) a vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of an antibody and an amino acid sequence comprising the VH of an antibody, or (2) a first vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of an antibody and a second vector comprising a nucleic acid encoding an amino acid sequence comprising the VH of an antibody.

[0297] In some embodiments, the cell can be used to prepare an anti-CD3 antibody or antigen-binding fragment according to the present disclosure. In some embodiments, the cell can be used to administer to a subject.

[0298] In some embodiments, the cell is eukaryotic. In certain embodiments, the cell is mammalian, such as Chinese hamster ovary (CHO) cells, human embryonic kidney (HEK) cells, such as HEK293 cells, or lymphocytes (e.g., Y0, NS0, Sp20 cells). In certain embodiments, the cell is yeast.

[0299] Production of anti-CD3 antibodies and thus cells

[0300] Any one of the anti-CD3 antibodies and / or antigen-binding fragments described herein can be produced or prepared using any suitable method, including recombinant methods, such as in vitro, ex vivo, or in vivo.

[0301] In some embodiments, methods of preparing an isolated, recombinant, and / or host cell or population of such cells comprising a nucleic acid encoding any of the anti-CD3 antibodies and antigen-binding fragments described herein may include introducing the nucleic acids and / or vectors described herein into one or more cells.

[0302] Physical methods for introducing nucleic acids into cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for generating cells comprising a vector and / or exogenous nucleic acid are well known in the art. See, e.g., Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York). A preferred method for introducing polynucleotides into a host cell is calcium phosphate transfection.

[0303] Biological methods for introducing nucleic acids of interest into cells include the use of DNA and RNA vectors. In certain embodiments, the nucleic acid encoding the antibody can be isolated and inserted into one or more vectors (e.g., viral vectors, plasmids, etc.) for further cloning and / or expression in isolated, recombinant, and / or cells. Such nucleic acids can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that specifically bind to the genes encoding the heavy and light chains of the antibody). Viral vectors, particularly retroviral vectors, have become the most widely used method for inserting genes into mammalian cells such as human cells. Other viral vectors can be derived from lentivirus, vaccinia virus, herpes simplex virus I, adenovirus, and adeno-associated virus, etc. See, e.g., U.S. Patent Nos. 5,350,674 and 5,585,362.

[0304] Chemical means for introducing nucleic acids into cells include colloidal dispersion systems such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems including water-in-oil emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system used as an in vitro and in vivo delivery vehicle is a liposome (e.g., an artificial membrane vesicle).

[0305] In the case of using a non-viral delivery system, an exemplary delivery vehicle is a liposome. Lipid formulations are considered for introducing nucleic acids into host cells (in vitro, ex vivo, or in vivo). In some cases, the nucleic acid can associate with the lipid. The nucleic acid associated with the lipid can be encapsulated in the aqueous interior of the liposome, dispersed within the lipid bilayer of the liposome, attached to the liposome via a linking molecule that associates with both the liposome and the oligonucleotide, entrapped within the liposome, complexed with the liposome, dispersed in a lipid-containing solution, mixed with the lipid, combined with the lipid, contained within the lipid as a suspension, contained within a micelle or complexed with a micelle, or otherwise associated with the lipid. The composition associated with the lipid, lipid / DNA, or lipid / expression vector is not limited to any particular structure in solution. For example, they can exist as bilayer structures, micelles, or together with "collapsed" structures. They can also simply be dispersed in solution, possibly forming aggregates of non-uniform size or shape. Lipids are fatty substances, which can be naturally occurring or synthetic lipids. For example, lipids include the fat droplets naturally present in the cytoplasm and a class of compounds containing long-chain aliphatic hydrocarbons and their derivatives such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.

[0306] In some embodiments, a method of preparing an anti-CD3 antibody or antigen-binding fragment according to the present disclosure can include culturing an isolated, recombinant, and / or host cell containing a nucleic acid encoding the antibody under conditions suitable for the expression of the antibody as described above, and optionally harvesting, recovering, and / or purifying the antibody or antigen-binding fragment from the cell (or host cell culture medium).

[0307] Suitable host cells for vectors encoding antibodies for cloning and / or expression include prokaryotic or eukaryotic cells. For example, antibodies can be produced in bacteria, especially when 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. (See also Charlton, Methods in Molecular Biology, Vol. 248 (B.K.C. Lo, ed., Humana Press, Totowa, N.J., 2003), pp. 245-254, which describes the expression of antibody fragments in Escherichia coli (E. coli).) After expression, the antibodies can be isolated in the soluble fraction from the bacterial cell paste and further purified. In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast are suitable cloning or expression hosts for vectors encoding antibodies, including fungal and yeast strains in which their glycosylation pathways have been "humanized" to produce antibodies with a partially or fully human glycosylation pattern. See, for example, Gerngross, Nat. Biotech. 22:1409-1414 (2004), and Li et al., Nat. Biotech. 24:210-215 (2006); WO 2009 / 036379; WO 2010 / 105256; and WO 2012 / 009568.

[0308] 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 (describing the PLANTIBODIES™ technology for producing antibodies in transgenic plants). Viral cells can also be used as hosts. For example, mammalian cell lines suitable for growth in suspension may be useful. Other examples of useful mammalian host cell lines are monkey kidney CV1 lines transformed by SV40 (COS-7); human embryonic kidney lines (e.g., 293 or 293 cells as described in Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK); mouse Sertoli cells (e.g., TM4 cells as 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); dog kidney cells (MDCK); buffalo rat hepatocytes (BRL 3A); human lung cells (W138); human hepatocytes (HepG2); mouse mammary tumor (MMT 060562); TRI cells, as described, for example, in Mather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982); MRC 5 cells; and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR-CHO cells (Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)) and myeloma cell lines such as Y0, NS0, and Sp2 / 0. For a review of certain mammalian host cell lines suitable for antibody production, see, for example, Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (B.K.C. Lo, ed., Humana Press, Totowa, N.J.), pp. 255-268 (2003).

[0309] Cells that produce the antibodies or antigen-binding fragments of the present disclosure, such as hybridomas or other recombinant cells, can be grown using standard methods in a medium suitable for this purpose (such as D-MEM or RPMI-1640), or grown in vivo as ascites. The antibodies or antigen-binding fragments expressed and / or secreted by the cells can be separated from the cells, medium, ascites, or serum using conventional immunoglobulin purification procedures, such as but not limited to protein A-agarose, hydroxyapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography (Ma H. et al., Methods. 2017 Mar 1;116:23-33.doi:10.1016 / j.ymeth.2016.11.008.Epub 2016 Nov 18;Shukla A.A. et al., Trends Biotechnol. 2010 May;28(5):253-61.doi:10.1016 / j.tibtech.2010.02.001.Epub 2010 Mar 19;Arora S. et al., Methods. 2017 Mar 1;116:84-94.doi:10.1016 / j.ymeth.2016.12.010.Epub 2016 Dec 22).

[0310] Methods for expressing, isolating, and evaluating multispecific and bispecific antibodies and antibody fragments are also known in the art (e.g., see Brinkmann U. et al., MAbs. 2017 Feb-Mar;9(2):182-212.Published online 2017 Jan 10.doi:10.1080 / 19420862.2016.1268307;Dimasi N. et al., Methods. 2018 Aug 11.pii:S1046-2023(18)30149-X.doi:10.1016 / j.ymeth.2018.08.004).

[0311] Pharmaceutical compositions

[0312] The present disclosure also encompasses pharmaceutical compositions comprising: (A) (i) any of the anti-CD3 antibodies and antigen-binding fragments described herein, (ii) a nucleic acid encoding such an anti-CD3 antibody or antigen-binding fragment, (iii) a vector encoding such an anti-CD3 antibody or antigen-binding fragment, and / or (iv) an isolated or recombinant cell comprising (i), (ii), and / or (iii); and (B) a pharmaceutically acceptable carrier and / or excipient.

[0313] Accordingly, in some embodiments, an anti-CD3 antibody or antigen-binding fragment can be the active ingredient; in some embodiments, a nucleic acid encoding an anti-CD3 antibody or antigen-binding fragment (e.g., DNA or RNA, such as mRNA) or a vector can be the active ingredient; and in some embodiments, a cell comprising a nucleic acid or vector encoding an anti-CD3 antibody or antigen-binding fragment can be the active ingredient. In certain embodiments, when mRNA is the active ingredient, the mRNA can be formulated into lipid nanoparticles, which can facilitate administration and delivery to the cells of a subject receiving the composition.

[0314] In some embodiments, a pharmaceutical composition comprising an anti-CD3 antibody and / or antigen-binding fragment as described herein can be prepared, for example, in the form of a lyophilized composition or an aqueous solution by mixing such an antibody having the desired degree of purity with one or more optional pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences, 16th Edition, Osol, A. Ed. (1980)), optionally prepared for modified (e.g., sustained) release. Exemplary lyophilized antibody compositions are described in U.S. Patent No. 6,267,958. Aqueous antibody formulations include those described in U.S. Patent No. 6,171,586 and WO2006 / 044908, the latter composition containing a histidine-acetate buffer.

[0315] Pharmaceutically acceptable carriers are generally non-toxic to the recipient at the dosages and concentrations employed and include, but are not limited to: buffers such as phosphates, citrates, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as cetyltrimethylammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butanol, or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates (including glucose, mannose, or 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 nonionic surfactants such as polyethylene glycol (PEG). Exemplary pharmaceutically acceptable carriers herein further include interstitial drug dispersants such as soluble neutral active hyaluronidase glycoprotein (sHASEGP), e.g., human soluble PH-20 hyaluronidase glycoprotein, such as rHuPH20( Baxter International, Inc.). Certain exemplary sHASEGPs, including rHuPH20, and methods of use are described in U.S. Patent Publication Nos. 2005 / 0260186 and 2006 / 0104968.

[0316] The active ingredient can be encapsulated in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization (such as hydroxymethylcellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules in colloidal drug delivery systems (such as liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules) or in macroemulsions, respectively). Such techniques are disclosed in Remington's Pharmaceutical Sciences, 16th Edition, edited by Osol, A. (1980).

[0317] In some embodiments, the pharmaceutical compositions according to the present disclosure can be used in any of the in vivo methods described herein.

[0318] Additional agent

[0319] In some embodiments, the pharmaceutical composition can be used alone without additional agents.

[0320] In some embodiments, the pharmaceutical composition can further comprise one or more additional agents (e.g., one or more therapeutic agents) or can be used in combination with one or more additional agents (e.g., one or more therapeutic agents) in a treatment regimen (co-administered or administered separately). The additional agents that can be combined or used together with the anti-CD3 antibody according to the present disclosure are preferably those having complementary activities that do not adversely affect each other and are present in an amount effective for the intended purpose.

[0321] In certain embodiments, the one or more additional agents can be or can comprise chemotherapeutic agents, gene therapy agents, DNA therapy agents, viral therapy agents, RNA therapy agents, immunotherapy agents, nanotherapy agents, monoclonal antibodies, or combinations of the foregoing. In certain embodiments, the one or more additional therapeutic agents can be or can include adjuvants or novel adjuvants. In certain embodiments, the one or more additional therapeutic agents can be or can include small molecule enzyme inhibitors or anti-metastatic agents.

[0322] In certain embodiments, the one or more additional therapeutic agents can be or can include side effect limiting agents (e.g., agents designed to reduce the occurrence and / or mitigate the severity of treatment side effects, such as anti-nausea agents, including but not limited to neurokinin-1 receptor antagonists (NK1RAs), 5-hydroxytryptamine receptor antagonists (5-HT3 RAs), dexamethasone, olanzapine, and palonosetron, etc.).

[0323] Non-limiting exemplary additional agents may include chemotherapeutic agents, antibody-drug conjugates (ADCs), immunotherapeutic agents, and / or biologic modifiers.

[0324] In certain embodiments, the chemotherapeutic agent may be selected from alkylating agents, antimetabolites, plant alkaloids, and anticancer antibiotics, further optionally selected from one or more of the following: cyclophosphamide, cisplatin, carboplatin, oxaliplatin, etoposide, irinotecan, lurbinectedin, paclitaxel, docetaxel, cabazitaxel, altretamine, capecitabine, gemcitabine, ifosfamide, melphalan, pemetrexed, topotecan, vinorelbine, mitoxantrone, ixabepilone, eribulin, estramustine, vinblastine, vincristine, 5-fluorouracil (5-FU), doxorubicin, epirubicin, dactinomycin, or derivatives thereof. In certain embodiments, the chemotherapeutic agent may be selected from cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP).

[0325] In certain embodiments, the ADC may be selected from anti-CD79b antibody-drug conjugates (such as anti-CD79b-MC-vc-PAB-MMAE or anti-CD79b antibody-drug conjugate, or polatuzumab vedotin, as described in either U.S. Patent No. 8,088,378 and / or US2014 / 0030280), anti-CD19 antibody-drug conjugates, anti-CD22 antibody-drug conjugates, anti-CD45 antibody-drug conjugates, and anti-CD32 drug conjugates. The biologic modifiers may be selected from BCL-2 inhibitors (such as GDC-0199 / ABT-199), lenalidomide PI3K-δ inhibitors (such as idelalisib, ), PD-1 axis-binding antagonists, agonists (e.g., agonist antibodies against co-stimulatory molecules) such as CD40, CD226, CD28, OX40 (e.g., AgonOX), GITR, CD137 (also known as TNFRSF9, 4-1BB, or ILA), CD27 (e.g., CDX-1127), HVEM, or CD127, antagonists (e.g., antagonist antibodies against inhibitory co-stimulatory 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 ) Trametinib (also known as ticilimumab or CP-675,206), urelumab (also known as BMS-663513), MGA271, antagonists against TGFβ such as metelimumab (also known as CAT-192), nonsulmonab (also known as GC1008), LY2157299k, and adoptive transfer of T cells expressing chimeric antigen receptors (CARs) (e.g., cytotoxic T cells or CTLs), e.g., adoptive transfer of T cells comprising a dominant-negative TGFβ receptor such as a dominant-negative TGFβ type II receptor.

[0326] In certain embodiments, the chemotherapeutic agent can be or can include an immune checkpoint inhibitor and / or a growth factor or growth factor receptor inhibitor, optionally an inhibitor of PD-L1, PD-1, CTLA-4, VISTA, EGF, EGFR, VEGF, and / or VEGFR, or an antibody or antigen-binding fragment against PD-L1, PD-1, CTLA-4, VISTA, EGF, EGFR, VEGF, and / or VEGFR, or an antibody or antigen-binding fragment against a cancer antigen.

[0327] In certain embodiments, the one or more additional agents can be or can include a chemotherapeutic agent, a cytotoxic agent, an antihormonal agent, a growth inhibitor, a cytotoxic agent, an agent for use in radiotherapy, an antiangiogenic agent, an apoptotic agent, an antitubulin agent, or other agents such as epidermal growth factor receptor (EGFR) antagonists (e.g., tyrosine kinase inhibitors), HER1 / EGFR inhibitors (e.g., erlotinib (TARCEVA TM )), platelet-derived growth factor inhibitors (e.g., GLEEVEC TM (imatinib mesylate)), COX-2 inhibitors (e.g., celecoxib), interferons, cytokines, antibodies other than the anti-CD3 antibody of the present disclosure, such as antibodies that bind to one or more of the following targets: ErbB2, ErbB3, ErbB4, PDGFR-β, BIyS, APRIL, BCMA VEGF or VEGF receptor, TRAIL / Apo2, PD-1, PD-L1, PD-L2, or another bioactive or organic chemical agent. In certain embodiments, the one or more additional therapeutic agents can be or can include a glucocorticoid, optionally dexamethasone.

[0328] Kits

[0329] In another aspect of the present disclosure, a kit is provided that can be used for treating, preventing, and / or diagnosing the diseases, disorders, or conditions described herein. The kit can comprise: (A) a container containing: (i) any one of the anti-CD3 antibodies and antigen-binding fragments described herein, (ii) a nucleic acid encoding such an anti-CD3 antibody or antigen-binding fragment, (iii) a vector encoding such an anti-CD3 antibody or antigen-binding fragment, (iv) an isolated or recombinant cell comprising (i), (ii), and / or (iii); and / or (v) any one of the pharmaceutical compositions described herein; and (B) a label or package insert on or associated with the container. In certain embodiments, the container can contain one or more additional agents, optionally a cytotoxic agent and / or a therapeutic agent or any one of the additional agents described herein.

[0330] In certain embodiments, the kit can comprise: (A-1) a first container containing: (i) any one of the anti-CD3 antibodies and antigen-binding fragments described herein, (ii) a nucleic acid encoding such an anti-CD3 antibody or antigen-binding fragment, (iii) a vector encoding such an anti-CD3 antibody or antigen-binding fragment, (iv) an isolated or recombinant cell comprising (i), (ii), and / or (iii); and / or (v) any one of the pharmaceutical compositions described herein; (A-2) a second container containing: one or more additional agents, optionally a cytotoxic agent and / or a therapeutic agent or any one of the additional agents described herein; and (B) a label or package insert on or associated with the container.

[0331] In certain embodiments, the kit can further comprise a second (or third) container containing a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and glucose solution. In certain embodiments, the kit can further comprise other materials desirable from a commercial and user perspective, including other buffers, diluents, filters, needles, and syringes.

[0332] Suitable containers can include, for example, bottles, vials, syringes, IV solution bags, etc. The container can be formed from various materials such as glass or plastic. The container contains a composition that is effective, either alone or in combination with another composition, for treating, preventing, and / or diagnosing a condition and can have a sterile access port (e.g., the container can be an intravenous infusion bag or a vial with a stopper penetrable by a subcutaneous injection needle). The label or package insert can indicate that the composition is for treating the selected condition.

[0333] In some embodiments, the kit according to the present disclosure can be used in any one of the in vivo methods described herein.

[0334] In certain embodiments, the kits disclosed herein can be used alone or in combination with one or more additional agents. In certain embodiments, the kits can be used to treat any of the diseases, disorders, or conditions described herein, such as proliferative disorders (e.g., cancer) or autoimmune disorders (e.g., arthritis, rheumatoid arthritis, colitis, inflammatory bowel disease, autoimmune type I diabetes, etc.).

[0335] In vivo methods and uses

[0336] The present disclosure also encompasses in vivo methods.

[0337] In some aspects, methods and uses for treating a subject in need thereof and methods and uses for treating or preventing a disease, disorder, or condition in a subject are provided. Such methods and uses can include administering to the subject an effective amount of (i) any of the anti-CD3 antibodies and antigen-binding fragments described herein, (ii) a nucleic acid encoding such an anti-CD3 antibody or antigen-binding fragment, (iii) a vector encoding such an anti-CD3 antibody or antigen-binding fragment, (iv) an isolated or recombinant cell comprising (i), (ii), and / or (iii); and / or (v) any of the pharmaceutical compositions described above.

[0338] In some aspects, methods and uses for inducing cytotoxicity against cells expressing a target molecule of interest are provided. Such methods and uses can include administering to the subject an effective amount of (i) a multispecific antibody or antibody fragment comprising at least one anti-CD3 antibody or antigen-binding fragment described herein, (ii) a nucleic acid encoding such a multispecific antibody or antibody fragment, (iii) a vector encoding such a multispecific antibody or antibody fragment, (iv) an isolated or recombinant cell comprising (i), (ii), and / or (iii); and / or (v) a pharmaceutical composition comprising (i), (ii), (iii), and / or (iv).

[0339] In any of the aspects and embodiments of the methods and uses described above, in some embodiments, the anti-CD3 antibody or antigen-binding fragment can be used to enhance immune function in an individual suffering from a proliferative disorder or an autoimmune disorder. After administration, such an antibody can enhance immune function in an individual suffering from a proliferative disorder or an autoimmune disorder by activating effector cells (e.g., T cells, such as CD8+ and / or CD4+ T cells, including Tregs), expanding (increasing) the population of effector cells, reducing the population of target cells (e.g., cells expressing a second biomolecule recognized by an anti-CD3 antibody (such as a bispecific antibody) of the present disclosure), and / or killing target cells (e.g., target tumor cells).

[0340] In yet another aspect, methods and uses are provided for detecting CD3 or CD3-expressing cells in a subject, such as in a disease site (e.g., a tumor site) or a potential disease site in a subject. Such methods and uses can include administering to the subject an effective amount of any one of the anti-CD3 antibodies and antigen-binding fragments described herein. In some embodiments, such methods and uses can be used for diagnosis and / or detection. In certain embodiments, the diagnosis and / or detection can include determining the stage, severity, or immune signature of a disease or potential disease. In some cases, the immune signature can include the number of CD3-expressing cells in a tumor site. Without wishing to be bound by theory, the higher the number of CD3-expressing cells in a tumor, the more likely the tumor is to be effectively treated by an anti-CD3 antibody or antigen-binding fragment according to the present disclosure or another immunotherapy.

[0341] In any one of the aspects and embodiments herein that include methods and uses, the subject can be a mammal, particularly a human.

[0342] In any one of the aspects and embodiments herein that include methods and uses, the subject can have or be at risk of having a disease, disorder, or condition.

[0343] In any one of the aspects and embodiments herein that include methods and uses, the disease, disorder, or condition can be any suitable disease, disorder, or condition, including but not limited to those described herein. In some embodiments, the disease, disorder, or condition can be a proliferative disorder, cancer, tumor disorder, immuno-oncology disorder, neurological disorder, cognitive disorder, neurodegenerative disorder, and / or an inflammatory and / or autoimmune disorder (e.g., rheumatoid arthritis, colitis, inflammatory bowel disease, autoimmune type I diabetes, etc.).

[0344] In certain embodiments, the disease, disorder, or condition can be cancer. Without wishing to be bound by theory, tumor cells generally have an extracellular pH of about 6.3 - 6.5, and the anti-CD3 antibodies and antigen-binding fragments described herein preferentially promote binding and activity in and around the tumor microenvironment due to their preferential CD3 binding at low (lower) pH values, such as around pH 6. In certain embodiments, the use of the anti-CD antibodies and their antigen-binding fragments can result in selective and sustained cytotoxic activity at or around the tumor site, thereby (i) reducing the effective amount required for a desired purpose (e.g., treatment) and / or (ii) reducing or eliminating off-target effects.

[0345] In certain embodiments, an RNA such as mRNA encoding an anti-CD3 antibody or antigen-binding fragment or a composition comprising such RNA can be administered. In some cases, the RNA (e.g., mRNA) can be formulated into lipid nanoparticles to facilitate administration and delivery to the cells of a subject receiving the RNA.

[0346] Route of administration and dosing

[0347] In any of the aspects and embodiments of the methods described herein, an effective amount of such an anti-CD3 antibody or antigen-binding fragment (and optionally any additional agent) or a pharmaceutical composition comprising such an anti-CD3 antibody or antigen-binding fragment can be administered by any suitable means, including parenterally, intralungally, and intranasally, and, if local treatment is desired, including intralesional administration. Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. In certain embodiments, the administration is subcutaneous administration, which may exhibit a lesser toxic response in a subject as compared to intravenous injection. The dosing can be by any suitable route (e.g., injection, such as intravenous or subcutaneous injection) and can depend in part on whether the administration is short-term or long-term. Various dosing schedules are contemplated herein, including but not limited to single or multiple administrations at different time points, bolus administration, and pulsed infusion.

[0348] The antibodies of the present disclosure will be formulated (e.g., formulated as a pharmaceutical composition), dosed, and administered in a manner consistent with good medical practice. Factors considered in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site to which the agent is to be delivered, the method of administration, the dosing schedule, and other factors known to the physician. The antibodies need not but may optionally be formulated (e.g., formulated as a pharmaceutical composition) together with one or more agents currently used to prevent or treat the disorder being discussed. The effective amount of such other agents depends on the amount of antibody present in the composition, the type of disorder or treatment, and the other factors discussed above. These are generally used at the same dose and route of administration as described herein, or at about 1% to 99% of the doses described herein, or at any dose and any route determined empirically / clinically to be appropriate.

[0349] For the prevention or treatment of a disease, disorder, or condition, the appropriate dose of the antibody of the present disclosure (when used alone or in combination with one or more other additional therapeutic agents) will depend on the type of disease to be treated, the type of antibody, the severity and course of the disease, whether the antibody is administered for prophylactic or therapeutic purposes, previous therapy, the clinical history of the patient and response to the antibody, and the judgment of the attending physician. In certain embodiments, the antibody can be appropriately administered to a patient one or in a series of treatments.

[0350] As a general proposition, an effective amount (e.g., a therapeutically effective amount) of an anti-CD3 antibody or antigen-binding fragment thereof administered to a human can be in the range of about 0.01 to about 100 mg / kg of patient body weight, whether administered as a single dose or multiple doses. In some embodiments, the antibody or antigen-binding fragment can be administered, for example, at 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 per day. In one embodiment, the anti-CD3 antibody described herein is administered to a human at a dose of about 100 mg, about 200 mg, about 300 mg, about 400 mg, about 500 mg, about 600 mg, about 700 mg, about 800 mg, about 900 mg, about 1000 mg, about 1100 mg, about 1200 mg, about 1300 mg, or about 1400 mg on day 1 of a 21-day cycle. The dose can be administered as a single dose or as multiple doses (e.g., 2 or 3 doses) such as by infusion. For repeated administration over several days or longer, depending on the condition, treatment generally will continue until the desired disease symptom suppression occurs. An exemplary dose of the antibody will be in the range of 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) can be administered to a patient. Such doses can be administered intermittently, for example, weekly or every three weeks (e.g., such that the patient receives about two to about twenty doses, or for example about six doses of the anti-CD3 antibody). An initial higher loading dose can be administered, followed by one or more lower doses. The progress of such therapy can be readily monitored by conventional techniques and assays.

[0351] Monotherapy and combination therapy

[0352] In any of the aspects and embodiments of the methods and uses described herein, an effective amount of such an anti-CD3 antibody or antigen-binding fragment can be administered to a subject alone.

[0353] In some embodiments, an effective amount of such an anti-CD3 antibody or antigen-binding fragment may be administered to a subject in combination with at least one additional agent. The one or more additional agents (e.g., therapeutic agents and / or adjuvants) may be or may include chemotherapeutic agents, gene therapy agents, DNA therapy agents, viral therapy agents, RNA therapy agents, nanotherapy agents, monoclonal antibodies, immunotherapy agents, and / or any agent such as but not limited to one or more additional agents specifically described herein. In certain embodiments, the one or more additional agents may be included in a pharmaceutical composition together with an anti-CD3 antibody or antigen-binding fragment according to the present disclosure. In certain embodiments, the one or more additional agents may not be included in a pharmaceutical composition of an anti-CD3 antibody or antigen-binding fragment according to the present disclosure, but may be administered (e.g., simultaneously) with the anti-CD3 antibody or antigen-binding fragment. In certain embodiments, the one or more additional agents may be administered separately from the anti-CD3 antibody or antigen-binding fragment (e.g., before or after administration of the anti-CD3 antibody or antigen-binding fragment). In some cases, administration of the anti-CD3 antibody or antigen-binding fragment and administration of the one or more additional agents may be performed within about one month of each other, or within about one, two, or three weeks, or within about one, two, three, four, five, or six days of each other.

[0354] In some embodiments, an effective amount of such an anti-CD3 antibody or antigen-binding fragment may be administered to a subject in combination with at least one additional therapy. The one or more additional therapies may be or may include radiation therapy (e.g., γ-irradiation), surgery, bone marrow transplantation, chemotherapy, or any combination of the foregoing. In certain embodiments, the one or more additional therapies may be administered (e.g., simultaneously) with the anti-CD3 antibody or antigen-binding fragment. In certain embodiments, the one or more additional therapies may be administered separately from the anti-CD3 antibody or antigen-binding fragment (e.g., before or after administration of the anti-CD3 antibody or antigen-binding fragment). In some cases, administration of the anti-CD3 antibody or antigen-binding fragment and administration of the one or more additional therapies may be performed within about one month of each other, or within about one, two, or three weeks, or within about one, two, three, four, five, or six days of each other.

[0355] Any anti-CD3 antibody and antigen-binding fragment, including bispecific anti-CD3 antibodies and antigen-binding fragments of the present disclosure that bind to CD3 and a second biomolecule (e.g., a cell surface antigen such as a tumor antigen), can be used in combination with the therapies described herein, such as in combination with radiation therapy.

[0356] Examples are provided below to illustrate embodiments of the present disclosure. These examples are not intended to limit the invention to any specific application or theory of operation.

[0357] Examples

[0358] Example 1: Novel pH-Dependent Anti-CD3 Antibodies

[0359] Previously, the pH-dependent anti-CD3 antibody ADI-48587 (also referred to herein as Antibody No. 2 and originally disclosed in PCT / US2020 / 036657) was identified from an antibody sequence library and is different from the non-pH-dependent anti-CD3 antibody ADI-26906 (also referred to herein as Antibody No. 1 and originally disclosed in PCT / US2018 / 031705).

[0360] To obtain improved pH-dependent anti-CD3 antibodies, the variable region sequences of ADI-48587 and ADI-26906 were altered. The novel anti-CD3 antibodies obtained include ADI-74968, ADI-74967, ADI-74966, ADI-74965, ADI-79842, ADI-79843, ADI-79848, ADI-79844, ADI-79845, ADI-79846, and ADI-79847 (referred to as Antibody Nos. 6, 5, 4, 3, 7, 8, 9, 10, 11, 12, and 13, respectively). The amino acid sequences of VH, VL, CDR, and FR and the nucleic acid sequences encoding VH and VL are shown in the Appendix (Tables A - P). The consensus sequences shared among such antibodies are also shown in the Appendix (Table Q).

[0361] Example 2: Monovalent Binding Affinity at Different pHs - IgG, SPR

[0362] Anti-CD3 antibodies (Antibody Nos. 1 - 13) were produced as human IgG1 antibodies in yeast or Chinese hamster ovary (CHO) cells and Fab fragments were generated from the IgG1 antibodies by papain digestion and purified. The IgG produced in yeast is non-glycosylated. The IgG produced by CHO has standard CH2 glycosylation. The products were confirmed to exhibit the expected mass by LC-MS analysis and >95% monomeric form by SEC-HPLC, with or without low pH stress (method described below). Using an 8K (Cytiva, previously GE Healthcare Life Sciences) system, the monovalent binding affinity for human or cynomolgus monkey CD3εδ-Fc at pH 7.4 and pH 6.0 was measured via surface plasmon resonance (SPR) and analyzed using evaluation software.

[0363] The results are shown in Table 1. As shown in the table, the Fab of Antibody Nos. 3 - 11 showed higher affinity at pH 6.0 than at pH 7.4 (i.e., lower K D)。The Fabs of antibodies numbered 3 - 6 produced by yeast bind to human CD3εδ-Fc at pH 6.0, but not at pH 7.4, where numbers 3, 5, and 6 exhibit higher affinity (i.e., lower K D )。Similar results were obtained with CHO-derived Fabs of antibodies numbered 3 - 6, where the affinity for human and cynomolgus monkey CD3εδ-Fc increased at pH 6.0 compared to the affinity at pH 7.4, and the affinity for human CD3εδ-Fc increased compared to Ab number 2.

[0364] Table 1: Binding affinities for CD3εδ-Fc analyzed by ·P.F. = Poor fit. Binding was observed under the conditions of this assay, but could not be adequately fit to a 1:1 binding model.

[0365]

[0366]

[0367] ·P.F. = Poor fit. Binding was observed under the conditions of this assay, but could not be adequately fit to a 1:1 binding model.

[0368] ·N.B. = No observable binding under the conditions of this assay; K could not be assigned D 。

[0369] ·N.A. = Not evaluated.

[0370] Example 3: Monovalent binding affinities at different pHs - IgG1, BLI

[0371] Anti-CD3 antibodies numbered 1 - 13 were produced as human IgG1 antibodies in yeast or CHO cells. The IgG produced in yeast is non-glycosylated. The IgG produced by CHO has standard CH2 glycosylation. The binding affinities for human or cynomolgus monkey CD3εδ-Fc at pH 7.4 and pH 6.0 were measured via Biolayer Interferometry (BLI) using the ForteBio HTX system and analyzed using ForteBio software.

[0372] The results are shown in Tables 2A and 2B. As shown in Table 2A, the antibodies numbered 3 - 12 produced by yeast showed higher affinity (i.e., lower K D ) for human CD3εδ-Fc at pH 6.0 than at pH 7.4, and had higher affinity (i.e., lower K D)。Similar results were observed for antibodies numbered 3 - 6 produced by CHO. As shown in Table 2B, yeast - produced antibodies numbered 3 - 10 and 12 bound to cynomolgus monkey CD3εδ - Fc with higher affinity at pH 6.0 than at pH 7.4. CHO - produced antibody 3 - 6 bound to cynomolgus monkey CD3εδ - Fc with higher affinity than Ab number 2 at pH 6.0 and did not bind to cynomolgus monkey CD3εδ - Fc at pH 7.4.

[0373] Table 2A: Binding affinities for human CD3εδ - Fc analyzed by

[0374]

[0375]

[0376] ·Equilibrium dissociation constant (K D = koff / kon); data were fit to a 1:1 binding model.

[0377] ·P.F. = goodness - of - fit difference. Binding was observed under the conditions of this assay, but could not be adequately fit to a 1:1 binding model.

[0378] ·N.B. = No observable binding under the conditions of this assay; K could not be assigned D .

[0379] ·N.A. = Not evaluated.

[0380] Table 2B: Binding affinities and kinetics for cynomolgus monkey CD3εδ - Fc analyzed by

[0381]

[0382]

[0383] ·Equilibrium dissociation constant (K D = koff / kon); data were fit to a 1:1 binding model.

[0384] ·N.B. = No observable binding under the conditions of this assay; K could not be assigned D .

[0385] ·N.A. = Not evaluated.

[0386] Example 4: Cell binding

[0387] Produce anti-CD3 antibodies numbered 1-13 as aglycosylated IgG containing the IgG1 Fc region in yeast cells. Binding to cells expressing human or cynomolgus monkey CD3 (human CD3+ Jurkat cells or cynomolgus monkey HSC-F cells, respectively) at pH 7.4 and pH 6.0 was measured by fluorescence-activated single-cell sorting (FACS). Normalized cell binding (NCB) values were calculated based on the median fluorescence intensity (MFI) values. CD3-negative Jurkat cells were used as a negative control.

[0388] The results are shown in Table 3. As shown in Table 3, antibodies numbered 3-12 showed higher binding to human CD3+ Jurkat cells and cynomolgus monkey HSC-F cells at pH 6.0 than at pH 7.4 (i.e., higher NCB values). In addition, compared to Ab number 2 (parent, pH-dependent antibody), antibodies numbered 3 and 5-12 showed higher binding to human CD3+ Jurkat cells at pH 6.0, and antibodies numbered 3 and 5-11 showed higher binding to cynomolgus monkey HSC-F cells at pH 6.0. None of the tested antibodies bound to the negative control cells.

[0389] Table 3: Cell binding measured by flow cytometry

[0390]

[0391] NCB = (sample MFI - secondary only MFI) / secondary only MFI Example 5: Developability - PSR

[0392] Evaluating the developability of anti-CD3 antibodies through multispecificity analysis. Antibodies with high affinity for the target may fail in the clinical setting because they also exhibit binding to multiple non-target entities. Antibody multispecificity is evaluated by measuring the interaction with a multispecific reagent (PSR). PSRs are prepared as described, for example, in WO 2014 / 179363 and Xu et al., Protein Eng Des Sel, 26(10):663-670 (2013). Briefly, 2.5 liters of CHO-S cells are used as starting material. The cells are pelleted at 2,400 x g for 5 minutes in 500 mL centrifuge bottles filled to 400 mL. The cell pellets are combined and then resuspended in 25 ml of buffer B and pelleted at 2,400 x g for 3 minutes. The buffer is decanted and the wash is repeated once. The cell pellets are resuspended in 3 x pellet volume of buffer B containing 1x protease inhibitor (Roche, complete, without EDTA) using a polytron homogenizer, with the cells kept on ice. The homogenate is then centrifuged at 2,400 x g for 5 minutes and the supernatant is retained and pelleted again (2,400 x g / 5 minutes) to ensure removal of unbroken cells, cell debris, and nuclei; the resulting supernatant is the total protein preparation. The supernatant is then transferred to two Nalgene Oak Ridge 45 mL centrifuge tubes and pelleted at 40,000 x g for 40 minutes at 4°C. The supernatant containing the soluble cytoplasmic protein (SCP) is then transferred to a clean Oak Ridge tube and centrifuged again at 40,000 x g. In parallel, the pellet containing the membrane fraction (EMF) is retained and centrifuged at 40,000 for 20 minutes to remove residual supernatant. The EMF pellet is then rinsed with buffer B. Then 8 mL of buffer B is added to the membrane pellet to dislodge the pellet and transferred to a Dounce homogenizer. After homogenizing the pellet, they are transferred to 50 mL conical tubes and represent the final EMF preparation.

[0393] Approximately 10 6 -10 7One billion mammalian cells (e.g., CHO, HEK293, Sf9) at a density of cells / mL were transferred from the tissue culture environment to 4 x 250 mL conical tubes and pelleted at 550 x g for 3 minutes. All subsequent steps were carried out at 4 °C or on ice with ice-cold buffers. The cells were washed with 100 mL PBSF (1x PBS + 1 mg / mL BSA) and pooled into one conical tube. After removing the supernatant, the cell pellet was then resuspended in 30 mL buffer B (50 mM HEPES, 0.15 M NaCl, 2 mM CaCl2, 5 mM KCl, 5 mM MgCl2, 10% glycerol, pH 7.2) and pelleted at 550 x g for 3 minutes. The buffer B supernatant was decanted, and the cells were resuspended in 3 x pellet volume of buffer B plus 2.5x protease inhibitor (Roche, complete, EDTA-free). The protease inhibitor in buffer B is included from here on. The cells were homogenized four times for 30-second pulses (Polytron homogenizer, PT1200E), and the membrane fraction was pelleted at 40,000 x g for 1 hour at 4 °C. The pellet was rinsed with 1 mL buffer B; the supernatant was retained and designated as s. The pellet was transferred to a Dounce homogenizer with 3 mL buffer B and resuspended by slowly moving the pestle up and down for 30 - 35 strokes. 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 the Dc-protein assay kit (BioRad). To solubilize the EMF, it was transferred to solubilization buffer (50 mM HEPES, 0.15 M NaCl, 2 mM CaCl2, 5 mM KCl, 5 mM MgCl2, 1% n-dodecyl-β-D-maltopyranoside (DDM), 1x protease inhibitor (pH 7.2) to a final concentration of 1 mg / mL. The mixture was rotated overnight at 4 °C, followed by centrifugation at 40,000 x g for 1 hour in a 50 mL Oak Ridge tube (Fisher Scientific, 050529-ID). The supernatant representing the soluble membrane protein (SMP) was collected, and the protein yield was quantified as described above.

[0394] For biotinylation, an NHS-LC-biotin stock solution was prepared according to the manufacturer's protocol (Pierce, Thermo Fisher). Briefly, 20 μl of biotin reagent was added per 1 mg of EMF sample and incubated at 4 °C for 3 h with gentle stirring. The volume was adjusted to 25 mL with buffer B and transferred to an Oak Ridge centrifuge tube. The biotinylated EMF (b-EMF) was precipitated at 40,000 x g for 1 h and washed twice with 3 mL of buffer C (buffer B minus glycerol) without disturbing the pellet. The residual solution was removed. The pellet was resuspended in 3 mL of buffer C using a Dounce homogenizer as previously described. The resuspended pellet now represents biotinylated EMF (b-EMF). Dissolve as described above to prepare b-SMP.

[0395] PSR binding analysis. Determination is generally performed as described, for example, in: Xu et al., Protein Eng Des Sel, 26(10):663 - 670 (2013). To characterize the PSR profile of monoclonal antibodies presented on yeast, 2 million IgG - presenting yeast were transferred to a 96 - well assay plate and pelleted at 3000 x g for 3 minutes to remove the supernatant. The pellet was resuspended in 50 μl of freshly prepared 1:10 diluted biotinylated PSR (b - PSR) stock solution and incubated on ice for 20 minutes. The cells were washed twice with 200 μl of cold PBSF and the pellet was resuspended in 50 μl of secondary labeling mixture (Extravidin - R - PE, anti - human LC - FITC, and propidium iodide). The mixture was incubated on ice for 20 minutes and then washed twice with 200 μl of ice - cold PBSF. The cells were resuspended in 100 μl of ice - cold PBSF and the plate was run on a FACSCanto (BD Biosciences) using an HTS sampler. The mean fluorescence intensity in the R - PE channel of the flow cytometry data was analyzed and normalized relative to an appropriate control (an antibody with an established PSR score) to assess non - specific binding. Many methods for presenting or displaying antibodies or antibody fragments on the surface of yeast have been previously described, all of which are consistent with this protocol (Blaise et al., Gene, 342(2):211 - 8 (2004); Boder and Wittrup, Nat Biotechnol., 15(6):553 - 7 (1997); Kuroda and Ueda, Biotechnol Lett., 33(1):1 - 9 (2011); Orcutt and Wittrup, Springer Protocols: Antibody Engineering, 1:207 - 233 (2010); Rakestraw et al., Protein Eng Des Sel., 24(6):525 - 30 (2011); Sazinsky et al., Proc Natl Acad Sci USA., 105(51):20167 - 72 (2008); Tasumi et al., Proc Natl Acad Sci USA., 106(31):12891 - 6 (2009).

[0396] The PSR score of parental antibody number 1 was the highest, indicating a relatively high level of non - target - specific binding. Antibodies numbered 2 - 6 exhibited PSR scores ≤0.1, which is considered a "no" PSR score, indicating a very low level of non - target - specific binding.

[0397] Example 6: Developability - HIC, AC - SINS, DLS, and Fab Tm

[0398] The developability of anti-CD3 antibodies was further determined by evaluating hydrophobicity, self-interaction, and stability. For these evaluations, antibodies numbered 1-6 were produced as human IgG1 antibodies in CHO cells. Fab fragments were generated from the IgG1 antibodies by papain digestion and purified.

[0399] Hydrophobicity according to HIC

[0400] Antibody hydrophobicity is a cause of antibody aggregation. To evaluate antibody hydrophobicity, hydrophobic interaction chromatography (HIC) analysis was performed on the IgG1-produced samples. Briefly, the IgG1 sample buffer was exchanged to 1 M ammonium sulfate and 0.1 M sodium phosphate (pH 6.5) using a Zeba 40 kDa 0.5 mL spin column (Thermo Pierce, catalog number 87766). A salt gradient from 1.8 M ammonium sulfate, 0.1 M sodium phosphate (pH 6.5) to the same conditions without ammonium sulfate was established on a Dionex ProPac HIC-10 column. The gradient was run at a flow rate of 0.75 mL / min for 17 minutes. An acetonitrile wash step was added at the end of the run to remove any residual protein, and the column was re-equilibrated over 7 column volumes prior to the next injection cycle. Peak retention times were monitored at A280 absorbance, and the ammonium sulfate concentration at elution was calculated based on the gradient and flow rate.

[0401] The HIC retention times of all tested IgG1s were < 10.5 minutes, indicating no to low hydrophobicity, i.e., a highly desirable developability profile.

[0402] Self-interaction according to AC-SINS

[0403] Measurement was performed in vitro by affinity capture self-interaction nanoparticle spectroscopy (AC-SINS) using the previously described protocol (Liu Y et al., MAbs. Mar-Apr 2014;6(2):483-92). Briefly, polyclonal goat anti-human IgG Fc antibody (capture; Jackson ImmunoResearch Laboratories) and polyclonal goat non-specific antibody (non-capture; Jackson ImmunoResearch Laboratories) were buffer-exchanged into 20 mM sodium acetate (pH 4.3) and concentrated to 0.4 mg / ml. A capture:non-capture volume ratio of 4:1 can be prepared and further incubated with 20 nm gold nanoparticles (AuNP; Ted Pella Inc.) at a volume ratio of 1:9 for 1 hour at room temperature. Then, thio-PEG (Sigma-Aldrich) was used to block the vacant sites on the AuNP and filtered through a 0.22 μm PVDF membrane (Millipore). Subsequently, the coated particles were added to the test IgG 1 antibody solution and incubated for 2 hours at room temperature, after which the absorbance at 510 nm to 570 nm was measured on a plate reader. The data points were fitted to a second-order polynomial in Excel to obtain the wavelength at the maximum absorbance. The value was reported as the difference (Δλmax) between the plasma wavelengths of the sample and the background. The self-interaction level was determined based on Δλmax. Self-interaction can be regarded as: low when Δλmax < 5.0 nm; medium when Δλmax ≥ 5.0 nm and < 20.0 nm; and high when Δλmax ≥ 20.0 nm.

[0404] Antibodies numbered 2-6 had a Δλmax < 5.0, indicating low self-interaction.

[0405] Self-interaction according to DLS

[0406] Self-interaction was also measured by dynamic light scattering (DLS). The diffusion interaction parameter (kD) of monoclonal antibodies measured at concentrations below 12 mg / mL had a strong correlation with their solution behavior at very high concentrations (>100 mg / mL). A positive kD value indicates repulsive interactions between molecules and has a positive correlation with low viscosity at high concentrations in the same formulation buffer. The kD value was obtained by measuring the mutual diffusion coefficient at a series of different concentrations by DLS. Specifically, DLS kD measurements were taken at various concentrations between 0.5 - 12 mg / mL in 10 mM histidine buffer at pH 6.0. A kD value < 20 mL / g was considered to be associated with high viscosity or high opalescence.

[0407] Antibodies numbered 1 - 6 produced kD values ≥ 20 mL / g, indicating low self - interaction.

[0408] Fab Tm according to DSF

[0409] The melting temperature (Tm) of the Fabs of antibodies numbered 1 - 6 was measured by differential scanning fluorimetry (DSF) using a CFX96 real - time system from Bio - Rad. Briefly, 20 μL of a 1 mg / mL sample was mixed with 10 μL of 20×SYPRO Orange. The plate was scanned from 40 °C to 95 °C at a rate of 0.5 °C / 2 minutes in a C1000 thermal cycler (BioRad) to collect the Fret signal. The first derivative of the raw data from the Bio - Rad analysis software was used to assign the Fab Tm.

[0410] All tested Fabs had Tm values above 65 °C, indicating high stability and thus desirable developability.

[0411] Materials and Methods

[0412] Unless otherwise specified, the following materials and methods were employed in the examples.

[0413] Production and purification of antibody yeast. Yeast clones were grown to saturation and then induced with shaking at 30 °C for 48 hours. After induction, the yeast cells were pelleted and the supernatant was harvested 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 on KappaSelect or CaptureSelect IgG - CH1 (GE Healthcare Life Sciences).

[0414] Antibody production and purification in CHO cells. Antibodies were produced as IgG1 by subcloning the antibody into a new expression vector and then transfecting and expressing in CHO cells. Fab fragments were generated by papain digestion and purified on KappaSelect or CaptureSelect IgG-CH1 (GE Healthcare Life Sciences). Gene fragments encoding VH and VL (Integrated DNA Technologies) were subcloned into heavy and light chain pcDNA 3.4+ vectors (ThermoFisher). The corresponding vectors were transiently co-transfected into CHO-K1 suspension cells using standard methods well known in the art. Typically, CHO-K1 cells grown to approximately 4x10*6 cells / mL were pelleted and resuspended in transfection medium. The DNA plasmid (1.5 ug total DNA / mL) was incubated with PEIpro (final 1:2, PolyPlus, catalog number 115-100) in transfection medium at room temperature before addition to the CHO-K1 cell suspension. The transfected cultures were fed and maintained at 32 °C with shaking until the supernatant was harvested (day 9) for purification. The cell culture supernatant was harvested by centrifugation and passed over protein A agarose (MabSelect SuRe; GE Healthcare Life Sciences). The bound antibody was then washed with PBS and eluted into 1 / 8 volume of 2M Hepes, pH 8.0 with buffer (200 mM acetic acid / 50 mM NaCl, pH 3.5). The final product was buffer exchanged into 25 mM Hepes and 150 mM sodium chloride, pH 7.3. Fab was generated using an overnight papain digestion followed by a CH1-resin purification step.

[0415] Hu and CyCD3εδFc heterodimer antigen production. Recombinant heterodimeric CD3 Fc fusion antigens were produced in HEK 293 cells by co-transfecting plasmids with heterologous signal peptide sequences (see Appendix Table U for sequences), the plasmids encoding Hu CD3g Fc (extracellular domain, ECD, residues 22-126) and CD3δ Fc-HIS (ECD residues 22-100) or Cy CD3ε Fc (ECD residues 22-117) and CD3δ Fc-HIS (ECD residues 22-100). In a computer-controlled Chromatographic separation was carried out on an Avant 150 preparative chromatography system (GE Healthcare Life Sciences), enabling online salt concentration monitoring during operation. The clarified culture supernatant was purified by removing CD3εε Fc homodimer using NiSepharose 6 Fast Flow (GE Healthcare Life Sciences). CD3εδFc HIS heterodimer was removed from CD3δδ Fc-HIS homodimer by MonoQ 10 / 100GL with a linear Tris-buffered KCl gradient at pH 8.5.

[0416] KD measurement (surface plasmon resonance; SPR). Affinity measurements were generally performed as previously described. Briefly, the human CD3εδ-Fc heterodimer generated as described above was immobilized onto a NiNTA sensor chip in an 8K (Cytiva, formerly GE Healthcare Life Sciences) at a response level of ~500 RU. Then Fab was injected at increasing concentrations ranging from 18.75 - 300 nM, 1.56 - 25 nM, 6.25 - 100 nM, or 1.25 - 20 nM. The sensor chip was regenerated doubly between cycles using 0.35 M EDTA and 0.1 M NaOH. The resulting data was subtracted from the double reference and fitted to a 1:1 binding model using evaluation software.

[0417] ForteBio K D ForteBio kinetics. ForteBio measurements (biolayer interferometry; BLI) were generally performed as previously described (Estep, P. et al., High throughput solution-based measurement of antibody-antigen affinity and epitope binning. MAbs, 2013.5(2): pp. 270 - 8). IgG was loaded online onto an AHC sensor. The sensor was equilibrated offline in the assay buffer for 30 minutes and then monitored online for 60 seconds for baseline establishment. The sensor with loaded IgG was exposed to 100 nM antigen (i.e., CD3) for 5 minutes, after which they were transferred to the assay buffer for 5 minutes for dissociation rate measurement. The kinetics were analyzed using a 1:1 binding model.

[0418] ForteBio kinetics. FortBio ​The HTX instrument is used with AHC, SA, or AHQ sensors in 12-channel mode (8 sensors per channel, 96 sensors per experiment). The instrument is driven by software (versions 8.2 and 9.0) supplied by the manufacturer. The sample name and concentration are entered into the plate data page, and the sensor-related proteins are identified in the "Information" column on the sensor data page. Kinetic experiments are collected with a 90 or 180 s baseline, 180 s association period, and 180 s dissociation period. All files are saved to a shared network drive in the naming convention of the identification experiment format.

[0419] Cell line propagation and cell labeling assays. Human Jurkat CD3+ cells (ATCC TIB-152) and Jurkat CD3- cells (ATCC TIB-153) were obtained from ATCC. Cyno HSC-F cells were obtained from the NIH Nonhuman Primate Reagent Resource. All cell lines were cultured in RPMI 1640 GlutaMax medium supplemented with 10% fetal bovine serum (FBS).

[0420] Cell binding assay. CD3+ and CD3- human Jurkat cells (ATCC) and cynomolgus macaque HSC-F cells (NIH) were thawed and washed with cold PBSF buffer pH 7.4 (PBS + 0.1% BSA, pH 7.4). Approximately 200,000 cells were aliquoted into each well of a 96-well plate (FACS assay plate, VWR BD 353263) and pelleted by centrifugation (500 x g for 5 minutes). The cells were washed with PBSF pH 7.4 or PBSF pH 6.0 (PBS + 0.1% BSA, pH 6.0) and then resuspended in 100 μl of PBSF pH 7.4 or PBSF pH 6.0 with the IgG antibody (100 nM) produced in yeast as described above. The mixture (cells + antibody) was incubated on ice for 20 minutes and then washed twice with PBSF pH 7.4 or PBSF pH 6.0. The cells were resuspended in 50 μl of propidium iodide (Roche; 1:500 dilution) and anti-human IgG-RPE (Southern Biotech; 1:100 dilution) prepared in PBSF pH 7.4 or PBSF pH 6.0 and then incubated on ice in the dark for 20 minutes, after which the cells were washed twice with PBSF pH 7.4 or PBSF pH 6.0. Binding was analyzed on a FACS Canto II.

[0421] PSR Preparation. Prepare the multispecific reactive reagent (PSR) as described, for example, in WO 2014 / 179363 and Xu et al., Protein Eng Des Sel, 26(10):663 - 670 (2013). Briefly, use 2.5 liters of CHO - S cells as the starting material. Centrifuge the cells at 2,400 x g for 5 minutes in 500 mL centrifuge bottles filled to 400 mL. Combine the cell pellets and then resuspend them in 25 ml of buffer B and centrifuge at 2,400 x g for 3 minutes. Decant the buffer and repeat the wash once. Resuspend the cell pellet in 3 x the pellet volume of buffer B containing 1x protease inhibitor (Roche, complete, without EDTA) using a polytron homogenizer, keeping the cells on ice. Then centrifuge the homogenate at 2,400 x g for 5 minutes and retain the supernatant and repellet it once (2,400 x g / 5 minutes) to ensure removal of unbroken cells, cell debris, and nuclei; the resulting supernatant is the total protein preparation. Then transfer the supernatant to two Nalgene Oak Ridge 45 mL centrifuge tubes and centrifuge at 40,000 x g for 40 minutes at 4°C. Then transfer the supernatant containing the soluble cytoplasmic protein (SCP) to a clean Oak Ridge tube and centrifuge again at 40,000 x g. In parallel, retain the pellet containing the membrane fraction (EMF) and centrifuge at 40,000 for 20 minutes to remove the residual supernatant. Then wash the EMF pellet with buffer B. Then add 8 mL of buffer B to the membrane pellet to dislodge the pellet and transfer it to a Dounce homogenizer. After homogenizing the pellet, transfer them to 50 mL conical tubes and represent the final EMF preparation.

[0422] Take about 10 6 - 10 71 billion mammalian cells (e.g., CHO, HEK293, Sf9) of each cell / mL are transferred to 4x250mL conical tubes from a tissue culture environment and precipitated at 550xg for 3 minutes. All subsequent steps are performed at 4°C or on ice with ice-cold buffer. The cells are washed with 100mL PBSF (1x PBS+1mg / mL BSA) and merged into a conical tube. After removing the supernatant, the cell pellet is then resuspended in 30mL buffer B (50mM HEPES, 0.15M NaCl, 2mM CaCl2, 5mM KCl, 5mM MgCl2, 10% glycerol, pH 7.2) and precipitated at 550x g for 3 minutes. The buffer B supernatant is decanted, and the cells are resuspended in the buffer B of 3x precipitation volume plus 2.5x protease inhibitors (Roche, completely, EDTA-free). The protease inhibitors in buffer B are all included from here onward. The cells were homogenized four times for 30 second pulses (Polyton homogenizer, PT1200E), and the membrane fraction was pelleted at 40,000 x g for 1 hour at 4°C. The pellet was rinsed with 1 mL of buffer B; the supernatant was retained and represented by s. The pellet was transferred to a Dounce homogenizer with 3 mL of buffer B and resuspended by slowly moving the pestle up and down for 30-35 strokes. The enriched membrane fraction (EMF) was moved 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 the Dc-Protein Assay Kit (BioRad). To solubilize EMF, transfer to solubilization buffer (50 mM HEPES, 0.15 M NaCl, 2 mM CaCl2, 5 mM KCl, 5 mM MgCl2, 1% n-dodecyl-bD-maltosylpyranoside (DDM), 1x protease inhibitor (pH 7.2) to a final concentration of 1 mg / mL. The mixture was rotated overnight at 4°C with rotation and then centrifuged at 40,000xg for 1 hour in a 50 mL Oak Ridge tube (Fisher Scientific, 050529-ID). The supernatant representing soluble membrane protein (SMP) was collected and the protein yield was quantified as described above.

[0423] For biotinylation, an NHS-LC-biotin stock solution was prepared according to the manufacturer's protocol (Pierce, Thermo Fisher). Briefly, 20 μl of biotin reagent was added per 1 mg of EMF sample and incubated at 4 °C for 3 h with gentle stirring. The volume was adjusted to 25 mL with buffer B and transferred to an Oak Ridge centrifuge tube. The biotinylated EMF (b-EMF) was precipitated at 40,000 x g for 1 h and washed twice with 3 mL of buffer C (buffer B minus glycerol) without disturbing the pellet. The residual solution was removed. The pellet was resuspended in 3 mL of buffer C using a Dounce homogenizer as previously described. The resuspended pellet now represents biotinylated EMF (b-EMF). It was dissolved as described above to prepare b-SMP.

[0424] PSR binding analysis. Determination is generally performed as described, for example, in: Xu et al., Protein Eng Des Sel, 26(10):663 - 670 (2013). To characterize the PSR profile of monoclonal antibodies presented on yeast, 2 million IgG - presenting yeast were transferred to a 96 - well assay plate and pelleted at 3000×g for 3 minutes to remove the supernatant. The pellet was resuspended in 50 μl of freshly prepared 1:10 diluted biotinylated PSR (b - PSR) stock solution and incubated on ice for 20 minutes. The cells were washed twice with 200 μl of cold PBSF and the pellet was resuspended in 50 μl of secondary labeling mixture (Extravidin - R - PE, anti - human LC - FITC, and propidium iodide). The mixture was incubated on ice for 20 minutes and then washed twice with 200 μl of ice - cold PBSF. The cells were resuspended in 100 μl of ice - cold PBSF and the plate was run on a FACSCanto (BD Biosciences) using an HTS sampler. The mean fluorescence intensity in the R - PE channel of the flow cytometry data was analyzed and normalized relative to an appropriate control (an antibody with an established PSR score) to assess non - specific binding. Many methods for presenting or displaying antibodies or antibody fragments on the yeast surface have been previously described, all of which are consistent with this protocol (Blaise et al., Gene, 342(2):211 - 8 (2004), Boder and Wittrup, Nat Biotechnol., 15(6):553 - 7 (1997), Kuroda and Ueda, Biotechnol Lett., 33(1):1 - 9 (2011), Orcutt and Wittrup, Springer Protocols. Antibody Engineering, 1:207 - 233 (2010), Rakestraw et al., Protein Eng Des Sel., 24(6):525 - 30 (2011), Sazinsky et al., Proc Natl Acad Sci USA., 105(51):20167 - 72 (2008), Tasumi et al., Proc Natl Acad Sci USA., 106(31):12891 - 6 (2009)). Multispecificity is evaluated based on the PSR score. The multispecificity of the antibody is considered as follows: when 0.0 ≤ PSR score < 0.10, it is none (no multispecificity); when 0.10 ≤ PSR score < 0.33, it is low; when 0.33 ≤ PSR score < 0.66, it is medium; when 0.66 ≤ PSR score ≤ 1.00, it is high.

[0425] HIC. The IgG1 sample buffer was exchanged into 1 M ammonium sulfate and 0.1 M sodium phosphate (pH 6.5) using a Zeba 40 kDa 0.5 mL spin column (Thermo Pierce, catalog number 87766). A salt gradient from 1.8 M ammonium sulfate, 0.1 M sodium phosphate (pH 6.5) to the same conditions without ammonium sulfate was established on a Dionex ProPac HIC-10 column. The gradient was run at a flow rate of 0.75 mL / min for 17 minutes. An acetonitrile wash step was added at the end of the run to remove any residual protein, and the column was re-equilibrated over 7 column volumes prior to the next injection cycle. Peak retention times were monitored at A280 absorbance, and the ammonium sulfate concentration at elution was calculated based on the gradient and flow rate. The hydrophobicity of the antibody was evaluated based on the HIC retention time. Hydrophobicity was considered as follows: none to low when the HIC retention time < 10.5 minutes; moderate when 10.5 minutes ≤ retention time < 11.5 minutes; and high when the retention time ≥ 11.5 minutes.

[0426] LC-MS. The IgG1 sample was reduced with DTT and then subjected to middledown 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 rate of 2 mL / min allowed separation of the sample and salts, elution of the sample, and regeneration of the column to be completed within a 2.1-minute cycle. A T-junction was used to deliver only 0.15 mL / min of the sample flow into the mass spectrometer for sample analysis. The Bruker maXis 4G mass spectrometer was operated in positive ion mode and detection was carried out in the range of 750 to 2500 m / z. The remaining source parameters were set as follows; the capillary was set to 5500 V, the nebulizer was set to 4.0 bar, the drying gas was set to 4.0 l / min, and the drying temperature was set to 200 °C. The MS spectra were analyzed using Bruker Data Analysis version 4.1, and deconvolution was completed using maximum entropy, with a mass range of 20 to 30 kDa. The tendency for heavy-chain / heavy-chain or heavy-chain / light-chain pairing failure was evaluated based on the LC-MS spectra. The presence of additional heavy-chain and / or light-chain peaks and / or half-antibody peaks indicated a tendency for pairing failure. LC-MS was also used to confirm whether the heavy-chain and light-chain masses matched the expected masses based on the amino acid sequences.

[0427] SEC. Agilent 1260 HPLC was used to monitor size exclusion chromatography (TSKgel Super SW mAb HTP column). Before use, the column was equilibrated with wash buffer (200 mM sodium phosphate, 250 mM sodium chloride at pH 6.8) at a flow rate adjusted to 0.400 mL / min. Approximately 2 - 5 μg of IgG1 or Fab protein sample was injected onto the column. Protein elution was monitored at a wavelength of 280 nm. The total assay time was approximately 6 minutes. Data were analyzed using ChemStation software. The tendency of antibody aggregation was evaluated based on the monomer % in the SEC chromatogram. Antibodies with a monomer % of 95% or higher were considered to be present essentially as monomers, i.e., not aggregated.

[0428] Acid stress tolerance. IgG1 samples were incubated at acidic or physiological pH and analyzed by SEC-HPLC. Briefly, 20 mg / mL IgG1 samples were buffer-exchanged into PBS (200 mM phosphate buffered with 250 mM sodium chloride, pH 7.0) or pH 3.5 buffer (50 mM sodium chloride, 200 mM acetic acid, pH 3.5). After 1 hour at room temperature (25 °C), the buffer-exchanged samples were diluted to 1 mg / mL in PBS (200 mM phosphate buffered with 250 mM sodium chloride at pH 7.0), and 2 μg of the sample was injected into an Agilent 1260 Infinity analytical HPLC (Agilent, Santa Clara, CA) equipped with a TSKgel SuperSW mAb HTP column (TOSOH Bioscience, King of Prussia, PA, product code 22855). SEC data were collected and analyzed using Agilent ChemStation software (Agilent, Santa Clara, CA). The tolerance of the antibody to acid stress was evaluated based on the tendency of the antibody to aggregate under acidic conditions, which was assessed by the monomer % in the SEC chromatogram.

[0429] AC-SINS. Measured in vitro by affinity capture self-interaction nanoparticle spectroscopy (AC-SINS) using the previously described protocol (Liu Y et al., MAbs. Mar-Apr 2014;6(2):483-92). Briefly, polyclonal goat anti-human IgG Fc antibodies (capture; Jackson ImmunoResearch Laboratories) and polyclonal goat non-specific antibodies (non-capture; Jackson ImmunoResearch Laboratories) were buffer-exchanged into 20 mM sodium acetate (pH 4.3) and concentrated to 0.4 mg / ml. A 4:1 volume ratio of capture:non-capture can be prepared and further incubated with 20 nm gold nanoparticles (AuNP; Ted Pella Inc.) at a 1:9 volume ratio for 1 hour at room temperature. Then thio-PEG (Sigma-Aldrich) was used to block the empty sites on the AuNP and filtered through a 0.22 μm PVDF membrane (Millipore). Subsequently, the coated particles were added to the test IgG1 antibody solution and incubated for 2 hours at room temperature, after which the absorbance from 510 nm to 570 nm was measured on a plate reader. The data points were fitted to a second-order polynomial in Excel to obtain the wavelength at the maximum absorbance. The value was reported as the difference (Δλmax) between the plasma wavelengths of the sample and the background. The self-interaction level was determined based on Δλmax. Self-interaction was considered as follows: low when Δλmax < 5.0 nm; medium when Δλmax ≥ 5.0 nm and < 20.0 nm; and high when Δλmax ≥ 20.0 nm.

[0430] DLS. Self-interaction was measured by dynamic light scattering (DLS). The diffusion interaction parameter (kD) of monoclonal antibodies measured at concentrations below 12 mg / mL has a strong correlation with its solution behavior at very high concentrations (>100 mg / mL). A positive kD value indicates repulsive interactions between molecules and has a positive correlation with low viscosity at high concentrations in the same formulation buffer. The kD value was obtained by measuring the mutual diffusion coefficient at a series of different concentrations by DLS. Specifically, DLS kD measurements were taken at various concentrations between 0.5 - 12 mg / mL in 10 mM histidine buffer at pH 6.0. A kD value < 20 mL / g was considered to be associated with high viscosity or high opalescence.

[0431] DSF. The melting temperature (Tm) was measured by differential scanning fluorimetry (DSF) using a Bio-Rad CFX96 real-time system. Briefly, 20 μL of a 1 mg / mL sample was mixed with 10 μL of 20× SYPRO Orange. The plate was scanned from 40 °C to 95 °C at a rate of 0.5 °C every 2 minutes in a C1000 thermal cycler (BioRad) to collect the Fret signal. The first derivative of the raw data from the Bio-Rad analysis software was used to assign the Fab Tm. Antibodies with a Tm higher than 65 °C were considered stable.

[0432] An informal sequence listing is provided in the appendix, which provides the amino acid sequences of VH, VL, CDR, and FR of the anti-CD3 antibodies analyzed in the examples, as well as the nucleic acid sequences encoding the VH and VL amino acid sequences.

[0433] Appendix

[0434] Table A: VH and VL Amino Acid Sequences

[0435]

[0436]

[0437]

[0438]

[0439]

[0440] Table B: VH and VL Nucleic Acid Sequences

[0441]

[0442]

[0443]

[0444]

[0445]

[0446]

[0447]

[0448]

[0449]

[0450]

[0451] Table CFR-H1 Amino Acid Sequence

[0452]

[0453] Table D: CDR-H1 Amino Acid Sequence

[0454] Antibody number ADI name Sequence SEQ ID NO: 1 ADI-26906 FNIKDYYMH 112 2 ADI-48587 FNIKDYYMH 212 3 ADI-74965 FNIKDYYMH 312 4 ADI-74966 FNIKDYYMH 412 5 ADI-74967 FNIKDYYMH 512 6 ADI-74968 FNIKDYYMH 612 7 ADI-79842 FNIKDYYMH 712 8 ADI-79843 FNIKDYYMH 812 9 ADI-79848 FNIKDYYMH 912 10 ADI-79844 FNIKDYYMH 1012 11 ADI-79845 FNIKDYYMH 1112 12 ADI-79846 FNIKDYYMH 1212 13 ADI-79847 FNIKDYYMH 1312

[0455] Bold residues indicate differences from the sequence encoded by the closest germline (VH1-03).

[0456] Table E: FR-H2 Amino Acid Sequence

[0457] Antibody number ADI name Sequence SEQ ID NO: 1 ADI-26906 WVRQAPGQRLEWMG 113 2 ADI-48587 WVRQAPGQRLEWMG 213 3 ADI-74965 WVRQAPGQRLEWMG 313 4 ADI-74966 WVRQAPGQRLEWMG 413 5 ADI-74967 WVRQAPGQRLEWMG 513 6 ADI-74968 WVRQAPGQRLEWMG 613 7 ADI-79842 WVRQAPGQRLEWMG 713 8 ADI-79843 WVRQAPGQRLEWMG 813 9 ADI-79848 WVRQAPGQRLEWMG 913 10 ADI-79844 WVRQAPGQRLEWMG 1013 11 ADI-79845 WVRQAPGQRLEWMG 1113 12 ADI-79846 WVRQAPGQRLEWMG 1213 13 ADI-79847 WVRQAPGQRLEWMG 1313

[0458] Table F: CDR-H2 Amino Acid Sequence

[0459] Antibody number ADI name Sequence SEQ ID NO: 1 ADI-26906 WIDLENANTIYDAKFQG 114 2 ADI-48587 WIDLENANTIYDAKFQG 214 3 ADI-74965 WIDLENANTIYDAKFQG 314 4 ADI-74966 WIDLENANTIYDAKFQG 414 5 ADI-74967 WIDLENANTIYDAKFQG 514 6 ADI-74968 WIDLENANTIYDAKFQG 614 7 ADI-79842 WIDLENANTIYDAKFQG 714 8 ADI-79843 WIDLENANTIYDAKFQG 814 9 ADI-79848 WIDLENANTIYDAKFQG 914 10 ADI-79844 WIDLENANTIYDAKFQG 1014 11 ADI-79845 WIDLENANTIYDAKFQG 1114 12 ADI-79846 WIDLENANTIYDAKFQG 1214 13 ADI-79847 WIDLENANTIYDAKFQG 1314

[0460] Bold residues indicate differences from the sequence encoded by the closest germline (VH1-03).

[0461] Table G: FR-H3 Amino Acid Sequence

[0462]

[0463] Bold residues indicate differences from the sequence encoded by the closest germline (VH1-03). Table H: CDR-H3 Amino Acid Sequence

[0464] Antibody Number ADI Name Sequence SEQ ID NO: 1 ADI-26906 ARDAYGRYFYDV 116 2 ADI-48587 ARDHYHRYFYDV 216 3 ADI-74965 ARDAYHRYFYDV 316 4 ADI-74966 ARDHYHRYFYDV 416 5 ADI-74967 ARDHYHRYFYDV 516 6 ADI-74968 ARDAYHRYFYDV 616 7 ADI-79842 ARDHYGRYFYDV 716 8 ADI-79843 ARDAYHRYFYDV 816 9 ADI-79848 ARDAYGRYFYDV 916 10 ADI-79844 ARDAYGRYFYDV 1016 11 ADI-79845 ARDAYGRYFYDV 1116 12 ADI-79846 ARDHYGRYFYDV 1216 13 ADI-79847 ARDHYHRYFYDV 1316

[0465] Italic residues indicate differences from the CDR-H3 sequence of ADI-26906.

[0466] Table I: FR-H4 Amino Acid Sequence

[0467] Antibody Number ADI Name Sequence SEQ ID NO: 1 ADI-26906 WGQGTLVTVSS 117 2 ADI-48587 WGQGTLVTVSS 217 3 ADI-74965 WGQGTLVTVSS 317 4 ADI-74966 WGQGTLVTVSS 417 5 ADI-74967 WGQGTLVTVSS 517 6 ADI-74968 WGQGTLVTVSS 617 7 ADI-79842 WGQGTLVTVSS 717 8 ADI-79843 WGQGTLVTVSS 817 9 ADI-79848 WGQGTLVTVSS 917 10 ADI-79844 WGQGTLVTVSS 1017 11 ADI-79845 WGQGTLVTVSS 1117 12 ADI-79846 WGQGTLVTVSS 1217 13 ADI-79847 WGQGTLVTVSS 1317

[0468] Table J: FR-L1 Amino Acid Sequence

[0469] Antibody Number ADI Name Sequence SEQ ID NO: 1 ADI-26906 DIVMTQSPDSLAVSLGERATINC 121 2 ADI-48587 DIVMTQSPDSLAVSLGERATINC 221 3 ADI-74965 DIVMTQSPDSLAVSLGERATINC 321 4 ADI-74966 DIVMTQSPDSLAVSLGERATINC 421 5 ADI-74967 GIVMTQSPDSLAVSLGERATINC 521 6 ADI-74968 DIVMTQSPDSLAVSLGERATINC 621 7 ADI-79842 DIVMTQSPDSLAVSLGERATINC 721 8 ADI-79843 DIVMTQSPDSLAVSLGERATINC 821 9 ADI-79848 DIVMTQSPDSLAVSLGERATINC 921 10 ADI-79844 DIVMTQSPDSLAVSLGERATINC 1021 11 ADI-79845 DIVMTQSPDSLAVSLGERATINC 1121 12 ADI-79846 DIVMTQSPDSLAVSLGERATINC 1221 13 ADI-79847 DIVMTQSPDSLAVSLGERATINC 1321

[0470] Bold residues indicate differences from the sequence encoded by the closest germline (VK4-01). Table K: CDR-L1 Amino Acid Sequence

[0471] Antibody Number ADI Name Sequence SEQ ID NO: 1 ADI-26906 KSSQSLLNARTGKNYLA 122 2 ADI-48587 KSSQSLLNARTGHNYLA 222 3 ADI-74965 KSSQSLLNARTGHNYLA 322 4 ADI-74966 KSSQSLLNARTGHNYLA 422 5 ADI-74967 KSSQSLLNARTGKNYLA 522 6 ADI-74968 KSSQSLLNARTGHNYLA 622 7 ADI-79842 KSSQSLLNARTGKNYLA 722 8 ADI-79843 KSSQSLLNARTGKNYLA 822 9 ADI-79848 KSSQSLLNARTGHNYLA 922 10 ADI-79844 KSSQSLLNARTGKNYLA 1022 11 ADI-79845 KSSQSLLNARTGKNYLA 1122 12 ADI-79846 KSSQSLLNARTGHNYLA 1222 13 ADI-79847 KSSQSLLNARTGHNYLA 1322

[0472] Bold residues indicate differences from the sequence encoded by the closest germline (VK4-01).

[0473] Italic residues indicate differences from the CDR-L1 sequence of ADI-26906.

[0474] Table L: FR-L2 Amino Acid Sequence

[0475] Antibody Number ADI Name Sequence SEQ ID NO: 1 ADI-26906 WYQQKPGQPPKLLIY 123 2 ADI-48587 WYQQKPGQPPKLLIY 223 3 ADI-74965 WYQQKPGQPPKLLIY 323 4 ADI-74966 WYQQKPGQPPKLLIY 423 5 ADI-74967 WYQQKPGQPPKLLIY 523 6 ADI-74968 WYQQKPGQPPKLLIY 623 7 ADI-79842 WYQQKPGQPPKLLIY 723 8 ADI-79843 WYQQKPGQPPKLLIY 823 9 ADI-79848 WYQQKPGQPPKLLIY 923 10 ADI-79844 WYQQKPGQPPKLLIY 1023 11 ADI-79845 WYQQKPGQPPKLLIY 1123 12 ADI-79846 WYQQKPGQPPKLLIY 1223 13 ADI-79847 WYQQKPGQPPKLLIY 1323

[0476] Table M: CDR-L2 Amino Acid Sequence

[0477] Antibody Number ADI Name Sequence SEQ ID NO: 1 ADI-26906 WASTRES 124 2 ADI-48587 WASTRES 224 3 ADI-74965 WASTRES 324 4 ADI-74966 WASTRES 424 5 ADI-74967 WASTRES 524 6 ADI-74968 WASTRES 624 7 ADI-79842 WASTRES 724 8 ADI-79843 WASTRES 824 9 ADI-79848 WASTRES 924 10 ADI-79844 WASTRES 1024 11 ADI-79845 WASTRES 1124 12 ADI-79846 WASTRES 1224 13 ADI-79847 WASTRES 1324

[0478] Table N: FR-L3 Amino Acid Sequence

[0479]

[0480] Table O: CDR-L3 Amino Acid Sequence

[0481] Antibody Number ADI Name Sequence SEQ ID NO: 1 ADI-26906 KQSYSRRT 126 2 ADI-48587 KQSHSHRT 226 3 ADI-74965 KQSHSHRT 326 4 ADI-74966 KQSYSHRT 426 5 ADI-74967 KQSHSHRT 526 6 ADI-74968 KQSYSHRT 626 7 ADI-79842 KQSYSRRT 726 8 ADI-79843 KQSYSRRT 826 9 ADI-79848 KQSYSRRT 926 10 ADI-79844 KQSHSRRT 1026 11 ADI-79845 KQSYSHRT 1126 12 ADI-79846 KQSHSHRT 1226 13 ADI-79847 KQSHSRRT 1326

[0482] Italic residues indicate differences from the CDR-L3 sequence of ADI-26906.

[0483] Table P: FR-L4 Amino Acid Sequence

[0484] Antibody Number ADI Name Sequence SEQ ID NO: 1 ADI-26906 FGGGTKVEIK 127 2 ADI-48587 FGGGTKVEIK 227 3 ADI-74965 FGGGTKVEIK 327 4 ADI-74966 FGGGTKVEIK 427 5 ADI-74967 FGGGTKVEIK 527 6 ADI-74968 FGGGTKVEIK 627 7 ADI-79842 FGGGTKVEIK 727 8 ADI-79843 FGGGTKVEIK 827 9 ADI-79848 FGGGTKVEIK 927 10 ADI-79844 FGGGTKVEIK 1027 11 ADI-79845 FGGGTKVEIK 1127 12 ADI-79846 FGGGTKVEIK 1227 13 ADI-79847 FGGGTKVEIK 1327

[0485] Table Q: Variable Region Amino Acid Sequences Common to Antibody Index Antibody Numbers 1 - 13

[0486]

[0487] Table R: SEQ ID NO Attributed to the Heavy Chain Variable Sequence

[0488]

[0489] Table S: SEQ ID NO Attributed to the Light Chain Variable Sequence

[0490]

[0491] Table T: Exemplary Constant Region Amino Acid Sequences

[0492]

[0493]

[0494] Table U: Human and Cynomolgus Macaque CD3ε Sequences

[0495]

Claims

1. An anti-cluster of differentiation 3 ("CD3") antibody or an antigen-binding fragment thereof, said anti-CD3 antibody or antigen-binding fragment thereof comprising: (A) A heavy chain variable domain (VH) polypeptide, said VH polypeptide comprising: (a) VH complementarity-determining region 1 (CDR-H1), said CDR-H1 comprising the amino acid sequence of: (i) The CDR-H1 contained in ADI-74968, ADI-74967, ADI-74966, ADI-74965, ADI-79842, ADI-79843, ADI-79848, ADI-79844, ADI-79845, ADI-79846 or ADI-79847; and / or (ii) FNIKDYYMH (SEQ ID NO:12, 612, 512, 412, 312, 712, 812, 912, 1012, 1112, 1212 or 1312); (b) VH complementarity-determining region 2 (CDR-H2), said CDR-H2 comprising the amino acid sequence of: (i) The CDR-H2 contained in ADI-74968, ADI-74967, ADI-74966, ADI-74965, ADI-79842, ADI-79843, ADI-79848, ADI-79844, ADI-79845, ADI-79846 or ADI-79847; and / or (ii) WIDLENANTIYDAKFQG (SEQ ID NO:14, 614, 514, 414, 314, 714, 814, 914, 1014, 1114, 1214 or 1314); and / or (c) VH complementarity-determining region 3 (CDR-H3), said CDR-H3 comprising the amino acid sequence of: (i) The CDR-H3 contained in ADI-74968, ADI-74967, ADI-74966, ADI-74965, ADI-79842, ADI-79843, ADI-79848, ADI-79844, ADI-79845, ADI-79846 or ADI-79847; (ii) ARDX2Y X3RYFYDV (SEQ ID NO:16), wherein X2 is A or H and X3 is H or G; and / or (iii) ARDAYHRYFYDV (SEQ ID NO:616, 316 or 816), ARDHYHRYFYDV (SEQ ID NO:516, 416 or 1316), ARDHYGRYFYDV (SEQ ID NO:716 or 1216) or ARDAYGRYFYDV (SEQ ID NO:916, 1016 or 1116); and / or (B) A light chain variable domain (VL) polypeptide, said VL polypeptide comprising: (a) VL Complementary Determining Region 1 (CDR-L1), said CDR-L1 comprising the following amino acid sequence: (i) the CDR-L1 contained in ADI-74968, ADI-74967, ADI-74966, ADI-74965, ADI-79842, ADI-79843, ADI-79848, ADI-79844, ADI-79845, ADI-79846 or ADI-79847; (ii) KSSQSLLNARTGX5NYLA (SEQ ID NO:22), where X5 is H or K; and / or (iii) KSSQSLLNARTGHNYLA (SEQ ID NO:622, 422, 322, 922, 1222 or 1322) or KSSQSLLNARTGKNYLA (SEQ ID NO:522, 722, 822, 1022 or 1122); (b) VL Complementary Determining Region 2 (CDR-L2), said CDR-L2 comprising the following amino acid sequence: (i) the CDR-L2 contained in ADI-74968, ADI-74967, ADI-74966, ADI-74965, ADI-79842, ADI-79843, ADI-79848, ADI-79844, ADI-79845, ADI-79846 or ADI-79847; and / or (ii) WASTRES (SEQ ID NO:24, 624, 524, 424, 324, 724, 824, 924, 1024, 1124, 1224 or 1324); and / or (c) VL Complementary Determining Region 3 (CDR-L3), said CDR-L3 comprising the following amino acid sequence: (i) the CDR-L3 contained in ADI-74968, ADI-74967, ADI-74966, ADI-74965, ADI-79842, ADI-79843, ADI-79848, ADI-79844, ADI-79845, ADI-79846 or ADI-79847; (ii) KQSX6SX7RT (SEQ ID NO:26), where X6 is Y or H and X7 is H or R; and / or (iii) KQSYSHRT (SEQ ID NO:626, 426 or 1126), KQSHSHRT (SEQ ID NO:526, 326 or 1226), KQSHSRRT (SEQ ID NO:1026 or 1326) or KQSYSRRT (SEQ ID NO:726, 826 or 926), Optionally, wherein said anti-CD3 antibody and / or antigen-binding fragment: (I) does not contain: (i) at least one of the CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 contained in ADI-48587; or (ii) at least one of the CDR-H1 containing FNIKDYYMH (SEQ ID NO:12 or 212), the CDR-H2 containing WIDLENANTIYDAKFQG (SEQ ID NO:14 or 214), the CDR-H3 containing ARDHYHRYFYDV (SEQ ID NO:216), the CDR-L1 containing KSSQSLLNARTGHNYLA (SEQ ID NO:222), the CDR-L2 containing WASTRES (SEQ ID NO:24 or 224), and the CDR-L3 containing KQSHSHRT (SEQ ID NO:226); and further optionally does not contain: (iii) at least one of the CDR-H3, CDR-L1, and CDR-L3 contained in ADI-48587; or (iv) at least one of the CDR-H3 containing ARDHYHRYFYDV (SEQ ID NO:216), the CDR-L1 containing KSSQSLLNARTGHNYLA (SEQ ID NO:222), and the CDR-L3 containing KQSHSHRT (SEQ ID NO:226); and (II) does not contain: (i) at least one of the CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 contained in ADI-26906; or (ii) at least one of the CDR-H1 containing FNIKDYYMH (SEQ ID NO:12 or 112), the CDR-H2 containing WIDLENANTIYDAKFQG (SEQ ID NO:14 or 114), the CDR-H3 containing ARDAYGRYFYDV (SEQ ID NO:116), the CDR-L1 containing KSSQSLLNARTGKNYLA (SEQ ID NO:122), the CDR-L2 containing WASTRES (SEQ ID NO:24 or 124), and the CDR-L3 containing KQSYSRRT (SEQ ID NO:126); and further optionally does not contain: (iii) the CDR-H3, CDR-L1, and CDR-L3 contained in ADI-26906; or (iv) the CDR-H3 containing ARDAYGRYFYDV (SEQ ID NO:116), the CDR-L1 containing KSSQSLLNARTGKNYLA (SEQ ID NO:122), and the CDR-L3 containing KQSYSRRT (SEQ ID NO:126).

2. The anti-CD3 antibody or antigen-binding fragment according to claim 1, wherein the anti-CD3 antibody or antigen-binding fragment comprises: (A) A VH polypeptide, wherein the VH polypeptide comprises: (a) CDR-H1, wherein the CDR-H1 comprises the following amino acid sequence: (i) The CDR-H1 contained in ADI-74968, ADI-74967, ADI-74966 or ADI-74965; and / or (ii) FNIKDYYMH (SEQ ID NO: 12, 612, 512, 412 or 312); (b) CDR-H2, wherein the CDR-H2 comprises the following amino acid sequence: (i) The CDR-H2 contained in ADI-74968, ADI-74967, ADI-74966 or ADI-74965; and / or (ii) WIDLENANTIYDAKFQG (SEQ ID NO: 14, 614, 514, 414 or 314); and / or (c) CDR-H3, wherein the CDR-H3 comprises the following amino acid sequence: (i) The CDR-H3 contained in ADI-74968, ADI-74967, ADI-74966 or ADI-74965; (ii) ARDX2Y X3RYFYDV (SEQ ID NO: 16), wherein X2 is A or H and X3 is H; and / or (iii) ARDAYHRYFYDV (SEQ ID NO: 616 or 316) or ARDHYHRYFYDV (SEQ ID NO: 516 or 416); and / or (B) A VL polypeptide, wherein the VL polypeptide comprises: (a) CDR-L1, wherein the CDR-L1 comprises the following amino acid sequence: (i) The CDR-L1 contained in ADI-74968, ADI-74967, ADI-74966 or ADI-74965; (ii) KSSQSLLNARTGX5NYLA (SEQ ID NO: 22), wherein X5 is H or K; and / or (iii) KSSQSLLNARTGHNYLA (SEQ ID NO: 622, 422 or 322) or KSSQSLLNARTGKNYLA (SEQ ID NO: 522); (b) CDR-L2, wherein the CDR-L2 comprises the following amino acid sequence: (i) The CDR-L2 contained in ADI-74968, ADI-74967, ADI-74966 or ADI-74965; and / or (ii) WASTRES (SEQ ID NO: 24, 624, 524, 424 or 324); and / or (c) CDR-L3, wherein the CDR-L3 comprises the following amino acid sequence: (i) The CDR-L3 contained in ADI-74968, ADI-74967, ADI-74966 or ADI-74965; (ii) KQSX6SX7RT (SEQ ID NO:26), where X6 is Y or H and X7 is H; and / or (iii) KQSYSHRT (SEQ ID NO:626 or 426) or KQSHSHRT (SEQ ID NO:526 or 326).

3. The anti-CD3 antibody or antigen-binding fragment according to any one of the preceding claims, wherein the anti-CD3 antibody or antigen-binding fragment comprises: (A) A VH polypeptide, the VH polypeptide comprising: (a) CDR-H1, the CDR-H1 comprising the following amino acid sequence: (i) the CDR-H1 contained in ADI-74968; and / or (ii) FNIKDYYMH (SEQ ID NO:12 or 612); (b) CDR-H2, the CDR-H2 comprising the following amino acid sequence: (i) the CDR-H2 contained in ADI-74968; and / or (ii) WIDLENANTIYDAKFQG (SEQ ID NO:14 or 614); and / or (c) CDR-H3, the CDR-H3 comprising the following amino acid sequence: (i) the CDR-H3 contained in ADI-74968; and / or (ii) ARDAYHRYFYDV (SEQ ID NO:616); and / or (B) A VL polypeptide, the VL polypeptide comprising: (a) CDR-L1, the CDR-L1 comprising the following amino acid sequence: (i) the CDR-L1 contained in ADI-74968; (ii) KSSQSLLNARTGHNYLA (SEQ ID NO:622); (b) CDR-L2, the CDR-L2 comprising the following amino acid sequence: (i) the CDR-L2 contained in ADI-74968; and / or (ii) WASTRES (SEQ ID NO:24 or 624); and / or (c) CDR-L3, the CDR-L3 comprising the following amino acid sequence: (i) the CDR-L3 contained in ADI-74968; (ii) KQSYSHRT (SEQ ID NO:626).

4. The anti-CD3 antibody or antigen-binding fragment according to any one of the preceding claims, wherein the anti-CD3 antibody or antigen-binding fragment comprises: (A) A VH polypeptide, the VH polypeptide comprising: (a) CDR-H1, the CDR-H1 comprising the following amino acid sequence: (i) the CDR-H1 contained in ADI-74967; and / or (ii) FNIKDYYMH (SEQ ID NO:12 or 512); (b) CDR-H2, the CDR-H2 comprising the following amino acid sequence: (i) the CDR-H2 contained in ADI-74967; and / or (ii) WIDLENANTIYDAKFQG (SEQ ID NO:14 or 514); and / or (c) CDR-H3, wherein the CDR-H3 comprises the following amino acid sequences: (i) the CDR-H3 contained in ADI-74967; (ii) ARDHYHRYFYDV (SEQ ID NO:516); and / or (B) A VL polypeptide, wherein the VL polypeptide comprises: (a) CDR-L1, wherein the CDR-L1 comprises the following amino acid sequences: (i) the CDR-L1 contained in ADI-74967; (ii) KSSQSLLNARTGKNYLA (SEQ ID NO:522); (b) CDR-L2, wherein the CDR-L2 comprises the following amino acid sequences: (i) the CDR-L2 contained in ADI-74967; and / or (ii) WASTRES (SEQ ID NO:24, 624, 524); and / or (c) CDR-L3, wherein the CDR-L3 comprises the following amino acid sequences: (i) the CDR-L3 contained in ADI-74967; (ii) KQSHSHRT (SEQ ID NO:526).

5. The anti-CD3 antibody or antigen-binding fragment according to any one of the preceding claims, wherein the anti-CD3 antibody or antigen-binding fragment comprises: (A) A VH polypeptide, wherein the VH polypeptide comprises the CDR-H1, the CDR-H2, and the CDR-H3; and / or (B) A VL polypeptide, wherein the VL polypeptide comprises the CDR-L1, the CDR-L2, and the CDR-L3.

6. The anti-CD3 antibody or antigen-binding fragment according to any one of the preceding claims, wherein the anti-CD3 antibody or antigen-binding fragment comprises: (A) A VH polypeptide, wherein the VH polypeptide comprises the CDR-H1, the CDR-H2, and the CDR-H3; and (B) A VL polypeptide, wherein the VL polypeptide comprises the CDR-L1, the CDR-L2, and the CDR-L3.

7. The anti-CD3 antibody or antigen-binding fragment according to any one of the preceding claims, wherein the anti-CD3 antibody or antigen-binding fragment comprises: (I) (i) the CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 contained in ADI-74968; or (ii) CDR-H1 comprising FNIKDYYMH (SEQ ID NO:12 or 612), CDR-H2 comprising WIDLENANTIYDAKFQG (SEQ ID NO:14 or 614), CDR-H3 comprising ARDAYHRYFYDV (SEQ ID NO:616), CDR-L1 comprising KSSQSLLNARTGHNYLA (SEQ ID NO:622), CDR-L2 comprising WASTRES (SEQ ID NO:24 or 624), and CDR-L3 comprising KQSYSHRT (SEQ ID NO:626); (II)(i) The CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 contained in ADI-74967; or (ii) CDR-H1 containing FNIKDYYMH (SEQ ID NO:12 or 512), CDR-H2 containing WIDLENANTIYDAKFQG (SEQ ID NO:14 or 514), CDR-H3 containing ARDHYHRYFYDV (SEQ ID NO:516), CDR-L1 containing KSSQSLLNARTGKNYLA (SEQ ID NO:522), CDR-L2 containing WASTRES (SEQ ID NO:24 or 524), and CDR-L3 containing KQSHSHRT (SEQ ID NO:526); (III)(i) The CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 contained in ADI-74966; or (ii) CDR-H1 containing FNIKDYYMH (SEQ ID NO:12 or 412), CDR-H2 containing WIDLENANTIYDAKFQG (SEQ ID NO:14 or 414), CDR-H3 containing ARDHYHRYFYDV (SEQ ID NO:416), CDR-L1 containing KSSQSLLNARTGHNYLA (SEQ ID NO:422), CDR-L2 containing WASTRES (SEQ ID NO:24 or 424), and CDR-L3 containing KQSYSHRT (SEQ ID NO:426); (IV)(i) The CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 contained in ADI-74965; or (ii) CDR-H1 containing FNIKDYYMH (SEQ ID NO:12 or 312), CDR-H2 containing WIDLENANTIYDAKFQG (SEQ ID NO:14 or 314), CDR-H3 containing ARDAYHRYFYDV (SEQ ID NO:316), CDR-L1 containing KSSQSLLNARTGHNYLA (SEQ ID NO:322), CDR-L2 containing WASTRES (SEQ ID NO:24 or 324), and CDR-L3 containing KQSHSHRT (SEQ ID NO:326); (V)(i) The CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 contained in ADI-79842; or (ii) CDR-H1 containing FNIKDYYMH (SEQ ID NO:12 or 712), CDR-H2 containing WIDLENANTIYDAKFQG (SEQ ID NO:14 or 714), CDR-H3 containing ARDHYGRYFYDV (SEQ ID NO:716), CDR-L1 containing KSSQSLLNARTGKNYLA (SEQ ID NO:722), CDR-L2 containing WASTRES (SEQ ID NO:24 or 724), and CDR-L3 containing KQSYSRRT (SEQ ID NO:726); (VI) (i) The CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 contained in ADI-79843; or (ii) CDR-H1 containing FNIKDYYMH (SEQ ID NO:12 or 812), CDR-H2 containing WIDLENANTIYDAKFQG (SEQ ID NO:14 or 814), CDR-H3 containing ARDAYHRYFYDV (SEQ ID NO:816), CDR-L1 containing KSSQSLLNARTGKNYLA (SEQ ID NO:822), CDR-L2 containing WASTRES (SEQ ID NO:24 or 824), and CDR-L3 containing KQSYSRRT (SEQ ID NO:826); (VII) (i) The CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 contained in ADI-79848; or (ii) CDR-H1 containing FNIKDYYMH (SEQ ID NO:12 or 912), CDR-H2 containing WIDLENANTIYDAKFQG (SEQ ID NO:14 or 914), CDR-H3 containing ARDAYGRYFYDV (SEQ ID NO:916), CDR-L1 containing KSSQSLLNARTGHNYLA (SEQ ID NO:922), CDR-L2 containing WASTRES (SEQ ID NO:24 or 924), and CDR-L3 containing KQSYSRRT (SEQ ID NO:926); (VIII) (i) The CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 contained in ADI-79844; or (ii) CDR-H1 comprising FNIKDYYMH (SEQ ID NO:12 or 1012), CDR-H2 comprising WIDLENANTIYDAKFQG (SEQ ID NO:14 or 1014), CDR-H3 comprising ARDAYGRYFYDV (SEQ ID NO:1016), CDR-L1 comprising KSSQSLLNARTGKNYLA (SEQ ID NO:1022), CDR-L2 comprising WASTRES (SEQ ID NO:24 or 1024), and CDR-L3 comprising KQSHSRRT (SEQ ID NO:1026); (IX)(i) said CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 contained in ADI-79845; or (ii) CDR-H1 comprising FNIKDYYMH (SEQ ID NO:12 or 1112), CDR-H2 comprising WIDLENANTIYDAKFQG (SEQ ID NO:14 or 1114), CDR-H3 comprising ARDAYGRYFYDV (SEQ ID NO:1116), CDR-L1 comprising KSSQSLLNARTGKNYLA (SEQ ID NO:1122), CDR-L2 comprising WASTRES (SEQ ID NO:24 or 1124), and CDR-L3 comprising KQSYSHRT (SEQ ID NO:1126); (X)(i) said CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 contained in ADI-79846; or (ii) CDR-H1 comprising FNIKDYYMH (SEQ ID NO:12 or 1212), CDR-H2 comprising WIDLENANTIYDAKFQG (SEQ ID NO:14 or 1214), CDR-H3 comprising ARDHYGRYFYDV (SEQ ID NO:1216), CDR-L1 comprising KSSQSLLNARTGHNYLA (SEQ ID NO:1222), CDR-L2 comprising WASTRES (SEQ ID NO:24 or 1224), and CDR-L3 comprising KQSHSHRT (SEQ ID NO:1226); (XI)(i) said CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 contained in ADI-79846; or (ii) CDR-H1 comprising FNIKDYYMH (SEQ ID NO: 12 or 1312), CDR-H2 comprising WIDLENANTIYDAKFQG (SEQ ID NO: 14 or 1314), CDR-H3 comprising ARDHYHRYFYDV (SEQ ID NO: 1316), CDR-L1 comprising KSSQSLLNARTGHNYLA (SEQ ID NO: 1322), CDR-L2 comprising WASTRES (SEQ ID NO: 24 or 1324), and CDR-L3 comprising KQSHSRRT (SEQ ID NO: 1326).

8. The anti-CD3 antibody or antigen-binding fragment according to any one of the preceding claims, wherein: (A) The VH polypeptide comprises: (a) VH framework region 1 (FR-H1), and the FR-H1 comprises the following amino acid sequence: (i) The FR-H1 contained in ADI-74968, ADI-74967, ADI-74966, ADI-74965, ADI-79842, ADI-79843, ADI-79848, ADI-79844, ADI-79845, ADI-79846 or ADI-79847; and / or (ii) QVQLVQSGAEVKKPGASVKVSCKASG (SEQ ID NO: 11, 611, 511, 411, 311, 711, 811, 911, 1011, 1111, 1211 or 1311); (b) VH framework region 2 (FR-H2), and the FR-H2 comprises the following amino acid sequence: (i) The FR-H2 contained in ADI-74968, ADI-74967, ADI-74966, ADI-74965, ADI-79842, ADI-79843, ADI-79848, ADI-79844, ADI-79845, ADI-79846 or ADI-79847; and / or (ii) WVRQAPGQRLEWMG (SEQ ID NO: 13, 613, 513, 413, 313, 713, 813, 913, 1013, 1113, 1213 or 1313); (c) VH framework region 3 (FR-H3), and the FR-H3 comprises the following amino acid sequence: (i) The FR-H3 contained in ADI-74968, ADI-74967, ADI-74966, ADI-74965, ADI-79842, ADI-79843, ADI-79848, ADI-79844, ADI-79845, ADI-79846 or ADI-79847; (ii) RVTITRDTSASTAYMX1LSSLRSEDTAVYYC (SEQ ID NO: 15), where X1 is E or G; and / or (iii) RVTITRDTSASTAYMELSSLRSEDTAVYYC (SEQ ID NO: 615, 515, 415, 315, 715, 815, 915, 1015, 1115 or 1315) or RVTITRDTSASTAYMGLSSLRSEDTAVYYC (SEQ ID NO: 1215); and / or (d) VH framework region 4 (FR-H4), the FR-H4 comprising the following amino acid sequence: (i) the FR-H4 contained in ADI-74968, ADI-74967, ADI-74966, ADI-74965, ADI-79842, ADI-79843, ADI-79848, ADI-79844, ADI-79845, ADI-79846 or ADI-79847; and / or (ii) WGQGTLVTVSS (SEQ ID NO: 17, 617, 517, 417, 317, 717, 817, 917, 1017, 1117, 1217 or 1317); and / or (B) the VL polypeptide comprises: (a) VL framework region 1 (FR-L1), the FR-L1 comprising the following amino acid sequence: (i) the FR-L1 contained in ADI-74968, ADI-74967, ADI-74966, ADI-74965, ADI-79842, ADI-79843, ADI-79848, ADI-79844, ADI-79845, ADI-79846 or ADI-79847; (ii) X4IVMTQSPDSLAVSLGERATINC (SEQ ID NO: 21), where X4 is D or G; and / or (iii) DIVMTQSPDSLAVSLGERATINC (SEQ ID NO: 621, 421, 321, 721, 821, 921, 1021, 1121, 1221 or 1321) or GIVMTQSPDSLAVSLGERATINC (SEQ ID NO: 521); (b) VL framework region 2 (FR-L2), the FR-L2 comprising the following amino acid sequence: (i) the FR-L2 contained in ADI-74968, ADI-74967, ADI-74966, ADI-74965, ADI-79842, ADI-79843, ADI-79848, ADI-79844, ADI-79845, ADI-79846 or ADI-79847; and / or (ii) WYQQKPGQPPKLLIY (SEQ ID NO: 23, 623, 523, 423, 323, 723, 823, 923, 1023, 1123, 1223 or 1323); (c) The VL framework region 3 (FR-L3), the FR-L3 comprising the following amino acid sequence: (i) The FR-L3 contained in ADI-74968, ADI-74967, ADI-74966, ADI-74965, ADI-79842, ADI-79843, ADI-79848, ADI-79844, ADI-79845, ADI-79846 or ADI-79847; and / or (ii) GVPDRFSGSGSGTDFTLTISSLQAEDVAVYYC (SEQ ID NO: 25, 625, 525, 425, 325, 725, 825, 925, 1025, 1125, 1225 or 1325); and / or (d) The VL framework region 4 (FR-L4), the FR-L4 comprising the following amino acid sequence: (i) The FR-L4 contained in ADI-74968, ADI-74967, ADI-74966, ADI-74965, ADI-79842, ADI-79843, ADI-79848, ADI-79844, ADI-79845, ADI-79846 or ADI-79847; and / or (ii) FGGGTKVEIK (SEQ ID NO: 27, 627, 527, 427, 327, 727, 827, 927, 1027, 1127, 1227 or 1327), or the anti-CD3 antibody or antigen-binding fragment comprises a VH and / or VL comprising any combination of the foregoing VH and VL framework regions.

9. The anti-CD3 antibody or antigen-binding fragment according to any one of the preceding claims, the anti-CD3 antibody or antigen-binding fragment comprising: (I)(i) The FR-H1, FR-H2, FR-H3, FLR-H4, FR-L1, FR-L2, FR-L3 and FR-L4 contained in ADI-74968, ADI-74966, ADI-74965, ADI-79842, ADI-79843, ADI-79848, ADI-79844, ADI-79845 or ADI-79847; or (ii) FR-H1 containing QVQLVQSGAEVKKPGASVKVSCKASG (SEQ ID NO: 11, 611, 411, 311, 711, 811, 911, 1011, 1111 or 1311), FR-H2 containing WVRQAPGQRLEWMG (SEQ ID NO: 13, 613, 413, 313, 713, 813, 913, 1013, 1113 or 1313), FR-H3 containing RVTITRDTSASTAYMELSSLRSEDTAVYYC (SEQ ID NO: 615, 415, 315, 715, 815, 915, 1015, 1115 or 1315), FR-H4 containing WGQGTLVTVSS (SEQ ID NO: 17, 617, 417, 317, 717, 817, 917, 1017, 1117 or 1317), FR-L1 containing DIVMTQSPDSLAVSLGERATINC (SEQ ID NO: 621, 421, 321, 721, 821, 921, 1021, 1121 or 1321), FR-L2 containing WYQQKPGQPPKLLIY (SEQ ID NO: 23, 623, 423, 323, 723, 823, 923, 1023, 1123 or 1323), FR-L3 containing GVPDRFSGSGSGTDFTLTISSLQAEDVAVYYC (SEQ ID NO: 25, 625, 425, 325, 725, 825, 925, 1025, 1125 or 1325) and FR-L4 containing FGGGTKVEIK (SEQ ID NO: 27, 627, 427, 327, 727, 827, 927, 1027, 1127 or 1327); (II) (i) the said FR-H1, FR-H2, FR-H3, FLR-H4, FR-L1, FR-L2, FR-L3 and FR-L4 contained in ADI-74967; or (ii) FR-H1 comprising QVQLVQSGAEVKKPGASVKVSCKASG (SEQ ID NO: 11 or 511), FR-H2 comprising WVRQAPGQRLEWMG (SEQ ID NO: 13 or 513), FR-H3 comprising RVTITRDTSASTAYMELSSLRSEDTAVYYC (SEQ ID NO: 515), FR-H4 comprising WGQGTLVTVSS (SEQ ID NO: 17 or 517), FR-L1 comprising GIVMTQSPDSLAVSLGERATINC (SEQ ID NO: 521), FR-L2 comprising WYQQKPGQPPKLLIY (SEQ ID NO: 23 or 523), FR-L3 comprising GVPDRFSGSGSGTDFTLTISSLQAEDVAVYYC (SEQ ID NO: 25 or 525), and FR-L4 comprising FGGGTKVEIK (SEQ ID NO: 27 or 527); or (III) (i) said FR-H1, FR-H2, FR-H3, FLR-H4, FR-L1, FR-L2, FR-L3, and FR-L4 contained in ADI-74968, ADI-74966, ADI-74965, ADI-79842, ADI-79843, ADI-79848, ADI-79844, ADI-79845, or ADI-79847; or (ii) FR-H1 comprising QVQLVQSGAEVKKPGASVKVSCKASG (SEQ ID NO: 11 or 1211), FR-H2 comprising WVRQAPGQRLEWMG (SEQ ID NO: 13 or 1213), FR-H3 comprising RVTITRDTSASTAYMGLSSLRSEDTAVYYC (SEQ ID NO: 1215), FR-H4 comprising WGQGTLVTVSS (SEQ ID NO: 17 or 1217), FR-L1 comprising DIVMTQSPDSLAVSLGERATINC (SEQ ID NO: 1221), FR-L2 comprising WYQQKPGQPPKLLIY (SEQ ID NO: 1223), FR-L3 comprising GVPDRFSGSGSGTDFTLTISSLQAEDVAVYYC (SEQ ID NO: 1225), and FR-L4 comprising FGGGTKVEIK (SEQ ID NO: 1227).

10. The anti-CD3 antibody or antigen-binding fragment according to any one of the preceding claims, wherein the anti-CD3 antibody or antigen-binding fragment comprises: (I)(i) The FR-H1, CDR-H1, FR-H2, CDR-H2, FR-H3, CDR-H3, FR-H4, FR-L1, CDR-L1, FR-L2, CDR-L2, FR-L3, CDR-L3, and FR-L4 contained in ADI-74968; or (ii) FR-H1, CDR-H1, FR-H2, CDR-H2, FR-H3, CDR-H3, FR-H4, FR-L1, CDR-L1, FR-L2, CDR-L2, FR-L3, CDR-L3, and FR-L4 respectively comprising SEQ ID NO: 611, 612, 613, 614, 615, 616, 617, 621, 622, 623, 624, 625, 626, and 627; (II)(i) The FR-H1, CDR-H1, FR-H2, CDR-H2, FR-H3, CDR-H3, FR-H4, FR-L1, CDR-L1, FR-L2, CDR-L2, FR-L3, CDR-L3, and FR-L4 contained in ADI-74967; or (ii) FR-H1, CDR-H1, FR-H2, CDR-H2, FR-H3, CDR-H3, FR-H4, FR-L1, CDR-L1, FR-L2, CDR-L2, FR-L3, CDR-L3, and FR-L4 respectively comprising SEQ ID NO: 511, 512, 513, 514, 515, 516, 517, 521, 522, 523, 524, 525, 526, and 527; (III)(i) The FR-H1, CDR-H1, FR-H2, CDR-H2, FR-H3, CDR-H3, FR-H4, FR-L1, CDR-L1, FR-L2, CDR-L2, FR-L3, CDR-L3, and FR-L4 contained in ADI-74966; or (ii) FR-H1, CDR-H1, FR-H2, CDR-H2, FR-H3, CDR-H3, FR-H4, FR-L1, CDR-L1, FR-L2, CDR-L2, FR-L3, CDR-L3, and FR-L4 respectively comprising SEQ ID NO: 411, 412, 413, 414, 415, 416, 417, 421, 422, 423, 424, 425, 426, and 427; (IV)(i) The FR-H1, CDR-H1, FR-H2, CDR-H2, FR-H3, CDR-H3, FR-H4, FR-L1, CDR-L1, FR-L2, CDR-L2, FR-L3, CDR-L3, and FR-L4 contained in ADI-74965; or (ii) FR-H1, CDR-H1, FR-H2, CDR-H2, FR-H3, CDR-H3, FR-H4, FR-L1, CDR-L1, FR-L2, CDR-L2, FR-L3, CDR-L3, and FR-L4 respectively containing SEQ ID NO: 311, 312, 313, 314, 315, 316, 317, 321, 322, 323, 324, 325, 326, and 327; (V)(i) The FR-H1, CDR-H1, FR-H2, CDR-H2, FR-H3, CDR-H3, FR-H4, FR-L1, CDR-L1, FR-L2, CDR-L2, FR-L3, CDR-L3, and FR-L4 contained in ADI-79842; or (ii) FR-H1, CDR-H1, FR-H2, CDR-H2, FR-H3, CDR-H3, FR-H4, FR-L1, CDR-L1, FR-L2, CDR-L2, FR-L3, CDR-L3, and FR-L4 respectively containing SEQ ID NO: 711, 712, 713, 714, 715, 716, 717, 721, 722, 723, 724, 725, 726, and 727; (VI)(i) The FR-H1, CDR-H1, FR-H2, CDR-H2, FR-H3, CDR-H3, FR-H4, FR-L1, CDR-L1, FR-L2, CDR-L2, FR-L3, CDR-L3, and FR-L4 contained in ADI-79843; or (ii) FR-H1, CDR-H1, FR-H2, CDR-H2, FR-H3, CDR-H3, FR-H4, FR-L1, CDR-L1, FR-L2, CDR-L2, FR-L3, CDR-L3, and FR-L4 respectively containing SEQ ID NO: 811, 812, 813, 814, 815, 816, 817, 821, 822, 823, 824, 825, 826, and 827; (VII)(i) The FR-H1, CDR-H1, FR-H2, CDR-H2, FR-H3, CDR-H3, FR-H4, FR-L1, CDR-L1, FR-L2, CDR-L2, FR-L3, CDR-L3, and FR-L4 contained in ADI-79848; or (ii) FR-H1, CDR-H1, FR-H2, CDR-H2, FR-H3, CDR-H3, FR-H4, FR-L1, CDR-L1, FR-L2, CDR-L2, FR-L3, CDR-L3, and FR-L4 respectively containing SEQ ID NO: 911, 912, 913, 914, 915, 916, 917, 921, 922, 923, 924, 925, 926, and 927; (VIII)(i) The FR-H1, CDR-H1, FR-H2, CDR-H2, FR-H3, CDR-H3, FR-H4, FR-L1, CDR-L1, FR-L2, CDR-L2, FR-L3, CDR-L3, and FR-L4 contained in ADI-79844; or (ii) FR-H1, CDR-H1, FR-H2, CDR-H2, FR-H3, CDR-H3, FR-H4, FR-L1, CDR-L1, FR-L2, CDR-L2, FR-L3, CDR-L3, and FR-L4 respectively comprising SEQ ID NO: 1011, 1012, 1013, 1014, 1015, 1016, 1017, 1021, 1022, 1023, 1024, 1025, 1026, and 1027; (IX)(i) The FR-H1, CDR-H1, FR-H2, CDR-H2, FR-H3, CDR-H3, FR-H4, FR-L1, CDR-L1, FR-L2, CDR-L2, FR-L3, CDR-L3, and FR-L4 contained in ADI-79845; or (ii) FR-H1, CDR-H1, FR-H2, CDR-H2, FR-H3, CDR-H3, FR-H4, FR-L1, CDR-L1, FR-L2, CDR-L2, FR-L3, CDR-L3, and FR-L4 respectively comprising SEQ ID NO: 1111, 1112, 1113, 1114, 1115, 1116, 1117, 1121, 1122, 1123, 1124, 1125, 1126, and 1127; (X)(i) The FR-H1, CDR-H1, FR-H2, CDR-H2, FR-H3, CDR-H3, FR-H4, FR-L1, CDR-L1, FR-L2, CDR-L2, FR-L3, CDR-L3, and FR-L4 contained in ADI-79846; or (ii) FR-H1, CDR-H1, FR-H2, CDR-H2, FR-H3, CDR-H3, FR-H4, FR-L1, CDR-L1, FR-L2, CDR-L2, FR-L3, CDR-L3, and FR-L4 respectively comprising SEQ ID NO: 1211, 1212, 1213, 1214, 1215, 1216, 1217, 1221, 1222, 1223, 1224, 1225, 1226, and 1227; or (XI)(i) The FR-H1, CDR-H1, FR-H2, CDR-H2, FR-H3, CDR-H3, FR-H4, FR-L1, CDR-L1, FR-L2, CDR-L2, FR-L3, CDR-L3, and FR-L4 contained in ADI-79847; or (ii) FR-H1, CDR-H1, FR-H2, CDR-H2, FR-H3, CDR-H3, FR-H4, FR-L1, CDR-L1, FR-L2, CDR-L2, FR-L3, CDR-L3 and FR-L4, which respectively contain SEQ ID NO:1311, 1312, 1313, 1314, 1315, 1316, 1317, 1321, 1322, 1323, 1324, 1325, 1326 and 1327.

11. An anti-CD3 antibody or antigen-binding fragment according to any one of the preceding claims, wherein the anti-CD3 antibody or antigen-binding fragment comprises: (A) a VH polypeptide comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO:610, 510, 410, 310, 710, 810, 910, 1010, 1110, 1210 or 1310; and / or (B) a VL polypeptide comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO:620, 520, 420, 320, 720, 820, 920, 1020, 1120, 1220 or 1320, optionally wherein the anti-CD3 antibody or antigen-binding fragment comprises: (I) (A) a VH polypeptide comprising (i) the CDR-H1, CDR-H2 and CFR-H3 sequences of SEQ ID NO:612, 614 and 616 respectively, and (ii) an amino acid sequence that is at least 90% identical to SEQ ID NO:610; and (B) a VL polypeptide comprising (i) the CDR-L1, CDR-L2 and CFR-L3 sequences of SEQ ID NO:622, 624 and 626 respectively; and (ii) an amino acid sequence that is at least 90% identical to SEQ ID NO:620; or (II) (A) a VH polypeptide comprising (i) the CDR-H1, CDR-H2 and CFR-H3 sequences of SEQ ID NO:512, 514 and 516 respectively, and (ii) an amino acid sequence that is at least 90% identical to SEQ ID NO:510; and (B) a VL polypeptide comprising (i) the CDR-L1, CDR-L2 and CFR-L3 sequences of SEQ ID NO:522, 524 and 526 respectively; and (ii) an amino acid sequence that is at least 90% identical to SEQ ID NO:

520.

12. The anti-CD3 antibody or antigen-binding fragment according to any one of the preceding claims, wherein the anti-CD3 antibody or antigen-binding fragment comprises VH and VL polypeptides comprising the amino acid sequences: (I) SEQ ID NO: 610 and 620, respectively; (II) SEQ ID NO: 510 and 520, respectively; (III) SEQ ID NO: 410 and 420, respectively; (IV) SEQ ID NO: 310 and 320, respectively; (V) SEQ ID NO: 710 and 720, respectively; (VI) SEQ ID NO: 810 and 820, respectively; (VII) SEQ ID NO: 910 and 920, respectively; (VIII) SEQ ID NO: 1010 and 1020, respectively; (IX) SEQ ID NO: 1110 and 1120, respectively; (X) SEQ ID NO: 1210 and 1220, respectively; or (XI) SEQ ID NO: 1310 and 1320, respectively.

13. The anti-CD3 antibody or antigen-binding fragment according to any one of the preceding claims, wherein the anti-CD3 antibody or antigen-binding fragment comprises one or more of the following: (i) an antibody constant region, CH1 domain, hinge, CH2 domain, and / or CH3 domain, which are optionally of or derived from IgG or human IgG, further optionally of or derived from human IgG1, IgG4, IgG2, or IgG3; (ii) a crystallizable fragment (Fc) region, which is optionally of: (1) human IgG1, which further optionally comprises one or more of the following amino acid modifications: N297A, N297Q, D265A, L234A, L235A, C226S, C229S, P238S, E233P, L234V, G236-deletion, P238A, A327Q, A327G, P329A, K322A, L234F, L235E, P331S, T394D, A330L, P331S, F243L, R292P, Y300L, V305I, P396L, S239D, I332E, S298A, E333A, K334A, L234Y, L235Q, G236W, S239M, H268D, D270E, K326D, A330M, K334E, G236A, K326W, S239D, E333S, S267E, H268F, S324T, E345R, E430G, S440Y M428L, N434S, L328F, M252Y, S254T, T256E or any combination thereof according to EU numbering; (2) Human IgG4, said human IgG4 further optionally comprising one or more of the following amino acid modifications: E233P, F234V, L235A, G237A, E318A, S228P, L236E, S241P, L248E, T394D, M252Y, S254T, T256E, N297A, N297Q according to EU numbering, or any combination thereof; (3) Human IgG2, said human IgG2 further optionally comprising one or more of the following amino acid modifications: P238S, V234A, G237A, H268A, H268Q, H268E, V309L, N297A, N297Q, A330S, P331S, C232S, C233S, M252Y, S254T, T256E according to EU numbering, or any combination thereof; and / or (4) Human IgG3, said human IgG3 further optionally comprising E235Y according to EU numbering; (iii) IgG, IgA, IgE, IgD or IgM, optionally IgG1, IgG4, IgG2 or IgG3; and / or (iv) Antibody fragments selected from: antigen-binding fragment (Fab); Fab2; Fab3; Fab' fragment; F(ab′)2; variable fragment (Fv); single-chain Fv (scFv) fragment; bispecific antibody; trispecific antibody; minibody; scFv-Fc; scFv2-Fc2; scFv-IgG; monovalent IgG (or semi-IgG); and / or chimeric antigen receptor (CAR), said CAR comprising an antigen-binding region comprising said VH polypeptide and / or said VL polypeptide, a transmembrane domain and at least one intracellular signaling domain (optionally derived from a T cell receptor, further optionally CD3ζ).

14. The anti-CD3 antibody or antigen-binding fragment according to any one of the preceding claims, said anti-CD3 antibody or antigen-binding fragment comprising a multispecific antibody or antibody fragment or being comprised in a multispecific antibody or antibody fragment, said multispecific antibody or antibody fragment having (a) a first antigen-binding region specific for CD3, said first antigen-binding region comprising said VH polypeptide and / or said VL polypeptide, and (b) one or more other antigen-binding regions, said other antigen-binding regions optionally comprising one or more of the following features: (i) The one or more other antigen-binding regions include antigen-binding regions specific for: oncology targets, target molecules expressed on cancer cells, immuno-oncology targets, target molecules expressed on immune cells, autoimmune disorder targets (optionally, autoreactive immune molecules or target molecules expressed on immune cells expressing autoreactive immune molecules), inflammatory disease targets (optionally, inflammatory cytokines or chemokines or their receptors), neurodegenerative disease targets, infectious disease targets (optionally, target molecules of viruses, bacteria or fungi), target molecules expressed on infected cells (optionally, infected with viruses, bacteria or fungi), metabolic disease targets, cognitive disorder targets, blood-brain barrier targets or blood disease targets; (ii) The one or more other antigen-binding regions include antigen-binding regions specific for a second antigen selected from: 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, ADAM 15, ADAM 17 / T ACE, ADAM8, ADAM9, ADAMTS, ADAMTS4, ADAMTS5, addressin, aFGF, ALCAM, ALK, ALK-1, ALK-7, α-1-antitrypsin, α-V / β-1 antagonist, ANG, Ang, APAF-1, APE, APJ, APP, APRIL, AR, ARC, ART, amphiregulin, anti-Id, ASPARTIC, atrial natriuretic factor, av / b3 integrin, Axl, b2M, B7-1, B7-2, B7-H, B-lymphocyte stimulator (BlyS), BACE, BACE-1, Bad, BAFF, BAFF-R, Bag-1, BAK, Bax, BCA-1, BCAM, Bel, BCMA, BDNF, b-ECGF, bFGF, BID, Bik, BFM, BLC, BL-CAM, BLK, BMP, BMP-2, BMP-2a, 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, b-NGF, BOK, bombesin, bone-derived neurotrophic factor, BPDE, BPDE-DNA, BTC, complement factor 3 (C3), C3a, C4, C5, C5a, C10, CA125, CAD-8, calcitonin, cAMP, carcinoembryonic antigen (CEA), cancer-associated antigen (CAA), cathepsin A, cathepsin B, cathepsin C / DPPI, cathepsin D, cathepsin E, cathepsin H, cathepsin L, cathepsin O, cathepsin S, cathepsin V, cathepsin X / Z / P, CBL, CCI, CCK2, CCL, CCL1, CCL11, CCL12, CCL13, CCL 14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL2, CCL20, CCL21, CCL22, CCL23, CCL24,CCL25, CCL26, CCL27, CCL28, CCL3, CCL4, CCL5, CCL6, CCL7, CCL8, CCL9 / 10, CCR, CCR1, CCR10, CCR10, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CD1, CD2, CD4, CD5, CD6, CD7, CD8, CD10, CD11a, CD1b, CD11c, 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, Clostridium botulinum toxin, Clostridium perfringens 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, 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, Eosinophil chemotactic factor 1, 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-α1, GFR-α2, GFR-α3, GITR, glucagon, Glut 4, glycoprotein IIb / IIIa (GP IIb / 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, hematopoietic 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, HIVIIIB gp120 V3 loop, HLA, HLA-DR, HM1.24, HMFGPEM, HRG, Hrk, human cardiac myosin, human cytomegalovirus (HCMV), human growth hormone (HGH), HVEM, 1-309 antigen, 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)-α, INF-β, INF-γ, inhibin, iNOS, insulin A chain, insulin B chain, insulin-like growth factor 1, integrin α2, integrin α3, integrin α4, integrin α4 / β1, integrin α4 / β7,Integrin α5(αV), Integrin α5 / β1, Integrin α5 / β3, Integrin α6, Integrin β1, Integrin β2, Interferon γ, 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, Luteinizing Hormone, Lymphotoxin β 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-α, 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 Substance, Mug, MuSK, NAIP, NAP, NCAD, N-Cadherin, NCA 90, NCAM, NCAM, Enkephalinase, Neurotrophin-3, -4 or -6, Neuregulin, Nerve Growth Factor (NGF), NGFR, NGF-β, nNOS, NO, NOS, Npn, NRG-3, NT, NTN, OB, OGG1, OPG, OPN, OSM, OX40L, OX40R, pl50, p95, PADPr, Parathyroid Hormone, PARC, PARP, PBR, PBSF, PCAD, P-Cadherin, PCNA, PDGF, PDGF, PDK-1, PECAM, PEM, PF4, PGE, PGF, PGI2, PGJ2, PIN, PLA2, Placental Alkaline Phosphatase (PLAP), PIGF, PLP, PP14, Proinsulin, Prorelaxin, 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 α / β), TdT, TECK, TEM1, TEM5, TEM7, TEM8, TERT, Testicular PLAP-like alkaline phosphatase, TfR, TGF, TGF-α, TGF-β, TGF-β pan-specific, TGF-βRI (ALK-5), TGF-βRII, TGF-βRIIb, TGF-βRIII, TGF-β1, TGF-α2, TGF-β3, TGF-α4, TGF-β5, Thrombin, Thymic Ck-1, Thyroid-stimulating hormone, Tie, TIMP, TIQ, Tissue factor, TMEFF2, Tmpo, TMPRSS2, TNF, TNF-α, TNF-αβ, TNF-β2, TNFc, TNF-RI, TNF-RII, TNFRSF10A (TRAIL R1 Apo-2, DR4), TNFRSFIOB (TRAIL R2 DR5, KILLER, TRICK-2A, TRICK-B), TNFRSF10C (TRAIL R3 DcR1, LIT, TRID), TNFRSF10D (TRAIL R4 DcR2, TRUNDD), TNFRSF11A (RANK ODF R, TRANCE R), TNFRSFIIB (OPG OCIF, TR1), TNFRSF12 (TWEAK R FN14), TNFRSF13B (TACI), TNFRSF13C (BAFFR), TNFRSF14 (HVEM ATAR, HveA, LIGHT R, TR2), TNFRSF16 (NGFR p75NTR), TNFRSF17 (BCMA), TNFRSF 18 (GITR AITR), TNFRSF19 (TROY TAJ, TRADE), TNFRSF19L (RELT), TNFRSFIA (TNF RICD120a, p55-60), TNFRSFIB (TNF RII CD120b, p75-80), TNFRSF26 (TNFRH3), TNFRSF3 (LTbRTNF RIII, TNFC R), TNFRSF4 (OX40 ACT35,TXGP1 R), TNFRSF 5 (CD40p50), TNFRSF6 (FasApo - 1, APT1, CD95), TNFRSF6B (DcR3 M68, TR6), TNFRSF7 (CD27), TNFRSF8 (CD30), TNFRSF9 (4 - 1BBCD137, ILA), TNFRSF21 (DR6), TNFRSF22 (DcTRAIL R2TNFRH2), TNFRST23 (DcTRAIL R1TNFRH 1), TNFRSF25 (DR3Apo - 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 (LIGHTHVEM ligand, LTg), TNFSF15 (TLIA / VEGI), TNFSF18 (GITR ligand AITR ligand, TL6), TNFSF1A (TNF - a titin, 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 CA 125, Lewis Y - related carbohydrate 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 Willebrand’s 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-3), and hormone receptors and growth factors; (iii) The one or more other antigen-binding regions include antigen-binding regions specific for antigens selected from: 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, FcγRIIIa (CD16), FcγRIIa (CD32a), FcγRIIb (CD32b), FcγRI (CD64), Toll-like receptor (TLR), 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; (iv) The multispecific antibody or antibody fragment is bispecific; (v) The multispecific antibody or antibody fragment comprises at least three antigen-binding regions; (vi) The multispecific antibody or antibody fragment is trispecific; (vii) The multispecific antibody or antibody fragment comprises a multispecific format selected from: Fab-Fc-scFv, scFv2-Fc2, scFv-IgG, "bottle-opener", Mab-scFv, Mab-Fv, bis-scFv, central Fv, central scFv, single-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 and / or (viii) The multispecific antibody or antibody fragment comprises one or more of the following: at least one CLκ-preferred variant CH1 domain, optionally the CLκ-preferred variant CH1 domain described in WO2021067404; at least one CLλ-preferred variant CH1 domain, optionally the CLλ-preferred variant CH1 domain described in WO2021067404; at least one pair of variant CH1 domains and variant CL domains that preferentially pair with each other, optionally the pair described in WO2022150787; and / or at least one pair of variant CH3 domains and another variant CH3 domain that preferentially pair with each other, optionally the pair described in WO2022150785.

15. The anti-CD3 antibody or antigen-binding fragment according to any one of the preceding claims, the anti-CD3 antibody or antigen-binding fragment comprising one or more of the following features: (I) It binds to CD3 with greater binding affinity or avidity at acidic pH, optionally about pH 6.0, than at physiological pH, optionally about pH 7.4, optionally wherein: (i) The binding affinity or avidity is measured by: (1) Surface plasmon resonance (SPR), optionally using system; (2) Biolayer interferometry (BLI), optionally using system; (3) Enzyme-linked immunosorbent assay (ELISA); and / or (4) Radioimmunoassay (RIA); (ii) The CD3 is: (1) Human CD3, optionally CD3εδ; and / or (2) Non-human primate CD3, optionally monkey CD3, further optionally cynomolgus monkey CD3, optionally CD3εδ; (iii) Based on the equilibrium dissociation constant (Kd) value, its binding to CD3 is at least ×1.2, at least ×1.5, at least ×2, at least ×5, at least ×10, at least ×20, at least ×50, at least ×100, at least ×10 3 times, at least ×10 4 times, at least ×10 5 times, at least ×10 6 times, at least ×10 7 times, at least ×10 8 times, or at least ×10 9 ; and / or (iv) The anti-CD3 antibody or its antigen-binding fragment does not bind to CD3 at physiological pH, optionally about pH 7.4; (II) It binds to human CD3, optionally CD3εδ, at acidic pH, optionally about pH 6.0, with the following Kd value: (i) Optionally via SPR measurement, further optionally using system, less than 1.57×10 8 (M), less than 1.0×10 8 (M), less than 9.0×10 9 (M), less than 8.0×10 9 (M), less than 7.0×10 9 (M), less than 6.0×10 9 (M), less than 5.0×10 9 (M), less than 4.0×10 9 (M), less than 3.0×10 9 (M), less than 2.0×10 9 (M), less than 1.0×10 9 (M), less than 9.0×10 10 (M) or less than 8.0×10 10 (M) of the Kd value; (ii) Optionally via SPR measurement, further optionally using system, 1.56×10 8 (M) to 7.0×10 10 (M), 1.0×10 8 (M) to 7.0×10 10 (M), 1.0×10 8 (M) to 1.0×10 9 (M), 2.0×10 8 (M) to 1.0×10 9 (M), or 5.0×10 8 (M) to 1.0×10 9 (M) for the Kd value; (iii) Optionally via BLI measurement, further optionally using system, less than 9.33×10 9 (M), less than 9.0×10 9 (M), less than 8.0×10 9 (M), less than 7.0×10 9 (M), less than 6.0×10 9 (M), less than 5.0×10 9 (M), less than 4.0×10 9 (M), less than 3.0×10 9 (M) or less than 2.0×10 9 (M) for the Kd value; and / or (iv) Optionally via BLI measurement, further optionally using system, 9.32×10 9 (M) to 1.0×10 9 (M), between 9.0×10 9 (M) to 1.0×10 9 (M), between 8.0×10 9 (M) to 3.0×10 9 (M), between 7.0×10 9 (M) to 4.0×10 9 or between 6.0×10 9 (M) to 5.0×10 9 (M); the Kd value (III) It binds to cynomolgus monkey CD3, optionally CD3εδ, at acidic pH, optionally about pH 6.0, optionally with the following Kd value: (i) Optionally via BLI measurement, further optionally using system, less than 2.0×10 8 (M), less than 1.0×10 8 (M), less than 9.0×10 9 (M), less than 8.0×10 9 (M), less than 7.0×10 9 (M), less than 6.0×10 9 (M), less than 5.0×10 9 (M), less than 4.0×10 9 (M), less than 3.0×10 9 (M) or less than 2.0×10 9 (M) of the Kd value; and / or (ii) Optionally via BLI measurement, further optionally using system, 2.0×10 8 (M) to 1.0×10 9 (M); between 1.0×10 8 (M) and 1.0×10 9 (M); between 9.0×10 9 (M) and 2.0×10 9 (M); or between 8.0×10 9 (M) and 5.0×10 9 for the Kd value; (IV) It binds to cells expressing CD3 with greater binding at acidic pH, optionally about pH 6.0, than at physiological pH, optionally about pH 7.4, optionally wherein: (i) The binding is measured by flow cytometry, further optionally based on the normalized cell binding (NCB) value calculated using the median fluorescence intensity (MFI) value; (ii) The cells express: (1) Human CD3, optionally CD3εδ; and / or (2) Non-human primate CD3, optionally monkey CD3, further optionally cynomolgus monkey CD3, optionally CD3εδ; (iii) The cells are: (1) Primary cells; (2) Cells of a cell line; (3) Human cells, optionally human T cells, further optionally Jurkat cells; (4) Non-human primate cells, optionally monkey cells, further optionally cynomolgus monkey cells, still further optionally HSC-F cells; (iv) Based on the NCB value when measured by flow cytometry, the binding to cells expressing CD3 is at least ×1.2, at least ×1.5, at least ×2, at least ×3, at least ×4, at least ×5, at least ×10, at least ×20, at least ×50, at least ×100, at least ×10 3 、at least ×10 4 、at least ×10 5 、at least ×10 6 、at least ×10 7 、at least ×10 8 or at least ×10 9 ; and / or (v) The anti-CD3 antibody or its antigen-binding fragment does not bind to cells expressing CD3 at physiological pH, optionally about pH 7.4 (V) When binding to CD3 on cells, optionally T cells, it triggers the activation of the cytotoxic function of said cells and / or enhances the cytotoxic function of said cells; (VI) When binding to CD3 on cells, optionally T cells, it does not induce cytokine production at a level capable of inducing cytokine release syndrome (CRS) upon binding to CD3; and / or (VII) It comprises a multispecific antibody or antibody fragment or is comprised in a multispecific antibody or antibody fragment, said multispecific antibody or antibody fragment having at least (a) a first antigen-binding region specific for CD3, said first antigen-binding region comprising said VH polypeptide and / or said VL polypeptide, and (b) one or more other antigen-binding regions, said one or more other antigen-binding regions including at least one antigen-binding region that binds to an antigen different from that bound by said first antigen-binding region; and when binding to (i) CD3 on a first cell, optionally a T cell, and (ii) said second antigen expressed on a second cell, said first cell exhibits cytotoxicity towards said second cell.

16. A nucleic acid encoding an anti-CD3 antibody or antigen-binding fragment according to any one of the preceding claims, said nucleic acid optionally comprising: (A) A nucleic acid sequence encoding a VH polypeptide that is at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 650, 550, 450, 350, 750, 850, 950, 1050, 1150, 1250 or 1350; and / or (B) A nucleic acid sequence encoding a VL polypeptide that is at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 660, 560, 460, 360, 760, 860, 960, 1060, 1160, 1260 or 1360, further optionally comprising a nucleic acid sequence encoding a VH polypeptide and a nucleic acid sequence encoding a VL polypeptide of the following: (I) Respectively, SEQ ID NO: 650 and 660; (II) Respectively, SEQ ID NO: 550 and 560; (III) Respectively, SEQ ID NO: 450 and 460; (IV) Respectively, SEQ ID NO: 350 and 360; (V) Respectively, SEQ ID NO: 750 and 760; (VI) Respectively, SEQ ID NO: 850 and 860; (VII) Respectively, SEQ ID NO: 950 and 960; (VIII) Respectively, SEQ ID NO: 1050 and 1060; (IX) Respectively, SEQ ID NO: 1150 and 1160; (X) Respectively, SEQ ID NO: 1250 and 1260; or (XI) Separately, SEQ ID NO: 1350 and 1360.

17. A vector, the vector comprising the nucleic acid according to claim 16, optionally wherein: (i) the vector is an expression vector; and / or (ii) the vector comprises a plasmid, a viral vector (optionally an adenovirus, a lentivirus or a retrovirus), a lipid-based vector, a self-replicating RNA vector, virus-like particles, a polymer-based vector and / or nanoparticles, optionally lipid-based nanoparticles.

18. An isolated or recombinant cell, the isolated or recombinant cell comprising the nucleic acid according to claim 16 or the vector according to claim 17, transfected with the nucleic acid according to claim 16 or the vector according to claim 17, transformed with the nucleic acid according to claim 16 or the vector according to claim 17 or transduced with the nucleic acid according to claim 16 or the vector according to claim 17, optionally wherein the isolated or recombinant cell is: (i) a mammal, optionally a human, a non-human primate, a monkey, a rabbit, a rodent, a hamster, a rat or a mouse; or (ii) a non-mammal, optionally a plant, a bacterium, a fungus, a yeast, a protozoan or an insect, and optionally wherein the isolated or recombinant cell is an immune cell or a hybridoma.

19. A pharmaceutical composition, the pharmaceutical composition comprising: (A) an anti-CD3 antibody or antigen-binding fragment according to any one of claims 1-15, the nucleic acid according to claim 16, the vector according to claim 17 and / or the isolated or recombinant cell according to claim 18; and (B) a pharmaceutically acceptable carrier and / or excipient.

20. A method of treating a subject in need of such treatment, the method comprising administering to the subject an effective amount of: (i) an anti-CD3 antibody or antigen-binding fragment according to any one of claims 1-15; (ii) the nucleic acid according to claim 16; (iii) the vector according to claim 17; (iv) the isolated or recombinant cell according to claim 18; and / or (v) the pharmaceutical composition according to claim 19, optionally wherein: (a) the subject is (i) a mammal, optionally a human, a non-human primate, a monkey, a horse, a cow, a sheep, a goat, a pig, a dog, a cat, a rabbit, a rodent, a hamster, a rat or a mouse; or (ii) a non-mammal vertebrate, optionally a bird, a fish, an amphibian or a reptile; (b) the subject has or is at risk of developing a disease, disorder or condition; and / or (c) the method further comprises administering to the subject an additional agent, the additional agent optionally being an adjuvant or a therapeutic agent.

21. A method of treating or preventing a disease, disorder or condition in a subject, the method comprising administering to the subject an effective amount of: (i) an anti-CD3 antibody or antigen-binding fragment according to any one of claims 1-15; (ii) the nucleic acid according to claim 16; (iii) The carrier according to claim 17; (iv) The isolated or recombinant cell according to claim 18; and / or (v) The pharmaceutical composition according to claim 19, Optionally wherein: (a) The subject is (i) A mammal, optionally a human, non-human primate, monkey, horse, cow, sheep, goat, pig, dog, cat, rabbit, rodent, hamster, rat or mouse; or (ii) A non-mammalian vertebrate, optionally a bird, fish, amphibian or reptile; and / or (b) The method further comprises administering to the subject an additional agent, which is optionally an adjuvant or therapeutic agent.

22. A method of inducing cytotoxicity in a cell expressing a target molecule of interest, the method comprising administering to the subject an effective amount of: (i) The anti-CD3 antibody or antigen-binding fragment according to claim 14; (ii) A nucleic acid encoding the anti-CD3 antibody or antigen-binding fragment according to claim 14; (iii) A carrier comprising the nucleic acid of (ii); (iv) An isolated or recombinant cell comprising the nucleic acid or the carrier, transfected with the nucleic acid or the carrier, transformed with the nucleic acid or the carrier, or transduced with the nucleic acid or the carrier; and / or (v) A pharmaceutical composition comprising (A) the anti-CD3 antibody or antigen-binding fragment according to claim 14, a nucleic acid encoding the anti-CD3 antibody or antigen-binding fragment according to claim 14, a carrier comprising the nucleic acid, and / or a cell comprising the nucleic acid or the carrier, transfected with the nucleic acid or the carrier, transformed with the nucleic acid or the carrier, or transduced with the nucleic acid or the carrier; and / or (B) a pharmaceutically acceptable carrier and / or excipient, Optionally wherein: (a) The subject is (i) A mammal, optionally a human, non-human primate, monkey, horse, cow, sheep, goat, pig, dog, cat, rabbit, rodent, hamster, rat or mouse; or (ii) A non-mammalian vertebrate, optionally a bird, fish, amphibian or reptile; (b) The subject has or is at risk of developing a disease, disorder or condition; and / or (c) The method further comprises administering to the subject an additional agent, which is optionally an adjuvant or therapeutic agent.

23. The method according to any one of claims 20-22, wherein the disease, disorder or condition includes cancer or neoplastic conditions, autoimmune diseases, neurodegenerative diseases, infectious diseases, inflammatory diseases or other diseases, optionally wherein: (i) The cancer is: (i-1) Solid cancer, optionally selected from one or more of the following: mesothelioma, malignant pleural mesothelioma, non-small cell lung cancer, small cell lung cancer, squamous cell lung cancer, large cell lung cancer, pancreatic cancer, pancreatic ductal adenocarcinoma, esophageal adenocarcinoma, breast cancer, glioblastoma, ovarian cancer, colorectal cancer, prostate cancer, cervical cancer, skin cancer, melanoma, kidney cancer, liver cancer, brain cancer, thymoma, sarcoma, carcinoma, uterine cancer, kidney cancer, gastrointestinal cancer, urothelial cancer, pharyngeal cancer, head and neck cancer, rectal cancer, esophageal cancer or bladder cancer or its metastasis; and / or (i-2) Liquid cancer, optionally selected from: chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), multiple myeloma, acute lymphoblastic leukemia (ALL), Hodgkin lymphoma, B-cell acute lymphoblastic leukemia (BALL), T-cell acute lymphoblastic leukemia (TALL), small lymphocyte leukemia (SLL), B-cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt lymphoma, diffuse large B-cell lymphoma (DLBCL), DLBCL associated with chronic inflammation, chronic myeloid leukemia, myeloproliferative neoplasm, follicular lymphoma, pediatric follicular lymphoma, hairy cell leukemia, small cell or large cell follicular lymphoma, malignant lymphoproliferative condition, MALT lymphoma (mucosa-associated lymphoid tissue extranodal marginal zone lymphoma), marginal zone lymphoma, myelodysplasia, myelodysplastic syndrome, non-Hodgkin lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenström macroglobulinemia, splenic marginal zone lymphoma, splenic lymphoma / leukemia, splenic diffuse red pulp small B-cell lymphoma, hairy cell leukemia variant, lymphoplasmacytic lymphoma, heavy chain disease, plasma cell myeloma, solitary plasmacytoma of bone, extramedullary plasmacytoma, nodal marginal zone lymphoma, pediatric nodal marginal zone lymphoma, primary cutaneous follicle center lymphoma, lymphomatoid granulomatosis, primary mediastinal (thymic) large B-cell lymphoma, intravascular large B-cell lymphoma, ALK+ large B-cell lymphoma, large B-cell lymphoma arising from HHV8-associated multicentric Castleman disease, primary effusion lymphoma, B-cell lymphoma, acute myeloid leukemia (AML) or lymphoma not otherwise specified; (ii) The autoimmune or inflammatory disease is psoriasis, rheumatoid arthritis, autoimmune arthritis, type I diabetes, systemic lupus erythematosus, myasthenia gravis, multiple sclerosis, scleroderma, inflammatory bowel disease, Crohn's disease, ulcerative colitis, Guillain-Barré syndrome, chronic inflammatory demyelinating polyneuropathy, pemphigus vulgaris, Sjogren's syndrome, Addison's disease, Behçet's disease, Schmidt syndrome, celiac disease, dermatomyositis, autoimmune vitiligo, Graves' disease, Hashimoto's thyroiditis, Kawasaki disease, pernicious anemia, autoimmune vasculitis or fibrosis; (iii) The neurodegenerative disease is Alzheimer's disease, Huntington's disease, Parkinson's disease, amyotrophic lateral sclerosis, Friedreich's ataxia, Lewy body disease, spinal muscular atrophy, motor neuron disease, multiple sclerosis, Batten disease, Creutzfeldt-Jakob disease; (iv) The infectious disease is a viral disease, a bacterial disease, a fungal disease, a yeast disease, a protozoal disease, a prion disease or a parasitic disease, optionally wherein (1) the viral disease is human immunodeficiency virus (HIV), hepatitis virus (optionally hepatitis A, B or C virus), human papillomavirus (HPV), herpes simplex virus (HSV) (optionally HSV-1 or HSV-2), enterovirus, human cytomegalovirus, adenovirus, rhinovirus, poxvirus, influenza virus, coronavirus (optionally MERS-CoV, SARS-CoV or SARS-CoV-2 or common human coronavirus), norovirus, West Nile virus, Zika virus, poliovirus, Ebola virus or dengue virus (DENV) infection, (2) the bacterial disease is Salmonella, Escherichia coli, Mycobacterium tuberculosis, methicillin-resistant Staphylococcus aureus (MRSA), Clostridium difficile, Streptococcus pneumoniae, Klebsiella pneumoniae, Pseudomonas aeruginosa, Helicobacter pylori, Neisseria gonorrhoeae, Vibrio vulnificus, and / or (3) the fungal disease is aspergillosis, Candida, Candida auris, Cryptococcus neoformans, Pneumocystis jirovecii, Mucorales, Talaromyces, tinea, Blastomyces, Coccidioides, Cryptococcus gattii, Histoplasma, Paracoccidioides or Sporothrix infection.

24. A method for preparing an anti-CD3 antibody or antigen-binding fragment according to any one of claims 1-15, the method comprising: (a) culturing a cell comprising the nucleic acid according to claim 16 under conditions permitting the expression of the antibody or antigen-binding fragment, and (b) harvesting and purifying the antibody or antigen-binding fragment from the cell culture from (a).

25. A method for preparing an isolated or recombinant cell or population of such cells according to claim 18, the method comprising introducing the nucleic acid according to claim 16 and / or the vector according to claim 17 into one or more cells, optionally wherein the introduction is carried out in vitro, ex vivo or in vivo.

26. The anti-CD3 antibody or antigen-binding fragment according to any one of claims 1-15, the nucleic acid according to claim 16, the vector according to claim 17, the cell or population of such cells according to claim 18 and / or the pharmaceutical composition according to claim 19, for use in medicine.

27. An anti-CD3 antibody or antigen-binding fragment thereof according to any one of claims 1-15, a nucleic acid according to claim 16, a vector according to claim 17, a cell according to claim 18 or a population of such cells and / or a pharmaceutical composition according to claim 19 for use in treating a disease, disorder or condition, optionally wherein the disease, disorder or condition comprises cancer or neoplastic condition, autoimmune disease, neurodegenerative disease, infectious disease, inflammatory disease or another disease, further optionally wherein: (i) the cancer is: (i-1) a solid cancer, optionally selected from one or more of the following: mesothelioma, malignant pleural mesothelioma, non-small cell lung cancer, small cell lung cancer, squamous cell lung cancer, large cell lung cancer, pancreatic cancer, pancreatic ductal adenocarcinoma, esophageal adenocarcinoma, breast cancer, glioblastoma, ovarian cancer, colorectal cancer, prostate cancer, cervical cancer, skin cancer, melanoma, renal cancer, liver cancer, brain cancer, thymoma, sarcoma, carcinoma, uterine cancer, renal cancer, gastrointestinal cancer, urothelial cancer, pharyngeal cancer, head and neck cancer, rectal cancer, esophageal cancer or bladder cancer or metastases thereof; and / or (i-2) a liquid cancer, optionally selected from: chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), multiple myeloma, acute lymphoblastic leukemia (ALL), Hodgkin lymphoma, B cell acute lymphoblastic leukemia (BALL), T cell acute lymphoblastic leukemia (TALL), small lymphocytic leukemia (SLL), B cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt lymphoma, diffuse large B cell lymphoma (DLBCL), DLBCL associated with chronic inflammation, chronic myeloid leukemia, myeloproliferative neoplasm, follicular lymphoma, pediatric follicular lymphoma, hairy cell leukemia, small cell or large cell follicular lymphoma, malignant lymphoproliferative condition, MALT lymphoma (mucosa-associated lymphoid tissue extranodal marginal zone lymphoma), marginal zone lymphoma, myelodysplasia, myelodysplastic syndrome, non-Hodgkin lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenström macroglobulinemia, splenic marginal zone lymphoma, splenic lymphoma / leukemia, splenic diffuse red pulp small B cell lymphoma, hairy cell leukemia variant, lymphoplasmacytic lymphoma, heavy chain disease, plasma cell myeloma, solitary plasmacytoma of bone, extramedullary plasmacytoma, nodal marginal zone lymphoma, pediatric nodal marginal zone lymphoma, primary cutaneous follicle center lymphoma, lymphomatoid granulomatosis, primary mediastinal (thymic) large B cell lymphoma, intravascular large B cell lymphoma, ALK+ large B cell lymphoma, HHV8-associated multicentric Castleman disease-derived large B cell lymphoma, primary effusion lymphoma, B cell lymphoma, acute myeloid leukemia (AML) or lymphoma not otherwise specified; (ii) The autoimmune or inflammatory disease is psoriasis, rheumatoid arthritis, autoimmune arthritis, type I diabetes, systemic lupus erythematosus, myasthenia gravis, multiple sclerosis, scleroderma, inflammatory bowel disease, Crohn's disease, ulcerative colitis, Guillain - Barré syndrome, chronic inflammatory demyelinating polyneuropathy, pemphigus vulgaris, Sjögren's syndrome, Addison's disease, Behçet's disease, Schmidt syndrome, celiac disease, dermatomyositis, autoimmune vitiligo, Graves' disease, Hashimoto's thyroiditis, Kawasaki disease, pernicious anemia, autoimmune vasculitis or fibrosis; (iii) The neurodegenerative disease is Alzheimer's disease, Huntington's disease, Parkinson's disease, amyotrophic lateral sclerosis, Friedreich's ataxia, Lewy body disease, spinal muscular atrophy, motor neuron disease, multiple sclerosis, Batten disease, Creutzfeldt - Jakob disease; (iv) The infectious disease is a viral disease, bacterial disease, fungal disease, yeast disease, protozoal disease, prion disease or parasitic disease. Optionally, (1) the viral disease is human immunodeficiency virus (HIV), hepatitis virus (optionally hepatitis A, B or C virus), human papillomavirus (HPV), herpes simplex virus (HSV) (optionally HSV - 1 or HSV - 2), enterovirus, human cytomegalovirus, adenovirus, rhinovirus, poxvirus, influenza virus, coronavirus (optionally MERS - CoV, SARS - CoV or SARS - CoV - 2 or common human coronavirus), norovirus, West Nile virus, Zika virus, poliovirus, Ebola virus or dengue virus (DENV) infection, (2) the bacterial disease is Salmonella, Escherichia coli, Mycobacterium tuberculosis, methicillin - resistant Staphylococcus aureus (MRSA), Clostridioides difficile, Streptococcus pneumoniae, Klebsiella pneumoniae, Pseudomonas aeruginosa, Helicobacter pylori, Neisseria gonorrhoeae, Vibrio vulnificus, and / or (3) the fungal disease is aspergillosis, Candida, Candida auris, Cryptococcus neoformans, Pneumocystis jirovecii, Mucorales, Talaromyces, tinea, Blastomyces, Coccidioides, Cryptococcus gattii, Histoplasma, Paracoccidioides or Sporothrix infection.

28. Use of the anti - CD3 antibody or antigen - binding fragment according to any one of claims 1 - 15, the nucleic acid according to claim 16, the vector according to claim 17, the isolated or recombinant cell or population of such cells according to claim 18, and / or the pharmaceutical composition according to claim 19 for the preparation of a medicament for the treatment of a disease, disorder or condition, optionally wherein the disease, disorder or condition includes cancer or neoplastic condition, autoimmune disease, neurodegenerative disease, infectious disease, inflammatory disease or another disease, further optionally wherein: (i) The cancer is: (i-1) Solid cancer, optionally selected from one or more of the following: mesothelioma, malignant pleural mesothelioma, non-small cell lung cancer, small cell lung cancer, squamous cell lung cancer, large cell lung cancer, pancreatic cancer, pancreatic ductal adenocarcinoma, esophageal adenocarcinoma, breast cancer, glioblastoma, ovarian cancer, colorectal cancer, prostate cancer, cervical cancer, skin cancer, melanoma, kidney cancer, liver cancer, brain cancer, thymoma, sarcoma, carcinoma, uterine cancer, kidney cancer, gastrointestinal cancer, urothelial cancer, pharyngeal cancer, head and neck cancer, rectal cancer, esophageal cancer or bladder cancer or its metastasis; and / or (i-2) Liquid cancer, optionally selected from: chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), multiple myeloma, acute lymphoblastic leukemia (ALL), Hodgkin lymphoma, B-cell acute lymphoblastic leukemia (BALL), T-cell acute lymphoblastic leukemia (TALL), small lymphocyte leukemia (SLL), B-cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt lymphoma, diffuse large B-cell lymphoma (DLBCL), DLBCL associated with chronic inflammation, chronic myeloid leukemia, myeloproliferative neoplasms, follicular lymphoma, pediatric follicular lymphoma, hairy cell leukemia, small cell or large cell follicular lymphoma, malignant lymphoproliferative conditions, MALT lymphoma (mucosa-associated lymphoid tissue extranodal marginal zone lymphoma), marginal zone lymphoma, myelodysplasia, myelodysplastic syndrome, non-Hodgkin lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenström macroglobulinemia, splenic marginal zone lymphoma, splenic lymphoma / leukemia, splenic diffuse red pulp small B-cell lymphoma, hairy cell leukemia variant, lymphoplasmacytic lymphoma, heavy chain disease, plasma cell myeloma, solitary plasmacytoma of bone, extramedullary plasmacytoma, nodal marginal zone lymphoma, pediatric nodal marginal zone lymphoma, primary cutaneous follicle center lymphoma, lymphomatoid granulomatosis, primary mediastinal (thymic) large B-cell lymphoma, intravascular large B-cell lymphoma, ALK+ large B-cell lymphoma, large B-cell lymphoma arising from HHV8-associated multicentric Castleman disease, primary effusion lymphoma, B-cell lymphoma, acute myeloid leukemia (AML) or lymphoma not otherwise specified; (ii) The autoimmune or inflammatory disease is psoriasis, rheumatoid arthritis, autoimmune arthritis, type I diabetes, sarcoidosis, systemic lupus erythematosus, myasthenia gravis, multiple sclerosis, scleroderma, inflammatory bowel disease, Crohn's disease, ulcerative colitis, Guillain-Barré syndrome, chronic inflammatory demyelinating polyneuropathy, pemphigus vulgaris, Sjögren's syndrome, Addison's disease, Behçet's disease, Schmidt syndrome, celiac disease, dermatomyositis, autoimmune vitiligo, Graves' disease, Hashimoto's thyroiditis, Kawasaki disease, pernicious anemia, autoimmune vasculitis or fibrosis; (iii) The neurodegenerative disease is Alzheimer's disease, Huntington's disease, Parkinson's disease, amyotrophic lateral sclerosis, Friedreich's ataxia, Lewy body disease, spinal muscular atrophy, motor neuron disease, multiple sclerosis, Batten disease, Creutzfeldt-Jakob disease; (iv) The infectious disease is a viral disease, a bacterial disease, a fungal disease, a yeast disease, a protozoal disease, a prion disease or a parasitic disease, optionally where (1) the viral disease is human immunodeficiency virus (HIV), hepatitis virus (optionally hepatitis A, B or C virus), human papillomavirus (HPV), herpes simplex virus (HSV) (optionally HSV-1 or HSV-2), enterovirus, human cytomegalovirus, adenovirus, rhinovirus, poxvirus, influenza virus, coronavirus (optionally MERS-CoV, SARS-CoV or SARS-CoV-2 or common human coronavirus), norovirus, West Nile virus, Zika virus, poliovirus, Ebola virus or dengue virus (DENV) infection, (2) the bacterial disease is Salmonella, Escherichia coli, Mycobacterium tuberculosis, methicillin-resistant Staphylococcus aureus (MRSA), Clostridioides difficile, Streptococcus pneumoniae, Klebsiella pneumoniae, Pseudomonas aeruginosa, Helicobacter pylori, Neisseria gonorrhoeae, Vibrio vulnificus, and / or (3) the fungal disease is aspergillosis, Candida, Candida auris, Cryptococcus neoformans, Pneumocystis jirovecii, Mucorales, Talaromyces, tinea, Blastomyces, Coccidioides, Cryptococcus gattii, Histoplasma, Paracoccidioides or Sporothrix infection.

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