pH-Dependent Anti-CD3 Antibodies and Related Methods

Abstract

The present disclosure provides anti-CD3 antibodies and antigen-binding fragments, as well as multispecific antibodies and antibody fragments thereof. The present disclosure further provides nucleic acids and vectors encoding such antibodies or antibody fragments, and cells containing such nucleic acids. Pharmaceutical compositions, in vivo methods, screening methods, and manufacturing methods for the anti-CD3 antibodies and antigen-binding fragments are also provided.

Description

[Technical Field]

[0001] (CROSS-REFERENCE TO RELATED APPLICATIONS) This application claims priority to U.S. Provisional Patent Application No. 63 / 417,118, filed October 18, 2022, entitled "PH-DEPENDENT ANTI-CD3 ANTIBODIES AND METHODS RELATING THERETO," the contents of which are incorporated herein by reference in their entirety.

[0002] (Reference to Electronic Sequence Listing) The contents of the Electronic Sequence Listing (1160430o003813.xml, size: 508,346 bytes, and creation date: October 11, 2023) are incorporated herein by reference in their entirety. BACKGROUND OF THE INVENTION

[0003] Cell proliferative disorders, such as cancer, are characterized by the uncontrolled growth of a cell subpopulation. Cell proliferative disorders are the leading cause of death in developed countries and the second leading cause of death in developing countries. The total number of newly diagnosed cases of cancer each year is predicted to reach 23.6 million by 2030. The National Cancer Institute predicts that approximately 2 million new cases of cancer will be diagnosed in the United States in 2020, and more than 600,000 Americans will die from cancer. Therefore, cancer treatment represents a significant and ever-increasing societal burden.

[0004] The idea of ​​harnessing the cytotoxic ability of T cells to kill tumor cells through the use of CD3-targeting bispecific antibodies dates back to the mid-1980s (Staerz et al. Nature 1985 314:628-32). Many bispecific antibodies developed to date contain a first binding site specific for CD3 for T cell recruitment and activation, and a second binding site for a target disease-associated antigen, such as an antigen produced by tumor cells. CD3 bispecific antibodies bind both the CD3 surface receptor on T cells and their second target antigen, such as a protein expressed on tumors, allowing competent T cells to bind to target-expressing cells via cross-linking by the CD3 bispecific antibody, regardless of the peptide / MHC specificity of their T cell receptors. (See, e.g., Bassan, 2012, Blood 120:5094-95.) Using CD3 bispecific antibodies to cross-link T cells and tumor cells can induce dramatic regression of advanced malignancies, even resulting in complete remission in some cases. More than 25 different CD3 bispecific antibodies are currently in clinical development for the treatment of hematological malignancies or solid cancers by targeting CD19, CD20, CD33, and CD123, or EpCAM, HER2, PSMA, and CEA, respectively (see, e.g., Liu et al. Front Immunol 2017 8:38).

[0005] Various anti-CD3 antibodies, including monoclonal and bispecific antibodies, are known in the art. See, for example, U.S. Patent Nos. 7,262,276, 7,635,472, 7,862,813, 9,587,021, and 10,174,124. However, many of these anti-CD3 antibodies induce excessive cytokine production, often resulting in harmful cytokine release syndrome (CRS). The anti-CD3 binding domain of bispecific antibodies associates with all T cells, thereby recruiting a high-cytokine-producing CD4 T cell subset. Therefore, there is an unmet need for safe and effective anti-CD3 antibodies that exhibit a desirable CRS risk profile, for example, in terms of binding specificity to CD3 expressed on T cells, T cell activation, and (re)directing activated T cells to kill target cells, while attenuating the risk of CRS.

[0006] One approach to developing CD3-binding regions that exhibit a desirable CRS risk profile is to engineer CD3-binding regions with pH-dependent antigen binding. In the past, pH-dependent antigen binding has been engineered by incorporating histidine and / or other ionizable residues into the binding interface of antibodies and other proteins (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, resulting in pH-dependent differences in binding affinity (Gera et al., PLOS ONE 7(11) e48928. doi:10.1371 / 2012). It is recognized that the pH range of human blood is approximately 7.6–7.8, whereas tumor cells have an extracellular pH of approximately 6.3–6.5. This tumor pH is at least partly due to the accumulation of metabolic acids that are not adequately removed due to poor tumor vascularization. Previously, the present applicant engineered pH-dependent CD3-binding antibodies to preferentially bind to CD3 at low pH values, promoting binding and activity within and around the tumor microenvironment ( WO2020247932A1 ). Without wishing to be bound by theory, it is believed that CD3-binding regions engineered to preferentially bind to CD3 at low pH, e.g., a pH of about 6, may generate selective and sustained cytotoxic activity at or around the tumor site, potentially reducing or eliminating on-target off-tumor effects and improving half-life, allowing for reduced dosages. Summary of the Invention

[0007] The present disclosure relates to improved pH-dependent anti-CD3 antibodies and antigen-binding fragments thereof, e.g., those that bind to cynomolgus monkey CD3 as well as human CD3 and / or provide improved binding to CD3 and / or CD3-expressing cells at pH 6.0, and methods of using or producing them.

[0008] In one aspect, the present disclosure provides pH-dependent anti-CD3 antibodies and antigen-binding fragments.

[0009] In some embodiments, the anti-CD3 antibody or antigen-binding fragment thereof comprises: (A) a heavy chain variable domain (VH) polypeptide comprising: (a) a VH complementarity determining region 1 (CDR-H1) comprising: (i) a 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) a VH comprising the amino acid sequence of 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) comprising: (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) the amino acid sequence of WIDLENANTIYDAKFQG (SEQ ID NO: 14, 614, 514, 414, 314, 714, 814, 914, 1014, 1114, 1214, or 1314). CDR-H2, and / or (c) VH complementarity-determining region 3 (CDR-H3), including: (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) a VH comprising the amino acid sequence of 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 comprising: (a) a VL complementarity determining region 1 (CDR-L1) of: (i) ADI-74968, ADI-74967, ADI-74966, ADI-74965, ADI-79842, ADI-79843, ADI-79848, ADI-79844, ADI-79845, ADI-79846, or ADI-79847. CDR-L1 contained in I-79847, (ii) KSSQSLLNARTGX5NYLA (SEQ ID NO: 22) (wherein X5 is H or K), and / or (iii) a VL comprising the amino acid sequence of KSSQSLLNARTGHNYLA (SEQ ID NO: 622, 422, 322, 922, 1222, or 1322) or KSSQSLLNARTGKNYLA (SEQ ID NO: 522, 722, 822, 1022, or 1122). (b) a VL complementarity-determining region 2 (CDR-L2) comprising: (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) the amino acid sequence of WASTRES (SEQ ID NO: 24, 624, 524, 424, 324, 724, 824, 924, 1024, 1124, 1224, or 1324).CDR-L2, and / or (c) VL complementarity determining region 3 (CDR-L3), including: (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 (sequence No. 26) (wherein X6 is Y or H and X7 is H or R), and / or (iii) a VL CDR-L3 comprising the amino acid sequence of 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.

[0010] In some cases, the anti-CD3 antibody and / or antigen-binding fragment does not include (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 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).

[0011] In certain cases, the anti-CD3 antibody and / or antigen-binding fragment does not include (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).

[0012] In some cases, the anti-CD3 antibody and / or antigen-binding fragment does not include (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).

[0013] In some cases, the anti-CD3 antibody and / or antigen-binding fragment does not include (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).

[0014] In certain embodiments, the anti-CD3 antibody or antigen-binding fragment comprises: (A) a VH polypeptide comprising: (a) a CDR-H1 comprising: (i) the CDR-H1 contained in ADI-74968, ADI-74967, ADI-74966, or ADI-74965, and / or (ii) the amino acid sequence of FNIKDYYMH (SEQ ID NO: 12, 612, 512, 412, or 312); (b) a CDR-H2 comprising: (i) the CDR-H1 contained in ADI-74968, ADI-74967, ADI-74966, or ADI-74965; , ADI-74967, ADI-74966, or ADI-74965, and / or (ii) a CDR-H2 comprising the amino acid sequence of WIDLENANTIYDAKFQG (SEQ ID NO: 14, 614, 514, 414, or 314), and / or (c) a CDR-H3 comprising: (i) a CDR-H3 contained in ADI-74968, ADI-74967, ADI-74966, or ADI-74965, (ii) ARDX2YX3RYFYDV (SEQ ID NO: 16) (wherein X2 is A or H and X3 is H), and / or (iii) a CDR-H3 comprising the amino acid sequence of ARDAYHRYFYDV (SEQ ID NO: 616 or 316) or ARDHYHRYFYDV (SEQ ID NO: 516 or 416), and / or (B) a VL polypeptide comprising: (a) a CDR-L1 comprising: (i) ADI the amino acid sequence of 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) a CDR-L2 comprising: (i) a CDR-L2 contained in ADI-74968, ADI-74967, ADI-74966, or ADI-74965, and / or (ii) a CDR-L2 comprising the amino acid sequence of WASTRES (SEQ ID NO: 24, 624, 524, 424, or 324), and / or (c) a CDR-L3 comprising the amino acid sequence of: (i) ADI-74968, ADI The VL polypeptide may comprise a CDR-L3 contained in ADI-74967, ADI-74966, or ADI-74965, (ii) KQSX6SX7RT (SEQ ID NO: 26) (wherein X6 is Y or H and X7 is H), and / or (iii) a CDR-L3 comprising the amino acid sequence of KQSYSHRT (SEQ ID NO: 626 or 426) or KQSHSHRT (SEQ ID NO: 526 or 326).

[0015] In certain embodiments, the anti-CD3 antibody or antigen-binding fragment comprises: (A) a VH polypeptide comprising: (a) a CDR-H1 comprising: (i) the CDR-H1 contained in ADI-74968, and / or (ii) the amino acid sequence of FNIKDYYMH (SEQ ID NO: 12 or 612); (b) a CDR-H2 comprising: (i) the CDR-H2 contained in ADI-74968, and / or (ii) the amino acid sequence of WIDLENANTIYDAKFQG (SEQ ID NO: 14 or 614); and / or (c) a CDR-H3 comprising: (i) the CDR-H3 contained in ADI-74968, and / or (ii) the amino acid sequence of ARDAYHRYFYDV (SEQ ID NO: 616). and / or (B) a VL polypeptide comprising: (a) a CDR-L1 comprising: (i) the CDR-L1 contained in ADI-74968, (ii) a CDR-L1 comprising the amino acid sequence of KSSQSLLNARTGHNYLA (SEQ ID NO: 622), (b) a CDR-L2 comprising: (i) the CDR-L2 contained in ADI-74968, and / or (ii) a CDR-L2 comprising the amino acid sequence of WASTRES (SEQ ID NO: 24 or 624), and / or (c) a CDR-L3 comprising: (i) the CDR-L3 contained in ADI-74968, (ii) a CDR-L3 comprising the amino acid sequence of KQSYSHRT (SEQ ID NO: 626).

[0016] In certain embodiments, the anti-CD3 antibody or antigen-binding fragment comprises: (A) a VH polypeptide comprising: (a) a CDR-H1 comprising: (i) the CDR-H1 contained in ADI-74967, and / or (ii) the amino acid sequence of FNIKDYYMH (SEQ ID NO: 12 or 512); (b) a CDR-H2 comprising: (i) the CDR-H2 contained in ADI-74967, and / or (ii) the CDR-H2 comprising the amino acid sequence of WIDLENANTIYDAKFQG (SEQ ID NO: 14 or 514); and / or (c) a CDR-H3 comprising: (i) the CDR-H3 contained in ADI-74967, and / or (ii) the CDR-H3 comprising the amino acid sequence of ARDHYHRYFYDV (SEQ ID NO: 516). -H3, and / or (B) a VL polypeptide comprising: (a) CDR-L1 comprising: (i) the CDR-L1 contained in ADI-74967, (ii) a CDR-L1 comprising the amino acid sequence of KSSQSLLNARTGKNYLA (SEQ ID NO: 522), (b) CDR-L2 comprising: (i) the CDR-L2 contained in ADI-74967, and / or (ii) a CDR-L2 comprising the amino acid sequence of WASTRES (SEQ ID NOs: 24, 624, 524), and / or (c) CDR-L3 comprising: (i) the CDR-L3 contained in ADI-74967, (ii) a CDR-L3 comprising the amino acid sequence of KQSHSHRT (SEQ ID NO: 526).

[0017] In some cases, the anti-CD3 antibody or antigen-binding fragment of any 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.

[0018] In certain cases, an anti-CD3 antibody or antigen-binding fragment according to any 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.

[0019] 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) a CDR-H1 comprising FNIKDYYMH (SEQ ID NO: 12 or 612), CDR-H3 comprising WIDLENANTIYDAKFQG (SEQ ID NO: 14 or 614), 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).

[0020] 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) a CDR-H1 comprising FNIKDYYMH (SEQ ID NO: 12 or 512), a CDR-H2 comprising WIDLENANTIYDAKFQG (SEQ ID NO: 14 or 514), a CDR-H3 comprising ARDHYHRYFYDV (SEQ ID NO: 516), a CDR-L1 comprising KSSQSLLNARTGKNYLA (SEQ ID NO: 522), a CDR-L2 comprising WASTRES (SEQ ID NO: 24 or 524), and a CDR-L3 comprising KQSHSHRT (SEQ ID NO: 526).

[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-74966, or (ii) a CDR-H1 comprising FNIKDYYMH (SEQ ID NO: 12 or 412), a CDR-H2 comprising WIDLENANTIYDAKFQG (SEQ ID NO: 14 or 414), a CDR-H3 comprising ARDHYHRYFYDV (SEQ ID NO: 416), a CDR-L1 comprising KSSQSLLNARTGHNYLA (SEQ ID NO: 422), a CDR-L2 comprising WASTRES (SEQ ID NO: 24 or 424), and a CDR-L3 comprising KQSYSHRT (SEQ ID NO: 426).

[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-74965, or (ii) a CDR-H1 comprising FNIKDYYMH (SEQ ID NO: 12 or 312), a CDR-H2 comprising WIDLENANTIYDAKFQG (SEQ ID NO: 14 or 314), a CDR-H3 comprising ARDAYHRYFYDV (SEQ ID NO: 316), a CDR-L1 comprising KSSQSLLNARTGHNYLA (SEQ ID NO: 322), a CDR-L2 comprising WASTRES (SEQ ID NO: 24 or 324), and a CDR-L3 comprising KQSHSHRT (SEQ ID NO: 326).

[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-79842, or (ii) a CDR-H1 comprising FNIKDYYMH (SEQ ID NO: 12 or 712), a CDR-H2 comprising WIDLENANTIYDAKFQG (SEQ ID NO: 14 or 714), a CDR-H3 comprising ARDHYGRYFYDV (SEQ ID NO: 716), a CDR-L1 comprising KSSQSLLNARTGKNYLA (SEQ ID NO: 722), a CDR-L2 comprising WASTRES (SEQ ID NO: 24 or 724), and a CDR-L3 comprising KQSYSRRT (SEQ ID NO: 726).

[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-79843, or (ii) a CDR-H1 comprising FNIKDYYMH (SEQ ID NO: 12 or 812), a CDR-H2 comprising WIDLENANTIYDAKFQG (SEQ ID NO: 14 or 814), a CDR-H3 comprising ARDAYHRYFYDV (SEQ ID NO: 816), a CDR-L1 comprising KSSQSLLNARTGKNYLA (SEQ ID NO: 822), a CDR-L2 comprising WASTRES (SEQ ID NO: 24 or 824), and a CDR-L3 comprising KQSYSRRT (SEQ ID NO: 826).

[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-79848, or (ii) a CDR-H1 comprising FNIKDYYMH (SEQ ID NO: 12 or 912), a CDR-H2 comprising WIDLENANTIYDAKFQG (SEQ ID NO: 14 or 914), a CDR-H3 comprising ARDAYGRYFYDV (SEQ ID NO: 916), a CDR-L1 comprising KSSQSLLNARTGHNYLA (SEQ ID NO: 922), a CDR-L2 comprising WASTRES (SEQ ID NO: 24 or 924), and a CDR-L3 comprising KQSYSRRT (SEQ ID NO: 926).

[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-79844, or (ii) a CDR-H1 comprising FNIKDYYMH (SEQ ID NO: 12 or 1012), a CDR-H2 comprising WIDLENANTIYDAKFQG (SEQ ID NO: 14 or 1014), a CDR-H3 comprising ARDAYGRYFYDV (SEQ ID NO: 1016), a CDR-L1 comprising KSSQSLLNARTGKNYLA (SEQ ID NO: 1022), a CDR-L2 comprising WASTRES (SEQ ID NO: 24 or 1024), and a CDR-L3 comprising KQSHSRRT (SEQ ID NO: 1026).

[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-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).

[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-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).

[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-79846, or (ii) a CDR-H1 comprising FNIKDYYMH (SEQ ID NO: 12 or 1312), a CDR-H2 comprising WIDLENANTIYDAKFQG (SEQ ID NO: 14 or 1314), a CDR-H3 comprising ARDHYHRYFYDV (SEQ ID NO: 1316), a CDR-L1 comprising KSSQSLLNARTGHNYLA (SEQ ID NO: 1322), a CDR-L2 comprising WASTRES (SEQ ID NO: 24 or 1324), and a CDR-L3 comprising KQSHSRRT (SEQ ID NO: 1326).

[0030] In some cases, in the anti-CD3 antibody or antigen-binding fragment of any of the above embodiments, (A) the VH polypeptide comprises (a) a VH framework region 1 (FR-H1) comprising: (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) the amino acid sequence of 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) comprising: (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) the amino acid sequence of WVRQAPGQRLEWMG (SEQ ID NO: 13, 613, 513, 413, 313, 713, 813, 913, 1013, 1113, 1213, or 1313). (c) VH framework region 3 (FR-H3), including: (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) RVTITRDTSASTAYMX1LSSLR SEDTAVYYC (SEQ ID NO: 15) (wherein X1 is E or G), and / or (iii) a VH comprising the amino acid sequence of RVTITRDTSASTAYMELSSLRSEDTAVYYC (SEQ ID NO: 615, 515, 415, 315, 715, 815, 915, 1015, 1115, or 1315) or RVTITRDTSASTAYMGLSSLRSEDTAVYYC (SEQ ID NO: 1215).and / or (d) a VH framework region 4 (FR-H4) comprising: (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) a VH comprising the amino acid sequence of 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) a VL framework region 1 (FR-L1) comprising: (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) a VL comprising the amino acid sequence of DIVMTQSPDSLAVSLGERATINC (SEQ ID NO: 621, 421, 321, 721, 821, 921, 1021, 1121, 1221, or 1321) or GIVMTQSPDSLAVSLGERATINC (SEQ ID NO: 521). (b) a VL framework region 2 (FR-L2) comprising: (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) the amino acid sequence of WYQQKPGQPPKLLIY (SEQ ID NO: 23, 623, 523, 423, 323, 723, 823, 923, 1023, 1123, 1223, or 1323).(c) a VL framework region 3 (FR-L3) comprising: (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) the amino acid sequence of GVPDRFSGSGSGTDFTLTISSLQAEDVAVYYC (SEQ ID NO: 25, 625, 525, 425, 325, 725, 825, 925, 1025, 1125, 1225, or 1325). and / or (d) a VL framework region 4 (FR-L4) comprising: (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) a VL comprising the amino acid sequence of FGGGTKVEIK (SEQ ID NO: 27, 627, 527, 427, 327, 727, 827, 927, 1027, 1127, 1227, or 1327). Alternatively, the anti-CD3 antibody or antigen-binding fragment comprising FR-L4 can comprise a VH and / or VL comprising any combination of the VH and VL framework regions described above.

[0031] In certain cases, the anti-CD3 antibody or antigen-binding fragment of any of the above embodiments is selected from the group consisting of (i) 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) QVQLVQSG FR-H1 containing AEVKKPGASVKVSCKASG (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, 1013, 1113, or 1313), FR-H3 containing the sequence SEQ ID NO: 17, 617, 417, 317, 717, 817, 917, 1017, 1117, or 1317), FR-H4 containing the sequence SEQ ID NO: 17, 617, 417, 317, 717, 817, 917, 1017, 1117, or 1317), FR-L1 containing the sequence SEQ ID NO: 17, 617, 417, 317, 717, 817, 917, 1017, 1117, or 1317), FR-L2 containing the sequence SEQ ID NO: 17, 617, 417, 317, 717, 817, 917, 1017, 1117, or 1317), FR-L3 containing the sequence SEQ ID NO: 17, 617, 417, 317, 717, 817, 917, 1017, 1117, or 1317), FR-L4 containing the sequence SEQ ID NO: 17, 617, 417, 317, 717, 817, 917, 1017, 1117, or 1317), FR-L5 containing the sequence SEQ ID NO: 17, 617, 417, 317, 717, 817, 917, 1017, 1117, or 1317), FR-L6 containing the sequence SEQ ID NO: 17, 617, 417, 317, 717, 817, 917, 1 , 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).

[0032] In certain cases, the anti-CD3 antibody or antigen-binding fragment of any of the above embodiments is selected from the group consisting of (i) 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), RVTITRDTSASTAYMELSSLRSEDTA The FR-L1 may include FR-H3 comprising VYYC (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).

[0033] In certain cases, the anti-CD3 antibody or antigen-binding fragment of any of the above embodiments comprises (i) 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, WVRQAPGQRLEWMG (sequence number 11 or 1211), including QVQLVQSGAEVKKPGASVKVSCKASG (sequence number 11 or 1211). The fragments may include FR-H2 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).

[0034] In certain embodiments, the anti-CD3 antibody or antigen-binding fragment comprises (i) 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

[0035] (ii) may comprise 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.

[0036] In certain embodiments, the anti-CD3 antibody or antigen-binding fragment comprises (i) 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) SEQ ID NO: 51, respectively. 1, 512, 513, 514, 515, 516, 517, 521, 522, 523, 524, 525, 526, and 527, including 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.

[0037] In certain embodiments, the anti-CD3 antibody or antigen-binding fragment comprises (i) 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) SEQ ID NO: 41, respectively. 1, 412, 413, 414, 415, 416, 417, 421, 422, 423, 424, 425, 426, and 427, including 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.

[0038] In certain embodiments, the anti-CD3 antibody or antigen-binding fragment comprises (i) 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) SEQ ID NO: 31, respectively. 1, 312, 313, 314, 315, 316, 317, 321, 322, 323, 324, 325, 326, and 327, including 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.

[0039] In certain embodiments, the anti-CD3 antibody or antigen-binding fragment comprises (i) 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) SEQ ID NO: 71, respectively. 1, 712, 713, 714, 715, 716, 717, 721, 722, 723, 724, 725, 726, and 727, including 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.

[0040] In certain embodiments, the anti-CD3 antibody or antigen-binding fragment comprises (i) 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) SEQ ID NO: 81, respectively. 1, 812, 813, 814, 815, 816, 817, 821, 822, 823, 824, 825, 826, and 827, including 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.

[0041] In certain embodiments, the anti-CD3 antibody or antigen-binding fragment comprises (i) 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) SEQ ID NO: 91, respectively. 1, 912, 913, 914, 915, 916, 917, 921, 922, 923, 924, 925, 926, and 927, including 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.

[0042] In certain embodiments, the anti-CD3 antibody or antigen-binding fragment comprises (i) 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) SEQ ID NOs: 1011, 1012, respectively. , 1013, 1014, 1015, 1016, 1017, 1021, 1022, 1023, 1024, 1025, 1026, and 1027, including 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.

[0043] In certain embodiments, the anti-CD3 antibody or antigen-binding fragment is selected from the group consisting of (i) 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) SEQ ID NOs: 1111, 1112, and 1113, respectively. , 1113, 1114, 1115, 1116, 1117, 1121, 1122, 1123, 1124, 1125, 1126, and 1127, including 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.

[0044] In certain embodiments, the anti-CD3 antibody or antigen-binding fragment is selected from the group consisting of (i) 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) SEQ ID NOs: 1211, 1212, 1213, 1214, 1215, 1216, 1217, 1218, 1219, 1220, 1221, 1222, 1223, 1224, 1225, 1226, 1227, 1228, 1229, 1230, 1231, 1232, 1233, 1234, 1235, 1236, 1237, 1238, 1239, 1240, 1241, 1242, 1243, 1244, 1245, 1246, 1247, 1248, 1249, 1250, 1251, 1252, 1253, 1254, 1255, 1256, 1257, 1258, 1259, 1260, 1261, 1262, 1263, 1264, 1265, 1266, 1267, 1268, 1269, 1270, 1271, 1272, 1273, 12 , 1213, 1214, 1215, 1216, 1217, 1221, 1222, 1223, 1224, 1225, 1226, and 1227, including 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.

[0045] In certain embodiments, the anti-CD3 antibody or antigen-binding fragment is selected from the group consisting of (i) 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) SEQ ID NOs: 1311, 1312, 1313, 1314, 1315, 1316, 1317, 1318, 1319, 1319, 1320, 1321, 1322, 1323, 1324, 1325, 1326, 1327, 1328, 1329, 1330, 1331, 1332, 1333, 1334, 1335, 1336, 1337, 1338, 1339, 1340, 1341, 1342, 1343, 1344, 1345, 1346, 1347, 1348, 1349, 1350, 1351, 1352, 1353, 1354, 1355, 1356, 1357, 1358, 1359, 1360, 1361, 1362, 1363, 1364, 1365, 1366, 1367, 1368, 1369, 1370, 1371, 1372, 13 2, 1313, 1314, 1315, 1316, 1317, 1321, 1322, 1323, 1324, 1325, 1326, and 1327, including 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.

[0046] In certain embodiments, the anti-CD3 antibody or antigen-binding fragment comprises: (A) an amino acid sequence 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 VH polypeptide, and / or a VL polypeptide comprising an amino acid sequence 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.

[0047] 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 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 at least 90% identical to SEQ ID NO: 620.

[0048] 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: 512, 514, and 516, respectively, and (ii) an amino acid sequence 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 NOs: 522, 524, and 526, respectively, and (ii) an amino acid sequence at least 90% identical to SEQ ID NO: 520.

[0049] In certain embodiments, the anti-CD3 antibody or antigen-binding fragment may comprise a VH polypeptide and a VL polypeptide comprising the amino acid sequences of SEQ ID NOs: 610 and 620, respectively.

[0050] In certain embodiments, the anti-CD3 antibody or antigen-binding fragment may comprise a VH polypeptide and a VL polypeptide comprising the amino acid sequences of SEQ ID NOs: 510 and 520, respectively.

[0051] In certain embodiments, the anti-CD3 antibody or antigen-binding fragment may comprise a VH polypeptide and a VL polypeptide comprising the amino acid sequences of SEQ ID NOs: 410 and 420, respectively.

[0052] In certain embodiments, the anti-CD3 antibody or antigen-binding fragment may comprise a VH polypeptide and a VL polypeptide comprising the amino acid sequences of SEQ ID NOs: 310 and 320, respectively.

[0053] In certain embodiments, the anti-CD3 antibody or antigen-binding fragment may comprise a VH polypeptide and a VL polypeptide comprising the amino acid sequences of SEQ ID NOs: 710 and 720, respectively.

[0054] In certain embodiments, the anti-CD3 antibody or antigen-binding fragment may comprise a VH polypeptide and a VL polypeptide comprising the amino acid sequences of SEQ ID NOs: 810 and 820, respectively.

[0055] In certain embodiments, the anti-CD3 antibody or antigen-binding fragment may comprise a VH polypeptide and a VL polypeptide comprising the amino acid sequences of SEQ ID NOs: 910 and 920, respectively.

[0056] In certain embodiments, the anti-CD3 antibody or antigen-binding fragment may comprise a VH polypeptide and a VL polypeptide comprising the amino acid sequences of SEQ ID NOs: 1010 and 1020, respectively.

[0057] In certain embodiments, the anti-CD3 antibody or antigen-binding fragment may comprise a VH polypeptide and a VL polypeptide comprising the amino acid sequences of SEQ ID NOs: 1110 and 1120, respectively.

[0058] In certain embodiments, the anti-CD3 antibody or antigen-binding fragment may comprise a VH polypeptide and a VL polypeptide comprising the amino acid sequences of SEQ ID NOs: 1210 and 1220, respectively.

[0059] In certain embodiments, the anti-CD3 antibody or antigen-binding fragment may comprise a VH polypeptide and a VL polypeptide comprising the amino acid sequences of SEQ ID NOs: 1310 and 1320, respectively.

[0060] 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, the CH1 domain, the hinge, the CH2 domain, and / or the CH3 domain may be, or may be derived from, IgG or human IgG, respectively. In certain cases, the antibody constant region, the CH1 domain, the hinge, the CH2 domain, and / or the CH3 domain may be, or may be derived from, human IgG1, IgG4, IgG2, or IgG3, respectively.

[0061] In some embodiments, the anti-CD3 antibody or antigen-binding fragment may comprise a fragment crystallizable (Fc) region.

[0062] In certain embodiments, the Fc region may be a region of human IgG1. In some cases, the Fc region may contain the following amino acid modifications according to EU numbering: 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.

[0063] In certain embodiments, the Fc region may be a region of human IgG4. In some cases, the Fc region may include one or more of the following amino acid modifications according to EU numbering: E233P, F234V, L235A, G237A, E318A, S228P, L236E, S241P, L248E, T394D, M252Y, S254T, T256E, N297A, N297Q, or any combination thereof.

[0064] In certain embodiments, the Fc region may be a region of human IgG2. In some cases, the Fc region may include one or more of the following amino acid modifications according to EU numbering: P238S, V234A, G237A, H268A, H268Q, H268E, V309L, N297A, N297Q, A330S, P331S, C232S, C233S, M252Y, S254T, T256E, or any combination thereof.

[0065] In certain embodiments, the Fc region may be that of human IgG3. In some cases, the Fc region may include E235Y according to EU numbering.

[0066] 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.

[0067] In some embodiments, the anti-CD3 antibody or antigen-binding fragment may comprise an antibody fragment selected from the group consisting of a fragment antigen-binding (Fab), Fab2, Fab3, a Fab' fragment, F(ab')2, a variable fragment (Fv), a single-chain Fv (scFv) fragment, a diabody, a triabody, a minibody, scFv-Fc, scFv2-Fc2, scFv-IgG, a monovalent IgG (or half-IgG), and / or a chimeric antigen receptor (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, and further optionally from CD3ζ).

[0068] In some embodiments, the anti-CD3 antibody or antigen-binding fragment may comprise or be included in a multispecific antibody or antibody fragment having at least (a) a first antigen-binding region specific for CD3, comprising said VH polypeptide and / or said VL polypeptide, and (b) a second antigen-binding region.

[0069] 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 viral, bacterial, or fungal target molecule), a target molecule expressed on an infected cell (optionally a cell infected with a virus, bacteria, or fungus), a metabolic disease target, a cognitive disorder target, a blood-brain barrier target, or a hematological disease target.

[0070] In certain embodiments, the second antigen-binding region is selected from the group consisting of 17-IA, 4-1BB, 4Dc, 6-keto-PGF1a, 8-iso-PGF2a, 8-oxo-dG, Al adenosine receptor, A33, ACE, ACE-2, activin, activin A, activin AB, activin B, activin C, activin RIA, activin RIA ALK-2, activin RIB ALK-4, activin RIIA, activin RUB, ADAM, ADAM10, ADAM12, ADAM 15, ADAM 17 / T ACE, ADAM8, ADAM9, ADAMTS, ADAMTS4, ADAMTS5, addressin, aFGF, ALCAM, ALK, ALK-1, ALK-7, alpha-l-antitrypsin, alpha-V / beta-1 antagonist, ANG, Ang, APAF-1, APE, APJ, APP, APRIL, AR, ARC, ART, artemin, anti-Id, ASPARTIC, atrial natriuretic factor, av / b3 integrin, Ax l, b2M, B7-1, B7-2, B7-H, B lymphocyte stimulating factor (BlyS), BACE, BACE-1, Bad, BAFF, BAFF-R, Bag-1, BAK, Bax, BCA-1, BCAM, Bel, BCMA , BDNF, b-ECGF, bFGF, BID, Bik, BFM, BLC, BL-CAM, BLK, BMP, BMP-2, BMP-2a, BMP-3 osteogenin, BMP-4, BMP-2b, BMP-5, BMP-6Vgr-1, BMP-7 (OP-1), BMP-8 (BMP-8a, OP-2), BMPR, BMPR-IA (ALK-3), BMPR-IB (ALK-6), BRK-2, RPK-1, BMPR-II (BRK-3), BMP, b-NGF, BOK, bombesin, bone-derived neurotrophic factor, BPDE, BPDE-DNA, BTC, complement factor 3 (C3), C3a, C4, C5, C5a, CIO, CA125, CAD-8, calcitonin, cAMP, carcinoembryonic antigen (CEA), cancer-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, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL2, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CCL3, CCL4, CCL5, CCL6, CCL7, C CL8, CCL9 / 10, CCR, CCR1, CCR10, CCR10, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CD1, CD2, CD4, CD5, CD6, CD7, CD8, CD10, CD11a, CD11 b, CD11c, CD13, CD14, CD15, CD16, CD18, CD19, CD20, CD21, CD22, CD23, CD25, CD27L, CD28, CD29, CD30, CD30L, CD32, CD33 (p67 protein), CD34, C D38, 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, CMVUL, CNTF, CNTN-1, COX, C-Ret, CRG-2, CT-1, CTACK, CTGF, CTLA-4, CX3CL1, CX3CR1, CXCL, CXCL1, CXCL2, CXCL3, CXCL4, CXC L5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCR, CXCR1, CXCR2, CXCR3, CX CR4, 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, EDA-A1, EGA-A2, EDAR, EGF, EGFR (ErbB-1), EMA, EMMPRIN, EN A, endothelin receptor, enkephalinase, eNOS, Eot, eotaxin, EpCAM, ephrin B2 / EphB4, EPO, ERCC, E-selectin, ET-1, factor Ila, factor VII, factor VIIIc, factor IX, fibroblast activation protein (FAP), Fas, FcRl, FEN-1, ferritin, FGF, FGF-19, FGF-2, FGF3, FGF-8, FGFR, FGFR-3, fibrin, FL, FLIP, Flt-3, Flt-4, follicle-stimulating hormone, fractalkine, FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, FZD10, G250, Gas 6, GCP-2, GCSF, GD2, GD3, GDF, GDF-1, GDF-3 (Vgr-2), GDF-5 (BMP-14, CDMP-1), GDF-6 (BMP-13, CDMP-2), GDF-7 (BMP-12, CDMP-3), GDF-8 (myostatin), GDF-9, GDF-15 (MIC-1), GDNF, GFAP, GFRa-1, GFR-alpha1, GFR-alpha2, GFR-alpha3, GITR, glucagon, Glut 4, glycoprotein IIb / IIIa (GP IIb / IIIa), GM-CSF, gpl30, gp72, GRO, growth hormone-releasing factor, hapten (NP-cap or NIP-cap), HB-EGF, HCC, HCMVgB 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, HIV IIIB gp120 V3 loop, HLA, HLA-DR, HM1.24, HMFG PEM, HRG, Hrk, human cardiac myosin, human cytomegalovirus (HCMV), human growth hormone (HGH), HVEM, 1-309, IAP, ICAM, ICAM-1, ICAM-3, ICE, ICOS, IFNg, Ig, IgA receptor, IgE, IGF, IGF-binding protein, IGF-1R, IGFBP, IGF-I, IGF-II, IL, IL-1, IL-1R, IL-2, IL-2R, IL-4, IL-4R, IL-5, IL-5R, IL-6, IL-6R, IL-8, IL-9, IL-10, IL-12, IL-13, IL-15, IL-18, IL-18R, IL-23, interferon (INF)-alpha, INF-beta, INF-gamma, inhibin, iNOS, insulin A chain, insulin B chain, insulin-like growth factor 1, interferon (INF)-alpha, interferon (INF)-beta, interferon (INF) ... Integrin alpha 2, integrin alpha 3, integrin alpha 4, integrin alpha 4 / beta 1, integrin, alpha 4 / beta 7, integrin alpha 5 (alpha V), integrin alpha 5 / beta 1, integrin alpha 5 / beta 3, integrin alpha 6, integrin beta 1, integrin beta 2, interferon gamma, IP-10, l-TAC, JE, kallikrein 2, kallikrein 5, kallikrein 6, kallikrein 11, kallikrein 12, kallikrein 14, kallikrein 15, kallikrein LI, kallikrein L2, kallikrein L3, kallikrein L4, KC, KDR, keratinocyte growth factor (KGF), laminin 5, LAMP, LAP, LAP (TGF-1), latent TGF-1, latent TGF-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 beta receptor, Mac-1, MAdCAM, MAG, MAP2, MARC, MCAM, MCAM, MCK-2, MCP, M-CSF, MDC, Mer, metalloproteinase, MGDF receptor, MGMT, MHC (HLA-DR), MIF, MIG, MIP, MIP-1-alpha, MK, MMAC1, MMP, MMP-1, MMP-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, neprilysin, neurotrophin-3, -4, or -6, neurturin, nerve cell growth factor (NGF), NGFR, NGF-beta, nNOS, NO, NOS, Npn, NRG-3, NT, NTN, OB, OGG1, OPG, OPN, OSM, OX40L, OX40R, pl50, p95, PADPr, parathyroid hormone, PARC, PARP, PBR, PBSF, PCAD, P-cadherin, PCNA, PDGF, PD K-1, PECAM, PEM, PF4, PGE, PGF, PGI2, PGJ2, PIN, PLA2, placental alkaline phosphatase (PLAP), PIGF, PLP, PP14, proinsulin, prorelaxin, protein C, PS, PSA, PSCA, prostate-specific membrane antigen (PSMA), PTEN, PTHrp, Ptk, PTN, R51, RANK, RANKL, RANTES, relaxin A chain, relaxin B chain, renin, respiratory syncytial virus (RSV) F, RSVFgp, Ret, rheumatoid factor, RLIP76, RPA2, RSK, S100, SCF / KL, SDF-1, SERINE, serum albumin, sFRP-3, Shh, SIGIRR, SK-1, SLAM, SLPI, SMAC, SMDF, SMOH, SOD, SPARC, Stat, STEAP, STEAP-II, TACE, TACI, TAG-72 (tumor-associated glycoprotein-72), TARC, TCA-3, T cell receptor (e.g., T cell receptor alpha / beta), TdT, TECK, TEM1, TEM5, TEM7, TEM8, TERT, testicular PLAP-like alkaline phosphatase, TfR, TGF, TGF-alpha, TGF-beta, TGF-beta Pan-specific, TGF-beta RI (ALK-5), TGF-beta RII, TGF-beta RIIb, TGF-beta RIII, TGF-beta 1, TGF-beta 2, TGF-beta 3, TGF-beta 4, TGF-beta 5, thrombin, thymic Ck-1, thyroid-stimulating hormone, Tie, TIMP, TIQ, tissue factor, TMEFF2, Tmpo, TMPRSS2, TNF, TNF-alpha, TNF-alpha beta, TNF-beta 2, TNFc, TNF-RI, TNF-RII, TNFRSF10A (TRAIL) 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(BAFF R)、TNFRSF14(WHO ATAR、HveA、LIGHT R、TR2)、TNFRSF16(NGFR p75NTR)、TNFRSF17(BCMA)、TNFRSF18(GITR AITR)、TNFRSF19(TROY CROWN、TRADE)、TNFRSF19L(RELT)、TNFRSFIA(TNF RI CD120a、p55-60)、TNFRSFIB(TNF RII CD120b、p75-80)、TNFRSF26(TNFRH3)、TNFRSF3(LTbR TNF RIII) TNFC R), TNFRSF4(OX40 ACT35, TXGP1 R), TNFRSF 5(CD40 p50), TNFRSF6(Fas Apo-1, APT1, CD95), TNFRSF6B(DcR3). M68, TR6, TNFRSF7(CD27), TNFRSF8(CD30), TNFRSF9(4-1BB CD137, ILA), TNFRSF21(DR6), TNFRSF22(DcTRAIL R2 TNFRH2), TNFRST23(DcTRAIL). R1, TNFRH1, TNFRSF25 (DR3 Apo-3, LARD, TR-3, TRAMP, WSL-1), TNFSF10 (TRAIL). Apo-2 polymer TL2) TNFSF11(TRANCE / RANK dimer ODF) TNFSF12(TWEAK). Apo-3リンド, DR3リンド, TNFSF13(APRIL TALL2), TNFSF13B(BAFF LIGHT, TALL1, THANK, TNFSF20), TNFSF14(LIGHT HVEM glycoprotein LTg, TNFSF15(TLIA / VEGI), TNFSF18 (GITR copy AITR copy TL6), TNFSF1A (TNF-a diphtheria, DIF, TNFSF2, TNFSF1B(TNF-b LTa, TNFSF1), TNFSF3(LTb TNFC, p33, TNFSF4(OX40 substrate gp34, TXGP1), TNFSF5(CD40 substrate, CD154, gp39, HIGM1, IMD3, TRAP), TNFSF6(Fasリガンド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, transferrin receptor, TRF, Trk, TROP-2, TSG, TSLP, tumor-associated antigen CA 125, tumor-associated antigen expressing Lewis Y-related carbohydrate, TWEAK, TXB2, Ung, uPAR, uPAR-1, urokinase, VCAM, VCAM-1, VECAD, VE-cadherin, VE-cadherin-2, VEFGR-1 (flt-1), VEGF, VEGFR, VEGFR-3 (flt-4), VEGI, VFM, viral antigen, VLA, VLA-1, VLA-4, VNR integrin, von Willebrand factor, WIF-1, WNT1, WNT2, WNT2B / 13, WNT3, WNT3A, WNT4, WNT5A, WNT5B, WNT6, WNT7 A, WNT7B, WNT8A, WNT8B, WNT9A, WNT9A, WNT9B, WNT10A, WNT10B, WNT11, WNT16, XCL1, XCL2, XCR1, XCR1, XEDAR, XIAP, XPD, CTLA4 (cytotoxic T lymphocyte antigen-4), PD1 (programmed cell death protein 1), PD-L1 (programmed cell death ligand 1), LAG-3 (lymphocyte activation gene-3), TIM-3 (T-cell immunoglobulin and mucin protein-3), and a hormone receptor and a growth factor.

[0071] In certain embodiments, the second antigen-binding region may 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, cytokine, IL-2, IL-5, IL-13, IL-6, IL-17, IL-12, IL-23, TNFa, TGFb, cytokine receptor, IL-2R, chemokine, chemokine receptor, growth factor, VEGF, and HGF.

[0072] In certain embodiments, a multispecific antibody or antibody fragment may be bispecific. In certain embodiments, a multispecific antibody or antibody fragment may further comprise a third antigen-binding region. In certain embodiments, a multispecific antibody or antibody fragment may be trispecific.

[0073] In certain embodiments, the multispecific antibody or antibody fragment may comprise a multispecific format selected from the group consisting of Fab-Fc-scFv, scFv2-Fc2, scFv-IgG, "bottle opener", Mab-scFv, Mab-Fv, dual-scFv, central Fv, central-scFv, one-arm central-scFv, Fab-Fab, Fab-Fv, mAb-Fv, mAb-Fab, DART, BiTE, common light chain-IgG, TandAb, crossoverMab, SEED, BEAT, TrioMab, and DuetMab.

[0074] In certain embodiments, the multispecific antibody or antibody fragment may comprise at least one CLκ-preferred variant CH1 domain, optionally a CLκ-preferred variant CH1 domain described in WO2021067404.

[0075] In certain embodiments, the multispecific antibody or antibody fragment may comprise at least one CLλ-preferred variant CH1 domain, optionally a CLλ-preferred variant CH1 domain described in WO2021067404.

[0076] In certain embodiments, the multispecific antibody or antibody fragment may comprise at least one pair of variant CH1 domain and variant CL domain, optionally a pair described in WO2022150787, that preferentially pair with each other.

[0077] In certain embodiments, the multispecific antibody or antibody fragment may comprise at least one pair of a variant CH3 domain and another variant CH3 domain, optionally a pair described in WO2022150785, that preferentially pair with each other.

[0078] In some embodiments, the anti-CD3 antibody or antigen-binding fragment may bind to CD3 with higher binding affinity or avidity at an 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 via (1) surface plasmon resonance (SPR), optionally using a BIACORE® system (GE healthcare or CYTIVA®), (2) biolayer interferometry (BLI), optionally using an OCTET® system, (3) enzyme-linked immunosorbent assay (ELISA), and / or (4) radioimmunoassay (RIA). In certain embodiments, the CD3 may be (1) human CD3, optionally CD3εδ, and / or (2) non-human primate, optionally monkey, and further optionally cynomolgus CD3, optionally CD3εδ. In certain embodiments, 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 at a pH more acidic than physiological pH (optionally, about pH 6.0) based on the equilibrium dissociation constant (Kd) value. 3 , at least x 10 4 , at least x 10 5 , at least x 10 6 , at least x 10 7 , at least x 10 8 , or at least x10 9 In certain embodiments, the anti-CD3 antibody or antigen-binding fragment is unable to bind to CD3 at physiological pH, optionally at about pH 7.4.

[0079] In some embodiments, the anti-CD3 antibody or antigen-binding fragment may bind to human CD3 (optionally CD3γδ) at an acidic pH (optionally about pH 6.0).

[0080] In certain embodiments, the anti-CD3 antibody or antigen-binding fragment has a cytotoxicity of 1.57×10 at an acidic pH (optionally about pH 6.0). 8 (M) Less than 1.0 × 10 8 (M) Less than 9.0 x 10 9 (M) Less than 8.0 x 10 9 (M) Less than 7.0 x 10 9 (M) Less than 6.0 x 10 9 (M) Less than 5.0 x 10 9 (M) Less than 4.0 × 10 9 (M) Less than 3.0 x 10 9 (M) Less than 2.0 x 10 9 (M) Less than 1.0 × 10 9 (M) Less than 9.0 x 10 10 (M) or less than 8.0 x 10 10 (M) or less. In some cases, the Kd value may be measured via SPR, optionally using a BIACORE® system.

[0081] In certain embodiments, the anti-CD3 antibody or antigen-binding fragment is 1.56 x 10 8 (M) ~ 7.0 x 10 10 (M), 1.0 × 10 8 (M) ~ 7.0 x 10 10 (M), 1.0 × 10 8 (M) ~ 1.0 × 10 9 (M), 2.0 × 10 8 (M) ~ 1.0 × 10 9 (M), or 5.0 × 10 8 (M) ~ 1.0 × 10 9 (M) can bind to human CD3 (optionally CD3εδ) at an acidic pH (optionally about pH 6.0) with a Kd value of (M). In some cases, the Kd value can be measured via SPR, optionally using a BIACORE® system.

[0082] In certain embodiments, the anti-CD3 antibody or antigen-binding fragment is 9.33 x 10 9 (M) Less than 9.0 x 10 9 (M) Less than 8.0 x 109 (M) Less than 7.0 x 10 9 (M) Less than 6.0 x 10 9 (M) Less than 5.0 x 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) or less. In some cases, the Kd value may be measured via BLI, optionally using an OCTET® system.

[0083] In certain embodiments, the anti-CD3 antibody or antigen-binding fragment is 9.32 x 10 9 (M) ~ 1.0 × 10 9 (M), 9.0 x 10 9 (M) ~ 1.0 × 10 9 (M), 8.0 x 10 9 (M) ~ 3.0 × 10 9 (M), 7.0 x 10 9 (M) ~ 4.0 × 10 9 , or 6.0 × 10 9 (M) ~ 5.0 × 10 9 (M) can bind to human CD3 (optionally CD3εδ) at an acidic pH (optionally about pH 6.0) with a Kd value of (M). In some cases, the Kd value can be measured via BLI, optionally using an OCTET® system.

[0084] In some embodiments, the anti-CD3 antibody or antigen-binding fragment may bind to cynomolgus monkey CD3 (optionally CD3εδ) at an acidic pH (optionally about pH 6.0).

[0085] In certain embodiments, the anti-CD3 antibody or antigen-binding fragment is 2.0 x 10 8 (M) Less than 1.0 × 10 8 (M) Less than 9.0 x 10 9 (M) Less than 8.0 x 10 9 (M) Less than 7.0 x 10 9 (M) Less than 6.0 x 10 9(M) Less than 5.0 x 10 9 (M) Less than 4.0 × 10 9 (M) Less than 3.0 x 10 9 (M) or less than 2.0 × 10 9 (M) or less. In some cases, the Kd value may further optionally be measured via BLI using an OCTET® system.

[0086] In certain embodiments, the anti-CD3 antibody or antigen-binding fragment is 2.0 x 10 8 (M) ~ 1.0 × 10 9 (M), 1.0 × 10 8 (M) ~ 1.0 × 10 9 (M), 9.0 x 10 9 (M) ~ 2.0 × 10 9 (M), or 8.0 × 10 9 (M) ~ 5.0 × 10 9 The antibody may bind to cynomolgus monkey CD3 (optionally CD3εδ) at an acidic pH (optionally about pH 6.0) with a Kd value of 0.01 to 0.02. In some cases, the Kd value may further optionally be measured via BLI using an OCTET® system.

[0087] In some embodiments, the anti-CD3 antibody or antigen-binding fragment may bind to CD3-expressing cells with stronger binding at an acidic pH (optionally about pH 6.0) than at physiological pH (optionally about pH 7.4).

[0088] In certain embodiments, cell binding may be measured via flow cytometry, and optionally based on median fluorescence intensity (MFI). In certain embodiments, the cells may express human CD3, optionally CD3εδ. In certain embodiments, the cells may express non-human primate (optionally monkey, and optionally cynomolgus monkey) CD3 (optionally CD3εδ). In certain embodiments, the cells may be primary cells. In certain embodiments, the cells may be cells of a cell line. In certain embodiments, the cells may be human cells. In certain embodiments, the cells may be human T cells. In certain embodiments, the cells may be Jurkat cells. In certain embodiments, the cells may be non-human primate (optionally monkey, and optionally cynomolgus monkey) cells. In certain embodiments, the cells may be HSC-F cells.

[0089] In certain embodiments, binding to CD3-expressing cells, as measured via flow cytometry, 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 at a pH more acidic than physiological pH (optionally about pH 6.0) based on an NCB value calculated using an MFI value. 3 , at least x 10 4 , at least x 10 5 , at least x 10 6 , at least x 10 7 , at least x 10 8 , or at least x10 9In some cases, the NCB value is calculated as {(sample MFI)-(secondary only MFI)} / (secondary only MFI), or in other words, {(MFI for "incubation with test antibody, then incubation with secondary antibody")-(MFI for incubation without primary antibody, then incubation with secondary antibody)} / (MFI for incubation without primary antibody, then incubation with secondary antibody).

[0090] In certain embodiments, the binding to CD3-expressing cells is incapable of binding to CD3-expressing cells at physiological pH (optionally about pH 7.4).

[0091] In some embodiments, the anti-CD3 antibody or antigen-binding fragment, upon binding to CD3 on a cell (optionally, a T cell), may induce activation of the cell and / or enhance the cytotoxic function of the cell.

[0092] In certain embodiments, while eliciting T cell activation or T cell killing, they may exhibit a reduced tendency to elicit cytokine production to levels that can induce cytokine release syndrome (CRS). In certain embodiments, upon binding to CD3 on a cell (optionally a T cell), an anti-CD3 antibody or antigen-binding fragment may not, upon binding to CD3, elicit cytokine production to levels that can induce CRS.

[0093] In some 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 comprising said VH polypeptide and / or said VL polypeptide, and (b) a second antigen-binding region specific for a second antigen, wherein upon binding to (i) CD3 on a first cell (optionally a T cell) and (ii) a second antigen expressed on the second cell, the first cell may exhibit cytotoxicity against the second cell.

[0094] In another aspect, the present disclosure provides nucleic acids (e.g., isolated or recombinant nucleic acids, such as a nucleic acid, a combination of two or more nucleic acids, one or more nucleic acids) encoding any of the anti-CD3 antibodies and antigen-binding fragments described herein. Such nucleic acids can include, for example, DNA, such as cDNA, or RNA, such as mRNA, for delivery to a cell and expression of the anti-CD3 antibody or antigen-binding fragment.

[0095] In some embodiments, a nucleic acid according to the disclosure comprises: (A) a nucleic acid 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.

[0096] In certain embodiments, the nucleic acid may comprise the nucleic acid sequences encoding the VH and VL polypeptides of SEQ ID NOs: 650 and 660, respectively.

[0097] In certain embodiments, the nucleic acid may comprise (I) a nucleic acid sequence encoding a VH polypeptide and a nucleic acid sequence encoding a VL polypeptide of SEQ ID NOs: 550 and 560, respectively.

[0098] In certain embodiments, the nucleic acid may comprise (I) a nucleic acid sequence encoding a VH polypeptide and a nucleic acid sequence encoding a VL polypeptide of SEQ ID NOs: 450 and 460, respectively.

[0099] In certain embodiments, the recombinant nucleic acid may comprise (I) a nucleic acid sequence encoding a VH polypeptide and a nucleic acid sequence encoding a VL polypeptide of SEQ ID NOs: 350 and 360, respectively.

[0100] In certain embodiments, the nucleic acid may comprise (I) a nucleic acid sequence encoding a VH polypeptide and a nucleic acid sequence encoding a VL polypeptide of SEQ ID NOs: 750 and 760, respectively.

[0101] In certain embodiments, the nucleic acid may comprise (I) a nucleic acid sequence encoding a VH polypeptide and a nucleic acid sequence encoding a VL polypeptide of SEQ ID NOs: 850 and 860, respectively.

[0102] In certain embodiments, the nucleic acid may comprise (I) a nucleic acid sequence encoding a VH polypeptide and a nucleic acid sequence encoding a VL polypeptide of SEQ ID NOs: 950 and 960, respectively.

[0103] In certain embodiments, the nucleic acid may comprise (I) a nucleic acid sequence encoding a VH polypeptide and a nucleic acid sequence encoding a VL polypeptide of SEQ ID NOs: 1050 and 1060, respectively.

[0104] In certain embodiments, the nucleic acid may comprise (I) a nucleic acid sequence encoding a VH polypeptide and a nucleic acid sequence encoding a VL polypeptide of SEQ ID NOs: 1150 and 1160, respectively.

[0105] In certain embodiments, the nucleic acid may comprise (I) a nucleic acid sequence encoding a VH polypeptide and a nucleic acid sequence encoding a VL polypeptide of SEQ ID NOs: 1250 and 1260, respectively.

[0106] In certain embodiments, the nucleic acid may comprise (I) a nucleic acid sequence encoding a VH polypeptide and a nucleic acid sequence encoding a VL polypeptide of SEQ ID NOs: 1350 and 1360, respectively.

[0107] In some cases, the nucleic acid may comprise RNA (e.g., mRNA), which may include an RNA sequence encoding a VH polypeptide and an RNA sequence encoding a VL polypeptide corresponding to any of the foregoing nucleic acid sequences.

[0108] In another aspect, the present disclosure provides a vector (e.g., one or more vectors, such as a vector, a combination of two or more vectors) encoding any of the anti-CD3 antibodies and antigen-binding fragments described herein.

[0109] In some embodiments, a vector may comprise any of the nucleic acids described herein.

[0110] In some embodiments, the vector may be an expression vector.

[0111] In some embodiments, the vector may comprise a plasmid, a viral vector (optionally adenovirus, lentivirus, or 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.

[0112] In another aspect, the disclosure provides isolated or recombinant cells comprising, transfected with, transformed with, or transduced with any of the nucleic acids and / or vectors described herein.

[0113] In some embodiments, the isolated or recombinant cell may be mammalian. In certain embodiments, the isolated or recombinant cell may be human, non-human primate, monkey, rabbit, rodent, hamster, rat, or mouse. In some embodiments, the isolated or recombinant cell may be non-mammalian, optionally plant, bacterial, fungal, yeast, protozoan, or insect. In some embodiments, the isolated or recombinant cell may be an immune cell or a hybridoma.

[0114] In another aspect, the present disclosure provides a pharmaceutical composition.

[0115] In some embodiments, pharmaceutical compositions according to the present disclosure may include (A) any of the anti-CD3 antibodies and antigen-binding fragments described herein, and (B) a pharmaceutically acceptable carrier and / or excipient. In some embodiments, pharmaceutical compositions according to the present disclosure may include (A) any of the nucleic acids described herein, and (B) a pharmaceutically acceptable carrier and / or excipient. In some embodiments, pharmaceutical compositions according to the present disclosure may include (A) any of the vectors described herein, and (B) a pharmaceutically acceptable carrier and / or excipient. In some embodiments, pharmaceutical compositions according to the present disclosure may include (A) any of the isolated or recombinant cells described herein, and (B) a pharmaceutically acceptable carrier and / or excipient.

[0116] In another aspect, the 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.

[0117] In some embodiments, the method may include administering to a subject an effective amount of any of the anti-CD3 antibodies and antigen-binding fragments described herein. In some embodiments, the method may include administering to a subject an effective amount of any of the nucleic acids described herein. In some embodiments, the method may include administering to a subject an effective amount of any of the vectors described herein. In some embodiments, the method may include administering to a subject an effective amount of any of the isolated or recombinant cells described herein. In some embodiments, the method may include administering to a subject an effective amount of any of the pharmaceutical compositions described herein.

[0118] In another aspect, the disclosure provides a method of inducing cytotoxicity against cells expressing a target molecule of interest.

[0119] In some embodiments, the method may include administering to a subject an effective amount of any of the anti-CD3 antibodies and antibody fragments comprising or included in the multispecific antibodies or antibody fragments described herein. In some embodiments, the method may include administering to a subject an effective amount of a nucleic acid encoding the 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 the nucleic acid and / or encoding the 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 comprising, transfected with, transformed with, or transduced with the nucleic acid or vector. In some embodiments, the method may comprise administering to the subject an effective amount of (A) any of the anti-CD3 antibodies and antigen-binding fragments comprising or included in the multispecific antibodies or antibody fragments described herein, a nucleic acid encoding the anti-CD3 antibody or antigen-binding fragment, a vector comprising the nucleic acid, and / or an isolated or recombinant cell comprising, transfected with, transformed with, or transduced with the nucleic acid or the vector, and / or (B) a pharmaceutical composition comprising a pharmaceutically acceptable carrier and / or excipient.

[0120] 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.

[0121] In any of the above method embodiments, in some cases the method may further comprise administering to the subject an additional agent, optionally an adjuvant or therapeutic agent.

[0122] In any of the above method embodiments, in some cases the subject may have or be at risk of developing a disease, disorder, or condition.

[0123] In any of the above method embodiments, in some cases the disease, disorder, or condition comprises a cancer or neoplastic condition, an autoimmune disease, a neurodegenerative disease, an infectious disease, an inflammatory disease, or another disease.

[0124] In certain embodiments, the cancer may be a solid cancer, optionally selected from one or more of mesothelioma, malignant pleural mesothelioma, non-small cell lung cancer, small cell lung cancer, squamous cell lung carcinoma, large cell lung carcinoma, 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, kidney cancer, gastrointestinal cancer, urothelial cancer, pharyngeal cancer, head and neck cancer, rectal cancer, esophageal cancer, or bladder cancer, or metastases thereof.

[0125] In certain embodiments, the cancer is a liquid cancer, optionally including chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), multiple myeloma, acute lymphocytic leukemia (ALL), Hodgkin's lymphoma, B-cell acute lymphocytic leukemia (BALL), T-cell acute lymphocytic leukemia (TALL), small lymphocytic leukemia (SLL), B-cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkina Fascia, bladder cancer, leukemia ... Diffuse large B-cell lymphoma (DLBCL), DLBCL associated with chronic inflammation, chronic myeloid leukemia, myeloproliferative disorders, follicular lymphoma, childhood follicular lymphoma, hairy cell leukemia, small cell or large cell follicular lymphoma, malignant lymphoproliferative conditions, MALT lymphoma (extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue), marginal zone lymphoma, myelodysplasia, myelodysplastic syndrome , non-Hodgkin's lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenstrom's hypergammaglobulinemia, 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, isolated plasmacytoma of bone, extraskeletal plasmacytoma, nodular marginal zone lymphoma, pediatric nodular marginal zone lymphoma The cancer may be a liquid cancer selected from 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 in HHV8-associated multicentric Castleman disease, primary effusion lymphoma, B-cell lymphoma, acute myeloid leukemia (AML), or unclassifiable lymphoma.

[0126] In certain embodiments, the autoimmune or inflammatory disease may be psoriasis, rheumatoid arthritis, autoimmune arthritis, type 1 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, Sjogren's syndrome, Addison's disease, Behcet's disease, Schmidt's syndrome, celiac disease, dermatomyositis, autoimmune vitiligo, Graves' disease, Hashimoto's thyroiditis, Kawasaki disease, pernicious anemia, autoimmune vasculitis, or fibrosis.

[0127] In certain embodiments, the neurodegenerative disease may be 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, or Creutzfeldt-Jakob disease.

[0128] In certain embodiments, the infectious disease may be a viral disease, a bacterial disease, a fungal disease, a yeast disease, a protozoan disease, a prion disease, or a parasitic disease, and optionally: (1) the viral disease is human immunodeficiency virus (HIV), hepatitis virus (optionally hepatitis A, B, or C), 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 a common human coronavirus); (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, or Vibrio vulnificus; and / or (3) the fungal disease is Aspergillosis, Candida, Candida auris, Cryptococcus neoformans, Pneumocystis jirovecii, Mucor, Talaromyces, Ringer's disease, Blastomyces, Coccidioides, Cryptococcus gattii, Histoplasma, Paracoccidioides, or Sporothrix infection.

[0129] In another aspect, the present disclosure provides methods of producing the anti-CD3 antibodies or antigen-binding fragments described herein.

[0130] In some embodiments, the method may include (a) culturing cells containing nucleic acid encoding an anti-CD3 antibody or antigen-binding fragment under conditions that allow for 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).

[0131] In another aspect, the present disclosure provides methods of producing isolated or recombinant cells or populations of such cells according to the present disclosure.

[0132] In some embodiments, the method may include introducing into one or more cells (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 a vector comprising such a nucleic acid.

[0133] In certain embodiments, the introduction can occur in vitro, ex vivo, or in vivo.

[0134] Any of the anti-CD3 antibodies and antigen-binding fragments according to this disclosure, any of the nucleic acids according to this disclosure, any of the vectors according to this disclosure, any of the isolated or recombinant cells or populations of such cells according to this disclosure, and / or any of the pharmaceutical compositions according to this disclosure may be for use in the preparation of a medicament or a medicament for use in medicine.

[0135] Any of the anti-CD3 antibodies and antigen-binding fragments according to this disclosure, any of the nucleic acids according to this disclosure, any of the vectors according to this disclosure, any of the isolated or recombinant cells or populations of such cells according to this disclosure, and / or any of the pharmaceutical compositions according to this disclosure may be for use in the treatment of a disease, disorder, or condition, optionally any of the diseases, disorders, or conditions described herein.

[0136] The present disclosure further encompasses the use of any of the anti-CD3 antibodies and antigen-binding fragments according to the present disclosure, any of the nucleic acids according to the present disclosure, any of the vectors according to the present disclosure, any of the isolated or recombinant cells or populations of such cells according to the present disclosure, and / or any of the pharmaceutical compositions according to the present disclosure for the manufacture of a medicament for the treatment of a disease, disorder, or condition, optionally any of the diseases, disorders, or conditions described herein. DETAILED DESCRIPTION OF THE INVENTION

[0137] definition 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.

[0138] It is to be understood that the terminology used herein is for the purpose of describing particular versions or embodiments only, and is not intended to limit the scope of the present invention, which will be limited only by the appended claims.

[0139] All references cited herein, including patent and non-patent literature, are incorporated herein by reference in their entirety. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.

[0140] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Thus, reference to a "cell" refers to one or more cells and equivalents thereof known to those of skill in the art, and so forth.

[0141] As used herein, the term "about," when used in reference to a specific recited numerical value, means that the value can vary by no more than 5% from the recited value. For example, as used herein, the expression "about 100" includes 95 and 105, and all values ​​therebetween (e.g., 96, 97, 98, 99, 99.1, 99.2, 99.3, 99.4, 100, 101, 102, 103, 104, etc.).

[0142] Aspects and embodiments of the disclosure described herein should be understood to include "comprising" aspects and embodiments, "consisting of" aspects and embodiments, and "consisting essentially of" aspects and embodiments. Transitional phrases such as "comprising," "including," "having," "containing," "involving," "composed of," and the like should be understood to be open-ended, i.e., not excluding additional, unrecited elements. Such transitional phrases thus encompass both open-ended embodiments and, where appropriate, closed and semi-closed embodiments (i.e., embodiments described as "consisting of" and "essentially consisting of," respectively). Only the transitional phrases "consisting of" and "essentially consisting of" shall be closed or semi-closed transitional phrases, respectively.

[0143] The term "antibody" is used herein in the broadest sense and encompasses a variety of antibody structures, including, but not limited to, intact antibodies and antibody fragments (preferably fragments thereof 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, and the like.

[0144] Terms such as "intact antibody" and "whole antibody" are used interchangeably herein to refer to antibodies having a structure substantially similar to that of a natural antibody. In some cases, antibodies comprise heavy (H) and light (L) chains interconnected by disulfide bonds. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and some of these can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. Heavy chain constant domains corresponding to different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. For example, an intact IgG (or IgD or IgE) antibody comprises two heavy immunoglobulin chains and two light immunoglobulin chains. Thus, in some cases, an antibody according to the present disclosure may 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 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, antibodies according to the present disclosure may instead comprise multiple (e.g., two, three, four, five, etc.) units each comprising two pairs of heavy and light chains, or antigen-binding fragments thereof, interconnected by disulfide bonds.

[0145] Each heavy chain is composed of a heavy chain variable domain (VH) and a heavy chain constant domain (CH), which typically consists of domains CH1, CH2, and CH3. Each light chain is composed of a light chain variable domain (VL) and a light chain constant domain (CL). Typically (with some exceptions, such as nanobodies, camelid heavy chain antibodies, and IgNARs), one VH and one VL can form an antigen-binding region. VH and VL can be further subdivided into regions of hypervariability called complementarity-determining regions (CDRs), with more conserved regions called framework regions (FRs) interposed between them. Each VH and VL polypeptide is composed of three CDRs and four FRs, arranged in the following order from amino-terminus to carboxy-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The CDRs in the heavy chain are designated "CDR-H1," "CDR-H2," and "CDR-H3," respectively, and the CDRs in the light chain are designated "CDR-L1," "CDR-L2," and "CDR-L3." The FRs in the heavy chain are designated "FR-H1," "FR-H2," "FR-3," and "FR-H4," respectively, and the FRs in the light chain are designated "FR-L1," "FR-L2," "FR-L3," and "FR-L4." In certain embodiments of the present disclosure, the FRs of an antibody (or antigen-binding fragment thereof) may be identical to human germline coding 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 may be naturally occurring or artificially modified. A consensus amino acid sequence may be defined based on a side-by-side comparative analysis of two or more CDRs.

[0146] Numbering of amino acid residues in antibody variable and / or constant domains may be done by any suitable numbering scheme, method, and definition, for example, by numbering schemes, methods, and definitions based on numbering schemes 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, for example, the NCBI online tool, IgBlast; Dondelinger et al., Front Immunol. 2018 Oct 16;9:2278).

[0147] According to IMGT (International ImMunoGeneTics Information System for Immunoglobulins or Antibodies, T-cell Receptors, MH, Immunoglobulin Superfamilies 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 broadly herein to encompass any naturally occurring corresponding heavy chain constant domain and / or region allotypes and variants thereof, which may contain fewer or more amino acids (e.g., a CH1 domain may include part of the hinge region) and / or amino acid modifications.

[0148] An exemplary CH1 domain of human IgG1 may comprise the amino acid sequence of SEQ ID NO: 41 or 42, an exemplary hinge of human IgG1 may comprise the amino acid sequence of SEQ ID NO: 51, and a CH2 domain of human IgG1 may comprise the amino acid sequence of SEQ ID NO: 61. An exemplary CH3 domain of human IgG1 may comprise the amino acid sequence 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 these exemplary sequences may be used in conjunction with the anti-CD3 variable sequences described herein.

[0149] The "Fc region" is the C-terminal region of an immunoglobulin heavy chain containing at least a portion of the constant region, including native-sequence Fc regions and variant Fc regions. The Fc region of a human IgG heavy chain can range from Cys226 or from Pro230 to the carboxyl terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may be present or absent. Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region follows the EU numbering system, also known as the EU index. This system is described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991.

[0150] The phrase "effector function" of an antibody refers to a biological activity attributable to the Fc region of the antibody and varies depending on 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, down-regulation of cell surface receptors (e.g., B cell receptors), and B cell activation.

[0151] There are two major light chain isotypes, kappa (κ) and lambda (λ), and the corresponding light chain constant domains are called kappa CL domains (CLκ domains) and lambda CL domains (CLλ domains), respectively.

[0152] According to IMGT, the CLκ domain is located at amino acid positions 108-214 (EU numbering). An exemplary CLκ domain of human IgG may comprise the amino acid sequence of SEQ ID NO: 81. According to IMGT, the CLλ domain is located at amino acid positions 107-215 (EU numbering). An exemplary CLλ domain of human IgG may comprise the amino acid sequence of SEQ ID NO: 82.

[0153] The terms "CLκ domain" and "CLλ domain" are used broadly herein to encompass any naturally occurring, corresponding light chain constant domain and / or region allotypes and variants thereof, which may contain fewer or more amino acids and / or amino acid modifications.

[0154] Various standard sequences of the constant domains of human IgG1, IgG2, IgG3, and IgG4 (corresponding to different allotypes) are known in the art and can be found, for example, in Vidarsson et al., Front Immunol. 2014 Oct 20;5:520 and U.S. Patent No. 9,150,663, the disclosures of which are incorporated herein by reference in their entireties. Again, these reference sequences are intended to be exemplary, as the applicant intends the human IgG1, IgG2, IgG3, and IgG4 sequences to include all naturally occurring human IgG1, IgG2, IgG3, and IgG4 allotypes.

[0155] An "antigen-binding fragment" or "antigen-binding antibody fragment" refers to a portion of an intact antibody or a combination of portions derived from one or more intact antibodies that bind to the antigen to which the intact antibody binds (in this case, CD3). Antigen-binding fragments of antibodies include any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex. Exemplary antigen-binding fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab'), diabodies, linear antibodies, single-chain antibody molecules (e.g., single-chain variable fragments (scFv), half antibodies, nanobodies, or VH-only or VL-only), and multispecific antibodies formed from antibody fragments. In some embodiments, the antigen-binding fragment of an anti-CD3 antibody described herein is an scFv. The term "half molecule" or "half antibody" when referring to IgG, IgE, or IgD, which may also be referred to as "half IgG," "half IgE," or "half IgD," respectively, refers to one set of heavy chains and one light chain of the referenced antibody.

[0156] "Antigen-binding region" refers to the portion of an antibody or antigen-binding fragment that has specificity for an antigen.

[0157] For multispecific antibodies (e.g., bispecific, trispecific, tetraspecific, etc.), such antibodies contain 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 present within the same antigen. A "bispecific antibody" is a type of multispecific antibody that contains two different antigen-binding regions that recognize and specifically bind to two different antigens or two epitopes. Bispecific antibodies can, for example, target two different surface receptors on the same or different cells (e.g., immune cells and cancer cells).

[0158] The phrase "different antigens" can refer to different and / or distinct proteins, polypeptides, or molecules, as well as different and / or distinct epitopes that may be contained within a single protein, a single polypeptide, or a single molecule.

[0159] The term "epitope" refers to an antigenic determinant that interacts with a specific antigen-binding site within the variable region of an antibody molecule, known as the paratope. A single antigen may have multiple epitopes. Thus, different antibodies may bind to different regions on an antigen and have different biological effects. The term "epitope" also refers to the site on an antigen to which B cells and / or T cells respond. It also refers to the region of an antigen that is bound by an antibody. Epitopes may be defined structurally or functionally. Functional epitopes are generally a subset of structural epitopes and contain residues that directly contribute to the affinity of the interaction. Epitopes may also be conformational, i.e., composed of nonlinear amino acids. In certain embodiments, epitopes may include determinants that are chemically active surface groups of molecules, such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and in certain embodiments, epitopes may have specific three-dimensional structural characteristics and / or specific charge characteristics.

[0160] A "monoclonal antibody" or "mAb" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical and / or bind to the same epitope, excluding potential variant antibodies (e.g., containing natural mutations or arising during generation of the monoclonal antibody preparation). Such variants are generally present in small amounts. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant.

[0161] "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), and includes, for example, the CD3ε chain, the CD3γ chain, the CD3α chain, and the CD3β chain, unless otherwise indicated. The term encompasses "full-length," unprocessed CD3 (e.g., unprocessed or unmodified CD3ε or CD3γ) as well as any form of CD3 that results from processing in cells. The term also encompasses naturally occurring variants of CD3, such as splice variants or allelic variants. CD3 includes, for example, the human CD3ε protein (NCBI Reference SEQ ID NO: NP - 000724), which is a 207 amino acid long protein, and human CD3γ protein (NCBI Reference SEQ ID NO: NP - 000064), which is 182 amino acids in length. The term also refers to human or cynomolgus monkey CD3ε proteins, SEQ ID NOs: 91 and 92, respectively (Table U). "CD3εN27" and "CD3εN13" refer to the N-terminal 27 and 13 amino acids of CD3, respectively, including any chemical modifications or linkages thereto.

[0162] "Anti-CD3 antibody" refers to an antibody or antigen-binding fragment thereof capable of binding to CD3, e.g., CD3ε and / or CD3γ, e.g., human CD3ε and / or CD3γ, with sufficient affinity and / or specificity such that the antibody is useful as a diagnostic and / or therapeutic agent in targeting CD3. In some embodiments, the anti-CD3 antibody has a binding affinity of about 100 x 10 -9 M or less, approximately 50 x 10 -9 M or less, approximately 25 x 10 -9 M or less, approximately 20 x 10 -9 M or less, or about 10 x 10 -9 The dissociation constant (K DIn some embodiments, the anti-CD3 antibody binds to CD3 at about 5×10 -9 The dissociation constant (K D In some embodiments, the anti-CD3 antibody binds to CD3 at about 2.5 x 10 -9 The dissociation constant (K D In some embodiments, the anti-CD3 antibody binds to CD3 at about 1 x 10 -10 The dissociation constant (K D ) binds to CD3. In some embodiments, K D is measured by SPR, e.g., BIACORE®, BLI measurements using, e.g., a FORTEBIO OCTET® HTX instrument (Pall Life Sciences), or solution-affinity ELISA. In some embodiments, the KD is measured using an scFv fragment of an anti-CD3 antibody. In some embodiments, a monovalent KD is measured. In some embodiments, the anti-CD3 antibody binds to an epitope of CD3 that is conserved among CD3 from various species, e.g., the cross-reactive species of human and cynomolgus monkey.

[0163] An "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 of the additional agents described herein.

[0164] The term "developable" or "developability" refers to the extent to which one or more polypeptides in a plurality of polypeptides possess desirable characteristics, such as, but not limited to, desirable binding specificity, e.g., binding to a cognate antigen with a desirable affinity and not significantly binding to non-cognate antigens, desirable expression, e.g., in mammalian cells, solubility, viscosity, aggregation, chemical and / or physical stability, desirable shelf-life, melting point, toxicity, pharmacokinetic profile, circulating half-life, and clearance characteristics. Such characteristics may be treated individually, as a combination of a subset of such characteristics, or collectively as markers for the likelihood that the one or more polypeptides will be successfully developed as therapeutic candidates and ultimately become approved drugs. Generally, polypeptides with desirable developability characteristics possess 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 point, relatively long circulating half-life, relatively low clearance rate, etc. In contrast, polypeptides with undesirable developability properties generally possess 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 point, relatively short circulatory half-life, relatively fast clearance rate, etc.

[0165] Methods and assays that can be employed to confirm the extent to which a polypeptide, such as, for example, an anti-CD3 antibody and / or antigen-binding fragment thereof described herein, possesses desirable developability properties are available in the art and may 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, pages 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 methodology, ELISA assays such as BVP ELISA assays, AC-SINS assays (Liu et al; MAbs, Vol. 6, 483-492 (2013)). (2014), melting point (Tm) assay, differential scanning calorimetry or differential scanning fluorometry (DSF), etc. (e.g., He et al., J. Pharm. Sci., Vol. 100(4), pp. 1330-1340 (2011); Wagner et al., Pharm. Develop. & Technol (posted online in 2012; hyper-text transfer protocol: informahealthcare.com / doi / abs / 10.3109 / 10837450.2011.649851); Hotzel et al., MAbs, Vol. 4(6), pages 753-7601 (2012); Weiqiang et al., J. Pharm. Sci., Vol. 101(5), pp. 1701-1720 (2012); Banks et al., J. Pharm. Sci., Vol. 101(8), pp. 2720-2732 (2012); Lie et al., J. Pharm. Sci., Vol. 94(9), pp. 1928-1948 (2005); and Payne et al., Biopolymers, Vol.85(5), pp. 527-533 (2006)).

[0166] A "developability profile" refers to an index that can be assigned to an antibody when assessing its developability. A developability profile is a scale or metric by which the developability of anti-CD3 antibodies can be evaluated, compared, and / or ranked. Such a developability profile serves as a measure of the degree of interaction between a CD3-binding substance and an antibody comprising the same. The degree of interaction may be assessed by any number of means available in the art that provide an output value that correlates with the strength or affinity of a polypeptide for a bound moiety. Exemplary means include flow cytometry, such as fluorescence-activated cell sorting (FACS), enzyme-linked immunosorbent assay (ELISA), quantitative immunoaffinity assay, or immunoprecipitation assay, mammalian two-hybrid assay, or yeast two-hybrid assay. In the case of FACS, as shown in the examples, the degree of interaction between a polypeptide in a plurality of polypeptides and PSR may be ascertained by generating a mean fluorescence intensity for each detected polypeptide-PSR interaction, then ordering the mean fluorescence intensities in either ascending or descending order, thereby ranking the polypeptides in the plurality of polypeptides according to the relative degree of interaction between each detected polypeptide and PSR. By performing such a ranking on a plurality of polypeptides, polypeptides with high developability can be easily identified, and polypeptides with low developability can also be easily identified.

[0167] The developability profile may also take the form of a normalized score, for example, by normalizing the developability of an anti-CD3 antibody described herein to the developability of a standard (or control) antibody, such as an anti-HEL antibody.

[0168] The term "cytokine release syndrome" (or CRS) refers to a pro-inflammatory positive feedback loop between cytokines and immune cells, resulting in the excessive and uncontrolled release of pro-inflammatory cytokines by cells within the immune system (see, for example, Lee et al., Blood, Vol. 124, pages 188-195 (2014) and Tisoncik et al., Microbiol Mol Biol Rev, Vol. 76, pages 16-32 (2012)). Upon stimulation and activation, T cells release a range of cytokines at levels and to an extent that produce adverse biological / physiological effects of varying degrees and severity. Such effects include, for example, acute inflammation characterized by redness (redness), swelling or edema, fever (hot flashes), pain (pain), and "loss of function" (loss of function). When localized to the skin or other tissues, biological / physiological effects include increased blood flow, allowing vascular leukocytes and plasma proteins to reach extravascular sites of injury, increased local temperature, and the development of pain, tissue edema and extravascular pressure, and decreased tissue perfusion. Other biological / physiological effects include organ and systemic dysfunction, such as heart failure, adult respiratory distress syndrome, neurotoxicity, renal and / or hepatic failure, and disseminated intravascular coagulation. Elevated levels of IFNγ, IL-6, TNFα, TGFβ, IL-2, granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-10, IL-8, IL-5, and / or fractalkine have been implicated as indicators and / or causes of CRS or a propensity for T cell stimulation to induce CRS.

[0169] The term "pH-dependent" antibody refers to an antibody with a modified amino acid sequence that allows for 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 exhibit pH-dependent binding. pH-dependent binding refers to the preference of an antibody to bind to an antigen at a given pH (or a given pH range) compared to a different pH (or pH range). In one embodiment, a pH-dependent antibody preferentially or selectively binds to an antigen at an acidic pH (e.g., a pH of approximately 6) compared to physiological pH (e.g., a pH of approximately 7 or 7.4). The antibody sequence may be modified by substitution with one or more ionizable amino acid residues, such as histidine, lysine, arginine, aspartic acid, and glutamic acid. Ionizable residues may be substituted into the CDRs and / or FRs. In some embodiments, there may be 1 to 10 substitutions per VH or VK of the variant. In some embodiments, there may be 1 to 6 substitutions per VH or VK of the variant. While the pH range of human blood is approximately 7.6-7.8, tumor cells have an extracellular pH of approximately 6.3-6.5, which is due, at least in part, to the accumulation of metabolic acidity that is not adequately removed due to poor tumor vascularization. Therefore, antibodies that preferentially bind to antigens at sub-physiological pH (e.g., pH approximately 6) may potentially generate selective and sustained cytotoxic activity at or near tumor sites, thereby reducing or eliminating off-target effects, minimizing CRS risk, and improving half-life and dosing. Thus, in certain embodiments, the anti-CD3 antibodies and / or antigen-binding fragments described herein may reduce the likelihood of or reduce the severity of potential CRS.

[0170] The tendency of an antibody to bind to multiple targets is called "multispecificity," which may be associated with target-specific therapeutic antibodies and negative clinical outcomes. The anti-CD3 antibodies and antigen-binding fragments of the present disclosure may exhibit reduced multispecificity (e.g., as assessed by interaction with a multispecificity reagent (PSR)). Antibodies with reduced multispecificity may be engineered from starting antibodies and antibody fragments by replacing various variable domain amino acid residues with residues having charged side chains. Residues may be replaced with amino acid residues having negatively charged side chains, such as Asp and Glu residues. Residues selected for substitution may be selected from residues that are not predicted to interact specifically with CD3 amino acid residues bound by anti-CD3 antibodies and antigen-binding fragments. Antibodies with high interaction with PSR may be referred to as "multispecific" antibodies and may be referred to as relatively "undevelopable" or relatively "non-developable."

[0171] A "conservative amino acid substitution" is one in which an amino acid residue is replaced with another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). Generally, conservative amino acid substitutions do not substantially change the functional properties of a protein. When two or more amino acid sequences differ from each other by conservative substitutions, the percent or degree of similarity may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are known to those of skill in the art. (See, e.g., Pearson (1994) Methods Mol. Biol. 24: 307-331.) Examples of groups of amino acids with side chains having similar chemical properties include: 1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine, 2) aliphatic-hydroxyl side chains: serine and threonine, 3) amide-containing side chains: asparagine and glutamine, 4) aromatic side chains: phenylalanine, tyrosine, and tryptophan, 5) basic side chains: lysine, arginine, and histidine, 6) acidic side chains: aspartic acid and glutamic acid, and 7) sulfur-containing side chains: cysteine ​​and methionine. In some embodiments, conservative amino acid substitution groups are valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid-aspartic acid, and asparagine-glutamine. Alternatively, in some embodiments, a conservative substitution includes any change that has a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science 256: 1443 45. In some embodiments, a "moderately conservative" substitution includes any change that has a non-negative value in the PAM250 log-likelihood matrix.

[0172] 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. The following are non-limiting examples of polynucleotides: 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, substitution of one or more analogs of natural nucleotides, and introduction of a means for attaching a polynucleotide to a protein, metal ion, labeling component, another polynucleotide, or solid support. Polynucleotides can be obtained by chemical synthesis, recombinantly, or derived from a microorganism.

[0173] A "vector" is a compound or substance composition that contains an isolated nucleic acid and can be used to deliver the isolated nucleic acid to the interior of a cell. Numerous vectors are known in the art, including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, viruses, and virus-like particles (VLPs). Thus, the term "vector" includes autonomously replicating plasmids, self-replicating RNA, or virus particles. This term should also be interpreted to include non-plasmid and non-viral compounds that facilitate the transfer of nucleic acids into cells, such as polylysine compounds and liposomes. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated viral vectors, retroviral vectors, lentiviral vectors, etc.

[0174] The term "host cell" refers to a cell into which exogenous nucleic acid has been introduced, including the progeny of such a cell. Host cells include transformants and transformed cells, which include the primary transformed cell and its progeny without regard to the number of transfers.

[0175] A "pharmaceutical formulation" refers to a preparation in a form that allows the biological activity of an active ingredient contained therein, such as an anti-CD3 antibody described herein, to be effective, and preferably does not contain additional ingredients that are unacceptably toxic to the subject to which the formulation is administered.

[0176] A "pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical formulation, other than the active ingredient, that is not toxic to a subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives. In another embodiment, a pharmaceutical formulation comprises any of the anti-CD3 antibodies provided herein and at least one additional therapeutic agent.

[0177] For example, an "effective amount" of an anti-CD3 antibody disclosed herein, or a composition (e.g., a pharmaceutical composition) comprising such an antibody, is at least the minimum amount required to achieve a desired therapeutic or prophylactic result, such as a measurable improvement or prevention of a particular disorder, e.g., a cell proliferative disorder such as cancer, preferably without or with minimal toxic or adverse consequences. An effective amount may vary depending, inter alia, on the patient's disease state, age, sex, and weight, and the ability of the antibody (or antigen-binding fragment thereof) to elicit a desired response in that individual, and in some cases, by being co-administered with one or more additional therapeutic agents.

[0178] "Disorder" refers to any condition or disease that would benefit from treatment, including, but not limited to, chronic and acute disorders or diseases, including pathological conditions that predispose the mammal to the disorder in question.

[0179] The terms "cell proliferative disorder" and "proliferative disorder" refer to disorders associated with some degree of abnormal cell proliferation. Cell proliferative disorders include cancers, e.g., tumors.

[0180] As used herein, the term "tumor" refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues.

[0181] "Cancer" refers to the physiological condition in mammals characterized by uncontrolled cell growth. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies, and more specific examples include squamous cell carcinoma (e.g., squamous cell carcinoma), small cell lung cancer, non-small cell lung cancer, lung cancer, including lung adenoma and lung squamous cell carcinoma, cancer of the peritoneum, hepatocellular carcinoma, gastric cancer (including gastrointestinal cancer and gastrointestinal stromal cancer), and gastrointestinal cancer (including gastrointestinal stromal cancer). cancer), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, cancer of the urinary tract, liver cancer (hepatoma), breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine cancer, salivary gland 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 lentigo melanoma, nodular melanoma, multiple myeloma and B-cell lymphoma (low-grade / follicular non-Hodgkin's lymphoma (NHL), small lymphocytic (SL) NHL, intermediate-grade / follicular NHL, intermediate-grade diffuse NHL, high-grade These include: high-grade immunoblastic NHL, high-grade lymphoblastic NHL, high-grade small noncleaved cell NHL, bulky disease NHL, mantle cell lymphoma, AIDS-related lymphoma, and Waldenstrom's macroglobulinemia), chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), hairy cell leukemia, chronic myeloblastic leukemia, and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal blood vessel proliferation associated with phacomatosis, edema (e.g., edema associated with brain tumors), Meigs' syndrome, brain tumors and head and neck cancers, and associated 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's lymphoma (NHL), renal cell carcinoma, prostate cancer, liver cancer, pancreatic cancer, soft tissue sarcoma, Kaposi's sarcoma, carcinoid carcinoma, head and neck cancer, ovarian cancer, mesothelioma, and multiple myeloma. In some embodiments, the cancer is selected from small cell lung cancer, glioblastoma, neuroblastoma, melanoma, breast cancer, gastric cancer, colorectal cancer (CRC), and hepatocellular carcinoma. However, in some embodiments, the cancer is selected from non-small cell lung cancer, colorectal cancer, glioblastoma, and breast cancer, including metastatic forms of these cancers.In other embodiments, the cancer excludes Hodgkin's lymphoma, but is selected from the group consisting of germinal center B-cell-like (GCB) DLBCL, activated B-cell-like (ABC) DLBCL, follicular lymphoma (FL), mantle cell lymphoma (MCL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), marginal zone lymphoma (MZL), small lymphocytic leukemia (SLL), lymphoplasmacytic lymphoma (LL), Waldenstrom's macroglobulinemia (WM), and leukemia-associated lymphoma (LEM). Central nervous system lymphoma (CNSL), Burkitt lymphoma (BL), B-cell prolymphocytic leukemia, splenic marginal zone lymphoma, hairy cell leukemia, splenic lymphoma / leukemia, unclassifiable, splenic diffuse red pulp small B-cell lymphoma, hairy cell leukemia variant, Waldenstrom's macroglobulinemia, heavy chain disease, alpha heavy chain disease, gamma heavy chain disease, mucous membrane-associated lymphoid tissue disease, plasma cell myeloma, isolated plasmacytoma of bone, extraskeletal plasmacytoma, extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue Zone lymphoma (MALT lymphoma), nodal marginal zone lymphoma, pediatric nodal marginal zone lymphoma, pediatric follicular lymphoma, primary cutaneous follicle-centered lymphoma, T-cell / histiocytic-rich large B-cell lymphoma, primary DLBCL of the CNS, primary cutaneous DLBCL, foot type, EBV-positive DLBCL in the elderly, DLBCL associated with chronic inflammation, lymphomatoid granulomatosis, primary mediastinal (thymic) large B-cell lymphoma, intravascular large B-cell lymphoma, AL A class of mature B-cell carcinomas including K-positive large B-cell lymphoma, plasmablastic lymphoma, large B-cell lymphoma occurring in HHV8-associated multicentric Castleman disease, primary effusion lymphoma; B-cell lymphoma, unclassifiable, with features intermediate between diffuse large B-cell lymphoma and Burkitt lymphoma; and B-cell lymphoma, unclassifiable, with features intermediate between diffuse large B-cell lymphoma and classical Hodgkin lymphoma.

[0182] As used herein, "treatment" or "treat" or "treating" refers to clinical intervention in an attempt to alter the natural course of the individual being treated, and can be performed either for prophylactic purposes or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, prevention of disease onset or recurrence, alleviation of symptoms, reduction and / or reversal of any direct or indirect pathological consequences of the disease, prevention of metastasis (e.g., of cancer), delay in progression of the disease / disorder / condition, reduction in the rate of disease progression, improvement or palliation of the disease state, and remission or improved prognosis.

[0183] As used herein, the terms "prevent," "preventing," and "prevention" refer to the prevention or inhibition of the development or occurrence of a disorder or disease.

[0184] As used herein, the terms "amelioration" and "alleviation" refer to a decrease or lessening in the severity of a condition or any of its symptoms.

[0185] As used herein, "delaying the progression" of a disorder or disease means postponing, preventing, slowing, inhibiting, stabilizing, and / or postponing the onset of the disorder or disease (e.g., a cell proliferative disorder, e.g., cancer). Delay can be for varying lengths of time, depending on the history of the disease and / or the individual being treated.

[0186] As used herein, "detection" includes quantitative or qualitative detection.

[0187] Anti-CD3 antibody variable region sequence Provided herein are anti-CD3 antibodies and antigen-binding fragments that exhibit pH-dependent binding, and optionally, a favorable developability profile.

[0188] In some embodiments, an anti-CD3 antibody or antigen-binding fragment may comprise one or more CDR sequences (i) contained in any of Antibody Nos. A003-A013, (ii) contained in the variable domain sequence set forth in Appendix Table A, and / or (iii) encoded in a nucleic acid sequence encoding the variable domain of any of Antibody Nos. A003-013 and / or set forth in Appendix Table B. In some embodiments, an anti-CD3 antibody or antigen-binding fragment may comprise one or more CDR sequences set forth in Appendix Tables D, F, H, K, M, and O.

[0189] 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 Nos. 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 Nos. 6 or 5. In certain embodiments, the anti-CD3 antibody or antigen-binding fragment may comprise the CDR-H3, CDR-L1, and CDR-L3 sequences of antibody No. 6.

[0190] In some embodiments, an 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 of antibody Nos. A003-A013. In certain embodiments, an 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 No. 6 or 5. In certain embodiments, an 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 No. 6.

[0191] The anti-CD3 antibodies and antigen-binding fragments according to the present disclosure can comprise any suitable FR sequences.

[0192] In some embodiments, the anti-CD3 antibody or antigen-binding fragment may comprise one or more heavy chain FR sequences encoded by 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.

[0193] In some embodiments, the anti-CD3 antibody or antigen-binding fragment may comprise one or more light chain FR sequences encoded by 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 sequence.

[0194] In some embodiments, an anti-CD3 antibody or antigen-binding fragment may comprise one or more FR sequences (i) contained in any of Antibody Nos. A003-A013, (ii) contained in the variable domain sequence set forth in Appendix Table A, and / or (iii) encoded in a nucleic acid sequence encoding the variable domain of any of Antibody Nos. A003-013 and / or set forth in Appendix Table B. In some embodiments, an anti-CD3 antibody or antigen-binding fragment may comprise one or more FR sequences set forth in Appendix Tables C, E, G, I, J, L, N, and P.

[0195] In some embodiments, an 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 of antibody Nos. A003-A013. In certain embodiments, an 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 No. 6 or 5. In certain embodiments, an 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 No. 6.

[0196] In some embodiments, the anti-CD3 antibody or antigen-binding fragment may comprise a VH polypeptide comprising an amino acid sequence 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 (i) the VH polypeptide sequence of any of antibody Nos. A003-A013, (ii) the VH polypeptide sequence encoded by the VH-coding sequence of any of antibody Nos. A003-A013, (iii) any of the VH polypeptide sequences set forth in Appendix Table A, and / or (iv) the VH polypeptide sequence encoded by the VH-coding sequence set forth in Appendix Table B.

[0197] In some embodiments, the anti-CD3 antibody or antigen-binding fragment may comprise a VL polypeptide comprising an amino acid sequence 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 (i) the VL polypeptide sequence of any of Antibody Nos. A003-A013, (ii) the VL polypeptide sequence encoded by the VL coding sequence of any of Antibody Nos. A003-A013, (iii) any of the VL polypeptide sequences set forth in Appendix Table A, and / or (iv) the VL polypeptide sequence encoded by the VL coding sequence set forth in Appendix Table B.

[0198] In certain embodiments, percent identity may be measured by any known algorithm for sequence identity, such as, for example, FASTA, BLAST, or GAP.

[0199] 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 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 at least 90% identical to SEQ ID NO: 620.

[0200] In certain embodiments, the anti-CD3 antibody or antigen-binding fragment may comprise: (i) a VH polypeptide comprising the CDR-H1, CDR-H2, and CFR-H3 sequences of SEQ ID NOs: 512, 514, and 516, respectively, and (ii) an amino acid sequence 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 NOs: 522, 524, and 526, respectively, and (ii) an amino acid sequence at least 90% identical to SEQ ID NO: 520.

[0201] In certain embodiments, the anti-CD3 antibody or antigen-binding fragment may comprise a VH polypeptide and a VL polypeptide comprising any of the VH and VL amino acid sequence combinations shown in Appendix Table A.

[0202] 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.

[0203] 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.

[0204] Antibody Constant Domains and Regions 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., a portion of a hinge, 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 CH1 and a portion of a hinge). Such constant domains, Fc regions, and / or constant regions or portions thereof may individually be or 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 variants thereof, and optionally include any of the modifications described herein.

[0205] In some embodiments, one or more amino acid modifications may be introduced into the Fc region of an anti-CD3 antibody of the disclosure, thereby generating an Fc region variant (see, e.g., US 2012 / 0251531). The Fc region variant may comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) containing an amino acid modification (e.g., a substitution) at one or more amino acid positions.

[0206] In certain embodiments, the present disclosure contemplates anti-CD3 antibody variants that retain some, but not all, effector functions, making them desirable candidates for applications in which in vivo antibody half-life is important but certain effector functions (e.g., complement and ADCC) are unnecessary or deleterious. In vitro and / or in vivo cytotoxicity assays can be performed to confirm reduced / loss of CDC and / or ADCC activity. For example, Fc receptor (FcR) binding assays can be performed to confirm that the antibody lacks FcγR binding (and thus likely lacks ADCC activity) but retains FcRn binding ability. Primary cells that mediate ADCC (e.g., NK cells) express only FcγIII, while monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression on hematopoietic cells is summarized on page 464, Table 3, of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991). Non-limiting examples of in vitro assays to assess ADCC activity of a molecule of interest are described in U.S. Pat. No. 5,500,362 (e.g., Hellstrom, I. et al. Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I. et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985); U.S. Pat. No. 5,821,337 (see Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assay methods may be employed (see, e.g., ACTI™ Non-Radioactive Cytotoxicity Assay for Flow Cytometry (Cell Technology, Inc., Mountain View, Calif.); and CYTOTOX 96® Non-Radioactive Cytotoxicity Assay (Promega, Madison, Wis.)). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer cells (NKs).Alternatively, or in addition, the ADCC activity of the molecule of interest may be assessed in vivo in an animal model, such as that disclosed in Clynes et al. Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). A C1q binding assay may be performed to confirm that the antibody is unable to bind to C1q and therefore lacks CDC activity. See, for example, the C1q and C3c binding ELISAs in WO2006 / 029879 and WO2005 / 100402. To assess complement activation, a CDC assay may be performed (see, e.g., Gazzano-Santoro et al. J. Immunol Methods 202:163 (1996); Cragg, MS et al. Blood. 101:1045-1052 (2003); and Cragg, MS and MJ Glennie Blood. 103:2738-2743 (2004)). FcRn binding and in vivo clearance / half-life determinations can also be performed using methods known in the art (see, e.g., Petkova, SB et al. Int'l. Immunol 18(12):1759-1769 (2006)).

[0207] In certain embodiments, antibodies with reduced effector function include antibodies with substitutions at one or more of residues 238, 265, 269, 270, 297, 327, and 329 in the Fc region (U.S. Patent Nos. 6,737,056 and 8,219,149). In some embodiments, Fc variants include Fc variants with substitutions at two or more of amino acid positions 265, 269, 270, 297, and 327, including the so-called "DANA" Fc variant in which residues 265 and 297 are substituted with alanine (U.S. Patent Nos. 7,332,581 and 8,219,149).

[0208] In some embodiments, the anti-CD3 antibodies and / or antigen-binding fragments described herein may comprise a silent Fc region (e.g., the Fc is removed entirely or the Fc region is modified to reduce or eliminate effector function) and / or may comprise a masking agent (e.g., a polypeptide mask positioned (e.g., attached via a cleavable linker) thereby reducing or inhibiting the ability of the antibody or antigen-binding fragment to induce effector function-inducing molecules such as complement (e.g., C1q)).

[0209] Antigen-binding fragments and multispecific antibodies Anti-CD3 antigen-binding fragments according to the present disclosure may take any suitable format, including but not limited to any of the formats described herein (e.g., Fab, scFv, etc.), so long as such modifications retain pH-dependent CD3 binding and do not substantially reduce the ability of the antibody to bind CD3.

[0210] In certain embodiments, the anti-CD3 antibodies and / or antigen-binding fragments thereof described herein may comprise or be included in multispecific antibodies or antibody fragments that may comprise at least two different variable regions, each capable of specifically binding to a different antigen or to a different epitope on the same antigen; specifically, bispecific antibodies have binding specificity for a second antigen. In some embodiments, the binding specificities are for two different epitopes of CD3 (e.g., CD3ε or CD3γ). In other embodiments, one of the binding specificities is for CD3 (e.g., CD3ε or CD3γ) and the other is for a different biological molecule (e.g., a cell surface antigen, e.g., a tumor antigen).

[0211] Multispecific antibodies comprising at least one anti-CD3 antibody and / or antigen-binding fragment disclosed herein can be constructed using a variety of techniques, including, but not limited to, recombinant coexpression of two immunoglobulin heavy chain-light chains with different specificities (see, e.g., Milstein and Cuello, Nature 305: 537 (1983)), WO 93 / 08829, and Traunecker et al., EMBO J. 10: 3655 (1991)), "knobs-in-holes" engineering (see, e.g., U.S. Pat. No. 5,731,168), immunoglobulin crossover (also known as Fab domain swapping or CrossMab format) technology (see, e.g., WO 2009 / 080253; Schaefer et al., Proc. Natl. Acad. Sci. USA, 108:11187-11192). (2011)), engineering electrostatic steering effects for creating antibody Fc heterodimeric molecules (WO2009 / 089004A1), cross-linking two or more antibodies or fragments (see, e.g., U.S. Pat. No. 4,676,980, and Brennan et al., Science, 229: 81 (1985)), leucine zippers (see, e.g., Kostelny et al., J. Immunol, 148(5):1547-1553 (1992)), "diabody" technology (see, e.g., Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)), single-chain Fv (sFv) dimers (see, e.g., Gruber et al., J. Immunol, 152:5368 (1994)), and trispecific antibodies, for example, as described in Tutt et al. J. Immunol 147: 60 (1991).

[0212] In the case of the antigen-binding fragments of the anti-CD3 antibodies described herein, a variety of multispecific antibody formats can be used. Non-limiting examples of multispecific and bispecific formats include, for example, Fab-Fc-scFv (bottle opener type) (XENCOR), Mab-scFv (XENCOR), Mab-Fv (XENCOR), dual scFv (XENCOR), central Fv (XENCOR), central scFv (XENCOR), one-arm central scFv (XENCOR), Fab-Fab (XENCOR), Fab-Fv (XENCOR), mAb-Fv (XENCOR), mAb-Fab (XENCOR), DART (MACROGENICS), BiTE (AMGEN / MICROMET), KiTE, common light chain-IgG (Genentech), TanDab (SFFIMED) Cross-Mab (ROCHE), SEED (EMD SERONO), BEAT (GLENMARK), TrioMab (TRION PHARMA / FRESENIUS), and others. BIOTECH), DuetMab (MEDIMMUNE), and the like, which are for example (WO2021067404; WO2022150787; WO2022150785; WO 95 / 09917; WO 2008 / 119566; WO 2008 / 119567; WO2011 / 121110; WO 2010 / 037835; WO 2007 / 042261; WO 2007 / 110205; WO 2011 / 121110; WO 2012 / 055961; WO 2012 / 16067; WO 2016 / 086189; WO 2016 / 182751; WO 2015 / 006749; WO 2014 / 049003; WO 2013 / 177101; WO 2015 / 128509; US 7,951,917; US 2009 / 0252729; US 2014 / 0348839; US 7,183,076; al., Mabs, Vol. 7, pages 377-389 (2015); Muda et al., Protein Engineering, Design, & Selection, Vol.24, pages 447-454 (2011); and Del Bano et al., Antibodies, Vol. 5, pages 1-23 (2016). In some embodiments, the anti-CD3 scFv fragments described herein may comprise one or more variable regions of a multispecific (e.g., bispecific) antibody.

[0213] In some embodiments, a multispecific antibody or antibody fragment may comprise one or more engineered variant constant domains that promote efficient polypeptide heterodimer formation (e.g., a first heavy chain and a second heavy chain different from the first heavy chain) for bispecific antibody formation.

[0214] In certain embodiments, a multispecific antibody or antibody fragment may comprise 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 those described in WO2021067404.

[0215] In certain embodiments, a multispecific antibody or antibody fragment may comprise at least one pair of a CH1 domain and a CL domain that preferentially pair with each other. In the preferentially paired pair of a CH1 domain and a CL domain, the CH1 domain prefers to pair with the CL domain over another given CL domain, such as a wild-type CL domain, and / or the CL domain prefers to pair with the CH1 domain over another given CH1 domain, such as a wild-type CH1 domain. One or both of the CH1 domain and the CL domain may be variant domains. In certain embodiments, such preferentially paired pairs of a CH1 domain and a CL domain may be selected from the pairs described in WO2022150787.

[0216] In certain embodiments, a multispecific antibody or antibody fragment may comprise at least one pair of a first CH3 domain and a second CH3 domain that is different from the first CH3, which preferentially pair with each other (i.e., form a heterodimer). In such a preferentially paired pair, the first CH3 domain prefers to pair with the second CH3 domain over another first CH3 domain, and / or the second CH3 domain prefers to pair with the first CH3 domain over another second CH3 domain. One or both of the CH3 domains may be variant domains. In certain embodiments, such a preferentially paired pair of first and second CH3 domains may be selected from the pairs described in WO2022150785.

[0217] Exemplary Technical Advantages Exemplary technical advantages provided by some of the embodiments according to the present disclosure are listed below. It should be noted that the technical advantages possessed by such embodiments may not be limited to the advantages specifically listed below. It should also be noted that embodiments other than the embodiments specifically mentioned below may have one or more of the advantages listed below.

[0218] Binding to cynomolgus monkey CD3 As shown in Table 2B, the parent pH-dependent antibody, antibody No. 2, does not bind to cynomolgus monkey CD3, whereas the novel pH-dependent antibodies, antibody Nos. 3 to 10 and 12, bind to cynomolgus monkey CD3.

[0219] Non-human primates, such as cynomolgus monkeys, are often similar to humans and are therefore ideal species for establishing the pharmacokinetics of antibodies and collecting efficacy data during preclinical trials. Without wishing to be bound by theory, the ability to bind to cynomolgus monkey CD3 is a technical advantage because it enables various in vitro studies using cynomolgus monkey CD3, thereby aiding in the design of such preclinical trials in cynomolgus monkeys.

[0220] In some embodiments, an anti-CD3 antibody or antigen-binding fragment according to the present disclosure may comprise one or more CDRs of any of antibody numbers 3-10 and 12. 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 of antibody numbers 3-10 and 12. In certain embodiments, an 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.

[0221] Improved binding to human CD3 at acidic pH As shown in Table 2A, the novel pH-dependent antibodies, Antibodies Nos. 3-12, bind to human CD3 at pH 6.0 with higher affinity than the parent pH-dependent antibody, Antibody No. 2, as measured via BLI using the ForteBio OCTET® system.

[0222] As discussed above, tumor cells typically have an extracellular pH of approximately 6.3-6.5, which is due, at least in part, to the accumulation of metabolic acid that is not adequately removed due to poor tumor vascularization. Thus, without wishing to be bound by theory, antibodies with higher affinity at an acidic pH, such as pH 6.0, exhibit more potent efficacy (e.g., higher cytotoxic activity) at or around the tumor site. Furthermore, without wishing to be bound by theory, this allows for the use of reduced dosages, thus minimizing toxicity.

[0223] In some embodiments, an anti-CD3 antibody or antigen-binding fragment according to the present disclosure may comprise one or more CDRs of any of antibody numbers 3 to 12. 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 of antibody numbers 3 to 12. In certain embodiments, an anti-CD3 antibody or antigen-binding fragment according to the present disclosure may comprise a combination of six CDRs of antibody numbers 6 or 5.

[0224] Improved binding to human CD3-expressing cells at acidic pH As shown in Table 3, the novel pH-dependent antibodies, Antibodies Nos. 3 and 5-11, bind to human CD3-expressing cells (CD3+ Jurkat cells) at a higher pH of 6.0 than the parent pH-dependent antibody, Antibody No. 2, based on NCB values ​​obtained from flow cytometry analysis.

[0225] As mentioned above, tumor cells typically have an extracellular pH of about 6.3-6.5, which is lower than physiological pH (approximately pH 7.4). Therefore, without wishing to be bound by theory, antibodies that have better binding to cells at an acidic pH, such as pH 6.0, may exhibit stronger effects (e.g., greater cytotoxicity) at or around the tumor site. For example, such antibodies may provide better binding to T cells while also binding to tumor cells, thereby enabling a stronger cytotoxic effect by the T cells against the tumor cells. Furthermore, without wishing to be bound by theory, this may allow for the use of reduced dosages, thus minimizing toxicity.

[0226] In some embodiments, an anti-CD3 antibody or antigen-binding fragment according to the present disclosure may comprise one or more CDRs of any of antibody numbers 3 to 12. 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 of antibody numbers 3 to 12. In certain embodiments, an anti-CD3 antibody or antigen-binding fragment according to the present disclosure may comprise a combination of six CDRs of antibody numbers 6 or 5.

[0227] Improved binding to cynomolgus monkey CD3-expressing cells at acidic pH As shown in Table 3, the novel pH-dependent antibodies, antibodies Nos. 3 and 5-11, bind to cynomolgus monkey CD3-expressing cells (HSC-F cells) at a higher pH of 6.0 than the parent pH-dependent antibody, antibody No. 2, based on NCB values ​​obtained from flow cytometry analysis.

[0228] As mentioned above, non-human primates such as cynomolgus monkeys are often similar to humans and are therefore ideal species for establishing the pharmacokinetics of antibodies and collecting efficacy data during preclinical trials. Without wishing to be bound by theory, improved binding to cynomolgus monkey CD3-expressing cells is a technical advantage because it increases the likelihood of success in such preclinical trials, which is important for bringing antibodies to the clinical stage.

[0229] In some embodiments, an anti-CD3 antibody or antigen-binding fragment according to the present disclosure may comprise one or more CDRs of antibody Nos. 3 and any of 5 to 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 antibody Nos. 3 and any of 5 to 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 antibody Nos. 6 or 5.

[0230] Further modifications Sequence modification The present disclosure also contemplates 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 aforementioned antibodies. Once obtained, such derived antibodies and / or antigen-binding fragments may be tested for one or more desirable properties, such as improved binding specificity, increased binding affinity, improved pH-dependent antigen binding, improved developability, etc.

[0231] In some embodiments, such modifications may be in the FR and / or CDR regions.

[0232] In some embodiments, one or more substitutions, insertions, or deletions can be made within one or more CDRs of the anti-CD3 antibodies described herein, so long as such modifications maintain pH sensitivity and do not substantially reduce the ability of the antibody to bind to its antigen. For example, conservative changes (e.g., conservative amino acid substitutions provided herein) that do not substantially reduce binding affinity can be made in a CDR. Such changes may be, for example, outside the antigen-contacting residues in the CDR. In certain embodiments, each of the six CDRs may contain one, two, or no more than three amino acid substitutions. In certain embodiments, CDR-H1, CDR-H2, and / or CDR-L2 may contain one, two, or no more than three amino acid substitutions relative to any of antibody Nos. 3-13, preferably antibody Nos. 6 or 5. In certain embodiments, the CDR-H3, CDR-L1, and CDR-L3 may not contain any substitutions, insertions, or deletions relative to the CDR-H3, CDR-L1, and CDR-L3 of any of antibodies nos. 3 to 13, preferably antibody nos. 6 or 5.

[0233] A useful method for identifying antibody residues or regions that can be targeted for mutagenesis is called "alanine scanning mutagenesis," described in Cunningham and Wells (1989) Science, 244:1081-1085. In this method, a residue or group of target residues (e.g., charged residues such as arg, asp, his, lys, and glu) is identified and replaced with neutral or negatively charged amino acids (e.g., alanine or polyalanine), and it is determined whether the antibody-antigen interaction is affected. Further substitutions can be introduced at amino acid positions that show functional sensitivity to the initial substitution. Alternatively, or in addition, crystal structures of antigen-antibody complexes can be used to identify contact points between the antibody and antigen. Such contact residues and adjacent residues can be targeted or excluded from substitution candidates. Variants can be screened to determine whether they contain desirable properties.

[0234] Amino acid sequence insertions include amino- and / or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing several hundred or more residues, as well as intersequence insertions of single or multiple amino acid residues. An example of a terminal insertion is an antibody with an N-terminal methionyl residue. Other insertional variants of antibody molecules include the fusion to the antibody N- or C-terminus to an enzyme (e.g., an enzyme for ADEPT) or a polypeptide that increases the serum half-life of the antibody.

[0235] In addition to substituting one or more CDR residues, omission of one or more CDRs is also possible. The scientific literature reports antibodies in which one or two CDRs can be deleted to alter binding. Padlan et al. (1995 FASEB J. 9:133-139) analyzed the contact regions between antibodies and their antigens based on published crystal structures and concluded that only approximately one-fifth to one-third of the CDR residues actually contact their associated antigen. Padlan also found that in many antibodies, one or two CDRs do not contain any amino acids that contact the antigen (see also Vajdos et al. 2002 J Mol Biol 320:415-428). CDR residues that do not contact the antigen can be identified by molecular modeling and / or empirically based on previous studies from regions of the Kabat CDRs outside the Chothia CDRs (e.g., residues H60-H65 of CDRH2 are often not required). When a CDR or residue thereof is omitted, it is usually replaced with an amino acid at the corresponding position in another human antibody sequence or a consensus of such sequences. The position of substitution within the CDR, and the amino acid to be substituted, can also be selected empirically.

[0236] Bond-mediated modification In some embodiments, the anti-CD3 antibodies and / or antigen-binding fragments thereof described herein are conjugated to a moiety or agent, thereby forming an immunoconjugate.

[0237] In certain embodiments, anti-CD3 antibodies according to the present disclosure may be further modified to contain additional nonproteinaceous moieties known and readily available in the art. Moieties suitable for derivatization of antibodies include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (either homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone), polyethylene glycol, polypropylene glycol homopolymer, polypropylene oxide / ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and combinations thereof. Polyethylene glycol propionaldehyde may be advantageous in manufacturing due to its stability in water. The polymer may be of any molecular weight and may be branched or unbranched. The number of polymers attached to an antibody can vary, and when multiple polymers are attached, they can be the same molecule or different molecules. Generally, the number and / or type of polymers used for derivatization can be determined depending on considerations including, but not limited to, the particular property or function of the antibody to be improved, whether the antibody derivative will be used therapeutically under defined conditions, etc.

[0238] In some embodiments, a labeled anti-CD3 antibody is provided. The anti-CD3 antibodies and / or antigen-binding fragments thereof described herein may contain a label or moiety that is directly (e.g., fluorescent, dye, electron-dense, chemiluminescent, and radioactive) or indirectly (e.g., enzyme or ligand) detectable. Non-limiting examples of labels include radioisotopes such as 32P, 14C, 125I, 3H, and 131I; fluorophores such as rare earth chelates or fluorescein and its derivatives; rhodamine and its derivatives; dansyl; umbelliferone; luciferases such as firefly luciferase and bacterial luciferase (U.S. Pat. No. 4,737,456); luciferin; 2,3-dihydrophthalazinedione; horseradish peroxidase (HRP); alkaline phosphatase; β-galactosidase; and the like. Examples of suitable oxidases include oxidases, glucoamylases, lysozymes, monosaccharide oxidases such as glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase, heterocyclic oxidases such as uricase and xanthine oxidase coupled with enzymes that use hydrogen peroxide to oxidize dye precursors such as HRP, lactoperoxidase, or microperoxidase, biotin / avidin, spin labels, bacteriophage labels, stable free radicals, and the like.

[0239] In certain embodiments, the anti-CD3 antibody or antigen-binding fragment of the present disclosure may be, comprise, or be included in an antibody-drug conjugate (ADC). The ADC may comprise (a) any anti-CD3 antibody or antigen-binding fragment described herein and (b) a drug conjugated thereto. In certain embodiments, the drug may be, for example, but not limited to, an anti-cancer agent, an anti-proliferative agent, a cytotoxic agent, an anti-angiogenic agent, an apoptotic agent, an immunostimulant, an antibacterial agent, an antibiotic, an antiviral agent, an anti-inflammatory agent, an enzyme, a hormone, a toxin, a radioisotope, a chemical 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, an siRNA, an shRNA, a microRNA, an oligonucleotide, or a diagnostic imaging agent.In some cases, the drug is doxorubicin, daunorubicin, cucurbitacin, chaetocin, chaetoglobocin, chlamydocin, calicheamicin, nemorubicin, cryptophycin, menthacarcin, ansamitocin, mitomycin C, geldanamycin, mequelcarmycin, rebeccamycin, safracin, oxilactomycin, oligomycin, actinomycin, sandramycin, hypothemycin, polyketomycin, hydroxyelliptic cin, thiocolchicine, methotrexate, triptolide, taltoburine, lactacystin, dolastatin, auristatin, monomethylauristatin E (MMAE), monomethylauristatin F (MMAF), telomestatin, tubastatin A, combretastatin, maytansinoid, MMAD, MMAF, DM1, DM4, DTT, 16-GMB-APA-GA, 17-DMAP-GA, JW55, pyrrolobenzodiazepine, SN-38, Ro 5-3335, pwainaphycin, duocarmycin, bafilomycin, taxoid, tubulysin, ferulenol, luciol A, fumagillin, hygrolysin, glucopyericidin, amanitin, ansatrienin, sinerubin, phallacidin, phalloidin, phytosphingosine, piericidin, polonetin, fodophyllotoxin, glumicidin A, sanguinarine, sinefungin, herboxidien, microcorrin B, microcystin, muscotoxin A, It may be selected from the group consisting of tryptoxin, trypolin A, myoseverin, mitoxin B, noqualin A, pseudodoralic acid B, xeurotin A, cyclopamine, curbulin, colchicine, aphidicolin, englerin, cordycepin, apoptolidin, epothilone A, rimaquinone, isotropolone, isofistulalin, quinaldopeptin, ixabepilone, aeroprisinin, arginosine, agrochelin, epothilone, and derivatives of any one of the foregoing.

[0240] Glycosylation In some embodiments, the anti-CD3 antibodies and / or antigen-binding fragments thereof described herein may be modified to increase or decrease the extent to which the antibodies are glycosylated. Adding or deleting glycosylation sites from an anti-CD3 antibody of the present disclosure can be readily accomplished by altering the amino acid sequence to create or remove one or more glycosylation sites. In certain embodiments, the addition or deletion of glycosylation sites may not be limited to the constant region of the anti-CD3 antibody or antigen-binding fragment.

[0241] Antibody characterization and further screening In some embodiments, the anti-CD3 antibodies and / or antigen-binding fragments described herein may be characterized for their physical / chemical properties and / or biological activity by various assays known in the art. In some embodiments, variants of any of the anti-CD3 antibodies and antigen-binding fragments thereof described herein may be identified, screened, selected, or characterized for their physical / chemical properties and / or biological activity by various assays known in the art. In some embodiments, multispecific antibodies or antibody fragments, including the anti-CD3 antibodies or antigen-binding fragments described herein, may be designed and screened, selected, or characterized for their physical / chemical properties and / or biological activity by various assays known in the art.

[0242] In certain embodiments, such assays may include SPR, BLI, ELISA, Western blot, flow cytometry, and the like.

[0243] 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 present disclosure for binding to CD3. In an example of a competitive assay, immobilized CD3 is incubated in a solution containing a first, labeled antibody that binds to CD3 and a second, unlabeled antibody that is tested for its ability to compete with the first antibody for binding to CD3. The second antibody may be present in hybridoma supernatant. As a control, immobilized CD3 is incubated in a solution containing the first, labeled antibody but not the second, unlabeled antibody. After incubation under conditions that allow the first antibody to bind to CD3, excess unbound antibody is removed, and the amount of label bound to immobilized CD3 is measured. A significant decrease in the amount of label bound to immobilized CD3 in the test sample compared to the control sample indicates that the first and second antibodies compete for binding to CD3. See, for example, Harlow and Lane (1988) Antibodies: A Laboratory Manual. Ch. 14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY).

[0244] Biologically active anti-CD3 antibodies and / or antigen-binding fragments thereof may be identified using standard methods. Biological activity may include, for example, binding to CD3 on the surface of T cells, either in vivo, in vitro, or ex vivo. In the case of multispecific anti-CD3 antibodies (e.g., bispecific antibodies having one arm that binds to CD3 and another arm that binds to a different target, e.g., a cell surface antigen, e.g., a tumor antigen), biological activity may also include effector cell activation (e.g., activation of CD8+ T cells and / or CD4+ T cells), expansion of the effector cell population (i.e., an increase in T cell numbers), depletion of the target cell population (i.e., a decrease in the population of cells expressing a second biological molecule on their cell surface), and / or target cell killing.

[0245] Target-binding-based assessment In some embodiments, the anti-CD3 antibodies or antigen-binding fragments described herein may exhibit binding levels to CD3 in a preferred range around the pH of interest.

[0246] In certain embodiments, antigen binding is measured using a K D In certain embodiments, the K D can be determined as described in the Examples.

[0247] In certain embodiments, the anti-CD3 antibodies or antigen-binding fragments described herein have a K for human CD3 of about 0.5 nM to about 50 nM at about pH 6.0. D In certain embodiments, the K value by SPR for human CD3 at pH 6.0 can be D In certain embodiments, the anti-CD3 antibodies or antigen-binding fragments described herein have a K by SPR against human CD3 of about 1 nM or greater at about pH 7.4. D In certain embodiments, the K value by SPR for human CD3 at pH 7.4 can be D may be about 10 nM or greater, or 100 nM or greater, 1000 nM or greater, or 10,000 nM or greater. In certain embodiments, no binding is observed at pH 7.4.

[0248] In certain embodiments, antigen binding is measured using a K D In certain embodiments, the K D can be determined as described in the Examples.

[0249] In certain embodiments, the anti-CD3 antibodies or antigen-binding fragments described herein have a K by BLI against human CD3 of about 0.5 nM to about 20 nM at about pH 6.0. D In certain embodiments, the K value by BLI for human CD3 at pH 6.0 can be D In certain embodiments, the anti-CD3 antibodies or antigen-binding fragments described herein have a K by BLI against human CD3 of about 5 nM or greater at about pH 7.4. D In certain embodiments, the K value by BLI for human CD3 at pH 7.4 D may be about 10 nM or greater.

[0250] In certain embodiments, the anti-CD3 antibodies or antigen-binding fragments described herein have a K by BLI against non-human primate (e.g., cynomolgus or rhesus) CD3 of about 0.5 nM to about 20 nM at about pH 6.0. D In certain embodiments, the K value by BLI for non-human primate CD3 at pH 6.0 can be D In certain embodiments, the K by BLI for non-human primate CD3 at about pH 7.4 may be about 5 nM to about 10 nM, or about 5 nM to about 8 nM. D In certain embodiments, the K value by BLI for non-human primate CD3 at pH 7.4 may be D may be about 20 nM or greater.

[0251] Without wishing to be bound by theory, it is believed that the higher the affinity (i.e., the lower the K D Antibodies having a specific agonist activity (e.g., a specific agonist activity) may induce stronger cytokine release by and / or activation of CD3-expressing cells upon antigen binding, and in some cases, the stronger cytokine release and / or activation may help reduce the dosage (e.g., effective amount) of the anti-CD3 antibody or antigen-binding fragment required for treatment.

[0252] In some embodiments, the anti-CD3 antibodies or antigen-binding fragments described herein may exhibit stronger binding to CD3-expressing cells around the pH of interest.

[0253] 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) and / or normal cell binding (NCB) values ​​calculated using the MFI values. In certain embodiments, the MFI and / or NCB values ​​can be determined by incubating cells with a test antibody followed by a secondary antibody that binds to the test antibody (a negative control includes incubation without the test antibody followed by incubation with the secondary antibody), as described in the Examples. In some cases, the NCB value is calculated as {(sample MFI)-(secondary only MFI)} / (secondary only MFI).

[0254] In certain embodiments, the described anti-CD3 antibodies or antigen-binding fragments may have an NCB value for binding to human CD3-expressing cells at about pH 6.0 that is at least 1.2 times greater than the NCB value for binding to human CD3-expressing cells at about pH 7.4. In certain embodiments, the NCB value at about pH 6.0 is 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, or at least 10 times greater than the NCB value at about pH 7.4. 3 , at least x 10 4 , at least x 10 5 , at least x 10 6 , at least x 10 7 , at least x 10 8 , or at least x10 9 In certain cases, the NCB value at about pH 6.0 may be at least 3, at least 4, or at least 5 times greater than the NCB value at about pH 7.4. Optionally, the human CD3-expressing cells may be CD3-expressing Jurkat cells.

[0255] In some embodiments, the anti-CD3 antibodies or antigen-binding fragments described herein may exhibit improved binding to CD3-expressing cells (e.g., human CD3-expressing cells) at an acidic pH (e.g., about pH 6.0) relative to known anti-CD3 antibodies.

[0256] In certain embodiments, improved binding to CD3-expressing cells can optionally be determined by FACS based on MFI value and / or NCB value calculated using MFI value.In certain embodiments, MFI value and / or NCB value can be determined as described in Examples.In some cases, NCB value can be calculated as described above.

[0257] Without wishing to be bound by theory, antibodies with stronger or improved binding to human CD3-expressing cells (e.g., T cells) at acidic pH (e.g., about pH 6.0) (i.e., higher NCB values) may induce stronger cytokine release by and / or activation of CD3-expressing cells upon antigen binding in an acidic environment, and in some cases, stronger cytokine release and / or activation may help reduce the dosage (e.g., effective amount) of the anti-CD3 antibody or antigen-binding fragment required for treatment.

[0258] Evaluation based on other development feasibility parameters In some embodiments, anti-CD3 antibodies and / or antigen-binding fragments, including multispecific antibodies and variants thereof provided herein (e.g., multispecific antibodies designed to include a second specificity of interest), may be identified for developability, screened for developability, selected for developability, or characterized for developability.

[0259] In certain embodiments, the anti-CD3 antibody or antigen-binding fragment may exhibit a favorable developability profile. The developability profile of the anti-CD3 antibody may be obtained by performing one or more of a PSR assay, an SCP assay, an AC-SINS assay, an ELISA, a DSF assay, a Tm assay, an HIC assay, a CIC assay, or a combination thereof.

[0260] In some embodiments, the anti-CD3 antibodies or antigen-binding fragments described herein may exhibit reduced multispecificity (tendency to bind to multiple molecules or epitopes). In certain embodiments, multispecificity may be determined based on a multispecificity reagent (PSR) score obtained by a PSR assay. In certain embodiments, the PSR score may be determined as described in the Examples. In some embodiments, the anti-CD3 antibodies or antigen-binding fragments described herein exhibit a PSR score of about 0.0 to about 0.45. In some embodiments, the PSR score is about 0.0 to about 0.4. In some embodiments, the PSR is about 0.0 to about 0.35. In some embodiments, the PSR is about 0.0 to about 0.3. In some embodiments, the PSR is about 0.0 to about 0.25. In some embodiments, the PSR is about 0.0 to about 0.2. In some embodiments, the PSR is about 0.0 to about 0.15. In some embodiments, the PSR is about 0.0 to about 0.1. In some embodiments, a score of 0.0 to 0.1 is a "clean PSR." In some embodiments, a score of 0.1 to 0.33 is a "low PSR." In some embodiments, a score of 0.33 to 0.66 is a "medium PSR." In some embodiments, a score of 0.66 to 1.00 is a "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.

[0261] In some embodiments, the anti-CD3 antibodies or antigen-binding fragments described herein may exhibit reduced hydrophobicity. In certain embodiments, hydrophobicity may be determined based on the retention time observed during HIC. In certain embodiments, HIC may be performed and retention times obtained as described in the Examples. In some embodiments, the anti-CD3 antibodies or antigen-binding fragments described herein exhibit an HIC score of less than about 10.5 minutes (clean 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 antibody's developability.

[0262] In some embodiments, the anti-CD3 antibodies or antigen-binding fragments described herein may exhibit a reduced tendency for self-interaction. In certain embodiments, the tendency for self-interaction may be determined based on the Δλmax value observed during affinity capture self-interaction nanoparticle spectroscopy (AC-SINS). In certain embodiments, AC-SINS may be performed and the Δλmax value obtained as described in the Examples. In some embodiments, the anti-CD3 antibodies or antigen-binding fragments described herein may exhibit a Δλmax of about 0.0 nm to about 15.0 nm. In some embodiments, the Δλmax may be about 0.0 nm to about 10.0 nm. In some embodiments, the Δλmax may be about 0.0 nm to about 7.5 nm. In some embodiments, the Δλmax may be about 0.0 nm to about 5.0 nm. In some embodiments, the Δλmax may be about 0.0 nm to about 3.0 nm. In some embodiments, the Δλmax may be about 0.0 nm to about 2.0 nm. In some embodiments, the Δλmax may be about 0.0 nm to 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." In general, the lower the propensity for self-interaction, the more favorable the developability of the antibody.

[0263] In some embodiments, the anti-CD3 antibodies or antigen-binding fragments described herein may exhibit reduced viscosity. In some embodiments, viscosity may be determined based on the diffusion interaction parameter (kD) value observed during dynamic light scattering (DLS). In some embodiments, DLS may be performed and kD values ​​obtained as described in the Examples, for example, using 10 mM histidine buffer (pH about 6). In some embodiments, the anti-CD3 antibodies or antigen-binding fragments described herein may exhibit a kD of about 5 mL / g or greater. In some embodiments, the kD may be about 10 mL / g or greater. In some embodiments, the kD may be about 15 mL / g or greater. In some embodiments, the kD may be about 20 mL / g or greater. In some embodiments, the kD may be about 25 mL / g or greater. In some embodiments, a Δλmax < 20 mL / g may be considered associated with high viscosity or high opalescence. Generally, the lower the viscosity, the more favorable the antibody's developability.

[0264] In some embodiments, the anti-CD3 antibodies or antigen-binding fragments described herein may exhibit a reduced chance of heavy chain-light chain mispairing (including failed pairing). In some embodiments, heavy chain-light chain mispairing may be determined based on the presence of a heavy chain peak (indicating a heavy chain that was not successfully paired with a light chain) and / or a light chain peak (indicating a light chain that was not successfully paired with a heavy chain) observed during liquid chromatography-mass spectrometry (LC-MS). In some embodiments, LC-MS may be performed as described in the Examples. In some embodiments, the anti-CD3 antibodies or antigen-binding fragments described herein may not exhibit a heavy chain peak or a light chain peak. Generally, the smaller the heavy chain and light chain peaks, the more favorable the antibody's developability.

[0265] In some embodiments, the anti-CD3 antibodies or antigen-binding fragments described herein may exhibit reduced aggregation propensity. In some embodiments, aggregation propensity may be determined based on the % monomer value observed during size exclusion chromatography (SEC) (i.e., the % of antibody species (e.g., IgG or Fab) present in their full size without aggregation or multimerization, of the protein from antibody production and, optionally, purification). In some embodiments, SEC may be performed as described in the Examples. In some embodiments, the anti-CD3 antibodies or antigen-binding fragments described herein may exhibit a % monomer in SEC of about 95% or greater, indicating that the antibody is substantially present as a monomer, i.e., without aggregation. In some embodiments, the % monomer may be about 97% or greater. In some embodiments, the % monomer may be about 98% or greater. In some embodiments, the % monomer may be about 99% or greater. In some embodiments, the % monomer may be about 99.5% or greater. Generally, the higher the % monomer value, the more favorable the developability of the antibody.

[0266] In some embodiments, the anti-CD3 antibodies or antigen-binding fragments described herein may exhibit improved resistance to acidic environments or acidic stress, such as a pH of about 6 or less, about 5 or less, about 4 or less, or about 3.5 or less. In some embodiments, resistance to low pH may be determined based on the tendency to aggregate when exposed to low pH. In some embodiments, the tendency to aggregate under low pH may 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, such SEC for low pH resistance testing may be performed as described in the Examples. In some embodiments, the anti-CD3 antibodies or antigen-binding fragments described herein may exhibit a % monomer in SEC of about 95% or greater after exposure to low pH, indicating that the antibody is substantially present as a monomer, i.e., without aggregation. In some embodiments, the % monomer may be about 96% or greater. In some embodiments, the % monomer may be about 97% or greater. In some embodiments, the % monomer may be about 98% or greater. In some embodiments, the % monomer may be about 99% or greater. Generally, the higher the % monomer value after exposure to low pH, the higher the resistance to acidic stress, i.e., the more favorable the antibody's developability. Without wishing to be bound by theory, improved resistance to acidic stress may help provide a longer shelf life and / or improved in vivo stability (e.g., in an acidic cancer microenvironment).

[0267] In some embodiments, the anti-CD3 antibodies or antigen-binding fragments described herein may exhibit improved stability. In certain embodiments, stability may be evaluated based on Tm values. In some cases, the anti-CD3 antibodies or antigen-binding fragments described herein may exhibit a Tm of about 65°C or higher. In some embodiments, Tm may be determined using DSF, which may be performed as described in the Examples, or other suitable methods. Generally, the higher the Tm, the more stable the antibody, i.e., the more preferred the mode of development.

[0268] Nucleic acids, vectors, and cells The present disclosure further encompasses nucleic acids (single nucleic acid or combinations 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.

[0269] In some embodiments, a single nucleic acid molecule may encode 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) may be encoded on the same strand of a nucleic acid molecule. In some cases, (i) and (ii) may be encoded under a single promoter. In certain cases, (i) and (ii) may be encoded in the same direction (in some cases, (i) and (ii) may be transcribed into a single transcript, and in some cases, (i) and (ii) may be transcribed into two separate transcripts). In certain cases, (i) and (ii) may be encoded in opposite directions. In some cases, (i) and (ii) may be encoded under separate promoters. In certain embodiments, (i) and (ii) may be encoded on different strands within a nucleic acid molecule.

[0270] In some embodiments, the nucleic acid encoding the antibody may comprise (i) a first nucleic acid encoding an amino acid sequence comprising a VH, and (ii) a second nucleic acid encoding an amino acid sequence comprising a VL.

[0271] In certain embodiments, the nucleic acid may comprise a sequence encoding a VH of SEQ ID NO: 650 and a sequence encoding a VL of SEQ ID NO: 660. In certain embodiments, the nucleic acid may comprise a sequence encoding a VH of SEQ ID NO: 550 and a sequence encoding a VL of SEQ ID NO: 560. In some cases, the nucleic acid may comprise RNA, which may comprise RNA versions of such sequences.

[0272] The present disclosure further encompasses vectors (one vector or a combination of two (or more) vectors) (e.g., expression vectors) encoding the VH and VL of any of the anti-CD3 antibodies and antigen-binding fragments described herein.

[0273] In some embodiments, a single vector may contain nucleic acids 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) may be encoded on the same strand of a nucleic acid molecule. In some cases, (i) and (ii) may be encoded under a single promoter. In certain cases, (i) and (ii) may be encoded in the same direction (in some cases, (i) and (ii) may be transcribed into a single transcript, and in some cases, (i) and (ii) may be transcribed into two separate transcripts). In certain cases, (i) and (ii) may be encoded in opposite directions. In some cases, (i) and (ii) may be encoded under separate promoters. In certain embodiments, (i) and (ii) may be encoded on different strands within a nucleic acid.

[0274] In some embodiments, the vector may comprise (i) a first vector comprising a nucleic acid encoding an amino acid sequence comprising a VH, and (ii) a second vector comprising a nucleic acid encoding an amino acid sequence comprising a VL.

[0275] In certain embodiments, a vector may comprise a nucleic acid comprising a sequence encoding a VH of SEQ ID NO: 650 and a nucleic acid comprising a sequence encoding a VL of SEQ ID NO: 660. In certain embodiments, a vector may comprise a nucleic acid comprising a sequence encoding a VH of SEQ ID NO: 550 and a nucleic acid comprising a sequence encoding a VL of SEQ ID NO: 560.

[0276] The present disclosure further provides isolated, recombinant, and / or host cells comprising any of the above-described anti-CD3 antibodies and antigen-binding fragments, comprising any of the above-described nucleic acids, and / or comprising, transfected with, transduced with, or transformed with any of the above-described vectors.

[0277] In some embodiments, the cells contain, are transfected with, are transduced with, and / or are transformed with: (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 the 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 the antibody.

[0278] In some embodiments, the cells may be for producing an anti-CD3 antibody or antigen-binding fragment according to the present disclosure. In some embodiments, the cells may be for administration to a subject.

[0279] In some embodiments, the cell is a eukaryotic cell. In certain embodiments, the cell is a mammalian cell, for example, a Chinese hamster ovary (CHO) cell, a human embryonic kidney (HEK) cell such as HEK293 cell, or a lymphoid cell (for example, Y0, NS0, Sp20 cell). In certain embodiments, the cell is a yeast cell.

[0280] Production of anti-CD3 antibodies and cells therefor Any of the anti-CD3 antibodies and / or antigen-binding fragments described herein may be produced or manufactured using any suitable method, including, for example, recombinant methods such as in vitro, ex vivo, or in vivo.

[0281] In some embodiments, isolated, recombinant, and / or host cells comprising nucleic acids encoding any of the anti-CD3 antibodies and antigen-binding fragments described herein, or populations of such cells, may include introducing the nucleic acids and / or vectors described herein into one or more cells.

[0282] Physical methods for introducing nucleic acids into cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, etc. Methods for generating cells containing vectors and / or exogenous nucleic acids are well known in the art. See, for example, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York). A preferred method for introducing polynucleotides into host cells is calcium phosphate transfection.

[0283] Biological methods for introducing nucleic acids of interest into cells include the use of DNA and RNA vectors. In certain embodiments, nucleic acids encoding antibodies may be isolated and inserted into one or more vectors (e.g., viral vectors, plasmids, etc.) for further cloning and / or expression in isolated cells, recombinant cells, and / or cells. The nucleic acids can be readily isolated and sequenced using conventional methods (e.g., by using oligonucleotide probes capable of specifically binding to genes encoding the heavy and light chains of antibodies). Viral vectors, particularly retroviral vectors, have become the most widely used method for inserting genes into mammalian, e.g., human, cells. Other viral vectors can be derived from lentiviruses, poxviruses, herpes simplex virus type I, adenoviruses, adeno-associated viruses, and the like. See, e.g., U.S. Patent Nos. 5,350,674 and 5,585,362.

[0284] Chemical means for introducing nucleic acids into cells include polymer complexes, nanocapsules, microspheres, beads, and colloidal dispersion systems such as lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (e.g., artificial membrane vesicle).

[0285] When a non-viral delivery system is utilized, an exemplary delivery vehicle is a liposome. The use of lipid formulations is intended for the introduction of nucleic acids into host cells (in vitro, ex vivo, or in vivo). In some cases, nucleic acids may be associated with lipids. The lipid-associated nucleic acid may 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 associated with both the liposome and the oligonucleotide, entrapped in the liposome, formed a complex with the liposome, dispersed in a solution containing lipids, mixed with lipids, combined with lipids, contained as a suspension in lipids, comprised in or complexed with micelles, or otherwise associated with lipids. Lipid, lipid / DNA, or lipid / expression vector-associated compositions are not limited to any particular structure in solution. For example, they may exist in a bilayer structure as micelles, or may have a "collapsed" structure. They may also simply be dispersed in solution, or may form aggregates that are not uniform in size or shape. Lipids are fatty substances that can be natural or synthetic. For example, lipids include the lipid droplets that naturally occur in the cytoplasm, as well as a class of compounds that contain long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, aminoalcohols, and aldehydes.

[0286] In some embodiments, methods of producing an anti-CD3 antibody or antigen-binding fragment according to the present disclosure may include culturing isolated, recombinant, and / or host cells containing nucleic acid encoding an antibody as provided above under conditions suitable for expression of the antibody, and optionally harvesting, recovering, and / or purifying the antibody or antigen-binding fragment from the cells (or host cell culture medium).

[0287] Suitable host cells for cloning and / or expressing antibody-encoding vectors include prokaryotic or eukaryotic cells. For example, antibodies can be produced in bacteria, particularly if glycosylation and Fc effector functions are not required. For expression of antibody fragments and polypeptides in bacteria, see, e.g., U.S. Patent Nos. 5,648,237, 5,789,199, and 5,840,523. (See also Charlton, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ, 2003), pp. 245-254, which describes the expression of antibody fragments in E. coli.) After expression, the antibody can be isolated from the bacterial cell paste in a soluble fraction and can be further purified. In addition to prokaryotes, eukaryotes such as filamentous fungi or yeast, including fungal and yeast strains whose glycosylation pathways have been "humanized" to produce antibodies with partially or fully human glycosylation patterns, are also suitable cloning or expression hosts for antibody-encoding vectors. See, e.g., Gerngross, Nat. Biotech. 22:1409-1414 (2004), and Li et al., Nat. Biotech. 24:210-215 (2006); WO 2009 / 036379; WO 2010 / 105256; and WO 2012 / 009568.

[0288] Plant cell cultures can also be used as hosts.See, for example, U.S. Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (which describe the PLANTIBODIES™ technology for producing antibodies in transgenic plants).Vertebrate cells can also be used as hosts.For example, mammalian cell lines adapted to grow in suspension may be useful. Other examples of useful mammalian host cell lines include SV40 (COS-7) transformed monkey kidney CV1 cells, human embryonic kidney cell lines (e.g., 293 or 293 cells described in Graham et al., J. Gen Virol. 36:59 (1977)), baby hamster kidney cells (BHK), mouse Sertoli cells (e.g., TM4 cells described in Mather, Biol. Reprod. 23:243-251 (1980)), monkey kidney cells (CV1), African green monkey kidney cells (VERO-76), human cervical carcinoma cells (HELA), canine kidney cells (MDCK; buffalo rat liver cells (BRL 3A), human lung cells (W138), human liver cells (Hep G2), and mouse mammary tumor (MMT 060562), e.g., Mather et al., Annals NY Acad. Sci. 383:44-68. (1982) are TRI cells, 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 specific mammalian host cell lines suitable for antibody production, see, for example, Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ), pp. 255-268 (2003).

[0289] Cells such as hybridomas or other recombinant cells that produce an antibody or antigen-binding fragment of the disclosure can be grown in a culture medium suitable for this purpose (such as D-MEM or RPMI-1640) or as ascites in vivo using standard methods. Antibodies or antigen-binding fragments expressed and / or secreted by the cells can be separated from the cells, culture medium, ascites fluid, or serum using conventional immunoglobulin purification procedures, including, but not limited to, protein A-Sepharose, 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 AA 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).

[0290] Methods for expressing, isolating, and evaluating multispecific and bispecific antibodies and antibody fragments are also known in the art (see, e.g., 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).

[0291] Pharmaceutical Composition The present disclosure further encompasses pharmaceutical compositions comprising: (A) (i) any of the anti-CD3 antibodies and antigen-binding fragments described herein; (ii) a nucleic acid encoding the anti-CD3 antibody or antigen-binding fragment; (iii) a vector encoding the 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.

[0292] Thus, in some embodiments, an anti-CD3 antibody or antigen-binding fragment may be the active ingredient, in some embodiments, a nucleic acid (e.g., DNA or RNA such as mRNA) or a vector encoding an anti-CD3 antibody or antigen-binding fragment may be the active ingredient, and in some embodiments, a cell containing a nucleic acid or a vector encoding an anti-CD3 antibody or antigen-binding fragment may be the active ingredient. In certain embodiments, when mRNA is the active ingredient, the mRNA may be formulated into lipid nanoparticles, which may facilitate administration and delivery to cells of a subject receiving the composition.

[0293] In some embodiments, pharmaceutical compositions comprising the anti-CD3 antibodies and / or antigen-binding fragments described herein can be prepared, for example, by combining the antibodies having the desired purity with one or more optional pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)) in the form of a lyophilized composition or an aqueous solution, optionally prepared for modified (e.g., sustained) release. Exemplary lyophilized antibody compositions are described in U.S. Pat. No. 6,267,958. Aqueous antibody formulations include those described in U.S. Pat. No. 6,171,586 and WO2006 / 044908, the latter compositions comprising a histidine-acetate buffer.

[0294] Pharmaceutically acceptable carriers are generally non-toxic to recipients at the dosages and concentrations employed and include, but are not limited to, buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzylammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl alcohol or benzyl alcohol, alkyl parabens such as methyl paraben or propyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol); low molecular weight (less than about 10 residues) polypeptides; Examples of pharmaceutically acceptable carriers include proteins such as serum albumin, gelatin, or immunoglobulins, hydrophilic polymers such as polyvinylpyrrolidone, amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine, monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin, chelating agents such as EDTA, sugars such as sucrose, mannitol, trehalose, or sorbitol, salt-forming counterions such as sodium, metal complexes (e.g., Zn-protein complexes), and / or non-ionic surfactants such as polyethylene glycol (PEG). Pharmaceutically acceptable carriers herein also include interstitial drug dispersion agents, such as soluble neutral-active hyaluronidase glycoprotein (sHASEGP), for example, human soluble PH-20 hyaluronidase glycoprotein, for example, rHuPH20 (HYLENEX®, Baxter International, Inc.). Certain exemplary sHASEGPs, including rHuPH20, and methods of using same are described in U.S. Patent Application Publications 2005 / 0260186 and 2006 / 0104968.

[0295] The active ingredient may be encapsulated in microcapsules prepared by droplet formation techniques or by interfacial polymerization, such as hydroxymethylcellulose or gelatin microcapsules, and poly(methyl methacrylate) microcapsules, in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules), or in macroemulsions, respectively. Such techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

[0296] In some embodiments, a pharmaceutical composition according to the present disclosure may be used in any of the in vivo methods described herein.

[0297] Additional medications In some embodiments, the pharmaceutical composition may be used alone, without additional agents.

[0298] In some embodiments, the pharmaceutical composition may further comprise or may be used in combination with (co-administered with or separately from) an additional agent (e.g., therapeutic agent) in a treatment regimen. Additional agents that may be combined with or used with an anti-CD3 antibody according to the present disclosure may preferably be agents with complementary activities that do not adversely affect each other and are present in amounts that are effective for the intended purpose.

[0299] In certain embodiments, the additional agent may be or include a chemotherapeutic agent, a gene therapy agent, a DNA therapy agent, a viral therapy agent, an RNA therapy agent, an immunotherapy agent, a nanotherapy agent, a monoclonal antibody, or a combination of the foregoing. In certain embodiments, the additional therapeutic agent may be or include an adjuvant or neoadjuvant. In certain embodiments, the additional therapeutic agent may be or include a small molecule enzyme inhibitor or an anti-metastatic agent.

[0300] In certain embodiments, the additional therapeutic agent may be or include a side effect limiting agent (e.g., an agent intended to reduce the occurrence and / or severity of therapeutic side effects such as anti-nausea agents, including but not limited to neurokinin-1 receptor antagonists (NK1 RAs), serotonin receptor antagonists (5-HT3 RAs), dexamethasone, olanzapine, and palonosetron).

[0301] Non-limiting examples of additional agents can include chemotherapeutic agents, antibody-drug conjugates (ADCs), immunotherapeutic agents, and / or biological modifiers.

[0302] In certain embodiments, the chemotherapeutic agent may be selected from alkylating agents, antimetabolites, plant alkaloids, and anticancer antibiotics, and may optionally be one or more chemotherapeutic agents selected from 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 prednisolone (CHOP).

[0303] In certain embodiments, the ADC may be selected from an anti-CD79b antibody drug conjugate (e.g., CD79b-MC-vc-PAB-MMAE or an anti-CD79b antibody drug conjugate described in any one of U.S. Pat. No. 8,088,378 and / or US 2014 / 0030280, or polatuzumab vedotin), an anti-CD19 antibody drug conjugate, an anti-CD22 antibody drug conjugate, an anti-CD45 antibody drug conjugate, and an anti-CD32 drug conjugate.Biological modifiers include BCL-2 inhibitors (e.g., GDC-0199 / ABT-199), lenalidomide (REVLIMID®), PI3K-delta inhibitors (e.g., idelalisib (ZYDELIG®)), PD-1 axis binding antagonists, such as CD40, CD226, CD28, OX40 (e.g., AgonOX), GITR, CD137 (TNFRSF9, 4-1 agonists, such as agonistic antibodies directed against activating costimulatory molecules such as CD27 (e.g., CDX-1127), HVEM, or CD127; antagonists, such as antagonist antibodies directed against inhibitory costimulatory molecules such as CTLA-4 (also known as CD152), PD-1, TIM-3, BTLA, VISTA, LAG-3, B7-H3, B7-H4, IDO (e.g., 1-methyl-D-tryptophan (also known as 1-D-MT)), TIGIT, MICA / B, GITR (e.g., TRX518), or arginase; ipilimumab (also known as MDX-010, MDX-101, or YERVOY®), tremelimumab (also known as ticilimumab or CP-675,206, urelumab (also known as BMS-663513), MGA271, antagonists directed against TGF beta, such as, for example, metelimumab (also known as CAT-192), fresolimumab (also known as GC1008), LY2157299k, and adoptive transfer of T cells (e.g., cytotoxic T cells or CTLs) expressing a chimeric antigen receptor (CAR), e.g., adoptive transfer of T cells comprising a dominant-negative TGF beta receptor, such as a dominant-negative TGF beta type II receptor.

[0304] In certain embodiments, the chemotherapeutic agent may be or may 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.

[0305] In certain embodiments, the additional agent is a chemotherapeutic agent, a cytotoxic agent, an antihormonal agent, a growth inhibitory agent, a cytotoxic agent, an agent used in radiation therapy, an anti-angiogenic agent, an apoptotic agent, an anti-tubulin agent, or other agent, such as an epidermal growth factor receptor (EGFR) antagonist (e.g., a tyrosine kinase inhibitor), a HER1 / EGFR inhibitor (e.g., erlotinib (TARCEVA™)), a platelet-derived growth factor inhibitor (e.g., GLEEVEC™ (imatinib mesylate)), a COX-2 inhibitor (e.g., celecoxib), an interferon, a cytokine, an antibody other than the anti-CD3 antibody of the disclosure, such as an antibody targeting the following: ErbB2, ErbB3, ErbB4, PDGFR-beta, BIyS, APRIL, BCMA. The additional therapeutic agent may be or may include an antibody that binds to one or more of VEGF or a VEGF receptor, TRAIL / Apo2, PD-1, PD-L1, PD-L2, or another bioactive or organic chemical agent. In certain embodiments, the additional therapeutic agent may be or may include a glucocorticoid, optionally dexamethasone.

[0306] kit In another aspect of the present disclosure, kits useful for treating, preventing, and / or diagnosing the diseases, disorders, or conditions described herein are provided. The kits may include (A) a container containing (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 herein, and (B) a label or package insert on or associated with the container. In certain embodiments, the container may contain an additional agent, optionally a cytotoxic agent and / or a therapeutic agent, or any of the additional agents described herein.

[0307] In certain embodiments, the kit may comprise: (A-1) a first container comprising: (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 herein; (A-2) a second container comprising an additional agent, optionally a cytotoxic agent and / or therapeutic agent, or any of the additional agents described herein; and (B) a label or package insert on or associated with the container.

[0308] In certain embodiments, the kit may further comprise a second (or third) container containing a pharmaceutically acceptable buffer, such as, for example, bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextrose solution. In certain embodiments, the kit may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

[0309] Suitable containers can include, for example, bottles, vials, syringes, IV solution bags, etc. The containers can be formed from a variety of materials, such as, for example, glass or plastic. The container can hold the composition alone or in combination with another composition effective for treating, preventing, and / or diagnosing a condition, and can have a sterile access port (e.g., the container can be an intravenous solution bag or a vial with a stopper pierceable by a hypodermic injection needle). A label or package insert can indicate that the composition is used for treating the condition of choice.

[0310] In some embodiments, a kit according to the present disclosure may be used in any of the in vivo methods described herein.

[0311] In certain embodiments, the kit disclosed herein can be used alone or in combination with additional drugs.In certain embodiments, the kit can be used to treat any disease, disorder, or condition described herein, such as cell proliferative disease (e.g., cancer) or autoimmune disease (e.g., arthritis, rheumatoid arthritis, colitis, inflammatory bowel disease, autoimmune type I diabetes, etc.).

[0312] In vivo methods and uses The present disclosure further encompasses in vivo methods.

[0313] In some aspects, methods and uses are provided for treating a subject in need of such treatment, and for treating or preventing a disease, disorder, or condition in a subject. Such methods and uses may include administering to a subject an effective amount of any 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) a pharmaceutical composition.

[0314] In some aspects, methods and uses are provided for inducing cytotoxicity against cells expressing a target molecule in a subject. Such methods and uses may comprise administering to a 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).

[0315] In any of the aspects and embodiments of the above methods and uses, in some embodiments, the anti-CD3 antibody or antigen-binding fragment may be used to enhance immune function in an individual with a cell proliferative disorder or an autoimmune disease. After administration, the antibody may enhance immune function in an individual with a cell proliferative disorder or an autoimmune disease by activating effector cells (e.g., T cells, such as CD8+ and / or CD4+ T cells, including Tregs), expanding (increasing) the effector cell population, depleting a target cell population (e.g., cells expressing a second biological molecule recognized by the anti-CD3 antibody, such as a bispecific antibody of the present disclosure), and / or killing target cells (e.g., target tumor cells).

[0316] In a further aspect, methods and uses are provided for detecting CD3 or CD3-expressing cells in a subject, for example, at a disease site (e.g., a tumor site) or a potential disease site in the subject. Such methods and uses may include administering to the subject an effective amount of any of the anti-CD3 antibodies and antigen-binding fragments described herein. In some embodiments, such methods and uses may be for diagnostic and / or detection purposes. In certain embodiments, diagnosis and / or detection may include determining the stage, severity, or immune profile of a disease or potential disease. In some cases, the immune profile may include a large number of CD3-expressing cells at the tumor site. Without wishing to be bound by theory, the greater the number of CD3-expressing cells in a tumor, the greater the likelihood that the tumor can be effectively treated with an anti-CD3 antibody or antigen-binding fragment of the present disclosure or another immunotherapy.

[0317] In any of the aspects and embodiments herein, including the methods and uses, the subject may be a mammal, in particular a human.

[0318] In any of the aspects and embodiments herein, including the methods and uses, the subject may have or be at risk of developing a disease, disorder, or condition.

[0319] In any of the aspects and embodiments herein, including methods and uses, the disease, disorder, or condition may be any suitable disease, disorder, or condition, including but not limited to those described herein. In some embodiments, the disease, disorder, or condition may be a proliferative disorder, cancer, neoplastic disorder, immune-neoplastic disorder, neurological disorder, cognitive disorder, neurodegenerative disorder, and / or inflammatory and / or autoimmune disease (e.g., rheumatoid arthritis, colitis, inflammatory bowel disease, autoimmune type 1 diabetes, etc.).

[0320] In certain embodiments, the disease, disorder, or condition may be cancer. Without wishing to be bound by theory, tumor cells typically have an extracellular pH of approximately 6.3 to 6.5, and the anti-CD3 antibodies and antigen-binding fragments described herein preferentially bind to CD3 at low pH values, e.g., about pH 6, promoting preferential binding and activity within and around the tumor microenvironment. In certain embodiments, the use of anti-CD3 antibodies and antigen-binding fragments thereof results in selective and sustained cytotoxic activity at or near the tumor site, which may (i) reduce the effective dose required for the intended purpose (e.g., treatment) and / or (ii) reduce or eliminate off-target effects.

[0321] In certain embodiments, RNA, such as mRNA encoding an anti-CD3 antibody or antigen-binding fragment, or a composition comprising such RNA, may be administered. In some cases, the RNA (e.g., mRNA) may be formulated into lipid nanoparticles to facilitate administration and delivery to the cells of the subject receiving the RNA.

[0322] Route of administration and dosage In any of the aspects and embodiments of the methods described herein, an effective amount of the anti-CD3 antibody or antigen-binding fragment (and optionally any additional agent), or a pharmaceutical composition comprising the same, may be administered by any suitable means, including parenteral, intrapulmonary, and intranasal administration, and, if desired for localized treatment, intralesional administration. Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. In certain embodiments, administration is subcutaneous, which may exhibit a lower toxic reaction in the subject compared to intravenous injection. Dosing can be administered by any suitable route (e.g., injection, such as intravenous or subcutaneous injection), and may depend, in part, on whether administration is temporary or chronic. Various dosing schedules are contemplated herein, including, but not limited to, single administration or multiple administrations over various time points, bolus administration, and pulse infusion.

[0323] The antibodies of the present disclosure are formulated (e.g., as pharmaceutical compositions), dosed, and administered in a manner consistent with sound medical practice. Factors to consider in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of drug delivery, the method of administration, the dosing schedule, and other factors known to medical professionals. Optionally, but not necessarily, the antibodies may be formulated (e.g., as pharmaceutical compositions) with one or more agents currently used to prevent or treat the disorder in question. The effective amount of such other agents will depend on the amount of antibody present in the composition, the type of disorder or treatment, and other factors discussed above. These will generally be used at the same doses and by the same routes of administration as those described herein, or at approximately 1-99% of the doses described herein, or at any dose and by any route empirically / clinically determined to be appropriate.

[0324] For the prevention or treatment of a disease, injury, or condition, the appropriate dose of the disclosed antibodies (when used alone or in combination with one or more other additional therapeutic agents) will depend on the type of disease being treated, the type of antibody, the severity and course of the disease, whether the antibody is being administered for prophylactic or therapeutic purposes, previous treatments, the patient's medical history and responsiveness to the antibody, and the discretion of the attending physician. In certain embodiments, the antibodies may be suitably administered to the patient at one time or over a series of treatments.

[0325] As a general rule, an effective amount (e.g., a therapeutically effective amount) of an anti-CD3 antibody or antigen-binding fragment administered to a human, whether administered as a single dose or multiple doses, can range from about 0.01 to about 100 mg / kg of patient body weight. In some embodiments, the antibody or antigen-binding fragment may be administered daily at, for example, about 0.01 to about 45 mg / kg, about 0.01 to about 40 mg / kg, about 0.01 to about 35 mg / kg, about 0.01 to about 30 mg / kg, about 0.01 to about 25 mg / kg, about 0.01 to about 20 mg / kg, about 0.01 to about 15 mg / kg, about 0.01 to about 10 mg / kg, about 0.01 to about 5 mg / kg, or about 0.01 to about 1 mg / kg. In one embodiment, an anti-CD3 antibody described herein is administered to a human at a dose of about 100 mg, about 200 mg, about 300 mg, about 400 mg, about 500 mg, about 600 mg, about 700 mg, about 800 mg, about 900 mg, about 1000 mg, about 1100 mg, about 1200 mg, about 1300 mg, or about 1400 mg on day 1 of a 21-day cycle. The dose may be administered as a single dose or multiple doses (e.g., two or three doses), e.g., by infusion. When administered repeatedly over several days or longer, treatment is generally sustained until a desired suppression of disease symptoms occurs, depending on the condition. One exemplary dose of the antibody ranges from about 0.05 mg / kg to about 10 mg / kg. Thus, one or more doses of about 0.5 mg / kg, 2.0 mg / kg, 4.0 mg / kg, or 10 mg / kg (or any combination thereof) may be administered to the patient. Such doses may be administered intermittently, for example, weekly or every three weeks (e.g., the patient receives from about 2 to about 20, or, for example, about 6, doses of anti-CD3 antibody). An initial higher loading dose may be administered, followed by one or more lower loading doses. The progress of this therapy is easily monitored by conventional techniques and assays.

[0326] Monotherapy and combination therapy In any of the aspects and embodiments of the methods and uses described herein, an effective amount of the anti-CD3 antibody or antigen-binding fragment may be administered alone to a subject.

[0327] In some embodiments, an effective amount of the anti-CD3 antibody or antigen-binding fragment may be administered to a subject in combination with at least one additional agent. The additional agent (e.g., a therapeutic agent and / or adjuvant) may be or include any agent, such as, but not limited to, a chemotherapeutic agent, a gene therapy agent, a DNA therapy agent, a viral therapy agent, an RNA therapy agent, a nanotherapy agent, a monoclonal antibody, an immunotherapy agent, and / or any additional agent specifically described herein. In certain embodiments, the additional agent may be included in a pharmaceutical composition together with the anti-CD3 antibody or antigen-binding fragment according to the present disclosure. In certain embodiments, the additional agent may not be included in a pharmaceutical composition of the anti-CD3 antibody or antigen-binding fragment according to the present disclosure, but may be administered together with (e.g., simultaneously with) the anti-CD3 antibody or antigen-binding fragment. In certain embodiments, the additional agent 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, the administration of the anti-CD3 antibody or antigen-binding fragment and the administration of the additional agent may occur within about one month, or within about one, two, or three weeks, or within about one, two, three, four, five, or six days of each other.

[0328] In some embodiments, an effective amount of the anti-CD3 antibody or antigen-binding fragment may be administered to a subject in combination with at least one additional therapy. The additional therapy may be or include radiation therapy (e.g., gamma irradiation), surgery, bone marrow transplant, chemotherapy, or any combination of the foregoing. In certain embodiments, the additional therapy may be administered together (e.g., simultaneously) with the anti-CD3 antibody or antigen-binding fragment. In certain embodiments, the additional therapy 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 the additional therapy may occur within about one month, or within about one week, two weeks, or three weeks, or within about one, two, three, four, five, or six days of each other.

[0329] 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 biological molecule (e.g., a cell surface antigen such as a tumor antigen) can be used in the combination therapies described herein, such as in combination with radiation therapy.

[0330] Examples are provided below to illustrate embodiments of the present disclosure. These examples are not intended to limit the invention to any particular application or theory of operation. [Example]

[0331] Example 1: Novel pH-dependent anti-CD3 antibodies The pH-dependent anti-CD3 antibody ADI-48587 (also referred to herein as Antibody No. 2 and first disclosed in PCT / US2020 / 036657) was previously identified from a library of antibody sequences distinct from the non-pH-dependent anti-CD3 antibody ADI-26906 (also referred to herein as Antibody No. 1 and first disclosed in PCT / US2018 / 031705).

[0332] The variable region sequences of ADI-48587 and ADI-26906 were altered to obtain improved pH-dependent anti-CD3 antibodies. The resulting novel anti-CD3 antibodies included ADI-74968, ADI-74967, ADI-74966, ADI-74965, ADI-79842, ADI-79843, ADI-79848, ADI-79844, ADI-79845, ADI-79846, and ADI-79847 (also referred to as antibody numbers 6, 5, 4, 3, 7, 8, 9, 10, 11, 12, and 13, respectively). The amino acid sequences of the VH, VL, CDR, and FR, as well as the nucleic acid sequences encoding the VH and VL, are listed in the Appendix (Tables A–P). The consensus sequences shared among the antibodies are also listed in the Appendix (Table Q).

[0333] Example 2: Monovalent Binding Affinity - IgG, SPR at Different pH Anti-CD3 antibodies (antibody numbers 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. IgG produced in yeast was nonglycosylated. CHO-produced IgG had standard CH2 glycosylation. The products exhibited the expected mass by LC-MS analysis, regardless of low pH stress (method described below), and were confirmed to be >95% monomeric by SEC-HPLC. Monovalent binding affinity to human or cynomolgus CD3εδ-Fc at pH 7.4 and pH 6.0 was measured via surface plasmon resonance (SPR) using a BIACORE® 8K (Cytiva, formerly GE Healthcare Life Sciences) system and analyzed using BIACORE® evaluation software.

[0334] The results are shown in Table 1. As shown in the table, the Fabs of antibody numbers 3 to 11 have higher affinity (i.e., lower K) at pH 6.0 than at pH 7.4. D ) The Fabs of antibodies nos. 3 to 6 produced in yeast bound to human CD3εδ-Fc at pH 6.0 but not at pH 7.4, and nos. 3, 5, and 6 exhibited higher affinity (i.e., lower K ) than Ab no. 2 (parent, pH-dependent antibody) at pH 6.0. D Similar results were obtained with CHO-derived Fabs of Abs Nos. 3 to 6, which showed increased affinity for human and cynomolgus monkey CD3εδ-Fc at pH 6.0 compared to the affinity at pH 7.4, and increased affinity for human CD3εδ-Fc compared to Ab No. 2. [Table 1]

[0335] Example 3: Monovalent binding affinity at different pH - IgG1, BLI Anti-CD3 antibodies (antibody numbers 1-13) were produced as human IgG1 antibodies in yeast or CHO cells. IgG produced in yeast was nonglycosylated. CHO-produced IgG had standard CH2 glycosylation. Binding affinity to human or cynomolgus CD3εδ-Fc at pH 7.4 and pH 6.0 was measured via biolayer interferometry (BLI) using a ForteBio OCTET® HTX system and analyzed using ForteBio software.

[0336] The results are shown in Tables 2A and 2B. As shown in Table 2A, antibodies Nos. 3 to 12 produced in yeast exhibited higher affinity (i.e., lower K) for human CD3εδ-Fc at pH 6.0 than at pH 7.4. D ) and exhibited higher affinity (i.e., lower K ) for human CD3εδ-Fc at pH 6.0 than Ab No. 2 (parent, pH-dependent antibody). D ) were observed. Similar results were observed with antibodies Nos. 3 to 6 produced in CHO. As shown in Table 2B, antibodies Nos. 3 to 10 and 12 produced in yeast bound to cynomolgus monkey CD3εδ-Fc with higher affinity at pH 6.0 than at pH 7.4. Antibodies Nos. 3 to 6 produced in CHO bound to cynomolgus monkey CD3εδ-Fc with higher affinity than Ab No. 2 at pH 6.0, but did not bind to cynomolgus monkey CD3εδ-Fc at pH 7.4. [Table 2A] [Table 2B]

[0337] Example 4: Cell Binding Anti-CD3 antibodies (antibodies no. 1-13) were produced in yeast cells as nonglycosylated IgG containing the IgG1 Fc region. Binding to human or cynomolgus monkey CD3-expressing cells (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 median fluorescence intensity (MFI). CD3-negative Jurkat cells were used as a negative control.

[0338] The results are shown in Table 3. As shown in Table 3, antibody numbers 3-12 exhibited higher binding (i.e., higher NCB values) to human CD3+ Jurkat cells and cynomolgus monkey HSC-F cells at pH 6.0 than at pH 7.4. Furthermore, compared to Ab number 2 (parent, pH-dependent antibody), antibody numbers 3 and 5-12 exhibited higher binding to human CD3+ Jurkat cells at pH 6.0, and antibody numbers 3 and 5-11 exhibited higher binding to cynomolgus monkey HSC-F cells at pH 6.0. None of the tested antibodies bound to the negative control cells. [Table 3]

[0339] Example 5: Development Potential - PSR The developability of anti-CD3 antibodies was evaluated by multispecificity analysis. Antibodies with high affinity for their target may otherwise fail in clinical settings, exhibiting binding to multiple non-target entities. Antibody multispecificity was assessed by measuring their interaction with a multispecificity reagent (PSR). PSR was 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 were used as starting material. In a 500 mL centrifuge bottle filled to 400 mL, the cells were pelleted at 2,400 × g for 5 minutes. The cell pellet was combined and then resuspended in 25 mL of buffer B and pelleted at 2,400 × g for 3 minutes. The buffer was decanted, and the wash was repeated once more. The cell pellet was resuspended in 3x the pellet volume of Buffer B containing 1x protease inhibitor (Roche, Complete, EDTA-free) using a Polytron homogenizer while keeping the cells on ice. The homogenate was then centrifuged at 2,400 x g for 5 minutes, and the supernatant was retained and pelleted once more (2,400 x g / 5 minutes) to ensure removal of unbroken cells, cell debris, and nuclei. The resulting supernatant was the total protein preparation. The supernatant was 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 soluble cytosolic proteins (SCP), was then transferred to a clean Oak Ridge tube and centrifuged once more at 40,000 x g. In parallel, the pellet containing the membrane fraction (EMF) was retained and centrifuged at 40,000 for 20 minutes, and the residual supernatant was removed. The EMF pellet was then rinsed with Buffer B. 8 mL of Buffer B was then added to the membrane pellet, and the pellet was scraped and transferred to a Dounce homogenizer. After the pellets were homogenized, they were transferred to a 50 mL conical tube, which represented the final EMF preparation.

[0340] about 10 6 ~107 Mammalian cells (e.g., CHO, HEK293, Sf9, etc.) at 1 billion cells / mL were transferred from tissue culture medium into 4 x 250 mL conical tubes and pelleted at 550 x g for 3 minutes. All subsequent steps were performed at 4 °C or on ice using ice-cold buffers. The cells were washed with 100 mL of PBSF (1 x PBS + 1 mg / mL BSA) and combined in one conical tube. After removing the supernatant, the cell pellet was resuspended in 30 mL of buffer B (50 mM HEPES, 0.15 M NaCl, 2 mM CaCl, 5 mM KCl, 5 mM HCl). The cells were resuspended in 10% MgCl2, 10% glycerol, pH 7.2, and pelleted at 550 x g for 3 min. The supernatant in Buffer B was decanted, and the cells were resuspended in 3 x pellet volume of Buffer B supplemented with 2.5 x protease inhibitors (Roche, cOmplete, EDTA-free). From this point on, protease inhibitors were included in Buffer B. The cells were homogenized (Polyton homogenizer, PT1200E) with four 30-second pulses, and the membrane fraction was pelleted at 40,000 x g for 1 h at 4°C. The pellet was collected in 1 mL of PBS. The pellet was transferred to a Dounce homogenizer with 3 mL of buffer B and resuspended by slowly moving the pestle up and down 30-35 times. The enriched membrane fraction (EMF) was transferred to a new collection tube, and the pestle was rinsed to collect all potential proteins. The protein concentration of the purified EMF was determined using a Dc-protein assay kit (BioRad). To solubilize the EMF, solubilization buffer (50 mM NaCl) was added. The mixture was transferred to a final concentration of 1 mg / mL in a 50 mL Oak Ridge tube (Fisher Scientific, 050529-ID) containing 0.15 M HEPES, 0.15 M NaCl, 2 mM CaCl, 5 mM KCl, 5 mM MgCl, 1% n-dodecyl-bD-maltopyranoside (DDM), 1x protease inhibitors, pH 7.2. The mixture was rotated overnight at 4°C and then centrifuged at 40,000 x g for 1 hour in a 50 mL Oak Ridge tube (Fisher Scientific, 050529-ID). The supernatant, which contains soluble membrane proteins (SMPs), was collected and protein production quantified as described above.

[0341] For biotinylation, prepare NHS-LC-biotin stock solution according to the manufacturer's protocol (Pierce, Thermo Fisher). Briefly, 20 μl of biotin reagent was added to every 1 mg of EMF sample and incubated at 4°C for 3 hours with gentle agitation. Adjust the volume to 25 mL with Buffer B and transfer to an Oak Ridge centrifuge tube. Precipitate biotinylated EMF (b-EMF) at 40,000 × g for 1 hour and rinse twice with 3 mL of Buffer C (Buffer B minus glycerol) without disturbing the pellet. Remove residual solution. Resuspend the pellet in 3 mL of Buffer C using a Dounce homogenizer as described above. The resuspended pellet is now biotinylated EMF (b-EMF). It was solubilized as described above for preparing b-SMP.

[0342] PSR binding analysis. Assays were 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 displayed on yeast, two million IgG-displaying yeast cells were transferred to a 96-well assay plate, pelleted at 3000 × g for 3 minutes, and the supernatant was removed. The pellet was resuspended in 50 μl of a 1:10 dilution of freshly prepared stock biotinylated PSR (b-PSR) and incubated on ice for 20 minutes. Cells were washed twice with 200 μl of cold PBSF and resuspended in 50 μl of secondary labeling mix (Extravidin-R-PE, anti-human LC-FITC, and propidium iodide). This mix was incubated on ice for 20 minutes, followed by two washes with 200 μl of ice-cold PBSF. Cells were resuspended in 100 μl of ice-cold PBSF and plated on a FACSCanto (BD Biosciences) using the HTS sample injector. Flow cytometry data were analyzed for mean fluorescence intensity in the R-PE channel and normalized to appropriate controls (antibodies with established PSR scores) for assessment of nonspecific binding.Numerous methods for displaying or directing antibodies or antibody fragments on the surface of yeast have been previously described, all of which are compatible with the present 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 U S A., 105(51):20167-72 (2008), Tasumi et al., Proc Natl Acad Sci US A., 106(31):12891-6 (2009)).

[0343] Parent antibody No. 1 had the highest PSR score, indicating a higher level of non-target specific binding. Antibodies Nos. 2-6 showed PSR scores ≤0.1, or "clean" PSR scores, indicating very low levels of non-target specific binding.

[0344] Example 6: Developability - HIC, AC-SINS, DLS, and Fab Tm The developability of the anti-CD3 antibodies was further determined by evaluating their hydrophobicity, self-interaction, and stability. For these evaluations, antibodies 1–6 were produced as human IgG1 antibodies in CHO cells. Fab fragments were generated from the IgG1 antibodies by papain digestion and purified.

[0345] Hydrophobicity by HIC Antibody hydrophobicity is one cause of antibody aggregation. To evaluate antibody hydrophobicity, IgG1 production samples were subjected to hydrophobic interaction chromatography (HIC) analysis. Briefly, the IgG1 samples were buffer-exchanged into 1 M ammonium sulfate and 0.1 M sodium phosphate at pH 6.5 using a Zeba 40 kDa 0.5 mL spin column (Thermo Pierce, catalog number 87766). A salt gradient was established on a Dionex ProPac HIC-10 column from 1.8 M ammonium sulfate and 0.1 M sodium phosphate at pH 6.5 to the same conditions without ammonium sulfate. The gradient was run for 17 min at a flow rate of 0.75 ml / min. An acetonitrile wash step was added at the end of the run to remove any remaining protein, and the column was re-equilibrated for over 7 column volumes before the next injection cycle. Peak retention times were monitored by absorbance at A280, and the ammonium sulfate concentration at elution was calculated based on the gradient and flow rate.

[0346] All of the IgG1s tested had HIC retention times <10.5 min, indicating clean to low hydrophobicity, a highly desirable developability profile.

[0347] Self-interaction by AC-SINS Self-interaction was measured in vitro by affinity capture self-interaction nanoparticle spectroscopy (AC-SINS) using a previously described protocol (Liu et al., MAbs. Mar-Apr 2014;6(2):483-92). Briefly, a polyclonal goat anti-human IgG Fc antibody (capture; Jackson ImmunoResearch Laboratories) and a polyclonal goat nonspecific 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 4:1 volume ratio of capture:non-capture was 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. Thiolated PEG (Sigma-Aldrich) was then used to block vacant sites on the AuNPs and filtered through a 0.22 μm PVDF membrane (Millipore). The coated particles were then added to the test IgG1 antibody solution and incubated for 2 hours at room temperature before measuring the absorbance at 510-570 nm on a plate reader. Data points were fitted with a second-order polynomial in Excel to obtain the wavelength at maximum absorbance. Values ​​were reported as the difference between the sample and background plasmon wavelengths (Δλmax). The self-interaction level was determined based on Δλmax. Self-interaction was considered as follows: low when Δλmax<5.0 nm, moderate when Δλmax≥5.0 nm and <20.0 nm, and high when Δλmax≥20.0 nm.

[0348] Antibodies Nos. 2 to 6 had a Δλmax of <5.0, indicating low self-interaction.

[0349] Self-interaction by DLS Self-interactions were also measured by dynamic light scattering (DLS). The diffusion interaction parameter (kD) of monoclonal antibodies, measured at concentrations lower than 12 mg / mL, correlates strongly with their solution behavior at very high concentrations (>100 mg / mL). Positive kD values ​​indicate repulsive interactions between molecules and correlate positively with low viscosity at high concentrations in the same formulation buffer. kD values ​​were obtained by measuring the mutual diffusion coefficients at a series of different concentrations using DLS. Specifically, DLS kD measurements were performed at multiple concentrations ranging from 0.5 to 12 mg / mL in a 10 mM histidine buffer at pH 6.0. kD values ​​<20 mL / g were considered to be associated with high viscosity or opalescence.

[0350] Antibodies Nos. 1-6 yielded kD values ​​of ≥ 20 mL / g, indicating low self-interaction.

[0351] Fab Tm by DSF Melting points (T m ) was measured by differential scanning fluorescence (DSF) using a Bio-Rad CFX96 real-time system. Briefly, 20 μL of 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 min in a C1000 thermocycler (Bio-Rad) to collect Fret signals. Fab Tms were assigned using the first derivative of the raw data from Bio-Rad analysis software.

[0352] All tested Fabs had Tm values ​​greater than 65°C, indicating high stability and therefore desirable developability.

[0353] material and method Unless otherwise stated, the following materials and methods were used in the examples.

[0354] Yeast production and purification of antibodies. Yeast clones were grown to saturation and then induced for 48 hours at 30°C with shaking. After induction, yeast cells were pelleted and the supernatant was collected for purification. IgG was purified using a Protein A column and eluted with acetic acid at pH 2.0. Fab fragments were generated by papain digestion and purified with KappaSelect or CaptureSelect IgG-CH1 (GE Healthcare LifeSciences).

[0355] Antibody production and purification in CHO cells. Antibodies were produced as IgG1 by subcloning the antibody into a new expression vector, followed by transfection and expression in CHO cells. Fab fragments were generated by papain digestion and purified using KappaSelect or CaptureSelect IgG-CH1 (GE Healthcare LifeSciences). VH- and VL-encoding gene fragments (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 4 x 10*6 cells / mL were pelleted and resuspended in transfection medium. DNA plasmids (1.5 μg total DNA / mL) were incubated with PEIpro (final 1:2, PolyPlus, catalog no. 115-100) in transfection medium at room temperature before being added to the CHO-K1 cell suspension. Transfected cultures were fed and maintained at 32°C with shaking until the supernatant was harvested for purification (day 9). Cell culture supernatants were collected by centrifugation and passed through Protein A agarose (MabSelect SuRe; GE Healthcare Life Sciences). Bound antibodies were then washed with PBS and eluted with buffer (200 mM acetic acid / 50 mM NaCl, pH 3.5) into 1 / 8 volume of 2 M Hepes, pH 8.0. The final product was buffer exchanged into 25 mM Hepes and 150 mM sodium chloride, pH 7.3. Fab was purified using overnight papain digestion, followed by a CH1-resin purification step.

[0356] Generation of Hu and Cy CD3εδ Fc Heterodimeric Antigens. Recombinant heterodimeric CD3 Fc fusion antigens were produced in HEK293 cells by cotransfection of plasmids encoding Hu CD3ε 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) with heterologous signal peptide sequences (see Appendix Table U for sequences). Chromatographic separation was performed on a computer-controlled AKTA Avant 150 preparative chromatography system (GE Healthcare Life Sciences) with an integrated conductivity sensor, allowing in-line salt concentration monitoring during operation. Clarified culture supernatants were purified with Ni Sepharose 6 Fast Flow (GE Healthcare Life Sciences) to remove CD3εε Fc homodimers. CD3εδ Fc-HIS heterodimers were separated from CD3δδ Fc-HIS homodimers by Mono Q 10 / 100 GL with a linear Tris-buffered KCl gradient at pH 8.5.

[0357] BIACORE® KD measurements (surface plasmon resonance; SPR). BIACORE® affinity measurements were performed generally as previously described. Briefly, human CD3εδ-Fc heterodimers generated as described above were immobilized on a NiNTA sensor chip in a BIACORE® 8K (Cytiva, formerly GE Healthcare Life Sciences) to a response level of approximately 500 RU. Fab was then injected at increasing concentrations ranging from 18.75 to 300 nM, 1.56 to 25 nM, 6.25 to 100 nM, or 1.25 to 20 nM. The sensor chip was regenerated in duplicate between cycles using 0.35 M EDTA and 0.1 M NaOH. The resulting data were double-reference subtracted and fitted to a 1:1 binding model using the BIACORE® evaluation software.

[0358] ForteBio K D Measurement (Biolayer Interferometry; BLI). ForteBio affinity measurements were generally performed as previously reported (Estep, P., et al., High throughput solution-based measurement of antibody-antigen affinity and epitope binning. MAbs, 2013. 5(2): p. 270-8). IgG was loaded online onto the AHC sensor. The sensor was equilibrated offline in assay buffer for 30 minutes and then monitored online for 60 seconds to establish a baseline. The IgG-loaded sensors were exposed to 100 nM antigen (i.e., CD3) for 5 minutes, after which they were transferred to assay buffer for 5 minutes and dissociation rate measurements were performed. A 1:1 binding model was used to analyze the kinetics.

[0359] ForteBio Kinetics. A ForteBio OCTET® HTX instrument was used in 12-channel mode (8 sensors per channel, 96 sensors per experiment) with either AHC, SA, or AHQ sensors. The instrument was operated with manufacturer-supplied software (versions 8.2 and 9.0). Sample names and concentrations were entered on the plate data page, and proteins bound to the sensors were identified in the "Information" column on the sensor data page. Kinetic experiments were collected with either a 90- or 180-second baseline, a 180-second association phase, and a 180-second dissociation phase. All files were saved to a shared network drive using a naming convention that identifies the experiment type.

[0360] Cell line expansion and cell labeling assay. Human Jurkat CD3+ cells (ATCC TIB-152) and Jurkat CD3- cells (ATCC TIB-153) were obtained from ATCC. Cynomolgus monkey HSC-F cells were obtained from the NIH Non-human Primate Reagent Resource. All cell lines were cultured in RPMI 1640 GlutaMax medium supplemented with 10% fetal bovine serum (FBS).

[0361] Cell binding assay. CD3+ and CD3+ human Jurkat cells (ATCC) and cynomolgus monkey 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 dispensed per well of a 96-well plate (FACS Assay Plate VWR BD 353263) and pelleted by centrifugation (500 × g for 5 minutes). Cells were washed with either PBSF pH 7.4 or PBSF pH 6.0 (PBS + 0.1% BSA, pH 6.0) and then resuspended in 100 μl of either PBSF pH 7.4 or PBSF pH 6.0 containing 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 either PBSF pH 7.4 or PBSF pH 6.0. 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 either PBSF pH 7.4 or PBSF pH 6.0. After incubation on ice in the dark for 20 minutes, cells were washed twice with either PBSF pH 7.4 or PBSF pH 6.0. Binding was analyzed on a FACS Canto II.

[0362] PSR Preparation. The polyspecific reaction reagent (PSR) was 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 were used as starting material. In a 500 mL centrifuge bottle filled to 400 mL, the cells were pelleted at 2,400 × g for 5 minutes. The cell pellets were combined and then resuspended in 25 mL of Buffer B and pelleted at 2,400 × g for 3 minutes. The buffer was decanted, and the wash was repeated once more. The cell pellets were resuspended in 3 volumes of Buffer B containing 1 × protease inhibitor (Roche, Complete, EDTA-free) using a Polytron homogenizer while keeping the cells on ice. The homogenate was then centrifuged at 2,400 × g for 5 minutes, and the supernatant was retained and pelleted once more (2,400 × g / 5 minutes) to ensure removal of unbroken cells, cell debris, and nuclei. The resulting supernatant was the total protein preparation. The supernatant was then transferred to two Nalgene Oak Ridge 45 mL centrifuge tubes and pelleted at 40,000 × g for 40 minutes at 4°C. The supernatant containing soluble cytosolic proteins (SCP) was then transferred to a clean Oak Ridge tube and centrifuged once more at 40,000 × g. In parallel, the pellet containing the membrane fraction (EMF) was retained and centrifuged at 40,000 × g for 20 minutes to remove any remaining supernatant. The EMF pellet was then rinsed with Buffer B. 8 mL of Buffer B was then added to the membrane pellet, which was then scraped and transferred to a Dounce homogenizer. After the pellets were homogenized, they were transferred to a 50 mL conical tube to represent the final EMF preparation.

[0363] about 10 6 ~10 7Mammalian cells (e.g., CHO, HEK293, Sf9, etc.) at 1 billion cells / mL were transferred from tissue culture medium into 4 x 250 mL conical tubes and pelleted at 550 x g for 3 minutes. All subsequent steps were performed at 4 °C or on ice using ice-cold buffers. The cells were washed with 100 mL of PBSF (1 x PBS + 1 mg / mL BSA) and combined in one conical tube. After removing the supernatant, the cell pellet was resuspended in 30 mL of buffer B (50 mM HEPES, 0.15 M NaCl, 2 mM CaCl, 5 mM KCl, 5 mM HCl). The cells were resuspended in 10% MgCl2, 10% glycerol, pH 7.2, and pelleted at 550 x g for 3 min. The supernatant in Buffer B was decanted, and the cells were resuspended in 3 x pellet volume of Buffer B supplemented with 2.5 x protease inhibitors (Roche, cOmplete, EDTA-free). From this point on, protease inhibitors were included in Buffer B. The cells were homogenized (Polyton homogenizer, PT1200E) with four 30-second pulses, and the membrane fraction was pelleted at 40,000 x g for 1 h at 4°C. The pellet was collected in 1 mL of PBS. The pellet was transferred to a Dounce homogenizer with 3 mL of buffer B and resuspended by slowly moving the pestle up and down 30-35 times. The enriched membrane fraction (EMF) was transferred to a new collection tube, and the pestle was rinsed to collect all potential proteins. The protein concentration of the purified EMF was determined using a Dc-protein assay kit (BioRad). To solubilize the EMF, solubilization buffer (50 mM NaCl) was added. The mixture was transferred to a final concentration of 1 mg / mL in a 50 mL Oak Ridge tube (Fisher Scientific, 050529-ID) containing 0.15 M HEPES, 0.15 M NaCl, 2 mM CaCl, 5 mM KCl, 5 mM MgCl, 1% n-dodecyl-bD-maltopyranoside (DDM), 1x protease inhibitors, pH 7.2. The mixture was rotated overnight at 4°C and then centrifuged at 40,000 x g for 1 hour in a 50 mL Oak Ridge tube (Fisher Scientific, 050529-ID). The supernatant, which contains soluble membrane proteins (SMPs), was collected and protein production quantified as described above.

[0364] For biotinylation, prepare NHS-LC-biotin stock solution according to the manufacturer's protocol (Pierce, Thermo Fisher). Briefly, 20 μl of biotin reagent was added to every 1 mg of EMF sample and incubated at 4°C for 3 hours with gentle agitation. Adjust the volume to 25 mL with Buffer B and transfer to an Oak Ridge centrifuge tube. Precipitate biotinylated EMF (b-EMF) at 40,000 × g for 1 hour and rinse twice with 3 mL of Buffer C (Buffer B minus glycerol) without disturbing the pellet. Remove residual solution. Resuspend the pellet in 3 mL of Buffer C using a Dounce homogenizer as described above. The resuspended pellet is now biotinylated EMF (b-EMF). It was solubilized as described above for preparing b-SMP.

[0365] PSR binding analysis. Assays were 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 displayed on yeast, two million IgG-displaying yeast cells were transferred to a 96-well assay plate, pelleted at 3000 × g for 3 minutes, and the supernatant was removed. The pellet was resuspended in 50 μl of a 1:10 dilution of freshly prepared stock biotinylated PSR (b-PSR) and incubated on ice for 20 minutes. Cells were washed twice with 200 μl of cold PBSF and resuspended in 50 μl of secondary labeling mix (Extravidin-R-PE, anti-human LC-FITC, and propidium iodide). This mix was incubated on ice for 20 minutes, followed by two washes with 200 μl of ice-cold PBSF. Cells were resuspended in 100 μl of ice-cold PBSF and plated on a FACSCanto (BD Biosciences) using the HTS sample injector. Flow cytometry data were analyzed for mean fluorescence intensity in the R-PE channel and normalized to appropriate controls (antibodies with established PSR scores) for assessment of nonspecific binding.Numerous methods for displaying or directing antibodies or antibody fragments on the surface of yeast have been previously described, all of which are compatible with the present 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 U S A., 105(51):20167-72 (2008), Tasumi et al., Proc Natl Acad Sci US A., 106(31):12891-6 (2009)). Polyspecificity was evaluated based on the PSR score. The polyspecificity of an antibody was considered as follows: clean (no polyspecificity) when 0.0≦PSR score<0.10, low when 0.10≦PSR score<0.33, moderate when 0.33≦PSR score<0.66, and high when 0.66≦PSR score≦1.00.

[0366] HIC. The IgG1 sample was buffer-exchanged into 1 M ammonium sulfate and 0.1 M sodium phosphate at pH 6.5 using a Zeba 40 kDa 0.5 mL spin column (Thermo Pierce, catalog number 87766). A salt gradient was established on a Dionex ProPac HIC-10 column from 1.8 M ammonium sulfate and 0.1 M sodium phosphate at pH 6.5 to the same conditions without ammonium sulfate. The gradient was run for 17 min at a flow rate of 0.75 ml / min. An acetonitrile wash step was added at the end of the run to remove any remaining protein, and the column was re-equilibrated for over 7 column volumes before the next injection cycle. Peak retention times were monitored by absorbance at A280, and the ammonium sulfate concentration at elution was calculated based on the gradient and flow rate. Antibody hydrophobicity was assessed based on HIC retention time. Hydrophobicity was considered as follows: clean to low when HIC retention time was <10.5 min, moderate when 10.5 min < retention time < 11.5 min, and high when 11.5 min < retention time.

[0367] LC-MS. The IgG1 sample was reduced with DTT and then subjected to mid-down LC-MS analysis on a Bruker maXis4G mass spectrometer coupled to an Agilent 1100 HPLC (Agilent). A POROS R2 10 μm (2.1 × 30 mm) reversed-phase column was used to remove salts from the sample. A fast LC flow of 2 mL / min allowed for separation of the sample and salts, and sample elution and column regeneration were completed within a 2.1-minute cycle. A T-junction was used to deliver a sample flow of only 0.15 mL / min to the mass spectrometer for sample analysis. The Bruker maXis 4G mass spectrometer was operated in positive ion mode, with detection in the 750–2500 m / z range. The remaining source parameters were set as follows: the capillary was set at 5500 V, the nebulizer at 4.0 bar, the drying gas at 4.0 L / min, and the drying temperature at 200 °C. MS spectra were analyzed using Bruker Data Analysis version 4.1, and deconvolution was performed using maximum entropy deconvolution in the 20-30 kDa mass range. The tendency for heavy-heavy or heavy-light chain pairing failure was assessed based on LC-MS spectrographs. The presence of additional heavy and / or light chain peaks and / or half-antibody peaks indicates a tendency for pairing failure. LC-MS was also used to confirm that the heavy and light chain masses matched the expected masses based on the amino acid sequence.

[0368] SEC. Column chromatography (TSKgel Super SW mAb HTP column) was monitored using an Agilent 1260 HPLC. The column was equilibrated with wash buffer (200 mM sodium phosphate, 250 mM sodium chloride, pH 6.8) at a flow rate adjusted to 0.400 mL / min prior to use. Approximately 2–5 μg of IgG1 or Fab protein sample was injected onto the column. Protein migration was monitored at a wavelength of 280 nm. Total assay time was approximately 6 min. Data were analyzed using ChemStation software. The tendency of antibodies to aggregate was assessed based on the percent monomer in the SEC chromatogram. Antibodies with a percent monomer of 95% or greater were considered to be substantially present as monomers, i.e., not aggregated.

[0369] Tolerance to acidic stress. IgG1 samples were incubated at acidic or physiological pH and subjected to SEC-HPLC analysis. Briefly, IgG1 samples at 20 mg / mL 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, pH 7.0), and 2 μg of sample was injected onto 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 resistance of antibodies to acidic stress was evaluated based on the tendency of the antibody to aggregate under acidic conditions, as assessed by the % monomer in the SEC chromatogram.

[0370] AC-SINS. Self-interaction was measured in vitro by affinity capture self-interaction nanoparticle spectroscopy (AC-SINS) using a previously described protocol (Liu et al., MAbs. Mar-Apr 2014;6(2):483-92). Briefly, a polyclonal goat anti-human IgG Fc antibody (capture; Jackson ImmunoResearch Laboratories) and a polyclonal goat nonspecific 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 4:1 volume ratio of capture:non-capture was 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. Thiolated PEG (Sigma-Aldrich) was then used to block vacant sites on the AuNPs, which were then filtered through a 0.22 μm PVDF membrane (Millipore). The coated particles were then added to the test IgG1 antibody solution and incubated for 2 hours at room temperature before measuring the absorbance between 510 and 570 nm on a plate reader. Data points were fitted with a second-order polynomial in Excel to obtain the wavelength at maximum absorbance. Values ​​were reported as the difference between the sample and background plasmon wavelengths (Δλmax). The self-interaction level was determined based on Δλmax. Self-interaction was considered as follows: low when Δλmax < 5.0 nm, moderate when Δλmax ≥ 5.0 nm and < 20.0 nm, and high when Δλmax ≥ 20.0 nm.

[0371] DLS. Self-interactions were measured by dynamic light scattering (DLS). The diffusion interaction parameter (kD) of a monoclonal antibody, measured at concentrations lower than 12 mg / mL, correlates strongly with its solution behavior at very high concentrations (>100 mg / mL). Positive kD values ​​indicate repulsive interactions between molecules and correlate positively with low viscosity at high concentrations in the same formulation buffer. kD values ​​were obtained by measuring the mutual diffusion coefficients at a series of different concentrations using DLS. Specifically, DLS kD measurements were performed at multiple concentrations ranging from 0.5 to 12 mg / mL in a 10 mM histidine buffer at pH 6.0. kD values ​​<20 mL / g were considered to be associated with high viscosity or opalescence.

[0372] DSF. Melting temperatures (Tm) were measured by differential scanning fluorescence (DSF) using a Bio-Rad CFX96 real-time system. Briefly, 20 μL of 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 min in a C1000 thermocycler (Bio-Rad) to collect Fret signals. Fab Tm was assigned using the first derivative of the raw data from Bio-Rad analysis software. Antibodies with a Tm greater than 65°C were considered stable.

[0373] An informal sequence listing is provided in the Appendix, providing the VH, VL, CDR, and FR amino acid sequences, as well as the nucleic acid sequences encoding the VH and VL amino acid sequences, of the anti-CD3 antibodies analyzed in the Examples. appendix [Table A-1] [Table A-2] [Table A-3] [Table A-4]

Table B-1

Table B-2

Table B-3

Table B-4

Table B-5

Table B-6

Table B-7

Table B-8

Table B-9

Table H

Table I

Table J

Table L

Table M

Table N

Table O

Table P

Table Q

Table R

Table S

Table T

Table U

Claims

1. An anti-cluster of differentiation 3 (CD3) antibody or antigen-binding fragment thereof, (A) a heavy chain variable domain (VH) polypeptide comprising: (a) a VH complementarity determining region 1 (CDR-H1) comprising: (i) 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) a VH CDR-H1 comprising the amino acid sequence of 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) comprising: (i) 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) a VH polypeptide comprising a VH CDR-H2 comprising the amino acid sequence of 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) comprising: (i) 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) ARDX ２ Y X ３ RYFYDV (SEQ ID NO: 16) ２ is A or H, and X ３ is H or G), and / or (iii) a VH CDR-H3 comprising the amino acid sequence of 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 comprising: (a) a VL complementarity determining region 1 (CDR-L1) comprising: (i) 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) KSSQSLLNARTGX ５ NYLA (SEQ ID NO: 22) ５ is H or K), and / or (iii) a VL CDR-L1 comprising the amino acid sequence of KSSQSLLNARTGHNYLA (SEQ ID NO: 622, 422, 322, 922, 1222, or 1322) or KSSQSLLNARTGKNYLA (SEQ ID NO: 522, 722, 822, 1022, or 1122); (b) a VL complementarity determining region 2 (CDR-L2) comprising: (i) 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) a VL CDR-L2 comprising the amino acid sequence of WASTRES (SEQ ID NO: 24, 624, 524, 424, 324, 724, 824, 924, 1024, 1124, 1224, or 1324); and / or (c) a VL complementarity-determining region 3 (CDR-L3) comprising: (i) 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) KQSX ６ SX ７ RT (SEQ ID NO: 26) ６ is Y or H, and X ７ is H or R), and / or (iii) a VL polypeptide comprising a VL CDR-L3 comprising the amino acid sequence of 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, the anti-CD3 antibody and / or antigen-binding fragment: (I) (i) at least one of CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 contained in ADI-48587; or (ii) does not contain 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 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); and Further optionally, (iii) at least one of the CDR-H3, CDR-L1, and CDR-L3 contained in ADI-48587; or (iv) does not contain at least one of CDR-H3 comprising ARDHYHRYFYDV (SEQ ID NO: 216), CDR-L1 comprising KSSQSLLNAARTGHNYLA (SEQ ID NO: 222), and CDR-L3 comprising KQSHSHRT (SEQ ID NO: 226); and (II) (i) at least one of CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 contained in ADI-26906; or (ii) does not contain 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); and Further optionally, (iii) the CDR-H3, CDR-L1, and CDR-L3 contained in ADI-26906, or (iv) An anti-CD3 antibody or antigen-binding fragment thereof that does not comprise CDR-H3 comprising ARDAYGRYFYDV (SEQ ID NO: 116), CDR-L1 comprising KSSQSLLNARTGKNYLA (SEQ ID NO: 122), and CDR-L3 comprising KQSYSRRT (SEQ ID NO: 126).

2. (A) a VH polypeptide comprising: (a) CDR-H1, comprising: (i) CDR-H1 contained in ADI-74968, ADI-74967, ADI-74966, or ADI-74965, and / or (ii) CDR-H1 comprising the amino acid sequence of FNIKDYYMH (SEQ ID NO: 12, 612, 512, 412, or 312); (b) a CDR-H2 comprising: (i) CDR-H2 contained in ADI-74968, ADI-74967, ADI-74966, or ADI-74965, and / or (ii) a CDR-H2 comprising the amino acid sequence of WIDLENANTIYDAKFQG (SEQ ID NO: 14, 614, 514, 414, or 314); and / or (c) a CDR-H3 comprising: (i) CDR-H3 contained in ADI-74968, ADI-74967, ADI-74966, or ADI-74965; (ii) ARDX ２ Y X ３ RYFYDV (SEQ ID NO: 16) ２ is A or H, and X ３ is H), and / or (iii) a VH polypeptide comprising a CDR-H3 comprising the amino acid sequence of ARDAYHRYFYDV (SEQ ID NO: 616 or 316) or ARDHYHRYFYDV (SEQ ID NO: 516 or 416); and / or (B) a VL polypeptide comprising: (a) CDR-L1, comprising: (i) CDR-L1 contained in ADI-74968, ADI-74967, ADI-74966, or ADI-74965; (ii) KSSQSLLNARTGX ５ NYLA (SEQ ID NO: 22) ５ is H or K), and / or (iii) CDR-L1 comprising the amino acid sequence of KSSQSLLNAARTGHNYLA (SEQ ID NO: 622, 422, or 322) or KSSQSLLNARTGKNYLA (SEQ ID NO: 522); (b) a CDR-L2 comprising: (i) CDR-L2 contained in ADI-74968, ADI-74967, ADI-74966, or ADI-74965, and / or (ii) a CDR-L2 comprising the amino acid sequence of WASTRES (SEQ ID NO: 24, 624, 524, 424, or 324); and / or (c) a CDR-L3 comprising: (i) CDR-L3 contained in ADI-74968, ADI-74967, ADI-74966, or ADI-74965; (ii) KQSX ６ SX ７ RT (SEQ ID NO: 26) ６ is Y or H, and X ７ is H), and / or The anti-CD3 antibody or antigen-binding fragment of claim 1, comprising a VL polypeptide comprising (iii) a CDR-L3 comprising the amino acid sequence of KQSYSHRT (SEQ ID NO: 626 or 426) or KQSHSHRT (SEQ ID NO: 526 or 326).

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

4. (A) a VH polypeptide comprising: (a) a CDR-H1 comprising: (i) the CDR-H1 contained in ADI-74967, and / or (ii) the amino acid sequence of FNIKDYYMH (SEQ ID NO: 12 or 512); (b) a CDR-H2 comprising: (i) the CDR-H2 contained in ADI-74967, and / or (ii) the amino acid sequence of WIDLENANTIYDAKFQG (SEQ ID NO: 14 or 514); and / or (c) a VH polypeptide comprising a CDR-H3, wherein the CDR-H3 comprises: (i) the CDR-H3 contained in ADI-74967; (ii) a CDR-H3 comprising the amino acid sequence of ARDHYHRYFYDV (SEQ ID NO:516); and / or (B) a VL polypeptide comprising: (a) a CDR-L1 comprising: (i) the CDR-L1 contained in ADI-74967; (ii) a CDR-L1 comprising the amino acid sequence of KSSQSLLNARTGKNYLA (SEQ ID NO: 522); (b) a CDR-L2 comprising: (i) the CDR-L2 contained in ADI-74967, and / or (ii) the amino acid sequence of WASTRES (SEQ ID NOs: 24, 624, 524); and / or (c) a VL polypeptide comprising: (i) a CDD-L3 contained in ADI-74967; and (ii) a CDR-L3 comprising the amino acid sequence of KQSHSHRT (SEQ ID NO: 526).

5. (A) a VH polypeptide comprising the CDR-H1, the CDR-H2, and the CDR-H3; and / or (B) The anti-CD3 antibody or antigen-binding fragment of any one of claims 1 to 4, comprising a VL polypeptide comprising the CDR-L1, the CDR-L2, and the CDR-L3.

6. (A) a VH polypeptide comprising the CDR-H1, the CDR-H2, and the CDR-H3; and (B) The anti-CD3 antibody or antigen-binding fragment of any one of claims 1 to 5, comprising a VL polypeptide comprising the CDR-L1, the CDR-L2, and the CDR-L3.

7. (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 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); (III) (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); (IV) (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); (V) (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); (VI) (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); (VII) (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); (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) the 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) 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 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) the CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 contained in ADI-79846, or (ii) The anti-CD3 antibody or antigen-binding fragment of any one of claims 1 to 6, comprising: 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. (A) the VH polypeptide is (a) VH framework region 1 (FR-H1) comprising: (i) 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) VH FR-H1 comprising the amino acid sequence of 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) comprising: (i) 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) VH FR-H2 comprising the amino acid sequence of 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) comprising: (i) 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) RVTITRDTSASTAYMX １ LSSLRSEDTAVYYC (SEQ ID NO: 15) １ is E or G), and / or (iii) a VH FR-H3 comprising the amino acid sequence of 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) comprising: (i) 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) a VH FR-H4 comprising the amino acid sequence of WGQGTLVTVSS (SEQ ID NO: 17, 617, 517, 417, 317, 717, 817, 917, 1017, 1117, 1217, or 1317); and / or (B) the VL polypeptide is (a) VL framework region 1 (FR-L1) comprising: (i) 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) X ４ IVMTQSPDSLAVSLGERATINC (SEQ ID NO: 21) ４ is D or G), and / or (iii) VL FR-L1 comprising the amino acid sequence of 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) comprising: (i) 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) VL FR-L2 comprising the amino acid sequence of WYQQKPGQPPKLLIY (SEQ ID NO: 23, 623, 523, 423, 323, 723, 823, 923, 1023, 1123, 1223, or 1323); (c) a VL framework region 3 (FR-L3) comprising: (i) 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) a VL FR-L3 comprising the amino acid sequence of 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) comprising: (i) 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) a VL FR-L4 comprising the amino acid sequence of FGGGTKVEIK (SEQ ID NO: 27, 627, 527, 427, 327, 727, 827, 927, 1027, 1127, 1227, or 1327); or Alternatively, the anti-CD3 antibody or antigen-binding fragment of any one of claims 1 to 7 comprises a VH and / or VL comprising any combination of the aforementioned VH and VL framework regions.

9. (I) (i) ADI-74968, ADI-74966, ADI-74965, ADI-79842, ADI-79843, ADI-79848, ADI-79844, ADI-7 9845, or the FR-H1, FR-H2, FR-H3, FLR-H4, FR-L1, FR-L2, FR-L3, and FR-L4 contained in ADI-79847, or (ii) FR-H1 including QVQLVQSGAEVKKPGASVKVSCKASG (SEQ ID NO: 11, 611, 411, 311, 711, 811, 911, 1011, 1111, or 1311), FR including WVRQAPGQRLEWMG (SEQ ID NO: 13, 613, 413, 313, 713, 813, 913, 1013, 1113, or 1313) -H2, FR-H3 including RVTITRDTSASTAYMELSSLRSEDTAVYYC (SEQ ID NO: 615, 415, 315, 715, 815, 915, 1015, 1115, or 1315), FR-H4 including WGQGTLVTVSS (SEQ ID NO: 17, 617, 417, 317, 717, 817, 917, 1017, 1117, or 1317) , FR-L1 including DIVMTQSPDSLAVSLGERATINC (SEQ ID NO: 621, 421, 321, 721, 821, 921, 1021, 1121, or 1321), FR-L2 including WYQQKPGQPPKLLIY (SEQ ID NO: 23, 623, 423, 323, 723, 823, 923, 1023, 1123, or 1323), GVPDR FR-L3 comprising FSGSGSGTDFTLTISSLQAEDVAVYYC (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); (II) (i) 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 containing QVQLVQSGAEVKKPGASVKVSCKASG (SEQ ID NO: 11 or 511), FR-H2 containing WVRQAPGQRLEWMG (SEQ ID NO: 13 or 513), FR-H3 containing RVTITRDTSASTAYMELSSLRSEDTAVYYC (SEQ ID NO: 515), FR-H4 containing WGQGTLVTVSS (SEQ ID NO: 17 or 517), G FR-L1 comprising IVMTQSPDSLAVSLGERATINC (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) ADI-74968, ADI-74966, ADI-74965, ADI-79842, ADI-79843, ADI-79848, ADI-79844, ADI- 79845, or the FR-H1, FR-H2, FR-H3, FLR-H4, FR-L1, FR-L2, FR-L3, and FR-L4 contained in ADI-79847, or (ii) FR-H1 containing QVQLVQSGAEVKKPGASVKVSCKASG (SEQ ID NO: 11 or 1211), FR-H2 containing WVRQAPGQRLEWMG (SEQ ID NO: 13 or 1213), FR-H3 containing RVTITRDTSASTAYMGLSSLRSEDTAVYYC (SEQ ID NO: 1215), FR-H4 containing WGQGTLVTVSS (SEQ ID NO: 17 or 1217), DIVMTQSP 9. The anti-CD3 antibody or antigen-binding fragment of any one of claims 1 to 8, comprising FR-L1 comprising DSLAVSLGERATINC (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. (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 comprising SEQ ID NOs: 611, 612, 613, 614, 615, 616, 617, 621, 622, 623, 624, 625, 626, and 627, respectively; (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 comprising SEQ ID NOs: 511, 512, 513, 514, 515, 516, 517, 521, 522, 523, 524, 525, 526, and 527, respectively; (III) (i) 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 comprising SEQ ID NOs: 411, 412, 413, 414, 415, 416, 417, 421, 422, 423, 424, 425, 426, and 427, respectively; (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-H4 comprising SEQ ID NOs: 311, 312, 313, 314, 315, 316, 317, 321, 322, 323, 324, 325, 326, and 327, respectively; (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 comprising SEQ ID NOs: 711, 712, 713, 714, 715, 716, 717, 721, 722, 723, 724, 725, 726, and 727, respectively; (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 comprising SEQ ID NOs: 811, 812, 813, 814, 815, 816, 817, 821, 822, 823, 824, 825, 826, and 827, respectively; (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 comprising SEQ ID NOs: 911, 912, 913, 914, 915, 916, 917, 921, 922, 923, 924, 925, 926, and 927, respectively; (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 comprising SEQ ID NOs: 1011, 1012, 1013, 1014, 1015, 1016, 1017, 1021, 1022, 1023, 1024, 1025, 1026, and 1027, respectively; (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 comprising SEQ ID NOs: 1111, 1112, 1113, 1114, 1115, 1116, 1117, 1121, 1122, 1123, 1124, 1125, 1126, and 1127, respectively; (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 comprising SEQ ID NOs: 1211, 1212, 1213, 1214, 1215, 1216, 1217, 1221, 1222, 1223, 1224, 1225, 1226, and 1227, respectively; 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) The anti-CD3 antibody or antigen-binding fragment of any one of claims 1 to 9, comprising 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.

11. (A) a VH polypeptide comprising an amino acid sequence 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 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, the anti-CD3 antibody or antigen-binding fragment comprises: (I)(A) (i) a VH polypeptide comprising the CDR-H1, CDR-H2, and CDR-H3 sequences of SEQ ID NOs: 612, 614, and 616, respectively, and (ii) an amino acid sequence at least 90% identical to SEQ ID NO: 610; (B) (i) the CDR-L1, CDR-L2, and CDR-L3 sequences of SEQ ID NOs: 622, 624, and 626, respectively, and (ii) a VL polypeptide comprising an amino acid sequence 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 NOs: 512, 514, and 516, respectively, and (ii) an amino acid sequence at least 90% identical to SEQ ID NO: 510; (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 at least 90% identical to SEQ ID NO:

520.

12. a VH polypeptide and a VL polypeptide (I) SEQ ID NOs: 610 and 620, respectively; (II) SEQ ID NOs: 510 and 520, respectively; (III) SEQ ID NOs: 410 and 420, respectively; (IV) SEQ ID NOs: 310 and 320, respectively; (V) SEQ ID NOs: 710 and 720, respectively; (VI) SEQ ID NOs: 810 and 820, respectively; (VII) SEQ ID NOs: 910 and 920, respectively; (VIII) SEQ ID NOs: 1010 and 1020, respectively; (IX) SEQ ID NOs: 1110 and 1120, respectively; (X) SEQ ID NOs: 1210 and 1220, respectively; or (XI) The anti-CD3 antibody or antigen-binding fragment of any one of the preceding claims, comprising the amino acid sequence of SEQ ID NOs: 1310 and 1320, respectively.

13. (i) an antibody constant region, CH1 domain, hinge, CH2 domain, and / or CH3 domain, optionally being or derived from IgG or human IgG, and further optionally being or derived from human IgG1, IgG4, IgG2, or IgG3; (ii) a fragment crystallizable (Fc) region, optionally comprising: (1) Human IgG1, optionally with the following amino acid modifications according to EU numbering: 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; (2) human IgG4, optionally comprising one or more of the following amino acid modifications according to EU numbering: E233P, F234V, L235A, G237A, E318A, S228P, L236E, S241P, L248E, T394D, M252Y, S254T, T256E, N297A, N297Q, or any combination thereof; (3) human IgG2, optionally comprising one or more of the following amino acid modifications according to EU numbering: P238S, V234A, G237A, H268A, H268Q, H268E, V309L, N297A, N297Q, A330S, P331S, C232S, C233S, M252Y, S254T, T256E, or any combination thereof; and / or (4) a fragment crystallizable (Fc) region that is a human IgG3, and optionally a human IgG3 that contains E235Y according to EU numbering; (iii) IgG, IgA, IgE, IgD, or IgM, optionally IgG1, IgG4, IgG2, or IgG3, and / or (iv) Fragment antigen binding (Fab), Fab ２ , Fab ３ , Fab' fragment, F(ab') ２ 13. The anti-CD3 antibody or antigen-binding fragment of any one of claims 1 to 12, comprising any one or more of the following antibody fragments selected from the group consisting of: a variable fragment (Fv), a single-chain Fv (scFv) fragment, a diabody, a triabody, a minibody, scFv-Fc, scFv2-Fc2, scFv-IgG, a monovalent IgG (or half-IgG), and / or a chimeric antigen receptor (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, and further optionally from CD3ζ).

14. (a) a first antigen-binding region specific for CD3, comprising said VH polypeptide and / or said VL polypeptide, and (b) one or more other antigen-binding regions, optionally having the following properties: (i) the one or more other antigen-binding regions comprise an antigen-binding region 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 disease 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 a receptor thereof), a neurodegenerative disease target, an infectious disease target (optionally a viral, bacterial, or fungal target molecule), a target molecule expressed on an infected cell (optionally a cell infected with a virus, bacteria, or fungus), a metabolic disease target, a cognitive impairment target, a blood-brain barrier target, or a hematological disease target; (ii) 17-IA, 4-1BB, 4Dc, 6-keto-PGF1a, 8-iso-PGF2a, 8-oxo-dG, Al adenosine receptor, A33, ACE, ACE-2, activin, activin A, activin AB, activin B, activin C, activin RIA, activin RIA ALK-2, activin RIB ALK-4, activin RIIA, activin RUB, ADAM, ADAM10, ADAM12, ADAM15, ADAM17 / T ACE, ADAM8, ADAM9, ADAMTS, ADAMTS4, ADAMTS5, addressin, aFGF, ALCAM, ALK, ALK-1, ALK-7, alpha-l-antitrypsin, alpha-V / beta-1 antagonist, ANG, Ang, APAF-1, APE, APJ, APP, APRIL, AR, ARC, ART, artemin, anti-Id, ASPARTIC, atrial natriuretic factor, av / b3 integrin, Ax l, b2M, B7-1, B7-2, B7-H, B lymphocyte stimulating factor (BlyS), BACE, BACE-1, Bad, BAFF, BAFF-R, Bag-1, BAK, Bax, BCA-1, BCAM, Bel, BCMA , BDNF, b-ECGF, bFGF, BID, Bik, BFM, BLC, BL-CAM, BLK, BMP, BMP-2, BMP-2a, BMP-3 osteogenin, BMP-4, BMP-2b, BMP-5, BMP-6Vgr-1, BMP-7 (OP-1), BMP-8 (BMP-8a, OP-2), BMPR, BMPR-IA (ALK-3), BMPR-IB (ALK-6), BRK-2, RPK-1, BMPR-II (BRK-3), BMP, b-NGF, BOK, bombesin, bone-derived neurotrophic factor, BPDE, BPDE-DNA, BTC, complement factor 3 (C3), C3a, C4, C5, C5a, CIO, CA125, CAD-8, calcitonin, cAMP, carcinoembryonic antigen (CEA), cancer-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, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL2, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CCL3, CCL4, CCL5, CCL6, CCL7, CCL8, CCL9 / 10, CCR, CCR1, CCR10, CCR10, CCR2, CCR3, CCR4, CCR5, CCR6 , CCR7, CCR8, CCR9, CD1, CD2, CD4, CD5, CD6, CD7, CD8, CD10, CD11a, CD1 b, CD11c, CD13, CD14, CD15, CD16, CD18, CD19, CD20, CD21, CD22, CD23, CD25, CD27L, CD28, CD29, CD30, CD30L, CD32, CD33 (p67 protein), CD34, C D38, 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, CMVUL, CNTF, CNTN-1, COX, C-Ret, CRG-2, CT-1, CTACK, CTGF, CTLA-4, CX3CL1, CX3CR1, CXCL, CXCL1, CXCL2, CXCL3, CXCL4, CXC L5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCR, CXCR1, CXCR2, CXCR3, CX CR4, 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, EDA-A1, EGA-A2, EDAR, EGF, EGFR (ErbB-1), EMA, EMMPRIN, EN A, endothelin receptor, enkephalinase, eNOS, Eot, eotaxin, EpCAM, ephrinB2 / EphB4, EPO, ERCC, E-selectin, ET-1, factor Ila, factor VII, factor VIIIc, factor IX, fibroblast activation protein (FAP), Fas, FcRl, FEN-1, ferritin, FGF, FGF-19, FGF-2, FGF3, FGF-8, FGFR, FGFR-3, fibrin, FL, FLIP, Flt-3, Flt-4, follicle-stimulating hormone, fractalkine, FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, FZD10, G250, Gas 6, GCP-2, GCSF, GD2, GD3, GDF, GDF-1, GDF-3 (Vgr-2), GDF-5 (BMP-14, CDMP-1), GDF-6 (BMP-13, CDMP-2), GDF-7 (BMP-12, CDMP-3), GDF-8 (myostatin), GDF-9, GDF-15 (MIC-1), GDNF, GFAP, GFRα-1, GFR-alpha1, GFR-alpha2, GFR-alpha3, GITR, glucagon, Glut 4, glycoprotein IIb / IIIa (GP IIb / IIIa), GM-CSF, gpl30, gp72, GRO, growth hormone-releasing factor, hapten (NP-cap or NIP-cap), HB-EGF, HCC, HCMVgB 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, HIV IIIB gpl20 V3 loop, HLA, HLA-DR, HM1.24, HMFG PEM, HRG, Hrk, human cardiac myosin, human cytomegalovirus (HCMV), human growth hormone (HGH), HVEM, 1-309, IAP, ICAM, ICAM-1, ICAM-3, ICE, ICOS, IFNg, Ig, IgA receptor, IgE, IGF, IGF-binding protein, IGF-1R, IGFBP, IGF-I, IGF-II, IL, IL-1, IL-1R, I L-2, IL-2R, IL-4, IL-4R, IL-5, IL-5R, IL-6, IL-6R, IL-8, IL-9, IL-10, IL-12, IL-13, IL-15, IL-18, IL-18R, IL-23, interferon (INF)-alpha, INF-beta, INF-gamma, inhibin, iNOS, insulin A chain, insulin B chain, insulin-like growth factor 1, interferon (IFN)-alpha, interferon (IFN)-beta, interferon (IFN)-gamma ... Integrin alpha 2, integrin alpha 3, integrin alpha 4, integrin alpha 4 / beta 1, integrin, alpha 4 / beta 7, integrin alpha 5 (alpha V), integrin alpha 5 / beta 1, integrin alpha 5 / beta 3, integrin alpha 6, integrin beta 1, integrin beta 2, interferon gamma, IP-10, 1-TAC, JE, kallikrein 2, kallikrein 5, kallikrein 6, kallikrein 11, kallikrein 12, kallikrein 14, kallikrein 15, kallikrein LI, kallikrein L2, kallikrein L3, kallikrein L4, KC, KDR, keratinocyte growth factor (KGF), laminin 5, LAMP, LAP, LAP (TGF-1), latent TGF-1, latent TGF-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 beta receptor, Mac-1, MAdCAM, MAG, MAP2, MARC, MCAM, MCK-2, MCP, M-CSF, MDC, Mer, metalloproteinase, MGDF receptor, MGMT, MHC (HLA-DR), MIF, MIG, MIP, MIP-1-alpha, MK, MMAC1, MMP, MMP-1, MMP-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, neprilysin, neurotrophin-3, -4, or -6, neurturin, nerve growth factor (NGF), NGFR, NGF-beta, nNOS, NO, NOS, Npn, NRG-3, NT, NTN, OB, OGG1, OPG, OPN, OSM, OX40L, OX40R, p150, p95, PADPr, parathyroid hormone, PARC, PARP, PBR, PBSF, PCAD, P-cadherin, PCNA, PDGF, PD K-1, PECAM, PEM, PF4, PGE, PGF, PGI2, PGJ2, PIN, PLA2, placental alkaline phosphatase (PLAP), PIGF, PLP, PP14, proinsulin, prorelaxin, protein C, PS, PSA, PSCA, prostate-specific membrane antigen (PSMA), PTEN, PTHrp, Ptk, PTN, R51, RANK, RANKL, RANTES, relaxin A chain, relaxin B chain, renin, respiratory syncytial virus (RSV) F, RSVFgp, Ret, rheumatoid factor, RLIP76, RPA2, RSK, S100, SCF / KL, SDF-1, SERINE, serum albumin, sFRP-3, Shh, SIGIRR, SK-1, SLAM, SLPI, SMAC, SMDF, SMOH, SOD, SPARC, Stat, STEAP, STEAP-II, TACE, TACI, TAG-72 (tumor-associated glycoprotein-72), TARC, TCA-3, T cell receptor (e.g., T cell receptor alpha / beta), TdT, TECK, TEM1, TEM5, TEM7, TEM8, TERT, testicular PLAP-like alkaline phosphatase, TfR, TGF, TGF-alpha, TGF-beta, TGF-beta Pan-specific, TGF-betaRI (ALK-5), TGF-betaRII, TGF-betaRIIb, TGF-betaRIII, TGF-beta1, TGF-beta2, TGF-beta3, TGF-beta4, TGF-beta5, thrombin, thymic Ck-1, thyroid-stimulating hormone, Tie, TIMP, TIQ, tissue factor, TMEFF2, Tmpo, TMPRSS2, TNF, TNF-alpha, TNF-alphabeta, TNF-beta2, 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 (BAFF R), TNFRSF14 (HVEM ATAR 、HYS、LOOKS 、ROS2)、SLOOKS16(106). 07SYS、SYSYS17(SYSYS)、SYSYSYS18(JSYS). SHYSY、SYSYS10(SYS3 WHY、WHYWH、WHYWHYWHYWHYWHYWHYWHYWHYWHY 1000000000000000000000000000000000000000000000000000000 H1200、F75806、SHOSH2 6(SYS3)、SYSYS3(SYS THIS SUCH、SHIS S6、SHOSCHS4(SYS)0 SIS33、SHIS1 S) 、WHYS 5(SH40 50)、SWHYS6 DA11、HAR1、DAY5)、DYSCHYS(DYS3 H68、S6)、S6S67(S027)、 FASHION(SHR30)、SHASHES(400) HR137、HARSH、SHRYSY21 DASH) 、SHASH22(DASH) 2 SHY2)、SHYSYS23(DYSYS 11、HARSH1)、HARSHY25(SY3 FASH3、SHASH、SHASH、SHASH 、SHAKE1)、SHASE100(SHARE). DAY2リガンド、LOY2)、SHAS11(SHAS SHASE RHリガンドDAY 、DAYリガンド)、DAYS12(SIR). DA3リガンド、DAY3リガンド)、DAYDAY13(SYS) LOVE2)、SIGNIFICANCE130 LOVE SY、 LOVE1 LOVE LOVE LOVE LOVE LOVE LOVE LIKEリガンド、CHES)、SHOSE 55(SHESHES)、SHE 18(SYSリガンドDYSリガンド 46) THYSYS1(SURコネクチン、DYS2)、DYSYS1 LIKE、SYSYS1)、SYSYS(LOVE DASH、D33)、SDASH4(S40リガンドN34 、SHA1)、SHASH54(4). 40リガンド、D154、N39、NO 1. CHR3リガンド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, transferrin receptor, TRF, Trk, TROP-2, TSG, TSLP, tumor-associated antigen CA 125, tumor-associated antigens expressing Lewis Y-related carbohydrate, TWEAK, TXB2, Ung, uPAR, uPAR-1, urokinase, VCAM, VCAM-1, VECAD, VE-cadherin, VE-cadherin-2, VEFGR-1 (flt-1), VEGF, VEGFR, VEGFR-3 (flt-4), VEGI, VFM, viral antigens, VLA, VLA-1, VLA-4, VNR integrin, von Willebrand factor, WIF-1, WNT1, WNT2, WNT2B / 13, WNT3, WNT3A, WNT4, WNT5A, WNT5B, WNT6, WNT7A, WNT7B, WNT8 the one or more other antigen-binding regions comprising an antigen-binding region specific for a second antigen selected from the group consisting of A, WNT8B, WNT9A, WNT9A, WNT9B, WNT10A, WNT10B, WNT11, WNT16, XCL1, XCL2, XCR1, XCR1, XEDAR, XIAP, XPD, CTLA4 (cytotoxic T-lymphocyte antigen-4), PD1 (programmed cell death protein 1), PD-L1 (programmed cell death ligand 1), LAG-3 (lymphocyte activation gene-3), TIM-3 (T-cell immunoglobulin and mucin protein-3), and hormone receptors and growth factors; (iii) the one or more other antigen-binding regions are 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), FcγRIIc (CD32c), FcγRIIb (CD32a), FcγRIIb (CD32b), FcγRIIb ... an antigen-binding region specific for an antigen selected from the group consisting of gammaRIIb (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 the group consisting of Fab-Fc-scFv, scFv2-Fc2, scFv-IgG, "bottle opener", Mab-scFv, Mab-Fv, dual-scFv, central Fv, central scFv, one-arm central scFv, Fab-Fab, Fab-Fv, mAb-Fv, mAb-Fab, DART, BiTE, common light chain-IgG, TandAb, crossMab, SEED, BEAT, TrioMab, and DuetMab; and / or 14. An anti-CD3 antibody or antigen-binding fragment according to any one of claims 1 to 13, wherein the multispecific antibody or antibody fragment comprises or consists of one or more other antigen-binding regions comprising one or more of the following properties: at least one CLK-preferred variant CH1 domain, optionally a CLK-preferred variant CH1 domain as described in WO2021067404; at least one CLλ-preferred variant CH1 domain, optionally a CLλ-preferred variant CH1 domain as described in WO2021067404; at least one pair of a variant CH1 domain and a variant CL domain that preferentially pair with each other, optionally a pair as described in WO2022150787; and / or at least one pair of a variant CH3 domain and another variant CH3 domain that preferentially pair with each other, optionally comprising one or more of the pairs as described in WO2022150785.

15. The following characteristics: (I) binds to CD3 with a higher binding affinity or avidity at an acidic pH (optionally, about pH 6.0) than at physiological pH (optionally, about pH 7.4), and optionally (i) the binding affinity or avidity is: (1) Surface Plasmon Resonance (SPR), optionally using the BIACORE® system; (2) Bio-Layer Interferometry (BLI), optionally using an OCTET® system; (3) enzyme-linked immunosorbent assay (ELISA), and / or (4) measured via radioimmunoassay (RIA); (ii) the CD3 is (1) human CD3, optionally CD3εδ, and / or (2) non-human primate CD3, optionally monkey CD3, further optionally cynomolgus CD3, optionally CD3εδ; (iii) 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 at a pH more acidic than physiological pH (optionally, about pH 7.4) based on an equilibrium dissociation constant (Kd) value 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 Large, and / or (iv) the anti-CD3 antibody or antigen-binding fragment thereof does not bind to CD3 at physiological pH, optionally at about pH 7.4; (II) binds to human CD3, optionally CD3εδ, at an acidic pH, optionally about pH 6.0, with the following Kd value: (i) 1.57×10, optionally further measured via SPR using a BIACORE® system ８ (M) Less than 1.0 x 10 ８ (M) Less than 9.0 x 10 ９ (M) Less than 8.0 x 10 ９ (M) Less than 7.0 x 10 ９ (M) Less than 6.0 x 10 ９ (M) Less than 5.0 x 10 ９ (M) Less than 4.0 x 10 ９ (M) Less than 3.0 x 10 ９ (M) Less than 2.0 x 10 ９ (M) Less than 1.0 x 10 ９ (M) Less than 9.0 x 10 １０ (M) or less than 8.0 x 10 １０ a Kd value less than (M); (ii) 1.56×10, optionally measured via SPR, further optionally using a BIACORE® system ８ (M) ~ 7.0 x 10 １０ (M), 1.0 x 10 ８ (M) ~ 7.0 x 10 １０ (M), 1.0 x 10 ８ (M) ~ 1.0 x 10 ９ (M), 2.0 x 10 ８ (M) ~ 1.0 x 10 ９ (M), or 5.0 x 10 ８ (M) ~ 1.0 x 10 ９ (M) Kd value, (iii) 9.33×10, as optionally measured via BLI, optionally further using an OCTET® system ９ (M) Less than 9.0 x 10 ９ (M) Less than 8.0 x 10 ９ (M) Less than 7.0 x 10 ９ (M) Less than 6.0 x 10 ９ (M) Less than 5.0 x 10 ９ (M) Less than 4.0 x 10 ９ (M) Less than 3.0 x 10 ９ (M) or less than 2.0 x 10 ９ a Kd value of less than (M), and / or (iv) 9.32×10, as optionally measured via BLI, further optionally using an OCTET® system ９ (M) ~ 1.0 x 10 ９ (M), 9.0 x 10 ９ (M) ~ 1.0 x 10 ９ (M), 8.0 x 10 ９ (M) ~ 3.0 x 10 ９ (M), 7.0 x 10 ９ (M) ~ 4.0 x 10 ９ , or 6.0 x 10 ９ (M) ~ 5.0 x 10 ９ Kd value of (M), (III) binds to cynomolgus monkey CD3, optionally CD3εδ, at an acidic pH, optionally about pH 6.0, optionally with the following Kd value: (i) 2.0 x 10, optionally measured via BLI, further optionally using an OCTET® system ８ (M) Less than 1.0 x 10 ８ (M) Less than 9.0 x 10 ９ (M) Less than 8.0 x 10 ９ (M) Less than 7.0 x 10 ９ (M) Less than 6.0 x 10 ９ (M) Less than 5.0 x 10 ９ (M) Less than 4.0 x 10 ９ (M) Less than 3.0 x 10 ９ (M) or less than 2.0 x 10 ９ a Kd value of less than (M), and / or (ii) 2.0 x 10, optionally measured via BLI using an OCTET® system ８ (M) ~ 1.0 x 10 ９ (M), 1.0 x 10 ８ (M) ~ 1.0 x 10 ９ (M), 9.0 x 10 ９ (M) ~ 2.0 x 10 ９ (M), or 8.0 x 10 ９ (M) ~ 5.0 x 10 ９ Kd value of (IV) binds to CD3-expressing cells with stronger binding at an acidic pH (optionally about pH 6.0) than at physiological pH (optionally about pH 7.4), and optionally (i) the binding is measured via flow cytometry, and optionally based on normal cell binding (NCB) values ​​calculated using median fluorescence intensity (MFI) values; (ii) the cells are (1) human CD3, optionally CD3εδ, and / or (2) expressing non-human primate CD3, optionally monkey CD3, further optionally cynomolgus CD3, optionally CD3εδ; (iii) the cells are (1) Primary cells, (2) cells of a cell line; (3) human cells, optionally human T cells, and further optionally Jurkat cells; (4) non-human primate cells, optionally monkey cells, further optionally cynomolgus cells, and even more optionally HSC-F cells; (iv) Binding to CD3-expressing cells, as measured via flow cytometry, 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 at a pH more acidic than physiological pH (optionally about pH 7.4) based on the NCB value. 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 Large, and / or (v) the anti-CD3 antibody or antigen-binding fragment thereof does not bind to CD3-expressing cells at physiological pH, optionally at about pH 7.4; (V) upon binding to CD3 on a cell, optionally a T cell, induces activation and / or enhances cytotoxic function of said cell; (VI) cells, optionally, that do not induce cytokine production upon binding to CD3 to a level that is capable of inducing cytokine release syndrome (CRS) upon binding to CD3 on T cells; and / or (VII) The anti-CD3 antibody or antigen-binding fragment of any one of claims 1 to 14, comprising or contained in a multispecific antibody or antibody fragment having at least (a) a first antigen-binding region specific for CD3, comprising the VH polypeptide and / or the VL polypeptide, and (b) one or more other antigen-binding regions comprising at least one antigen-binding region that binds to an antigen different from the first antigen-binding region, and comprising one or more of the following properties: (i) CD3 on a first cell, optionally a T cell, and (ii) the second antigen expressed on a second cell, the first cell exhibits cytotoxicity against the second cell.

16. A nucleic acid encoding the anti-CD3 antibody or antigen-binding fragment of any one of claims 1 to 15, 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) comprising a nucleic acid sequence encoding a VL polypeptide 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, (I) SEQ ID NOs: 650 and 660, respectively; (II) SEQ ID NOs: 550 and 560, respectively; (III) SEQ ID NOs: 450 and 460, respectively; (IV) SEQ ID NOs: 350 and 360, respectively; (V) SEQ ID NOs: 750 and 760, respectively; (VI) SEQ ID NOs: 850 and 860, respectively; (VII) SEQ ID NOs: 950 and 960, respectively; (VIII) SEQ ID NOs: 1050 and 1060, respectively; (IX) SEQ ID NOs: 1150 and 1160, respectively; (X) SEQ ID NOs: 1250 and 1260, respectively; or (XI) A nucleic acid comprising a nucleic acid sequence encoding a VH polypeptide and a nucleic acid sequence encoding a VL polypeptide of SEQ ID NOs: 1350 and 1360, respectively.

17. 17. A vector comprising the nucleic acid of claim 16, optionally comprising: (i) is an expression vector; and / or (ii) Vectors, including plasmids, viral vectors (optionally adenoviral, lentiviral, or retroviral), lipid-based vectors, self-replicating RNA vectors, virus-like particles, polymer-based vectors, and / or nanoparticles, optionally lipid-based nanoparticles.

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

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

20. A method of treating a subject in need of such treatment, comprising administering to said subject an effective amount of: (i) the anti-CD3 antibody or antigen-binding fragment of any one of claims 1 to 15; (ii) a nucleic acid according to claim 16; (iii) the vector according to claim 17 ; (iv) an isolated or recombinant cell according to claim 18, and / or (v) administering the pharmaceutical composition of claim 19, Optionally, (a) the object 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 comprising administering to said subject an additional agent, optionally an adjuvant or therapeutic agent.

21. A method of treating or preventing a disease, disorder, or condition in a subject, comprising administering to the subject an effective amount of: (i) the anti-CD3 antibody or antigen-binding fragment of any one of claims 1 to 15; (ii) a nucleic acid according to claim 16; (iii) the vector according to claim 17 ; (iv) an isolated or recombinant cell according to claim 18, and / or (v) administering the pharmaceutical composition of claim 19, Optionally, (a) the object 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 comprising administering to said subject an additional agent, optionally an adjuvant or therapeutic agent.

22. 1. A method for inducing cytotoxicity against cells expressing a target molecule in a subject, comprising administering to the subject an effective amount of: (i) the anti-CD3 antibody or antigen-binding fragment of claim 14; (ii) a nucleic acid encoding the anti-CD3 antibody or antigen-binding fragment of claim 14; (iii) a vector comprising the nucleic acid of (ii); (iv) an isolated or recombinant cell containing, transfected with, transformed with, or transduced with said nucleic acid or vector, and / or (v) administering (A) the anti-CD3 antibody or antigen-binding fragment of claim 14, a nucleic acid encoding the anti-CD3 antibody or antigen-binding fragment of claim 14, a vector comprising said nucleic acid, and / or a cell comprising, transfected with, transformed with, or transduced with said nucleic acid or said vector, and / or (B) a pharmaceutically acceptable carrier and / or excipient; Optionally, (a) the object 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 comprising administering to said subject an additional agent, optionally an adjuvant or therapeutic agent.

23. the disease, disorder, or condition comprises cancer or a neoplastic condition, an autoimmune disease, a neurodegenerative disease, an infectious disease, an inflammatory disease, or another disease, and optionally (i) the cancer is (i-1) a solid cancer, optionally selected from one or more of mesothelioma, malignant pleural mesothelioma, non-small cell lung cancer, small cell lung cancer, squamous cell lung carcinoma, large cell lung carcinoma, 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, kidney 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) Liquid cancers, optionally including chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), multiple myeloma, acute lymphocytic leukemia (ALL), Hodgkin's lymphoma, B-cell acute lymphocytic leukemia (BALL), T-cell acute lymphocytic leukemia (TALL), small lymphocytic leukemia (SLL), B-cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt's lymphoma, Diffuse large B-cell lymphoma (DLBCL), DLBCL associated with chronic inflammation, chronic myeloid leukemia, myeloproliferative disorders, follicular lymphoma, childhood follicular lymphoma, hairy cell leukemia, small cell or large cell follicular lymphoma, malignant lymphoproliferative conditions, MALT lymphoma (extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue), marginal zone lymphoma, myelodysplasia, myelodysplastic syndrome, non-Hodgkin lymphoma Kidney lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenstrom's hypergammaglobulinemia, 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, isolated plasmacytoma of bone, extraskeletal plasmacytoma, nodular marginal zone lymphoma, childhood nodular marginal zone lymphoma a liquid cancer selected from 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 in HHV8-associated multicentric Castleman disease, primary effusion lymphoma, B-cell lymphoma, acute myeloid leukemia (AML), or unclassifiable lymphoma; (ii) the autoimmune disease 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, Behcet's disease, Schmidt's 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, or Creutzfeldt-Jakob disease; (iv) the infectious disease is a viral disease, a bacterial disease, a fungal disease, a yeast disease, a protozoan disease, a prion disease, or a parasitic disease, and optionally, (1) the viral disease is human immunodeficiency virus (HIV), hepatitis virus (optionally hepatitis A, B, or C), 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 a 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, or Vibrio vulnificus; and / or (3) the fungal disease is aspergillosis, candidiasis, Candida auris infection, Cryptococcus neoformans infection, Pneumocystis jiroveci infection, mucormycosis, talaromycosis, ringworm, blastomycosis, coccidioidomycosis, Cryptococcus gattii infection, histoplasmosis, paracoccidioidomycosis, or sporotrichosis.

24. A method for producing the anti-CD3 antibody or antigen-binding fragment of any one of claims 1 to 15, comprising: (a) culturing a cell containing the nucleic acid of claim 16 under conditions that allow expression of the antibody or antigen-binding fragment; and (b) recovering and purifying said antibody or antigen-binding fragment from said cell culture of (a).

25. 19. A method for producing an isolated or recombinant cell or a population of such cells according to claim 18, comprising introducing a nucleic acid according to claim 16 and / or a vector according to claim 17 into one or more cells, optionally wherein said introduction occurs in vitro, ex vivo or in vivo.

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

27. 20. An anti-CD3 antibody or antigen-binding fragment of any one of claims 1 to 15, a nucleic acid of claim 16, a vector of claim 17, a cell or a population of such cells of claim 18, and / or a pharmaceutical composition of claim 19 for use in the treatment of a disease, disorder, or condition, optionally wherein the disease, disorder, or condition comprises a cancer or neoplastic condition, an autoimmune disease, a neurodegenerative disease, an infectious disease, an inflammatory disease, or another disease, and further optionally (i) the cancer is (i-1) a solid cancer, optionally selected from one or more of mesothelioma, malignant pleural mesothelioma, non-small cell lung cancer, small cell lung cancer, squamous cell lung carcinoma, large cell lung carcinoma, 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, kidney 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) Liquid cancers, optionally including chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), multiple myeloma, acute lymphocytic leukemia (ALL), Hodgkin's lymphoma, B-cell acute lymphocytic leukemia (BALL), T-cell acute lymphocytic leukemia (TALL), small lymphocytic leukemia (SLL), B-cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt's lymphoma, Diffuse large B-cell lymphoma (DLBCL), DLBCL associated with chronic inflammation, chronic myeloid leukemia, myeloproliferative disorders, follicular lymphoma, childhood follicular lymphoma, hairy cell leukemia, small cell or large cell follicular lymphoma, malignant lymphoproliferative conditions, MALT lymphoma (extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue), marginal zone lymphoma, myelodysplasia, myelodysplastic syndrome, non-Hodgkin lymphoma Kidney lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenstrom's hypergammaglobulinemia, 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, isolated plasmacytoma of bone, extraskeletal plasmacytoma, nodular marginal zone lymphoma, childhood nodular marginal zone lymphoma a liquid cancer selected from 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 in HHV8-associated multicentric Castleman disease, primary effusion lymphoma, B-cell lymphoma, acute myeloid leukemia (AML), or unclassifiable lymphoma; (ii) the autoimmune disease 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, Behcet's disease, Schmidt's 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, or Creutzfeldt-Jakob disease; (iv) the infectious disease is a viral disease, a bacterial disease, a fungal disease, a yeast disease, a protozoan disease, a prion disease, or a parasitic disease, and optionally, (1) the viral disease is selected from the group consisting of human immunodeficiency virus (HIV), hepatitis virus (optionally hepatitis A, B, or C), 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 a common human coronavirus), norovirus, West Nile virus, Zika virus, poliovirus, (1) the bacterial disease is a 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 (2) the fungal disease is an Aspergillosis, Candida, Candida auris, Cryptococcus neoformans, Pneumocystis jirovecii, Mucor, Talaromyces, Tinea, Blastomyces, Coccidioides, Cryptococcus gattii, Histoplasma, Paracoccidioides, or Sporothrix infection.

28. 20. Use of the anti-CD3 antibody or antigen-binding fragment of any one of claims 1 to 15, the nucleic acid of claim 16, the vector of claim 17, the isolated or recombinant cell or population of such cells of claim 18, and / or the pharmaceutical composition of claim 19 for the manufacture of a medicament for the treatment of a disease, disorder, or condition, optionally wherein the disease, disorder, or condition comprises a cancer or neoplastic condition, an autoimmune disease, a neurodegenerative disease, an infectious disease, an inflammatory disease, or another disease, and further optionally (i) the cancer is (i-1) a solid cancer, optionally selected from one or more of mesothelioma, malignant pleural mesothelioma, non-small cell lung cancer, small cell lung cancer, squamous cell lung carcinoma, large cell lung carcinoma, 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, kidney 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) Liquid cancers, optionally including chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), multiple myeloma, acute lymphocytic leukemia (ALL), Hodgkin's lymphoma, B-cell acute lymphocytic leukemia (BALL), T-cell acute lymphocytic leukemia (TALL), small lymphocytic leukemia (SLL), B-cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt's lymphoma, Diffuse large B-cell lymphoma (DLBCL), DLBCL associated with chronic inflammation, chronic myeloid leukemia, myeloproliferative disorders, follicular lymphoma, childhood follicular lymphoma, hairy cell leukemia, small cell or large cell follicular lymphoma, malignant lymphoproliferative conditions, MALT lymphoma (extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue), marginal zone lymphoma, myelodysplasia, myelodysplastic syndrome, non-Hodgkin lymphoma Kidney lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenstrom's hypergammaglobulinemia, 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, isolated plasmacytoma of bone, extraskeletal plasmacytoma, nodular marginal zone lymphoma, childhood nodular marginal zone lymphoma a liquid cancer selected from 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 in HHV8-associated multicentric Castleman disease, primary effusion lymphoma, B-cell lymphoma, acute myeloid leukemia (AML), or unclassifiable lymphoma; (ii) the autoimmune disease 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, Sjogren's syndrome, Addison's disease, Behcet's disease, Schmidt's 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, or Creutzfeldt-Jakob disease; (iv) the infectious disease is a viral disease, a bacterial disease, a fungal disease, a yeast disease, a protozoan disease, a prion disease, or a parasitic disease, and optionally, (1) the viral disease is selected from the group consisting of human immunodeficiency virus (HIV), hepatitis virus (optionally hepatitis A, B, or C), 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 a common human coronavirus), norovirus, West Nile virus, (1) the bacterial disease is selected from the group consisting of Salmonella, Escherichia coli, Mycobacterium tuberculosis, Methicillin-resistant Staphylococcus aureus (MRSA), Clostridium difficile, Streptococcus pneumoniae, Klebsiella pneumoniae, Pseudomonas aeruginosa, Helicobacter pylori, Neisseria gonorrhoeae, and Vibrio vulnificus; and / or (2) the fungal disease is selected from the group consisting of aspergillosis, candidiasis, Candida auris infection, Cryptococcus neoformans infection, Pneumocystis jiroveci infection, mucormycosis, talaromycosis, ringworm, blastomycosis, coccidioidomycosis, Cryptococcus gattii infection, histoplasmosis, paracoccidioidomycosis, and sporotrichosis.

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