Anti-CD3 Antibodies

Abstract

The present disclosure includes anti-CD3 binding domains and antibodies and / or antigen-binding fragments that contain such domains. Multispecific antibodies and antibody fragments are also included. The present disclosure further includes nucleic acids and vectors that encode such antibodies or antibody fragments and cells that contain such nucleic acids. Pharmaceutical compositions, in vivo methods, and manufacturing methods for anti-CD3 antibodies or antigen-binding fragments are also included.

Description

[Background technology]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 245,499, entitled "Anti-CD3 Antibodies," filed September 17, 2021, the contents of which are incorporated by reference in their entirety herein. Disclosure of sequences

[0002] The contents of the electronic sequence listing (1160430.003213.xml, size: 964,969 bytes, and creation date: September 15, 2022) are incorporated herein by reference in their entirety.

[0003] Cellular proliferative disorders, such as cancer, are characterized by the uncontrolled proliferation of a subpopulation of cells. Cellular 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 new cases of cancer diagnosed each year is projected to reach 23.6 million by 2030. The National Cancer Institute predicts that approximately 2 million new cases of cancer will be diagnosed in the United States in 2018, and more than 600,000 Americans will die from cancer. Cancer treatment therefore represents a significant and ever-increasing societal burden.

[0004] The idea of ​​harnessing the cytotoxic potential of T cells to kill tumor cells through the use of CD3-targeting bispecific antibodies dates back to the mid-1980s. (Staerz et al. Nature 1985 314:628-32). Many bispecific antibodies developed to date contain a first binding site specific for CD3 for T cell recruitment and activation, and a second binding site for a target disease-associated antigen, such as an antigen produced by tumor cells. By binding to its second target protein expressed on tumors, CD3 bispecific antibodies trigger the CD3 surface receptor on T cells, so that competent T cells can bind to target-expressing cells via cross-linking by CD3 bispecific antibodies, 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 with tumor cells can induce dramatic regression of advanced malignancies, and in some cases even complete remission. More than 25 different CD3 bispecific antibodies are currently in clinical development by targeting CD19, CD20, CD33 and CD123, or EpCAM, HER2, PSMA and CEA, respectively, for the treatment of hematological malignancies or solid cancers (see, for example, Liu et al. Front Immunol 2017 8:38).

[0005] While bispecific antibodies have shown significant advantages over monospecific antibodies for cancer treatment and detection, their widespread commercial application has been hindered by the lack of efficient / low-cost production methods, the lack of stability of bispecific polypeptides, and their short half-life in humans. Many methods for generating bispecific monoclonal antibodies have been developed over the past decades. However, many bispecific antibody candidates with excellent selectivity and high potency towards their target of interest often suffer challenges in downstream development and clinical efficacy, including: multispecific binding, off-target binding, non-specific binding, poor expression levels or profiles in eukaryotic host cells, e.g., mammalian host cells or yeast cells, poor chemical and physical properties, e.g., poor / low "shelf-life" stability, poor (low) solubility, poor (high) viscosity, tendency to aggregate, and poor clinical and biophysical profiles, e.g., poor pharmacokinetic profile, poor pharmacological profile, fast or poor in vivo clearance rate, short circulatory half-life, some of which cause them to be discontinued in development.

[0006] Specific techniques and assays exist to evaluate many of the aforementioned developability characteristics of discovered antibodies in the context of downstream development activities ("post-development antibodies"), e.g., CIC, SIC, BVP-ELISA, TMA, and other assays. However, such assays are often not compatible with the high-throughput format of early antibody discovery platforms. Furthermore, these characterizations often require milligram- to gram-scale quantities of protein, which often imposes practical limitations on the number of leads that can be pragmatically considered for development, and as a result, the likelihood of program success is reduced. As a result, significant resources are often spent attempting to qualify poorly characterized lead candidates, with few back-ups available at later development stages.

[0007] A variety of anti-CD3 antibodies are known in the art, including monoclonal and bispecific antibody formats. For example, see 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 have the developability problems described above. Therefore, such anti-CD3 antibodies are not ideal candidates for designing multispecific antibodies for clinical purposes, for example. Thus, there is an unmet need to provide an anti-CD3 antibody that exhibits desirable developability characteristics, such as binding specificity to CD3 expressed on T cells, activation of T cells, and (re)directing activated T cells to kill target cells, and is safe and effective. Summary of the Invention

[0008] One aspect of the present disclosure provides anti-CD3 antibodies and antigen-binding fragments, eg, those that exhibit desirable developability characteristics.

[0009] In some embodiments, the disclosure provides antibodies or antigen-binding fragments that comprise (i) a complementarity determining region (CDR) that comprises the amino acid sequence of a CDR contained in any of the variable domain sequences listed in Table 1A or 1 B. In some embodiments, the disclosure provides antibodies or antigen-binding fragments that comprise a CDR that comprises an amino acid sequence selected from any of those listed in Table 2A or 2B.

[0010] In some embodiments, the disclosure provides: (A)(a)(i) a CDRH1 contained in any one of antibody numbers V002 to V019 and A001 to A005; (ii) a CDRH1 contained in any one of SEQ ID NOs: 210, 310, 410, 510, 610, 710, 810, 910, 1010, 1110, 1210, 1310, 1410, 1510, 1610, 1710, 1810, 1910, 2110, 2210, 2310, 2410, and 2510; (iii) FX1X2X3DYYMH (SEQ ID NO: 112), wherein Xi is N or D, X2 is I or D, and X3 is K or D; and / or (iv) a VH comprising the amino acid sequence of any one of SEQ ID NOs: 212, 312, 412, 512, 612, 712, 812, 912, 1012, 1112, 1212, 1312, 1412, 1512, 1612, 1712, 1812, 1912, 2112, 2212, 2312, 2412, and 2512. CDR1 (CDRH1), (b) (i) CDRH2 contained in any one of antibody numbers V002 to V019 and A001 to A005, (ii) CDRH2 contained in any one of SEQ ID NOs: 210, 310, 410, 510, 610, 710, 810, 910, 1010, 1110, 1210, 1310, 1410, 1510, 1610, 1710, 1810, 1910, 2110, 2210, 2310, 2410, and 2510, (iii) WIX4LEX5X6X7TX8X9DAKFQX 10 (SEQ ID NO: 114) (wherein X4 is D or E, X5 is N or E, X6 is A, D, or G, X7 is N, E, or D, X8 is I or V, X9 is Y or D, and X 10is G or D), and / or (iv) a VH comprising any one of the amino acid sequences of SEQ ID NOs: 214, 314, 414, 514, 614, 714, 814, 914, 1014, 1114, 1214, 1314, 1414, 1514, 1614, 1714, 1814, 1914, 2114, 2214, 2314, 2414, and 2514. CDR2 (CDRH2), and / or (c) (i) a CDRH3 contained in any one of antibody numbers V002 to V019 and A001 to A005; (ii) a CDRH3 contained in any one of SEQ ID NOs: 210, 310, 410, 510, 610, 710, 810, 910, 1010, 1110, 1210, 1310, 1410, 1510, 1610, 1710, 1810, 1910, 2110, 2210, 2310, 2410, and 2510; (iii) X 11 RDX 12 YGRYFYDX 13 (SEQ ID NO: 116) (wherein X 11 is A or G, and X 12 is A or Q, and X 13is V or E), and / or (iv) a VH comprising any one of the amino acid sequences of SEQ ID NOs: 216, 316, 416, 516, 616, 716, 816, 916, 1016, 1116, 1216, 1316, 1416, 1516, 1616, 1716, 1816, 1916, 2116, 2216, 2316, 2416, and 2516. (B) a heavy chain variable domain (VH) polypeptide comprising CDR3 (CDRH3), and / or (a) (i) a CDRL1 contained in any one of antibody numbers V002 to V019 and A001 to A005; (ii) SEQ ID NOs: 220, 320, 420, 520, 620, 720, 820, 920, 1020, 1120, 1220, 1320, 1420, 1520, 1620, 1720, 1820, 1920, 2120; 2220, 2320, 2420, and 2520, and / or (iii) a VL comprising the amino acid sequence of any one of SEQ ID NOs: 122, 222, 322, 422, 522, 622, 722, 822, 922, 1022, 1122, 1222, 1322, 1422, 1522, 1622, 1722, 1822, 1922, 2122, 2222, 2322, 2422, and 2522. (b) (i) a CDRL2 contained in any one of antibody numbers V002 to V019 and A001 to A005; (ii) a CDRL2 contained in any one of SEQ ID NOs: 220, 320, 420, 520, 620, 720, 820, 920, 1020, 1120, 1220, 1320, 1420, 1520, 1620, 1720, 1820, 1920, 2120, 2220, 2320, 2420, and 2520; and (iii) WASTRX. 14 S (SEQ ID NO: 124) (wherein X 14is E or S), and / or (iv) a VL comprising any one of the amino acid sequences of SEQ ID NOs: 224, 324, 424, 524, 624, 724, 824, 924, 1024, 1124, 1224, 1324, 1424, 1524, 1624, 1724, 1824, 1924, 2124, 2224, 2324, 2424, and 2524. CDR2 (CDRL2), and / or (c) (i) a CDRL3 contained in any one of antibody numbers V002 to V019 and A001 to A005; (ii) a CDRL3 contained in any one of SEQ ID NOs: 220, 320, 420, 520, 620, 720, 820, 920, 1020, 1120, 1220, 1320, 1420, 1520, 1620, 1720, 1820, 1920, 2120, 2220, 2320, 2420, and 2520; (iii) X 15 QSYX 16 X 17 RT (SEQ ID NO: 126), 15 is K or V, and X 16 is S or F, and X 17 is R or L), and / or (iv) an antibody or antigen-binding fragment comprising a light chain variable domain (VL) polypeptide comprising a VL CDR3 (CDRL3) comprising the amino acid sequence of any one of SEQ ID NOs: 226, 326, 426, 526, 626, 726, 826, 926, 1026, 1126, 1226, 1326, 1426, 1526, 1626, 1726, 1826, 1926, 2126, 2226, 2326, 2426, and 2526, or an antibody or antigen-binding fragment comprising a combination of one or more of the foregoing CDRs.

[0011] In certain embodiments, the anti-CD3 antibody or antigen-binding fragment may comprise (i) at least one of CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 contained in A001, or (ii) at least one of SEQ ID NOs: 2112, 2114, 2116, 2122, 2124, and 2126. In certain embodiments, the anti-CD3 antibody or antigen-binding fragment may not comprise (i) at least one of CDRH1 and CDRH2 contained in A001, or (ii) at least one of SEQ ID NOs: 2112 and 2114.

[0012] In certain embodiments, the anti-CD3 antibody or antigen-binding fragment may not include (i) at least one of CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 contained in A002, or (ii) at least one of SEQ ID NOs: 2212, 2214, 2216, 2222, 2224, and 2226. In certain embodiments, the anti-CD3 antibody or antigen-binding fragment may not include (i) at least one of CDRH1 and CDRH2 contained in A002, or (ii) at least one of SEQ ID NOs: 2212 and 2214.

[0013] In certain embodiments, the anti-CD3 antibody or antigen-binding fragment may not include (i) at least one of CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 contained in A003, or (ii) at least one of SEQ ID NOs: 2312, 2314, 2316, 2322, 2324, and 2326. In certain embodiments, the anti-CD3 antibody or antigen-binding fragment may not include (i) at least one of CDRH1 and CDRH2 contained in A003, or (ii) at least one of SEQ ID NOs: 2312 and 2314.

[0014] In certain embodiments, the anti-CD3 antibody or antigen-binding fragment may not include (i) at least one of CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 contained in A004, or (ii) at least one of SEQ ID NOs: 2412, 2414, 2416, 2422, 2424, and 2426. In certain embodiments, the anti-CD3 antibody or antigen-binding fragment may not include (i) at least one of CDRH1 and CDRH2 contained in A004, or (ii) at least one of SEQ ID NOs: 2412 and 2414.

[0015] In certain embodiments, the anti-CD3 antibody or antigen-binding fragment may not include (i) at least one of CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 contained in A005, or (ii) at least one of SEQ ID NOs: 2512, 2514, 2516, 2522, 2524, and 2526. In certain embodiments, the anti-CD3 antibody or antigen-binding fragment may not include (i) at least one of CDRH1 and CDRH2 contained in A005, or (ii) at least one of SEQ ID NOs: 2512 and 2514.

[0016] In certain embodiments, an anti-CD3 antibody or antigen-binding fragment may comprise (A) a VH polypeptide comprising said CDRH1, said CDRH2, and said CDRH3, as described above, and / or (B) a VL polypeptide comprising said CDRL1, said CDRL2, and said CDRL3, as described above. In certain embodiments, an anti-CD3 antibody or antigen-binding fragment may comprise (A) a VH polypeptide comprising said CDRH1, said CDRH2, and said CDRH3, as described above, and (B) a VL polypeptide comprising said CDRL1, said CDRL2, and said CDRL3, as described above.

[0017] In certain embodiments, the anti-CD3 antibody or antigen-binding fragment may comprise (i) CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDR-L3 contained in antibody no. V002, or (ii) CDRH1 comprising SEQ ID NO: 212, CDRH2 comprising SEQ ID NO: 214, CDRH3 comprising SEQ ID NO: 216, CDRL1 comprising SEQ ID NO: 222, CDRL2 comprising SEQ ID NO: 224, and CDRL3 comprising SEQ ID NO: 226.

[0018] In certain embodiments, the anti-CD3 antibody or antigen-binding fragment may comprise (i) CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDR-L3 contained in antibody no. V003, or (ii) CDRH1 comprising SEQ ID NO: 312, CDRH2 comprising SEQ ID NO: 314, CDRH3 comprising SEQ ID NO: 316, CDRL1 comprising SEQ ID NO: 322, CDRL2 comprising SEQ ID NO: 324, and CDRL3 comprising SEQ ID NO: 326.

[0019] In certain embodiments, the anti-CD3 antibody or antigen-binding fragment may comprise (i) CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDR-L3 contained in antibody no. V004, or (ii) CDRH1 comprising SEQ ID NO: 412, CDRH2 comprising SEQ ID NO: 414, CDRH3 comprising SEQ ID NO: 416, CDRL1 comprising SEQ ID NO: 422, CDRL2 comprising SEQ ID NO: 424, and CDRL3 comprising SEQ ID NO: 426.

[0020] In certain embodiments, the anti-CD3 antibody or antigen-binding fragment may comprise (i) CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDR-L3 contained in antibody no. V005, or (ii) CDRH1 comprising SEQ ID NO:512, CDRH2 comprising SEQ ID NO:514, CDRH3 comprising SEQ ID NO:516, CDRL1 comprising SEQ ID NO:522, CDRL2 comprising SEQ ID NO:524, and CDRL3 comprising SEQ ID NO:526.

[0021] In certain embodiments, the anti-CD3 antibody or antigen-binding fragment may comprise (i) CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDR-L3 contained in antibody no. V006, or (ii) CDRH1 comprising SEQ ID NO: 612, CDRH2 comprising SEQ ID NO: 614, CDRH3 comprising SEQ ID NO: 616, CDRL1 comprising SEQ ID NO: 622, CDRL2 comprising SEQ ID NO: 624, and CDRL3 comprising SEQ ID NO: 626.

[0022] In certain embodiments, the anti-CD3 antibody or antigen-binding fragment may comprise (i) CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDR-L3 contained in antibody no. V007, or (ii) CDRH1 comprising SEQ ID NO: 712, CDRH2 comprising SEQ ID NO: 714, CDRH3 comprising SEQ ID NO: 716, CDRL1 comprising SEQ ID NO: 722, CDRL2 comprising SEQ ID NO: 724, and CDRL3 comprising SEQ ID NO: 726.

[0023] In certain embodiments, the anti-CD3 antibody or antigen-binding fragment may comprise (i) CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDR-L3 contained in antibody no. V008, or (ii) CDRH1 comprising SEQ ID NO: 812, CDRH2 comprising SEQ ID NO: 814, CDRH3 comprising SEQ ID NO: 816, CDRL1 comprising SEQ ID NO: 822, CDRL2 comprising SEQ ID NO: 824, and CDRL3 comprising SEQ ID NO: 826.

[0024] In certain embodiments, the anti-CD3 antibody or antigen-binding fragment may comprise (i) CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDR-L3 contained in antibody no. V009, or (ii) CDRH1 comprising SEQ ID NO: 912, CDRH2 comprising SEQ ID NO: 914, CDRH3 comprising SEQ ID NO: 916, CDRL1 comprising SEQ ID NO: 922, CDRL2 comprising SEQ ID NO: 924, and CDRL3 comprising SEQ ID NO: 926.

[0025] In certain embodiments, the anti-CD3 antibody or antigen-binding fragment may comprise (i) CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDR-L3 contained in antibody No. V010, or (ii) CDRH1 comprising SEQ ID NO:1012, CDRH2 comprising SEQ ID NO:1014, CDRH3 comprising SEQ ID NO:1016, CDRL1 comprising SEQ ID NO:1022, CDRL2 comprising SEQ ID NO:1024, and CDRL3 comprising SEQ ID NO:1026.

[0026] In certain embodiments, the anti-CD3 antibody or antigen-binding fragment may comprise (i) CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDR-L3 contained in antibody No. V011, or (ii) CDRH1 comprising SEQ ID NO:1112, CDRH2 comprising SEQ ID NO:1114, CDRH3 comprising SEQ ID NO:1116, CDRL1 comprising SEQ ID NO:1122, CDRL2 comprising SEQ ID NO:1124, and CDRL3 comprising SEQ ID NO:1126.

[0027] In certain embodiments, the anti-CD3 antibody or antigen-binding fragment may comprise (i) CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDR-L3 contained in antibody No. V012, or (ii) CDRH1 comprising SEQ ID NO: 1212, CDRH2 comprising SEQ ID NO: 1214, CDRH3 comprising SEQ ID NO: 1216, CDRL1 comprising SEQ ID NO: 1222, CDRL2 comprising SEQ ID NO: 1224, and CDRL3 comprising SEQ ID NO: 1226.

[0028] In certain embodiments, the anti-CD3 antibody or antigen-binding fragment may comprise (i) CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDR-L3 contained in antibody no. V013, or (ii) CDRH1 comprising SEQ ID NO: 1312, CDRH2 comprising SEQ ID NO: 1314, CDRH3 comprising SEQ ID NO: 1316, CDRL1 comprising SEQ ID NO: 1322, CDRL2 comprising SEQ ID NO: 1324, and CDRL3 comprising SEQ ID NO: 1326.

[0029] In certain embodiments, the anti-CD3 antibody or antigen-binding fragment may comprise (i) CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDR-L3 contained in antibody No. V014, or (ii) CDRH1 comprising SEQ ID NO: 1412, CDRH2 comprising SEQ ID NO: 1414, CDRH3 comprising SEQ ID NO: 1416, CDRL1 comprising SEQ ID NO: 1422, CDRL2 comprising SEQ ID NO: 1424, and CDRL3 comprising SEQ ID NO: 1426.

[0030] In certain embodiments, the anti-CD3 antibody or antigen-binding fragment may comprise (i) CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDR-L3 contained in antibody no. V015, or (ii) CDRH1 comprising SEQ ID NO: 1512, CDRH2 comprising SEQ ID NO: 1514, CDRH3 comprising SEQ ID NO: 1516, CDRL1 comprising SEQ ID NO: 1522, CDRL2 comprising SEQ ID NO: 1524, and CDRL3 comprising SEQ ID NO: 1526.

[0031] In certain embodiments, the anti-CD3 antibody or antigen-binding fragment may comprise (i) CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDR-L3 contained in antibody No. V016, or (ii) CDRH1 comprising SEQ ID NO: 1612, CDRH2 comprising SEQ ID NO: 1614, CDRH3 comprising SEQ ID NO: 1616, CDRL1 comprising SEQ ID NO: 1622, CDRL2 comprising SEQ ID NO: 1624, and CDRL3 comprising SEQ ID NO: 1626.

[0032] In certain embodiments, the anti-CD3 antibody or antigen-binding fragment may comprise (i) CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDR-L3 contained in antibody no. V017, or (ii) CDRH1 comprising SEQ ID NO: 1712, CDRH2 comprising SEQ ID NO: 1714, CDRH3 comprising SEQ ID NO: 1716, CDRL1 comprising SEQ ID NO: 1722, CDRL2 comprising SEQ ID NO: 1724, and CDRL3 comprising SEQ ID NO: 1726.

[0033] In certain embodiments, the anti-CD3 antibody or antigen-binding fragment may comprise (i) CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDR-L3 contained in antibody No. V018, or (ii) CDRH1 comprising SEQ ID NO: 1812, CDRH2 comprising SEQ ID NO: 1814, CDRH3 comprising SEQ ID NO: 1816, CDRL1 comprising SEQ ID NO: 1822, CDRL2 comprising SEQ ID NO: 1824, and CDRL3 comprising SEQ ID NO: 1826.

[0034] In certain embodiments, the anti-CD3 antibody or antigen-binding fragment may comprise (i) CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDR-L3 contained in antibody no. V019, or (ii) CDRH1 comprising SEQ ID NO: 1912, CDRH2 comprising SEQ ID NO: 1914, CDRH3 comprising SEQ ID NO: 1916, CDRL1 comprising SEQ ID NO: 1922, CDRL2 comprising SEQ ID NO: 1924, and CDRL3 comprising SEQ ID NO: 1926.

[0035] In some embodiments, the anti-CD3 antibody or antigen-binding fragment comprises: (A)(a)(i) an FRH1 contained in any one of antibody numbers V002 to V019 and A001 to A005; (ii) an FRH1 contained in any one of SEQ ID NOs: 210, 310, 410, 510, 610, 710, 810, 910, 1010, 1110, 1210, 1310, 1410, 1510, 1610, 1710, 1810, 1910, 2110, 2210, 2310, 2410, and 2510; and / or (iii) an FRH1 contained in any one of SEQ ID NOs: 111, 211, 311, 411, 511, 611, 711, 811, 911, 1011, 1111, 1211, 1311, 1411, 1510, 1610, 1710, 1810, 1910, 2110, 2210, 2310, 2410, and 2510. (b) a VH framework region 1 (FRH1) comprising any one of the amino acid sequences of SEQ ID NOs: 11, 1511, 1611, 1711, 1811, 1911, 2111, 2211, 2311, 2411, and 2511; (b) (i) an FRH2 contained in any one of antibody numbers V002 to V019 and A001 to A005; (ii) an FRH2 contained in any one of SEQ ID NOs: 210, 310, 410, 510, 610, 710, 810, 910, 1010, 1110, 1210, 1310, 1410, 1510, 1610, 1710, 1810, 1910, 2110, 2210, 2310, 2410, and 2510; and (iii) WVRQAPGQRLEWX. 18 G (SEQ ID NO: 113) (wherein X 18is M or I), and / or (iv) a VH framework region 2 (FRH2) comprising any one of the amino acid sequences of SEQ ID NOs: 213, 313, 413, 513, 613, 713, 813, 913, 1013, 1113, 1213, 1313, 1413, 1513, 1613, 1713, 1813, 1913, 2113, 2213, 2313, 2413, and 2513; (c) (i) an FRH3 contained in any one of antibody Nos. V002 to V019 and A001 to A005; (ii) an FRH4 contained in any one of SEQ ID NOs: 210, 310, 410, 510, 61 and / or (iii) an FRH3 sequence selected from the group consisting of SEQ ID NOs: 115, 215, 315, 415, 515, 615, 715, 815, 915, 1015, 1115, 1215, 1315, 1415, 1515, 1615, 1715, 1815, 1915, 2115, 2215, 2315, 2415, and 2515. and / or (d) (i) an FRH4 contained in any one of antibody numbers V002 to V019 and A001 to A005; (ii) an FRH4 contained in any one of SEQ ID NOs: 210, 310, 410, 510, 610, 710, 810, 910, 1010, 1110, 1210, 1310, 1410, 1510, 1610, 1710, 1810, 1910, 2110, 2210, 2310, 2410, and 2510; and / or (iii) an FRH4 contained in any one of SEQ ID NOs: 117, 217, 317, 417, 510, 610, 710, 810, 910, 1010, 1110, 1210, 1310, 1410, 1510, 1610, 1710, 1810, 1910, 2110, 2210, 2310, 2410, and 2510; and / or (B)(a)(i) an FRL1 contained in any one of antibody numbers V002 to V019 and A001 to A005; (ii) SEQ ID NOs: 220, 320, 420, 520, 620, 720, 820, 920,FRL1 contained in any one of 1020, 1120, 1220, 1320, 1420, 1520, 1620, 1720, 1820, 1920, 2120, 2220, 2320, 2420, and 2520; (iii) DIVMX; 19 QSPDSLAVSLGERATINC (SEQ ID NO: 121), 19is T or S), and / or (iv) a VL framework region 1 (FRL1) comprising any one of the amino acid sequences of SEQ ID NOs: 221, 321, 421, 521, 621, 721, 821, 921, 1021, 1121, 1221, 1321, 1421, 1521, 1621, 1721, 1821, 1921, 2121, 2221, 2321, 2421, and 2521; (b) (i) an FRL2 contained in any one of antibody Nos. V002 to V019 and A001 to A005; (ii) an FRL3 contained in any one of SEQ ID NOs: 220, 320, 420, 520, 62 and / or (iii) an FRL2 sequence selected from the group consisting of SEQ ID NOs: 123, 223, 323, 423, 523, 623, 723, 823, 923, 1023, 1123, 1223, 1323, 1423, 1523, 1623, 1723, 1823, 1923, 2123, 2223, 2323, 2423, and 2523. (c) (i) an FRL3 contained in any one of antibody numbers V002 to V019 and A001 to A005; (ii) an FRL3 contained in any one of SEQ ID NOs: 220, 320, 420, 520, 620, 720, 820, 920, 1020, 1120, 1220, 1320, 1420, 1520, 1620, 1720, 1820, 1920, 2120, 2220, 2320, 2420, and 2520; and / or (iii) an FRL3 contained in any one of SEQ ID NOs: 125, 225, 325, 425, 525, 620, 720, 820, 920, 1020, 1120, 1220, 1320, 1420, 1520, 1620, 1720, 1820, 1920, 2120, 2220, 2320, 2420, and 2520; and / or (d) (i) a VL framework region 3 (FRL3) comprising any one of the amino acid sequences of SEQ ID NOs: 220, 320, 420, 520, 620, 720, 820, 920, 1020, 1120, 1220, 1325, 1425, 1525, 1625, 1725, 1825, 1925, 2125, 2225, 2325, 2425, and 2525; and / or (d) (i) an FRL4 contained in any one of antibody numbers V002 to V019 and A001 to A005;1520, 1620, 1720, 1820, 1920, 2120, 2220, 2320, 2420, and 2520, and / or (iii) a VL framework region 4 (FRL4) comprising the amino acid sequence of any one of SEQ ID NOs: 127, 227, 327, 427, 527, 627, 727, 827, 927, 1027, 1127, 1227, 1327, 1427, 1527, 1627, 1727, 1827, 1927, 2127, 2227, 2327, 2427, and 2527, or the antibody or antigen-binding fragment may comprise a VH and / or VL comprising any combination of the foregoing VH and VL framework regions.

[0036] In some embodiments, the anti-CD3 antibody or antigen-binding fragment comprises: (i) FRH1, FRH2, FRH3, FLRH4, FRL1, FRL2, FRL3, and FRL4 contained in any one of antibody numbers A001, V002 to V007, A003 to A005, and V014 to V019; and / or (ii) any one of SEQ ID NOs: 111, 211, 311, 411, 511, 611, 711, 1411, 1511, 1611, 1711, 1811, 1911, 2111, 2311, 2411, and 2511. FR-H1 including SEQ ID NOs: 213, 313, 413, 513, 613, 713, 1413, 1513, 1613, 1713, 1813, 1913, 2113, 2313, 2413, and 2513; FR-H2 including any one of SEQ ID NOs: 115, 215, 315, 415, 515, 615, 715, 1415, 1515, 1615, 1715, 1815, 1915, 2115, 2315, 2415, and 2515; FR-H3 including any one of SEQ ID NOs: 117, 217, 317, 417, 517, 617, 717; FR-H4 including any one of SEQ ID NOs: 1417, 1517, 1617, 1717, 1817, 1917, 2117, 2317, 2417, and 2517; FR-L1 including any one of SEQ ID NOs: 221, 321, 421, 521, 621, 721, 1421, 1521, 1621, 1721, 1821, 1921, 2121, 2321, 2421, and 2521; SEQ ID NOs: 123, 223, 323, 423, 523, 623, 723, 1423, 1523, 1623, 1723, 1823, 1923, 2123, 2323; FR-L2 comprising any one of SEQ ID NOs: 125, 225, 325, 425, 525, 625, 725, 1425, 1525, 1625, 1725, 1825, 1925, 2125, 2325, 2425, and 2525; FR-L3 comprising any one of SEQ ID NOs: 127, 227, 327, 427, 527, 627, 727, 1427, 1527, 1627, 1727, 1827, 1927, 2127, 2327, 2427, and 2527.

[0037] In certain embodiments, the anti-CD3 antibody or antigen-binding fragment comprises (i) FRH1, FRH2, FRH3, FLRH4, FRL1, FRL2, FRL3, and FRL4 contained in any one of antibody numbers A002 and V008 to V013, and / or (ii) FR-H1 comprising any one of SEQ ID NOs: 111, 811, 911, 1011, 1111, 1211, 1311, and 2211, FR-H2 comprising any one of SEQ ID NOs: 813, 913, 1013, 1113, 1213, 1313, and 2213, FR-H3 comprising any one of SEQ ID NOs: 115, 815, 915, 1015, 1115, 1215, 1315, and 2215, and / or (iii) any one of SEQ ID NOs: 115, 815, 915, 1015, 1115, 1215, 1315, and 2215. FR-H4 including any one of sequence numbers 117, 817, 917, 1017, 1117, 1217, 1317, and 2217; FR-L1 including any one of sequence numbers 821, 921, 1021, 1121, 1221, 1321, and 2221; FR-L2 including any one of sequence numbers 123, 823, 923, 1023, 1123, 1223, 1323, and 2223; FR-L3 including any one of sequence numbers 125, 825, 925, 1025, 1125, 1225, 1325, and 2225; and FR-L4 including any one of sequence numbers 127, 827, 927, 1027, 1127, 1227, 1327, and 2227.

[0038] In certain embodiments, the anti-CD3 antibody or antigen-binding fragment may include (i) FRH1, CDRH1, FRH2, CDRH2, FRH3, CDRH3, FRH4, FRL1, CDRL1, FRL2, CDRL2, FRL3, CDRL3, and FRL4 contained in antibody number V002, or (ii) FRH1, CDRH1, FRH2, CDRH2, FRH3, CDRH3, FRH4, FRL1, CDRL1, FRL2, CDRL2, FRL3, CDRL3, and FRL4 comprising SEQ ID NOs: 211, 212, 213, 214, 215, 216, 217, 221, 222, 223, 224, 225, 226, and 227, respectively.

[0039] In certain embodiments, the anti-CD3 antibody or antigen-binding fragment may include (i) FRH1, CDRH1, FRH2, CDRH2, FRH3, CDRH3, FRH4, FRL1, CDRL1, FRL2, CDRL2, FRL3, CDRL3, and FRL4 contained in antibody number V003, or (ii) FRH1, CDRH1, FRH2, CDRH2, FRH3, CDRH3, FRH4, FRL1, CDRL1, FRL2, CDRL2, FRL3, CDRL3, and FRL4 comprising SEQ ID NOs: 311, 312, 313, 314, 315, 316, 317, 321, 322, 323, 324, 325, 326, and 327, respectively.

[0040] In certain embodiments, the anti-CD3 antibody or antigen-binding fragment may include (i) FRH1, CDRH1, FRH2, CDRH2, FRH3, CDRH3, FRH4, FRL1, CDRL1, FRL2, CDRL2, FRL3, CDRL3, and FRL4 contained in antibody number V004, or (ii) FRH1, CDRH1, FRH2, CDRH2, FRH3, CDRH3, FRH4, FRL1, CDRL1, FRL2, CDRL2, FRL3, CDRL3, and FRL4 comprising SEQ ID NOs: 411, 412, 413, 414, 415, 416, 417, 421, 422, 423, 424, 425, 426, and 427, respectively.

[0041] In certain embodiments, the anti-CD3 antibody or antigen-binding fragment may include (i) FRH1, CDRH1, FRH2, CDRH2, FRH3, CDRH3, FRH4, FRL1, CDRL1, FRL2, CDRL2, FRL3, CDRL3, and FRL4 contained in antibody number V005, or (ii) FRH1, CDRH1, FRH2, CDRH2, FRH3, CDRH3, FRH4, FRL1, CDRL1, FRL2, CDRL2, FRL3, CDRL3, and FRL4 comprising SEQ ID NOs: 511, 512, 513, 514, 515, 516, 517, 521, 522, 523, 524, 525, 526, and 527, respectively.

[0042] In certain embodiments, the anti-CD3 antibody or antigen-binding fragment may include (i) FRH1, CDRH1, FRH2, CDRH2, FRH3, CDRH3, FRH4, FRL1, CDRL1, FRL2, CDRL2, FRL3, CDRL3, and FRL4 contained in antibody number V006, or (ii) FRH1, CDRH1, FRH2, CDRH2, FRH3, CDRH3, FRH4, FRL1, CDRL1, FRL2, CDRL2, FRL3, CDRL3, and FRL4 comprising SEQ ID NOs: 611, 612, 613, 614, 615, 616, 617, 621, 622, 623, 624, 625, 626, and 627, respectively.

[0043] In certain embodiments, the anti-CD3 antibody or antigen-binding fragment may include (i) FRH1, CDRH1, FRH2, CDRH2, FRH3, CDRH3, FRH4, FRL1, CDRL1, FRL2, CDRL2, FRL3, CDRL3, and FRL4 contained in antibody number V007, or (ii) FRH1, CDRH1, FRH2, CDRH2, FRH3, CDRH3, FRH4, FRL1, CDRL1, FRL2, CDRL2, FRL3, CDRL3, and FRL4 comprising SEQ ID NOs: 711, 712, 713, 714, 715, 716, 717, 721, 722, 723, 724, 725, 726, and 727, respectively.

[0044] In certain embodiments, the anti-CD3 antibody or antigen-binding fragment may include (i) FRH1, CDRH1, FRH2, CDRH2, FRH3, CDRH3, FRH4, FRL1, CDRL1, FRL2, CDRL2, FRL3, CDRL3, and FRL4 contained in antibody number V008, or (ii) FRH1, CDRH1, FRH2, CDRH2, FRH3, CDRH3, FRH4, FRL1, CDRL1, FRL2, CDRL2, FRL3, CDRL3, and FRL4 comprising SEQ ID NOs: 811, 812, 813, 814, 815, 816, 817, 821, 822, 823, 824, 825, 826, and 827, respectively.

[0045] In certain embodiments, the anti-CD3 antibody or antigen-binding fragment may include (i) FRH1, CDRH1, FRH2, CDRH2, FRH3, CDRH3, FRH4, FRL1, CDRL1, FRL2, CDRL2, FRL3, CDRL3, and FRL4 contained in antibody number V009, or (ii) FRH1, CDRH1, FRH2, CDRH2, FRH3, CDRH3, FRH4, FRL1, CDRL1, FRL2, CDRL2, FRL3, CDRL3, and FRL4 comprising SEQ ID NOs: 911, 912, 913, 914, 915, 916, 917, 921, 922, 923, 924, 925, 926, and 927, respectively.

[0046] In certain embodiments, the anti-CD3 antibody or antigen-binding fragment may include (i) FRH1, CDRH1, FRH2, CDRH2, FRH3, CDRH3, FRH4, FRL1, CDRL1, FRL2, CDRL2, FRL3, CDRL3, and FRL4 contained in antibody number V010, or (ii) FRH1, CDRH1, FRH2, CDRH2, FRH3, CDRH3, FRH4, FRL1, CDRL1, FRL2, CDRL2, FRL3, CDRL3, and FRL4 comprising SEQ ID NOs: 1011, 1012, 1013, 1014, 1015, 1016, 1017, 1021, 1022, 1023, 1024, 1025, 1026, and 1027, respectively.

[0047] In certain embodiments, the anti-CD3 antibody or antigen-binding fragment may include (i) FRH1, CDRH1, FRH2, CDRH2, FRH3, CDRH3, FRH4, FRL1, CDRL1, FRL2, CDRL2, FRL3, CDRL3, and FRL4 contained in antibody number V011, or (ii) FRH1, CDRH1, FRH2, CDRH2, FRH3, CDRH3, FRH4, FRL1, CDRL1, FRL2, CDRL2, FRL3, CDRL3, and FRL4 comprising SEQ ID NOs: 1111, 1112, 1113, 1114, 1115, 1116, 1117, 1121, 1122, 1123, 1124, 1125, 1126, and 1127, respectively.

[0048] In certain embodiments, the anti-CD3 antibody or antigen-binding fragment may include (i) FRH1, CDRH1, FRH2, CDRH2, FRH3, CDRH3, FRH4, FRL1, CDRL1, FRL2, CDRL2, FRL3, CDRL3, and FRL4 contained in antibody number V012, or (ii) FRH1, CDRH1, FRH2, CDRH2, FRH3, CDRH3, FRH4, FRL1, CDRL1, FRL2, CDRL2, FRL3, CDRL3, and FRL4 comprising SEQ ID NOs: 1211, 1212, 1213, 1214, 1215, 1216, 1217, 1221, 1222, 1223, 1224, 1225, 1226, and 1127, respectively.

[0049] In certain embodiments, the anti-CD3 antibody or antigen-binding fragment may include (i) FRH1, CDRH1, FRH2, CDRH2, FRH3, CDRH3, FRH4, FRL1, CDRL1, FRL2, CDRL2, FRL3, CDRL3, and FRL4 contained in antibody number V013, or (ii) FRH1, CDRH1, FRH2, CDRH2, FRH3, CDRH3, FRH4, FRL1, CDRL1, FRL2, CDRL2, FRL3, CDRL3, and FRL4 comprising SEQ ID NOs: 1311, 1312, 1313, 1314, 1315, 1316, 1317, 1321, 1322, 1323, 1324, 1325, 1326, and 1327, respectively.

[0050] In certain embodiments, the anti-CD3 antibody or antigen-binding fragment may include (i) FRH1, CDRH1, FRH2, CDRH2, FRH3, CDRH3, FRH4, FRL1, CDRL1, FRL2, CDRL2, FRL3, CDRL3, and FRL4 contained in antibody number V014, or (ii) FRH1, CDRH1, FRH2, CDRH2, FRH3, CDRH3, FRH4, FRL1, CDRL1, FRL2, CDRL2, FRL3, CDRL3, and FRL4 comprising SEQ ID NOs: 1411, 1412, 1413, 1414, 1415, 1416, 1417, 1421, 1422, 1423, 1424, 1425, 1426, and 1427, respectively.

[0051] In certain embodiments, the anti-CD3 antibody or antigen-binding fragment may include (i) FRH1, CDRH1, FRH2, CDRH2, FRH3, CDRH3, FRH4, FRL1, CDRL1, FRL2, CDRL2, FRL3, CDRL3, and FRL4 contained in antibody number V015, or (ii) FRH1, CDRH1, FRH2, CDRH2, FRH3, CDRH3, FRH4, FRL1, CDRL1, FRL2, CDRL2, FRL3, CDRL3, and FRL4 comprising SEQ ID NOs: 1511, 1512, 1513, 1514, 1515, 1516, 1517, 1521, 1522, 1523, 1524, 1525, 1526, and 1527, respectively.

[0052] In certain embodiments, the anti-CD3 antibody or antigen-binding fragment may include (i) FRH1, CDRH1, FRH2, CDRH2, FRH3, CDRH3, FRH4, FRL1, CDRL1, FRL2, CDRL2, FRL3, CDRL3, and FRL4 contained in antibody number V016, or (ii) FRH1, CDRH1, FRH2, CDRH2, FRH3, CDRH3, FRH4, FRL1, CDRL1, FRL2, CDRL2, FRL3, CDRL3, and FRL4 comprising SEQ ID NOs: 1611, 1612, 1613, 1614, 1615, 1616, 1617, 1621, 1622, 1623, 1624, 1625, 1626, and 1627, respectively.

[0053] In certain embodiments, the anti-CD3 antibody or antigen-binding fragment may include (i) FRH1, CDRH1, FRH2, CDRH2, FRH3, CDRH3, FRH4, FRL1, CDRL1, FRL2, CDRL2, FRL3, CDRL3, and FRL4 contained in antibody number V017, or (ii) FRH1, CDRH1, FRH2, CDRH2, FRH3, CDRH3, FRH4, FRL1, CDRL1, FRL2, CDRL2, FRL3, CDRL3, and FRL4 comprising SEQ ID NOs: 1711, 1712, 1713, 1714, 1715, 1716, 1717, 1721, 1722, 1723, 1724, 1725, 1726, and 1727, respectively.

[0054] In certain embodiments, the anti-CD3 antibody or antigen-binding fragment may include (i) FRH1, CDRH1, FRH2, CDRH2, FRH3, CDRH3, FRH4, FRL1, CDRL1, FRL2, CDRL2, FRL3, CDRL3, and FRL4 contained in antibody number V018, or (ii) FRH1, CDRH1, FRH2, CDRH2, FRH3, CDRH3, FRH4, FRL1, CDRL1, FRL2, CDRL2, FRL3, CDRL3, and FRL4 comprising SEQ ID NOs: 1811, 1812, 1813, 1814, 1815, 1816, 1817, 1821, 1822, 1823, 1824, 1825, 1826, and 1827, respectively.

[0055] In certain embodiments, the anti-CD3 antibody or antigen-binding fragment may include (i) FRH1, CDRH1, FRH2, CDRH2, FRH3, CDRH3, FRH4, FRL1, CDRL1, FRL2, CDRL2, FRL3, CDRL3, and FRL4 contained in antibody number V019, or (ii) FRH1, CDRH1, FRH2, CDRH2, FRH3, CDRH3, FRH4, FRL1, CDRL1, FRL2, CDRL2, FRL3, CDRL3, and FRL4 comprising SEQ ID NOs: 1911, 1912, 1913, 1914, 1915, 1916, 1917, 1921, 1922, 1923, 1924, 1925, 1926, and 1927, respectively.

[0056] In some embodiments, the antibody or antigen-binding fragment may comprise a VH comprising an amino acid sequence selected from any of the amino acid sequences listed in Table 1A, or 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 thereto, and / or a VL comprising an amino acid sequence selected from any of the amino acid sequences listed in Table 1B, or 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 thereto.

[0057]

[0058] In certain embodiments, (A) the VH polypeptide comprises 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:210, and / or (B) the VL polypeptide comprises 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:220.

[0059] In certain embodiments, (A) the VH polypeptide comprises 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:310, and / or (B) the VL polypeptide comprises 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:320.

[0060] In certain embodiments, (A) the VH polypeptide comprises 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:410, and / or (B) the VL polypeptide comprises 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:420.

[0061] In certain embodiments, (A) the VH polypeptide comprises 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:510, and / or (B) the VL polypeptide comprises 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:520.

[0062] In certain embodiments, (A) the VH polypeptide comprises 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, and / or (B) the VL polypeptide comprises 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.

[0063] In certain embodiments, (A) the VH polypeptide comprises 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:710, and / or (B) the VL polypeptide comprises 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:720.

[0064] In certain embodiments, (A) the VH polypeptide comprises 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:810, and / or (B) the VL polypeptide comprises 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:820.

[0065] In certain embodiments, (A) the VH polypeptide comprises 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:910, and / or (B) the VL polypeptide comprises 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:920.

[0066] In certain embodiments, (A) the VH polypeptide comprises 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:1010, and / or (B) the VL polypeptide comprises 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:1020.

[0067] In certain embodiments, (A) the VH polypeptide comprises 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:1110, and / or (B) the VL polypeptide comprises 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:1120.

[0068] In certain embodiments, (A) the VH polypeptide comprises 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:1210, and / or (B) the VL polypeptide comprises 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:1220.

[0069] In certain embodiments, (A) the VH polypeptide comprises 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:1310, and / or (B) the VL polypeptide comprises 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:1320.

[0070] In certain embodiments, (A) the VH polypeptide comprises 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:1410, and / or (B) the VL polypeptide comprises 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:1420.

[0071] In certain embodiments, (A) the VH polypeptide comprises 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:1510, and / or (B) the VL polypeptide comprises 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:1520.

[0072] In certain embodiments, (A) the VH polypeptide comprises 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: 1610, and / or (B) the VL polypeptide comprises 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: 1620.

[0073] In certain embodiments, (A) the VH polypeptide comprises 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:1710, and / or (B) the VL polypeptide comprises 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:1720.

[0074] In certain embodiments, (A) the VH polypeptide comprises 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: 1810, and / or (B) the VL polypeptide comprises 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: 1820.

[0075] In certain embodiments, (A) the VH polypeptide comprises 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:1910, and / or (B) the VL polypeptide comprises 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:1920.

[0076] In certain embodiments, the anti-CD3 antibody or antigen-binding fragment may comprise VH and VL polypeptides comprising the amino acid sequences of SEQ ID NOs: 210 and 220, respectively. In certain embodiments, the anti-CD3 antibody or antigen-binding fragment may comprise VH and VL polypeptides comprising the amino acid sequences of SEQ ID NOs: 310 and 320, respectively. In certain embodiments, the anti-CD3 antibody or antigen-binding fragment may comprise VH and VL polypeptides comprising the amino acid sequences of SEQ ID NOs: 410 and 420, respectively. In certain embodiments, the anti-CD3 antibody or antigen-binding fragment may comprise VH and VL polypeptides comprising the amino acid sequences of SEQ ID NOs: 510 and 520, respectively. In certain embodiments, the anti-CD3 antibody or antigen-binding fragment may comprise VH and VL polypeptides comprising the amino acid sequences of SEQ ID NOs: 610 and 620, respectively. In certain embodiments, the anti-CD3 antibody or antigen-binding fragment may comprise VH and VL polypeptides comprising the amino acid sequences of SEQ ID NOs: 710 and 720, respectively. In certain embodiments, the anti-CD3 antibody or antigen-binding fragment may comprise VH and VL polypeptides comprising the amino acid sequences of SEQ ID NOs: 810 and 820, respectively. In certain embodiments, the anti-CD3 antibody or antigen-binding fragment may comprise VH and VL polypeptides comprising the amino acid sequences of SEQ ID NOs: 910 and 920, respectively. In certain embodiments, the anti-CD3 antibody or antigen-binding fragment may comprise VH and VL polypeptides comprising the amino acid sequences of SEQ ID NOs: 1010 and 1020, respectively. In certain embodiments, the anti-CD3 antibody or antigen-binding fragment may comprise VH and VL polypeptides comprising the amino acid sequences of SEQ ID NOs: 1110 and 1120, respectively. In certain embodiments, the anti-CD3 antibody or antigen-binding fragment may comprise VH and VL polypeptides comprising the amino acid sequences of SEQ ID NOs: 1210 and 1220, respectively. In certain embodiments, the anti-CD3 antibody or antigen-binding fragment may comprise VH and VL polypeptides comprising the amino acid sequences of SEQ ID NOs: 1310 and 1320, respectively.In certain embodiments, the anti-CD3 antibody or antigen-binding fragment may comprise VH and VL polypeptides comprising the amino acid sequences of SEQ ID NOs: 1410 and 1420, respectively. In certain embodiments, the anti-CD3 antibody or antigen-binding fragment may comprise VH and VL polypeptides comprising the amino acid sequences of SEQ ID NOs: 1510 and 1520, respectively. In certain embodiments, the anti-CD3 antibody or antigen-binding fragment may comprise VH and VL polypeptides comprising the amino acid sequences of SEQ ID NOs: 1610 and 1620, respectively. In certain embodiments, the anti-CD3 antibody or antigen-binding fragment may comprise VH and VL polypeptides comprising the amino acid sequences of SEQ ID NOs: 1710 and 1720, respectively. In certain embodiments, the anti-CD3 antibody or antigen-binding fragment may comprise VH and VL polypeptides comprising the amino acid sequences of SEQ ID NOs: 1810 and 1820, respectively. In certain embodiments, the anti-CD3 antibody or antigen-binding fragment may comprise VH and VL polypeptides comprising the amino acid sequences of SEQ ID NOs: 1910 and 1920, respectively.

[0077] In some embodiments, an anti-CD3 antibody or antigen-binding fragment may comprise one or more of an antibody constant region, a CH1 domain, a hinge, a CH2 domain, and / or a CH3 domain, which optionally are or are derived from IgG or human IgG, individually, and further optionally are or are derived from human IgG1, IgG4, IgG2, or IgG3, respectively.

[0078] In some embodiments, the anti-CD3 antibody or antigen-binding fragment may optionally comprise a human IgG1, IgG4, IgG2, or IgG3, or a fragment crystallizable (Fc) region derived from human IgG1, IgG4, IgG2, or IgG3.

[0079] In certain embodiments, when an anti-CD3 antibody or antigen-binding fragment comprises one or more of an antibody constant region, constant domain, and / or Fc region of or derived from a human IgG1, the antibody constant region, constant domain, and / or Fc region may contain one or more of the following amino acid modifications according to EU numbering: N297A, N297Q, D265A, L234A, L235A, C226S, C229S, P238S, E233P, L234V, G236 deletion, P238A, A327Q, A32 7G, P329A, K322A, L234F, L235E, P331S, T394D, A330L, P331S, F243L, R292P, Y300L, V305I, P396L, S239D, I332E, S298A, E333A, K334A, L 234Y, L235Q, G236W, S239M, H268D, D270E, K326D, A330M, K334E, G236A, K326W, S239D, E333S, S267E, H268F, S324T, E345R, E430G, S440Y The interleaved channel may include one or more of M428L, N434S, L328F, M252Y, S254T, T256E, or any combination thereof.

[0080] In certain embodiments, when an anti-CD3 antibody or antigen-binding fragment comprises one or more of an antibody constant region, constant domain, and / or Fc region of or derived from human IgG4, the antibody constant region, constant domain, and / or Fc region may comprise 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.

[0081] In certain embodiments, when an anti-CD3 antibody or antigen-binding fragment comprises one or more of an antibody constant region, constant domain, and / or Fc region of or derived from human IgG2, the antibody constant region, constant domain, and / or Fc region may comprise 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.

[0082] In certain embodiments, when the anti-CD3 antibody or antigen-binding fragment comprises one or more of an antibody constant region, constant domain, and / or Fc region of or derived from human IgG3, the antibody constant region, constant domain, and / or Fc region may comprise E235Y according to EU numbering.

[0083] In some embodiments, the anti-CD3 antibody or antigen-binding fragment may comprise IgG, IgA, IgE, IgD, or IgM, optionally, IgG1, IgG4, IgG2, or IgG3.

[0084] In some embodiments, the anti-CD3 antibody or antigen-binding fragment may comprise an antibody fragment selected from the group consisting of a fragment, an antigen-binding (Fab) region, a Fab2, a Fab3, a Fab' fragment, a F(ab')2, a variable fragment (Fv), a single-chain Fv (scFv) fragment, a bispecific antibody, a triabody, a minibody, a scFv-Fc, a scFv2-Fc2, a 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).

[0085] In some embodiments, the anti-CD3 antibody or antigen-binding fragment may bind to (i) human CD3, (ii) non-human primate CD3, optionally monkey, and further optionally cynomolgus and / or rhesus CD3, and / or (iii) rodent CD3, optionally mouse CD3. In some embodiments, the anti-CD3 antibody or antigen-binding fragment may bind to CD3εδ. In some embodiments, the anti-CD3 antibody or antigen-binding fragment may comprise or be included in a multispecific (e.g., bispecific, trispecific, tetraspecific, etc.) 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. In certain embodiments, the multispecific antibody or antibody fragment may comprise or be included in one or more scFvs.

[0086] In certain embodiments, the second antigen-binding region is specific for or specifically binds to a tumor target, a target molecule expressed on a cancer cell, an immune tumor target, a target molecule expressed on an immune cell, a neurodegenerative disease target, an autoimmune disorder target (optionally an autoreactive immune molecule or a target molecule expressed on an immune cell expressing an autoreactive immune molecule), an infectious disease target, an inflammatory disease target (optionally an inflammatory cytokine or chemokine or its receptor), an infectious disease target (optionally a viral, bacterial, or fungal target molecule), a target molecule expressed on an infected cell (optionally 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.

[0087] In certain embodiments, the second antigen-binding region is selected from the group consisting of 17-IA, 4-1BB, 4Dc, 6-keto-PGFla, 8-iso-PGF2a, 8-oxo-dG, Al adenosine receptor, A33, ACE, ACE-2, activin, activin A, activin AB, activin B, activin C, activin RIA, activin RIA ALK-2, activin RIB ALK-4, activin RIIA, activin RUB, ADAM, ADAM10, ADAM12, ADAM15, ADAM17 / T ACE, ADAM8, ADAM9, ADAMTS, ADAMTS4, ADAMTS5, addressin, aFGF, ALCAM, ALK, ALK-1, ALK-7, alpha-l-antitrypsin, alpha-V / beta-1 antagonist, ANG, Ang, APAF-1, APE, APJ, APP, APRIL, AR, ARC, ART, artemin, anti-Id, ASPARTIC, atrial natriuretic factor, av / b3 integrin, Axl, b2M, B7-1, B7-2, B7-H, B-lymphocyte stimulatory factor (BlyS), BACE, BACE-1, Bad, BAFF, BAFF-R, Bag-1, BAK, Bax, BCA-1, BCAM, Bel, BCMA, BDNF, b-ECGF, bFGF, BID, Bik, BFM, BLC, BL-CAM, BLK, BMP, BMP-2 BMP-2a, BMP-3 osteogenin, BMP-4 BMP-2b, BMP-5, BMP-6Vgr-1, BMP-7 (OP-1), BMP-8 (BMP-8a, OP-2), BMPR, BMPR-IA (ALK-3), BMPR-IB (ALK-6), BRK-2, RPK-1, BMPR-II (BRK-3), 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), carcinoma-associated antigen, cathepsin A, cathepsin B, 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, CCL10, CCL11, CCL12, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL2, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CCL3, CCL4, CCL5, CCL6, CCL7 ... CL8, CCL9 / 10, CCR, CCR1, CCR10, CCR10, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CD1, CD2, CD4, CD5, CD6, CD7, CD8, CD10, CDlla, CDll b, CDllc, 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, CXCL 4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCR, CXCR1, CXCR 2, CXCR3, CXCR4, CXCR5, CXCR6, cytokeratin tumor-associated antigen, DAN, DCC, DcR3, DC-SIGN, decay-accelerating factor, des(l-3)-IGF-I (brain IGF-1), Dhh, digoxin, DNAM-1, Dnase, Dpp, DPPIV / CD26, Dtk, ECAD, EDA, EDA-A1, EDA-A2, EDAR, EGF, EGFR (ErbB-1), EMA, EMMPRIN, EN A, endothelin receptor, enkephalinase, eNOS, Eot, eotaxin l, EpCAM, ephrin B2 / EphB4, EPO, ERCC, E-selectin, ET-1, factor Ila, factor VII, factor VIIIc, factor IX, fibroblast activation protein (FAP), Fas, FcRl, FEN-1, ferritin, FGF, FGF-19, FGF-2, FGF3, FGF-8, FGFR, FGFR-3, fibrin, FL, FLIP, Flt-3, Flt-4, follicle-stimulating hormone, fractalkine, FZD1, FZD2, FZD3, FZD4, FZD5 , FZD6, FZD7, FZD8, FZD9, FZD10, G250, Gas6, GCP-2, GCSF, GD2, GD3, GDF, GDF-1, GDF-3 (Vgr-2), GDF-5 (BMP-14, CDMP-1), GDF-6 (BMP-13, CDMP-2), GDF-7 (BMP-12, CDMP-3), GDF-8 (myostatin), GDF-9, GDF-15 (MIC-1), GDNF, GFAP, GFRa-1, GFR-alphal, GFR-alpha2, GFR-alpha3, GITR, glucagon, Glut4, glycoprotein Ilb / IIIa (GPI), Ilb / IIIa), GM-CSF, gpl30, gp72, GRO, growth hormone releasing factor, hapten (NP-cap or NIP-cap), HB-EGF, HCC, 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, INF-beta, INF-gamma, inhibin, iNOS, insulin A chain, insulin B chain, insulin-like growth factor 1, interferon (INF)-alpha, INF-beta, INF-gamma, interferon (INF) ... integrin alpha 2, integrin alpha 3, integrin alpha 4, integrin alpha 4 / beta l, integrin, alpha 4 / beta 7, integrin alpha 5 (alpha V), integrin alpha 5 / beta l, integrin alpha 5 / beta 3, integrin alpha 6, integrin beta l, integrin beta 2, interferon gamma, IP-10, 1-TAC, JE, kallikrein 2, kallikrein 5, kallikrein 6, kallikrein 11, kallikrein 12, kallikrein 14, kallikrein 15, kallikrein LI, kallikrein L2, kallikrein L3, kallikrein L4, KC, KDR, keratinocyte growth factor (KGF), laminin 5, LAMP, LAP, LAP (TGF-1), latent TGF-1, latent TGF-1bpl, LBP, LDGF, LECT2, Lefty, Lewis-Y antigen, Lewis-Y related antigen, LFA-1, LFA-3, Lfo, LIF, LIGHT, lipoprotein, LIX, LKN, Lptn, L-selectin, LT-a, LT-b, LTB4, LTBP-1, pulmonary surfactant, luteinizing hormone, lymphotoxin beta receptor, Mac-1, MAdCAM, MAG, MAP2, MARC, MCAM, MCAM, MCK-2, MCP, M-CSF, MDC, Mer, metalloproteinase ase, MGDF receptor, MGMT, MHC (HLA-DR), MIF, MIG, MIP, MIP-1-alpha, MK, MMAC1, MMP, MMP-1, MMP-10, MMP-11, MMP-12, MMP-13, MMP-14, MMP-15, MMP-2, MMP-24, MMP-3, MMP-7, MMP-8, MMP-9, MPIF, Mpo, MSK, MSP, Mucin (Mucl), MUC18, Mullerian inhibitor, Mug, MuSK, NAIP, NAP, NCAD, N-cadherin, NCA90, NCAM, NCAM, neprilysin, neurotrophin-3, -4, or -6, neurturin, neuron 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 GF, PDK-1, PECAM, PEM, PF4, PGE, PGF, PGI2, PGJ2, PIN, PLA2, placental alkaline phosphatase (PLAP), PIGF, PLP, PP14, proinsulin, prorelaxin, protein C, PS, PSA, PSCA, prostate-specific membrane antigen (PSMA), PTEN, PTHrp, Ptk, PTN, R51, RANK, RANKL, RANTES, relaxin A-chain, relaxin B-chain, renin, respiratory syncytial virus (RSV)F, RSVFgp, Ret, rheumatoid factor, RLIP76, RPA2, RSK, S100, SCF / KL, SDF-1, serine, serum albumin, sFRP-3, Shh, SIGIRR, SK-1, SLAM, SLPI, SMAC, SMDF, SMOH, SOD, SPARC, Stat, STEAP, STEAP-II, TACE, TACI, TAG-72 (tumor-associated glycoprotein-72), TARC, TCA-3, T cell receptor (e.g., T cell receptor alpha / beta), TdT, TECK, TEM1, TEM5, TEM7, TEM8, TERT, testicular PLAP-like alkaline phosphatase, T fR, TGF, TGF-alpha, TGF-beta, TGF-beta Pan specific, TGF-beta RI (ALK-5), TGF-beta RII, TGF-beta Rllb, TGF-beta RIII, TGF-beta l, 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 Rl Apo-2, DR4), TNFRSFIOB(TRAIL R2 DR5, KILLER, TRICK-2A, TRICK-B), TNFRSF10C(TRAIL R3 DcRl, LIT, TRID), TNFRSF10D(TRAIL R4 DcR2, TRUNDD), TNFRSF11A(RANK ODF R, TRANCE R), TNFRSFllB(OPG OCIF, TR1), TNFRSF12(TWEAK R FN14), TNFRSF13B(TACI), TNFRSF13C(BAFF R), TNFRSF14(HVEM ATAR、HveA、LIGHT R、TR2)、TNFRSF16(NGFR p75NTR)、TNFRSF17(BCMA)、TNFRSF18(GITR AITR)、TNFRSF19(TROY TAJ、TRADE)、TNFRSF19L(RELT)、TNFRSFIA(TNF RI). CD120a, p55-60, TNFRSFIB(TNF RII CD120b, p75-80), TNFRSF26(TNFRH3), TNFRSF3(LTbR TNF RIII, TNFC R), TNFRSF4(OX40 ACT35, TXGP1). R), TNFRSF5(CD40 p50), TNFRSF6(Fas Apo-1, APT1, CD95), TNFRSF6B(DcR3 M68, TR6), TNFRSF7(CD27), TNFRSF8(CD30), TNFRSF9(4-1BB). CD137, ILA, TNFRSF21(DR6), TNFRSF22(DcTRAIL R2 TNFRH2), TNFRST23(DcTRAIL Rl TNFRH1), TNFRSF25(DR3 Apo-3, LARD, TR-3, TRAMP, WSL-1), TNFSF10(TRAIL). Apo-2 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 WHO LINES LTg TNFSF15(TLIA / VEGI) TNFSF18(GITR LINES AITR TL6, TNFSFIA (TNF-a, DIF, TNFSF2), TNFSF1B (TNF-b). LTa、TNFSF1)、TNFSF3(LTbTNFC, p33), TNFSF4 (OX40 ligand gp34, TXGP1), TNFSF5 (CD40 ligand CD154, gp39, HIGM1, IMD3, TRAP), TNFSF6 (Fas ligand Apo-1 ligand, APT1 ligand), TNFSF7 (CD27 ligand CD70), TNFSF8 (CD30 ligand CD153), TNFSF9 (4-1BB ligand CD137 ligand), TP-1, t-PA, Tpo, TRAIL, TRAIL R, TRAIL-R1, TRAIL-R2, TRANCE, transferrin receptor, TRF, Trk, TROP-2, TSG, TSLP, tumor-associated antigen CA125, Lewis Y-related carbohydrate expressed tumor-associated antigen, TWEAK, TXB2, Ung, uPAR, uPAR-1, urokinase, VCAM, VCAM-1, VECAD, VE-cadherin, VE-cadherin-2, VEFGR-1 (flt-1), VEGF, VEGFR, VEGFR-3 (flt-4), VEGI, VFM, viral antigen, VLA, VLA-1, VLA-4, VNR integrin, von Willebrand factor, WIF-1, WNT1, WNT2, WNT2B / 13, WNT3, W The antibodies are specific for or specifically bind to one or more of NT3A, WNT4, WNT5A, WNT5B, WNT6, WNT7A, WNT7B, WNT8A, WNT8B, WNT9A, WNT9A, WNT9B, WNT10A, WNT10B, WNT11, WNT16, XCL1, XCL2, XCR1, XCR1, XEDAR, XIAP, XPD, CTLA4 (cytotoxic T-lymphocyte antigen-4), PD1 (programmed cell death protein 1), PD-L1 (programmed cell death ligand 1), LAG-3 (lymphocyte activation gene-3), TIM-3 (T-cell immunoglobulin and mucin protein-3), hormone receptors, and growth factors.

[0088] In certain embodiments, the second antigen-binding region is specific for or specifically binds to one or more of BCMA, CTLA4 (cytotoxic T lymphocyte antigen-4), PD1 (programmed cell death protein 1), PD-L1 (programmed cell death ligand 1), LAG-3 (lymphocyte activation gene-3), TIM-3, CD20, CD2, CD19, Her2, EGFR, EpCAM, FcyRIIIa (CD16), FcyRIIa (CD32a), FcyRIIb (CD32b), FcyRI (CD64), Toll-like receptors (TLRs), TLR4, TLR9, cytokines, IL-2, IL-5, IL-13, IL-6, IL-17, IL-12, IL-23, TNFα, TGFβ, cytokine receptors, IL-2R, chemokines, chemokine receptors, growth factors, VEGF, and HGF.

[0089] In certain embodiments, the multispecific antibody or antibody fragment further comprises a third antigen-binding region, hi certain embodiments, the multispecific antibody or antibody fragment is trispecific.

[0090] In certain embodiments, 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.

[0091] In certain embodiments, the multispecific antibody or antibody fragment comprises one or more of the following: at least one CLκ-preferred variant CH1 domain, optionally a CLκ-preferred variant CH1 domain described in WO2021067404; at least one CLλ-preferred variant CH1 domain, optionally a CLλ-preferred variant CH1 domain 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 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 a pair described in WO2022150785.

[0092] In some embodiments, the anti-CD3 antibody or antigen-binding fragment may optionally exhibit a reduced PSR score compared to its parent antibody, optionally any one or more or all of antibody numbers A001, A002, A003, A004, and / or A005.

[0093] In some embodiments, the anti-CD3 antibody or antigen-binding fragment may exhibit a PSR score of less than 0.33, less than about 0.30, less than about 0.20, less than about 0.15, less than about 0.12, less than about 0.10, less than about 0.08, less than about 0.06, less than about 0.05, less than about 0.04, less than about 0.03, less than about 0.02, or less than about 0.01. In some embodiments, the anti-CD3 antibody or antigen-binding fragment may exhibit a PSR score of ≧about 0.10 and < 0.33 or a PSR score of about < 0.10.

[0094] In some embodiments, the anti-CD3 antibody or antigen-binding fragment may exhibit a hydrophobic interaction chromatography (HIC) retention time of < about 10.5 minutes, < about 10.0 minutes, < about 9.5 minutes, < about 9.0 minutes, or < about 8.5 minutes.

[0095] In some embodiments, the anti-CD3 antibody or antigen-binding fragment may exhibit (i) a reduced affinity capture self-interacting nanoparticle spectroscopy (AC-SINS) Δλmax compared to any one or more or all of Antibody Nos. A001-A005; (ii) an AC-SINS Δλmax of less than about 20.0 nm, less than about 15.0 nm, less than about 12.0 nm, less than about 10.0 nm, less than about 8.0 nm, less than about 6.0 nm, less than about 5.0 nm, less than about 4.0 nm, less than about 3.0 nm, less than about 2.0 nm, or less than about 1.0 nm; and / or (iii) an AC-SINS Δλmax of ≧5.0 nm and <20.0 or an AC-SINS Δλmax of about <5.0 nm.

[0096] In some embodiments, the anti-CD3 antibody or antigen-binding fragment may exhibit (i) a higher dynamic light scattering (DLS) diffusion interaction parameter (kD) than any one or more or all of Antibody Nos. A001-A005, (ii) a DLS kD of about 5 mL / g or more, about 10 mL / g or more, about 15 mL / g or more, about 20 mL / g or more, about 25 mL / g or more, about 30 mL / g or more, or about 35 mL / g or more, and / or (iii) a DLS kD of about 10 mL / g to about 40 mL / g, about 15 mL / g to about 40 mL / g, or about 20 mL / g to about 40 mL / g. In certain embodiments, the DLS kD is measured using 10 mM histidine buffer, optionally at a pH of 6.0.

[0097] In some embodiments, the anti-CD3 antibody or antigen-binding fragment may comprise (i) a melting temperature (Tm) of about 65° C. or higher, or (ii) a Tm of about 70° C. or higher, about 75° C. or higher, about 80° C. or higher, and / or (iii) a Tm of about 70° C. to about 90° C., about 75° C. to about 85° C. In certain embodiments, the anti-CD3 antigen-binding fragment may be a Fab.

[0098] In some embodiments, the anti-CD3 antibody or antigen-binding fragment may not significantly aggregate or multimerize. In certain embodiments, the anti-CD3 antibody or antigen-binding fragment may exhibit (i) about 90% or more, about 95% or more, about 97% or more, about 98% or more, about 99% or more percent monomer by size exclusion chromatography (SEC), and / or (ii) about 90% to about 100%, about 95% to about 100%, about 97% to about 100%, about 98% to about 100%, about 99% to about 100% percent monomer by SEC. In certain embodiments, the anti-CD3 antigen-binding fragment may be a Fab or IgG, optionally an IgG1.

[0099] In certain embodiments, the anti-CD3 antibodies or antigen-binding fragments may not significantly aggregate or multimerize when exposed to acidic conditions or acidic stress, optionally at a pH of about 6 or less, about 5 or less, about 4 or less, or about 3.5 or less. In certain embodiments, aggregation or multimerization under acidic conditions may be assessed by SEC, as described above and herein.

[0100] In some embodiments, the anti-CD3 antibody or antigen-binding fragment may, optionally, not exhibit a significant amount of heavy chain-light chain mispairing, as determined by the absence of an unpaired heavy chain peak and / or an unpaired light chain peak, as measured by liquid chromatography mass spectrometry (LC-MS), when the anti-CD3 antibody or antigen-binding fragment is, optionally, recombinantly produced in mammalian cells, and even more optionally, in Chinese Hamster Ovary (CHO) cells.

[0101] In certain embodiments, the anti-CD3 antibody or antigen-binding fragment may bind to a CD3-expressing cell, which optionally is a (i) T cell, (ii) a human cell, a non-human primate (optionally, monkey, and optionally, cynomolgus and / or rhesus) cell, and / or a rodent (optionally, mouse) cell, and / or (iii) a primary cell or a cell line cell.

[0102] In certain embodiments, the anti-CD3 antibody or antigen-binding fragment has (i) about 1.0×10 6 M or less, approximately 5.0×10 7 M or less, approximately 1.0×10 7 M or less, approximately 5.0×10 8 M or less, approximately 1.0×10 8 M or less, approximately 5.0×10 9 M or less, approximately 1.0×10 9 M or less, approximately 5.0×10 10 M or less, or about 1.0 x 10 10 M or less, (ii) approximately 1.0×10 6 M ~ approx. 1.0×10 11 M, approx. 1.0×10 7 M ~ approx. 1.0×10 11 M, or approximately 1.0 x 10 8 M ~ approx. 1.0×10 10 It may bind to CD3 with an equilibrium dissociation constant (Kd) of M. In some cases, such binding may be measured by surface plasmon resonance (SPR), optionally using a BIACORE® system, or by biolayer interferometry (BLI), further optionally using an Octet® system. In some cases, the CD3 is human, non-human primate (optionally monkey, further optionally cynomolgus and / or rhesus), and / or rodent (optionally mouse) CD3.

[0103] In certain embodiments, the anti-CD3 antibody or antigen-binding fragment, upon binding to CD3 on a cell, may induce activation of the cell, optionally a T cell, and / or enhance the cytotoxic function of the cell, optionally a T cell.

[0104] In certain embodiments, the anti-CD3 antibody or antigen-binding fragment, upon binding to CD3 on a cell, may not induce cytokine production by the cell to a level that could induce cytokine release syndrome (CRS).

[0105] In certain embodiments, the anti-CD3 antibody or antigen-binding fragment may comprise or may comprise 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 exhibits cytotoxicity against the second cell.

[0106] One aspect of the disclosure provides a nucleic acid (eg, one or more nucleic acids).

[0107] In some embodiments, a nucleic acid (e.g., one or more isolated or recombinant nucleic acids, such as a nucleic acid or a combination of two or more nucleic acids) is provided and can encode an anti-CD3 antibody or antigen-binding fragment according to any of the embodiments described herein. Such a nucleic acid can include, for example, an mRNA for delivery to a cell and expression on the anti-CD3 antibody or antigen-binding fragment.

[0108] In some embodiments, the nucleic acid is: (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: 250, 350, 450, 550, 650, 750, 850, 950, 1050, 1150, 1250, 1350, 1450, 1550, 1650, 1750, 1850, or 1950, or an 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: 260, 360, 460, 560, 660, 760, 860, 960, 1060, 1160, 12560, 1360, 1460, 1560, 1660, 1760, 1860, or 1960, or an mRNA version of any of the foregoing.

[0109] In certain embodiments, the nucleic acid is selected from the group consisting of: (I) SEQ ID NOs: 250 and 260, respectively; (II) SEQ ID NOs: 350 and 360, respectively; (III) SEQ ID NOs: 450 and 460, respectively; (IV) SEQ ID NOs: 550 and 560, respectively; (V) SEQ ID NOs: 650 and 660, respectively; (VI) SEQ ID NOs: 750 and 760, respectively; (VII) SEQ ID NOs: 850 and 860, respectively; (VIII) SEQ ID NOs: 950 and 960, respectively; (IX) SEQ ID NOs: 1050 and 1060, respectively; (X) SEQ ID NOs: 1150 and 1160, respectively; (XI) SEQ ID NOs: (XII) SEQ ID NOs: 1350 and 1360, respectively; (XIII) SEQ ID NOs: 1450 and 1460, respectively; (XIV) SEQ ID NOs: 1550 and 1560, respectively; (XV) SEQ ID NOs: 1650 and 1660, respectively; (XVI) SEQ ID NOs: 1750 and 1760, respectively; (XVII) SEQ ID NOs: 1850 and 1860, respectively; or (XVIII) SEQ ID NOs: 1950 and 1960, respectively; or the nucleic acid sequences encoding the VH and VL polypeptides of the mRNA versions of any of the foregoing.

[0110] Another aspect of the disclosure provides vectors (e.g., one or more vectors, such as a vector or a combination of two or more vectors) and / or constructs comprising any of the nucleic acids according to the disclosure.

[0111] In certain embodiments, the vector may be an expression vector. In certain embodiments, the vector may include 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] Another aspect of the disclosure provides cells containing, transfected, transformed or transduced with any of the nucleic acids and / or vectors and / or constructs.

[0113] In some embodiments, the cell is (i) a mammalian cell, optionally a human, non-human primate, monkey, rabbit, rodent, hamster, rat, or mouse cell or a yeast cell, or (ii) a non-mammalian cell, optionally a plant, bacterial, fungal, yeast, protozoan, or insect cell.

[0114] In certain embodiments, the isolated or recombinant cell is an immune cell or a hybridoma.

[0115] Another aspect of the present disclosure provides a pharmaceutical composition comprising (i) an anti-CD3 antibody or antigen-binding fragment according to any of the embodiments described herein, optionally including an antibody or antigen-binding fragment according to any of the embodiments described herein, a nucleic acid according to any of the embodiments described herein, a vector according to any of the embodiments described herein, and / or a cell according to any of the embodiments described herein, and (ii) a pharmaceutically acceptable carrier and / or excipient.

[0116] Another aspect of the disclosure provides in vivo methods using at least one of an anti-CD3 antibody or antigen-binding fragment or an encoding nucleic acid or a cell capable of containing and / or expressing any of the anti-CD3 antibodies or antigen-binding fragments or the nucleic acids encoding them disclosed herein.

[0117] In some embodiments, the method is a method of treating a subject in need of such treatment. In some embodiments, the method is a method of treating or preventing a disease, disorder, or condition in a subject in need of such treatment (e.g., in a mammal). In any of such methods, in certain embodiments, the method may include administering an effective amount of (i) an anti-CD3 antibody or antigen-binding fragment according to any of the embodiments described herein, (ii) a nucleic acid according to any of the embodiments described herein, (iii) a vector according to any of the embodiments described herein, (iv) an isolated or recombinant cell (optionally an immune cell, T cell, and / or natural killer (NK) cell) according to any of the embodiments described herein, and / or (v) a pharmaceutical composition according to any of the embodiments described herein.

[0118] In some embodiments, the method is a method of inducing cytotoxicity against a cell expressing a target molecule of a subject. In certain embodiments, the method may comprise administering to the subject an effective amount of (i) any of the multispecific antibodies or antibody fragments described herein, (ii) a nucleic acid encoding such a multispecific antibody or antibody fragment, (iii) a vector comprising such a nucleic acid, (iv) one or more isolated or recombinant cells comprising, transfected with, transformed with, or transduced with such a nucleic acid or vector, and / or (v) a pharmaceutical composition comprising (A) such a multispecific antibody or antibody fragment, such a nucleic acid, such a vector, such one or more cells, and (B) a pharma- ceutically acceptable carrier and / or excipient.

[0119] In certain embodiments of any of the above methods, the subject may be (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. In certain embodiments, the subject may have or be at risk of developing a disease, disorder, or condition. In certain embodiments of any of the above methods, the method may further include administering to the subject an additional agent (e.g., any of the agents described herein), optionally an adjuvant or therapeutic agent.

[0120] In certain embodiments of any of the above methods, the disorder may include one or more of a proliferative disorder, a neoplastic disorder, a cancer or neoplastic condition, an immune-neoplastic disorder, a neurological disorder, a neurodegenerative disorder, an infectious disease, and an autoimmune disorder, an autoimmune disorder, or another disease.

[0121] In certain embodiments of any of the above methods, the disease, disorder, or condition may include cancer.

[0122] 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.

[0123] 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, Burkitt's lymphoma, 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 lymphoma, myelodysplastic syndrome ... J.D. 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, Nodal Marginal Zone Lymphoma, Childhood Nodal Marginal Zone Lymphoma, The liquid cancer may be selected from 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.

[0124] In certain embodiments of any of the above methods, the disease, disorder, or condition may comprise an autoimmune or inflammatory disease.

[0125] In certain embodiments, the autoimmune or inflammatory disease may be 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-Barre 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.

[0126] In certain embodiments of any of the above methods, the disease, disorder, or condition may include a neurodegenerative disease.

[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, Creutzfeldt-Jakob disease.

[0128] In certain embodiments of any of the above methods, the disease, disorder, or condition may comprise an infectious disease.

[0129] In certain embodiments, the infectious disease may be a viral disease, a bacterial disease, a fungal disease, a yeast disease, a protozoal disease, a prion disease, or a parasitic disease. In some cases, the viral disease may be a human immunodeficiency virus (HIV), a hepatitis virus (optionally Hepatitis A, B, or C), a human papilloma virus (HPV), a herpes simplex virus (HSV) (optionally HSV-1 or HSV-2), an enterovirus, a human cytomegalovirus, an adenovirus, a rhinovirus, a poxvirus, an influenza virus, a coronavirus (optionally MERS-CoV, SARS-CoV, or SARS-CoV-2, or a common human coronavirus), a norovirus, a West Nile virus, a Zika virus, a poliovirus, an Ebola virus, or a dengue virus (DENV) infection. In some cases, the bacterial disease may be 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. In some cases, the fungal disease may be an Aspergillosis, Candida, Candida auris, Cryptococcus neoformans, Pneumocystis jiroveccii, Mucor, Talaromyces, Ringworm, Blastomyces, Coccidioides, Cryptococcus gattii, Histoplasma, Paracoccidioides, or Sporothrix infection.

[0130] One aspect of the disclosure provides a method of producing an anti-CD3 antibody or antigen-binding fragment according to any of the embodiments described herein.

[0131] In some embodiments, the method may include (a) culturing cells that contain nucleic acid encoding an anti-CD3 antibody or antigen-binding fragment under conditions that allow for expression of the anti-CD3 antibody or antigen-binding fragment, and (b) harvesting and purifying the anti-CD3 antibody or antigen-binding fragment from the cell culture from (a).

[0132] Another aspect of the disclosure provides a method of producing an isolated or recombinant cell or a population of such cells according to any of the embodiments described herein.

[0133] In some embodiments, the method may include introducing a nucleic acid according to any of the embodiments described herein and / or a vector according to any of the embodiments described herein into one or more cells. In certain embodiments, the introduction may 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 for use in a medicine or pharmaceutical product.

[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. [Brief description of the drawings]

[0137] The foregoing and other objects, features, and advantages of particular embodiments of the present disclosure will be apparent from the following description and accompanying drawing figures.

[0138] [Figure 1] FIG. 1 is a graph showing normalized PSR scores of anti-CD3 antibodies plotted against antibody KD values.

[0139] [Diagram 2] FIG. 2 is a graph showing AC-SINS values ​​for anti-CD3 antibodies plotted against antibody KD values.

[0140] [Diagram 3] FIG. 3 is a graph showing normalized PSR scores for anti-CD3 antibodies plotted against antibody KD values.

[0141] [Figure 4] FIG. 4 is a graph showing AC-SINS values ​​for anti-CD3 antibodies plotted against antibody KD values. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0142] The present disclosure relates generally to anti-CD3 antibodies and antigen-binding fragments that may have one or more improved developability properties.

[0143] Some embodiments of the present disclosure may relate to anti-CD3 antibodies and antigen-binding fragments with reduced multispecificity. Some embodiments of the present disclosure may relate to anti-CD3 antibodies and antigen-binding fragments with reduced tendency for self-interaction. Some embodiments of the present disclosure may relate to anti-CD3 antibodies and antigen-binding fragments with improved resistance to the stress of low pH. Some embodiments of the present disclosure may relate to anti-CD3 antibodies and antigen-binding fragments with reduced aggregation or multimerization tendency. Some embodiments of the present disclosure may relate to anti-CD3 antibodies and antigen-binding fragments with improved binding to target cells (CD3-expressing cells). Some embodiments of the present disclosure may relate to anti-CD3 antibodies and antigen-binding fragments with increased binding preference at acidic pH. Some embodiments of the present disclosure may relate to anti-CD3 antibodies and antigen-binding fragments with improved cytokine release syndrome (CRS) risk profile, in some cases due to pH-dependent antigen-binding profile.

[0144] Potential development of anti-CD3 antibodies and antigen-binding fragments The term "developable" or "developability" refers to the extent to which one or more polypeptides in a plurality of polypeptides have desirable characteristics for manufacturing, storage, off-target binding, such as, for example, 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, pharmacokinetic profile, circulating half-life, and clearance characteristics. Such characteristics may be treated independently, as a combination of a subset of such characteristics, or collectively, as landmarks for the likelihood that the one or more polypeptides may be successfully developed as therapeutic candidates and ultimately become approved drugs. Generally, a polypeptide having desirable developability characteristics possesses 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 slow clearance rate, and the like. In contrast, polypeptides with undesirable developability properties generally possess one or more of, e.g., 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 slow clearance rate, and the like.

[0145] 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 CD3 binding domains of the present disclosure may exhibit reduced multispecificity (e.g., as assessed by interaction with a multispecificity reagent (PSR)). Such domains may be engineered from the starting domain by replacing various domain amino acid residues with residues having charged side chains. Residues may be replaced with amino acid residues having negatively charged side chains, e.g., Asp and Glu residues. Residues selected for replacement may be selected from residues that are not predicted to specifically interact with the CD3 amino acid residues targeted by the CD3 binding domain.

[0146] Methods and assays that may be employed to confirm the extent to which a polypeptide, such as, for example, the anti-CD3 antibodies and / or antigen-binding fragments described herein, possesses desirable developability properties are available in the art and include, for example, polyspecific reagent (PSR) assays (WO 2014 / 179363 and Xu et al., Protein Eng Des Sel, Vol. 26, pages 663-670 (2013)), such as SMP assays and SCP assays, 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), viscosimetry, whole cell binding, tissue microarray methodologies, ELISA assays such as the BVP ELISA assay, AC-SINS assays (Liu et al. al;MAbs,Vol.6,483-492(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),753-7601(2012); Weiqiang et al. al., J. Pharm. Sci., Vol. 101(5), pp. 1701-1720 (2012); Banks et al., J. Pharm. Sci., Vol. 101(8), pp. 2720-2732 (2012); Lie et al., J. Pharm. Sci., Vol. 94(9), pp. 1928-1948 (2005); and Payne et al., Biopolymers, Vol. 85(5), pp. 527-533 (2006)).

[0147] In some embodiments, antibodies identified as having low developability are so detected by their interaction with PSR, and are therefore referred to as "multispecific" polypeptides. Such multispecific antibodies may also be referred to as relatively "undevelopable" or relatively "non-developable."

[0148] "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 assessed, compared, and / or ranked. Such a developability profile serves as a measure of the degree of interaction of the CD3 binding agent with the 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 the polypeptide for the moiety to which it is bound. Exemplary means include, for example, 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, etc. In the case of FACS, as shown in the Examples, the degree of interaction between a polypeptide in the plurality of polypeptides and PSR may be ascertained by generating a mean fluorescence intensity (MFI) for each detected polypeptide-PSR interaction, and then ordering the MFIs in either ascending or descending order, thereby ranking the polypeptides in the plurality of polypeptides according to the relative degree of interaction between each detected polypeptide and PSR. By performing such a ranking on the ranking of the plurality of polypeptides, polypeptides possessing high developability are readily identified, as well as polypeptides possessing low developability.

[0149] 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.

[0150] In some embodiments, the anti-CD3 antibodies or antigen-binding fragments described herein may exhibit multispecificity or reduced tendency to bind multiple molecules or epitopes. In some embodiments, multispecificity may be determined based on a multispecificity reagent (PSR) score obtained by a PSR assay. In some embodiments, the PSR score may be determined as described in the Examples. In some embodiments, the anti-CD3 antibodies and / or antigen-binding fragments described herein may exhibit a PSR score of about 0.0 to about 0.45. In some embodiments, the PSR 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 PSR score of 0.0≦<0.10 is considered a "clean PSR." In some embodiments, a PSR score of 0.10≦<0.33 is considered a "low PSR." In some embodiments, a PSR score of 0.33≦<0.66 is considered a "medium PSR." In some embodiments, a PSR score of 0.66≦<1.00 is considered a "high PSR." In some embodiments, a high PSR score indicates reduced (or poor) developability. In general, the lower the PSR score, the more favorable the developability of the antibody.

[0151] In some embodiments, the anti-CD3 antibodies or antigen-binding fragments described herein may exhibit reduced hydrophobicity. In some embodiments, hydrophobicity may be determined based on retention times observed during HIC. In some 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 may exhibit HIC retention times of less than about 10.5 minutes (clean to low HIC score). In some embodiments, the anti-CD3 antibodies or antigen-binding fragments described herein may exhibit 10.5 minutes ≦ HIC retention times < 11.5 minutes (medium HIC score). A HIC retention time of 11.5 minutes ≦ may be considered a high HIC score. In general, the shorter the HIC retention time, the more favorable the developability of the antibody.

[0152] In some embodiments, the anti-CD3 antibodies or antigen-binding fragments described herein may exhibit a reduced propensity for self-interaction. In some embodiments, the propensity for self-interaction may be determined based on Δλmax values ​​observed during affinity capture self-interaction nanoparticle spectroscopy (AC-SINS). In some embodiments, AC-SINS may be performed and Δλmax values ​​obtained as described in the Examples. In some embodiments, the anti-CD3 antibodies and / or antigen-binding fragments described herein may exhibit a Δλmax of about 0.0 nm to about 15.0 nm. In some embodiments, a Δλmax of about 0.0 nm to about 10.0 nm. In some embodiments, a Δλmax of about 0.0 nm to about 7.5 nm. In some embodiments, a Δλmax of about 0.0 nm to about 5.0 nm. In some embodiments, a Δλmax of about 0.0 nm to about 3.0 nm. In some embodiments, a Δλmax of about 0.0 nm to about 2.0 nm. In some embodiments, a Δλmax of 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.

[0153] In some embodiments, the anti-CD3 antibodies or antigen-binding fragments described herein may exhibit reduced viscosity. In some embodiments, the 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 and / or antigen-binding fragments described herein may exhibit a kD of about 5 mL / g or more. In some embodiments, the kD may be about 10 mL / g or more. In some embodiments, the kD may be about 15 mL / g or more. In some embodiments, the kD may be about 20 mL / g or more. In some embodiments, the kD may be about 25 mL / g or more. In some embodiments, a Δλmax<20 mL / g may be considered to be associated with high viscosity or high opalescence. In general, the lower the viscosity, the more favorable the developability of the antibody.

[0154] In some embodiments, the anti-CD3 antibodies or antigen-binding fragments described herein may exhibit a reduced chance of heavy-light chain mispairing (including failure of pairing). In some embodiments, heavy-light chain mispairing may be determined based on the presence of a heavy chain peak (indicative of a heavy chain that was not successfully paired with a light chain) and / or a light chain peak (indicative of 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 and / or antigen-binding fragments described herein may not exhibit a heavy or light chain peak. In general, the smaller the heavy and light chain peaks, the more favorable the developability of the antibody.

[0155] 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 the protein from antibody production and, optionally, purification, that is present in its full size (e.g., IgG or Fab) without aggregation). In some embodiments, SEC may be performed as described in the Examples. In some embodiments, the anti-CD3 antibodies and / 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. In general, the higher the % monomer value, the more favorable the developability of the antibody.

[0156] 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 aggregation tendency when exposed to low pH. In some embodiments, aggregation tendency under low pH may be determined based on % monomer values ​​(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 and / or antigen-binding fragments described herein may exhibit a % monomer in SEC of about 95% or more 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 more. In some embodiments, the % monomer may be about 97% or more. In some embodiments, the % monomer may be about 98% or more. In some embodiments, the % monomer may be about 99% or more. In general, the higher the % monomer value after exposure to low pH, the higher the resistance to acidic stress, i.e., the more favorable the developability of the antibody. Without wishing to be bound by theory, improved resistance to acidic stress may result in a longer shelf life and / or improved in vivo stability (e.g., in an acidic cancer microenvironment).

[0157] In some embodiments, the anti-CD3 antibodies and / or antigen-binding fragments described herein may exhibit a Tm of about 65° C. or greater. In some embodiments, the 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 is, i.e., the more exploitable the mode.

[0158] In some embodiments, the anti-CD3 antibodies and / or antigen-binding fragments described herein may be further modified to minimize effector function, e.g., a silenced Fc.

[0159] The term "cytokine release syndrome" (or CRS) refers to a pro-inflammatory positive feedback loop between cytokines and immune cells, resulting in excessive and uncontrolled release of pro-inflammatory cytokines by cells in 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 to levels and extents 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, as well as decreased tissue perfusion. Other biological / physiological effects include organ and systemic dysfunction, including cardiac 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 the propensity for T cell stimulation to cause CRS.

[0160] In certain embodiments, the anti-CD3 antibodies and / or antigen-binding fragments described herein are degraded and / or modified to reduce the likelihood or severity of antibody-induced CRS. Non-limiting examples of modifications include silent Fc regions (e.g., removing the Fc entirely or modifying the Fc region to reduce or eliminate effector function) and / or masking (e.g., a polypeptide mask positioned to reduce or inhibit the ability of the antibody or antigen-binding fragment to specifically bind CD3).

[0161] Anti-CD3 antibodies and antigen-binding fragments "Cluster of Differentiation 3" or "CD3" generally refers to any native CD3 from any vertebrate source, including mammals such as primates (e.g., humans and non-human primates) and rodents (e.g., mice and rats), and includes, for example, the CD3ε, CD3γ, CD3α, and CD3β chains, 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 native 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 protein of 207 amino acids in length. _ 000064), which is a protein that is 182 amino acids in length. "CD3εN27" and "CD3εN13" refer to the N-terminal 27 amino acids and the N-terminal 13 amino acids of CD3, respectively, including any chemical modifications or linkages thereto.

[0162] "Anti-CD3 antibody" refers to an antibody or antigen-binding fragment having the ability to bind 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 concentration of about 100×10 -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 D In 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 2.5×10 -9 The dissociation constant (K D In some embodiments, the anti-CD3 antibody binds to CD3 at about 1×10 -10 The dissociation constant (K D ) binds to CD3. In some embodiments, K D is measured by surface plasmon resonance, e.g., using a BIACORE® system, biolayer interferometry, e.g., using an ORTEBIO Octet® HTX instrument (Pall Life Sciences), or solution-affinity ELISA. D is measured using an scFv fragment of an anti-CD3 antibody. In some embodiments, the monovalent K D In some embodiments, the anti-CD3 antibody binds to an epitope of CD3 that is conserved among CD3 from various species, such as the cross-reactive species of human and cynomolgus monkey.

[0163] The term "antibody" is used herein in the broadest sense and encompasses a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), whole antibodies, and antibody fragments, preferably those fragments thereof that exhibit the desired antigen-binding activity (i.e., antigen-binding fragments).

[0164] 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 the generation of the monoclonal antibody preparation). Such variants are generally present in small amounts. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen.

[0165] For multispecific antibodies (e.g., bispecific, trispecific, tetraspecific, etc.), such antibodies contain at least two different antigen-binding regions that recognize and specifically bind at least two different antigens. For bispecific antibodies, such antibodies contain two different antigen-binding regions that recognize and specifically bind at least two different antigens or epitopes. A "bispecific antibody" is a type of multispecific antibody that contains two different antigen-binding regions that recognize and specifically bind at least two different antigens or at least two epitopes. The at least two epitopes may or may not be within the same antigen. Bispecific antibodies can, for example, target two different surface receptors on the same or different cells (e.g., immune cells and cancer cells).

[0166] "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, polypeptide, or single molecule.

[0167] The term "epitope" refers to an antigenic determinant that interacts with a specific antigen-binding site in the variable region of an antibody molecule, known as the paratope. An 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 a site on an antigen to which B cells and / or T cells react. It also refers to the region of an antigen that is bound by an antibody. Epitopes may be structurally or functionally defined. Functional epitopes are generally a subset of structural epitopes, and have residues that directly contribute to the affinity of the interaction. Epitopes may also be conformational; that is, they may be 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.

[0168] Terms such as "intact antibody" and "whole antibody" are used interchangeably herein to refer to an antibody having a structure substantially similar to that of a natural antibody. In some cases, an antibody comprises a heavy (H) and a light (L) chain interconnected by disulfide bonds. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, some of which may be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to the 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 immunoglobulin heavy chains and two immunoglobulin light 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 contain multiple units each comprising two pairs of heavy and light chains interconnected by disulfide bonds. For example, intact IgA contains two units and intact IgM contains five units. Thus, in other cases, an antibody according to the present disclosure may instead contain multiple (e.g., two, three, four, five, etc.) units each comprising two pairs of heavy and light chains interconnected by disulfide bonds, or antigen-binding fragments thereof.

[0169] Each heavy chain is composed of a heavy chain variable domain (VH) and a heavy chain constant region (CH), which is typically composed of the domains CH1, CH2 and CH3. Each light chain is composed of a light chain variable domain (VL) and a light chain constant domain (CL). VH and VL can be further subdivided into regions of hypervariability called complementarity determining regions (CDRs) between which are located more conserved regions called framework regions (FRs). Each VH and VL polypeptide is composed of three CDRs and four FRs arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The CDRs in the heavy chain are designated "CDRH1", "CDRH2" and "CDRH3", respectively, and the CDRs in the light chain are designated "CDRL1", "CDRL2" and "CDRL3". In certain embodiments of the present disclosure, the FRs of the antibody (or antigen-binding fragment) may be identical to human germline sequences or may be naturally or artificially modified. A consensus sequence of amino acids may be defined based on a side-by-side comparative analysis of two or more CDRs.

[0170] 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, a numbering scheme, method, and definition based on a numbering scheme such as EU numbering (as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)), IMGT numbering, Kabat numbering, Chothia numbering, Martin numbering, Gelfand numbering, or Honneger numbering), or structurally (see, for example, NCBI online tool, IgBlast; Dondelinger et al., Front Immunol. 2018 Oct 16;9:2278).

[0171] 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 in a broad sense 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., the CH1 domain may include a portion of the hinge region) and / or amino acid modifications.

[0172] An exemplary CH1 domain of human IgG1 may comprise the amino acid sequence of SEQ ID NO: 91 or 92, 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.

[0173] "Fc region" is the C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region, including native sequence Fc regions and variant Fc regions. The Fc region of a human IgG heavy chain 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 is according to the EU numbering system, also called the EU index. The 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.

[0174] 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.

[0175] According to IMGT, the CLκ domain is 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, and according to IMGT, the CLλ domain is at amino acid positions 107-215 (EU numbering). An exemplary CL λ domain of human IgG may comprise the amino acid sequence of SEQ ID NO: 82,

[0176] The terms "CLκ domain", "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.

[0177] 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 entirety. Again, these reference sequences are intended to be exemplary, as the human IgG1, IgG2, IgG3, and IgG4 sequences are intended by the applicant to include all naturally occurring human IgG1, IgG2, IgG3, and IgG4 allotypes.

[0178] "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 engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex, including antibody fragments. Exemplary antigen-binding fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2, diabodies, linear antibodies, single-chain antibody molecules (e.g., single-chain variable fragments ("scFv"), half antibodies, nanobodies, or VHH, or only VH or VL domains), and multispecific antibodies formed from antibody fragments. In some embodiments, the antigen-binding fragment of an anti-CD3 antibody described herein is a 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 heavy chain and one light chain set of the referenced antibody.

[0179] "Antigen-binding region" refers to the portion of an antibody or antigen-binding fragment having specificity for an antigen.

[0180] Multispecific antibodies and antibody fragments Multispecific antibodies comprising at least one anti-CD3 antibody and / or antigen-binding fragment disclosed herein can be produced by a variety of techniques, including, but not limited to, recombinant co-expression of two immunoglobulin heavy chain-light chains with different specificities (see, e.g., Milstein and Cuello, Nature 305:537 (1983)), WO 93 / 08829, and Traunecker et al., EMBO J. 10:3655 (1991)), "knobs-in-holes" engineering (see, e.g., U.S. Pat. No. 5,731,168), immunoglobulin crossover (also known as Fab domain swapping or CrossMab format) technology (see, e.g., WO 2009 / 080253; Schaefer et al., J. Immunol. 1999, 11:1112 (1999)), and / or other techniques. al., Proc. Natl. Acad. Sci. USA, 108:11187-11192 (2011)), engineering electrostatic steering effects for the creation of antibody Fc heterodimeric molecules (WO 2009 / 089004 A1), cross-linking of two or more antibodies or fragments (see, e.g., U.S. Pat. No. 4,676,980, and Brennan et al., Science, 229:81 (1985)), leucine zippers (see, e.g., Kostelny et al., J. Immunol, 148(5):1547-1553 (1992)), "diabody" technology (see, e.g., Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)), single-chain Fv (scFv) dimers (see, e.g., Gruber et al., J. Immunol., 1999, 2002, 2003, 2004, 2005, 2006, 2007, 2009, 2010, 2011, 2012, 2013, 2014, 2015, 2016, 2017, 2018, 2019, 2020, 2030, 2020, 2030, 2030, 2040, 2040, 2050, 2060, 2070, 2080, 2090, 2100, 2110, 2120, 2130, 2140, 2150, 2160, 2170, 218 al., J. Immunol, 152:5368 (1994)), as well as trispecific antibodies, as described, for example, in Tutt et al. J. Immunol 147:60 (1991).

[0181] In the case of the multispecific antibodies and antibody fragments described herein, a variety of multispecific antibody formats can be used.Non-limiting examples of multispecific and bispecific formats include Fab-Fc-scFv (bottle opener) (XENCOR), Mab-scFv (XENCOR), Mab-Fv (XENCOR), dual scFv (XENCOR), central Fv (XENCOR), central scFv (XENCOR), one-arm central scFv (XENCOR), Fab-Fab (XENCOR), Fab-Fv (XENCOR), mAb-Fv (XENCOR), mAb-Fab (XENCOR), DART (MACROGENICS), BiTE (AMGEN / MICROMET), KiTE, common light chain-IgG (Genentech), TandAb (SFIMED), Cross-Mab (ROCHE), SEED (EMD Serono), BEAT (Glenmark), TrioMab (Trion Pharma / Fresenius Biotech), DuetMab (Medimune), and the like, which are, for example, (WO2021067404; WO2022150787; WO2022150785; WO95 / 09917; WO2008 / 119566; WO2008 / 119567; WO2011 / 121110; WO2010 / 037835; WO2007 / 042261; WO2007 / 110205; WO 2011 / 121110;WO2012 / 055961;WO2012 / 16067;WO2016 / 086189;WO2016 / 182751;WO2015 / 006749;WO2014 / 049 003;WO2013 / 177101;WO2015 / 128509;US7,951,917;US2009 / 0252729;US2014 / 0348839;US7,183,076;Mazor et al., Mabs, Vol. 7, pages 377-389 (2015); Muda et al., Protein Engineering, Design, & Selection, Vol. 24, pages 447-454 (2011); and Del Bano et al., Antibodies, Vol. 5, pages 1-23 (2016).In some embodiments, the anti-CD3 scFv fragments described herein comprise one or more variable domains of a multispecific (eg, bispecific) antibody.

[0182] In some embodiments, a multispecific antibody or antibody fragment may comprise one or more engineered variant constant domains that promote efficient polypeptide dimer formation (e.g., a first heavy chain and a second heavy chain different from the first heavy chain) for bispecific antibody formation.

[0183] In certain embodiments, the 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.

[0184] In certain embodiments, the multispecific antibody or antibody fragment may comprise at least one pair of CH1 domain and CL domain that preferentially pair with each other. In the preferential pairing pair of CH1 domain and 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 preferential pairing pairs of CH1 domain and CL domain may be selected from pairs described in WO2022150787.

[0185] In certain embodiments, the multispecific antibody or antibody fragment may comprise at least one pair of a first CH3 domain and a second CH3 domain different from the first CH3, which preferentially pair with each other (i.e., form a dimer). In such preferentially paired pairs, the first CH3 domain prefers to pair with the second CH3 domain rather than with another first CH3 domain, and / or the second CH3 domain prefers to pair with the first CH3 domain rather than with another second CH3 domain. One or both of the CH3 domains may be variant domains. In certain embodiments, such preferentially paired pairs of first and second CH3 domains may be selected from pairs described in WO2022150785. target

[0186] In certain embodiments, the anti-CD3 antibodies and / or antigen-binding fragments described herein are contained in multispecific antibodies, particularly bispecific antibodies, having binding specificity for a second antigen. Such a second antigen may be a target entirely different from the first target, or may be a different epitope present on the same target. In some embodiments, the binding specificity is for two different epitopes of CD3 (e.g., CD3ε or CD3γ). In other embodiments, one of the binding specificities is for 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).

[0187] Non-limiting examples of second antigens for bispecific antibodies comprising the anti-CD3 antibodies and / or antigen-binding fragments described herein include 17-IA, 4-1BB, 4Dc, 6-keto-PGFla, 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, activin ALK-2 ... ALK-4, activin RIIA, activin RUB, ADAM, ADAM10, ADAM12, ADAM15, ADAM17 / TACE, ADAM8, ADAM9, ADAMTS, ADAMTS4, ADAMTS5, addressin, aFGF, ALCAM, ALK, ALK-1, ALK-7, alpha-l-antitrypsin, alpha-V / beta-1 antagonist, ANG, Ang, APAF-1, APE, APJ, APP, APRIL, A R, ARC, ART, Artemin, Anti-Id, ASPARTIC, Atrial natriuretic factor, av / b3 integrin, Axl, b2M, B7-1, B7-2, B7-H, B-lymphocyte stimulatory factor (BlyS), BACE, BACE-1, Bad, BAFF, BAFF-R, Bag-1, BAK, Bax, BCA-1, BCAM, Bel, BCMA, BDNF, b-ECGF, bFGF, BID, Bik, BIM, BLC, BL-CAM, BLK, BM P, BMP-2, BMP-2a, BMP-3 osteogenin, BMP-4, BMP-2b, BMP-5, BMP-6, Vgr-1, BMP-7 (OP-1), BMP-8 (BMP-8a, OP-2), BMPR, BMPR-IA (ALK-3), BMPR-IB (ALK-6), BRK-2, RPK-1, BMPR-II (BRK-3), BMP, b-NGF, BOK, bombesin, bone-derived neurotrophic factor, BPDE, BPDE-DNA, BTC, supplement C3, C3a, C4, C5, C5a, CIO, CA125, CAD-8, calcitonin, cAMP, carcinoembryonic antigen (CEA), carcinoma-associated antigen, cathepsin A, cathepsin B, cathepsin C / DPPI, cathepsin D, cathepsin E, cathepsin H, cathepsin L, cathepsin O, cathepsin S, cathepsin V, cathepsin X / Z / P, CBL, CCI, CCK2, CCL, CCLl, CCLll, 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, CDlla, CDllb, CDllc, CD13, CD14, CD15, CD16, CD18, CD19, CD20, CD21, CD22, CD23, CD25, CD27L, CD28, CD29, CD30, CD30L, CD32, CD33 (p67 protein), CD34, CD38, CD40, CD40L, CD44, CD45, CD46, CD49a, CD52, CD54, CD55, CD56, CD61, CD64, CD66e, CD74, CD80 (B7-1), CD89, CD95, CD123, CD137, CD138, CD140a, CD146, CD147, CD148, CD152, CD164, CEACAM5, CFTR, cGMP, CINC, Clostridium botulinum toxin, Clostridium perfringens toxin, CKb8-l, CLC, CMV, CMV UL, CNTF, CNTN-1, COX, C-Ret, CRG-2, CT-1, CTACK, CTGF, CTLA-4, CX3CL1, CX3CR1, CXCL, CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCR, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, Cytokeratin tumor-related antigen, DAN, DCC, DcR3, DC-SIGN, Decay accelerating factor, Des(l-3)-IGF-I (Brain IGF-1), Dhh, Digoxin, DNAM-1, Dnase, Dpp, DPPIV / CD26, Dtk, ECAD, EDA, EDA-A1, EDA-A2, EDAR, EGF, EGFR (ErbB-1), EMA, EMMPRIN, EN A, Endothelin receptor, Enkephalinase, eNOS, Eot,Eotaxin l, EpCAM, ephrin B2 / EphB4, EPO, ERCC, E-selectin, ET-1, factor Ila, factor VII, factor VIIIc, factor IX, fibroblast activation protein (FAP), Fas, FcRl, FEN-1, ferritin, FGF, FGF-19, FGF-2, FGF3, FGF-8, FGFR, FGFR-3, fibrin, FL, FLIP, Flt-3, Flt-4, follicle-stimulating hormone, fractalkine, FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, FZD10, G250, Gas6, GCP-2, GCSF, GD2, GD3, GDF, GDF-1, GDF-3 (Vgr-2), GDF-5 (BMP-14, CDMP-1), GDF-6 (BMP-13, CDMP-2), GDF-7 (BMP-12, CDMP-3), GDF-8 (myostatin), GDF-9, GDF-15 (MIC-1), GDNF, GFAP, GFRa-1, GFR-alpha1, GFR-alpha2, GFR-alpha3, GITR, glucagon, Glut 4, glycoprotein Ilb / IIIa (GP Ilb / IIIa), GM-CSF, gpl30, gp72, GRO, growth hormone releasing factor, hapten (NP-cap or NIP-cap), HB-EGF, HCC, HCMV gB envelope glycoprotein, HCMV gH envelope glycoprotein, HCMV UL, hematopoietic growth factor (HGF), Hep B gpl20, heparanase, Her2, Her2 / neu (ErbB-2), Her3 (ErbB-3), Her4 (ErbB-4), herpes simplex virus (HSV) gB glycoprotein, HSV gD glycoprotein, HGFA, high molecular weight melanoma-associated antigen (HMW-MAA), HIV gpl20, HIV IIIB gp 120 V3 loop, HLA, HLA-DR, HM1.24, HMFG PEM, HRG, Hrk, human cardiac myosin, human cytomegalovirus (HCMV), human growth hormone (HGH), HVEM, 1-309, IAP, ICAM, ICAM-1, ICAM-3, ICE, ICOS, IFNg, Ig, IgA receptor, IgE, IGF, IGF binding protein, IGF-1R, IGFBP, IGF-I, IGF-II, IL, IL-1, IL-1R, IL-2, IL-2R, IL-4, IL-4R,IL-5, IL-5R, IL-6, IL-6R, IL-8, IL-9, IL-10, IL-12, IL-13, IL-15, IL-18, IL-18R, IL-23, interferon (INF)-alpha, INF-beta, INF-gamma, inhibin, iNOS, insulin A chain, insulin B chain, insulin-like growth factor 1, integrin alpha 2, integrin alpha 3, integrin alpha 4, integrin alpha 4 / beta l, integrin, alpha 4 / beta 7, integrin alpha 5 (alpha V), integrin alpha 5 / beta l, integrin alpha 5 / beta 3, integrin alpha 6, integrin beta l, integrin beta 2, interferon gamma, IP-10, 1-TAC, JE, kallikrein 2, kallikrein 5, kallikrein 6, , kallikrein 11, kallikrein 12, kallikrein 14, kallikrein 15, kallikrein LI, kallikrein L2, kallikrein L3, kallikrein L4, KC, KDR, keratinocyte growth factor (KGF), laminin 5, LAMP, LAP, LAP (TGF-1), latent TGF-1, latent TGF-1 bpl, LBP, LDGF, LECT2, Lefty, Lewis-Y antigen, Lewis-Y related antigen, LFA-1, LFA-3, Lfo, LIF, LIGHT, lipoprotein, LIX, LKN, Lptn, L-selectin, LT-a, LT-b, LTB4, LTBP-1, pulmonary surfactant, luteinizing hormone, lymphotoxin beta receptor, Mac-1, MAdCAM, MAG, MAP2, MARC, MCAM, MCAM, MCK-2, MCP, M-CSF, MDC, Mer, metalloplast protease, MGDF receptor, MGMT, MHC (HLA-DR), MIF, MIG, MIP, MIP-1-alpha, MK, MMAC1, MMP, MMP-1, MMP-10, MMP-11, MMP-12, MMP-13, MMP-14, MMP-15, MMP-2, MMP-24, MMP-3, MMP-7, MMP-8, MMP-9, MPIF, Mpo, MSK, MSP, mucin (Mucl), MUC18, Mullerian-inhibitin substance, Mug, MuSK, NAIP, NAP, NCAD, N-cadherin, NCA90, NCAM, NCAM, neprilysin,Neurotrophin-3, -4, or -6, Neurturin, Neuron Growth Factor (NGF), NGFR, NGF-beta, nNOS, NO, NOS, Npn, NRG-3, NT, NTN, OB, OGG1, OPG, OPN, OSM, OX40L, OX40R, pl50, p95, PADPr, Parathyroid Hormone, PARC, PARP, PBR, PBSF, PCAD, P-cadherin, PCNA, PDGF, PDK-1, PECAM, PEM, P F4, PGE, PGF, PGI2, PGJ2, PIN, PLA2, placental alkaline phosphatase (PLAP), P1GF, PLP, PP14, proinsulin, prorelaxin, protein C, PS, PSA, PSCA, prostate-specific membrane antigen (PSMA), PTEN, PTHrp, Ptk, PTN, R51, RANK, RANKL, RANTES, RANTES, relaxin A-chain, relaxin B-chain, renin, respiratory syncytial virus (RSV)F, RSV Fgp, Ret, rheumatoid factor, RLIP76, RPA2, RSK, S100, SCF / KL, SDF-1, serine, serum albumin, sFRP-3, Shh, SIGIRR, SK-1, SLAM, SLPI, SMAC, SMDF, SMOH, SOD, SPARC, Stat, STEAP, STEAP-II, TACE, TACI, TAG-72 (tumor-associated glycoprotein-72), TARC, TCA-3, T cell receptor (e.g., T cell receptor alpha / beta), TdT, TECK, TEM1, TEM5, TEM7, TEM8, TERT, testicular PLAP-like alkaline phosphatase, T fR, TGF, TGF-alpha, TGF-beta, TGF-beta Pan specific, TGF-beta RI (ALK-5), TGF-beta RII, TGF-beta Rllb, TGF-beta RIII, TGF-beta l, 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 Rl Apo-2, DR4), TNFRSFIOB (TRAIL R2 DR5, KILLER, TRICK-2A,TRICK-B)、TNFRSF10C(TRAIL R3 DcRl、LIT、TRID)、TNFRSF10D(TRAIL R4 DcR2、TRUNDD)、TNFRSF11A(RANK ODF R、TRANCE R)、TNFRSFllB(OPG OCIF、TR1)、 TNFRSF12(TWEAK R FN14)、TNFRSF13B(TACI)、TNFRSF13C(BAFF R)、TNFRSF14(WHEM ATAR、HveA、LIGHT R、TR2)、TNFRSF16(NGFR p75NTR), TNFRSF17(BCMA), TNFRSF18(GITR AITR), TNFRSF19(TROY CROWN), TNFRSF19L(RELT), TNFRSFIA(TNF RI CD120a, p55-60), TNFRSFIB(TNF RII). CD120b, p75-80, TNFRSF26(TNFRH3), TNFRSF3(LTbR TNF RIII, TNFC R), TNFRSF4(OX40 ACT35, TXGP1 R), TNFRSF5(CD40 p50), TNFRSF6(Fas). Apo-1, APT1, CD95, TNFRSF6B(DcR3 M68, TR6), TNFRSF7(CD27), TNFRSF8(CD30), TNFRSF9(4-lBB CD137, ILA), TNFRSF21(DR6), TNFRSF22(DcTRAIL R2). TNFRH2, TNFRST23(DcTRAIL Rl TNFRH1), TNFRSF25(DR3 Apo-3, LARD, TR-3, TRAMP, WSL-1), TNFSF10(TRAIL Apo-2 polymer TL2) TNFSF11(TRANCE / RANK dimer ODF) TNFSF12(TWEAK). Apo-3リンド, DR3リンド, TNFSF13(APRIL TALL2), TNFSF13B(BAFF LIGHT, TALL1, THANK, TNFSF20), TNFSF14(LIGHT HVEM LINK, LTg, TNFSF15(TL1A / VEGI), TNFSF18(GITR LINK, AITR TL6, TNFSFIA (TNF-a, DIF, TNFSF2), TNFSF1B (TNF-b). LTa、TNFSF1)、TNFSF3(LTbTNFC, p33), TNFSF4 (OX40 ligand gp34, TXGP1), TNFSF5 (CD40 ligand CD154, gp39, HIGM1, IMD3, TRAP), TNFSF6 (Fas ligand Apo-1 ligand, APT1 ligand), TNFSF7 (CD27 ligand CD70), TNFSF8 (CD30 ligand CD153), TNFSF9 (4-lBB 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 CA125, Lewis Y-related carbohydrate expressed tumor-associated antigen, TWEAK, TXB2, Ung, uPAR, uPAR-1, urokinase, VCAM, VCAM-1, VECAD, VE-cadherin, VE-cadherin-2, VEFGR-1 (flt-1), VEGF, VEGFR, VEGFR-3 (flt-4), VEGI, VIM, viral antigen, VLA, VLA-1, VLA-4, VNR integrin, von Willebrand factor, WIF- and targets selected from the group consisting of: 1, WNT1, WNT2, WNT2B / 13, WNT3, WNT3A, WNT4, WNT5A, WNT5B, WNT6, WNT7A, WNT7B, WNT8A, WNT8B, WNT9A, WNT9A, WNT9B, WNT10A, WNT10B, WNT11, WNT16, XCL1, XCL2, XCR1, XCR1, XEDAR, XIAP, XPD, CTLA4 (cytotoxic T lymphocyte antigen-4), PD1 (programmed cell death protein 1), PD-L1 (programmed cell death ligand 1), LAG-3 (lymphocyte activation gene-3), TIM-3 (T cell immunoglobulin and mucin protein-3), receptors for hormones, and growth factors.

[0188] Potential for developing multispecific antibodies and antibody fragments As described throughout, the anti-CD3 antibodies and / or antigen-binding fragments thereof provided herein have one or more favorable developability characteristics and are therefore relatively developable. Thus, to effectively and efficiently develop multispecific antibodies or antibody fragments, including anti-CD3 antibodies or antigen-binding fragments according to the present disclosure, one or more candidate multispecific antibodies or antibody fragments are designed (e.g., including multispecific antibody formats and antibody sequences to provide a second specificity) and one or more developability parameters (e.g., multispecificity, hydrophobicity, self-interaction, viscosity, stability, shelf life, tendency to heavy-light chain mispairing, tendency to aggregate, and / or resistance to acidic stress) and / or any other properties (e.g., antigen binding, target cell binding, etc.) can be tested. Assays that may be used to evaluate developability and / or other antibody properties include, but are not limited to, one or more of: PSR assays, CIC, SIC, HIC, SEC, DLS spectroscopy, photon correlation spectroscopy, quasi-elastic light scattering, CD, viscometry, whole cell binding, tissue microarray methodology, ELISA assays such as BVP ELISA assays, AC-SINS assays, Tm assays; differential scanning calorimetry or DSF, and the like.

[0189] Anti-CD3 antibody variable sequence In some embodiments, the anti-CD3 antibodies and antigen-binding fragments of the present disclosure may comprise one or more CDR sequences contained in the variable domain amino acid sequence of any one of those associated with antibody numbers A001-A005 and V002-V0019 shown in Tables 1A and 1B. In some embodiments, the anti-CD3 antibodies and antigen-binding fragments of the present disclosure may comprise one or more CDR sequences encoded in a nucleic acid sequence encoding the variable domain of any one of those associated with antibody numbers A001-A005 and V002-V0019 shown in Tables 1C and 1D. In some embodiments, the anti-CD3 antibodies and antigen-binding fragments of the present disclosure may comprise one or more CDR sequences of those associated with antibody numbers A001-A005 and V002-V0019 shown in Tables 2A and 2B.

[0190] In certain embodiments, the anti-CD3 antibodies and antigen-binding fragments of the disclosure may comprise CDRH1, CDRH2, and CDRH3 sequences individually selected from the CDRH1, CDRH2, and CDRH3 sequences contained in any VH sequence associated with antibody Nos. A001-A005 and V002-V0019 shown in Table 1A, encoded by a nucleic acid sequence encoding any VH associated with antibody Nos. A001-A005 and V002-V0019 shown in Table 1C, or associated with any of antibody Nos. A001-A005 and V002-V0019 shown in Table 2A.

[0191] In certain embodiments, the anti-CD3 antibodies and antigen-binding fragments of the present disclosure may comprise a set of CDRH1, CDRH2, and CDRH3 sequences contained in any one of the VH sequences associated with antibody Nos. A001-A005 and V002-V0019 shown in Table 1A, encoded by any one of the nucleic acid sequences encoding the VHs associated with antibody Nos. A001-A005 and V002-V0019 shown in Table 1C, or associated with antibody Nos. A001-A005 and V002-V0019 shown in Table 2A.

[0192] In certain embodiments, the anti-CD3 antibodies and antigen-binding fragments of the present disclosure may comprise CDRL1, CDRL2, and CDRL3 sequences individually selected from those contained in any VL sequence associated with antibody Nos. A001-A005 and V002-V0019 shown in Table 1B, encoded by a nucleic acid sequence encoding any VL associated with antibody Nos. A001-A005 and V002-V0019 shown in Table 1D, or associated with any of antibody Nos. A001-A005 and V002-V0019 shown in Table 2B.

[0193] In certain embodiments, the anti-CD3 antibodies and antigen-binding fragments of the present disclosure may comprise a set of CDRL1, CDRL2, and CDRL3 sequences contained in any one of the VL sequences associated with antibody Nos. A001-A005 and V002-V0019 shown in Table 1B, encoded by any one of the nucleic acid sequences encoding the VLs associated with antibody Nos. A001-A005 and V002-V0019 shown in Table 1D, or associated with antibody Nos. A001-A005 and V002-V0019 shown in Table 2B.

[0194] In certain embodiments, the anti-CD3 antibodies and antigen-binding fragments of the present disclosure may comprise a set of CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 sequences contained in any of the VH and VL sequences associated with antibody Nos. A001-A005 and V002-V0019 shown in Tables 1A and 1B, encoded by any one of the VH-encoding nucleic acid sequences and VL-encoding nucleic acid sequences associated with antibody Nos. A001-A005 and V002-V0019 shown in Tables 1C and 1D, or associated with antibody Nos. A001-A005 and V002-V0019 shown in Tables 2A and 2B.

[0195] In some embodiments, the anti-CD3 antibodies and antigen-binding fragments of the present disclosure may comprise one or more FR amino acid sequences contained in any known germline-encoded variable domain sequence (e.g., a mammalian germline sequence, e.g., a mouse, human, or non-human germline sequence) or a variant thereof. Such germline sequences may include human VH1-03 and / or VK4-01 germline sequences.

[0196] In some embodiments, the anti-CD3 antibodies and antigen-binding fragments of the present disclosure may include one or more of the FR sequences contained in the variable domain amino acid sequence of any one of those associated with antibody numbers A001-A005 and V002-V0019 shown in Tables 1A and 1B. In some embodiments, the anti-CD3 antibodies and antigen-binding fragments of the present disclosure may include one or more FR sequences encoded by a nucleic acid sequence encoding the variable domain of any one of those associated with antibody numbers A001-A005 and V002-V0019 shown in Tables 1C and 1D. In some embodiments, the anti-CD3 antibodies and antigen-binding fragments of the present disclosure may include one or more FR sequences of those associated with antibody numbers A001-A005 and V002-V0019 shown in Tables 3A and 3B.

[0197] In certain embodiments, the anti-CD3 antibodies and antigen-binding fragments of the present disclosure may comprise one or more of the FRH1, FRH2, FRH3, and FRH4 sequences individually selected from the FRH1, FRH2, FRH3, and FRH4 sequences contained in any VH sequence associated with antibody Nos. A001-A005 and V002-V0019 shown in Table 1A, encoded by a nucleic acid sequence encoding any VH associated with antibody Nos. A001-A005 and V002-V0019 shown in Table 1C, or associated with any of antibody Nos. A001-A005 and V002-V0019 shown in Table 3A.

[0198] In certain embodiments, the anti-CD3 antibodies and antigen-binding fragments of the present disclosure may comprise a set of FRH1, FRH2, FRH3, and FRH4 sequences contained in any one of the VH sequences associated with antibody Nos. A001-A005 and V002-V0019 shown in Table 1A, encoded by any one of the nucleic acid sequences encoding the VHs associated with antibody Nos. A001-A005 and V002-V0019 shown in Table 1C, or associated with antibody Nos. A001-A005 and V002-V0019 shown in Table 3A.

[0199] In certain embodiments, the anti-CD3 antibodies and antigen-binding fragments of the present disclosure may comprise one or more of the FRL1, FRL2, FRL3, and FRL4 sequences individually selected from the FRL1, FRL2, FRL3, and FRL4 sequences contained in any VL sequence associated with antibody Nos. A001-A005 and V002-V0019 shown in Table 1B, encoded by a nucleic acid sequence encoding any VL associated with antibody Nos. A001-A005 and V002-V0019 shown in Table 1D, or associated with any of antibody Nos. A001-A005 and V002-V0019 shown in Table 3B.

[0200] In certain embodiments, the anti-CD3 antibodies and antigen-binding fragments of the present disclosure may comprise a set of FRL1, FRL2, FRL3, and FRL4 sequences contained in any one of the VL sequences associated with antibody Nos. A001-A005 and V002-V0019 shown in Table 1B, encoded by any one of the nucleic acid sequences encoding the VLs associated with antibody Nos. A001-A005 and V002-V0019 shown in Table 1D, or associated with antibody Nos. A001-A005 and V002-V0019 shown in Table 3B.

[0201] In certain embodiments, the anti-CD3 antibodies and antigen-binding fragments of the present disclosure may comprise a set of FRH1, FRH2, FRH3, FRH4, FRL1, FRL2, FRL3, and FRL4 sequences contained in any of the VH and VL sequences associated with antibody numbers A001-A005 and V002-V0019 shown in Tables 1A and 1B, encoded by any one of the VH-encoding nucleic acid sequences and VL-encoding nucleic acid sequences associated with antibody numbers A001-A005 and V002-V0019 shown in Tables 1C and 1D, or associated with antibody numbers A001-A005 and V002-V0019 shown in Tables 2A and 2B.

[0202] In certain embodiments, the anti-CD3 antibodies and antigen-binding fragments of the disclosure may comprise a set of FRH1, CDRH1, FRH2, CDRH2, FRH3, CDRH3, FRH4, FRL1, CDRL1, FRL2, CDRL2, FRL3, CDRL3, and FRL4 sequences contained in a combination of VH and VL sequences associated with any one of antibody numbers A001-A005 and V002-V0019 shown in Tables 1A and 1B, encoded by a combination of VH- and VL-encoding nucleic acid sequences associated with any one of antibody numbers A001-A005 and V002-V0019 shown in Tables 1C and 1D, or associated with any one of antibody numbers V002-V0019 shown in Tables 2A, 2B, 3A, and 3B.

[0203] In certain embodiments, the anti-CD3 antibodies and antigen-binding fragments of the present disclosure may comprise a VH polypeptide sequence associated with any one of antibody numbers A001-A005 and V002-V0019 shown in Table 1A, or a 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 thereto. In certain embodiments, the anti-CD3 antibodies and antigen-binding fragments of the present disclosure may comprise a VL polypeptide sequence associated with any one of antibody numbers A001-A005 and V002-V0019 shown in Table 1B, or a 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 thereto. In certain embodiments, the anti-CD3 antibodies and antigen-binding fragments of the present disclosure may comprise a set of VH and VL sequences associated with any one of antibody numbers V002-V0019 shown in Tables 1A and 1B.

[0204] In certain embodiments, the present disclosure provides nucleic acids encoding the anti-CD3 antibodies and antigen-binding fragments of the present disclosure. The nucleic acids may encode a VH polypeptide sequence associated with any one of antibody numbers A001-A005 and V002-V0019 shown in Table 1A, or a 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 thereto. Such nucleic acids may include a VH nucleic acid sequence associated with any one of antibody numbers A001-A005 and V002-V0019 shown in Table 1C, or a 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 thereto, or an mRNA version of any of the foregoing. In some embodiments, the nucleic acids of the disclosure include variants listed in Table 1C, where such variants include alternative codons (e.g., codon-optimized variants). The nucleic acids may encode a VL polypeptide sequence associated with any one of antibody numbers A001-A005 and V002-V0019 shown in Table 1B, or a 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 thereto. Such nucleic acids can comprise a VL nucleic acid sequence associated with any one of antibody numbers A001-A005 and V002-V0019 shown in Table ID, or a 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 thereto, or an mRNA version of any of the foregoing. In some embodiments, the nucleic acids of the disclosure include variants listed in Table ID, where such variants include alternative codons (e.g., codon-optimized variants).

[0205] Modifications to the anti-CD3 antibody sequence The present disclosure also contemplates modifications of anti-CD3 antibodies, including one or more amino acid substitutions, insertions and / or deletions in the FR and / or CDR regions of the heavy and light chain variable domains, as compared to any of the anti-CD3 antibody sequences disclosed herein, including, but not limited to, antibody numbers V002-V0019. Once obtained, such derived antibodies and / or antigen-binding fragments may be validated for one or more desirable properties, such as, for example, improved binding specificity, increased binding affinity, improved developability, etc.

[0206] In some embodiments, the VH and / or VL of an anti-CD3 antibody and / or antigen-binding fragment according to the present disclosure may have at least about 100%, at least about 99%, at least about 98%, at least about 97%, at least about 96%, at least about 95%, at least about 94%, at least about 93%, at least about 92%, at least about 91%, at least about 90%, at least about 89%, at least about 88%, at least about 87%, at least about 86%, at least about 85%, at least about 84%, at least about 83%, at least about 82%, at least about 80%, at least about 75%, at least about 70%, at least about 65%, at least about 60%, at least about 55%, or at least about 50% amino acid sequence identity to the VH and / or VL, respectively, of any one of antibody numbers V002-V19 disclosed in Tables 1A and / or 1B. In some embodiments, the percent identity is measured by any known algorithm for sequence identity, such as, for example, FASTA, Basic Local Alignment Search Tool (BLAST®) or GAP.

[0207] In some embodiments, residue positions that are not identical differ by conservative amino acid substitutions. A "conservative amino acid substitution" is one in which an amino acid residue is replaced by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). 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 with similar chemical properties include: 1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine, 2) aliphatic-hydroxyl side chains: serine and threonine, 3) amide-containing side chains: asparagine and glutamine, 4) aromatic side chains: phenylalanine, tyrosine, and tryptophan, 5) basic side chains: lysine, arginine, and histidine, 6) acidic side chains: aspartic acid and glutamic acid, and 7) sulfur-containing side chains: cysteine ​​and methionine. In some embodiments, the conservative amino acid substitution groups are valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, 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.

[0208] Substitution of one or more CDR residues or omission of one or more CDRs are also possible. In the scientific literature, there are reports of 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 areas between an antibody and its antigen based on published crystal structures and concluded that only about one-fifth to one-third of the CDR residues actually contact their associated antigen. Padlan also found that in many antibodies, one or two CDRs do not have 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 based on previous studies, by molecular modeling and / or empirically, from regions of the Kabat CDRs that are 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.

[0209] 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) to determine whether antibody-antigen interaction is affected. Further substitutions may be introduced at amino acid positions that show functional sensitivity to the initial substitution. Alternatively, or in addition, a crystal structure of an antigen-antibody complex identifies contact points between the antibody and antigen. Such contact and adjacent residues may be targeted or excluded from substitution candidates. Variants may be screened to determine whether they contain the desired properties.

[0210] 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 one or more 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 of the antibody N-terminus or C-terminus to an enzyme (e.g., an enzyme for ADEPT) or a polypeptide that extends the serum half-life of the antibody.

[0211] As described throughout, the anti-CD3 antibodies and / or antigen-binding fragments provided herein have favorable developability and are therefore relatively developable. Thus, when the sequence (variable and / or constant sequence) of the anti-CD3 antibody is modified, the developability profile, including one or more developability parameters (e.g., polyspecificity, hydrophobicity, self-interaction, viscosity, stability, shelf life, heavy-light chain mispairing tendency, aggregation tendency, and / or resistance to acid stress) and / or any other properties of the derivative antibody, may be tested. Assays that may be used to evaluate developability and / or other antibody properties include, but are not limited to, one or more of PSR assay, CIC, SIC, HIC, SEC, DLS spectroscopy, photon correlation spectroscopy, quasi-elastic light scattering, CD, viscometry, whole cell binding, tissue microarray methodology, ELISA assays such as BVP ELISA assay, AC-SINS assay, Tm assay; differential scanning calorimetry or DSF, etc. Further antibody selection may be performed based on the antibody exhibiting the desired developability parameters or profile.

[0212] Antibody Modification via Conjugation In certain embodiments, the engineered CD3 binding domain and antibodies comprising same may be further modified to contain additional non-proteinaceous 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, but are not limited to, 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.

[0213] In some embodiments, the anti-CD3 antibodies and / or antigen-binding fragments described herein are conjugated to a therapeutic moiety, thereby forming an immunoconjugate. An "immunoconjugate" is an antibody conjugated to one or more heterologous molecules, such as, for example, an antibiotic, a second anti-CD3 antibody, a vaccine, or a toxoid, or any other therapeutic moiety.

[0214] In certain embodiments, the other therapeutic agent may be a chemotherapeutic agent, optionally one or more chemotherapeutic agents selected from alkylating agents, antimetabolites, plant alkaloids, and anticancer antibiotics, and further optionally 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.

[0215] In certain embodiments, the other therapeutic agent may be a therapeutic agent, optionally an immune checkpoint inhibitor or a growth factor or growth factor receptor inhibitor, further 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.

[0216] In certain embodiments, the other therapeutic agent may be an antiemetic agent, optionally one or more antiemetic agents selected from neurokinin-1 receptor antagonists (NK1 RA), serotonin receptor antagonists (5-HT3 RA), dexamethasone, olanzapine, and palonosetron.

[0217] In some embodiments, the anti-CD3 antibodies and / or antigen-binding fragments described herein may include a label or moiety that is directly (e.g., fluorescent labels, dye labels, electron-dense labels, chemiluminescent labels, and radioactive labels) or indirectly (e.g., enzymes or ligands) 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 ... Examples of suitable oxidases include saccharide oxidases, such as glucoamylase, lysozyme, monosaccharide oxidases, e.g., 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.

[0218] Sequence modifications in the constant region In some embodiments, the anti-CD3 antibodies and / or antigen-binding fragments described herein may be further modified to minimize effector function, e.g., to minimize silent Fc or to enhance one or more effector functions.

[0219] "Effector function" refers to the biological activity attributable to the Fc region of an 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, downregulation of cell surface receptors (e.g., B cell receptors), and B cell activation.

[0220] In certain embodiments, one or more amino acid modifications may be introduced into the Fc region of an anti-CD3 antibody of the disclosure, thereby generating an Fc region variant (see, e.g., US2012 / 0251531). The Fc region variant may comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) that includes an amino acid modification (e.g., a substitution) at one or more amino acid positions.

[0221] 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 where in vivo antibody half-life is important but certain effector functions (e.g., complement and ADCC) are unnecessary or detrimental. In vitro and / or in vivo cytotoxicity assays can be performed to confirm the reduction / loss of CDC and / or ADCC activity. For example, Fc receptor (FcR) binding assays can be performed to confirm that the antibody lacks FcγR binding (and thus likely lacks ADCC activity) but retains FcRn binding ability. Primary cells mediating ADCC (e.g., NK cells) express only FcγIII, while monocytes express FcγRI, FcγRII and FcγRIII. FcR expression on hematopoietic cells is summarized in Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991), page 464, Table 3. Non-limiting examples of in vitro assays for assessing ADCC activity of a molecule of interest are described in U.S. Patent No. 5,500,362 (e.g., Hellstrom, I. et al. Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985); U.S. Patent No. 5,821,337 (see Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assay methods 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 (PBMC) and natural killer cells (NK).Alternatively, or in addition, the ADCC activity of the molecule of interest may be evaluated in vivo in an animal model, such as the animal model 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 cannot bind to C1q and therefore lacks CDC activity. See, for example, the C1q and C3c binding ELISA 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)).

[0222] In some embodiments, antibodies with reduced effector function include antibodies with substitutions at one or more of residues 238, 265, 269, 270, 297, 327, and 329 in the Fc region (U.S. Patent Nos. 6,737,056 and 8,219,149). In some embodiments, Fc variants include Fc variants with substitutions at two or more of amino acid positions 265, 269, 270, 297, and 327, including the so-called "DANA" Fc variant in which residues 265 and 297 have been substituted to alanine (U.S. Patent Nos. 7,332,581 and 8,219,149).

[0223] In some embodiments, the anti-CD3 antibodies and antigen-binding fragments described herein may comprise an Fc region derived from a human IgG1, the Fc region comprising 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, Y 300L, 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. In some embodiments, the anti-CD3 antibodies and antigen-binding fragments described herein may comprise an Fc region derived from human IgG4, which may optionally comprise 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.

[0224] In some embodiments, the anti-CD3 antibodies and antigen-binding fragments described herein may comprise an Fc region derived from human IgG2, which may optionally 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.

[0225] In some embodiments, the anti-CD3 antibodies and antigen-binding fragments described herein may comprise an Fc region derived from human IgG3, which may optionally comprise E235Y according to EU numbering.

[0226] In other embodiments, the anti-CD3 antibodies and / or antigen-binding fragments described herein are further modified to include a masking agent, such as a polypeptide mask attached via a cleavable linker.

[0227] In certain embodiments, the anti-CD3 antibodies and / or antigen-binding fragments described herein are modified to increase or decrease the extent to which the antibodies are glycosylated. Adding or deleting glycosylation sites from the anti-CD3 antibodies of the present disclosure can be readily accomplished by altering the amino acid sequence such that one or more glycosylation sites are created or removed. In certain embodiments, the addition or deletion of glycosylation sites may not be limited to the constant region of the anti-CD3 antibody or antigen-binding fragment.

[0228] Generation of anti-CD3 antibodies and antigen-binding fragments Anti-CD3 antibodies and / or antigen-binding fragments may be produced using any suitable method, including, but not limited to, recombinant methods.

[0229] For example, provided is one or more isolated nucleic acids encoding an anti-CD3 antibody or antigen-binding fragment described herein. The nucleic acid may encode an amino acid sequence comprising the VL of the antibody and / or an amino acid sequence comprising the VH of the antibody (e.g., the light and / or heavy chains of the antibody).

[0230] In some embodiments, one 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.

[0231] 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.

[0232] The disclosure provides one or more vectors (eg, expression vectors) that contain such nucleic acids.

[0233] In some embodiments, a single vector may include a nucleic acid encoding both (i) an amino acid sequence comprising the VH of an antibody and (ii) an amino acid sequence comprising the VL of an antibody. In certain embodiments, (i) and (ii) 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.

[0234] In some embodiments, the one or more vectors may include (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.

[0235] The disclosure provides isolated recombinant and / or host cells that include one or more of such above-mentioned nucleic acids, optionally contained in one or more of such above-mentioned vectors.

[0236] In one such embodiment, the host cell contains and / or has (e.g., is 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 an antibody.

[0237] In one embodiment, the host cell is eukaryotic, optionally mammalian (e.g., Chinese hamster ovary (CHO) cells, human embryonic kidney (HEK) cells such as HEK293 cells, or lymphoid cells (e.g., Y0, NS0, Sp20 cells)) or yeast.

[0238] The present disclosure provides a method of making an anti-CD3 antibody or antigen-binding fragment, the method comprising culturing a cell described herein, such as a host cell comprising a nucleic acid encoding an antibody provided above, under conditions suitable for expression of the antibody, and, optionally, recovering the antibody from the host cell (or host cell culture medium).

[0239] 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, regardless of the number of transfers.

[0240] For recombinant production of an anti-CD3 antibody, nucleic acid encoding the antibody is isolated and inserted into one or more vectors, further cloned, and / or expressed in a host cell, e.g., as described above. The nucleic acid can be readily isolated and sequenced by conventional methods (e.g., by using oligonucleotide probes capable of binding specifically to genes encoding the antibody heavy and light chains).

[0241] Suitable host cells for cloning or expressing vectors encoding antibodies include prokaryotic or eukaryotic cells. For example, antibodies can be produced in bacteria, especially when glycosylation and Fc effector functions are not required. For expression of antibody fragments and polypeptides in bacteria, see, for example, U.S. Patent Nos. 5,648,237, 5,789,199 and 5,840,523. (See also Charlton, Methods in Molecular Biology, Vol. 248 (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 also be further purified. In addition to prokaryotes, eukaryotes such as filamentous fungi or yeasts, including fungal and yeast strains whose glycosylation pathways have been "humanized" to result in the production of 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); WO2009 / 036379; WO2010 / 105256; and WO2012 / 009568.

[0242] 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 PLANTIBODY™ technology for producing antibodies in transgenic plants).Vertebrate cells can also be used as hosts.For example, mammalian trunk cell lines adapted to grow in suspension may be useful. Other examples of useful mammalian host cell lines include monkey kidney CV1 line transformed with SV40 (COS-7), human embryonic kidney cell lines (e.g., 293 or 293 cells described in Graham et al., J. Gen Virol. 36:59 (1977)), baby hamster kidney cells (BHK), mouse Sertoli cells (e.g., TM4 cells described in Mather, Biol. Reprod. 23:243-251 (1980)), monkey kidney cells (CV1), African green monkey kidney cells (VERO-76), human cervical carcinoma cells (HELA), canine kidney cells (MDCK; buffalo rat liver cells (BRL 3A), human lung cells (W138), human liver cells (Hep G2), mouse mammary tumor (MMT 060562), e.g., Mather et al., Annals TRI cells, MRC 5 cells, and FS4 cells, described in NYAcad.Sci.383:44-68(1982). Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR-CHO cells (Urlaub et al., Proc.Natl.Acad.Sci.USA 77:4216(1980)), and myeloma cell lines, such as Y0, NS0, and Sp2 / 0. For a review of certain 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).

[0243] Identification and / or characterization of additional antibodies Anti-CD3 antibodies and / or antigen-binding fragments may be identified, screened, selected, or characterized for their physical / chemical performance and / or biological activity by various assays known in the art, such as, for example, ELISA and Western blotting. Alternatively, a competitive assay may be used to identify antibodies that compete with the anti-CD3 antibodies 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 a 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 reduction in the amount of label bound to immobilized CD3 in the test sample compared to the control sample indicates that the first and second antibodies compete for binding to CD3 (see, e.g., Harlow and Lane (1988) Antibodies: A Laboratory Manual. Ch. 14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY).

[0244] Biologically active anti-CD3 antibodies and / or antigen-binding fragments 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 tumor antigen, a cell surface antigen, etc.), biological activity may also include activation of effector cells (e.g., activation of CD8+T and / or CD4+T cells), expansion of the effector cell population (i.e., increase in the number of T cells), depletion of the target cell population (i.e., reduction of the cell population expressing a second biological molecule on the cell surface) and / or killing of target cells.

[0245] Diagnostic and Therapeutic Uses of Anti-CD3 Antibodies and Antigen-Binding Fragments In some embodiments, the anti-CD3 antibodies and / or antigen-binding fragments described herein may be used for therapy and / or diagnosis and / or detection.

[0246] As used herein, "detection" includes quantitative or qualitative detection.

[0247] In certain embodiments, anti-CD3 antibodies and antigen-binding fragments that contain the labels or detection moieties described herein may be used.

[0248] The CD3 antibodies and / or antigen-binding fragments described herein, as well as pharmaceutical compositions of the antibodies, may be used in therapeutic methods. In one embodiment, the anti-CD3 antibodies and / or antigen-binding fragments described herein, or pharmaceutical compositions comprising the antibodies, may be used to treat or delay progression of a cell proliferative disorder or an autoimmune disorder. In some embodiments, the anti-CD3 antibodies and antigen-binding fragments may be used to treat cancer. Typically, tumor cells have an extracellular pH of about 6.3 to 6.5. In some embodiments, the anti-CD3 antibodies and antigen-binding fragments described herein promote preferential CD3 binding at low pH values, such as about pH 6 or lower. Accordingly, binding and activity in and around the tumor microenvironment may be promoted. In some embodiments, the use of anti-CD antibodies and antigen-binding fragments may generate selective and sustained cytotoxic activity at or around the tumor site, with associated reduction or elimination of off-target effects.

[0249] "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.

[0250] The terms "cell proliferative disorder" and "proliferative disorder" refer to disorders associated with some degree of abnormal cell proliferation. Cell proliferative disorders can include cancer and / or tumors.

[0251] 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.

[0252] "Cancer" refers to a physiological condition in mammals characterized by unregulated cell proliferation. 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 carcinoma, non-small cell lung carcinoma, 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. 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 Examples of lymphomas that may be present include: advanced 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 dysplasia associated with phacomatosis, edema (e.g., edema associated with brain tumors), Meigs' syndrome, brain tumors and head and neck cancers and associated metastases. In some 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 is selected from the group consisting of, but not limited to, Hodgkin's lymphoma, 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 (WMCL), and / or AML. WM), central nervous system lymphoma (CNSL), Burkitt lymphoma (BL), B-cell prolymphocytic leukemia, splenic marginal zone lymphoma, hairy cell leukemia, splenic lymphoma / leukemia, unclassified, splenic diffuse red pulp small B-cell lymphoma, hairy cell leukemia variant, heavy chain disease, alpha heavy chain disease, gamma heavy chain disease, mu heavy chain disease, plasma cell myeloma, isolated plasmacytoma of bone, extraosseous plasmacytoma, extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue (MALT) lymphoma), nodal marginal zone lymphoma, pediatric nodal marginal zone lymphoma, pediatric follicular lymphoma, primary cutaneous follicular center cell lymphoma, T cell / histiocyte-rich large B cell lymphoma, primary DLBCL of the CNS, primary cutaneous DLBCL, foot type, EBV-positive DLBCL of the elderly, DLBCL associated with chronic inflammation, lymphomatoid granulomatosis, primary mediastinal (thymic) large B cell lymphoma, intravascular large B cell lymphoma, ALK-positive large cell lymphoma and selected from follicular B-cell lymphoma, plasmablastic lymphoma, large B-cell lymphoma arising in HHV8-associated multicentric Castleman disease, a class of mature B-cell carcinomas including primary effusion lymphoma, B-cell lymphoma, unclassified, with features intermediate between diffuse large B-cell lymphoma and Burkitt lymphoma, and B-cell lymphoma, unclassified, with features intermediate between diffuse large B-cell lymphoma and classical Hodgkin lymphoma.

[0253] As used herein, "treatment" or "treat" or "treating" refers to clinical intervention in an attempt to change the natural course of the individual being treated, and can be performed either for preventive purposes or during the course of clinical pathology. The desired effects of treatment include, but are not limited to, prevention of disease onset or recurrence, relief of symptoms, reduction of any direct or indirect pathological consequences of the disease, prevention of metastasis, reduction in the rate of disease progression, improvement or palliation of the disease state, and remission or improvement of prognosis.

[0254] 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.

[0255] As used herein, the terms "amelioration" and "alleviation" refer to a decrease or diminution in the severity of a condition or any of its symptoms.

[0256] In some embodiments, the antibodies of the present disclosure are used to delay the onset of a disorder or disease or to delay the progression of a disorder or disease. As used herein, "delaying the progression" of a disorder or disease means to postpone, hinder, slow, inhibit, stabilize, and / or postpone the onset of the disorder or disease (e.g., a cell proliferative disorder, e.g., cancer). Delay can be of varying lengths of time, depending on the history of the disease and / or the individual being treated.

[0257] An effective amount of the antibody or composition may be administered to an individual suffering from cancer, or arthritis, rheumatoid arthritis, colitis, inflammatory bowel disease, autoimmune type I diabetes, etc. For example, an "effective amount" of an anti-CD3 antibody disclosed herein, or a composition (e.g., a pharmaceutical composition) comprising the antibody, is at least the minimum amount required to achieve a desired therapeutic or prophylactic result, such as a measurable amelioration or prevention of a particular disorder, e.g., a cell proliferative disorder, such as cancer, preferably without or with minimal toxic or adverse consequences. The effective amount may vary depending, inter alia, on the disease state, age, sex, and weight of the patient, and the ability of the antibody (or antigen-binding fragment) to elicit a desired response in the individual, and in some cases by co-administration with one or more additional therapeutic agents.

[0258] In some embodiments, the anti-CD3 antibodies and / or antigen-binding fragments described herein may be used to enhance immune function in individuals with cell proliferative or autoimmune disorders. After administration, the antibodies or compositions may enhance immune function in individuals with cell proliferative or autoimmune disorders by activating effector cells (e.g., T cells, such as CD8+ and / or CD4+ T cells, including Tregs), expanding (increasing) the effector cell population, depleting the target cell population (e.g., cells expressing a second biological molecule recognized by an anti-CD3 antibody, such as a bispecific antibody of the invention), and / or killing target cells (e.g., target tumor cells).

[0259] The anti-CD3 antibodies and / or antigen-binding fragments disclosed herein may be used to treat disorders including, but not limited to, proliferative disorders, oncological disorders, immuno-oncological disorders, neurological disorders, cognitive disorders, neurodegenerative disorders, and autoimmune disorders. In one embodiment, an effective amount of the anti-CD3 antibodies, alone or in combination with at least one additional agent, may be administered to an individual with such a disorder. Such an "individual" may be a mammal, particularly a human.

[0260] One or more of the antibodies of the present disclosure may be used alone or in combination with other agents in a treatment. For example, an anti-CD3 antibody and / or antigen-binding fragment may be co-administered with at least one additional therapeutic agent. Non-limiting examples of additional therapeutic agents include chemotherapeutic agents, antibody-drug conjugates (ADCs), and / or biological modifiers.

[0261] The chemotherapeutic agent may be selected from cyclophosphamide, doxorubicin, vincristine, and prednisolone (CHOP).

[0262] 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 US2014 / 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.

[0263] 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), for example, adoptive transfer of T cells comprising a dominant negative TGF beta receptor, such as a dominant negative TGF beta type II receptor.

[0264] Some more non-limiting exemplary additional therapeutic agents are growth inhibitors, cytotoxic agents, agents used in radiation therapy, anti-angiogenic agents, apoptotic agents, anti-tubulin agents, or other agents, such as epidermal growth factor receptor (EGFR) antagonists (e.g., tyrosine kinase inhibitors), HER1 / EGFR inhibitors (e.g., erlotinib ((TARCEVA™)), platelet derived growth factor inhibitors (e.g., GLEEVAC™ (imatinib mesylate)), COX-2 inhibitors (e.g., celecoxib), interferons, cytokines, antibodies other than the anti-CD3 antibodies of the present disclosure, such as antibodies that bind to one or more of the following targets: ErbB2, ErbB3, ErbB4, PDGFR-beta, BIyS, APRIL, BCMA, VEGF or a VEGF receptor, TRAIL / Apo2, PD-1, PD-L1, or PD-L2, or other bioactive or organic chemical agents.

[0265] In some embodiments, the disclosure provides methods wherein the additional therapeutic agent is a glucocorticoid. In one embodiment, the glucocorticoid is dexamethasone.

[0266] The anti-CD3 antibodies and / or antigen-binding fragments disclosed herein can be used to enhance immune function in individuals, such as humans, with such disorders. In one embodiment, a method of enhancing immune function comprises administering to the individual an anti-CD3 antibody in an amount effective to activate effector cells (e.g., T cells, e.g., CD8+ T cells and / or CD4+ T cells), expand (increase) an effector cell population, deplete a target cell population, and / or kill a target cell (e.g., a target tumor cell).

[0267] In a further aspect, pharmaceutical formulations are also provided that include the anti-CD3 antibodies and / or antigen-binding fragments described herein, e.g., for use in any of the therapeutic and / or diagnostic methods described above. A "pharmaceutical formulation" refers to a preparation in a form such that the biological activity of an active ingredient contained therein, such as, e.g., an anti-CD3 antibody described herein, is effective, and preferably does not contain additional ingredients that are unacceptably toxic to the subject to which the formulation is administered.

[0268] In one embodiment, a pharmaceutical formulation comprises any of the anti-CD3 antibodies disclosed herein and a pharma- ceutically acceptable carrier.

[0269] "Pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, that is not toxic to a subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0270] In some embodiments, at least one additional therapeutic agent administered in the treatment method may be included in a pharmaceutical composition or formulation together with the anti-CD3 antibody. Thus, in certain embodiments, a pharmaceutical formulation includes any of the anti-CD3 antibodies provided herein and at least one additional therapeutic agent. In certain embodiments, the at least one additional therapeutic agent may be one or more of the additional therapeutic agents described above.

[0271] Combination therapy as described above includes mixed administration (two or more therapeutic agents in the same formulation or in separate formulations) and separate administration, where administration of the antibody of the present disclosure can occur before, simultaneously, and / or after administration of the additional therapeutic agent. In one embodiment, administration of the anti-CD3 antibody and administration of the additional therapeutic agent occur within about one month, or within about one, two, or three weeks, or within about one, two, three, four, five, or six days of each other. Anti-CD3 antibodies of the present disclosure (e.g., bispecific anti-CD3 antibodies of the present disclosure that bind to CD3 and a second biological molecule, e.g., a cell surface antigen such as a tumor antigen, e.g., a TDB antibody or variant thereof of the present disclosure) can also be used in combination with radiation therapy.

[0272] The disclosed antibodies (and / or any additional therapeutic agents) can be administered by any suitable means, including parenterally, intrapulmonary, intranasally, or, if localized treatment is desired, intralesional administration. Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. In some embodiments, the antibodies are administered subcutaneously. In some embodiments, an anti-CD3 antibody administered by subcutaneous injection exhibits a lower toxic reaction in a patient than the same anti-CD3 antibody administered by intravenous injection. Dosing can be by any suitable route, such as by injection, e.g., intravenous or subcutaneous injection, depending in part on whether administration is temporary or chronic. A variety of dosing schedules are contemplated herein, including, but not limited to, a single dose or multiple doses over various time points, bolus administration, and pulse infusion. In some embodiments, the antibodies of the disclosure may be administered via delivery of mRNA encoding such antibodies. Such administration may include formulation of the mRNA into lipid nanoparticles to facilitate administration and delivery to the cells of the subject being treated.

[0273] The disclosed antibodies are formulated, dosed, and administered in a manner consistent with sound medical practice. Factors to be considered in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the location of delivery of the agent, the method of administration, the dosing schedule, and other factors known to medical practitioners. Optionally, but not required, the antibody may be formulated with one or more agents currently used to prevent or treat the disorder of interest. The effective amount of such other agents will depend on the amount of antibody present in the formulation, the type of disorder or treatment, and other factors discussed above. These will generally be used in the same dosages and routes of administration as described herein, or at approximately 1-99% of the dosages described herein, or at any dosage and via any route empirically / clinically determined to be appropriate.

[0274] For the prevention or treatment of disease, the appropriate dosage 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 administered for prophylactic or therapeutic purposes, previous treatments, the patient's medical history and responsiveness to the antibody, and the discretion of the attending physician. The antibody is suitably administered to the patient at one time or over a series of treatments.

[0275] As a general rule, a therapeutically effective amount of an anti-CD3 antibody administered to a human, whether administered as a single dose or multiple doses, will be in the range of about 0.01 to about 100 mg / kg of patient body weight. In some embodiments, the antibody used is administered daily at, for example, about 0.01 to about 45 mg / kg, about 0.01 to about 40 mg / kg, about 0.01 to about 35 mg / kg, about 0.01 to about 30 mg / kg, about 0.01 to about 25 mg / kg, about 0.01 to about 20 mg / kg, about 0.01 to about 15 mg / kg, about 0.01 to about 10 mg / kg, about 0.01 to about 5 mg / kg, or about 0.01 to about 1 mg / kg. In one embodiment, the anti-CD3 antibodies described herein are administered to a human at a dosage 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 dosage may be administered as a single dose or as multiple doses (e.g., 2 or 3 doses), e.g., by infusion. When administered repeatedly over several days or more, depending on the condition, treatment is generally sustained until a desired suppression of disease symptoms occurs. 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 every week 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.

[0276] In some embodiments, the disclosed method may further comprise an additional therapy. The additional therapy may be radiation therapy, surgery, chemotherapy, gene therapy, DNA therapy, viral therapy, RNA therapy, immunotherapy, bone marrow transplant, nanotherapy, monoclonal antibody therapy, or a combination of the above. The additional therapy may be in the form of adjuvant therapy or neoadjuvant therapy. In some embodiments, the additional therapy is administration of a small molecule enzyme inhibitor or an anti-metastatic agent. In some embodiments, the additional therapy is administration of a side effect limiting agent (e.g., an agent aimed at reducing the occurrence and / or severity of side effects of the treatment, such as an antiemetic agent). In some embodiments, the additional therapy is radiation therapy. In some embodiments, the additional therapy is surgery. In some embodiments, the additional therapy is a combination of radiation therapy and surgery. In some embodiments, the additional therapy is gamma irradiation. In some embodiments, the additional therapy may be individual administration of one or more of the above-mentioned therapeutic agents.

[0277] In another aspect of the disclosure, an article of manufacture is provided that contains materials useful for the treatment, prevention, and / or diagnosis of the above-mentioned disorders. The article of manufacture includes a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, and the like. The container may be formed from a variety of materials, such as, for example, glass or plastic. The container may hold the composition alone or in combination with another composition effective for the treatment, prevention, and / or diagnosis of a condition, and may have a sterile access port (e.g., the container may be an intravenous solution bag or a vial with a stopper pierceable by a hypodermic needle). At least one active agent in the composition is an antibody of the disclosure. The label or package insert indicates that the composition is used for the treatment of a selected condition. Additionally, the article of manufacture may include (a) a first container with a composition contained therein, the composition comprising an antibody of the disclosure, and (b) a second container with a composition contained therein, the composition comprising an additional cytotoxic agent or another therapeutic agent. The article of manufacture in this embodiment of the disclosure may further include a package insert indicating that the composition may be used to treat a particular condition. Alternatively, or in addition, the article of manufacture may further include a second (or third) container containing a pharma- ceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextrose solution. It may further include other items desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

[0278] Thus, the manufacture and / or preparation of pharmaceutical compositions comprising the anti-CD3 antibodies and / or antigen-binding fragments disclosed herein are also contemplated. The compositions may be used alone or in combination with other active agents to treat cell proliferative disorders (e.g., cancer) or autoimmune disorders (e.g., arthritis, rheumatoid arthritis, colitis, inflammatory bowel disease, autoimmune type I diabetes, etc.).

[0279] In some embodiments, pharmaceutical compositions comprising the anti-CD3 antibodies and / or antigen-binding fragments described herein are prepared, for example, by mixing the antibodies having the desired purity with one or more optional pharma- ceutically acceptable carriers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)) in the form of a lyophilized formulation or an aqueous solution, optionally prepared for modified (e.g., sustained) release. Exemplary lyophilized antibody formulations are described in U.S. Pat. No. 6,267,958. Aqueous antibody formulations include those described in U.S. Pat. No. 6,171,586 and WO2006 / 044908, the latter formulations including histidine-acetate buffers.

[0280] Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed and include, but are not limited to, buffers such as phosphates, citrates, and other organic acids, antioxidants including ascorbic acid and methionine, preservatives (e.g., octadecyldimethylbenzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl alcohol or benzyl alcohol, alkyl parabens such as methyl paraben or propyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol), low molecular weight (less than about 10 residues) polypeptides, For example, 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, including glucose, mannose, or dextrin, and other carbohydrates, 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). As used herein, examples of pharma-ceutically acceptable carriers further include interstitial drug dispersion agents, such as soluble neutral-active hyaluronidase glycoproteins (sHASEGPs), such as human soluble PH-20 hyaluronidase glycoproteins, such as 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.

[0281] Such formulations may contain more than one active ingredient, if necessary for the particular indication being treated, preferably ingredients with complementary activities that do not adversely affect each other, and that are present in amounts effective for the purpose intended. For example, it may be desirable to further provide additional therapeutic agents, such as chemotherapeutic agents, cytotoxic agents, growth inhibitory agents, and / or anti-hormonal agents.

[0282] 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 (such as liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules, etc.), or in the form of macroemulsions, respectively. Such techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

[0283] 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.

[0284] Also, it should be noted that, as used herein and in 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. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0285] As used herein, the term "about," when used in reference to a particular recited numerical value, means that the value may vary by no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes 99 and 101, and all values ​​therebetween (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0286] It should be understood that the aspects and embodiments of the disclosure described herein include "comprising," "consisting of," and "consisting essentially of" aspects and embodiments.

[0287] Although various embodiments and examples of the invention have been described with reference to certain particular molecules, compositions, methods, or protocols, it should be understood that the invention is not limited to the particular molecules, compositions, methods, or protocols described herein, as these may vary. It should be understood that in any method disclosed or claimed herein that includes more than one step, the order of the steps to be performed is not limited by the order of the steps recited, unless expressly indicated otherwise.

[0288] It is also 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 is limited only by the appended claims.

[0289] 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.

[0290] Examples are provided below to illustrate embodiments of the present disclosure and are not intended to limit the invention to any particular application or theory of operation. EXAMPLES

[0291] Example 1: Anti-CD3 antibodies Anti-CD3 antibodies A001 and A002 were isolated based on their affinity for CD3. A001 is described in U.S. Publication No. 2020 / 0190189 (referenced therein by clone number ADI-26906), which is incorporated by reference in its entirety. Antibodies V002-V007 contain various amino acid substitutions compared to A001. These substitutions were used to prepare variants of parent antibody A002 to obtain antibodies V008-V013. Amino acid and nucleic acid sequences associated with antibodies A001, A002 and V002-V013 are provided in Tables 1-4.

[0292] affinity The affinity of anti-CD3 antibodies to CD3 was measured using the rate constant (k on , k off , K D ) was determined. ForteBio affinity measurements were performed generally as previously reported (Estep et al., MAbs. 2013 5(2):270-8). Briefly, ForteBio affinity measurements were performed by online loading of human or cynomolgus monkey (cyno) CD3εδFc (300 nM) onto the AHC sensor. The sensor was equilibrated offline for 30 min in assay buffer and then monitored online for 60 s to establish a baseline. For monovalent binding measurements, the sensors with loaded CD3εδFc were exposed to 100 nM anti-CD3 Fab for 3 min, as further described in Example 2 below, and then they were transferred to assay buffer for 3 min for off-rate measurements. The kinetic data were fitted using a 1:1 binding model in the data analysis software provided by ForteBio. The resulting kinetic values ​​are provided in Table 5.

[0293] All antibodies tested showed affinity for both human and cynomolgus CD3.

[0294] cell binding The specificity of anti-CD3 antibodies for human CD3+ Jurkat cells and cynomolgus HSC-F cells was determined using a FACS cell binding assay. Briefly, cells were thawed and washed with cold PBSF buffer (PBS + 0.1% BSA, pH 7.4). Approximately 200,000 cells were dispensed per well of a 96-well plate and pelleted by centrifugation (500 x g for 5 min). Cells were washed and resuspended in 100 μl PBSF containing CHO-produced anti-CD3 IgG antibody (100 nM). The mixture (cells + antibody) was incubated on ice for 20 min and then washed twice with PBSF. Cells were resuspended in 50 μl propidium iodide (1:500 dilution) and anti-human IgG-RPE (1:100 dilution), then incubated on ice in the dark for 20 min before cells were washed twice with PBSF. Binding was analyzed on a FACS Canto II. The mean fluorescence intensity (MFI) is shown in Table 6. Also shown for comparison are results using cells that do not contain CD3 [CHO-S cells, Jurkat CD3 knockout (CD3-) cells, and HEK cells], including results using Jurkat CD3- cells in combination with anti-CD3 antibody Fab fragments (100 nM).

[0295] The results showed preferred binding by the tested antibodies to cells expressing CD3, as indicated by fluorescence intensity values ​​several fold higher than those for cells not expressing CD3.

[0296] Further FACS analysis was performed using 3-fold serial dilutions of anti-CD3 antibody Fab fragments. Fab fragments were generated from whole IgG antibodies by papain digestion and purified with KappaSelect or CaptureSelect IgG-CH1 (GE Healthcare LifeSciences) before preparation of serial dilutions. Binding to human CD3+ Jurkat cells and cynomolgus monkey CD3 HSC-F cells was assessed with each diluted sample, and the results were used to construct titration curves and calculate half-maximal effective concentration (EC50) values ​​for Fab binding. The results are shown in Table 7.

[0297] Fab fragments from all antibodies tested demonstrated binding to both human and cynomolgus CD3 as demonstrated by low EC50 values.

[0298] Development Potential - PSR The developability of anti-CD3 antibodies was determined using multiple assessments, including polyspecificity analysis. Antibodies with high affinity for a target may otherwise fail in a clinical setting, exhibiting binding to multiple non-target entities as well. Antibody polyspecificity was assessed by measuring the interaction with a polyspecific reagent (PSR). The 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 precipitated at 2,400 x g for 5 min. The cell pellet was combined and then resuspended in 25 ml of buffer B and precipitated at 2,400 x g for 3 min. The buffer was decanted and the wash was repeated once more. The cell pellet was resuspended in 3x 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 2,400xg for 5 min, the supernatant was retained and pelleted once more (2,400xg / 5 min) to ensure removal of unbroken cells, cell debris and nuclei. The resulting supernatant is the total protein preparation. The supernatant was then transferred to two Nalgene Oak Ridge 45mL centrifuge tubes and pelleted at 40,000xg for 40 min at 4°C. The supernatant containing the separated cytosolic proteins (SCP) was then transferred to a clean Oak Ridge tube and centrifuged once more at 40,000xg. In parallel, the pellet containing the membrane fraction (EMF) was retained and centrifuged at 40,000 for 20 minutes to remove the residual 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 off 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.

[0299] about 10 6 ~107 Cells / mL of mammalian cells (e.g., CHO, HEK293, Sf9, etc.) were transferred from tissue culture into 4x250mL conical tubes and pelleted at 550xg for 3 min. All subsequent steps were performed at 4°C or on ice using ice-cold buffers. Cells were washed with 100mL PBSF ((1xPBS + 1mg / mL BSA) and combined in one conical tube. After removing the supernatant, the cell pellet was resuspended in 30mL of buffer B (50mM HEPES, 0.15M NaCl, 2mM CaCl2, 5mM KCl, 5mM PBS ... The cells were resuspended in 10% MgCl2, 10% glycerol, pH 7.2) and pelleted at 550xg for 3 min. The supernatant in Buffer B was decanted and the cells were resuspended in 3x pellet volume of Buffer B supplemented with 2.5x protease inhibitors (Roche, cOmplete, EDTA-free). From this point on, Buffer B contained protease inhibitors. The cells were homogenized (Polyton homogenizer, PT1200E) with four 30 s pulses and the membrane fraction was pelleted at 40,000xg 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. The supernatant was retained and left as s. The pellet was transferred to a Dounce homogenizer with 3 mL of buffer B and resuspended by gently moving the pestle up and down for 30–35 strokes. The enriched membrane fraction (EMF) was transferred to a new collection tube and the pestle was rinsed to collect all potential proteins. The protein concentration of the purified EMF was determined using a Dc-Protein Assay Kit (BioRad). To solubilize the EMF, solubilization buffer (50 mM HEPES, 0.15M NaCl, 2mM CaCl2, 5mM KCl, 5mM MgCl2, 1% n-dodecyl-bD-maltopyranoside (DDM), 1x protease inhibitors, pH 7.2) to a final concentration of 1mg / mL. The mixture was rotated overnight at 4°C and then centrifuged at 40,000xg for 1 hour in a 50mL Oak Ridge tube (Fisher Scientific, 050529-ID). The supernatant, which is soluble membrane protein (SMP), was collected and protein production was quantified as described above.

[0300] For biotinylation, prepare NHS-LC-biotin stock solution according to the manufacturer's protocol (Pierce, Thermo Fisher). Briefly, 20 ul 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 x 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 pellet in 3 mL of Buffer C using a Dounce homogenizer as described above. The resuspended pellet is now biotinylated EMF (b-EMF). Solubilize as described above to prepare b-SMP.

[0301] PSR binding analysis. The assay was 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 were transferred to a 96-well assay plate, pelleted at 3000xg for 3 min, and the supernatant was removed. The pellet was resuspended in 50 μl of a 1:10 dilution of freshly prepared stock biotinylated PSR (b-PSRs) and incubated on ice for 20 min. The cells were washed twice with 200 μl of ice-cold PBSF and the pellet was resuspended in 50 μl of secondary labeling mix (Extravidin-R-PE, anti-human LC-FITC, and propidium iodide). The mixture was incubated on ice for 20 min, followed by washing twice with 200 ul of ice-cold PBSF. Cells were resuspended in 100 μl ice-cold PBSF and plates were run 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 non-specific binding.Numerous methods for displaying or directing antibodies or antibody fragments on the yeast surface 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 USA., 105(51):20167-72(2008); Tasumi et al., Proc Natl Acad Sci USA., 106(31):12891-6(2009)). The final PSR scores of the tested antibodies are listed in Table 8.

[0302] The parent antibodies A001 and A002 had the highest PSR scores and showed high levels of non-target specific binding. The A001 variants V002-V007 and A002 variants V008-V013 all showed reduced PSR scores compared to A001 and A002, respectively, with many showing PSR scores ≦0.1, "clean" PSR scores, and showing very low levels of non-target specific binding. Of the new variant antibodies, V002-V004, V006, and V007 showed "clear" PSR scores, while V005, V008, V010, V012, and V013 showed "low" PSR scores.

[0303] Example 2. Method The following paragraphs include methods used in anti-CD3 antibody evaluation, including many of those described above.

[0304] Generation of Hu and Cy CD3εδFc heterodimeric antigens. Recombinant heterodimeric CD3 Fc fusion antigens were produced in HEK293 cells by cotransfection of plasmids encoding Hu CD3εFc (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) utilizing heterologous signal peptide 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 by Ni Sepharose 6 Fast Flow (GE Healthcare Life Sciences) to remove CD3δδ Fc homodimers. CD3εδ Fc-HIS heterodimers were separated from CD3εεFc-HIS homodimers by a linear Tris-buffered KCl gradient at pH 8.5 with Mono Q 10 / 100 GL. Rhesus and mouse CD3εδ Fc heterodimers were generated in a similar manner.

[0305] Peptides. C-terminally biotinylated CD3ε N-terminal peptide was obtained from New England Peptide. All peptides were delivered with a purity of 95% or higher. Peptides were designed based on the primary sequence of Hu CD3ε and the crystal structure of Hu CD3εδ bound to OKT3 (Kjer-Nielsen L. et al. PNAS 2004). CD3ε N27 peptide has the sequence H2N-QDGNEEMGSITQTPYQVSISGTTVILT[K / SCBiot(dPEG4)]-amide (SEQ ID NO: 40) and CD3ε N13 peptide has the sequence H2N-QDGNEEMGGITQT[K / SCBiot(dPEG4)]-amide (SEQ ID NO: 41).

[0306] Antigen biotinylation. CD3 antigen was biotinylated using Pierce's EZ-Link Sulfo-NHS-Biotinylation Kit. Goat anti-human F(ab')2 kappa-FITC (LC-FITC), extravidin-PE (EA-PE) and streptavidin-633 (SA-633) were obtained from Southern Biotech, Sigma and Molecular Probes, respectively. Streptavidin microbeads and MACS LC separation columns were purchased from Miltenyi Biotec.

[0307] 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. Cyno 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).

[0308] Labeling of cells was performed by dispensing 100,000-200,000 cells per well in a 96-well assay plate. Cells were centrifuged at 500×g for 5 min at 4°C. They were then resuspended in 100 μl of 100 nM IgG and incubated at room temperature for 20 min. Cells were then washed three times with buffer (phosphate-buffered saline (PBS) / 0.1% bovine serum albumin (BSA)) and resuspended in secondary reagent, typically goat anti-human R-PE (Southern Biotech). Plates were analyzed on a FACSCanto (BD Biosciences) using the HTS sample injector. Flow cytometry data were analyzed relative to median fluorescence intensity in the R-PE channel.

[0309] FACS affinity pressure selection method. Briefly, yeast cells (at least approximately 2 x 10 7Cells / labeling condition) were incubated with biotinylated antigen in sufficient stoichiometric excess with respect to the average IgG presentation. Antigen labeling conditions are 100-1 nM under equilibration conditions and are typically performed for 20 min to several hours at room temperature in FACS wash buffer (phosphate-buffered saline (PBS) / 0.1% bovine serum albumin (BSA)). After washing three times with wash buffer, yeast are stained for 15 min at 4 °C with the secondary reagent anti-human light chain FITC conjugate (LC-FITC) diluted 1:100 and either streptavidin-633 (SA-633) diluted 1:500 or extravidin-phycoerythrin (EA-PA) diluted 1:50. After washing twice with ice-cold wash buffer, the cell pellet was stained with 1 × 10 7 Yeast are resuspended in a volume of at least 1 mL of wash buffer per yeast and transferred to a sort tube capped with a strainer. Sorting is performed using a FACS ARIA sorter (BD Biosciences) to determine sort gating and select for binders. After the final round of sorting, yeast cells are plated and individual colonies are picked and characterized.

[0310] 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 precipitated 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).

[0311] HEK production and purification of antibodies. Mammalian expression of IgG was performed by subcloning the antibody into a new expression vector followed by transient transfection and expression in HEK293ADI1. Monoclonal cell lines derived from HEK293 (DSMZ) were selected for aggregation-free growth, growth rate and transfectability. Briefly, expression vectors containing the antibody of interest were transfected by complexation with transfection reagent followed by exposure to HEK cells for 1 h before dilution of culture medium to a final density of 4 million cells per mL. Cells were then cultured for 7 days with fresh feed medium changes every 48 h. After 7 days, supernatants were collected after centrifugation and purification was performed using Protein A. CHT column purification was added to reach >95% monomer, if necessary.

[0312] Antibody production and purification in CHO cells. Antibodies were produced as IgG1 by subcloning the antibodies into new expression vectors followed by transfection and expression in CHO cells. Fab fragments were produced by papain digestion and purified with 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. Generally, CHO-K1 cells grown to approximately 4×10*6 cells / mL were pelleted and resuspended in transfection medium. DNA plasmids (1.5ug total DNA / mL) were incubated with PEIpro (final 1:2, PolyPlus, catalogue no. 115-100) in transfection medium at room temperature before being added to the CHO-K1 cell suspension. The transfected cultures were fed and maintained at 32°C with shaking until the supernatant was harvested for purification (day 9). The cell culture supernatant was 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 (200mM acetic acid / 50mM NaCl, pH 3.5) into 1 / 8 volume of 2M Hepes, pH 8.0. The final product was buffer exchanged into 25mM Hepes and 150mM sodium chloride, pH 7.3. Fabs were generated using overnight papain digestion followed by a CH1 resin purification step.

[0313] Cell Binding Assay. CD3+ human Jurkat cells (ATCC) and CHO-S cells (Invitrogen / ThermoFisher) were lysed 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 (500xg for 5 min). 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 100ul of either PBSF pH 7.4 or PBSF pH 6.0 containing IgG antibodies (100nM) produced in yeast or CHO cells 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 ul 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, then incubated for 20 minutes on ice in the dark, after which the cells were washed twice with either PBSF pH 7.4 or PBSF pH 6.0. Binding was analyzed on a FACS Canto II.

[0314] ForteBio K DMeasurement (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). Briefly, ForteBio affinity measurements were performed by loading IgG or CD3gδ Fc-HIS heterodimers onto AHC sensors. The sensors were equilibrated offline in assay buffer for 30 min and then monitored online for 60 s to establish a baseline. IgG-loaded sensors were exposed to 100 nM antigen (e.g., CD3) for 3 min, after which they were transferred to assay buffer for 10 min and dissociation rate measurements were performed. CD3εδ Fc-HIS heterodimer loaded sensors were exposed to 100 nM anti-CD3 Fab for 3 min, after which they were transferred to assay buffer for 3 or 10 min and dissociation rate measurements were performed. A 1:1 binding model was used to analyze the kinetics. To measure binding at different pH, measurements were performed at a given pH (e.g., pH 7.4 and pH 6.0).

[0315] 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 into 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 or 600 second dissociation phase. All files were saved in a shared network drive using a naming convention that identifies the experiment type.

[0316] HIC. The IgG1 sample was buffer exchanged into 1M ammonium sulfate and 0.1M sodium phosphate at pH 6.5 using a Zeba 40kDa 0.5mL spin column (Thermo Pierce, Cat. No. 87766). A salt gradient was established on a Dionex ProPac HIC-10 column from 1.8M ammonium sulfate, 0.1M sodium phosphate at pH 6.5 to the same conditions without ammonium sulfate. The gradient was run for 17 minutes at a flow rate of 0.75ml / 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.

[0317] LC-MS. IgG1 samples were reduced with DTT and then subjected to mid-down liquid chromatography mass spectrometry (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 in the samples. A fast LC flow of 2 mL / min allowed 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 only 0.15 mL / min of sample flow to the mass spectrometer for sample analysis. The Bruker maXis 4G mass spectrometer was run in positive ion mode with detection in the range of 750–2500 m / z. The remaining source parameters were set as follows: the capillary was set at 5500 V, the nebulizer was set at 4.0 bar, the drying gas was set at 4.0 L / min, and the drying temperature was set at 200°C.

[0318] MS spectra were analyzed using Bruker Data Analysis version 4.1, and deconvolution was performed using maximum entropy deconvolution in the mass range of 20–30 kDa.

[0319] Example 3: Additional anti-CD3 antibodies Anti-CD3 antibodies A003, A004, and A005 were previously isolated as described in U.S. Publication No. 2020 / 0190189 (referred to therein as ADI-26913, ADI-26920, and ADI-26921), which is incorporated by reference in its entirety.

[0320] Antibodies V014 and V015 were newly derived by incorporating various amino acid substitutions into one or more of the CDRs of A003. Antibodies V016 and V017 were newly derived by incorporating various amino acid substitutions into one or more of the CDRs of A004. Antibodies V018 and V019 were newly derived by incorporating various amino acid substitutions into one or more of the CDRs of A005. The amino acid and nucleic acid sequences associated with antibodies A003-A005 and V014-V019 are provided in Tables 1-4.

[0321] Example 4: Monovalent binding affinity at different pH - Fab, SPR Anti-CD3 antibodies A001-A005, V002, V004, V007, V009, V010, V013, and V014-V019 were produced in CHO cells and Fab fragments were generated from IgG1 antibodies by papain digestion and purification as described in Example 2. Monovalent binding affinity at pH 7.4 and pH 6.0 was measured via surface plasmon resonance (SPR). Briefly, human or cynomolgus CD3εδ Fc dimers generated as described in Example 2 were immobilized to a CM5 sensor chip in a Biacore® X100 (Cytiva, formerly GE Healthcare Life Sciences) to a response level of approximately 500 RU. Fabs were then injected at 3-fold increasing concentrations ranging from 0.74 to 180 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 Biacore® evaluation software.

[0322] The results are shown in Table 9. As shown in the table, all Fabs tested bound to both human and cynomolgus CD3εδ at both pH 7.4 and 6.0.

[0323] Example 5: Affinity and avidity at different pH - Fab or IgG1, BLI Anti-CD3 antibodies A001-A005, V002, V004, V007, V009, V010, V013, and V014-V019 were produced in CHO cells as IgG1 and Fab fragments were generated from the IgG1 antibodies by papain digestion and purification as described in Example 2. Human, cynomolgus, rhesus, and mouse CD3εδ Fc heterodimers were generated and monovalent binding affinity and avidity at pH 7.4 and pH 6.0 were measured using an Octet® HTX instrument (ForteBio) as described in Example 2.

[0324] The results are shown in Table 10. As shown in the table, all tested Fabs bound to both human and cynomolgus CD3εδ at both pH 7.4 and 6.0, and all tested Fabs (K D All IgGs tested (except for V017, which was indeterminable) bound to rhesus monkey CD3εδ at pH 7.4. Some of the IgGs tested (A001-A003, V002, V004, V009, V010, and V013-V015) bound to mouse CD3εδ to some extent at pH 7.4, but some of the IgGs tested (A004, A005, V007, and V016-V019) did not bind to mouse CD3εδ at pH 7.4.

[0325] Example 6: Cell-binding - IgG1, flow cytometry Anti-CD3 antibodies A001-A005, V002, V004, V007, V009, V010, V013, and V014-V019 were produced as IgG1 in CHO cells and analyzed for binding to human CD3+ Jurkat cells and cynomolgus monkey CD3+ HSC-F cells at pH 7.4 by flow cytometry as described in Example 2. NCB (normalized cell binding) values ​​were calculated based on control and sample MFI values ​​as NCB={(sample MFI)-(secondary only MFI)} / (secondary only MFI).

[0326] NCB values ​​for the tested IgG1s are shown in Table 11. As shown in the table, all antibodies tested bound to both human and cynomolgus CD3 expressing cells. Increased binding to human CD3+ Jurkat cells was observed for V014-V019 compared to their parent antibodies. For example, V014 and V015 showed higher binding than their parent antibodies A003, V016, V017 showed higher binding than their parent antibody A004, and V018 and V019 showed higher binding than their parent antibody A005.

[0327] Example 7: Developability - Fab or IgG1, Chromatography Anti-CD3 antibodies A001-A005, V002, V004, V007, V009, V010, V013, and V014-V019 were produced in CHO cells as IgG1 and Fab fragments were generated from the IgG1 antibodies by papain digestion and purification as described in Example 2.

[0328] Sequence confirmation To test whether the heavy and light chain masses matched the expected masses based on the amino acid sequence, IgG1 samples were subjected to LC-MS analysis as described in Example 2. Samples that matched the expected masses are marked as "PASS", as shown in Table 12.

[0329] purity To test the purity of the produced IgG1 and Fab (determined by the percentage of total IgG or Fab monomers, respectively, in the total antibody product (i.e., present without aggregation or multimerization)), the IgG1 and Fab production samples were subjected to size-exclusion chromatography (SEC)-high performance liquid chromatography (HPLC) analysis. Briefly, an Agilent 1260 HPLC was employed to monitor column chromatography (TSKgel Super SW mAb HTP column). 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 before 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. The total assay time was approximately 6 min. Data was analyzed using ChemStation software.

[0330] The % monomer values ​​obtained by SEC-HPLC are shown in Table 12. As shown in the table, % monomer greater than 95% was observed for all Fabs tested and nearly all IgG1s tested.

[0331] pH stress tolerance To test the tolerance to low pH stress, IgG1 samples were incubated at acidic pH or physiological pH and subjected to SEC-HPLC analysis. Briefly, IgG1 samples at 15 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 acetate, pH 3.5). After 1 h 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 into an Agilent 1260 Infinity analytical HPLC (Agilent, Santa Clara, CA) equipped with a TSKgel SuperSW mAb HTP column (TOSOH Bioscience, King of Prussia, PA, product code 22855). SEC data were collected and analyzed using Agilent ChemStation software (Agilent, Santa Clara, Calif.).

[0332] The % monomer values ​​obtained by SEC-HPLC are shown in Table 12. As shown in the table, the % monomer values ​​were not significantly affected by low pH and remained >95% for all IgG1s tested, indicating good tolerance to the stress of low pH.

[0333] Example 8: Development Potential - PSR Multispecificity by PSR binding assay The anti-CD3 antibodies A001-A005, V002, V004, V007, V009, V010, V013, and V014-V019 were subjected to PSR binding analysis as described in Example 1.

[0334] The PSR scores are shown in Table 13. As shown in the table, many of the IgG1s tested (A003-A005, V002, V004, V007, and V014-V019) showed clean PSR scores and no or negligible polyspecificity, while some of the IgG1s tested (A001, V009, V010, and V013) showed low PSR scores, indicating low polyspecificity.

[0335] Example 9: Developability - HIC, AC-SINS, and DLS Anti-CD3 antibodies A001-A005 and V002, V004, V007, V009, V010, V013, and V014-V019 were produced as IgG1 in CHO cells as described in Example 2.

[0336] Hydrophobicity by HIC The hydrophobicity of antibodies is one cause of antibody aggregation. IgG1 production samples were subjected to hydrophobic interaction chromatography (HIC) analysis as described in Example 2.

[0337] The observed HIC retention times are shown in Table 13. As shown in the table, all of the IgG1s tested had retention times <10.5 minutes, indicating clean to low hydrophobicity, i.e. a highly desirable developability profile.

[0338] 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 y et al., MAbs. Mar-Apr 2014;6(2):483-92). Briefly, polyclonal goat anti-human IgG Fc antibody (capture; Jackson ImmunoResearch Laboratories) and polyclonal goat non-specific antibody (non-capture; Jackson ImmunoResearch Laboratories) were buffer exchanged into 20 mM sodium acetate (pH 4.3) and concentrated to 0.4 mg / ml. A 4:1 volume ratio of capture:non-capture was prepared and further incubated with 20 nm gold nanoparticles (AuNP, Ted Pella Inc.) in a 1:9 volume ratio for 1 h at room temperature. Thiolated PEG (Sigma-Aldrich) was then used to block vacant sites on the AuNP 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). Self-interaction levels were determined based on Δλmax. Self-interaction is low when Δλmax<5.0 nm, moderate when Δλmax≧5.0 nm and <20.0 nm, and high when Δλmax≧20.0 nm.

[0339] The resulting Δλmax are shown in Table 13. As shown in the table, many of the IgG1s tested (A003, V004, V007, V010, and V013-V015) had a Δλmax of <5.0, and many of the IgG1s tested (A001, A002, A004, A005, V002, V007, V009, and V016-V019) had a Δλmax of ≧5.0 nm and <20.0 nM.

[0340] Self-interaction by 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 positively correlate with low viscosity at high concentrations in the same formulation buffer. kD values ​​were obtained by measuring the mutual diffusion coefficient (D) at a series of different concentrations by DLS. Specifically, DLS kD measurements were performed at multiple concentrations from 0.5 to 12 mg / mL in 10 mM histidine buffer at pH 6.0. kD values ​​<20 mL / g were considered to be associated with high viscosity or high opalescence.

[0341] The kD values ​​obtained are shown in Table 13. As shown in the table, many of the IgG1s tested (A001-A005 and V014-V016) had kD values ​​of ≧20 mL / g. Example 10: Developability - Fab Tm by DSF.

[0342] Anti-CD3 antibodies A001-A005, and V002, V004, V007, V009, V010, V013, and V014-V019 were produced in CHO cells as IgG1, and Fab fragments were generated from the IgG1 antibodies by papain digestion and purification as described in Example 2. 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 (BioRad) to collect Fret signals. Fab Tm was assigned using the first derivative of the raw data from Bio-Rad analysis software.

[0343] The Tm values ​​obtained are shown in Table 14. As shown in the table, all Fabs had Tm values ​​much higher than 65° C., indicating high stability and therefore favorable developability.

[0344] Exemplary embodiments Described herein below are several exemplary embodiments according to the present disclosure.

[0345] Embodiment 1. An antibody or antigen-binding fragment comprising a complementarity determining region (CDR) comprising an amino acid sequence selected from any of the amino acid sequences listed in Table 2 (Tables 2A and 2B).

[0346] Embodiment 2. An antibody or antigen-binding fragment comprising a heavy chain variable domain (VH) comprising an amino acid sequence selected from any of the amino acid sequences listed in Table 1 (Table 1A), and / or a light chain variable domain (VL) comprising an amino acid sequence selected from any of the amino acid sequences listed in Table 1 (Table 1B).

[0347] Embodiment 3. VH comprises the amino acid sequence of SEQ ID NO:1 and VL comprises the amino acid sequence of SEQ ID NO:15, or VH comprises the amino acid sequence of SEQ ID NO:2 and VL comprises the amino acid sequence of SEQ ID NO:16, or VH comprises the amino acid sequence of SEQ ID NO:3 and VL comprises the amino acid sequence of SEQ ID NO:15, or VH comprises the amino acid sequence of SEQ ID NO:4 and VL comprises the amino acid sequence of SEQ ID NO:15, or VH comprises the amino acid sequence of SEQ ID NO:5 and VL comprises the amino acid sequence of SEQ ID NO:15, or VH comprises the amino acid sequence of SEQ ID NO:6 and VL comprises the amino acid sequence of SEQ ID NO:15, or VH comprises the amino acid sequence of SEQ ID NO:7 and VL comprises the amino acid sequence of SEQ ID NO:15, or VH comprises the amino acid sequence of SEQ ID NO:8. and wherein the VL comprises the amino acid sequence of SEQ ID NO: 15, or wherein the VH comprises the amino acid sequence of SEQ ID NO: 9 and the VL comprises the amino acid sequence of SEQ ID NO: 16, or wherein the VH comprises the amino acid sequence of SEQ ID NO: 10 and the VL comprises the amino acid sequence of SEQ ID NO: 16, or wherein the VH comprises the amino acid sequence of SEQ ID NO: 11 and the VL comprises the amino acid sequence of SEQ ID NO: 16, or wherein the VH comprises the amino acid sequence of SEQ ID NO: 12 and the VL comprises the amino acid sequence of SEQ ID NO: 16, or wherein the VH comprises the amino acid sequence of SEQ ID NO: 13 and the VL comprises the amino acid sequence of SEQ ID NO: 16, or wherein the VH comprises the amino acid sequence of SEQ ID NO: 14 and the VL comprises the amino acid sequence of SEQ ID NO: 16.

[0348] Embodiment 4. The antibody or antigen-binding fragment of embodiment 1, comprising a VH CDR3 (CDRH3) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 33 and 34, a VH CDR2 (CDRH2) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 21 to 32, a VH CDR1 (CDRH1) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 17 to 20, a VH comprising a CDR set according to any of the CDR sets listed in Table 2A, a VL CDR3 (CDRL3) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 38 or 39, a VL CDR2 (CDRL2) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 36 or 37, a VL CDR1 (CDRL1) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 33 or 34, and / or a VL comprising a CDR set according to any of the CDR sets listed in Table 2B.

[0349] Embodiment 5. The antibody or antigen-binding fragment of any one of embodiments 1 to 4, wherein the antibody or antigen-binding fragment binds to cluster of differentiation 3 (CD3).

[0350] Embodiment 6. The antibody or antigen-binding fragment of embodiment 5, wherein the antibody or antigen-binding fragment binds to human and / or non-human primate CD3.

[0351] Embodiment 7. a second antibody that specifically binds to a tumor target, an immune-oncology target, a neurodegenerative disease target, an autoimmune disorder target, an infectious disease target, a metabolic disease target, a cognitive disorder target, a blood-brain barrier target, or a hematological disease target; and wherein the antibody or antigen-binding fragment comprises at least an antigen-binding domain having any one or more of the second binding specificities described herein, and the antibody or antigen-binding fragment comprises at least a second antigen-binding domain having any one or more of the second binding specificities described herein, and the antibody or antigen-binding fragment is selected from the group consisting of BCMA, CTLA4 (cytotoxic T lymphocyte antigen-4), PD1 (programmed cell death protein 1), PD-L1 (programmed cell death ligand 1), LAG-3 (lymphocyte activation gene-3), TIM-3, CD20, CD2, CD19, Her2, EGFR, EpCAM, FcyRIIIa (CD16), FcyRIIa (CD32a), and optionally a second antigen-binding domain that specifically binds one or more of the following: FcyRIIb (CD32b), FcyRI (CD64), Toll-like receptor (TLR), TLR4, TLR9, cytokines, IL-2, IL-5, IL-13, IL-6, IL-17, IL-12, IL-23, TNFα, TGFβ, cytokine receptors, IL-2R, chemokines, chemokine receptors, growth factors, VEGF, and HGF; and optionally the antibody or antigen-binding fragment further comprises at least one transmembrane domain, and a T cell receptor; and wherein the antibody or antigen-binding fragment comprises at least one intracellular domain derived from the CD3 ζ subunit and at least one costimulatory domain, and the antibody or antigen-binding fragment comprises scFv2-Fc2 and / or scFv-IgG, the antibody or antigen-binding fragment comprises an IgG constant domain, and / or the antibody or antigen-binding fragment comprises at least a second antigen-binding domain that specifically binds to an antigen, and the antibody or antigen-binding fragment is selected from the group consisting of Fab-Fc-scFv, "bottle-opener", Mab-scFv, Mab-Fv, dual-scFv,The antibody or antigen-binding fragment of any one of embodiments 1 to 6, comprising a multispecific format selected from the group consisting of 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.

[0352] Embodiment 8. The antibody or antigen-binding fragment of any one of embodiments 1 to 7, wherein the antibody or antigen-binding fragment exhibits a reduced PSR score compared to antibody A001 and / or A002.

[0353] EMBODIMENT 9. A nucleic acid comprising the antibody or antigen-binding fragment according to any one of embodiments 1 to 8.

[0354] Embodiment 10. A construct comprising the nucleic acid sequence of embodiment 9.

[0355] Embodiment 11. A cell comprising a nucleic acid according to embodiment 9 and / or a construct according to embodiment 10, wherein the cell is optionally a mammalian cell or a yeast cell.

[0356] Embodiment 12. A pharmaceutical composition comprising the antibody or antigen-binding fragment according to any one of embodiments 1 to 7 or the cell according to embodiment 11, and a pharma- ceutically acceptable carrier and / or excipient.

[0357] Embodiment 13. A method for treating a disorder in a mammal in need of such treatment, comprising administering an effective amount of an antibody or antigen-binding fragment of any one of embodiments 1-7, a cell of embodiment 11, and, optionally, an immune cell, a T cell, and / or a natural killer (NK) cell.

[0358] Embodiment 14. The method of embodiment 13, wherein the disorder comprises one or more of a proliferative disorder, an oncological disorder, an immune-oncological disorder, a neurological disorder, a neurodegenerative disorder, and an autoimmune disorder.

[0359] EMBODIMENT 15. The method of embodiment 13 or 14, wherein the method further comprises administering an additional therapeutic agent.

[0360] EMBODIMENT 16. The method of any one of embodiments 13-15, wherein the mammal is a human. [Table 1-1] [Table 1-2] [Table 2-1] [Table 2-2] [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4] [Table 4-1] [Table 4-2]

Table 4-3

Table 4-4

Table 5-1

Table 5-2

Table 6-1

Table 6-2

Table 7-1

Table 7-2

Table 8-1

Table 8-2

Table 9

Table 10

Table 11

Table 12

Table 13

Table 14

Table 15

Table 16

Table 17

Table 18

Table 19

Table 20

Table 21

Claims

1. An anti-cluster of differentiation 3 (CD3) antibody or antigen-binding antibody fragment, comprising: (A) (a) (i) Antibody No. V013, or a CDRH1 contained in any one of antibody Nos. V002 to V012, A0014 to A019, and A001 to A005; (ii) a CDRH1 contained in any one of SEQ ID NOs: 1310, 210, 310, 410, 510, 610, 710, 810, 910, 1010, 1110, 1210, 1410, 1510, 1610, 1710, 1810, 1910, 2110, 2210, 2310, 2410, and 2510; (iii) FX １ X ２ X ３ DYYMH (SEQ ID NO: 112) １ is N or D, and X ２ is I or D, and X ３ is D or K); and / or (iv) a VH CDR1 (CDRH1) comprising any one of the amino acid sequences of SEQ ID NOs: 1312, 212, 312, 412, 512, 612, 712, 812, 912, 1012, 1112, 1212, 1412, 1512, 1612, 1712, 1812, 1912, 2112, 2212, 2312, 2412, and 2512; (b) (i) CDRH2 contained in antibody No. V013, or any one of antibody Nos. V002 to V012, A0014 to A019, and A001 to A005; (ii) a CDRH2 contained in any one of SEQ ID NOs: 1310, 210, 310, 410, 510, 610, 710, 810, 910, 1010, 1110, 1210, 1410, 1510, 1610, 1710, 1810, 1910, 2110, 2210, 2310, 2410, and 2510; (iii) WIX ４ LEX ５ X ６ X ７ TX ８ X ９ DAKFQX １０ (SEQ ID NO: 114) (wherein X ４ is D or E, and X ５ is N or E, and X ６ is A, D, or G, and X ７ is N, E, or D, and X ８ is V or I, and X ９ is D or Y, and X １０ is G or D), and / or (iv) a VH CDR2 (CDRH2) comprising the amino acid sequence of any one of SEQ ID NOs: 1314, 214, 314, 414, 514, 614, 714, 814, 914, 1014, 1114, 1214, 1414, 1514, 1614, 1714, 1814, 1914, 2114, 2214, 2314, 2414, and 2514; and / or (c) (i) a CDRH3 contained in antibody No. V013, or any one of antibody Nos. V002 to V012, A0014 to A019, and A001 to A005; (ii) a CDRH3 contained in any one of SEQ ID NOs: 1310, 210, 310, 410, 510, 610, 710, 810, 910, 1010, 1110, 1210, 1410, 1510, 1610, 1710, 1810, 1910, 2110, 2210, 2310, 2410, and 2510; (iii) X １１ RDX １２ YGRYFYDX １３ (SEQ ID NO: 116) (wherein X １１ is A or G, and X １２ is A or Q, and X １３ is V or E), and / or (iv) a heavy chain variable domain (VH) polypeptide comprising a VH CDR3 (CDRH3) comprising the amino acid sequence of any one of SEQ ID NOs: 1316, 216, 316, 416, 516, 616, 716, 816, 916, 1016, 1116, 1216, 1416, 1516, 1616, 1716, 1816, 1916, 2116, 2216, 2316, 2416, and 2516; and / or (B) (a) (i) CDRL1 contained in antibody No. V013, or any one of antibody Nos. V002 to V012, A0014 to A019, and A001 to A005; (ii) a CDRL1 contained in any one of SEQ ID NOs: 1320, 220, 320, 420, 520, 620, 720, 820, 920, 1020, 1120, 1220, 1420, 1520, 1620, 1720, 1820, 1920, 2120, 2220, 2320, 2420, and 2520; and / or (iii) a VL CDR1 (CDRL1) comprising the amino acid sequence of any one of SEQ ID NOs: 1322, 122, 222, 322, 422, 522, 622, 722, 822, 922, 1022, 1122, 1222, 1422, 1522, 1622, 1722, 1822, 1922, 2122, 2222, 2322, 2422, and 2522; (b) (i) CDRL2 contained in antibody No. V013, or any one of antibody Nos. V002 to V012, A0014 to A019, and A001 to A005; (ii) a CDRL2 contained in any one of SEQ ID NOs: 1320, 220, 320, 420, 520, 620, 720, 820, 920, 1020, 1120, 1220, 1420, 1520, 1620, 1720, 1820, 1920, 2120, 2220, 2320, 2420, and 2520; (iii) WASTRX １４ S (SEQ ID NO: 124) １４ is S or E), and / or (iv) a VL CDR2 (CDRL2) comprising the amino acid sequence of any one of SEQ ID NOs: 1324, 224, 324, 424, 524, 624, 724, 824, 924, 1024, 1124, 1224, 1424, 1524, 1624, 1724, 1824, 1924, 2124, 2224, 2324, 2424, and 2524; and / or (c) (i) CDRL3 contained in antibody No. V013, or any one of antibody Nos. V002 to V012, A0014 to A019, and A001 to A005; (ii) a CDRL3 contained in any one of SEQ ID NOs: 1320, 220, 320, 420, 520, 620, 720, 820, 920, 1020, 1120, 1220, 1420, 1520, 1620, 1720, 1820, 1920, 2120, 2220, 2320, 2420, and 2520; (iii) X １５ QSYX １６ X １７ RT (SEQ ID NO: 126), (wherein X １５ is K or V, and X １６ is S or F, and X １７ is R or L), and / or (iv) a light chain variable domain (VL) polypeptide comprising a VL CDR3 (CDRL3) comprising the amino acid sequence of any one of SEQ ID NOs: 1326, 226, 326, 426, 526, 626, 726, 826, 926, 1026, 1126, 1226, 1426, 1526, 1626, 1726, 1826, 1926, 2126, 2226, 2326, 2426, and 2526; Optionally, (A) a VH polypeptide comprising the CDRH1, the CDRH2, and the CDRH3; and / or (B) a VL polypeptide comprising the CDRL1, the CDRL2, and the CDRL3, or a combination of one or more of the foregoing CDRs; Optionally, (I) (i) at least one of CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 contained in A001; or (ii) does not include at least one of SEQ ID NOs: 2112, 2114, 2116, 2122, 2124, and 2126; Further optionally, (i) at least one of the CDRH1 and CDRH2 contained in A001; or (ii) does not contain at least one of SEQ ID NOs: 2112 and 2114; (II) (i) at least one of CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 contained in A002; or (ii) does not include at least one of SEQ ID NOs: 2212, 2214, 2216, 2222, 2224, and 2226; Further optionally, (i) at least one of the CDRH1 and CDRH2 contained in A002; or (ii) does not contain at least one of SEQ ID NOs: 2212 and 2214; (III) (i) at least one of CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 contained in A003; or (ii) does not include at least one of SEQ ID NOs: 2312, 2314, 2316, 2322, 2324, and 2326; Further optionally, (i) at least one of the CDRH1 and CDRH2 contained in A003; or (ii) does not contain at least one of SEQ ID NOs: 2312 and 2314; (IV) (i) at least one of CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 contained in A004; or (ii) does not include at least one of SEQ ID NOs: 2412, 2414, 2416, 2422, 2424, and 2426; Further optionally, (i) at least one of the CDRH1 and CDRH2 contained in A004; or (ii) does not contain at least one of SEQ ID NOs: 2412 and 2414; and (V) (i) at least one of CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 contained in A005; or (ii) does not include at least one of SEQ ID NOs: 2512, 2514, 2516, 2522, 2524, and 2526; Further optionally, (i) at least one of the CDRH1 and CDRH2 contained in A005; or (ii) does not contain at least one of SEQ ID NOs: 2512 and 2514; Optionally, (i) human CD3, (ii) non-human primate CD3, optionally monkey, further optionally cynomolgus and / or rhesus CD3, and / or (iii) binds to rodent CD3, optionally mouse CD3; Further optionally, the anti-CD3 antibody or antigen-binding antibody fragment, wherein said CD3 is CD3εδ.

2. (I) (i) the CDRH1, the CDRH2, the CDRH3, the CDRL1, the CDRL2, and the CDR-L3 contained in antibody number V013, or (ii) a CDRH1 comprising SEQ ID NO: 1312, a CDRH2 comprising SEQ ID NO: 1314, a CDRH3 comprising SEQ ID NO: 1316, a CDRL1 comprising SEQ ID NO: 1322, a CDRL2 comprising SEQ ID NO: 1324, and a CDRL3 comprising SEQ ID NO: 1326; (II) (i) the CDRH1, the CDRH2, the CDRH3, the CDRL1, the CDRL2, and the CDR-L3 contained in antibody No. V002; or (ii) a CDRH1 comprising SEQ ID NO:212, a CDRH2 comprising SEQ ID NO:214, a CDRH3 comprising SEQ ID NO:216, a CDRL1 comprising SEQ ID NO:222, a CDRL2 comprising SEQ ID NO:224, and a CDRL3 comprising SEQ ID NO:226; (III) (i) the CDRH1, the CDRH2, the CDRH3, the CDRL1, the CDRL2, and the CDR-L3 contained in antibody No. V003, or (ii) a CDRH1 comprising SEQ ID NO:312, a CDRH2 comprising SEQ ID NO:314, a CDRH3 comprising SEQ ID NO:316, a CDRL1 comprising SEQ ID NO:322, a CDRL2 comprising SEQ ID NO:324, and a CDRL3 comprising SEQ ID NO:326; (IV) (i) the CDRH1, the CDRH2, the CDRH3, the CDRL1, the CDRL2, and the CDR-L3 contained in antibody No. V004, or (ii) a CDRH1 comprising SEQ ID NO:412, a CDRH2 comprising SEQ ID NO:414, a CDRH3 comprising SEQ ID NO:416, a CDRL1 comprising SEQ ID NO:422, a CDRL2 comprising SEQ ID NO:424, and a CDRL3 comprising SEQ ID NO:426; (V) (i) the CDRH1, the CDRH2, the CDRH3, the CDRL1, the CDRL2, and the CDR-L3 contained in antibody No. V005; or (ii) a CDRH1 comprising SEQ ID NO:512, a CDRH2 comprising SEQ ID NO:514, a CDRH3 comprising SEQ ID NO:516, a CDRL1 comprising SEQ ID NO:522, a CDRL2 comprising SEQ ID NO:524, and a CDRL3 comprising SEQ ID NO:526; (VI) (i) the CDRH1, the CDRH2, the CDRH3, the CDRL1, the CDRL2, and the CDR-L3 contained in antibody No. V006, or (ii) a CDRH1 comprising SEQ ID NO:612, a CDRH2 comprising SEQ ID NO:614, a CDRH3 comprising SEQ ID NO:616, a CDRL1 comprising SEQ ID NO:622, a CDRL2 comprising SEQ ID NO:624, and a CDRL3 comprising SEQ ID NO:626; (VII) (i) the CDRH1, the CDRH2, the CDRH3, the CDRL1, the CDRL2, and the CDR-L3 contained in antibody No. V007; or (ii) a CDRH1 comprising SEQ ID NO:712, a CDRH2 comprising SEQ ID NO:714, a CDRH3 comprising SEQ ID NO:716, a CDRL1 comprising SEQ ID NO:722, a CDRL2 comprising SEQ ID NO:724, and a CDRL3 comprising SEQ ID NO:726; (VIII) (i) the CDRH1, the CDRH2, the CDRH3, the CDRL1, the CDRL2, and the CDR-L3 contained in antibody No. V008, or (ii) a CDRH1 comprising SEQ ID NO:812, a CDRH2 comprising SEQ ID NO:814, a CDRH3 comprising SEQ ID NO:816, a CDRL1 comprising SEQ ID NO:822, a CDRL2 comprising SEQ ID NO:824, and a CDRL3 comprising SEQ ID NO:826; (IX) (i) the CDRH1, the CDRH2, the CDRH3, the CDRL1, the CDRL2, and the CDR-L3 contained in antibody No. V009, or (ii) a CDRH1 comprising SEQ ID NO:912, a CDRH2 comprising SEQ ID NO:914, a CDRH3 comprising SEQ ID NO:916, a CDRL1 comprising SEQ ID NO:922, a CDRL2 comprising SEQ ID NO:924, and a CDRL3 comprising SEQ ID NO:926; (X) (i) the CDRH1, the CDRH2, the CDRH3, the CDRL1, the CDRL2, and the CDR-L3 contained in antibody No. V010, or (ii) a CDRH1 comprising SEQ ID NO: 1012, a CDRH2 comprising SEQ ID NO: 1014, a CDRH3 comprising SEQ ID NO: 1016, a CDRL1 comprising SEQ ID NO: 1022, a CDRL2 comprising SEQ ID NO: 1024, and a CDRL3 comprising SEQ ID NO: 1026; (XI) (i) the CDRH1, the CDRH2, the CDRH3, the CDRL1, the CDRL2, and the CDR-L3 contained in antibody No. V011; or (ii) a CDRH1 comprising SEQ ID NO: 1112, a CDRH2 comprising SEQ ID NO: 1114, a CDRH3 comprising SEQ ID NO: 1116, a CDRL1 comprising SEQ ID NO: 1122, a CDRL2 comprising SEQ ID NO: 1124, and a CDRL3 comprising SEQ ID NO: 1126; (XII) (i) the CDRH1, the CDRH2, the CDRH3, the CDRL1, the CDRL2, and the CDR-L3 contained in antibody No. V012, or (ii) a CDRH1 comprising SEQ ID NO: 1212, a CDRH2 comprising SEQ ID NO: 1214, a CDRH3 comprising SEQ ID NO: 1216, a CDRL1 comprising SEQ ID NO: 1222, a CDRL2 comprising SEQ ID NO: 1224, and a CDRL3 comprising SEQ ID NO: 1226; (XIII) (i) the CDRH1, the CDRH2, the CDRH3, the CDRL1, the CDRL2, and the CDR-L3 contained in antibody No. V014, or (ii) a CDRH1 comprising SEQ ID NO: 1412, a CDRH2 comprising SEQ ID NO: 1414, a CDRH3 comprising SEQ ID NO: 1416, a CDRL1 comprising SEQ ID NO: 1422, a CDRL2 comprising SEQ ID NO: 1424, and a CDRL3 comprising SEQ ID NO: 1426; (XIV) (i) the CDRH1, the CDRH2, the CDRH3, the CDRL1, the CDRL2, and the CDR-L3 contained in antibody No. V015, or (ii) a CDRH1 comprising SEQ ID NO: 1512, a CDRH2 comprising SEQ ID NO: 1514, a CDRH3 comprising SEQ ID NO: 1516, a CDRL1 comprising SEQ ID NO: 1522, a CDRL2 comprising SEQ ID NO: 1524, and a CDRL3 comprising SEQ ID NO: 1526; (XV)(i) the CDRH1, the CDRH2, the CDRH3, the CDRL1, the CDRL2, and the CDR-L3 contained in antibody No. V016, or (ii) a CDRH1 comprising SEQ ID NO: 1612, a CDRH2 comprising SEQ ID NO: 1614, a CDRH3 comprising SEQ ID NO: 1616, a CDRL1 comprising SEQ ID NO: 1622, a CDRL2 comprising SEQ ID NO: 1624, and a CDRL3 comprising SEQ ID NO: 1626; (XVI) (i) the CDRH1, the CDRH2, the CDRH3, the CDRL1, the CDRL2, and the CDR-L3 contained in antibody No. V017, or (ii) a CDRH1 comprising SEQ ID NO: 1712, a CDRH2 comprising SEQ ID NO: 1714, a CDRH3 comprising SEQ ID NO: 1716, a CDRL1 comprising SEQ ID NO: 1722, a CDRL2 comprising SEQ ID NO: 1724, and a CDRL3 comprising SEQ ID NO: 1726; (XVII) (i) the CDRH1, the CDRH2, the CDRH3, the CDRL1, the CDRL2, and the CDR-L3 contained in antibody No. V018, or (ii) a CDRH1 comprising SEQ ID NO: 1812, a CDRH2 comprising SEQ ID NO: 1814, a CDRH3 comprising SEQ ID NO: 1816, a CDRL1 comprising SEQ ID NO: 1822, a CDRL2 comprising SEQ ID NO: 1824, and a CDRL3 comprising SEQ ID NO: 1826; or (XVIII) (i) the CDRH1, the CDRH2, the CDRH3, the CDRL1, the CDRL2, and the CDR-L3 contained in antibody No. V019, or (ii) The anti-CD3 antibody or antigen-binding antibody fragment of claim 1, comprising a CDRH1 comprising SEQ ID NO: 1912, a CDRH2 comprising SEQ ID NO: 1914, a CDRH3 comprising SEQ ID NO: 1916, a CDRL1 comprising SEQ ID NO: 1922, a CDRL2 comprising SEQ ID NO: 1924, and a CDRL3 comprising SEQ ID NO: 1926.

3. (A) the VH polypeptide is (a) (i) FRH1 contained in antibody No. V013, or any one of antibody Nos. V002 to V012, A0014 to A019, and A001 to A005; (ii) an FRH1 contained in any one of SEQ ID NOs: 1310, 210, 310, 410, 510, 610, 710, 810, 910, 1010, 1110, 1210, 1410, 1510, 1610, 1710, 1810, 1910, 2110, 2210, 2310, 2410, and 2510; and / or (iii) a VH framework region 1 (FRH1) comprising any one of the amino acid sequences of SEQ ID NOs: 1311, 111, 211, 311, 411, 511, 611, 711, 811, 911, 1011, 1111, 1211, 1411, 1511, 1611, 1711, 1811, 1911, 2111, 2211, 2311, 2411, and 2511; (b) (i) FRH2 contained in antibody No. V013, or any one of antibody Nos. V002 to V012, A0014 to A019, and A001 to A005; (ii) FRH2 contained in any one of SEQ ID NOs: 1310, 210, 310, 410, 510, 610, 710, 810, 910, 1010, 1110, 1210, 1410, 1510, 1610, 1710, 1810, 1910, 2110, 2210, 2310, 2410, and 2510; (iii) WVRQAPGQRLEWX １８ G (SEQ ID NO: 113) １８ is M or I), and / or (iv) a VH framework region 2 (FRH2) comprising any one of the amino acid sequences of SEQ ID NOs: 1313, 213, 313, 413, 513, 613, 713, 813, 913, 1013, 1113, 1213, 1413, 1513, 1613, 1713, 1813, 1913, 2113, 2213, 2313, 2413, and 2513; (c) (i) FRH3 contained in antibody No. V013, or any one of antibody Nos. V002 to V012, A0014 to A019, and A001 to A005; (ii) an FRH3 contained in any one of SEQ ID NOs: 1310, 210, 310, 410, 510, 610, 710, 810, 910, 1010, 1110, 1210, 1410, 1510, 1610, 1710, 1810, 1910, 2110, 2210, 2310, 2410, and 2510; and / or (iii) a VH framework region 3 (FRH3) comprising the amino acid sequence of any one of SEQ ID NOs: 1315, 115, 215, 315, 415, 515, 615, 715, 815, 915, 1015, 1115, 1215, 1415, 1515, 1615, 1715, 1815, 1915, 2115, 2215, 2315, 2415, and 2515; and / or (d) (i) FRH4 contained in antibody No. V013, or any one of antibody Nos. V002 to V012, A0014 to A019, and A001 to A005; (ii) FRH4 contained in any one of SEQ ID NOs: 1310, 210, 310, 410, 510, 610, 710, 810, 910, 1010, 1110, 1210, 1410, 1510, 1610, 1710, 1810, 1910, 2110, 2210, 2310, 2410, and 2510; and / or (iii) a VH framework region 4 (FRH4) comprising the amino acid sequence of any one of SEQ ID NOs: 1317, 117, 217, 317, 417, 517, 617, 717, 817, 917, 1017, 1117, 1217, 1417, 1517, 1617, 1717, 1817, 1917, 2117, 2217, 2317, 2417, and 2517; and / or (B) the VL polypeptide is (a) (i) FRL1 contained in antibody No. V013, or any one of antibody Nos. V002 to V012, A0014 to A019, and A001 to A005; (ii) FRL1 contained in any one of SEQ ID NOs: 1320, 220, 320, 420, 520, 620, 720, 820, 920, 1020, 1120, 1220, 1420, 1520, 1620, 1720, 1820, 1920, 2120, 2220, 2320, 2420, and 2520; (iii) DIVMX １９ QSPDSLAVSLGERATINC (SEQ ID NO: 121) １９ is S or T), and / or (iv) a VL framework region 1 (FRL1) comprising any one of the amino acid sequences of SEQ ID NOs: 221, 321, 421, 521, 621, 721, 821, 921, 1021, 1121, 1221, 1321, 1421, 1521, 1621, 1721, 1821, 1921, 2121, 2221, 2321, 2421, and 2521; (b) (i) FRL2 contained in antibody No. V013, or any one of antibody Nos. V002 to V012, A0014 to A019, and A001 to A005; (ii) an FRL2 contained in any one of SEQ ID NOs: 1320, 220, 320, 420, 520, 620, 720, 820, 920, 1020, 1120, 1220, 1420, 1520, 1620, 1720, 1820, 1920, 2120, 2220, 2320, 2420, and 2520; and / or (iii) a VL framework region 2 (FRL2) comprising any one of the amino acid sequences of SEQ ID NOs: 1323, 123, 223, 323, 423, 523, 623, 723, 823, 923, 1023, 1123, 1223, 1423, 1523, 1623, 1723, 1823, 1923, 2123, 2223, 2323, 2423, and 2523; (c) (i) FRL3 contained in antibody No. V013, or any one of antibody Nos. V002 to V012, A0014 to A019, and A001 to A005; (ii) an FRL3 contained in any one of SEQ ID NOs: 1320, 220, 320, 420, 520, 620, 720, 820, 920, 1020, 1120, 1220, 1420, 1520, 1620, 1720, 1820, 1920, 2120, 2220, 2320, 2420, and 2520; and / or (iii) a VL framework region 3 (FRL3) comprising the amino acid sequence of any one of SEQ ID NOs: 1325, 125, 225, 325, 425, 525, 625, 725, 825, 925, 1025, 1125, 1225, 1425, 1525, 1625, 1725, 1825, 1925, 2125, 2225, 2325, 2425, and 2525; and / or (d) (i) FRL4 contained in antibody No. V013, or any one of antibody Nos. V002 to V012, A0014 to A019, and A001 to A005; (ii) FRL4 contained in any one of SEQ ID NOs: 1320, 220, 320, 420, 520, 620, 720, 820, 920, 1020, 1120, 1220, 1420, 1520, 1620, 1720, 1820, 1920, 2120, 2220, 2320, 2420, and 2520, and / or (iii) a VL framework region 4 (FRL4) comprising the amino acid sequence of any one of SEQ ID NOs: 1327, 127, 227, 327, 427, 527, 627, 727, 827, 927, 1027, 1127, 1227, 1427, 1527, 1627, 1727, 1827, 1927, 2127, 2227, 2327, 2427, and 2527; or Alternatively, the anti-CD3 antibody or antigen-binding antibody fragment of claim 1 comprises a VH and / or VL comprising any combination of the aforementioned VH and VL framework regions.

4. (I) (i) the FRH1, FRH2, FRH3, FLRH4, FRL1, FRL2, FRL3, and FRL4 contained in antibody No. V013, or any one of antibody Nos. A002 and V008 to V012, and / or (ii) FR-H1 comprising any one of SEQ ID NOs: 1311, 111, 811, 911, 1011, 1111, 1211, and 2211; FR-H2 comprising any one of SEQ ID NOs: 1313, 813, 913, 1013, 1113, 1213, and 2213; FR-H3 comprising any one of SEQ ID NOs: 1315, 115, 815, 915, 1015, 1115, 1215, and 2215; FR-H4 comprising any one of SEQ ID NOs: 1317, 117, 817, 917, 1017, 1117, 1217, and 2217; FR-L1 comprising any one of sequence numbers 1321, 821, 921, 1021, 1121, 1221, and 2221; FR-L2 comprising any one of SEQ ID NOs: 1323, 123, 823, 923, 1023, 1123, 1223, and 2223; FR-L3 comprising any one of SEQ ID NOs: 1325, 125, 825, 925, 1025, 1125, 1225, and 2225; and FR-L4 comprising any one of SEQ ID NOs: 1327, 127, 827, 927, 1027, 1127, 1227, and 2227; or (II) (i) the FRH1, FRH2, FRH3, FLRH4, FRL1, FRL2, FRL3, and FRL4 contained in any one of antibody numbers A001, V002 to V007, A003 to A005, and V014 to V019, and / or (ii) FR-H1 comprising any one of SEQ ID NOs: 111, 211, 311, 411, 511, 611, 711, 1411, 1511, 1611, 1711, 1811, 1911, 2111, 2311, 2411, and 2511; FR-H2 comprising any one of SEQ ID NOs: 213, 313, 413, 513, 613, 713, 1413, 1513, 1613, 1713, 1813, 1913, 2113, 2313, 2413, and 2513 , FR-H3 including any one of SEQ ID NOs: 115, 215, 315, 415, 515, 615, 715, 1415, 1515, 1615, 1715, 1815, 1915, 2115, 2315, 2415, and 2515; FR-H4 including any one of SEQ ID NOs: 117, 217, 317, 417, 517, 617, 717, 1417, 1517, 1617, 1717, 1817, 1917, 2117, 2317, 2417, and 2517 , FR-L1 including any one of SEQ ID NOs: 221, 321, 421, 521, 621, 721, 1421, 1521, 1621, 1721, 1821, 1921, 2121, 2321, 2421, and 2521; FR-L2 including any one of SEQ ID NOs: 123, 223, 323, 423, 523, 623, 723, 1423, 1523, 1623, 1723, 1823, 1923, 2123, 2323, 2423, and 2523; FR-L3 comprising any one of SEQ ID NOs: 125, 225, 325, 425, 525, 625, 725, 1425, 1525, 1625, 1725, 1825, 1925, 2125, 2325, 2425, and 2525, and FR-L4 comprising any one of SEQ ID NOs: 127, 227, 327, 427, 527, 627, 727, 1427, 1527, 1627, 1727, 1827, 1927, 2127, 2327, 2427, and 2527.

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

5. (I) (i) The FRH1, the CDRH1, the FRH2, the CDRH2, the FRH3, the CDRH3, the FRH4, the FRL1, the CDRL1, the FRL2, the CDRL2, the FRL3, the CDRL3, and the FRL4 contained in antibody number V013, or (ii) FRH1, CDRH1, FRH2, CDRH2, FRH3, CDRH3, FRH4, FRL1, CDRL1, FRL2, CDRL2, FRL3, CDRL3, and FRL4 comprising SEQ ID NOs: 1311, 1312, 1313, 1314, 1315, 1316, 1317, 1321, 1322, 1323, 1324, 1325, 1326, and 1327, respectively; (II) (i) the FRH1, the CDRH1, the FRH2, the CDRH2, the FRH3, the CDRH3, the FRH4, the FRL1, the CDRL1, the FRL2, the CDRL2, the FRL3, the CDRL3, and the FRL4 contained in antibody number V002, or (ii) FRH1, CDRH1, FRH2, CDRH2, FRH3, CDRH3, FRH4, FRL1, CDRL1, FRL2, CDRL2, FRL3, CDRL3, and FRL4 comprising SEQ ID NOs: 211, 212, 213, 214, 215, 216, 217, 221, 222, 223, 224, 225, 226, and 227, respectively; (III) (i) the FRH1, the CDRH1, the FRH2, the CDRH2, the FRH3, the CDRH3, the FRH4, the FRL1, the CDRL1, the FRL2, the CDRL2, the FRL3, the CDRL3, and the FRL4 contained in antibody No. V003, or (ii) FRH1, CDRH1, FRH2, CDRH2, FRH3, CDRH3, FRH4, FRL1, CDRL1, FRL2, CDRL2, FRL3, CDRL3, and FRL4 comprising SEQ ID NOs: 311, 312, 313, 314, 315, 316, 317, 321, 322, 323, 324, 325, 326, and 327, respectively; (IV) (i) the FRH1, the CDRH1, the FRH2, the CDRH2, the FRH3, the CDRH3, the FRH4, the FRL1, the CDRL1, the FRL2, the CDRL2, the FRL3, the CDRL3, and the FRL4 contained in antibody number V004, or (ii) FRH1, CDRH1, FRH2, CDRH2, FRH3, CDRH3, FRH4, FRL1, CDRL1, FRL2, CDRL2, FRL3, CDRL3, and FRL4 comprising SEQ ID NOs: 411, 412, 413, 414, 415, 416, 417, 421, 422, 423, 424, 425, 426, and 427, respectively; (V) (i) the FRH1, the CDRH1, the FRH2, the CDRH2, the FRH3, the CDRH3, the FRH4, the FRL1, the CDRL1, the FRL2, the CDRL2, the FRL3, the CDRL3, and the FRL4 contained in antibody number V005, or (ii) FRH1, CDRH1, FRH2, CDRH2, FRH3, CDRH3, FRH4, FRL1, CDRL1, FRL2, CDRL2, FRL3, CDRL3, and FRL4 comprising SEQ ID NOs: 511, 512, 513, 514, 515, 516, 517, 521, 522, 523, 524, 525, 526, and 527, respectively; (VI) (i) the FRH1, the CDRH1, the FRH2, the CDRH2, the FRH3, the CDRH3, the FRH4, the FRL1, the CDRL1, the FRL2, the CDRL2, the FRL3, the CDRL3, and the FRL4 contained in antibody No. V006, or (ii) FRH1, CDRH1, FRH2, CDRH2, FRH3, CDRH3, FRH4, FRL1, CDRL1, FRL2, CDRL2, FRL3, CDRL3, and FRL4 comprising SEQ ID NOs: 611, 612, 613, 614, 615, 616, 617, 621, 622, 623, 624, 625, 626, and 627, respectively; (VII) (i) the FRH1, the CDRH1, the FRH2, the CDRH2, the FRH3, the CDRH3, the FRH4, the FRL1, the CDRL1, the FRL2, the CDRL2, the FRL3, the CDRL3, and the FRL4 contained in antibody No. V007, or (ii) FRH1, CDRH1, FRH2, CDRH2, FRH3, CDRH3, FRH4, FRL1, CDRL1, FRL2, CDRL2, FRL3, CDRL3, and FRL4 comprising SEQ ID NOs: 711, 712, 713, 714, 715, 716, 717, 721, 722, 723, 724, 725, 726, and 727, respectively; (VIII) (i) The FRH1, the CDRH1, the FRH2, the CDRH2, the FRH3, the CDRH3, the FRH4, the FRL1, the CDRL1, the FRL2, the CDRL2, the FRL3, the CDRL3, and the FRL4 contained in antibody No. V008, or (ii) FRH1, CDRH1, FRH2, CDRH2, FRH3, CDRH3, FRH4, FRL1, CDRL1, FRL2, CDRL2, FRL3, CDRL3, and FRL4 comprising SEQ ID NOs: 811, 812, 813, 814, 815, 816, 817, 821, 822, 823, 824, 825, 826, and 827, respectively; (IX) (i) the FRH1, the CDRH1, the FRH2, the CDRH2, the FRH3, the CDRH3, the FRH4, the FRL1, the CDRL1, the FRL2, the CDRL2, the FRL3, the CDRL3, and the FRL4 contained in antibody number V009, or (ii) FRH1, CDRH1, FRH2, CDRH2, FRH3, CDRH3, FRH4, FRL1, CDRL1, FRL2, CDRL2, FRL3, CDRL3, and FRL4 comprising SEQ ID NOs: 911, 912, 913, 914, 915, 916, 917, 921, 922, 923, 924, 925, 926, and 927, respectively; (X) (i) the FRH1, the CDRH1, the FRH2, the CDRH2, the FRH3, the CDRH3, the FRH4, the FRL1, the CDRL1, the FRL2, the CDRL2, the FRL3, the CDRL3, and the FRL4 contained in antibody No. V010, or (ii) FRH1, CDRH1, FRH2, CDRH2, FRH3, CDRH3, FRH4, FRL1, CDRL1, FRL2, CDRL2, FRL3, CDRL3, and FRL4 comprising SEQ ID NOs: 1011, 1012, 1013, 1014, 1015, 1016, 1017, 1021, 1022, 1023, 1024, 1025, 1026, and 1027, respectively; (XI) (i) the FRH1, the CDRH1, the FRH2, the CDRH2, the FRH3, the CDRH3, the FRH4, the FRL1, the CDRL1, the FRL2, the CDRL2, the FRL3, the CDRL3, and the FRL4 contained in antibody No. V011, or (ii) FRH1, CDRH1, FRH2, CDRH2, FRH3, CDRH3, FRH4, FRL1, CDRL1, FRL2, CDRL2, FRL3, CDRL3, and FRL4 comprising SEQ ID NOs: 1111, 1112, 1113, 1114, 1115, 1116, 1117, 1121, 1122, 1123, 1124, 1125, 1126, and 1127, respectively; (XII) (i) the FRH1, the CDRH1, the FRH2, the CDRH2, the FRH3, the CDRH3, the FRH4, the FRL1, the CDRL1, the FRL2, the CDRL2, the FRL3, the CDRL3, and the FRL4 contained in antibody No. V012, or (ii) FRH1, CDRH1, FRH2, CDRH2, FRH3, CDRH3, FRH4, FRL1, CDRL1, FRL2, CDRL2, FRL3, CDRL3, and FRL4 comprising SEQ ID NOs: 1211, 1212, 1213, 1214, 1215, 1216, 1217, 1221, 1222, 1223, 1224, 1225, 1226, and 1127, respectively; (XIII) (i) the FRH1, the CDRH1, the FRH2, the CDRH2, the FRH3, the CDRH3, the FRH4, the FRL1, the CDRL1, the FRL2, the CDRL2, the FRL3, the CDRL3, and the FRL4 contained in antibody No. V014, or (ii) FRH1, CDRH1, FRH2, CDRH2, FRH3, CDRH3, FRH4, FRL1, CDRL1, FRL2, CDRL2, FRL3, CDRL3, and FRL4 comprising SEQ ID NOs: 1411, 1412, 1413, 1414, 1415, 1416, 1417, 1421, 1422, 1423, 1424, 1425, 1426, and 1427, respectively; (XIV) (i) the FRH1, the CDRH1, the FRH2, the CDRH2, the FRH3, the CDRH3, the FRH4, the FRL1, the CDRL1, the FRL2, the CDRL2, the FRL3, the CDRL3, and the FRL4 contained in antibody No. V015, or (ii) FRH1, CDRH1, FRH2, CDRH2, FRH3, CDRH3, FRH4, FRL1, CDRL1, FRL2, CDRL2, FRL3, CDRL3, and FRL4 comprising SEQ ID NOs: 1511, 1512, 1513, 1514, 1515, 1516, 1517, 1521, 1522, 1523, 1524, 1525, 1526, and 1527, respectively; (XV) (i) the FRH1, the CDRH1, the FRH2, the CDRH2, the FRH3, the CDRH3, the FRH4, the FRL1, the CDRL1, the FRL2, the CDRL2, the FRL3, the CDRL3, and the FRL4 contained in antibody No. V016, or (ii) FRH1, CDRH1, FRH2, CDRH2, FRH3, CDRH3, FRH4, FRL1, CDRL1, FRL2, CDRL2, FRL3, CDRL3, and FRL4 comprising SEQ ID NOs: 1611, 1612, 1613, 1614, 1615, 1616, 1617, 1621, 1622, 1623, 1624, 1625, 1626, and 1627, respectively; (XVI) (i) the FRH1, the CDRH1, the FRH2, the CDRH2, the FRH3, the CDRH3, the FRH4, the FRL1, the CDRL1, the FRL2, the CDRL2, the FRL3, the CDRL3, and the FRL4 contained in antibody No. V017, or (ii) FRH1, CDRH1, FRH2, CDRH2, FRH3, CDRH3, FRH4, FRL1, CDRL1, FRL2, CDRL2, FRL3, CDRL3, and FRL4 comprising SEQ ID NOs: 1711, 1712, 1713, 1714, 1715, 1716, 1717, 1721, 1722, 1723, 1724, 1725, 1726, and 1727, respectively; (XVII) (i) the FRH1, the CDRH1, the FRH2, the CDRH2, the FRH3, the CDRH3, the FRH4, the FRL1, the CDRL1, the FRL2, the CDRL2, the FRL3, the CDRL3, and the FRL4 contained in antibody No. V018, or (ii) FRH1, CDRH1, FRH2, CDRH2, FRH3, CDRH3, FRH4, FRL1, CDRL1, FRL2, CDRL2, FRL3, CDRL3, and FRL4 comprising SEQ ID NOs: 1811, 1812, 1813, 1814, 1815, 1816, 1817, 1821, 1822, 1823, 1824, 1825, 1826, and 1827, respectively; or (XVIII) (i) the FRH1, the CDRH1, the FRH2, the CDRH2, the FRH3, the CDRH3, the FRH4, the FRL1, the CDRL1, the FRL2, the CDRL2, the FRL3, the CDRL3, and the FRL4 contained in antibody No. V019, or (ii) The anti-CD3 antibody or antigen-binding antibody fragment of claim 1, comprising FRH1, CDRH1, FRH2, CDRH2, FRH3, CDRH3, FRH4, FRL1, CDRL1, FRL2, CDRL2, FRL3, CDRL3, and FRL4, which comprise SEQ ID NOs: 1911, 1912, 1913, 1914, 1915, 1916, 1917, 1921, 1922, 1923, 1924, 1925, 1926, and 1927, respectively. (I), wherein: (A) the VH polypeptide comprises 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: 1310; and / or (B) the VL polypeptide comprises 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: 1320; (II), (A) the VH polypeptide comprises 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: 210; and / or (B) the VL polypeptide comprises 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: 220; (III), wherein (A) the VH polypeptide comprises 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: 310; and / or (B) the VL polypeptide comprises 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: 320; (IV), (A) the VH polypeptide comprises 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: 410; and / or (B) the VL polypeptide comprises 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: 420; (V), wherein (A) the VH polypeptide comprises 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: 510; and / or (B) the VL polypeptide comprises 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: 520; (VI), wherein (A) the VH polypeptide comprises 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; and / or (B) the VL polypeptide comprises 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; (VII), wherein (A) the VH polypeptide comprises 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: 710; and / or (B) the VL polypeptide comprises 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: 720; (VIII), wherein (A) the VH polypeptide comprises 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: 810; and / or (B) the VL polypeptide comprises 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: 820; (IX), wherein (A) the VH polypeptide comprises 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: 910; and / or (B) the VL polypeptide comprises 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: 920; In (X), (A) the VH polypeptide comprises 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: 1010; and / or (B) the VL polypeptide comprises 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: 1020; (XI), wherein (A) the VH polypeptide comprises 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: 1110; and / or (B) the VL polypeptide comprises 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: 1120; (XII), wherein (A) the VH polypeptide comprises 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: 1210; and / or (B) the VL polypeptide comprises 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: 1220; (XIII), wherein (A) the VH polypeptide comprises 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: 1410; and / or (B) the VL polypeptide comprises 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: 1420; (XIV), wherein (A) the VH polypeptide comprises 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: 1510; and / or (B) the VL polypeptide comprises 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: 1520; (XV), wherein (A) the VH polypeptide comprises 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: 1610; and / or (B) the VL polypeptide comprises 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: 1620; (XVI), wherein (A) the VH polypeptide comprises 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: 1710; and / or (B) the VL polypeptide comprises 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: 1720; (XVII), wherein (A) the VH polypeptide comprises 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: 1810; and / or (B) the VL polypeptide comprises 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: 1820; or (XVIII), wherein (A) the VH polypeptide comprises 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: 1910; and / or (B) The anti-CD3 antibody or antigen-binding antibody fragment of claim 2, wherein the VL polypeptide comprises 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: 1920.

7. the VH polypeptide and the VL polypeptide (I) SEQ ID NOs: 1310 and 1320, respectively; (II) SEQ ID NOs: 210 and 220, respectively; (III) SEQ ID NOs: 310 and 320, respectively; (IV) SEQ ID NOs: 410 and 420, respectively; (V) SEQ ID NOs: 510 and 520, respectively; (VI) SEQ ID NOs: 610 and 620, respectively; (VII) SEQ ID NOs: 710 and 720, respectively; (VIII) SEQ ID NOs: 810 and 820, respectively; (IX) SEQ ID NOs: 910 and 920, respectively; (X) SEQ ID NOs: 1010 and 1020, respectively; (XI) SEQ ID NOs: 1110 and 1120, respectively; (XII) SEQ ID NOs: 1210 and 1220, respectively; (XIII) SEQ ID NOs: 1410 and 1420, respectively; (XIV) SEQ ID NOs: 1510 and 1520, respectively; (XV) SEQ ID NOs: 1610 and 1620, respectively; (XVI) SEQ ID NOs: 1710 and 1720, respectively; (XVII) SEQ ID NOs: 1810 and 1820, respectively; or (XVIII) The anti-CD3 antibody or antigen-binding antibody fragment of claim 1, comprising the amino acid sequences of SEQ ID NOs: 1910 and 1920, respectively.

8. below, (i) one or more antibody constant regions, CH1 domains, hinges, CH2 domains, and / or CH3 domains, optionally individually 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 of, or derived from, a human IgG3, optionally comprising 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) region, Fab ２ , Fab ３ , Fab' fragment, F(ab') ２ 10. The anti-CD3 antibody or antigen-binding antibody fragment of claim 1, 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 bispecific antibody, 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).

9. (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, optionally having the following properties: (i) the second antigen-binding region is specific for a tumor target, a target molecule expressed on a cancer cell, an immune tumor target, a target molecule expressed on an immune cell, a neurodegenerative disease target, 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 a receptor thereof), an infectious disease target (optionally a viral, bacterial, or fungal target molecule), a target molecule expressed on an infected cell (optionally 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; (ii) 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, ADAM15, and ADAM17 / T ACE, ADAM8, ADAM9, ADAMTS, ADAMTS4, ADAMTS5, addressin, aFGF, ALCAM, ALK, ALK-1, ALK-7, alpha-l-antitrypsin, alpha-V / beta-1 antagonist, ANG, Ang, APAF-1, APE, APJ, APP, APRIL, AR, ARC, ART, artemin, anti-Id, ASPARTIC, atrial natriuretic factor, av / b3 integrin, A xl, 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), carcinoma-associated antigen, cathepsin A, cathepsin B, Cathepsin C / DPPI, cathepsin D, cathepsin E, cathepsin H, cathepsin L, cathepsin O, cathepsin S, cathepsin V, cathepsin X / Z / P, CBL, CCI, CCK2, CCL, CCL1, CCL11, CCL12, CCL13, 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, CDlla, CDll b, CDllc, 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, CXCL 4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCR, CXCR1, CXCR 2, CXCR3, CXCR4, CXCR5, CXCR6, cytokeratin tumor-associated antigen, DAN, DCC, DcR3, DC-SIGN, decay-accelerating factor, des(l-3)-IGF-I (brain IGF-1), Dhh, digoxin, DNAM-1, Dnase, Dpp, DPPIV / CD26, Dtk, ECAD, EDA, EDA-A1, EDA-A2, EDAR, EGF, EGFR (ErbB-1), EMA, EMMPRIN, EN A, endothelin receptor, enkephalinase, eNOS, Eot, eotaxin 1, EpCAM, ephrin B2 / EphB4, EPO, ERCC, E-selectin, ET-1, factor Ila, factor VII, factor VIIIc, factor IX, fibroblast activation protein (FAP), Fas, FcR1, FEN-1, ferritin, FGF, FGF-19, FGF-2, FGF3, FGF-8, FGFR, FGFR-3, fibrin, FL, FLIP, Flt-3, Flt-4, follicle-stimulating hormone, fractalkine, FZD1, FZD2, FZD3, FZD4, FZD5 , FZD6, FZD7, FZD8, FZD9, FZD10, G250, Gas6, GCP-2, GCSF, GD2, GD3, GDF, GDF-1, GDF-3 (Vgr-2), GDF-5 (BMP-14, CDMP-1), GDF-6 (BMP-13, CDMP-2), GDF-7 (BMP-12, CDMP-3), GDF-8 (myostatin), GDF-9, GDF-15 (MIC-1), GDNF, GFAP, GFRα-1, GFR-alpha1, GFR-alpha2, GFR-alpha3, GITR, glucagon, Glut4, glycoprotein Ilb / IIIa (GP Ilb / 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 gp 120 V3 loop, HLA, HLA-DR, HM1.24, HMFG PEM, HRG, Hrk, human cardiac myosin, human cytomegalovirus (HCMV), human growth hormone (HGH), HVEM, 1-309, IAP, ICAM, ICAM-1, ICAM-3, ICE, ICOS, IFNg, Ig, IgA receptor, IgE, IGF, IGF-binding protein, IGF-1R, IGFBP, IGF-I, IGF-II, IL, IL-1, IL-1R, 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, metalloprotein ase, MGDF receptor, MGMT, MHC (HLA-DR), MIF, MIG, MIP, MIP-1-alpha, MK, MMAC1, MMP, MMP-1, MMP-10, MMP-11, MMP-12, MMP-13, MMP-14, MMP-15, MMP-2, MMP-24, MMP-3, MMP-7, MMP-8, MMP-9, MPIF, Mpo, MSK, MSP, mucin (Mucl), MUC18, Müllerian inhibitory substance, Mug, MuSK, NAIP, NAP, NCAD, N-cadherin, NCA90, NCAM, NCAM, neprilysin, neurotrophin-3, -4, or -6, neurturin, neuron 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 GF, PDK-1, PECAM, PEM, PF4, PGE, PGF, PGI2, PGJ2, PIN, PLA2, placental alkaline phosphatase (PLAP), PIGF, PLP, PP14, proinsulin, prorelaxin, protein C, PS, PSA, PSCA, prostate-specific membrane antigen (PSMA), PTEN, PTHrp, Ptk, PTN, R51, RANK, RANKL, RANTES, relaxin A-chain, relaxin B-chain, renin, respiratory syncytial virus (RSV) F, RSVFgp, Ret, rheumatoid factor, RLIP76, RPA2, RSK, S100, SCF / KL, SDF-1, serine, serum albumin, sFRP-3, Shh, SIGIRR, SK-1, SLAM, SLPI, SMAC, SMDF, SMOH, SOD, SPARC, Stat, STEAP, STEAP-II, TACE, TACI, TAG-72 (tumor-associated glycoprotein-72), TARC, TCA-3, T cell receptor (e.g., T cell receptor alpha / beta), TdT, TECK, TEM1, TEM5, TEM7, TEM8, TERT, testicular PLAP-like alkaline phosphatase, T fR, TGF, TGF-alpha, TGF-beta, TGF-beta Pan specific, TGF-beta RI (ALK-5), TGF-beta RII, TGF-beta R11b, 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 Rl Apo-2, DR4), TNFRSFIOB (TRAIL R2 DR5, KILLER, TRICK-2A, TRICK-B), TNFRSF10C (TRAIL R3 DcRl, LIT, TRID), TNFRSF10D (TRAIL R4 DcR2, TRUNDD), TNFRSF11A (RANK ODF R, TRANCE R), TNFRSFllB (OPG OCIF, TR1), TNFRSF12 (TWEAK R FN14), TNFRSF13B (TACI), TNFRSF13C (BAFF R), TNFRSF14 (HVEM ATAR, H THIS、LOVES 100000000000000000000000000000 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) 、WHISCH5(CH040 50)、SWHWH(CH DA11、HAR1、DAY5)、DYSCHYS(DYS3 H68、S6)、S6S67(S027)、 FASHION(SHR30)、SHASHES(400) HR137、HARSH、SHRYSY21 DASH) 、SHASH22(DASH) 2 SHY2)、SYSYS23(SYSYS) SHYS1)、SHYSYS25(GY3 FASH3、SHASH、SHASH、SHASH 、SHAKE1)、SHASE100(SHARE). DAY2リガンド、LOY2)、SHAS11(SHAS SHASE RHリガンドDAY 、DAYリガンド)、DAYS12(SIR). DA3リガンド、DAY3リガンド)、DAYDAY13(SYS) LOVE2)、SIGNIFICANCE130 LOVE SY、 LOVE1 LOVE LOVE LOVE LOVE LOVE LOVE LIKEリガンド、THE)、SHOSE 15(THE) LIKE)、DYSYS18(SYSリガンドDAYSリガンドン、DYS、DYSYS2)、DYSYSYS LIKE、SYSYS1)、SYSYS(LOVETNFC, p33), TNFSF4 (OX40 ligand gp34, TXGP1), TNFSF5 (CD40 ligand CD154, gp39, HIGM1, IMD3, TRAP), TNFSF6 (Fas ligand Apo-1 ligand, APT1 ligand), TNFSF7 (CD27 ligand CD70), TNFSF8 (CD30 ligand CD153), TNFSF9 (4-1BB ligand CD137 ligand), TP-1, t-PA, Tpo, TRAIL, TRAIL R, TRAIL-R1, TRAIL-R2, TRANCE, transferrin receptor, TRF, Trk, TROP-2, TSG, TSLP, tumor-associated antigen CA 125, Lewis Y-related carbohydrate-expressing tumor-associated antigen, TWEAK, TXB2, Ung, uPAR, uPAR-1, urokinase, VCAM, VCAM-1, VECAD, VE-cadherin, VE-cadherin-2, VEFGR-1 (flt-1), VEGF, VEGFR, VEGFR-3 (flt-4), VEGI, VFM, viral antigen, VLA, VLA-1, VLA-4, VNR integrin, von Willebrand factor, WIF-1, WNT1, WNT2, WNT2B / 13, WNT3, WNT3A, WNT4, WNT5A, WNT5B, WNT6, specific for one or more of WNT7A, WNT7B, WNT8A, WNT8B, WNT9A, WNT9A, WNT9B, WNT10A, WNT10B, WNT11, WNT16, XCL1, XCL2, XCR1, XCR1, XEDAR, XIAP, XPD, CTLA4 (cytotoxic T lymphocyte antigen-4), PD1 (programmed cell death protein 1), PD-L1 (programmed cell death ligand 1), LAG-3 (lymphocyte activation gene-3), TIM-3 (T-cell immunoglobulin and mucin protein-3), hormone receptors, and growth factors; (iii) the second antigen-binding region is specific for one or more of BCMA, CTLA4 (cytotoxic T lymphocyte antigen-4), PD1 (programmed cell death protein 1), PD-L1 (programmed cell death ligand 1), LAG-3 (lymphocyte activation gene-3), TIM-3, CD20, CD2, CD19, Her2, EGFR, EpCAM, FcyRIIIa (CD16), FcyRIIa (CD32a), FcyRIIb (CD32b), FcyRI (CD64), Toll-like receptor (TLR), TLR4, TLR9, cytokine, IL-2, IL-5, IL-13, IL-6, IL-17, IL-12, IL-23, TNFα, TGFβ, cytokine receptor, IL-2R, chemokine, chemokine receptor, growth factor, VEGF, and HGF; (iv) the multispecific antibody or antibody fragment is bispecific; (v) the multispecific antibody or antibody fragment further comprises a third antigen-binding region; (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 (viii) the multispecific antibody or antibody fragment comprises one or more of the following: at least one CLK-preferred variant CH1 domain, optionally a CLK-preferred variant CH1 domain described in WO2021067404; at least one CLλ-preferred variant CH1 domain, optionally a CLλ-preferred variant CH1 domain 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 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 a pair described in WO2022150785; 2. The anti-CD3 antibody or antigen-binding antibody fragment of claim 1, comprising or consisting of a multispecific antibody or antibody fragment having at least a second antigen-binding region comprising one or more of:

10. The following characteristics: (I) (i) a reduced PSR score compared to any one or more or all of Antibody Nos. A001-A005; and / or (ii) a PSR score of less than 0.33, less than about 0.30, less than about 0.20, less than about 0.15, less than about 0.12, less than about 0.10, less than about 0.08, less than about 0.06, less than about 0.05, less than about 0.04, less than about 0.03, less than about 0.02, or less than about 0.01; and / or (iii) exhibits a PSR score of ≧ about 0.10 and < 0.33 or a PSR score of about < 0.10; (II) exhibits a hydrophobic interaction chromatography (HIC) retention time of less than about 10.5 minutes, less than about 10.0 minutes, less than about 9.5 minutes, less than about 9.0 minutes, or less than about 8.5 minutes; (III) (i) a reduced affinity capture self-interacting nanoparticle spectroscopy (AC-SINS) Δλmax compared to any one or more or all of Antibody Nos. A001-A005; (ii) an AC-SINS Δλmax of less than about 20.0 nm, less than about 15.0 nm, less than about 12.0 nm, less than about 10.0 nm, less than about 8.0 nm, less than about 6.0 nm, less than about 5.0 nm, less than about 4.0 nm, less than about 3.0 nm, less than about 2.0 nm, or less than about 1.0 nm; and / or (iii) exhibits an AC-SINS Δλmax of ≧about 5.0 nm and <20.0 or an AC-SINS Δλmax of about <5.0 nm; (IV) (i) a higher dynamic light scattering (DLS) diffusion interaction parameter (kD) compared to any one or more or all of Antibody Nos. A001 to A005; (ii) a DLS kD of about 5 mL / g or greater, about 10 mL / g or greater, about 15 mL / g or greater, about 20 mL / g or greater, about 25 mL / g or greater, about 30 mL / g or greater, or about 35 mL / g or greater; and / or (iii) exhibits a DLS kD of about 10 mL / g to about 40 mL / g, about 15 mL / g to about 40 mL / g, or about 20 mL / g to about 40 mL / g; Optionally, the DLS kD is measured using 10 mM histidine buffer, optionally at pH 6.

0. (V) (i) a melting point (Tm) of about 65°C or greater; or (ii) a Tm of about 70°C or greater, about 75°C or greater, or about 80°C or greater; and / or (iii) a Tm of about 70°C to about 90°C, about 75°C to about 85°C; Optionally, the anti-CD3 antibody or antigen-binding antibody fragment is a Fab. (VI) optionally, (i) a percent monomer of about 90% or greater, about 95% or greater, about 97% or greater, about 98% or greater, or about 99% or greater by size exclusion chromatography (SEC); and / or (ii) does not significantly aggregate or multimerize as determined by SEC with a percent monomer of about 90% to about 100%, about 95% to about 100%, about 97% to about 100%, about 98% to about 100%, or about 99% to about 100%; Optionally, the anti-CD3 antibody or antigen-binding antibody fragment is a Fab or IgG, optionally an IgG1; Optionally, the anti-CD3 antibody or antigen-binding antibody fragment does not significantly aggregate or multimerize under acidic conditions, optionally at a pH of about 6 or less, about 5 or less, about 4 or less, or about 3.5 or less; (VII) optionally, the anti-CD3 antibody or antigen-binding antibody fragment, when recombinantly produced, further optionally in mammalian cells, even more optionally in Chinese hamster ovary (CHO) cells, optionally does not exhibit a significant amount of heavy chain-light chain mispairing, as determined by the absence of unpaired heavy chain peaks and / or unpaired light chain peaks, as measured by liquid chromatography mass spectrometry (LC-MS). (VIII) binding to a CD3-expressing cell, and optionally, the CD3-expressing cell: (i) T cells, (ii) human cells, non-human primate (optionally monkey, and further optionally cynomolgus and / or rhesus) cells, and / or rodent (optionally mouse) cells, and / or (iii) is a primary cell or a cell line cell; (IX) optionally measured by surface plasmon resonance (SPR), further optionally by surface plasmon resonance (SPR) using a BIACORE® system, or optionally measured by biolayer interferometry (BLI), further optionally by biolayer interferometry (BLI) using an Octet® system; (i) About 1.0×10 ６ M or less, about 5.0×10 ７ M or less, about 1.0×10 ７ M or less, about 5.0×10 ８ M or less, about 1.0×10 ８ M or less, about 5.0×10 ９ M or less, about 1.0×10 ９ M or less, about 5.0×10 １０ Below M, about 1.0×10 １０ Below M, (ii) Approximately 1.0×10 ６ M ~ approx. 1.0 x 10 １１ M, approximately 1.0 x 10 ７ M ~ approx. 1.0 x 10 １１ M, or approximately 1.0 x 10 ８ M ~ approx. 1.0 x 10 １０ binds to CD3 with an equilibrium dissociation constant (Kd) of M Optionally, the CD3 is human, non-human primate (optionally monkey, and further optionally cynomolgus and / or rhesus), and / or rodent (optionally mouse) CD3; (X) upon binding to CD3 on a cell, induces activation and / or enhances cytotoxic function of said cell, optionally a T cell; (X) upon binding to CD3 on a cell, does not induce cytokine production by said cell to a level capable of inducing cytokine release syndrome (CRS); and / or (XI) A multispecific antibody or antibody fragment comprising 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) said second antigen expressed on a second cell, said first cell exhibits cytotoxicity against said second cell.

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

11. 2. A nucleic acid encoding the anti-CD3 antibody or antigen-binding antibody fragment of claim 1, 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: 1350, 250, 350, 450, 550, 650, 750, 850, 950, 1050, 1150, 1250, 1450, 1550, 1650, 1750, 1850, or 1950; 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: 1360, 260, 360, 460, 560, 660, 760, 860, 960, 1060, 1160, 12560, 1460, 1560, 1660, 1760, 1860, or 1960; Further optionally, (I) SEQ ID NOs: 1350 and 1360, respectively; (II) SEQ ID NOs: 250 and 260, respectively; (III) SEQ ID NOs: 350 and 360, respectively; (IV) SEQ ID NOs: 450 and 460, respectively; (V) SEQ ID NOs: 550 and 560, respectively; (VI) SEQ ID NOs: 650 and 660, respectively; (VII) SEQ ID NOs: 750 and 760, respectively; (VIII) SEQ ID NOs: 850 and 860, respectively; (IX) SEQ ID NOs: 950 and 960, respectively; (X) SEQ ID NOs: 1050 and 1060, respectively; (XI) SEQ ID NOs: 1150 and 1160, respectively; (XII) SEQ ID NOs: 1250 and 1260, respectively; (XIII) SEQ ID NOs: 1450 and 1460, respectively; (XIV) SEQ ID NOs: 1550 and 1560, respectively; (XV) SEQ ID NOs: 1650 and 1660, respectively; (XVI) SEQ ID NOs: 1750 and 1760, respectively; (XVII) SEQ ID NOs: 1850 and 1860, respectively; or (XVIII) 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: 1950 and 1960, respectively; a vector comprising said nucleic acid, optionally comprising: (i) an expression vector; and / or (ii) a vector comprising a plasmid, a viral vector (optionally adenoviral, lentiviral, or retroviral), 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; or an isolated or recombinant cell comprising, transfected with or transduced with said nucleic acid and / or said vector, 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, said immune cell further optionally being a T cell, and / or a natural killer (NK) cell.

12. 1. A pharmaceutical composition comprising: (A) an anti-CD3 antibody or antigen-binding antibody fragment according to any one of claims 1 to 10, and / or a nucleic acid, vector, and / or isolated or recombinant cell according to claim 11, and (B) A pharmaceutical composition comprising a pharmaceutically acceptable carrier and / or excipient. 。

13. A method for producing the anti-CD3 antibody or antigen-binding antibody fragment of any one of claims 1 to 10, comprising: (a) culturing a cell containing the nucleic acid of claim 11 under conditions that allow expression of the antibody or antigen-binding antibody fragment; and (b) recovering and purifying said antibody or antigen-binding antibody fragment from said culturing cell culture of (a).

14. 12. A method for producing an isolated or recombinant cell or a population of such cells according to claim 11, comprising introducing a nucleic acid and / or vector according to claim 11 into one or more cells, optionally wherein said introduction occurs in vitro, ex vivo or in vivo.

15. for use in the treatment of a disease, disorder, or condition; and / or To induce cytotoxicity against cells expressing a target molecule of interest 13. The pharmaceutical composition according to claim 12, Optionally, (1) 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; (iii) has or is at risk of developing a disease, disorder, or condition; and / or (iv) has been administered an additional agent, optionally an adjuvant or therapeutic agent; (2) 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 intestine, leukemia ... 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 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 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 common human coronavirus), norovirus, urinary tract infection ... (2) 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, or Vibrio vulnificus infection; and / or (3) 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.

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