Heterodimeric tetravalency and specificity antibody and uses thereof

Abstract

To provide immunoglobulin-related compositions (e.g., heterodimeric trivalent / tetravalent multispecific antibodies) that specifically bind to three or four distinct target antigens, where the immunoglobulin-related compositions described herein are useful in methods for detecting and treating cancer in a subject in need thereof.SOLUTION: A heterodimeric multispecific antibody comprises a first polypeptide chain, a second polypeptide chain, a third polypeptide chain and a fourth polypeptide chain, wherein the first and second polypeptide chains are covalently bonded to one another, the second and third polypeptide chains are covalently bonded to one another, and the third and fourth polypeptide chain are covalently bonded to one another and respective polupeptide chains have a specific sequence.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Cross-reference to related patent applications This application claims the benefit of and priority to U.S. Provisional Application No. 62 / 774,111, filed November 30, 2018, and U.S. Provisional Application No. 62 / 794,523, filed January 18, 2019, which are incorporated herein by reference in their entireties. Technical Field The present technology generally relates to the preparation of heterodimeric trivalent / tetravalent multispecific antibodies that specifically bind to three or four distinct target antigens, and their use. The heterodimeric trivalent / tetravalent multispecific antibodies described herein are useful in methods for detecting and treating cancer in subjects in need thereof. [Background technology]

[0002] The following background of the present technology is presented merely as an aid to understanding the present technology and is not admitted to describe or constitute prior art to the present technology. Many antibody platforms exist, including heterodimeric IgG and BiTEs. See Spiess et al., Mol Immunol 67:95-106 (2015); Shima et al., N Engl J Med 374:2044-2053 (2016); Topp et al., Lancet Oncol 16:57-66 (2015). However, no single antibody platform currently demonstrates clear and significant functional advantages over other platforms in the clinic.

[0003] For multispecific antibodies that engage immune cells, such as BiTEs, the ideal structure that maximizes antitumor activity is not defined and is likely to vary based on the target antigen or parent antibody (Wu & Cheung, Pharmacology & Therapeutics 182:161-175 (2018)). Important properties may include antigen size and proximity to the cell membrane, as well as serum half-life. See Bluemel et al., Cancer Immunol Immunother 59:1197-1209 (2010); Suzuki et al., J Immunol 184:1968-1976 (2010); Yang et al., Cancer Res 64:6673-6678 (2004). The spatial orientation, strength of each individual specific interaction, or number of interactions conferred by antibodies at the cell-cell interface is even less understood. Furthermore, the size of the antibody format, the flexibility of each binding domain, and their relative orientation to one another can also affect the ability to properly or effectively engage multiple antigens at once. Given these different complexities, it is crucial to understand whether a given platform design is properly optimized for therapeutic function. Summary of the Invention [Problem to be solved by the invention]

[0004] The present invention provides the preparation of heterodimeric trivalent / tetravalent multispecific antibodies that specifically bind to three or four distinct target antigens, and uses thereof. The heterodimeric trivalent / tetravalent multispecific antibodies described herein are useful in methods for detecting and treating cancer in subjects in need thereof. [Means for solving the problem]

[0005] In one aspect, the disclosure provides a polypeptide comprising a first polypeptide chain, a second polypeptide chain, a third polypeptide chain, and a fourth polypeptide chain, wherein the first polypeptide chain and the second polypeptide chain are covalently coupled to each other, the second polypeptide chain and the third polypeptide chain are covalently coupled to each other, and the third polypeptide chain and the fourth polypeptide chain are covalently coupled to each other, and wherein (a) the first polypeptide chain comprises, in an N-terminal to C-terminal direction: (i) a light chain variable domain of a first immunoglobulin capable of specifically binding to a first epitope; (ii) a light chain constant domain (CL-1) of a first immunoglobulin; (iii) a flexible peptide linker comprising the amino acid sequence (GGGGS)3; and (iv) a light chain variable domain (VL-2) of a second immunoglobulin linked to a heavy chain variable domain (VH-2) of a complementary second immunoglobulin, or a heavy chain variable domain (VH-2) of a second immunoglobulin linked to a light chain variable domain (VL-2) of a complementary second immunoglobulin, wherein the VL-2 and VH-2 specifically bind to a second epitope. (b) a second polypeptide comprising, from N-terminal to C-terminal direction: (i) a heavy chain variable domain (VH-1) of a first immunoglobulin capable of specifically binding to a first epitope; (ii) a first CH1 domain (CH1-1) of the first immunoglobulin; and (iii) a first heterodimerization domain of the first immunoglobulin, the first heterodimerization domain being capable of specifically binding to another first heterodimerization domain. (c) a third polypeptide comprising, from N-terminal to C-terminal direction: (i) a heavy chain variable domain (VH-3) of a third immunoglobulin capable of specifically binding to a third epitope; (ii) a second CH1 domain (CH1-3) of the third immunoglobulin; and (iii) a second heterodimerization domain of the third immunoglobulin, which comprises an amino acid sequence or nucleic acid sequence distinct from the first heterodimerization domain of the first immunoglobulin;(d) a fourth polypeptide comprising, from N-terminal to C-terminal direction: (i) a light chain variable domain (VL-3) of a third immunoglobulin capable of specifically binding to a third epitope; (ii) a light chain constant domain (CL-3) of the third immunoglobulin; (iii) a flexible peptidic linker comprising the amino acid sequence (GGGGS)3; and (iv) a light chain variable domain (VL-4) of a fourth immunoglobulin linked to a heavy chain variable domain (VH-4) of a complementary fourth immunoglobulin, or a heavy chain variable domain (VH-4) of a fourth immunoglobulin linked to a light chain variable domain (VL-4) of a complementary fourth immunoglobulin, wherein the VL-4 and VH-4 are capable of specifically binding to a second epitope, and the VL-4 or VH-4 are linked together via a flexible peptide linker comprising the amino acid sequence (GGGGS)6 to form a single-chain variable fragment. , VL-1 and VL-3 are each independently selected from the group consisting of SEQ ID NOs: 1, 9, 17, 25, 33, 41, 49, 57, 65, 73, 81, 89, 97, 105, 113, 121, 129, 145, 153, 161, 169, 177, 193, 201, 233, 241, 257, 273, 281, 289, 297, 305, 313, 321, 329, 337, 345, 353, 361, 369, 377, 385, 393, 401, 409, 417, 425, 433, 441, 449, 457, 465, 481, 489, 497, 521, 529, 537, 545, 553 , 561, 609, 617, 681, 689, 697, 705, 713, 721, 729, 737, 745, 753, 761, 769, 777, 785, 793, 801, 809, 817, 825, 833, 841, 849, 857, 865, 873, 881, 889, 945, 953, 961, 977, 985, 993, 1001, 1009, 1017, 1025, 1033, 1041, 1049, 1065, 1073, 1081, 1089, 1097, 1105, 1113, 1121, 1129, 1137, 1145, 1153, 1161, 1169,1177, 1185, 1193, 1201, 1209, 1217, 1225, 1233, 1241, 1249, 1257, 1265, 1273, 1281, 1289, 1297, 1305, 1313, 1321, 1329, 1337, 1345, 1353, 1361, 1369, 1377, 1385, 1393, 1401, 1409, 1417, 1425, 1433, 1441, 1449, 1450 57, 1465, 1473, 1481, 1489, 1497, 1505, 1513, 1521, 1529, 1545, 1553, 1561, 1569, 1577, 1585, 1593, 1601, 1609, 1617, 1625, 1633, 1649, 1657, 1673, 1681, 1689, 1697, 1705, 1713, 1721, 1729, 1737, 1745, 1753, 1761, 1769, 1777, 1785, 1793, 1801, 1809, 1817, 1833, 1841, 1849, 1857, 1865, 1873, 1881, 1889, 1913, 1937, 1945, 1953, 1961, 1969, 1977, 1985, 1993, 2001, 2009, 2017, 2025, 2033, 2041, 2049, 2057, 2065, 2073, 2081, 2082, 2083, 2084, 2085, 2086, 2087, 2088, 2089, 2090, 2091, 2092, 2093, 2094, 2095, 2096, 2097, 2098, 2099, 2000, 2001, 2002, 2003, 2004, 2005, 2006, 2007, 2008, 2009, 2010, 2011, 2012, 2013, 2014, 2015, 2016, 2017, 2018, 2019, 2020, 2021, 2022, 2023, 2024, 2025, 2026, 2027, 2028, 2029, 2030, 2031, V selected from any one of 89, 2097, 2105, 2113, 2121, 2129, 2137, 2145, 2153, 2161, 2169, 2177, 2185, 2193, 2201, 2209, 2217, 2225, 2233, 2241, 2249, 2257, 2265, 2273, 2281, 2297, 2305, 2313, 2321, 2329, 2337, and 2345; Land / or each of VH-1 and VH-3 independently comprises a CDR1 sequence, a CDR2 sequence, and a CDR3 sequence of the amino acid sequences SEQ ID NOs: 5, 13, 21, 29, 37, 45, 53, 61, 69, 77, 85, 93, 101, 109, 117, 125, 133, 149, 157, 165, 173, 181, 197, 205, 237, 245, 261, 277, 285, 293, 301, 309, 317, 325, 333, 341, 349, 357, 365, 373, 381, 389, 397, 405, 413, 421, 429, 437, 445, 453 , 461, 469, 485, 493, 501, 525, 533, 541, 549, 557, 565, 613, 621, 685, 693, 701, 709, 717, 725, 733, 741, 749, 757, 765, 773, 781, 789, 797, 805, 813, 821, 829, 837, 845, 853, 861, 869, 877, 885, 893, 949, 957, 965, 981, 989, 997, 1005, 1013, 1021, 1029, 1037, 1045, 1053, 1069, 1077, 1085, 1093, 1101, 1109, 1117, 1125, 1133, 1141, 1149, 1157, 1165, 1173, 1181, 1189, 1197, 1205, 1213, 1221, 1229, 1237, 1245, 1253, 1261, 1269, 1277, 1285, 1293, 1301, 1309, 1317, 1325, 1333, 1341, 1349, 1357, 1365, 1373, 1381, 1389, 1397, 1405, 1413, 1421, 1429, 1437, 1445, 1453, 1461, 1469, 1477, 1485, 1493, 1501, 1509, 1517, 1525, 1533, 1549, 1557, 1565, 1573, 1581, 1589, 1597, 1605, 1613, 1621, 1629, 1637, 1653, 1661, 1677, 1685, 1693, 1701, 1709, 1717, 1725, 1733, 1741, 1749, 1757, 1765, 1773, 1781, 1789, 1797, 1805, 1813, 1821, 1837, 1845, 1853, 1861, 1869, 1877, 1885, 1893, 1917, 1941, 1949, 1957, 1965, 1973,V selected from any one of 1981, 1989, 1997, 2005, 2013, 2021, 2029, 2037, 2045, 2053, 2061, 2069, 2077, 2085, 2093, 2101, 2109, 2117, 2125, 2133, 2141, 2149, 2157, 2165, 2173, 2181, 2189, 2197, 2205, 2213, 2221, 2229, 2237, 2245, 2253, 2261, 2269, 2277, 2285, 2301, 2309, 2317, 2325, 2333, 2341, and 2349; H Heterodimeric multispecific antibodies are provided, comprising amino acid sequences of CDR1, CDR2, and CDR3 sequences.

[0006] In one aspect, the disclosure provides a polypeptide comprising a first polypeptide chain, a second polypeptide chain, a third polypeptide chain, and a fourth polypeptide chain, wherein the first polypeptide chain and the second polypeptide chain are covalently coupled to each other, the second polypeptide chain and the third polypeptide chain are covalently coupled to each other, and the third polypeptide chain and the fourth polypeptide chain are covalently coupled to each other, and wherein (a) the first polypeptide chain specifically binds, in an N-terminal to C-terminal direction: (i) to a first epitope; (ii) a light chain constant domain (CL-1) of the first immunoglobulin; (iii) a flexible peptide linker comprising the amino acid sequence (GGGGS)3; and (iv) a light chain variable domain (VL-2) of a second immunoglobulin linked to a heavy chain variable domain (VH-2) of a complementary second immunoglobulin, or a heavy chain variable domain (VL-2) of a second immunoglobulin linked to a light chain variable domain (VH-2) of a complementary second immunoglobulin. (b) a second polypeptide comprising, from N-terminal to C-terminal direction: (i) a heavy chain variable domain (VH-1) of a first immunoglobulin capable of specifically binding to a first epitope; (ii) a heavy chain variable domain (VH-2) of a first immunoglobulin capable of specifically binding to a second epitope; (iii) a heavy chain variable domain (VH-3) of a first immunoglobulin capable of specifically binding to a second epitope; (iv) a heavy chain variable domain (VH-4) of a first immunoglobulin capable of specifically binding to a second epitope; (v) a heavy chain variable domain (VH-5) of a first immunoglobulin capable of specifically binding to a second epitope; (v) a heavy chain variable domain (VH-6) of a first immunoglobulin capable of specifically binding to a second epitope; (v) a heavy chain variable domain (VH-7) of a first immunoglobulin capable of specifically binding to a second epitope; (v) a heavy chain variable domain (VH-8) of a first immunoglobulin capable of specifically binding to a second epitope; (v) a heavy chain variable domain (VH-9) of a first immunoglobulin capable of specifically binding to a second epitope; (v) a heavy chain variable domain (VH-1 ... first CH1 domain (VH-1) of a first immunoglobulin capable of specifically binding to a second epitope; (v) a first CH2 domain (VH-1) of a first immunoglobulin capable of specifically binding to a second epitope; (v) a first CH3 domain (VH-1) of a first immunoglobulin capable of specifically binding to a second epitope; (v) a first CH4 domain (VH-1) of a first immunoglobulin capable of specifically binding to a second epi (iii) a first heterodimerization domain of a first immunoglobulin, the first heterodimerization domain being incapable of forming a stable homodimer with another first heterodimerization domain; and (c) a third polypeptide comprising, from N-terminal to C-terminal direction: (i) a heavy chain variable domain (VH-3) of a third immunoglobulin capable of specifically binding to a first epitope; (ii) a second CH1 domain (CH1-3) of the third immunoglobulin;and (iii) a second heterodimerization domain of a third immunoglobulin, the second heterodimerization domain comprising an amino acid sequence or nucleic acid sequence distinct from the first heterodimerization domain of the first immunoglobulin, the second heterodimerization domain being incapable of forming stable homodimers with another second heterodimerization domain, and being configured to form a heterodimer with the first heterodimerization domain of the first immunoglobulin; (d) the fourth polypeptide comprising, from N-terminal to C-terminal direction: (i) a light chain variable domain (VL-3) of the third immunoglobulin capable of specifically binding to the first epitope; (ii) a light chain constant domain (CL-3) of the third immunoglobulin; (iii) a flexible peptide linker comprising the amino acid sequence (GGGGS)3;and (iv) a light chain variable domain of a fourth immunoglobulin (VL-4) linked to a heavy chain variable domain of a complementary fourth immunoglobulin (VH-4), or a heavy chain variable domain of a fourth immunoglobulin (VH-4) linked to a light chain variable domain of a complementary fourth immunoglobulin (VL-4), wherein the VL-4 and VH-4 are capable of specifically binding to a third epitope, and wherein the VL-4 and VH-4 are linked together via a flexible peptide linker comprising the amino acid sequence (GGGGS) to form a single-chain variable fragment, wherein each of VL-2 and VL-4 is independently selected from the group consisting of SEQ ID NOs: 17, 25, 33, 41, 121, 137, 169, 177, 185, 193, 201, 209, 217, 225, 233, 24 1, 249, 257, 265, 321, 329, 337, 393, 401, 409, 473, 481, 489, 497, 505, 513, 545, 553, 561, 569, 577, 585, 593, 601, 625, 633, 641, 649, 657, 665, 673, 681, 689, 697, 705, 713, 721, 729, 737, 745, 753, 76 V selected from any one of 1, 769, 785, 793, 801, 809, 817, 849, 857, 865, 873, 881, 889, 897, 905, 913, 921, 929, 937, 945, 969, 977, 1009, 1057, 1537, 1569, 1601, 1641, 1665, 1825, 1865, 1897, 1905, 1913, 1921, 1929, 2265, 2281 2289, 2329, and 2345; Land / or each of VH-2 and VH-4 independently comprises the amino acid sequences of SEQ ID NOs: 21, 29, 37, 45, 125, 141, 173, 181, 189, 197, 205, 213, 221, 229, 237, 245, 253, 261, 269, 325, 333, 341, 397, 405, 413, 477, 485, 493, 501, 509, 517, 549, 557, 565, 573, 581, 589, 597, 605, 629, 637, 645, 653, 661, 669, 677 , 685, 693, 701, 709, 717, 725, 733, 741, 749, 757, 765, 773, 789, 797, 805, 813, 821, 853, 861, 869, 877, 885, 893, 901, 909, 917, 925, 933, 941, 949, 973, 981, 1013, 1061, 1541, 1573, 1605, 1645, 1669, 1829, 1869, 1901, 1909, 1917, 1925, 1933, 2269, 2285, 2293, 2333, and 2349. H Heterodimeric multispecific antibodies are provided, comprising amino acid sequences of CDR1, CDR2, and CDR3 sequences.

[0007] In another aspect, the disclosure provides a polypeptide comprising a first polypeptide chain, a second polypeptide chain, a third polypeptide chain, and a fourth polypeptide chain, wherein the first polypeptide chain and the second polypeptide chain are covalently linked to each other, the second polypeptide chain and the third polypeptide chain are covalently linked to each other, and the third polypeptide chain and the fourth polypeptide chain are covalently linked to each other, and wherein (a) the first polypeptide chain comprises, in an N-terminal to C-terminal direction: (i) a light chain variable domain of a first immunoglobulin capable of specifically binding to a first epitope; (ii) a light chain constant domain (CL-1) of a first immunoglobulin; (iii) a flexible peptide linker comprising the amino acid sequence (GGGGS)3; and (iv) a light chain variable domain (VL-2) of a second immunoglobulin linked to a heavy chain variable domain (VH-2) of a complementary second immunoglobulin, or a heavy chain variable domain (VH-2) of a second immunoglobulin linked to a light chain variable domain (VL-2) of a complementary second immunoglobulin, wherein the VL-2 and VH-2 specifically bind to a second epitope. (b) a second polypeptide comprising, from N-terminal to C-terminal direction: (i) a heavy chain variable domain (VH-1) of a first immunoglobulin capable of specifically binding to a first epitope; (ii) a first CH1 domain (CH1-1) of the first immunoglobulin; and (iii) a first heterodimerization domain of the first immunoglobulin, the first heterodimerization domain being linked together via a flexible peptide linker comprising the amino acid sequence (GGGGS)6 to form a single-chain variable fragment. (c) a third polypeptide comprising, from N-terminal to C-terminal direction: (i) a heavy chain variable domain (VH-3) of a third immunoglobulin capable of specifically binding to a third epitope; (ii) a second CH1 domain (CH1-3) of the third immunoglobulin; and (iii) a second heterodimerization domain of the third immunoglobulin, which comprises an amino acid sequence or nucleic acid sequence distinct from the first heterodimerization domain of the first immunoglobulin;(d) a fourth polypeptide comprising, from N-terminal to C-terminal direction: (i) a light chain variable domain (VL-3) of a third immunoglobulin capable of specifically binding to a third epitope; (ii) a light chain constant domain (CL-3) of the third immunoglobulin; (iii) a flexible peptidic linker comprising the amino acid sequence (GGGGS)3; and (iv) a light chain variable domain of a fourth immunoglobulin (VL-4) linked to a heavy chain variable domain (VH-4) of a complementary fourth immunoglobulin, or a heavy chain variable domain of a fourth immunoglobulin (VH-4) linked to a light chain variable domain of a fourth immunoglobulin (VL-4), wherein the VL-4 and VH-4 are capable of specifically binding to a fourth epitope, and the VL-4 or VH-4 are linked together via a flexible peptide linker comprising the amino acid sequence (GGGGS)6 to form a single-chain variable fragment. , VL-1 and VL-3 are each independently selected from the group consisting of SEQ ID NOs: 1, 9, 17, 25, 33, 41, 49, 57, 65, 73, 81, 89, 97, 105, 113, 121, 129, 145, 153, 161, 169, 177, 193, 201, 233, 241, 257, 273, 281, 289, 297, 305, 313, 321, 329, 337, 345, 353, 361, 369, 377, 385, 393, 401, 409, 417, 425, 433, 441, 449, 457, 465, 481, 489, 497, 521, 529, 537, 545, 553 , 561, 609, 617, 681, 689, 697, 705, 713, 721, 729, 737, 745, 753, 761, 769, 777, 785, 793, 801, 809, 817, 825, 833, 841, 849, 857, 865, 873, 881, 889, 945, 953, 961, 977, 985, 993, 1001, 1009, 1017, 1025, 1033, 1041, 1049, 1065, 1073, 1081, 1089, 1097, 1105, 1113, 1121, 1129, 1137, 1145, 1153, 1161, 1169,1177, 1185, 1193, 1201, 1209, 1217, 1225, 1233, 1241, 1249, 1257, 1265, 1273, 1281, 1289, 1297, 1305, 1313, 1321, 1329, 1337, 1345, 1353, 1361, 1369, 1377, 1385, 1393, 1401, 1409, 1417, 1425, 1433, 1441, 1449, 1450 57, 1465, 1473, 1481, 1489, 1497, 1505, 1513, 1521, 1529, 1545, 1553, 1561, 1569, 1577, 1585, 1593, 1601, 1609, 1617, 1625, 1633, 1649, 1657, 1673, 1681, 1689, 1697, 1705, 1713, 1721, 1729, 1737, 1745, 1753, 1761, 1769, 1777, 1785, 1793, 1801, 1809, 1817, 1833, 1841, 1849, 1857, 1865, 1873, 1881, 1889, 1913, 1937, 1945, 1953, 1961, 1969, 1977, 1985, 1993, 2001, 2009, 2017, 2025, 2033, 2041, 2049, 2057, 2065, 2073, 2081, 2082, 2083, 2084, 2085, 2086, 2087, 2088, 2089, 2090, 2091, 2092, 2093, 2094, 2095, 2096, 2097, 2098, 2099, 2000, 2001, 2002, 2003, 2004, 2005, 2006, 2007, 2008, 2009, 2010, 2011, 2012, 2013, 2014, 2015, 2016, 2017, 2018, 2019, 2020, 2021, 2022, 2023, 2024, 2025, 2026, 2027, 2028, 2029, 2030, 2031, V selected from any one of 89, 2097, 2105, 2113, 2121, 2129, 2137, 2145, 2153, 2161, 2169, 2177, 2185, 2193, 2201, 2209, 2217, 2225, 2233, 2241, 2249, 2257, 2265, 2273, 2281, 2297, 2305, 2313, 2321, 2329, 2337, and 2345; Land / or each of VH-1 and VH-3 independently comprises a CDR1 sequence, a CDR2 sequence, and a CDR3 sequence of the amino acid sequences SEQ ID NOs: 5, 13, 21, 29, 37, 45, 53, 61, 69, 77, 85, 93, 101, 109, 117, 125, 133, 149, 157, 165, 173, 181, 197, 205, 237, 245, 261, 277, 285, 293, 301, 309, 317, 325, 333, 341, 349, 357, 365, 373, 381, 389, 397, 405, 413, 421, 429, 437, 445, 453 , 461, 469, 485, 493, 501, 525, 533, 541, 549, 557, 565, 613, 621, 685, 693, 701, 709, 717, 725, 733, 741, 749, 757, 765, 773, 781, 789, 797, 805, 813, 821, 829, 837, 845, 853, 861, 869, 877, 885, 893, 949, 957, 965, 981, 989, 997, 1005, 1013, 1021, 1029, 1037, 1045, 1053, 1069, 1077, 1085, 1093, 1101, 1109, 1117, 1125, 1133, 1141, 1149, 1157, 1165, 1173, 1181, 1189, 1197, 1205, 1213, 1221, 1229, 1237, 1245, 1253, 1261, 1269, 1277, 1285, 1293, 1301, 1309, 1317, 1325, 1333, 1341, 1349, 1357, 1365, 1373, 1381, 1389, 1397, 1405, 1413, 1421, 1429, 1437, 1445, 1453, 1461, 1469, 1477, 1485, 1493, 1501, 1509, 1517, 1525, 1533, 1549, 1557, 1565, 1573, 1581, 1589, 1597, 1605, 1613, 1621, 1629, 1637, 1653, 1661, 1677, 1685, 1693, 1701, 1709, 1717, 1725, 1733, 1741, 1749, 1757, 1765, 1773, 1781, 1789, 1797, 1805, 1813, 1821, 1837, 1845, 1853, 1861, 1869, 1877, 1885, 1893, 1917, 1941, 1949, 1957, 1965, 1973,V selected from any one of 1981, 1989, 1997, 2005, 2013, 2021, 2029, 2037, 2045, 2053, 2061, 2069, 2077, 2085, 2093, 2101, 2109, 2117, 2125, 2133, 2141, 2149, 2157, 2165, 2173, 2181, 2189, 2197, 2205, 2213, 2221, 2229, 2237, 2245, 2253, 2261, 2269, 2277, 2285, 2301, 2309, 2317, 2325, 2333, 2341, and 2349; H and / or each of VL-2 and VL-4 independently comprises a CDR1 sequence, a CDR2 sequence, and a CDR3 sequence of the amino acid sequences SEQ ID NOs: 17, 25, 33, 41, 121, 137, 169, 177, 185, 193, 201, 209, 217, 225, 233, 241, 249, 257, 265, 321, 329, 337, 393, 401, 409, 473, 481, 489, 497, 505, 513, 545, 553, 561, 569, 577, 585, 593, 601, 625, 633, 640, 650, 660, 670, 680, 691, 702, 710, 720, 730, 740, 750, 760, 771, 782, 791, 802, 810, 825, 830, 840, 850, 860, 870, 882, 891, 902, 910, 920, 930, 940, 950, 960, 970, 982, 991, 1002, 1010, 1020, 1030, 1040, 1050, 1060, 1070, 1080, 1090, 1102, 1150, 1160, 1170, 41, 649, 657, 665, 673, 681, 689, 697, 705, 713, 721, 729, 737, 745, 753, 761, 769, 785, 793, 801, 809, 817, 849, 857, 865, 873, 881, 889, 897, 905, 913, 921, 929, 937, 945, 969, 977, 1009, 1057, 1537, 1569, 1601, 1641, 1665, 1825, 1865, 1897, 1905, 1913, 1921, 1929, 2265, 2281 V selected from any one of 2289, 2329, and 2345 Land / or each of VH-2 and VH-4 independently comprises the amino acid sequences of SEQ ID NOs: 21, 29, 37, 45, 125, 141, 173, 181, 189, 197, 205, 213, 221, 229, 237, 245, 253, 261, 269, 325, 333, 341, 397, 405, 413, 477, 485, 493, 501, 509, 517, 549, 557, 565, 573, 581, 589, 597, 605, 629, 637, 645, 653, 661, 669, 677 , 685, 693, 701, 709, 717, 725, 733, 741, 749, 757, 765, 773, 789, 797, 805, 813, 821, 853, 861, 869, 877, 885, 893, 901, 909, 917, 925, 933, 941, 949, 973, 981, 1013, 1061, 1541, 1573, 1605, 1645, 1669, 1829, 1869, 1901, 1909, 1917, 1925, 1933, 2269, 2285, 2293, 2333, and 2349. H Heterodimeric multispecific antibodies are provided, comprising amino acid sequences of CDR1, CDR2, and CDR3 sequences.

[0008] In another aspect, the disclosure provides a polypeptide comprising a first polypeptide chain, a second polypeptide chain, a third polypeptide chain, and a fourth polypeptide chain, wherein the first polypeptide chain and the second polypeptide chain are covalently coupled to each other, the second polypeptide chain and the third polypeptide chain are covalently coupled to each other, and the third polypeptide chain and the fourth polypeptide chain are covalently coupled to each other, and wherein (a) the first polypeptide chain specifically binds, in an N-terminal to C-terminal direction: (i) a first epitope; (ii) a light chain constant domain (CL-1) of the first immunoglobulin; (iii) a flexible peptide linker comprising the amino acid sequence (GGGGS)3; and (iv) a light chain variable domain (VL-2) of a second immunoglobulin linked to a heavy chain variable domain (VH-2) of a complementary second immunoglobulin, or a heavy chain variable domain (VL-2) of a second immunoglobulin linked to a light chain variable domain (VH-2) of a complementary second immunoglobulin. (b) a second polypeptide comprising, from N-terminal to C-terminal direction: (i) a heavy chain variable domain (VH-1) of a first immunoglobulin capable of specifically binding to a first epitope; (ii) a heavy chain variable domain (VH-2) of a first immunoglobulin capable of specifically binding to a second epitope; (iii) a heavy chain variable domain (VH-3) of a first immunoglobulin capable of specifically binding to a second epitope; (iv) a heavy chain variable domain (VH-4) of a first immunoglobulin capable of specifically binding to a second epitope; (v) a heavy chain variable domain (VH-5) of a first immunoglobulin capable of specifically binding to a second epitope; (v) a heavy chain variable domain (VH-6) of a first immunoglobulin capable of specifically binding to a second epitope; (v) a heavy chain variable domain (VH-7) of a first immunoglobulin capable of specifically binding to a second epitope; (v) a heavy chain variable domain (VH-8) of a first immunoglobulin capable of specifically binding to a second epitope; (v) a heavy chain variable domain (VH-9) of a first immunoglobulin capable of specifically binding to a second epitope; (v) a heavy chain variable domain (VH-1 ... first CH1 domain (VH-1) of a first immunoglobulin capable of specifically binding to a second epitope; (v) a first CH2 domain (VH-1) of a first immunoglobulin capable of specifically binding to a second epitope; (v) a first CH3 domain (VH-1) of a first immunoglobulin capable of specifically binding to a second epitope; (v) a first CH4 domain (VH-1) of a first immunoglobulin capable of specifically binding to a second epi (iii) a first heterodimerization domain of a first immunoglobulin, the first heterodimerization domain being incapable of forming a stable homodimer with another first heterodimerization domain; and (c) a third polypeptide comprising, from N-terminal to C-terminal direction: (i) a heavy chain variable domain (VH-3) of a third immunoglobulin capable of specifically binding to a first epitope; (ii) a second CH1 domain (CH1-3) of the third immunoglobulin;and (iii) a second heterodimerization domain of a third immunoglobulin, the second heterodimerization domain comprising an amino acid sequence or nucleic acid sequence distinct from the first heterodimerization domain of the first immunoglobulin, the second heterodimerization domain being incapable of forming stable homodimers with another second heterodimerization domain, and being configured to form heterodimers with the first heterodimerization domain of the first immunoglobulin; (d) the fourth polypeptide comprising, from N-terminal to C-terminal direction: (i) a light chain variable domain (VL-3) of the third immunoglobulin capable of specifically binding to a first epitope; and (ii) a light chain constant domain (CL-3) of the third immunoglobulin, wherein VL-2 is selected from the group consisting of SEQ ID NOs: 17, 25, 33, 41, 121, 137, 169, 177, 185, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 335, 340, 350, 360, 370, 380, 390, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 93, 201, 209, 217, 225, 233, 241, 249, 257, 265, 321, 329, 337, 393, 401, 409, 473, 481, 489, 497, 505, 513, 545, 553, 561, 569, 577, 585, 593, 601, 625, 633, 641, 649, 657, 665, 673, 681, 689, 697, 705, 713, 721, 729, 73 V selected from any one of 7, 745, 753, 761, 769, 785, 793, 801, 809, 817, 849, 857, 865, 873, 881, 889, 897, 905, 913, 921, 929, 937, 945, 969, 977, 1009, 1057, 1537, 1569, 1601, 1641, 1665, 1825, 1865, 1897, 1905, 1913, 1921, 1929, 2265, 2281 2289, 2329, and 2345; Land / or VH-2 comprises the CDR1 sequence, CDR2 sequence, and CDR3 sequence of the amino acid sequences SEQ ID NOs: 21, 29, 37, 45, 125, 141, 173, 181, 189, 197, 205, 213, 221, 229, 237, 245, 253, 261, 269, 325, 333, 341, 397, 405, 413, 477, 485, 493, 501, 509, 517, 549, 557, 565, 573, 581, 589, 597, 605, 629, 637, 645, 653, 661, 669, 677, 685, 69 V selected from any one of 3, 701, 709, 717, 725, 733, 741, 749, 757, 765, 773, 789, 797, 805, 813, 821, 853, 861, 869, 877, 885, 893, 901, 909, 917, 925, 933, 941, 949, 973, 981, 1013, 1061, 1541, 1573, 1605, 1645, 1669, 1829, 1869, 1901, 1909, 1917, 1925, 1933, 2269, 2285, 2293, 2333, and 2349 HHeterodimeric multispecific antibodies are provided, comprising amino acid sequences of CDR1, CDR2, and CDR3 sequences. In some embodiments, both the VH-1 and VH-3 are selected from the group consisting of SEQ ID NOs: 5, 13, 21, 29, 37, 45, 53, 61, 69, 77, 85, 93, 101, 109, 117, 125, 133, 149, 157, 165, 173, 181, 197, 205, 237, 245, 261, 277, 285, 293, 301, 309, 317, 325, 333, 341, 349, 357, 365, 373, 381, 389, 397, 405, 413, 421, 429, 437, 445, 453, 461, 469, 485, 493, 501, 502, 503, 504, 505, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, , 525, 533, 541, 549, 557, 565, 613, 621, 685, 693, 701, 709, 717, 725, 733, 741, 749, 757, 765, 773, 781, 789, 797, 805, 813, 821, 829, 837, 845, 853, 861, 869, 877, 885, 893, 949, 957, 965, 981, 989, 997, 1005, 1013, 1021, 1029, 1037, 1045, 1053, 1069, 1077, 1085, 1093, 1101, 1109, 1117, 11 25, 1133, 1141, 1149, 1157, 1165, 1173, 1181, 1189, 1197, 1205, 1213, 1221, 1229, 1237, 1245, 1253, 1261, 1269, 1277, 1285, 1293, 1301, 1309, 1317, 1325, 1333, 1341, 1349, 1357, 1365, 1373, 1381, 1389, 1397, 1405, 1413, 1421, 1429, 1437, 1445, 1453, 1461, 1469, 1477, 1485, 1493, 150 1, 1509, 1517, 1525, 1533, 1549, 1557, 1565, 1573, 1581, 1589, 1597, 1605, 1613, 1621, 1629, 1637, 1653, 1661, 1677, 1685, 1693, 1701, 1709, 1717, 1725, 1733, 1741, 1749, 1757, 1765, 1773, 1781, 1789, 1797, 1805, 1813, 1821, 1837, 1845, 1853, 1861, 1869, 1877, 1885, 1893, 1917, 1941,1949, 1957, 1965, 1973, 1981, 1989, 1997, 2005, 2013, 2021, 2029, 2037, 2045, 2053, 2061, 2069, 2077, 2085, 2093, 2101, 2109, 2117, 2125, 2133, 2141, 2149, 2157, V selected from any one of 2165, 2173, 2181, 2189, 2197, 2205, 2213, 2221, 2229, 2237, 2245, 2253, 2261, 2269, 2277, 2285, 2301, 2309, 2317, 2325, 2333, 2341, and 2349; Hand / or both VL-1 and VL-3 comprise the amino acid sequences CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 1, 9, 17, 25, 33, 41, 49, 57, 65, 73, 81, 89, 97, 105, 113, 121, 129, 145, 153, 161, 169, 177, 193, 201, 233, 241, 257, 273, 281, 289, 297, 305, 313, 321, 329, 337, 345, 353, 361, 369, 377, 385, 393, 401, 409, 417, 425, 433, 441, 449, 457, 465, 481, 489, 497, 521, 529, 537, 545, 553, 561, 609, 617, 681, 689, 697, 705, 713, 721, 729, 737, 745, 753, 761, 769, 777, 785, 793, 801, 809, 817, 825, 8 33, 841, 849, 857, 865, 873, 881, 889, 945, 953, 961, 977, 985, 993, 1001, 1009, 1017, 1025, 1033, 1041, 1049, 1065, 1073, 1081, 1089, 1097, 1105, 1113, 1121, 1129, 1137, 1145, 1153, 1161, 1169, 1177, 1185, 1193, 1201, 1209, 1217, 1225, 1233, 1241, 1249, 1257, 1265, 1273, 1281, 1289, 1297, 1305, 1313, 1321, 1329, 1337, 1345, 1353, 1361, 1369, 1377, 1385, 1393, 1401, 1409, 1417, 1425, 1433, 1441, 1449, 1457, 1465, 1473, 1481, 1489, 1497, 1505, 1513, 1521, 1529, 1545, 1553, 1561, 1569, 1577, 1585, 1593, 1601, 1609, 1617, 1625, 1633, 1649, 1657, 1673, 1681, 1689, 1697, 1705, 1713, 1721, 1729, 1737, 1745, 1753, 1761, 1769, 1777, 1785, 1793, 1801, 1809, 1817, 1833, 1841, 1849, 1857, 1865, 1873, 1881, 1889, 1913, 1937, 1945, 1953, 1961, 1969, 1977,V selected from any one of 1985, 1993, 2001, 2009, 2017, 2025, 2033, 2041, 2049, 2057, 2065, 2073, 2081, 2089, 2097, 2105, 2113, 2121, 2129, 2137, 2145, 2153, 2161, 2169, 2177, 2185, 2193, 2201, 2209, 2217, 2225, 2233, 2241, 2249, 2257, 2265, 2273, 2281, 2297, 2305, 2313, 2321, 2329, 2337, and 2345; L It includes CDR1, CDR2, and CDR3 amino acid sequences.

[0009] In yet another embodiment, the disclosure provides a polypeptide comprising a first polypeptide chain, a second polypeptide chain, a third polypeptide chain, and a fourth polypeptide chain, wherein the first polypeptide chain and the second polypeptide chain are covalently linked to each other, the second polypeptide chain and the third polypeptide chain are covalently linked to each other, and the third polypeptide chain and the fourth polypeptide chain are covalently linked to each other, and wherein (a) the first polypeptide chain comprises, in an N-terminal to C-terminal direction: (i) a first immunoglobulin polypeptide capable of specifically binding to a first epitope; (ii) a light chain constant domain (CL-1) of a first immunoglobulin; (iii) a flexible peptide linker comprising the amino acid sequence (GGGGS)3; and (iv) a light chain variable domain (VL-2) of a second immunoglobulin linked to a heavy chain variable domain (VH-2) of a complementary second immunoglobulin, or a heavy chain variable domain (VH-2) of a second immunoglobulin linked to a light chain variable domain (VL-2) of a complementary second immunoglobulin, wherein the VL-2 and VH-2 are specific for a second epitope. (b) a second polypeptide comprising, from N-terminal to C-terminal direction: (i) a heavy chain variable domain (VH-1) of a first immunoglobulin capable of specifically binding to a first epitope; (ii) a first CH1 domain (CH1-1) of the first immunoglobulin; and (iii) a first heterodimerization domain of the first immunoglobulin, the first heterodimerization domain being linked together via a flexible peptide linker comprising the amino acid sequence (GGGGS)6 to form a single-chain variable fragment. (c) a third polypeptide comprising, from N-terminal to C-terminal direction: (i) a heavy chain variable domain (VH-3) of a third immunoglobulin capable of specifically binding to a third epitope; (ii) a second CH1 domain (CH1-3) of the third immunoglobulin; and (iii) a second heterodimerization domain of the third immunoglobulin, which comprises an amino acid sequence or nucleic acid sequence distinct from the first heterodimerization domain of the first immunoglobulin;(d) a fourth polypeptide comprising, from N-terminal to C-terminal direction: (i) a light chain variable domain (VL-3) of a third immunoglobulin capable of specifically binding to a third epitope; and (ii) a light chain constant domain (CL-3) of the third immunoglobulin, wherein each of VL-1 and VL-3 independently has the sequence of SEQ ID NO: 1, 9, 17, 25, 33, 41, 49, 57, 65, 73, 81, 89, 97, 105, 113, 121, 129, 145, 153, 161, 169, 177, 193, 201, 233, 241, 257, 273, 281, 289, 297, 305, 313, 321, 329, 337, 345, 353, 361, 369, 377, 385, 393, 401, 409, 417, 425, 433, 441, 449, 457, 465, 481, 489, 497, 521, 529, 537, 545, 553, 561, 609, 617, 681, 689, 697, 7 05, 713, 721, 729, 737, 745, 753, 761, 769, 777, 785, 793, 801, 809, 817, 825, 833, 841, 849, 857, 865, 873, 881, 889, 945, 953, 961, 977, 985, 993, 1001, 1009, 1017, 1025, 1033, 1041, 1049, 1065, 1073, 1081, 1089, 1097, 1105, 1113, 1121, 1129, 1137, 1145, 1153, 1161, 1169, 1177, 1185, 1193, 1201, 1209, 1217, 1225, 1233, 1241, 1249, 1257, 1265, 1273, 1281, 1289, 1297, 1305, 1313, 1321, 1329, 1337, 1345, 1353, 1361, 1369, 1377, 1385, 1393, 1401, 1409, 1417, 1425, 1433, 1441, 1449, 1457, 1465, 1473, 1481, 1489, 1497, 1505, 1513, 1521, 1529, 1545, 1553, 1561, 1569, 1577, 1585, 1593, 1601, 1609, 1617,1625, 1633, 1649, 1657, 1673, 1681, 1689, 1697, 1705, 1713, 1721, 1729, 1737, 1745, 1753, 1761, 1769, 1777, 1785, 1793, 1801, 1809, 1817, 1833, 1841, 1849, 1857, 1865, 1873, 1881, 1889, 1913, 1937, 1945, 1953, 1961, 1969, 1977, 1985, 1993, 2001, 2009, 2017, 2018 V selected from any one of 25, 2033, 2041, 2049, 2057, 2065, 2073, 2081, 2089, 2097, 2105, 2113, 2121, 2129, 2137, 2145, 2153, 2161, 2169, 2177, 2185, 2193, 2201, 2209, 2217, 2225, 2233, 2241, 2249, 2257, 2265, 2273, 2281, 2297, 2305, 2313, 2321, 2329, 2337, and 2345; Land / or each of VH-1 and VH-3 independently comprises a CDR1 sequence, a CDR2 sequence, and a CDR3 sequence of the amino acid sequences SEQ ID NOs: 5, 13, 21, 29, 37, 45, 53, 61, 69, 77, 85, 93, 101, 109, 117, 125, 133, 149, 157, 165, 173, 181, 197, 205, 237, 245, 261, 277, 285, 293, 301, 309, 317, 325, 333, 341, 349, 357, 365, 373, 381, 389, 397, 405, 413, 421, 429, 437, 445, 453 , 461, 469, 485, 493, 501, 525, 533, 541, 549, 557, 565, 613, 621, 685, 693, 701, 709, 717, 725, 733, 741, 749, 757, 765, 773, 781, 789, 797, 805, 813, 821, 829, 837, 845, 853, 861, 869, 877, 885, 893, 949, 957, 965, 981, 989, 997, 1005, 1013, 1021, 1029, 1037, 1045, 1053, 1069, 1077, 1085, 1093, 1101, 1109, 1117, 1125, 1133, 1141, 1149, 1157, 1165, 1173, 1181, 1189, 1197, 1205, 1213, 1221, 1229, 1237, 1245, 1253, 1261, 1269, 1277, 1285, 1293, 1301, 1309, 1317, 1325, 1333, 1341, 1349, 1357, 1365, 1373, 1381, 1389, 1397, 1405, 1413, 1421, 1429, 1437, 1445, 1453, 1461, 1469, 1477, 1485, 1493, 1501, 1509, 1517, 1525, 1533, 1549, 1557, 1565, 1573, 1581, 1589, 1597, 1605, 1613, 1621, 1629, 1637, 1653, 1661, 1677, 1685, 1693, 1701, 1709, 1717, 1725, 1733, 1741, 1749, 1757, 1765, 1773, 1781, 1789, 1797, 1805, 1813, 1821, 1837, 1845, 1853, 1861, 1869, 1877, 1885, 1893, 1917, 1941, 1949, 1957, 1965, 1973,V selected from any one of 1981, 1989, 1997, 2005, 2013, 2021, 2029, 2037, 2045, 2053, 2061, 2069, 2077, 2085, 2093, 2101, 2109, 2117, 2125, 2133, 2141, 2149, 2157, 2165, 2173, 2181, 2189, 2197, 2205, 2213, 2221, 2229, 2237, 2245, 2253, 2261, 2269, 2277, 2285, 2301, 2309, 2317, 2325, 2333, 2341, and 2349; H and / or VL-2 comprises the CDR1 sequence, CDR2 sequence, and CDR3 sequence of the amino acid sequences SEQ ID NOs: 17, 25, 33, 41, 121, 137, 169, 177, 185, 193, 201, 209, 217, 225, 233, 241, 249, 257, 265, 321, 329, 337, 393, 401, 409, 473, 481, 489, 497, 505, 513, 545, 553, 561, 569, 577, 585, 593, 601, 625, 633, 641, 649, V selected from any one of 657, 665, 673, 681, 689, 697, 705, 713, 721, 729, 737, 745, 753, 761, 769, 785, 793, 801, 809, 817, 849, 857, 865, 873, 881, 889, 897, 905, 913, 921, 929, 937, 945, 969, 977, 1009, 1057, 1537, 1569, 1601, 1641, 1665, 1825, 1865, 1897, 1905, 1913, 1921, 1929, 2265, 2281, 2289, 2329, and 2345 Land / or VH-2 comprises the CDR1 sequence, CDR2 sequence, and CDR3 sequence of the amino acid sequences SEQ ID NOs: 21, 29, 37, 45, 125, 141, 173, 181, 189, 197, 205, 213, 221, 229, 237, 245, 253, 261, 269, 325, 333, 341, 397, 405, 413, 477, 485, 493, 501, 509, 517, 549, 557, 565, 573, 581, 589, 597, 605, 629, 637, 645, 653, 661, 669, 677, 685, 69 V selected from any one of 3, 701, 709, 717, 725, 733, 741, 749, 757, 765, 773, 789, 797, 805, 813, 821, 853, 861, 869, 877, 885, 893, 901, 909, 917, 925, 933, 941, 949, 973, 981, 1013, 1061, 1541, 1573, 1605, 1645, 1669, 1829, 1869, 1901, 1909, 1917, 1925, 1933, 2269, 2285, 2293, 2333, and 2349 H Heterodimeric multispecific antibodies are provided, comprising amino acid sequences of CDR1, CDR2, and CDR3 sequences.

[0010] Additionally or alternatively, in some embodiments of the heterodimeric multispecific antibodies disclosed herein, VH-1 or VH-3 is selected from the group consisting of SEQ ID NOs: 5, 13, 21, 29, 37, 45, 53, 61, 69, 77, 85, 93, 101, 109, 117, 125, 133, 149, 157, 165, 173, 181, 197, 205, 237, 245, 261, 277, 285, 293, 301, 309, 317, 325, 333, 341, 349, 357, 365, 373, 381, 389, 397, 405, 413, 421, 429, 437, 445 , 453, 461, 469, 485, 493, 501, 525, 533, 541, 549, 557, 565, 613, 621, 685, 693, 701, 709, 717, 725, 733, 741, 749, 757, 765, 773, 781, 789, 797, 805, 813, 821, 829, 837, 845, 853, 861, 869, 877, 885, 893, 949, 957, 965, 981, 989, 997, 1005, 1013, 1021, 1029, 1037, 1045, 1053, 1069, 1077, 1085, 1093, 1101, 1102, 1103, 1104, 1105, 1106, 1107, 1108, 1109, 1110, 1111, 1112, 1113, 1114, 1115, 1116, 1117, 1118, 109, 1117, 1125, 1133, 1141, 1149, 1157, 1165, 1173, 1181, 1189, 1197, 1205, 1213, 1221, 1229, 1237, 1245, 1253, 1261, 1269, 1277, 1285, 1293, 1301, 1309, 1317, 1325, 1333, 1341, 1349, 1357, 1365, 1373, 1381, 1389, 1397, 1405, 1413, 1421, 1429, 1437, 1445, 1453, 1461, 1469, 1477, 1485, 1493, 1501, 1509, 1517, 1525, 1533, 1549, 1557, 1565, 1573, 1581, 1589, 1597, 1605, 1613, 1621, 1629, 1637, 1653, 1661, 1677, 1685, 1693, 1701, 1709, 1717, 1725, 1733, 1741, 1749, 1757, 1765, 1773, 1781, 1789, 1797, 1805, 1813, 1821, 1837, 1845, 1853, 1861, 1869, 1877, 1885, 1893, 1917, 1941, 1949, 1957, 1965,V selected from any one of 1973, 1981, 1989, 1997, 2005, 2013, 2021, 2029, 2037, 2045, 2053, 2061, 2069, 2077, 2085, 2093, 2101, 2109, 2117, 2125, 2133, 2141, 2149, 2157, 2165, 2173, 2181, 2189, 2197, 2205, 2213, 2221, 2229, 2237, 2245, 2253, 2261, 2269, 2277, 2285, 2301, 2309, 2317, 2325, 2333, 2341, and 2349; Hand / or VL-1 or VL-3 comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NOs: 1, 9, 17, 25, 33, 41, 49, 57, 65, 73, 81, 89, 97, 105, 113, 121, 129, 145, 153, 161, 169, 177, 193, 201, 233, 241, 257, 273, 281, 289, 297, 305, 313, 321, 329, 337, 345, 353, 361, 369, 377, 380, 381, 382, ​​383, 384, 385, 386, 387, 388, 389, 390, 400, 401, 402, 403, 404, 405, 406, 407, 408, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 440, 441, 85, 393, 401, 409, 417, 425, 433, 441, 449, 457, 465, 481, 489, 497, 521, 529, 537, 545, 553, 561, 609, 617, 681, 689, 697, 705, 713, 721, 729, 737, 745, 75 3, 761, 769, 777, 785, 793, 801, 809, 817, 825, 833, 841, 849, 857, 865, 873, 881, 889, 945, 953, 961, 977, 985, 993, 1001, 1009, 1017, 1025, 1033, 1041, 1 049, 1065, 1073, 1081, 1089, 1097, 1105, 1113, 1121, 1129, 1137, 1145, 1153, 1161, 1169, 1177, 1185, 1193, 1201, 1209, 1217, 1225, 1233, 1241, 1249, 1257, 1265, 1273, 1281, 1289, 1297, 1305, 1313, 1321, 1329, 1337, 1345, 1353, 1361, 1369, 1377, 1385, 1393, 1401, 1409, 1417, 1425, 1433, 1441, 1449, 1457, 1465, 1473, 1481, 1489, 1497, 1505, 1513, 1521, 1529, 1545, 1553, 1561, 1569, 1577, 1585, 1593, 1601, 1609, 1617, 1625, 1633, 1649, 1657, 1673, 1681, 1689, 1697, 1705, 1713, 1721, 1729, 1737, 1745, 1753, 1761, 1769, 1777, 1785, 1793, 1801, 1809, 1817, 1833, 1841, 1849, 1857, 1865, 1873, 1881,1889, 1913, 1937, 1945, 1953, 1961, 1969, 1977, 1985, 1993, 2001, 2009, 2017, 2025, 2033, 2041, 2049, 2057, 2065, 2073, 2081, 2089, 2097, 2105, 2113, 2121, 2129, 2137, 214 V selected from any one of 5, 2153, 2161, 2169, 2177, 2185, 2193, 2201, 2209, 2217, 2225, 2233, 2241, 2249, 2257, 2265, 2273, 2281, 2297, 2305, 2313, 2321, 2329, 2337, and 2345; L It includes an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to the amino acid sequence.

[0011] Additionally or alternatively, in some embodiments of the heterodimeric multispecific antibodies disclosed herein, VH-2 or VH-4 is selected from the group consisting of SEQ ID NOs: 21, 29, 37, 45, 125, 141, 173, 181, 189, 197, 205, 213, 221, 229, 237, 245, 253, 261, 269, 325, 333, 341, 397, 405, 413, 477, 485, 493, 501, 509, 517, 549, 557, 565, 573, 581, 589, 597, 605, 629, 637, 645, 653, 661, 669, 67 V selected from any one of 7, 685, 693, 701, 709, 717, 725, 733, 741, 749, 757, 765, 773, 789, 797, 805, 813, 821, 853, 861, 869, 877, 885, 893, 901, 909, 917, 925, 933, 941, 949, 973, 981, 1013, 1061, 1541, 1573, 1605, 1645, 1669, 1829, 1869, 1901, 1909, 1917, 1925, 1933, 2269, 2285, 2293, 2333, and 2349 Hand / or VL-2 or VL-4 comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NOs: 17, 25, 33, 41, 121, 137, 169, 177, 185, 193, 201, 209, 217, 225, 233, 241, 249, 257, 265, 321, 329, 337, 393, 401, 409, 473, 481, 489, 497, 505, 513, 545, 553, 561, 569, 577, 585, 593, 601, 625, 633, 641, 649, 657, 665, 673, 681, 689, 697, 705, 713, 721, 729, 737, 745, 753, 761, 769, 785, 793, 801, 809, 817, 849, 857, 865, 873, 881, 889, 897, 905, 913, 921, 929, 937, 945, 969, 977, 1009, 1057, 1537, 1569, 1601, 1641, 1665, 1825, 1865, 1897, 1905, 1913, 1921, 1929, 2265, 2281 V selected from any one of 2289, 2329, and 2345 L It includes an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to the amino acid sequence.

[0012] Additionally or alternatively, in some embodiments of the heterodimeric multispecific antibodies disclosed herein, each of the VL-1 and VH-1 is SEQ ID NOs: 1 and 5, respectively; SEQ ID NOs: 9 and 13, respectively; SEQ ID NOs: 17 and 21, respectively; SEQ ID NOs: 25 and 29, respectively; SEQ ID NOs: 33 and 37, respectively; SEQ ID NOs: 41 and 45, respectively; SEQ ID NOs: 49 and 53, respectively; SEQ ID NOs: 57 and 61, respectively; SEQ ID NOs: 73 and 77, respectively; SEQ ID NOs: 89 and 93, respectively; SEQ ID NOs: 97 and 100, respectively. 01; SEQ ID NOs: 105 and 109, respectively; SEQ ID NOs: 113 and 117, respectively; SEQ ID NOs: 121 and 125, respectively; SEQ ID NOs: 129 and 133, respectively; SEQ ID NOs: 145 and 149, respectively; SEQ ID NOs: 161 and 165, respectively; SEQ ID NOs: 169 and 173, respectively; SEQ ID NOs: 177 and 181, respectively; SEQ ID NOs: 193 and 197, respectively; SEQ ID NOs: 201 and 205, respectively; SEQ ID NOs: 233 and 237, respectively; SEQ ID NOs: 241 and 245, respectively; SEQ ID NOs: 257 and 26, respectively 1; SEQ ID NOs: 273 and 277, respectively; SEQ ID NOs: 281 and 285, respectively; SEQ ID NOs: 289 and 293, respectively; SEQ ID NOs: 297 and 301, respectively; SEQ ID NOs: 305 and 309, respectively; SEQ ID NOs: 313 and 317, respectively; SEQ ID NOs: 321 and 325, respectively; SEQ ID NOs: 329 and 333, respectively; SEQ ID NOs: 337 and 341, respectively; SEQ ID NOs: 345 and 349, respectively; SEQ ID NOs: 353 and 357, respectively; SEQ ID NOs: 361 and 365, respectively; SEQ ID NOs: 369 and 373, respectively. SEQ ID NOs: 377 and 381, respectively; SEQ ID NOs: 385 and 389, respectively; SEQ ID NOs: 393 and 397, respectively; SEQ ID NOs: 401 and 405, respectively; SEQ ID NOs: 409 and 413, respectively; SEQ ID NOs: 417 and 421, respectively; SEQ ID NOs: 425 and 429, respectively; SEQ ID NOs: 433 and 437, respectively; SEQ ID NOs: 441 and 445, respectively; SEQ ID NOs: 449 and 453, respectively; SEQ ID NOs: 457 and 461, respectively; SEQ ID NOs: 465 and 469, respectively; SEQ ID NOs: 481 and 485, respectively;SEQ ID NOs: 489 and 493, respectively; SEQ ID NOs: 497 and 501, respectively; SEQ ID NOs: 521 and 525, respectively; SEQ ID NOs: 529 and 533, respectively; SEQ ID NOs: 537 and 541, respectively; SEQ ID NOs: 545 and 549, respectively; SEQ ID NOs: 553 and 557, respectively; SEQ ID NOs: 561 and 565, respectively; SEQ ID NOs: 609 and 613, respectively; SEQ ID NOs: 617 and 621, respectively; SEQ ID NOs: 681 and 685, respectively; SEQ ID NOs: 689 and 693, respectively; SEQ ID NOs: 697 and 701, respectively. SEQ ID NOs:705 and 709, respectively; SEQ ID NOs:713 and 717, respectively; SEQ ID NOs:721 and 725, respectively; SEQ ID NOs:729 and 733, respectively; SEQ ID NOs:737 and 741, respectively; SEQ ID NOs:745 and 749, respectively; SEQ ID NOs:753 and 757, respectively; SEQ ID NOs:761 and 765, respectively; SEQ ID NOs:769 and 773, respectively; SEQ ID NOs:785 and 789, respectively; SEQ ID NOs:793 and 797, respectively; SEQ ID NOs:801 and 805, respectively; SEQ ID NOs:809 and 813, respectively. SEQ ID NOs: 817 and 821, respectively; SEQ ID NOs: 825 and 829, respectively; SEQ ID NOs: 833 and 837, respectively; SEQ ID NOs: 841 and 845, respectively; SEQ ID NOs: 849 and 853, respectively; SEQ ID NOs: 857 and 861, respectively; SEQ ID NOs: 865 and 869, respectively; SEQ ID NOs: 873 and 877, respectively; SEQ ID NOs: 881 and 885, respectively; SEQ ID NOs: 889 and 893, respectively; SEQ ID NOs: 945 and 949, respectively; SEQ ID NOs: 953 and 957, respectively; SEQ ID NOs: 961 and 965, respectively. SEQ ID NOs: 977 and 981, respectively; SEQ ID NOs: 985 and 989, respectively; SEQ ID NOs: 993 and 997, respectively; SEQ ID NOs: 1001 and 1005, respectively; SEQ ID NOs: 1009 and 1013, respectively; SEQ ID NOs: 1017 and 1021, respectively; SEQ ID NOs: 1025 and 1029, respectively; SEQ ID NOs: 1033 and 1037, respectively; SEQ ID NOs: 1041 and 1045, respectively; SEQ ID NOs: 1065 and 1069, respectively; SEQ ID NOs: 1073 and 1077, respectively; SEQ ID NOs: 1081 and 1085, respectively;SEQ ID NOs: 1089 and 1093, respectively; SEQ ID NOs: 1097 and 1101, respectively; SEQ ID NOs: 1113 and 1117, respectively; SEQ ID NOs: 1121 and 1125, respectively; SEQ ID NOs: 1129 and 1133, respectively; SEQ ID NOs: 1137 and 1141, respectively; SEQ ID NOs: 1145 and 1149, respectively; SEQ ID NOs: 1153 and 1157, respectively; SEQ ID NOs: 1161 and 1165, respectively; SEQ ID NOs: 1169 and 1173, respectively; SEQ ID NOs: 1185 and 1189, respectively; SEQ ID NOs: 1193 and 1198, respectively. and 1197; SEQ ID NOs: 1201 and 1205, respectively; SEQ ID NOs: 1209 and 1213, respectively; SEQ ID NOs: 1217 and 1221, respectively; SEQ ID NOs: 1225 and 1229, respectively; SEQ ID NOs: 1233 and 1237, respectively; SEQ ID NOs: 1241 and 1245, respectively; SEQ ID NOs: 1249 and 1253, respectively; SEQ ID NOs: 1257 and 1261, respectively; SEQ ID NOs: 1265 and 1269, respectively; SEQ ID NOs: 1273 and 1277, respectively; SEQ ID NOs: 1281 and 1285, respectively; SEQ ID NOs: 1 SEQ ID NOs: 1329 and 1333, respectively; SEQ ID NOs: 1337 and 1341, respectively; SEQ ID NOs: 1345 and 1349, respectively; SEQ ID NOs: 1353 and 1357, respectively; SEQ ID NOs: 1361 and 1365, respectively; SEQ ID NOs: 1369 and 1373, respectively; SEQ ID NOs: 1377 and 1381, respectively; SEQ ID NOs: 1385 and 1389; SEQ ID NOs: 1393 and 1397, respectively; SEQ ID NOs: 1401 and 1405, respectively; SEQ ID NOs: 1409 and 1413, respectively; SEQ ID NOs: 1417 and 1421, respectively; SEQ ID NOs: 1433 and 1437, respectively; SEQ ID NOs: 1441 and 1445, respectively; SEQ ID NOs: 1457 and 1461, respectively; SEQ ID NOs: 1465 and 1469, respectively; SEQ ID NOs: 1473 and 1477, respectively; SEQ ID NOs: 1481 and 1485, respectively; SEQ ID NOs: 1489 and 1493, respectively;SEQ ID NOs: 1497 and 1501, respectively; SEQ ID NOs: 1505 and 1509, respectively; SEQ ID NOs: 1513 and 1517, respectively; SEQ ID NOs: 1521 and 1525, respectively; SEQ ID NOs: 1529 and 1533, respectively; SEQ ID NOs: 1545 and 1549, respectively; SEQ ID NOs: 1553 and 1557, respectively; SEQ ID NOs: 1561 and 1565, respectively; SEQ ID NOs: 1569 and 1573, respectively; SEQ ID NOs: 1577 and 1581, respectively; SEQ ID NOs: 1585 and 1589, respectively; SEQ ID NOs: 1593 and 1598, respectively. and 1597; SEQ ID NOs: 1601 and 1605, respectively; SEQ ID NOs: 1609 and 1613, respectively; SEQ ID NOs: 1617 and 1621, respectively; SEQ ID NOs: 1625 and 1629, respectively; SEQ ID NOs: 1633 and 1637, respectively; SEQ ID NOs: 1649 and 1653, respectively; SEQ ID NOs: 1657 and 1661, respectively; SEQ ID NOs: 1673 and 1677, respectively; SEQ ID NOs: 1681 and 1685, respectively; SEQ ID NOs: 1689 and 1693, respectively; SEQ ID NOs: 1697 and 1701, respectively; SEQ ID NOs: 1 SEQ ID NOs: 1705 and 1709; SEQ ID NOs: 1713 and 1717, respectively; SEQ ID NOs: 1721 and 1725, respectively; SEQ ID NOs: 1729 and 1733, respectively; SEQ ID NOs: 1737 and 1741, respectively; SEQ ID NOs: 1745 and 1749, respectively; SEQ ID NOs: 1753 and 1757, respectively; SEQ ID NOs: 1761 and 1765, respectively; SEQ ID NOs: 1769 and 1773, respectively; SEQ ID NOs: 1777 and 1781, respectively; SEQ ID NOs: 1785 and 1789, respectively; SEQ ID NOs: 1793 and 1797, respectively; SEQ ID NOs: 1801 and 1805; SEQ ID NOs: 1809 and 1813, respectively; SEQ ID NOs: 1817 and 1821, respectively; SEQ ID NOs: 1833 and 1837, respectively; SEQ ID NOs: 1841 and 1845, respectively; SEQ ID NOs: 1849 and 1853, respectively; SEQ ID NOs: 1857 and 1861, respectively; SEQ ID NOs: 1865 and 1869, respectively; SEQ ID NOs: 1873 and 1877, respectively; SEQ ID NOs: 1881 and 1885, respectively; SEQ ID NOs: 1889 and 1893, respectively; SEQ ID NOs: 1913 and 1917, respectively;SEQ ID NOs: 1937 and 1941, respectively; SEQ ID NOs: 1945 and 1949, respectively; SEQ ID NOs: 1953 and 1957, respectively; SEQ ID NOs: 1961 and 1965, respectively; SEQ ID NOs: 1969 and 1973, respectively; SEQ ID NOs: 1977 and 1981, respectively; SEQ ID NOs: 1985 and 1989, respectively; SEQ ID NOs: 1993 and 1997, respectively; SEQ ID NOs: 2001 and 2005, respectively; SEQ ID NOs: 2009 and 2013, respectively; SEQ ID NOs: 2017 and 2021, respectively; SEQ ID NOs: 2025 and 2026, respectively. and 2029; SEQ ID NOs: 2033 and 2037, respectively; SEQ ID NOs: 2041 and 2045, respectively; SEQ ID NOs: 2049 and 2053, respectively; SEQ ID NOs: 2057 and 2061, respectively; SEQ ID NOs: 2065 and 2069, respectively; SEQ ID NOs: 2073 and 2077, respectively; SEQ ID NOs: 2081 and 2085, respectively; SEQ ID NOs: 2089 and 2093, respectively; SEQ ID NOs: 2097 and 2101, respectively; SEQ ID NOs: 2105 and 2109, respectively; SEQ ID NOs: 2113 and 2117, respectively; SEQ ID NOs: 2114 and 2118, respectively. SEQ ID NOs: 2161 and 2165, respectively; SEQ ID NOs: 2169 and 2173, respectively; SEQ ID NOs: 2177 and 2181, respectively; SEQ ID NOs: 2185 and 2189, respectively; SEQ ID NOs: 2193 and 2197, respectively; SEQ ID NOs: 2201 and 2205, respectively; SEQ ID NOs: 2209 and 2213, respectively; SEQ ID NOs: 2217 and 2221; SEQ ID NOs: 2225 and 2229, respectively; SEQ ID NOs: 2233 and 2237, respectively; SEQ ID NOs: 2241 and 2245, respectively; SEQ ID NOs: 2249 and 2253, respectively; SEQ ID NOs: 2257 and 2261, respectively; SEQ ID NOs: 2273 and 2277, respectively; SEQ ID NOs: 2281 and 2285, respectively; SEQ ID NOs: 2305 and 2309, respectively; SEQ ID NOs: 2313 and 2317, respectively; SEQ ID NOs: 2321 and 2325, respectively; SEQ ID NOs: 2329 and 2333, respectively;V selected from the group consisting of SEQ ID NOs: 2337 and 2341, respectively; and SEQ ID NOs: 2345 and 2349, respectively; L Amino acid sequence and V H Contains the amino acid sequence.

[0013] Additionally or alternatively, in some embodiments of the heterodimeric multispecific antibodies disclosed herein, each of the VL-3 and VH-3 is SEQ ID NOs: 1 and 5, respectively; SEQ ID NOs: 9 and 13, respectively; SEQ ID NOs: 17 and 21, respectively; SEQ ID NOs: 25 and 29, respectively; SEQ ID NOs: 33 and 37, respectively; SEQ ID NOs: 41 and 45, respectively; SEQ ID NOs: 49 and 53, respectively; SEQ ID NOs: 57 and 61, respectively; SEQ ID NOs: 73 and 77, respectively; SEQ ID NOs: 89 and 93, respectively; SEQ ID NOs: 97 and 100, respectively. 01; SEQ ID NOs: 105 and 109, respectively; SEQ ID NOs: 113 and 117, respectively; SEQ ID NOs: 121 and 125, respectively; SEQ ID NOs: 129 and 133, respectively; SEQ ID NOs: 145 and 149, respectively; SEQ ID NOs: 161 and 165, respectively; SEQ ID NOs: 169 and 173, respectively; SEQ ID NOs: 177 and 181, respectively; SEQ ID NOs: 193 and 197, respectively; SEQ ID NOs: 201 and 205, respectively; SEQ ID NOs: 233 and 237, respectively; SEQ ID NOs: 241 and 245, respectively; SEQ ID NOs: 257 and 26, respectively 1; SEQ ID NOs: 273 and 277, respectively; SEQ ID NOs: 281 and 285, respectively; SEQ ID NOs: 289 and 293, respectively; SEQ ID NOs: 297 and 301, respectively; SEQ ID NOs: 305 and 309, respectively; SEQ ID NOs: 313 and 317, respectively; SEQ ID NOs: 321 and 325, respectively; SEQ ID NOs: 329 and 333, respectively; SEQ ID NOs: 337 and 341, respectively; SEQ ID NOs: 345 and 349, respectively; SEQ ID NOs: 353 and 357, respectively; SEQ ID NOs: 361 and 365, respectively; SEQ ID NOs: 369 and 373, respectively. SEQ ID NOs: 377 and 381, respectively; SEQ ID NOs: 385 and 389, respectively; SEQ ID NOs: 393 and 397, respectively; SEQ ID NOs: 401 and 405, respectively; SEQ ID NOs: 409 and 413, respectively; SEQ ID NOs: 417 and 421, respectively; SEQ ID NOs: 425 and 429, respectively; SEQ ID NOs: 433 and 437, respectively; SEQ ID NOs: 441 and 445, respectively; SEQ ID NOs: 449 and 453, respectively; SEQ ID NOs: 457 and 461, respectively; SEQ ID NOs: 465 and 469, respectively; SEQ ID NOs: 481 and 485, respectively;SEQ ID NOs: 489 and 493, respectively; SEQ ID NOs: 497 and 501, respectively; SEQ ID NOs: 521 and 525, respectively; SEQ ID NOs: 529 and 533, respectively; SEQ ID NOs: 537 and 541, respectively; SEQ ID NOs: 545 and 549, respectively; SEQ ID NOs: 553 and 557, respectively; SEQ ID NOs: 561 and 565, respectively; SEQ ID NOs: 609 and 613, respectively; SEQ ID NOs: 617 and 621, respectively; SEQ ID NOs: 681 and 685, respectively; SEQ ID NOs: 689 and 693, respectively; SEQ ID NOs: 697 and 701, respectively. SEQ ID NOs:705 and 709, respectively; SEQ ID NOs:713 and 717, respectively; SEQ ID NOs:721 and 725, respectively; SEQ ID NOs:729 and 733, respectively; SEQ ID NOs:737 and 741, respectively; SEQ ID NOs:745 and 749, respectively; SEQ ID NOs:753 and 757, respectively; SEQ ID NOs:761 and 765, respectively; SEQ ID NOs:769 and 773, respectively; SEQ ID NOs:785 and 789, respectively; SEQ ID NOs:793 and 797, respectively; SEQ ID NOs:801 and 805, respectively; SEQ ID NOs:809 and 813, respectively. SEQ ID NOs: 817 and 821, respectively; SEQ ID NOs: 825 and 829, respectively; SEQ ID NOs: 833 and 837, respectively; SEQ ID NOs: 841 and 845, respectively; SEQ ID NOs: 849 and 853, respectively; SEQ ID NOs: 857 and 861, respectively; SEQ ID NOs: 865 and 869, respectively; SEQ ID NOs: 873 and 877, respectively; SEQ ID NOs: 881 and 885, respectively; SEQ ID NOs: 889 and 893, respectively; SEQ ID NOs: 945 and 949, respectively; SEQ ID NOs: 953 and 957, respectively; SEQ ID NOs: 961 and 965, respectively. SEQ ID NOs: 977 and 981, respectively; SEQ ID NOs: 985 and 989, respectively; SEQ ID NOs: 993 and 997, respectively; SEQ ID NOs: 1001 and 1005, respectively; SEQ ID NOs: 1009 and 1013, respectively; SEQ ID NOs: 1017 and 1021, respectively; SEQ ID NOs: 1025 and 1029, respectively; SEQ ID NOs: 1033 and 1037, respectively; SEQ ID NOs: 1041 and 1045, respectively; SEQ ID NOs: 1065 and 1069, respectively; SEQ ID NOs: 1073 and 1077, respectively; SEQ ID NOs: 1081 and 1085, respectively;SEQ ID NOs: 1089 and 1093, respectively; SEQ ID NOs: 1097 and 1101, respectively; SEQ ID NOs: 1113 and 1117, respectively; SEQ ID NOs: 1121 and 1125, respectively; SEQ ID NOs: 1129 and 1133, respectively; SEQ ID NOs: 1137 and 1141, respectively; SEQ ID NOs: 1145 and 1149, respectively; SEQ ID NOs: 1153 and 1157, respectively; SEQ ID NOs: 1161 and 1165, respectively; SEQ ID NOs: 1169 and 1173, respectively; SEQ ID NOs: 1185 and 1189, respectively; SEQ ID NOs: 1193 and 1198, respectively. and 1197; SEQ ID NOs: 1201 and 1205, respectively; SEQ ID NOs: 1209 and 1213, respectively; SEQ ID NOs: 1217 and 1221, respectively; SEQ ID NOs: 1225 and 1229, respectively; SEQ ID NOs: 1233 and 1237, respectively; SEQ ID NOs: 1241 and 1245, respectively; SEQ ID NOs: 1249 and 1253, respectively; SEQ ID NOs: 1257 and 1261, respectively; SEQ ID NOs: 1265 and 1269, respectively; SEQ ID NOs: 1273 and 1277, respectively; SEQ ID NOs: 1281 and 1285, respectively; SEQ ID NOs: 1 SEQ ID NOs: 1329 and 1333, respectively; SEQ ID NOs: 1337 and 1341, respectively; SEQ ID NOs: 1345 and 1349, respectively; SEQ ID NOs: 1353 and 1357, respectively; SEQ ID NOs: 1361 and 1365, respectively; SEQ ID NOs: 1369 and 1373, respectively; SEQ ID NOs: 1377 and 1381, respectively; SEQ ID NOs: 1385 and 1389; SEQ ID NOs: 1393 and 1397, respectively; SEQ ID NOs: 1401 and 1405, respectively; SEQ ID NOs: 1409 and 1413, respectively; SEQ ID NOs: 1417 and 1421, respectively; SEQ ID NOs: 1433 and 1437, respectively; SEQ ID NOs: 1441 and 1445, respectively; SEQ ID NOs: 1457 and 1461, respectively; SEQ ID NOs: 1465 and 1469, respectively; SEQ ID NOs: 1473 and 1477, respectively; SEQ ID NOs: 1481 and 1485, respectively; SEQ ID NOs: 1489 and 1493, respectively;SEQ ID NOs: 1497 and 1501, respectively; SEQ ID NOs: 1505 and 1509, respectively; SEQ ID NOs: 1513 and 1517, respectively; SEQ ID NOs: 1521 and 1525, respectively; SEQ ID NOs: 1529 and 1533, respectively; SEQ ID NOs: 1545 and 1549, respectively; SEQ ID NOs: 1553 and 1557, respectively; SEQ ID NOs: 1561 and 1565, respectively; SEQ ID NOs: 1569 and 1573, respectively; SEQ ID NOs: 1577 and 1581, respectively; SEQ ID NOs: 1585 and 1589, respectively; SEQ ID NOs: 1593 and 1598, respectively. and 1597; SEQ ID NOs: 1601 and 1605, respectively; SEQ ID NOs: 1609 and 1613, respectively; SEQ ID NOs: 1617 and 1621, respectively; SEQ ID NOs: 1625 and 1629, respectively; SEQ ID NOs: 1633 and 1637, respectively; SEQ ID NOs: 1649 and 1653, respectively; SEQ ID NOs: 1657 and 1661, respectively; SEQ ID NOs: 1673 and 1677, respectively; SEQ ID NOs: 1681 and 1685, respectively; SEQ ID NOs: 1689 and 1693, respectively; SEQ ID NOs: 1697 and 1701, respectively; SEQ ID NOs: 1 SEQ ID NOs: 1705 and 1709; SEQ ID NOs: 1713 and 1717, respectively; SEQ ID NOs: 1721 and 1725, respectively; SEQ ID NOs: 1729 and 1733, respectively; SEQ ID NOs: 1737 and 1741, respectively; SEQ ID NOs: 1745 and 1749, respectively; SEQ ID NOs: 1753 and 1757, respectively; SEQ ID NOs: 1761 and 1765, respectively; SEQ ID NOs: 1769 and 1773, respectively; SEQ ID NOs: 1777 and 1781, respectively; SEQ ID NOs: 1785 and 1789, respectively; SEQ ID NOs: 1793 and 1797, respectively; SEQ ID NOs: 1801 and 1805; SEQ ID NOs: 1809 and 1813, respectively; SEQ ID NOs: 1817 and 1821, respectively; SEQ ID NOs: 1833 and 1837, respectively; SEQ ID NOs: 1841 and 1845, respectively; SEQ ID NOs: 1849 and 1853, respectively; SEQ ID NOs: 1857 and 1861, respectively; SEQ ID NOs: 1865 and 1869, respectively; SEQ ID NOs: 1873 and 1877, respectively; SEQ ID NOs: 1881 and 1885, respectively; SEQ ID NOs: 1889 and 1893, respectively; SEQ ID NOs: 1913 and 1917, respectively;SEQ ID NOs: 1937 and 1941, respectively; SEQ ID NOs: 1945 and 1949, respectively; SEQ ID NOs: 1953 and 1957, respectively; SEQ ID NOs: 1961 and 1965, respectively; SEQ ID NOs: 1969 and 1973, respectively; SEQ ID NOs: 1977 and 1981, respectively; SEQ ID NOs: 1985 and 1989, respectively; SEQ ID NOs: 1993 and 1997, respectively; SEQ ID NOs: 2001 and 2005, respectively; SEQ ID NOs: 2009 and 2013, respectively; SEQ ID NOs: 2017 and 2021, respectively; SEQ ID NOs: 2025 and 2026, respectively. and 2029; SEQ ID NOs: 2033 and 2037, respectively; SEQ ID NOs: 2041 and 2045, respectively; SEQ ID NOs: 2049 and 2053, respectively; SEQ ID NOs: 2057 and 2061, respectively; SEQ ID NOs: 2065 and 2069, respectively; SEQ ID NOs: 2073 and 2077, respectively; SEQ ID NOs: 2081 and 2085, respectively; SEQ ID NOs: 2089 and 2093, respectively; SEQ ID NOs: 2097 and 2101, respectively; SEQ ID NOs: 2105 and 2109, respectively; SEQ ID NOs: 2113 and 2117, respectively; SEQ ID NOs: 2114 and 2118, respectively. SEQ ID NOs: 2161 and 2165, respectively; SEQ ID NOs: 2169 and 2173, respectively; SEQ ID NOs: 2177 and 2181, respectively; SEQ ID NOs: 2185 and 2189, respectively; SEQ ID NOs: 2193 and 2197, respectively; SEQ ID NOs: 2201 and 2205, respectively; SEQ ID NOs: 2209 and 2213, respectively; SEQ ID NOs: 2217 and 2221; SEQ ID NOs: 2225 and 2229, respectively; SEQ ID NOs: 2233 and 2237, respectively; SEQ ID NOs: 2241 and 2245, respectively; SEQ ID NOs: 2249 and 2253, respectively; SEQ ID NOs: 2257 and 2261, respectively; SEQ ID NOs: 2273 and 2277, respectively; SEQ ID NOs: 2281 and 2285, respectively; SEQ ID NOs: 2305 and 2309, respectively; SEQ ID NOs: 2313 and 2317, respectively; SEQ ID NOs: 2321 and 2325, respectively; SEQ ID NOs: 2329 and 2333, respectively;V selected from the group consisting of SEQ ID NOs: 2337 and 2341, respectively; and SEQ ID NOs: 2345 and 2349, respectively; L Amino acid sequence and V H Contains the amino acid sequence.

[0014] Additionally or alternatively, for the heterodimeric multispecific antibodies disclosed herein, in some embodiments, each of the VL-1 and VH-1 is selected from the group consisting of SEQ ID NOs: 9 and 13, respectively; SEQ ID NOs: 49 and 53, respectively; SEQ ID NOs: 57 and 61, respectively; SEQ ID NOs: 65 and 69, respectively; SEQ ID NOs: 81 and 85, respectively; SEQ ID NOs: 153 and 157, respectively; SEQ ID NOs: 161 and 165, respectively; SEQ ID NOs: 193 and 197, respectively; SEQ ID NOs: 201 and 205, respectively; SEQ ID NOs: 273 and 277, respectively; SEQ ID NOs: 281 and 285, respectively; SEQ ID NOs: 289 and 293, respectively; SEQ ID NOs: 297 and 301, respectively; SEQ ID NOs: 305 and 309, respectively; SEQ ID NOs: 313 and 317, respectively; SEQ ID NOs: 361 and 365, respectively; SEQ ID NOs: 377 and 381, respectively; SEQ ID NOs: 393 and 397, respectively; SEQ ID NOs: 401 and 405, respectively; SEQ ID NOs: 409 and 413, respectively; SEQ ID NOs: 417 and 421, respectively; SEQ ID NOs: 425 and 429, respectively; SEQ ID NOs: 433 and 437, respectively. , SEQ ID NOs: 441 and 445, respectively; SEQ ID NOs: 449 and 453, respectively; SEQ ID NOs: 457 and 461, respectively; SEQ ID NOs: 465 and 469, respectively; SEQ ID NOs: 681 and 685, respectively; SEQ ID NOs: 689 and 693, respectively; SEQ ID NOs: 697 and 701, respectively; SEQ ID NOs: 705 and 709, respectively; SEQ ID NOs: 713 and 717, respectively; SEQ ID NOs: 721 and 725, respectively; SEQ ID NOs: 737 and 741, respectively; SEQ ID NOs: 745 and 749, respectively; SEQ ID NOs: 753 and 757, respectively; Sequence numbers 761 and 765; SEQ ID NOs: 777 and 781, respectively; SEQ ID NOs: 825 and 829, respectively; SEQ ID NOs: 833 and 837, respectively; SEQ ID NOs: 841 and 845, respectively; SEQ ID NOs: 953 and 957, respectively; SEQ ID NOs: 961 and 965, respectively; SEQ ID NOs: 977 and 981, respectively; SEQ ID NOs: 993 and 997, respectively; SEQ ID NOs: 1001 and 1005, respectively; SEQ ID NOs: 1009 and 1013, respectively; SEQ ID NOs: 1017 and 1021, respectively; SEQ ID NOs: 1033 and 1037, respectively;SEQ ID NOs: 1049 and 1053, respectively; SEQ ID NOs: 1073 and 1077, respectively; SEQ ID NOs: 1081 and 1085, respectively; SEQ ID NOs: 1089 and 1093, respectively; SEQ ID NOs: 1105 and 1109, respectively; SEQ ID NOs: 1129 and 1133, respectively; SEQ ID NOs: 1137 and 1141, respectively; SEQ ID NOs: 1153 and 1157, respectively; SEQ ID NOs: 1161 and 1165, respectively; SEQ ID NOs: 1177 and 1181, respectively; SEQ ID NOs: 1225 and 1229, respectively; SEQ ID NOs: 1241 and 1242, respectively. and 1245; SEQ ID NOs: 1257 and 1261, respectively; SEQ ID NOs: 1265 and 1269, respectively; SEQ ID NOs: 1297 and 1301, respectively; SEQ ID NOs: 1393 and 1397, respectively; SEQ ID NOs: 1409 and 1413, respectively; SEQ ID NOs: 1425 and 1429, respectively; SEQ ID NOs: 1441 and 1445, respectively; SEQ ID NOs: 1449 and 1453, respectively; SEQ ID NOs: 1457 and 1461, respectively; SEQ ID NOs: 1465 and 1469, respectively; SEQ ID NOs: 1473 and 1477, respectively; SEQ ID NOs: 1 SEQ ID NOs: 1545 and 1549, respectively; SEQ ID NOs: 1553 and 1557, respectively; SEQ ID NOs: 1561 and 1565, respectively; SEQ ID NOs: 1569 and 1573, respectively; SEQ ID NOs: 1577 and 1581, respectively; SEQ ID NOs: 1585 and 1589, respectively; SEQ ID NOs: 1609 and 1613; SEQ ID NOs: 1617 and 1621, respectively; SEQ ID NOs: 1649 and 1653, respectively; SEQ ID NOs: 1657 and 1661, respectively; SEQ ID NOs: 1673 and 1677, respectively; SEQ ID NOs: 1689 and 1693, respectively; SEQ ID NOs: 1697 and 1701, respectively; SEQ ID NOs: 1705 and 1709, respectively; SEQ ID NOs: 1713 and 1717, respectively; SEQ ID NOs: 1721 and 1725, respectively; SEQ ID NOs: 1729 and 1733, respectively; SEQ ID NOs: 1745 and 1749, respectively;SEQ ID NOs: 1753 and 1757, respectively; SEQ ID NOs: 1761 and 1765, respectively; SEQ ID NOs: 1769 and 1773, respectively; SEQ ID NOs: 1777 and 1781, respectively; SEQ ID NOs: 1785 and 1789, respectively; SEQ ID NOs: 1793 and 1797, respectively; SEQ ID NOs: 1817 and 1821, respectively; SEQ ID NOs: 1833 and 1837, respectively; SEQ ID NOs: 1841 and 1845, respectively; SEQ ID NOs: 1849 and 1853, respectively; SEQ ID NOs: 1857 and 1861, respectively; SEQ ID NOs: 1865 and 1869, respectively. V selected from the group consisting of SEQ ID NOs: 1889 and 1893, respectively; SEQ ID NOs: 2257 and 2261, respectively; SEQ ID NOs: 2265 and 2269, respectively; SEQ ID NOs: 2281 and 2285, respectively; SEQ ID NOs: 2297 and 2301, respectively; SEQ ID NOs: 2305 and 2309, respectively; SEQ ID NOs: 2313 and 2317, respectively; SEQ ID NOs: 2321 and 2325, respectively; SEQ ID NOs: 2329 and 2333, respectively; SEQ ID NOs: 2337 and 2341, respectively; and SEQ ID NOs: 2345 and 2349, respectively; L Amino acid sequence and V H Contains the amino acid sequence.

[0015] Additionally or alternatively, for the heterodimeric multispecific antibodies disclosed herein, in some embodiments, each of the VL-3 and VH-3 is selected from the group consisting of SEQ ID NOs: 9 and 13, respectively; SEQ ID NOs: 49 and 53, respectively; SEQ ID NOs: 57 and 61, respectively; SEQ ID NOs: 65 and 69, respectively; SEQ ID NOs: 81 and 85, respectively; SEQ ID NOs: 153 and 157, respectively; SEQ ID NOs: 161 and 165, respectively; SEQ ID NOs: 193 and 197, respectively; SEQ ID NOs: 201 and 205, respectively; SEQ ID NOs: 273 and 277, respectively; SEQ ID NOs: 281 and 285, respectively; SEQ ID NOs: 289 and 293, respectively; SEQ ID NOs: 297 and 301, respectively; SEQ ID NOs: 305 and 309, respectively; SEQ ID NOs: 313 and 317, respectively; SEQ ID NOs: 361 and 365, respectively; SEQ ID NOs: 377 and 381, respectively; SEQ ID NOs: 393 and 397, respectively; SEQ ID NOs: 401 and 405, respectively; SEQ ID NOs: 409 and 413, respectively; SEQ ID NOs: 417 and 421, respectively; SEQ ID NOs: 425 and 429, respectively; SEQ ID NOs: 433 and 437, respectively. , SEQ ID NOs: 441 and 445, respectively; SEQ ID NOs: 449 and 453, respectively; SEQ ID NOs: 457 and 461, respectively; SEQ ID NOs: 465 and 469, respectively; SEQ ID NOs: 681 and 685, respectively; SEQ ID NOs: 689 and 693, respectively; SEQ ID NOs: 697 and 701, respectively; SEQ ID NOs: 705 and 709, respectively; SEQ ID NOs: 713 and 717, respectively; SEQ ID NOs: 721 and 725, respectively; SEQ ID NOs: 737 and 741, respectively; SEQ ID NOs: 745 and 749, respectively; SEQ ID NOs: 753 and 757, respectively; Sequence numbers 761 and 765; SEQ ID NOs: 777 and 781, respectively; SEQ ID NOs: 825 and 829, respectively; SEQ ID NOs: 833 and 837, respectively; SEQ ID NOs: 841 and 845, respectively; SEQ ID NOs: 953 and 957, respectively; SEQ ID NOs: 961 and 965, respectively; SEQ ID NOs: 977 and 981, respectively; SEQ ID NOs: 993 and 997, respectively; SEQ ID NOs: 1001 and 1005, respectively; SEQ ID NOs: 1009 and 1013, respectively; SEQ ID NOs: 1017 and 1021, respectively; SEQ ID NOs: 1033 and 1037, respectively;SEQ ID NOs: 1049 and 1053, respectively; SEQ ID NOs: 1073 and 1077, respectively; SEQ ID NOs: 1081 and 1085, respectively; SEQ ID NOs: 1089 and 1093, respectively; SEQ ID NOs: 1105 and 1109, respectively; SEQ ID NOs: 1129 and 1133, respectively; SEQ ID NOs: 1137 and 1141, respectively; SEQ ID NOs: 1153 and 1157, respectively; SEQ ID NOs: 1161 and 1165, respectively; SEQ ID NOs: 1177 and 1181, respectively; SEQ ID NOs: 1225 and 1229, respectively; SEQ ID NOs: 1241 and 1242, respectively. and 1245; SEQ ID NOs: 1257 and 1261, respectively; SEQ ID NOs: 1265 and 1269, respectively; SEQ ID NOs: 1297 and 1301, respectively; SEQ ID NOs: 1393 and 1397, respectively; SEQ ID NOs: 1409 and 1413, respectively; SEQ ID NOs: 1425 and 1429, respectively; SEQ ID NOs: 1441 and 1445, respectively; SEQ ID NOs: 1449 and 1453, respectively; SEQ ID NOs: 1457 and 1461, respectively; SEQ ID NOs: 1465 and 1469, respectively; SEQ ID NOs: 1473 and 1477, respectively; SEQ ID NOs: 1 SEQ ID NOs: 1545 and 1549, respectively; SEQ ID NOs: 1553 and 1557, respectively; SEQ ID NOs: 1561 and 1565, respectively; SEQ ID NOs: 1569 and 1573, respectively; SEQ ID NOs: 1577 and 1581, respectively; SEQ ID NOs: 1585 and 1589, respectively; SEQ ID NOs: 1609 and 1613; SEQ ID NOs: 1617 and 1621, respectively; SEQ ID NOs: 1649 and 1653, respectively; SEQ ID NOs: 1657 and 1661, respectively; SEQ ID NOs: 1673 and 1677, respectively; SEQ ID NOs: 1689 and 1693, respectively; SEQ ID NOs: 1697 and 1701, respectively; SEQ ID NOs: 1705 and 1709, respectively; SEQ ID NOs: 1713 and 1717, respectively; SEQ ID NOs: 1721 and 1725, respectively; SEQ ID NOs: 1729 and 1733, respectively; SEQ ID NOs: 1745 and 1749, respectively;SEQ ID NOs: 1753 and 1757, respectively; SEQ ID NOs: 1761 and 1765, respectively; SEQ ID NOs: 1769 and 1773, respectively; SEQ ID NOs: 1777 and 1781, respectively; SEQ ID NOs: 1785 and 1789, respectively; SEQ ID NOs: 1793 and 1797, respectively; SEQ ID NOs: 1817 and 1821, respectively; SEQ ID NOs: 1833 and 1837, respectively; SEQ ID NOs: 1841 and 1845, respectively; SEQ ID NOs: 1849 and 1853, respectively; SEQ ID NOs: 1857 and 1861, respectively; SEQ ID NOs: 1865 and 1869, respectively. V selected from the group consisting of SEQ ID NOs: 1889 and 1893, respectively; SEQ ID NOs: 2257 and 2261, respectively; SEQ ID NOs: 2265 and 2269, respectively; SEQ ID NOs: 2281 and 2285, respectively; SEQ ID NOs: 2297 and 2301, respectively; SEQ ID NOs: 2305 and 2309, respectively; SEQ ID NOs: 2313 and 2317, respectively; SEQ ID NOs: 2321 and 2325, respectively; SEQ ID NOs: 2329 and 2333, respectively; SEQ ID NOs: 2337 and 2341, respectively; and SEQ ID NOs: 2345 and 2349, respectively; L Amino acid sequence and V H Contains the amino acid sequence.

[0016] Additionally or alternatively, for the heterodimeric multispecific antibodies disclosed herein, in some embodiments, each of the VL-2 and VH-2 is SEQ ID NOs: 17 and 21, respectively; SEQ ID NOs: 25 and 29, respectively; SEQ ID NOs: 33 and 37, respectively; SEQ ID NOs: 41 and 45, respectively; SEQ ID NOs: 121 and 125, respectively; SEQ ID NOs: 137 and 141, respectively; SEQ ID NOs: 169 and 173, respectively; SEQ ID NOs: 177 and 181, respectively; SEQ ID NOs: 185 and 189, respectively; SEQ ID NOs: 193 and 194, respectively. 7; SEQ ID NOs:201 and 205, respectively; SEQ ID NOs:209 and 213, respectively; SEQ ID NOs:217 and 221, respectively; SEQ ID NOs:225 and 229, respectively; SEQ ID NOs:233 and 237, respectively; SEQ ID NOs:241 and 245, respectively; SEQ ID NOs:249 and 253, respectively; SEQ ID NOs:257 and 261, respectively; SEQ ID NOs:265 and 269, respectively; SEQ ID NOs:321 and 325, respectively; SEQ ID NOs:329 and 333, respectively; SEQ ID NOs:337 and 341, respectively; SEQ ID NOs:393 and 397, respectively SEQ ID NOs: 401 and 405, respectively; SEQ ID NOs: 409 and 413, respectively; SEQ ID NOs: 473 and 477, respectively; SEQ ID NOs: 481 and 485, respectively; SEQ ID NOs: 489 and 493, respectively; SEQ ID NOs: 497 and 501, respectively; SEQ ID NOs: 505 and 509, respectively; SEQ ID NOs: 513 and 517, respectively; SEQ ID NOs: 545 and 549, respectively; SEQ ID NOs: 553 and 557, respectively; SEQ ID NOs: 561 and 565, respectively; SEQ ID NOs: 569 and 573, respectively; SEQ ID NOs: 577 and 581, respectively SEQ ID NOs: 585 and 589, respectively; SEQ ID NOs: 593 and 597, respectively; SEQ ID NOs: 601 and 605, respectively; SEQ ID NOs: 625 and 629, respectively; SEQ ID NOs: 633 and 637, respectively; SEQ ID NOs: 641 and 645, respectively; SEQ ID NOs: 649 and 653, respectively; SEQ ID NOs: 657 and 661, respectively; SEQ ID NOs: 665 and 669, respectively; SEQ ID NOs: 673 and 677, respectively; SEQ ID NOs: 681 and 685, respectively; SEQ ID NOs: 689 and 693, respectively; SEQ ID NOs: 697 and 701, respectively;SEQ ID NOs: 705 and 709, respectively; SEQ ID NOs: 713 and 717, respectively; SEQ ID NOs: 721 and 725, respectively; SEQ ID NOs: 729 and 733, respectively; SEQ ID NOs: 737 and 741, respectively; SEQ ID NOs: 745 and 749, respectively; SEQ ID NOs: 753 and 757, respectively; SEQ ID NOs: 761 and 765, respectively; SEQ ID NOs: 769 and 773, respectively; SEQ ID NOs: 785 and 789, respectively; SEQ ID NOs: 793 and 797, respectively; SEQ ID NOs: 801 and 805, respectively; SEQ ID NOs: 809 and 809, respectively. and 813; SEQ ID NOs: 817 and 821, respectively; SEQ ID NOs: 849 and 853, respectively; SEQ ID NOs: 857 and 861, respectively; SEQ ID NOs: 865 and 869, respectively; SEQ ID NOs: 873 and 877, respectively; SEQ ID NOs: 881 and 885, respectively; SEQ ID NOs: 889 and 893, respectively; SEQ ID NOs: 897 and 901, respectively; SEQ ID NOs: 905 and 909, respectively; SEQ ID NOs: 913 and 917, respectively; SEQ ID NOs: 921 and 925, respectively; SEQ ID NOs: 929 and 933, respectively; SEQ ID NOs: SEQ ID NOs: 937 and 941, respectively; SEQ ID NOs: 945 and 949, respectively; SEQ ID NOs: 969 and 973, respectively; SEQ ID NOs: 977 and 981, respectively; SEQ ID NOs: 1009 and 1013, respectively; SEQ ID NOs: 1057 and 1061, respectively; SEQ ID NOs: 1537 and 1541, respectively; SEQ ID NOs: 1569 and 1573, respectively; SEQ ID NOs: 1601 and 1605, respectively; SEQ ID NOs: 1641 and 1645, respectively; SEQ ID NOs: 1665 and 1669, respectively; SEQ ID NOs: 1825 and 1829, respectively; SEQ ID NOs: 1865 and 1869; SEQ ID NOs: 1897 and 1901, respectively; SEQ ID NOs: 1905 and 1909, respectively; SEQ ID NOs: 1913 and 1917, respectively; SEQ ID NOs: 1921 and 1925, respectively; SEQ ID NOs: 1929 and 1933, respectively; SEQ ID NOs: 2265 and 2269, respectively; SEQ ID NOs: 2281 and 2285, respectively; SEQ ID NOs: 2289 and 2293, respectively; SEQ ID NOs: 2329 and 2333, respectively; and SEQ ID NOs: 2345 and 2349, respectively; L Amino acid sequence and VH Contains the amino acid sequence.

[0017] Additionally or alternatively, for the heterodimeric multispecific antibodies disclosed herein, in some embodiments, each of the VL-4 and VH-4 is SEQ ID NOs: 17 and 21, respectively; SEQ ID NOs: 25 and 29, respectively; SEQ ID NOs: 33 and 37, respectively; SEQ ID NOs: 41 and 45, respectively; SEQ ID NOs: 121 and 125, respectively; SEQ ID NOs: 137 and 141, respectively; SEQ ID NOs: 169 and 173, respectively; SEQ ID NOs: 177 and 181, respectively; SEQ ID NOs: 185 and 189, respectively; SEQ ID NOs: 193 and 194, respectively. 7; SEQ ID NOs:201 and 205, respectively; SEQ ID NOs:209 and 213, respectively; SEQ ID NOs:217 and 221, respectively; SEQ ID NOs:225 and 229, respectively; SEQ ID NOs:233 and 237, respectively; SEQ ID NOs:241 and 245, respectively; SEQ ID NOs:249 and 253, respectively; SEQ ID NOs:257 and 261, respectively; SEQ ID NOs:265 and 269, respectively; SEQ ID NOs:321 and 325, respectively; SEQ ID NOs:329 and 333, respectively; SEQ ID NOs:337 and 341, respectively; SEQ ID NOs:393 and 397, respectively SEQ ID NOs: 401 and 405, respectively; SEQ ID NOs: 409 and 413, respectively; SEQ ID NOs: 473 and 477, respectively; SEQ ID NOs: 481 and 485, respectively; SEQ ID NOs: 489 and 493, respectively; SEQ ID NOs: 497 and 501, respectively; SEQ ID NOs: 505 and 509, respectively; SEQ ID NOs: 513 and 517, respectively; SEQ ID NOs: 545 and 549, respectively; SEQ ID NOs: 553 and 557, respectively; SEQ ID NOs: 561 and 565, respectively; SEQ ID NOs: 569 and 573, respectively; SEQ ID NOs: 577 and 581, respectively SEQ ID NOs: 585 and 589, respectively; SEQ ID NOs: 593 and 597, respectively; SEQ ID NOs: 601 and 605, respectively; SEQ ID NOs: 625 and 629, respectively; SEQ ID NOs: 633 and 637, respectively; SEQ ID NOs: 641 and 645, respectively; SEQ ID NOs: 649 and 653, respectively; SEQ ID NOs: 657 and 661, respectively; SEQ ID NOs: 665 and 669, respectively; SEQ ID NOs: 673 and 677, respectively; SEQ ID NOs: 681 and 685, respectively; SEQ ID NOs: 689 and 693, respectively; SEQ ID NOs: 697 and 701, respectively;SEQ ID NOs: 705 and 709, respectively; SEQ ID NOs: 713 and 717, respectively; SEQ ID NOs: 721 and 725, respectively; SEQ ID NOs: 729 and 733, respectively; SEQ ID NOs: 737 and 741, respectively; SEQ ID NOs: 745 and 749, respectively; SEQ ID NOs: 753 and 757, respectively; SEQ ID NOs: 761 and 765, respectively; SEQ ID NOs: 769 and 773, respectively; SEQ ID NOs: 785 and 789, respectively; SEQ ID NOs: 793 and 797, respectively; SEQ ID NOs: 801 and 805, respectively; SEQ ID NOs: 809 and 809, respectively. and 813; SEQ ID NOs: 817 and 821, respectively; SEQ ID NOs: 849 and 853, respectively; SEQ ID NOs: 857 and 861, respectively; SEQ ID NOs: 865 and 869, respectively; SEQ ID NOs: 873 and 877, respectively; SEQ ID NOs: 881 and 885, respectively; SEQ ID NOs: 889 and 893, respectively; SEQ ID NOs: 897 and 901, respectively; SEQ ID NOs: 905 and 909, respectively; SEQ ID NOs: 913 and 917, respectively; SEQ ID NOs: 921 and 925, respectively; SEQ ID NOs: 929 and 933, respectively; SEQ ID NOs: SEQ ID NOs: 937 and 941, respectively; SEQ ID NOs: 945 and 949, respectively; SEQ ID NOs: 969 and 973, respectively; SEQ ID NOs: 977 and 981, respectively; SEQ ID NOs: 1009 and 1013, respectively; SEQ ID NOs: 1057 and 1061, respectively; SEQ ID NOs: 1537 and 1541, respectively; SEQ ID NOs: 1569 and 1573, respectively; SEQ ID NOs: 1601 and 1605, respectively; SEQ ID NOs: 1641 and 1645, respectively; SEQ ID NOs: 1665 and 1669, respectively; SEQ ID NOs: 1825 and 1829, respectively; SEQ ID NOs: 1865 and 1869; SEQ ID NOs: 1897 and 1901, respectively; SEQ ID NOs: 1905 and 1909, respectively; SEQ ID NOs: 1913 and 1917, respectively; SEQ ID NOs: 1921 and 1925, respectively; SEQ ID NOs: 1929 and 1933, respectively; SEQ ID NOs: 2265 and 2269, respectively; SEQ ID NOs: 2281 and 2285, respectively; SEQ ID NOs: 2289 and 2293, respectively; SEQ ID NOs: 2329 and 2333, respectively; and SEQ ID NOs: 2345 and 2349, respectively; L Amino acid sequence and VH Contains the amino acid sequence.

[0018] Additionally or alternatively, in some embodiments of the heterodimeric multispecific antibodies disclosed herein, the first immunoglobulin or the third immunoglobulin is selected from the group consisting of a2b b3 (glycoprotein IIb / IIIa), a4, a4b7, a4b7 + aEb7, a5, activin receptor type 2B, ALK1, alpha-synuclein, amyloid beta, APP, AXL, blood group A, CAIX, CCL-2, CD105 (endoglin), CD115 (CSF1R), CD116a (CSF2Ra), CD123, CD152 (CTLA4), CD184 (CXCR4), CD19, CD192 (CCR2), CD194 (CCR4), CD195 (CCR5), CD20, CD200, CD22, CD221 (IGF1R), C D248, CD25, CD257(BAFF), CD26, CD262(DR5), CD276(B7H3), CD3, CD30(TNFRSF8), CD319(SLAMF7), CD33, CD332(FGFR2), CD350(FZ D10), CD37, CD371(CLEC12A), CD38, CD4, CD49b(a2), CD51(a5), CD52, CD56, CD61(a4b3), CD70, CD73(NT5E), CD74, CEA, Claudin 18.2, cMET, CRLR, DLL3, DLL4, DNA / histone (H1) complex, EGFR, EpCAM, EGFR-HER3, EGFRvIII, EphA3, ERGT(GalNAc)Tn antigen, FLT1, FOLR1, frizzled family receptor (FZD), Lewis Y, Lewis X, GCGR, GD2, GD2 α-acetyl, GD3, GM1, GM1 fucosyl, GM2, GPA33, GPNMB, GUCY2C, HER2, HER3, HGFR (cMET), IgHe, IGLF2, kallikrein, LINGO1, LOXL2, Ly6 / PLAUR domain-containing protein 3, MADCAM1, MAG, mesothelin, MT1-MMP (MMP14), MUC1, mucin 5AC, NaPi2b, NeuGc-GM3, notch, NOTCH2 / NOTCH3 receptor, oxLDL, P-selectin, PCSK9, PDGFRA, PDGFRa, phosphatidylserine, polysialic acid, PSMA, PVRL4, RGMA, and CD240D, a blood antigen of type D The first immunoglobulin binds to a cell surface antigen selected from the group consisting of: root plate-specific spondin 3, serum amyloid P component, STEAP-1, TACSTD2, TGFb, TWEAKR, TYRP1, VEGFR2, VSIR, CD171 (L1CAM), CD19, CD47, pMHC[NY-ESO1], pMHC[MART1], pMHC[MAGEA1], pMHC[tyrosinase], pMHC[gp100], pMHC[MUC1], pMHC[tax], pMHC[WT-1], pMHC[EBNA-1], pMHC[LMP2], pMHC[hTERT], GPC3, CD80, CD23, and fibronectin extracellular domain B. The first immunoglobulin and the third immunoglobulin may bind to the same epitope on the target cell, or may bind to two different epitopes on the target cell. In some embodiments, the target cell is a cancer cell.

[0019] Additionally or alternatively, in some embodiments of the heterodimeric multispecific antibodies disclosed herein, the second immunoglobulin or fourth immunoglobulin binds to an epitope on a leukocyte, on a monocyte, on a lymphocyte, on a granulocyte, on a macrophage, on a T cell, on an NK cell, on a B cell, on an NKT cell, on an ILC, or on a neutrophil.

[0020] In any of the above embodiments of the heterodimeric multispecific antibodies disclosed herein, the second immunoglobulin or the fourth immunoglobulin is selected from the group consisting of dabigatran, a4, a4b7, a4b7+aEb7, a5, AXL, BnDOTA, CD11a (LFA-1), CD3, CD4, CD8, CD16, CD19, CD22, CD23, CD25, CD28, CD30 (TNFRSF8), CD33, CD38, CD40, CD40L, CD47, CD49b(a2), CD54 (ICAM-1), CD56, CD74, CD80, CD115 (CSF1R), CD116a (CSF2Ra), CD123, CD134 (OX40), CD137 (41BB), CD1 52 (CTLA4), CD184 (CXCR4), CD192 (CCR2), CD194 (CCR4), CD195 (CCR5), CD223 (LAG-3), CD252 (OX40L), CD254 (RANKL), CD262 (DR5), CD27, CD200, CD221 (IGF1R), CD248, CD274 (PD-L1), CD275 (ICOS-L), CD278 (ICOS), CD279 (PD-1), CD319 (SLAMF7), CD371 (CLEC12A), MADCAM1, MT1-MMP (MMP14), NKG2A, NRP1, TIGIT, VSIR, KIRDL1 / 2 / 3, and KIR2DL2. The second immunoglobulin and the fourth immunoglobulin may bind to the same or different epitopes on leukocytes, monocytes, lymphocytes, granulocytes, macrophages, T cells, NK cells, B cells, NKT cells, ILCs, or neutrophils. In some embodiments, the second immunoglobulin binds to CD3 and the fourth immunoglobulin binds to an immune cell receptor selected from the group consisting of CD4, CD8, CD25, CD28, CTLA4, OX40, ICOS, PD-1, PD-L1, 41BB, CD2, CD69, and CD45. In other embodiments, the second immunoglobulin binds to CD16 and the fourth immunoglobulin binds to an immune cell receptor selected from the group consisting of CD56, NKG2D, and KIRDL1 / 2 / 3.In certain embodiments, the fourth immunoglobulin binds to an agent selected from the group consisting of a cytokine, a nucleic acid, a hapten, a small molecule, a radionuclide, an antitoxin, a vitamin, a peptide, a lipid, a carbohydrate, biotin, digoxin, or a conjugated variant of any of these.

[0021] Additionally or alternatively, in some embodiments of the heterodimeric multispecific antibodies disclosed herein, the first and third immunoglobulins bind to their respective epitopes with a monovalent or effective affinity of between about 100 nM and about 100 pM, hi certain embodiments, the first and third immunoglobulins bind to cell surface epitopes that are 60 to 120 angstroms apart.

[0022] Additionally or alternatively, in some embodiments of the heterodimeric multispecific antibodies disclosed herein, the first and third immunoglobulins bind to their respective epitopes with a monovalent or effective affinity of less than 100 pM, hi certain embodiments, the first and third immunoglobulins bind to cell surface epitopes that are at most 180 angstroms apart. Additionally or alternatively, in some embodiments of the heterodimeric multispecific antibodies disclosed herein, the first heterodimerization domain of the first immunoglobulin and / or the second heterodimerization domain of the third immunoglobulin is a CH2-CH3 domain and has an isotype selected from the group consisting of IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgM, IgD, and IgE.

[0023] Additionally or alternatively, in some embodiments of the heterodimeric multispecific antibodies disclosed herein, the first heterodimerization domain of the first immunoglobulin and / or the second heterodimerization domain of the third immunoglobulin comprise a constant region of IgG1 comprising one or more amino acid substitutions selected from the group consisting of N297A and K322A. Additionally or alternatively, in some embodiments of the heterodimeric multispecific antibodies disclosed herein, the first heterodimerization domain of the first immunoglobulin is a CH2-CH3 domain comprising a K409R mutation and the second heterodimerization domain of the third immunoglobulin is a CH2-CH3 domain comprising a F405L mutation. Also disclosed herein are recombinant nucleic acid sequences encoding any of the antibodies described herein. In another aspect, the technology provides host cells or vectors that express any nucleic acid sequence encoding any of the antibodies described herein.

[0024] In any of the above-described embodiments of the immunoglobulin analog constructs of the present technology, the HDTVS antibody may be conjugated to an agent selected from the group consisting of an isotope, a dye, a chromogen, an imaging agent, a drug, a toxin, a cytokine, an enzyme, an enzyme inhibitor, a hormone, a hormone antagonist, a growth factor, a radionuclide, a metal, a liposome, a nanoparticle, RNA, DNA, or any combination thereof.

[0025] In one aspect, the present disclosure provides a method for treating cancer in a subject in need thereof, comprising administering to the subject an effective amount of a heterodimeric multispecific antibody disclosed herein. The cancer can be lung cancer, colorectal cancer, skin cancer, breast cancer, ovarian cancer, leukemia, pancreatic cancer, or gastric cancer. Additionally or alternatively, in some embodiments, the heterodimeric multispecific antibody is administered to the subject separately, sequentially, or simultaneously with an additional therapeutic agent. Also disclosed herein are kits for the detection and / or treatment of disease (e.g., cancer), comprising at least one heterodimeric trivalent / tetravalent multispecific antibody of the present technology and instructions for use. [Brief explanation of the drawings]

[0026] [Figure 1a] Figure 1 shows the basic design strategy of each heterodimeric tetravalent specificity (HDTVS) variant compared to the parental 2+2 IgG-[L]-scFv. Five heterodimeric IgG-L-scFv designs display novel biological activities. Each construct achieves tri- or tetraspecificity using heterodimerization. [Figure 1b] FIG. 1 shows an overview of the 1+1+2Lo low affinity design and how it can be used to distinguish single antigen-positive healthy cells from double antigen-positive target cells. Single antigen positivity will result in less immune cell activation than double antigen positivity. [Figure 1c] FIG. 1 shows an overview of the 1+1+2Hi high affinity design and how it can be used to target one (or both) of two different cellular antigens. [Figure 1d]

[0023] Figure 1 shows an overview of the 2+1+1 design and how it can be used to improve immune cell activation. Targeting two different immune cell receptors can be used to more specifically recruit immune cell populations or to result in greater activation or inhibition of immune cells through cross-linking of multiple receptors. [Figure 1e]

[0013] Figure 1 shows an overview of the 2+1+1 design and how it can be used to expand immune cell recruitment or combine payload delivery with immunotherapy. Each HDTVS antibody requires only one immune cell receptor for recruitment and activation. Additional domains can then be used to attach payloads (for diagnostics, therapy, recruitment, etc.) or additional effector cells. [Figure 1f] Figure 1 shows an overview of the 1+1+1+1 design and how it can be used to combine the benefits of 1+1+2 with the benefits of 2+1+1. In this embodiment, the tetraspecificity may also result in improved specificity or target expansion and improved immune cell activation or payload delivery. [Figure 2a-1] Figure 1 shows the superior cytotoxicity, binding, and in vivo efficacy of the IgG-[L]-scFv design over the IgG-Het and BiTE formats. A 4-hour Cr51 release assay was used to assess the cytotoxicity of activated T cells against M14 melanoma tumor cells. Flow cytometry was used to assess the differential antigen binding of each bispecific antibody to huCD3 or GD2 on activated T cells or M14 melanoma tumor cells, respectively. Affinity was measured using SPR on a GD2-coated streptavidin chip. To assess in vivo efficacy, two mouse models were used: a syngeneic transgenic model with mouse T cells expressing huCD3, and a humanized xenograft model using activated human T cells engrafted into immunodeficient IL2-rg- / -Rag2- / - BALB / c mice. Mice were implanted subcutaneously with GD2(+) tumors and treated intravenously with the specific bispecific antibodies tested. [Figure 2a-2]Figure 1 shows the superior cytotoxicity, binding, and in vivo efficacy of the IgG-[L]-scFv design over the IgG-Het and BiTE formats. A 4-hour Cr51 release assay was used to assess the cytotoxicity of activated T cells against M14 melanoma tumor cells. Flow cytometry was used to assess the differential antigen binding of each bispecific antibody to huCD3 or GD2 on activated T cells or M14 melanoma tumor cells, respectively. Affinity was measured using SPR on a GD2-coated streptavidin chip. To assess in vivo efficacy, two mouse models were used: a syngeneic transgenic model with mouse T cells expressing huCD3, and a humanized xenograft model using activated human T cells engrafted into immunodeficient IL2-rg- / -Rag2- / - BALB / c mice. Mice were implanted subcutaneously with GD2(+) tumors and treated intravenously with the specific bispecific antibodies tested. [Figure 2a-3] Figure 1 shows the superior cytotoxicity, binding, and in vivo efficacy of the IgG-[L]-scFv design over the IgG-Het and BiTE formats. A 4-hour Cr51 release assay was used to assess the cytotoxicity of activated T cells against M14 melanoma tumor cells. Flow cytometry was used to assess the differential antigen binding of each bispecific antibody to huCD3 or GD2 on activated T cells or M14 melanoma tumor cells, respectively. Affinity was measured using SPR on a GD2-coated streptavidin chip. To assess in vivo efficacy, two mouse models were used: a syngeneic transgenic model with mouse T cells expressing huCD3, and a humanized xenograft model using activated human T cells engrafted into immunodeficient IL2-rg- / -Rag2- / - BALB / c mice. Mice were implanted subcutaneously with GD2(+) tumors and treated intravenously with the specific bispecific antibodies tested. [Figure 2b]FIG. 1 shows the superior cytotoxic effect of the IgG-[L]-scFv design over IgG-het using two additional anti-GD2 sequences. [Figure 3] Schematic diagrams for four IgG-[L]-scFv heterodimer variants, along with the parental and IgG-Het formats. The designs are ranked by their relative potency. [Figure 4-1] Figure 1 shows the in vitro binding activity of various IgG-[L]-scFv mutants. Affinity for GD2 and CD3 was measured using SPR with GD2- or huCD3de-coated chips, respectively. Binding to cells was assayed by flow cytometry using activated human T cells or M14 melanoma cells. Formation of T cell:tumor cell conjugates was measured by flow cytometry using differentially labeled activated human T cells and M14 melanoma tumor cells. [Figure 4-2] Figure 1 shows the in vitro binding activity of various IgG-[L]-scFv mutants. Affinity for GD2 and CD3 was measured using SPR with GD2- or huCD3de-coated chips, respectively. Binding to cells was assayed by flow cytometry using activated human T cells or M14 melanoma cells. Formation of T cell:tumor cell conjugates was measured by flow cytometry using differentially labeled activated human T cells and M14 melanoma tumor cells. [Figure 5] Figure 1 shows the in vitro cytotoxicity of each IgG-[L]-scFv mutant against two cell lines: M14 melanoma cell line and IMR32 neuroblastoma cell line. Cytotoxicity was measured using a Cr51 release assay and activated human T cells over a 4-hour period. [Figure 6-1]Figure 1 shows the in vitro activation of immune cells by each IgG-[L]-scFv mutant. Activation was measured by flow cytometry. Naive purified T cells and M14 melanoma cells were co-cultured for 24 or 96 hours, harvested, and stained for CD69 or CD25, respectively. T cells for the 96-hour time point were also labeled with Cell Trace Violet (CTV). Culture supernatants were also collected at the 24-hour time point for cytokine measurements. [Figure 6-2] Figure 1 shows the in vitro activation of immune cells by each IgG-[L]-scFv mutant. Activation was measured by flow cytometry. Naive purified T cells and M14 melanoma cells were co-cultured for 24 or 96 hours, harvested, and stained for CD69 or CD25, respectively. T cells for the 96-hour time point were also labeled with Cell Trace Violet (CTV). Culture supernatants were also collected at the 24-hour time point for cytokine measurements. [Figure 6-3] Figure 1 shows the in vitro activation of immune cells by each IgG-[L]-scFv mutant. Activation was measured by flow cytometry. Naive purified T cells and M14 melanoma cells were co-cultured for 24 or 96 hours, harvested, and stained for CD69 or CD25, respectively. T cells for the 96-hour time point were also labeled with Cell Trace Violet (CTV). Culture supernatants were also collected at the 24-hour time point for cytokine measurements. [Figure 7] Figure 1 shows the in vivo activity of each IgG-[L]-scFv mutant. To evaluate in vivo efficacy, two mouse models were used: a syngeneic transgenic model with mouse T cells expressing huCD3, and a humanized xenograft model using activated human T cells engrafted into immunodeficient IL2-rg- / -Rag2- / - BALB / c mice. Mice were implanted subcutaneously with GD2(+) tumors and treated intravenously with the specific bispecific antibodies tested. [Figure 8-1] Figure 1 shows various dual bivalent, bispecific antibody formats compared with an IgG-[L]-scFv design. Cytotoxicity was assessed using a 4-hour Cr51 release assay using activated human T cells and M14 melanoma cells. Conjugation activity was measured using flow cytometry. Cell binding was assessed by flow cytometry using activated human T cells and M14 melanoma cells. [Figure 8-2] Figure 1 shows various dual bivalent, bispecific antibody formats compared with an IgG-[L]-scFv design. Cytotoxicity was assessed using a 4-hour Cr51 release assay using activated human T cells and M14 melanoma cells. Conjugation activity was measured using flow cytometry. Cell binding was assessed by flow cytometry using activated human T cells and M14 melanoma cells. [Figure 9-1] Figure 1 shows IgG-[L]-scFv mutants that bind to CD33 or HER2. Cell binding activity was measured by flow cytometry using Molm13 cells, SKMEL28 cells, or MCF7 cells. Cytotoxicity was assessed using Molm13 cells and activated human T cells in a 4-hour Cr51 release assay. [Figure 9-2] Figure 1 shows IgG-[L]-scFv mutants that bind to CD33 or HER2. Cell binding activity was measured by flow cytometry using Molm13 cells, SKMEL28 cells, or MCF7 cells. Cytotoxicity was assessed using Molm13 cells and activated human T cells in a 4-hour Cr51 release assay. [Figure 10a-1] Figure 1 shows two 1+1+2 designs (high affinity and low affinity variants). Cell binding and cytotoxicity assays were performed using the GD2(+)HER2(+) cell line, U2OS. Cytotoxicity was measured using Cr51 release over a 4-hour period, and cell binding was assessed using flow cytometry. [Figure 10a-2] Figure 1 shows two 1+1+2 designs (high affinity and low affinity variants). Cell binding and cytotoxicity assays were performed using the GD2(+)HER2(+) cell line, U2OS. Cytotoxicity was measured using Cr51 release over a 4-hour period, and cell binding was assessed using flow cytometry. [Figure 10b-1] Figure 1 shows two 1+1+2 designs (high-affinity and low-affinity mutants). Cell binding and cytotoxicity assays were performed using GD2(+) IMR32 neuroblastoma cells or HER2(+) HCC1954 breast cancer cells. Cytotoxicity was measured using Cr51 release over a 4-hour period, and cell binding was assessed using flow cytometry. [Figure 10b-2] Figure 1 shows two 1+1+2 designs (high-affinity and low-affinity mutants). Cell binding and cytotoxicity assays were performed using GD2(+) IMR32 neuroblastoma cells or HER2(+) HCC1954 breast cancer cells. Cytotoxicity was measured using Cr51 release over a 4-hour period, and cell binding was assessed using flow cytometry. [Figure 11a] FIG. 1 shows exemplary Fc variants capable of heterodimerization. [Figure 11b] FIG. 1 shows exemplary Fc variants capable of heterodimerization. [Figure 11c] FIG. 1 shows exemplary Fc variants capable of heterodimerization. [Figure 11d] FIG. 1 shows exemplary Fc variants capable of heterodimerization. [Figure 11e] FIG. 1 shows exemplary Fc variants capable of heterodimerization. [Figure 12a] Figure 1 shows various dual bivalent, bispecific antibody formats compared to the IgG-[L]-scFv design in vivo. The schematic shows all four dual bivalent, bispecific antibodies expressed. [Figure 12b]Figure 1 shows mean tumor growth for the in vivo huDKO arming model. Tumor responses were assessed using a T cell arming model in which T cells were preincubated with each BsAb for 20 minutes at concentrations that achieved equivalent anti-GD2 binding domains (verified by flow cytometry). These pre-labeled or "armed" T cells were intravenously injected into tumor-bearing DKO mice. Each line represents one BsAb. Filled triangles represent doses of BsAb-armed human activated T cells (huATC) and IL-2. The dotted line indicates the absence of measurable tumors, and asterisks indicate tumor implantation. Error bars represent standard deviation. [Figure 12c] Figure 1 shows tumor growth from individual mice. Each figure represents one treatment group, with a schematic diagram (see above) for reference. Each solid line represents a single mouse, and the dotted line represents the group mean. [Figure 13] This figure demonstrates the combinatorial binding effect of L1CAM / GD2 1+1+2 Lo, a heterodimeric 1+1+2 Lo format antibody that can simultaneously bind to the ganglioside GD2 and the adhesion protein L1CAM. The design of the 1+1+2 Lo format antibody is shown on the left. The homodimeric formats for GD2 and L1CAM are included for reference. For this binding assay, neuroblastoma cells (IMR32) were incubated with each antibody for 30 minutes at 4°C, washed, and incubated with a fluorescent anti-human secondary antibody. After the final wash, the cells were analyzed using flow cytometry. In this example, the binding of the low-affinity 1+1+2 HDTVS antibody was stronger than the anti-L1CAM homodimeric antibody but weaker than the anti-GD2 homodimeric antibody, demonstrating improved targeting specificity for tumors expressing both GD2 and L1CAM. [Figure 14]This figure demonstrates the combinatorial binding effect of HER2 / EGFR 1+1+2 Hi, a heterodimeric 1+1+2 Hi format antibody that can bind both HER2 and EGFR simultaneously or individually. The design of the 1+1+2 Hi format antibody is shown on the right. The homodimeric formats for HER2 and EGFR are included for reference. For this binding assay, desmoplastic small round cell tumor cells (JN-DSRCT1) were incubated with each antibody for 30 minutes at 4°C, washed, and incubated with a fluorescent anti-human secondary antibody. After the final wash, the cells were analyzed using flow cytometry. In this example, the binding of the high-affinity 1+1+2 HDTVS antibody was stronger than either the anti-HER2 or anti-EGFR homodimeric antibodies, while maintaining specificity for both antigens, demonstrating cooperative binding. [Figure 15] This figure demonstrates the combinatorial binding effect of GD2 / B7H3 1+1+2 Lo, a heterodimeric 1+1+2 Lo format antibody capable of simultaneously binding both GD2 and B7H3. The design of the 1+1+2 Lo format antibody is shown on the left. The homodimeric formats for GD2 and B7H3, as well as monovalent control antibodies for GD2 or B7H3 (GD2 control or B7H3 control, respectively), were included for reference. For this binding assay, osteosarcoma cells (U2OS) were incubated with each antibody for 30 minutes at 4°C, washed, and incubated with a fluorescent anti-human secondary antibody. After a final wash, the cells were analyzed using flow cytometry. In this example, the binding of the low-affinity 1+1+2 HDTVS antibody was similar to that of the anti-B7H3 homodimeric antibody but weaker than that of the anti-GD2 homodimeric antibody. Importantly, GD2 / B7H3 1+1+2 Lo also showed improved binding over the monovalent control antibody, supporting cooperative binding. [Figure 16]This figure demonstrates the cytotoxic selectivity of HER2 / GD2 1+1+2 Lo, a heterodimeric 1+1+2 Lo format capable of simultaneously binding both GD2 and HER2. This format utilizes a low-affinity HER2 sequence. The design of the 1+1+2 Lo format antibody is shown below the line graph. The homodimeric formats for GD2 and HER2, as well as monovalent control antibodies for GD2 or HER2 (GD2 control and HER2 control, respectively), were included for reference. For this cytotoxicity assay, osteosarcoma cells (U2OS) were first incubated with 51Cr for 1 hour. After incubation, 51Cr-labeled target cells were mixed with the indicated antibodies and serial dilutions of activated human T cells at 37°C for 4 hours. After 4 hours, supernatants were collected and analyzed on a gamma counter to quantify 51Cr release. Cytotoxicity was measured as the % 51Cr release relative to maximum release. In this example, the low-affinity 1+1+2 heterodimeric antibody killed target cells as effectively as anti-GD2 and anti-HER2 homodimeric antibodies, but showed clear superiority over the monovalent control format. This confirms the selectivity possible with the 1+1+2 Lo design: target cells expressing each individual antigen will be targeted with 10- to 100-fold less cytotoxic potency than targets expressing both antigens simultaneously. Using a homodimeric design against GD2 or HER2 would lose this selectivity. [Figure 17a]Figure 1 demonstrates the cytotoxic bispecificity of HER2 / GPA33 1+1+2 Hi, a heterodimeric 1+1+2 Hi format capable of simultaneously binding both GPA33 and HER2. The design of the 1+1+2 Hi format antibody is shown below the line graph. Homodimeric formats for GPA33 and HER2, as well as monovalent control antibodies for GPA33 or HER2, were included for reference. For this cytotoxicity assay, colon cancer cells (Colo205) were first incubated with 51Cr for 1 hour. After incubation, 51Cr-labeled target cells were mixed with the indicated antibodies and serial dilutions of activated human T cells at 37°C for 4 hours. After 4 hours, supernatants were collected and read on a gamma counter to quantify 51Cr release. Cytotoxicity was measured as the % 51Cr release relative to maximum release. In this example, the high-affinity 1+1+2 heterodimeric antibody killed target cells as effectively as the anti-GPA33 homodimeric antibody, but with greater potency than the anti-HER2 homodimeric antibody and a monovalent control antibody. These data support the functional cooperativity of the HER2 and GPA33 antigen-binding domains and illustrate that the bispecificity of the 1+1+2Hi format does not significantly impair its cytotoxic activity against either antigen individually. [Figure 17b] This figure demonstrates the combinatorial binding effect of HER2 / GPA33 1+1+2 Hi, a heterodimeric 1+1+2 Hi format antibody that can bind both HER2 and GPA33 simultaneously or individually. The design of the 1+1+2 Hi format antibody is shown on the right. For this binding assay, colon cancer cells (Colo205) were incubated with each antibody for 30 minutes at 4°C, washed, and incubated with a fluorescent anti-human secondary antibody. After a final wash, the cells were analyzed using flow cytometry. In this example, the affinity binding of the 1+1+2 heterodimeric antibody was stronger than that of the anti-HER2 or anti-GPA33 homodimeric antibody and the monovalent control antibody, while maintaining specificity for both antigens, demonstrating cooperative binding. [Figure 18] This figure demonstrates the utility of CD3 / CD28 2+1+1, a heterodimeric 2+1+1 design capable of binding both CD3 and CD28 on T cells. The heterodimeric 1+1+2 format antibody design is shown below the line graph. Homodimeric formats for CD3 and CD28 are included for reference. For the cytokine assay, naive human T cells and melanoma tumor cells (M14) were cocultured with the indicated BsAbs for 20 hours, after which culture supernatants were collected and analyzed for the secreted cytokine, IL-2, by flow cytometry. Data were normalized to cytokine release by T cells without target cells or antibody after 20 hours. The CD3 / CD28 2+1+1 design clearly demonstrated stronger cytokine release activity than CD3 or CD28 engagement alone, illustrating the cooperative activity of CD3 / CD28 dual engagement. [Figure 19] Figure 1 demonstrates the combinatorial binding effect of CD3 / CD4 2+1+1, a heterodimeric 2+1+1 format antibody that can simultaneously bind both CD3 and CD4. The design of the heterodimeric 2+1+1 format antibody is shown on the right. For this binding assay, activated human T cells were incubated with each antibody for 30 minutes at 4°C, washed, and incubated with a fluorescent anti-human secondary antibody. After a final wash, the cells were analyzed using flow cytometry. In this example, the 2+1+1 heterodimer exhibits enhanced binding compared to a bivalent CD4 and monomeric CD3 binder (2+1), demonstrating cooperative binding. [Figure 20]This figure demonstrates the combinatorial binding effect of CD3 / PD-1 2+1+1, a heterodimeric 2+1+1 format antibody that can simultaneously bind to both CD3 and PD-1. The design of the heterodimeric 2+1+1 format antibody is shown on the right. For this binding assay, activated human T cells were incubated with each antibody for 30 minutes at 4°C, washed, and incubated with a fluorescent anti-human secondary antibody. After a final wash, the cells were analyzed using flow cytometry. In this example, the binding of the 2+1+1 heterodimer was better than either the anti-PD-1 homodimer or the anti-CD3 monomer (2+1) binder, demonstrating cooperative binding. [Figure 21a]Figure 21a shows the unique features of the IgG-L-scFv design compared to two other common bivalent design strategies: BiTE-Fc and IgG-H-scFv. Figure 21a confirms the potent T cell functional activity of the IgG-L-scFv design compared to other bivalent T cell bispecific antibody formats. The IgG-L-scFv, BiTE-Fc, and IgG-H-scFv format antibody designs are shown below the line graph. For the cytokine assay, naive T cells and melanoma tumor cells (M14) were co-cultured with each BsAb for 20 hours, and then the culture supernatants were collected and analyzed for the secreted cytokine, IL-2, by flow cytometry. Data were normalized to cytokine release by T cells after 20 hours without target cells or antibody. In contrast to the IgG-H-scFv (2+2HC) and BiTE-Fc (2+2B) designs, the IgG-L-scFv format (2+2) demonstrated a significant in vitro cytokine IL-2 response (shown in Figure 21c), which correlated with strong in vivo activity. Figure 21b illustrates the unusually weak T cell binding activity of the IgG-L-scFv design compared to other dual bivalent T cell bispecific antibody formats. For this binding assay, T cells and melanoma tumor cells (M14) were incubated with each antibody for 30 minutes at 4°C, washed, and incubated with a fluorescent anti-human secondary antibody. After the final wash, the cells were analyzed using flow cytometry. CD3-specific binding (Figure 21b, upper panel) and GD2-specific binding (Figure 21b, middle panel) are shown. The IgG-L-scFv, BiTE-Fc, and IgG-H-scFv format antibody designs are shown in Figure 21b (lower panel). In contrast to their GD2 binding activities, each BsAb exhibited distinct T cell binding activities. These data demonstrate how the IgG-L-scFv design is uniquely different from other double bivalent designs in which each scFv exhibits incomplete bivalent binding.While the incorporation of two scFv domains within the IgG-L-scFv represents an improvement over the monovalent design, it still does not match the avidity of the 2+2 HC or 2+2 B designs, illustrating the steric hindrance to binding of this format. Figure 21c illustrates the superiority of the IgG-L-scFv design in vivo. In contrast to other double bivalent designs, the IgG-L-scFv format was the only format capable of controlling tumor growth in mice. Here, immunodeficient mice (Balb / c IL-2Rgc- / -, Rag2- / -) were subcutaneously implanted with neuroblastoma cells (IMR32) and then treated intravenously with activated T cells and antibodies (twice weekly). Tumor size was measured with a caliper. [Figure 21b]Figure 21a shows the unique features of the IgG-L-scFv design compared to two other common bivalent design strategies: BiTE-Fc and IgG-H-scFv. Figure 21a confirms the potent T cell functional activity of the IgG-L-scFv design compared to other bivalent T cell bispecific antibody formats. The IgG-L-scFv, BiTE-Fc, and IgG-H-scFv format antibody designs are shown below the line graph. For the cytokine assay, naive T cells and melanoma tumor cells (M14) were co-cultured with each BsAb for 20 hours, and then the culture supernatants were collected and analyzed for the secreted cytokine, IL-2, by flow cytometry. Data were normalized to cytokine release by T cells after 20 hours without target cells or antibody. In contrast to the IgG-H-scFv (2+2HC) and BiTE-Fc (2+2B) designs, the IgG-L-scFv format (2+2) demonstrated a significant in vitro cytokine IL-2 response (shown in Figure 21c), which correlated with strong in vivo activity. Figure 21b illustrates the unusually weak T cell binding activity of the IgG-L-scFv design compared to other dual bivalent T cell bispecific antibody formats. For this binding assay, T cells and melanoma tumor cells (M14) were incubated with each antibody for 30 minutes at 4°C, washed, and incubated with a fluorescent anti-human secondary antibody. After the final wash, the cells were analyzed using flow cytometry. CD3-specific binding (Figure 21b, upper panel) and GD2-specific binding (Figure 21b, middle panel) are shown. The IgG-L-scFv, BiTE-Fc, and IgG-H-scFv format antibody designs are shown in Figure 21b (lower panel). In contrast to their GD2 binding activities, each BsAb exhibited distinct T cell binding activities. These data demonstrate how the IgG-L-scFv design is uniquely different from other double bivalent designs in which each scFv exhibits incomplete bivalent binding.While the incorporation of two scFv domains within the IgG-L-scFv represents an improvement over the monovalent design, it still does not match the avidity of the 2+2 HC or 2+2 B designs, illustrating the steric hindrance to binding of this format. Figure 21c illustrates the superiority of the IgG-L-scFv design in vivo. In contrast to other double bivalent designs, the IgG-L-scFv format was the only format capable of controlling tumor growth in mice. Here, immunodeficient mice (Balb / c IL-2Rgc- / -, Rag2- / -) were subcutaneously implanted with neuroblastoma cells (IMR32) and then treated intravenously with activated T cells and antibodies (twice weekly). Tumor size was measured with a caliper. [Figure 21c]Figure 21a shows the unique features of the IgG-L-scFv design compared to two other common bivalent design strategies: BiTE-Fc and IgG-H-scFv. Figure 21a confirms the potent T cell functional activity of the IgG-L-scFv design compared to other bivalent T cell bispecific antibody formats. The IgG-L-scFv, BiTE-Fc, and IgG-H-scFv format antibody designs are shown below the line graph. For the cytokine assay, naive T cells and melanoma tumor cells (M14) were co-cultured with each BsAb for 20 hours, and then the culture supernatants were collected and analyzed for the secreted cytokine, IL-2, by flow cytometry. Data were normalized to cytokine release by T cells after 20 hours without target cells or antibody. In contrast to the IgG-H-scFv (2+2HC) and BiTE-Fc (2+2B) designs, the IgG-L-scFv format (2+2) demonstrated a significant in vitro cytokine IL-2 response (shown in Figure 21c), which correlated with strong in vivo activity. Figure 21b illustrates the unusually weak T cell binding activity of the IgG-L-scFv design compared to other dual bivalent T cell bispecific antibody formats. For this binding assay, T cells and melanoma tumor cells (M14) were incubated with each antibody for 30 minutes at 4°C, washed, and incubated with a fluorescent anti-human secondary antibody. After the final wash, the cells were analyzed using flow cytometry. CD3-specific binding (Figure 21b, upper panel) and GD2-specific binding (Figure 21b, middle panel) are shown. The IgG-L-scFv, BiTE-Fc, and IgG-H-scFv format antibody designs are shown in Figure 21b (lower panel). In contrast to their GD2 binding activities, each BsAb exhibited distinct T cell binding activities. These data demonstrate how the IgG-L-scFv design is uniquely different from other double bivalent designs in which each scFv exhibits incomplete bivalent binding.While the incorporation of two scFv domains within the IgG-L-scFv represents an improvement over the monovalent design, it still does not match the avidity of the 2+2 HC or 2+2 B designs, illustrating the steric hindrance to binding of this format. Figure 21c illustrates the superiority of the IgG-L-scFv design in vivo. In contrast to other double bivalent designs, the IgG-L-scFv format was the only format capable of controlling tumor growth in mice. Here, immunodeficient mice (Balb / c IL-2Rgc- / -, Rag2- / -) were subcutaneously implanted with neuroblastoma cells (IMR32) and then treated intravenously with activated T cells and antibodies (twice weekly). Tumor size was measured with a caliper. [Figure 22] Figure 1 supports the in vitro characterization and design of the anti-CD33 / CD3 IgG-[L]-scFv panel. The in vitro cytotoxicity EC50, EC50 fold difference, antigen binding valency, heterodimeric design, and protein purity by SEC-HPLC for the anti-CD33 / CD3 IgG-[L]-scFv panel are summarized. Fold changes are based on 2 + 2 EC50. Purity was calculated as the protein fraction at the appropriate elution time from the total protein by the area under the curve of the SEC-HPLC chromatogram. For the cytotoxicity assay, CD33-transfected cells (Nalm6) were first incubated with 51Cr for 1 hour. 51Cr-labeled target cells were then mixed with the indicated antibodies and serial titrations of activated human T cells at 37°C for 4 hours. Supernatants were collected and analyzed on a gamma counter to quantify 51Cr release. Cytotoxicity was measured as the % 51Cr release relative to maximum release. These results confirm the relative importance of cis-oriented binding domains in a further antigen system (CD33) that is much more distal to the membrane than GD2 (see Figure 5). [Figure 23-1]Figure 1 provides an overview of the various HDTVS antibodies tested in the Examples disclosed herein. The table summarizes all successfully generated HDTVS format multispecific antibodies across various antigen models. All clones were expressed in Expi293 cells and heterodimerized using the controlled Fab arm exchange method. The HDTVS type indicates the type of each clone. Fab1 and scFv1 (and corresponding Ag1 and Ag3) are joined in cis orientation on a single heavy chain (linked by the Fab light chain), while Fab2 and scFv2 (and corresponding Ag2 and Ag4) are joined in cis orientation on separate heavy chain molecules (linked by the Fab light chain). [Figure 23-2] Figure 1 provides an overview of the various HDTVS antibodies tested in the Examples disclosed herein. The table summarizes all successfully generated HDTVS format multispecific antibodies across various antigen models. All clones were expressed in Expi293 cells and heterodimerized using the controlled Fab arm exchange method. The HDTVS type indicates the type of each clone. Fab1 and scFv1 (and corresponding Ag1 and Ag3) are joined in cis orientation on a single heavy chain (linked by the Fab light chain), while Fab2 and scFv2 (and corresponding Ag2 and Ag4) are joined in cis orientation on separate heavy chain molecules (linked by the Fab light chain). DETAILED DESCRIPTION OF THE INVENTION

[0027] It will be understood that certain aspects, modes, embodiments, variations, and features of the present methods are described below in varying levels of detail in order to provide a substantial understanding of the present technology.

[0028] The practice of the present methods uses many conventional techniques in molecular biology, protein biochemistry, cell biology, immunology, microbiology, and recombinant DNA. For example, Sambrook and Russell eds. (2001) Molecular Cloning: A Laboratory Manual, 3rd edition; the series Ausubel et al. eds. (2007) Current Protocols in Molecular Biology; the series Methods in Enzymology (Academic Press, Inc., NY); MacPherson et al. (1991) PCR 1: A Practical Approach (IRL Press at Oxford University Press); MacPherson et al. (1995) PCR 2: A Practical Approach;Harlow and Lane eds. (1999) Antibodies, A Laboratory Manual;Freshney (2005) Culture of Animal Cells: A Manual of Basic Technique, 5th edition;Gait ed. (1984) Oligonucleotide Synthesis;US Patent No. 4,683,195;Hames and Higgins eds. (1984) Nucleic Acid Hybridization;Anderson (1999) Nucleic Acid Hybridization; Hames and Higgins eds. (1984) Transcription and Translation; Immobilized Cells and Enzymes (IRL Press (1986)); Perbal (1984) A Practical Guide to Molecular Cloning; Miller and Calos eds.(1987) Gene Transfer Vectors for Mammalian Cells (Cold Spring Harbor Laboratory); Makrides ed. (2003) Gene Transfer and Expression in Mammalian Cells; Mayer and Walker eds. (1987) Immunochemical Methods in Cell and Molecular Biology (Academic Press, London); and Herzenberg et al. eds. (1996) Weir's Handbook of Experimental Immunology. Methods for detecting polypeptide gene expression products and measuring their levels (i.e., gene translation levels) are well known in the art and include the use of polypeptide detection methods, such as antibody detection and quantification methods (see also Strachan & Read, Human Molecular Genetics, Second Edition (John Wiley and Sons, Inc., NY, 1999)).

[0029] Advances in protein engineering can enhance the functional output of proteins by linking different peptides in sequence or placing them in complexes that do not occur in nature. Antibodies have been used as a platform for such enhancement, where binding to antigens can be modulated through affinity maturation (Boder et al., Proc Natl Acad Sci USA 97:10701-10705 (2000)) or increased valency (Cuesta et al., Trends Biotechnol 28:355-362 (2010)). Binding to Fc receptors can be modulated through point mutations (Leabman et al., MAbs 5:896-903 (2013)) or altered glycosylation (Xu et al., Cancer Immun Res 4: 631-638 (2016)), whereas pharmacokinetics can be affected by ablation of binding to FcR(n) (Suzuki et al., J Immunol 184:1968-1976 (2010)) or removal of entire antibody domains. However, no single antibody platform currently demonstrates a clear and significant functional advantage over others in the clinic.

[0030] The present disclosure presents an antibody platform in which up to four different antigen-binding domains simultaneously engage up to four different cellular targets, thereby increasing the avidity and modulating the specificity of therapeutic antibodies. This platform is based on the heterodimerization of two IgG half molecules (i.e., the IgG-[L]-scFv platform), where each IgG half molecule contains a heavy chain and a light chain and an scFv is linked to the C-terminus of at least one light chain. The resulting heterodimers are both trivalent / tetravalent and multispecific and are collectively referred to as HDTVS antibodies.

[0031] The native form of the IgG-[L]-scFv platform has bivalent binding (2 + 2) to two different targets (each integer represents a different specificity, while the value represents the valency). This disclosure presents five HDTVS platform variants that vary four functional domains (two Fabs and two scFvs) within the IgG(L)-scFv format: (1) a Lo1 + 1 + 2 HDTVS variant that achieves improved tumor cell specificity; (2) a Hi1 + 1 + 2 HDTVS variant that achieves expanded tumor cell selectivity; (3) a 2 + 1 + 1 HDTVS variant that achieves improved immune cell activation; (4) a 2 + 1 + 1 HDTVS variant that enables recruitment of different cells and / or payloads; and (5) a 1 + 1 + 1 + 1 HDTVS variant (Figures 1a-1f) that combines designs (1) or (2) with designs (3) or (4) to achieve more effective immune activation or payload delivery by refining specificity or expanding selectivity. To test the functional output of these HDTVS variants, an unrelated Fab that does not bind to either tumor cells or effector immune cells (e.g., T cells) can be used to neutralize one of the two Fab domains, creating monovalency for tumors. Alternatively, one of the scFv domains can be removed to create monovalency for effector immune cells (e.g., T cells).

[0032] As described herein, the biological potency of each design depends on the biophysical characteristics of the antigen-binding domain of the HDTVS variant. Unexpectedly, changes in valency did not perfectly correlate with changes in functional output. As shown in the examples described herein, the biological activity of trispecific and tetraspecific variants of the HDTVS platform depends on the antigen / epitope combination as well as the relative binding affinity for each target antigen (up to a total of four targets). The Lo1+1+2 HDTVS variant (b) binds two distinct tumor antigens in its Fab domains that are within 60-120 angstroms of each other (thus allowing simultaneous binding); and (c) exhibits monovalent and / or effective binding affinity (K) in the range of about 100 nM to about 100 pM to reduce bystander reactivity with healthy cells. D On the other hand, Hi1+1+2 HDTVS variants require the Fab domain to have high monovalent binding affinity and / or effective binding affinity (K D <100 pM), resulting in monovalent antibodies being nearly as effective as bivalent ones. Furthermore, the 2+1+1 HDTVS variant exhibited in vivo tumor-cleaving activity comparable to that observed with the native 2+2 form of the IgG-[L]-scFv platform. These results were unexpected given that the binding activity of the 2+1+1 HDTVS variant was approximately six-fold lower than that of the native 2+2 form of the IgG-[L]-scFv platform.

[0033] Therefore, orientation (cis vs. trans), valency (monovalent vs. bivalent), and affinity for the target (K DBiophysical properties such as ≒ nM or <pM) had an unpredictable impact on the functionality of HDTVS variants (e.g., an enhancement of several orders of magnitude in therapeutic efficacy). Furthermore, the HDTVS antibodies of the present technology exhibit excellent therapeutic efficacy compared to other conventional antibody platforms such as BiTE or heterodimeric IgG (IgG-Het). These results also support that different multispecific antibody platforms result in antibodies with substantially different biological properties. Without being bound by theory, it is considered that, in addition to the spatial distance between the antigen-binding domains of a multispecific antibody, the relative flexibility and orientation of the individual antigen-binding domains can determine their ability to drive cell-cell interactions.

[0034] Definitions Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. As used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. For example, reference to "a cell" includes combinations of two or more cells. In general, the terminology used herein and the laboratory procedures in cell culture, molecular genetics, organic chemistry, and analytical chemistry, and nucleic acid chemistry, as well as hybridization methods described below, are well known and commonly employed in the art.

[0035] As used herein, the "2+1+1" design refers to an HDTVS antibody in which two Fab domains recognize and bind to the same target antigen, and two scFvs recognize and bind to two separate target antigens. In some embodiments, the two scFvs of the 2+1+1 HDTVS antibody bind to two separate target antigens that are up to 180 angstroms apart from each other in order to engage two individual molecules on the same cell. As used herein, the term "about" in reference to a number is generally understood to include numbers that fall within a range of 1%, 5%, or 10% in either direction (greater than or less than) of the number (except when such number is less than 0% or greater than 100% of a possible value), unless stated otherwise or clear from the context.

[0036] As used herein, "administration" of an agent or drug to a subject includes any route that introduces or delivers a compound to a subject to perform its intended function. Administration can be performed by any suitable route, including, but not limited to, oral, intranasal, parenteral (intravenous, intramuscular, intraperitoneal, or subcutaneous), rectal, intrathecal, intratumoral, or topical. Administration includes self-administration and administration by another person.

[0037] The term "antibody," as used herein, collectively refers to immunoglobulin or immunoglobulin-like molecules, including, by way of example and not limitation, IgA, IgD, IgE, IgG, and IgM, combinations thereof, and similar molecules produced during the immune response of any vertebrate, e.g., mammals such as humans, goats, rabbits, and mice, as well as non-mammalian species, such as shark immunoglobulins. As used herein, "antibody" (including intact immunoglobulins) and "antigen-binding fragment" specifically bind to a molecule of interest (or a group of highly similar molecules of interest) to the substantial exclusion of binding to other molecules (e.g., with a binding constant for the molecule of interest that is at least 10 times higher than that for other molecules in a biological sample). 3 M -1 Large, at least 10 4 M -1 Greater than or at least 10 5 M -1(Antibodies and antibody fragments with large binding constants). The term "antibody" also includes genetically engineered forms such as chimeric antibodies (e.g., humanized murine antibodies), heteroconjugate antibodies (such as bispecific antibodies), etc. See also Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, Ill.); Kuby, J., Immunology, 3rd Ed., W.H. Freeman & Co., New York, 1997.

[0038] More specifically, an antibody refers to a polypeptide ligand comprising at least one light or heavy immunoglobulin chain variable region that specifically recognizes and binds to an epitope of an antigen. H ) region and the light chain variable (V L It is composed of heavy and light chains with variable regions called V H Area and V LThe regions are responsible for binding to the antigen recognized by the antibody. Typically, immunoglobulins have heavy (H) and light (L) chains interconnected by disulfide bonds. There are two types of light chains: lambda (λ) and kappa (κ). There are five major heavy chain classes (or isotypes) that determine the functional activity of antibody molecules: IgM, IgD, IgG, IgA, and IgE. Each heavy and light chain contains a constant region and a variable region (regions are also known as "domains"). When combined, the heavy and light chain variable regions specifically bind to the antigen. The light and heavy chain variable regions contain "framework" regions that flank three hypervariable regions, also called "complementarity-determining regions" or "CDRs." The extent of framework regions and CDRs has been defined (see Kabat et al., Sequences of Proteins of Immunological Interest, US Department of Health and Human Services, 1991, incorporated herein by reference). Today, the Kabat database is maintained online. The sequences of framework regions of different light or heavy chains are relatively conserved within a species. The framework regions of antibodies, which are the combined framework regions of the constituent light and heavy chains, largely adopt a β-sheet conformation, and the CDRs form loops that connect and, in some cases, form part of the β-sheet structure. Thus, the framework regions act to form a scaffold that allows the CDRs to be positioned in the correct orientation through non-covalent interactions between the chains.

[0039] CDRs are primarily responsible for binding to an epitope of an antigen. The CDRs of each chain are typically referred to as CDR1, CDR2, and CDR3, numbered sequentially starting from the N-terminus, and are also typically identified by the chain in which the particular CDR is located. Thus, V H CDR3 is located in the variable domain of the heavy chain of the antibody in which it is found, whereas V LCDR1 is derived from the variable domain of the light chain of the antibody in which it is found. Antibodies that bind to a target antigen have specific V H Area and V L Since each CDR has a sequence of distinct regions, it will have a unique CDR sequence. Antibodies with different specificities (i.e., different binding sites for different antigens) have different CDRs. Although it is the CDRs that vary between antibodies, only a limited number of amino acid positions within the CDRs are directly involved in binding to the antigen. These positions within the CDRs are called specificity-determining residues (SDRs). As used herein, "immunoglobulin analog constructs" refer to antibodies (including monoclonal antibodies, polyclonal antibodies, humanized antibodies, chimeric antibodies, recombinant antibodies, multispecific antibodies, bispecific antibodies, etc.), as well as antibody fragments. An antibody or an antigen-binding fragment thereof specifically binds to an antigen.

[0040] As used herein, the term "antibody-related polypeptide" refers to antigen-binding antibody fragments, including single-chain antibodies, which may comprise the variable region, alone or in combination with all or a portion of the following polypeptide elements: hinge region, CH1 domain, CH2 domain, and CH3 domain of an antibody molecule. The technology also includes any combination of the variable region with the hinge region, CH1, CH2, and CH3 domain. Antibody-related molecules useful in the present methods include Fab, Fab', and F(ab')2, Fd, single-chain Fv (scFv), single-chain antibodies, disulfide-linked Fv (sdFv), and V L Domain or V H Examples include, but are not limited to, fragments containing the V domain. L Domain, V H Domain, C L (ii) a Fab fragment, which is a monovalent fragment consisting of a V domain, a V domain, and a CH1 domain; (iii) a F(ab')2 fragment, which is a bivalent fragment comprising two Fab fragments linked by disulfide bridges in the hinge region; H (iv) a Fd fragment consisting of a V domain and a CH1 domain; L Domains and V H(v) an Fv fragment consisting of a V domain; H and (vi) isolated complementarity-determining regions (CDRs). Thus, an "antibody fragment" or "antigen-binding fragment" can include a portion of a full-length antibody, generally the antigen-binding or variable region thereof; diabodies; linear antibodies; single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.

[0041] As used herein, a "bispecific antibody" or "BsAb" refers to an antibody that can simultaneously bind to two targets with distinct structures, e.g., two different target antigens, two different epitopes on the same target antigen, or a hapten and a target antigen or an epitope on a target antigen. A variety of different bispecific antibody structures are known in the art. In some embodiments, each antigen-binding moiety in a bispecific antibody is a V H Area and / or V L In some such embodiments, the V H Area and / or V L The region is a V region found within a particular monoclonal antibody. H Area and / or V L In some embodiments, bispecific antibodies comprise V regions, each derived from a different monoclonal antibody. H Area and / or V L In some embodiments, a bispecific antibody comprises two antigen-binding moieties comprising a V region, one of which contains the CDRs from a first monoclonal antibody. H Area and / or V L and an immunoglobulin molecule having a V region, wherein the other antigen-binding portion comprises a V region containing CDRs derived from a second monoclonal antibody. H Area and / or V LAntibody fragments having regions (e.g., Fab, F(ab'), F(ab')2, Fd, Fv, dAB, scFv, etc.) contain two antigen-binding portions.

[0042] As used herein, the term "diabody" refers to a small antibody fragment with two antigen-binding sites, each of which is separated into two light chain variable domains (V L ) connected to a heavy chain variable domain (V H )(V H V L (Diabodies refer to fragments containing two domains, each of which is a fragment of a diabody.) By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with complementary domains on another chain, creating two antigen-binding sites. Diabodies are more fully described, for example, in EP 404,097; WO 93 / 11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA, 90: 6444-6448 (1993).

[0043] As used herein, the term "single chain antibody" or "single chain Fv (scFv)" refers to a single chain antibody comprising two domains of an Fv fragment, V L and V H Single-chain antibody molecules may comprise polymers with multiple individual molecules, such as dimers, trimers, or other polymers. Additionally, the two domains of the Fv fragment, V, L and V H are encoded by separate genes, but using recombinant methods they can be combined into V L Area and V HThese domains are connected by synthetic linkers that allow them to pair into single protein chains (known as single-chain Fvs (scFvs)) that form monovalent molecules (Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad Sci. USA 85:5879-5883). Such single-chain antibodies can be prepared by recombinant techniques or by enzymatic or chemical cleavage of intact antibodies.

[0044] Any of the above-mentioned antibody fragments are obtained using conventional techniques known to those skilled in the art, and the fragments are screened for binding specificity and neutralizing activity in the same manner as are intact antibodies. As used herein, "antigen" refers to a molecule to which an antibody (or antigen-binding fragment thereof) can selectively bind. Target antigens can be proteins, carbohydrates, nucleic acids, lipids, haptens, or other naturally occurring or synthetic compounds. In some embodiments, target antigens can be polypeptides. Antigens can also be administered to animals to generate an immune response in the animal. The term "antigen-binding fragment" refers to a fragment of the entire immunoglobulin structure that contains a portion of the polypeptide that is responsible for binding to an antigen. Examples of antigen-binding fragments useful in the present technology include, but are not limited to, scFv, (scFv)2, scFvFc, Fab, Fab', and F(ab')2.

[0045] "Binding affinity" refers to the strength of the total noncovalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). The affinity of a molecule X for its partner Y is generally determined by the dissociation constant (K D) Affinity can be measured by standard methods known in the art, including those described herein. Low affinity complexes generally contain antibodies that tend to dissociate readily from the antigen, whereas high affinity complexes generally contain antibodies that tend to remain bound to the antigen for extended periods of time.

[0046] As used herein, the term "biological sample" refers to sample material derived from living cells. Biological samples can include tissues, cells, protein or membrane extracts of cells, and biological fluids (e.g., ascites or cerebrospinal fluid (CSF)) isolated from a subject, as well as tissues, cells, and fluids present in a subject. Biological samples of the present technology include, but are not limited to, samples taken from breast tissue, renal tissue, cervix, endometrium, head or neck, gallbladder, parotid tissue, prostate, brain, pituitary gland, kidney tissue, muscle, esophagus, stomach, small intestine, colon, liver, spleen, pancreas, thyroid tissue, heart tissue, lung tissue, bladder, adipose tissue, lymph node tissue, uterus, ovarian tissue, adrenal tissue, testicular tissue, tonsils, thymus, blood, hair, oral cavity, skin, serum, plasma, CSF, semen, prostatic fluid, seminal fluid, urine, feces, sweat, saliva, sputum, mucus, bone marrow, lymph, and tears. Biological samples may also be obtained from biopsies of internal organs or from cancer. Biological samples may be obtained from subjects for diagnostic or research studies, or from unaffected individuals as controls or for basic research studies. Samples can be obtained by standard methods, including, for example, venipuncture and surgical biopsy. In certain embodiments, the biological sample is a breast, lung, colon, or prostate tissue sample obtained by needle biopsy.

[0047] As used herein, the term "cancer" refers to a neoplasm or tumor that results from the abnormal, uncontrolled growth of cells. In some embodiments, cancer refers to a benign or malignant tumor. In some embodiments, cancer is associated with a specific cancer antigen. As used herein, the term "CDR-grafted antibody" refers to an antibody in which at least one CDR of an "acceptor" antibody is replaced with a CDR "graft" from a "donor" antibody having the desired antigen specificity.

[0048] As used herein, the term "chimeric antibody" refers to an antibody in which, using recombinant DNA techniques, the Fc constant region of a monoclonal antibody derived from one species (e.g., a murine Fc constant region) has been replaced with the Fc constant region derived from an antibody of another species (e.g., a human Fc constant region). See generally, Robinson et al., PCT / US86 / 02269; Akira et al., European Patent Application No. 184,187; Taniguchi, European Patent Application No. 171,496; Morrison et al., European Patent Application No. 173,494; Neuberger et al., WO 86 / 01533; Cabilly et al., U.S. Pat. No. 4,816,567; Cabilly et al., European Patent Application No. 0125,023; Better et al., Science 240: 1041-1043, 1988; Liu et al., Proc. Natl. Acad. Sci. USA 84: 3439-3443, 1987; Liu et al., J. Immunol 139: 3521-3526, 1987; Sun et al., Proc. Natl. Acad. See Sci. USA 84: 214-218, 1987; Nishimura et al., Cancer Res 47: 999-1005, 1987; Wood et al., Nature 314: 446-449, 1985; and Shaw et al., J. Natl. Cancer Inst. 80: 1553-1559, 1988.

[0049] As used herein, the term "consensus FR" refers to an antibody framework (FR) region within the consensus sequence of an immunoglobulin. The FR region of an antibody does not contact the antigen. As used herein, a "control" is a substitute sample used in an experiment for comparison purposes. A control can be either "positive" or "negative." For example, if the purpose of an experiment is to determine the correlation of the effectiveness of a therapeutic agent to treatment for a particular type of disease, a positive control (a compound or composition known to exhibit the desired therapeutic effect) and a negative control (a subject or sample that is not treated or is given a placebo) are typically utilized. As used herein, the term "effective affinity" refers to a binding constant derived from measuring the overall binding kinetics of a compound through two or more simultaneous binding interactions (e.g., with an IgG, IgM, IgA, IgD, or IgE molecule, but not with an Fab domain).

[0050] As used herein, the term "effective amount" refers to an amount sufficient to achieve the desired therapeutic and / or prophylactic effect, e.g., an amount resulting in the prevention or alleviation of a disease or condition described herein, or one or more signs or symptoms associated with a disease or condition described herein. In the context of therapeutic or prophylactic applications, the amount of a composition administered to a subject will vary depending on the composition, the extent, type, and severity of the disease, and individual characteristics such as general health, age, sex, weight, and tolerance to drugs. Those skilled in the art will determine the appropriate dosage depending on these and other factors. The composition may also be administered in combination with one or more additional therapeutic compounds. In the methods described herein, a therapeutic composition may be administered to a subject with one or more signs or symptoms of a disease or condition described herein. As used herein, a "therapeutically effective amount" of a composition refers to a level of the composition at which the physiological effects of the disease or condition are ameliorated or eliminated. A therapeutically effective amount may be administered in one or more administrations.

[0051] As used herein, the term "effector cell" refers to an immune cell that is involved in the effector phase of an immune response, as opposed to the cognitive and activation phases of the immune response. Exemplary immune cells include cells of bone marrow or lymphatic origin, such as lymphocytes (e.g., B cells and T cells, including cytolytic T cells (CTLs)), killer cells, natural killer cells, macrophages, monocytes, eosinophils, neutrophils, polymorphonuclear cells, granulocytes, mast cells, and basophils. Effector cells express specific Fc receptors and perform specific immune functions. Effector cells, such as neutrophils capable of inducing ADCC, can induce antibody-dependent cell-mediated cytotoxicity (ADCC). For example, monocytes, macrophages, neutrophils, eosinophils, and lymphocytes that express FcαR are involved in the specific killing of target cells and in presenting antigens, or binding to cells that present antigens, to other components of the immune system. As used herein, "effector function" refers to a biochemical event resulting from the interaction of the Fc region of an antibody with an Fc receptor or an antigen. Effector functions include, but are not limited to, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), and complement-dependent cytotoxicity (CDC). Effector functions include both effector functions that act after antigen binding and effector functions that act independently of antigen binding.

[0052] As used herein, the term "epitope" refers to an antigenic determinant (site on an antigen) capable of specific binding to an antibody. Epitopes typically comprise chemically active surface groupings of molecules, such as amino acids or sugar side chains, and may have specific three-dimensional structural characteristics as well as specific charge characteristics. Conformational and nonconformational epitopes are distinguished in that the binding to the former is lost in the presence of denaturing solvents, whereas the binding to the latter is not. Thus, in some embodiments, the heterodimeric trivalent / tetravalent multispecific antibodies disclosed herein may bind to nonconformational epitopes and / or conformational epitopes. To screen for antibodies that bind to an epitope, a routine cross-blocking assay can be performed, such as that described in Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, Ed Harlow and David Lane (1988). This assay can be used to determine whether the antibody binds to the same site or epitope as the heterodimeric trivalent / tetravalent multispecific antibody of the present technology. Alternatively, or in addition, epitope mapping can be performed by methods known in the art. For example, the antibody sequence can be mutagenized, such as by alanine scanning, to identify contact residues. In a different method, peptides corresponding to different regions of the target protein antigen can be used in competition assays with the test antibody, or with the test antibody and an antibody whose epitope is characterized or known.

[0053] As used herein, "expression" includes one or more of the following: transcription of a gene into precursor mRNA; splicing and other processing of the precursor mRNA to produce a mature mRNA; stability of the mRNA; translation of the mature mRNA into protein (including codon usage and tRNA availability); and glycosylation and / or other modifications of the translation product, as required for proper expression and function. As used herein, the term "gene" refers to a segment of DNA that contains all the information to regulate the biosynthesis of an RNA product, including promoters, exons, introns, and other untranslated regions that control expression.

[0054] As used herein, a "heterodimerization domain incapable of forming stable homodimers" refers to a member of a pair of distinct but complementary chemical motifs (e.g., amino acids, nucleotides, sugars, lipids, synthetic chemical structures, or any combination thereof) that either self-assembles exclusively as a heterodimer with the second complementary member of the pair, or exhibits at least 10% or more resistance to assembly into a heterodimer with the second complementary member of the pair. 4 This refers to a pair of members that exhibits a 2-fold preference or forms unstable homodimers with the same member under reducing conditions, such as >2 mM 2-MEA, at room temperature for 90 minutes (see, e.g., Labrijn, AF et al., Proc. Natl. Acad. Sci. 110, 5145-50 (2013)). Examples of such heterodimerization domains include, but are not limited to, CH2-CH3, WinZip-A1B1, complementary oligonucleotide pairs, and CH-1 and CL pairs, including any of the Fc variants / mutations described herein.

[0055] As used herein, "Hi1+1+2" refers to a molecule that (a) binds to two distinct target epitopes and (b) has a monovalent binding affinity or effective affinity (K) that is <100 pM. D ) refers to a heterodimeric tetravalent multispecific antibody having Fab domains.

[0056] As used herein, the term "humanized" forms of non-human (e.g., murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins in which recipient hypervariable region residues are replaced by hypervariable region residues from a non-human species (donor antibody), such as mouse, rat, rabbit, or non-human primate, having the desired specificity, affinity, and capacity. In some embodiments, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or the donor antibody. These modifications are made to further refine antibody performance, such as binding affinity. Generally, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains (e.g., Fab, Fab', F(ab')2, or Fv) in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin, and the FR regions may contain one or more amino acid substitutions that improve binding affinity, but all or substantially all of the FR regions are FR regions according to consensus FR sequences of human immunoglobulins. The number of these amino acid substitutions in the FRs is typically no more than six in the H chain and no more than three in the L chain. The humanized antibody may also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones et al., Nature 321:522-525 (1986); Reichmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992). See, e.g., Ahmed & Cheung, FEBS Letters 588(2):288-297 (2014).

[0057] The term "hypervariable region" as used herein refers to the amino acid residues of an antibody which are responsible for binding to an antigen. Hypervariable regions generally include "complementarity determining regions" or "CDRs" (e.g., V L Residues approximately 24–34 (L1), 50–56 (L2), and 89–97 (L3) within V H amino acid residues from approximately 31-35B (H1), 50-65 (H2), and 95-102 (H3) within the H1 region (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)), and / or "hypervariable loops" (e.g., V L Residues 26–32 (L1), 50–52 (L2), and 91–96 (L3) in V H The residues are derived from approximately 26-32 (H1), 52A-55 (H2), and 96-101 (H3) of the ribosomal nucleotide sequence (Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)).

[0058] As used herein, the term "intact antibody" or "intact immunoglobulin" refers to an antibody having at least two heavy (H) chain polypeptides and two light (L) chain polypeptides interconnected by disulfide bonds. Each heavy chain contains a heavy chain variable region (herein referred to as HCVR or V H The heavy chain constant region is composed of three domains, CH1, CH2, and CH3. Each light chain contains a light chain variable region (herein referred to as LCVR or V L The light chain constant region is composed of one domain, C L It consists of: V H and V L The regions can be further subdivided into regions of hypervariability, called complementarity determining regions (CDRs), interspersed with regions of high conservation, called framework regions (FRs).H and V L is composed of three CDRs and four FRs, arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region of the antibody may mediate the binding of the immunoglobulin to host tissues or factors, including various cellular immune system (e.g., effector cells) and the first component (C1q) of the classical complement system.

[0059] As used herein, the terms "individual," "patient," or "subject" can refer to an individual organism, vertebrate, mammal, or human. In some embodiments, the individual, patient, or subject is a human. As used herein, "Lo1+1+2" refers to an antibody that (a) binds to two distinct target epitopes that are within 60-120 angstroms of each other (thus allowing simultaneous binding), and (b) has a monovalent binding affinity or effective affinity (K) in the range of about 100 nM to about 100 pM. D ) refers to a heterodimeric tetravalent multispecific antibody having Fab domains.

[0060] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., a population containing individual antibodies that are identical except for possible naturally occurring mutations that may be present in minor amounts. For example, a monoclonal antibody can be derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, and not the method by which it is produced. A monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. The modifier "monoclonal" indicates the character of the antibody obtained from a substantially homogeneous population of antibodies and is not intended to require production of the antibody by any particular method. Monoclonal antibodies can be prepared using a variety of techniques known in the art, including, but not limited to, hybridoma, recombinant, and phage display techniques. For example, the monoclonal antibodies used in accordance with the present methods may be made by the hybridoma method first described by Kohler et al., Nature 256:495 (1975), or may be made by recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567). "Monoclonal antibodies" can also be isolated from phage antibody libraries using the techniques described, for example, in Clackson et al., Nature 352:624-628 (1991); and Marks et al., J. Mol. Biol. 222:581-597 (1991).

[0061] As used herein, the term "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal compounds, isotonic and absorption delaying compounds, etc., that are compatible with pharmaceutical administration. Pharmaceutically acceptable carriers and their formulations are known to those skilled in the art and are described, for example, in Remington's Pharmaceutical Sciences (20th edition, ed. A. Gennaro, 2000, Lippincott, Williams & Wilkins, Philadelphia, Pa.). As used herein, the term "polyclonal antibody" refers to a preparation of antibodies derived from at least two different antibody-producing cell lines. Use of this term includes at least two antibody preparations that contain antibodies that specifically bind to different epitopes or regions of an antigen.

[0062] As used herein, the term "polynucleotide" or "nucleic acid" refers to any RNA or DNA, which may be unmodified or modified.Polynucleotides include, but are not limited to, single-stranded and double-stranded DNA, DNA that is a mixture of single-stranded and double-stranded regions, single-stranded and double-stranded RNA, RNA that is a mixture of single-stranded and double-stranded regions, and hybrid molecules containing DNA and RNA, which may be single-stranded, more typically double-stranded, or a mixture of single-stranded and double-stranded regions.In addition, polynucleotide refers to a triple-stranded region containing RNA or DNA, or both RNA and DNA.The term "polynucleotide" also includes DNA or RNA containing one or more modified bases, and DNA or RNA whose backbone has been modified for stability or other reasons.

[0063] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to a polymer comprising two or more amino acids connected to each other by peptide bonds or modified peptide bonds, i.e., peptide isosteres. Polypeptide refers to both short chains, commonly referred to as peptides, glycopeptides, or oligomers, and to longer chains, commonly referred to as proteins. Polypeptides can contain amino acids other than the 20 gene-encoded amino acids. Polypeptides include amino acid sequences modified by natural processes, such as post-translational processing, or by chemical modification techniques that are well known in the art. Such modifications are well described in basic texts and more detailed treatises, as well as in a voluminous research literature.

[0064] As used herein, the term "recombinant," e.g., when used in reference to a cell, or a nucleic acid, protein, or vector, indicates that the cell, nucleic acid, protein, or vector has been modified by the introduction of a heterologous nucleic acid or protein, or the alteration of a naturally occurring nucleic acid or protein, or that the material is derived from a cell so modified. Thus, for example, a recombinant cell expresses genes that are not found within the native (non-recombinant) form of the cell, or expresses naturally occurring genes that would otherwise be abnormally expressed, poorly expressed, or not expressed at all. As used herein, the term "separate" therapeutic use refers to the simultaneous or substantially simultaneous administration of at least two active ingredients by different routes. As used herein, the term " sequential " therapeutic use refers to the administration of at least two active ingredients at different times, with the administration routes being the same or different.More specifically, sequential use refers to the administration of one of the active ingredients before the administration of one or more other active ingredients.Therefore, one of the active ingredients can be administered for several minutes, several hours, or several days before the administration of one or more other active ingredients.In this case, simultaneous treatment is not performed.

[0065] As used herein, "specifically binds to" refers to a molecule (e.g., an antibody or antigen-binding fragment thereof) that recognizes and binds to a particular molecule (e.g., an antigen) but does not substantially recognize or substantially bind to other molecules. As used herein, the terms "specifically binding to," "specifically binding to," or being "specific for" a particular molecule (e.g., a polypeptide, or an epitope on a polypeptide) refer to, for example, a specificity of about 10 to the molecule to which it binds. -4 M, 10 -5 M, 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, or 10 -12 K of M D The term "specifically binds to" can also refer to binding when a molecule (e.g., an antibody or antigen-binding fragment thereof) binds to a particular polypeptide or an epitope on a particular polypeptide without substantially binding to any other polypeptides or polypeptide epitopes.

[0066] As used herein, the term "concurrent" therapeutic use refers to the simultaneous or substantially simultaneous administration of at least two active ingredients by the same route. As used herein, the term "therapeutic agent" is intended to mean a compound that, when present in an effective amount, provides a desired therapeutic effect to a subject in need thereof. As used herein, "treating" or "treatment" refers to the treatment of a disease or disorder described herein in a subject, such as a human, and includes: (i) inhibiting the disease or disorder, i.e., halting its development; (ii) alleviating the disease or disorder, i.e., causing regression of the disorder; (iii) slowing the progression of the disorder; and / or (iv) inhibiting, alleviating, or slowing the progression of one or more symptoms of the disease or disorder. In some embodiments, treating means that the symptoms associated with the disease are, for example, alleviated, relieved, cured, or placed in remission.

[0067] It should also be understood that the various modes of treatment for disorders described herein are intended to mean "substantial" treatment, including total treatment, but also including less-than-total treatment in which some biologically or medically relevant result is achieved. Treatment can be continuous, long-term treatment for chronic illnesses, or single or few doses for the treatment of acute conditions.

[0068] Heterodimeric trivalent / tetravalent multispecific antibodies of the present technology The heterodimeric trivalent / tetravalent multispecific antibodies of the present technology can bind to three or four targets with distinct structures, for example, three to four different target antigens, three to four different epitopes on the same target antigen, or a combination of a hapten and a target antigen or an epitope on a target antigen. Various HDTVS antibodies can be generated using molecular engineering. For example, the HDTVS antibodies disclosed herein utilize a combination of a complete immunoglobulin framework (e.g., IgG) and a single-chain variable fragment (scFv).

[0069] HDTVS antibodies can be made, for example, by combining and / or engineering heavy and / or light chains that recognize different epitopes of the same antigen or different epitopes of different antigens. In some embodiments, the HDTVS proteins are trivalent and trispecific, e.g., for a first antigen (one V H / V L pair) and a binding site for a second antigen (a different V H / V L In some embodiments, the HDTVS protein comprises an immunoglobulin (e.g., IgG) with a binding site for a first antigen (two V pairs) and an scFv for a third antigen. In some embodiments, the HDTVS protein is trivalent and bispecific, e.g., a binding site for a first antigen (two V pairs) and an scFv for a third antigen. H / V L In some embodiments, the HDTVS protein comprises an immunoglobulin (e.g., IgG) with two binding sites for a first antigen (one V pair) and an scFv for a second antigen. In some embodiments, the HDTVS protein is tetravalent and trispecific, e.g., with two binding sites for a first antigen (one V pair) and an scFv for a second antigen. H / V L pair) and a binding site for a second antigen (a different V H / V L In some embodiments, the HDTVS protein comprises an immunoglobulin (e.g., IgG) with a binding site for a first antigen (two V pairs) and two identical scFvs for a third antigen. In some embodiments, the HDTVS protein is tetravalent and trispecific, e.g., a binding site for a first antigen (two V pairs) and two identical scFvs for a third antigen. H / V L In some embodiments, the HDTVS protein comprises an immunoglobulin (e.g., IgG) with two binding sites for a first antigen (one V pair), an scFv for a second antigen, and an scFv for a third antigen. In some embodiments, the HDTVS protein is tetravalent and tetraspecific, e.g., with two binding sites for a first antigen (one V pair), an scFv for a second antigen, and an scFv for a third antigen. H / V L A binding site for a second antigen (a different V H / V L The scFv comprises an immunoglobulin (e.g., IgG) with binding sites for a first antigen (antigen pair), an scFv for a third antigen, and an scFv for a fourth antigen.

[0070] In some embodiments, at least one scFv of the HDTVS antibody of the present technology binds to an antigen or epitope of a B cell, a T cell, a myeloid cell, a plasma cell, or a mast cell. Additionally or alternatively, in certain embodiments, at least one scFv of the HDTVS antibody of the present technology is selected from the group consisting of dabigatran, a4, a4b7, a4b7+aEb7, a5, AXL, BnDOTA, CD11a (LFA-1), CD3, CD4, CD8, CD16, CD19, CD22, CD23, CD25, CD28, CD30 (TNFRSF8), CD33, CD38, CD40, CD40L, CD47, CD49b (a2), CD54 (ICAM-1), CD56, CD74, CD80, CD115 (CSF1R), CD116a (CSF2Ra), CD123, CD134 (OX40), CD137 (41BB), CD152 (CTLA4), C The antibody binds to an antigen selected from the group consisting of D184 (CXCR4), CD192 (CCR2), CD194 (CCR4), CD195 (CCR5), CD223 (LAG-3), CD252 (OX40L), CD254 (RANKL), CD262 (DR5), CD27, CD200, CD221 (IGF1R), CD248, CD274 (PD-L1), CD275 (ICOS-L), CD278 (ICOS), CD279 (PD-1), CD319 (SLAMF7), CD371 (CLEC12A), MADCAM1, MT1-MMP (MMP14), NKG2A, NRP1, TIGIT, VSIR, KIRDL1 / 2 / 3, and KIR2DL2.

[0071] Additionally or alternatively, in certain embodiments, the HDTVS antibodies disclosed herein are selected from the group consisting of a2b b3 (glycoprotein IIb / IIIa), a4, a4b7, a4b7+aEb7, a5, activin receptor type 2B, ALK1, alpha-synuclein, amyloid beta, APP, AXL, blood group A, CAIX, CCL-2, CD105 (endoglin), CD115 (CSF1R), CD116a (CSF2Ra), CD123, CD152 (CTLA4), CD184 (CXCR4), CD19, CD192 (CCR2), CD194 (CCR4), CD195 (CCR5), CD20, CD200, CD22 , CD221(IGF1R), CD248, CD25, CD257(BAFF), CD26, CD262(DR5), CD276(B7H3), CD3, CD30(TNFRSF8), CD319(SLAMF7), CD33, CD332(FGFR2), C D350(FZD10), CD37, CD371(CLEC12A), CD38, CD4, CD49b(a2), CD51(a5), CD52, CD56, CD61(a4b3), CD70, CD73(NT5E), CD74, CEA, claudin-18.2, cMET, CRLR, DLL3, DLL4, DNA / histone (H1) complex, EGFR, EpCAM, EGFR-HER3, EGFRvIII, EphA3, ERGT(GalNAc)Tn antigen, FLT1, FOLR1, frizzled family receptor (FZD), Lewis Y, Lewis X, GCGR, GD2, GD2 α-acetyl, GD3, GM1, GM1 fucosyl, GM2, GPA33, GPNMB, GUCY2C, HER2, HER3, HGFR (cMET), IgHe, IGLF2, kallikrein, LINGO1, LOXL2, Ly6 / PLAUR domain-containing protein 3, MADCAM1, MAG, mesothelin, MT1-MMP (MMP14), MUC1, mucin 5AC, NaPi2b, NeuGc-GM3, notch, NOTCH2 / NOTCH3 receptor, oxLDL, P-selectin, PCSK9, PDGFRA, PDGFRa, phosphatidylserine, polysialic acid, PSMA, PVRL4, RGMA, CD240D, a blood type D antigen, and root plate-specific stromal growth factor receptor (RGF). The antibody is capable of binding to a cell (e.g., a tumor cell) that expresses a cell surface antigen selected from the group consisting of: phosphodiesterase 3, serum amyloid P component, STEAP-1, TACSTD2, TGFb, TWEAKR, TYRP1, VEGFR2, VSIR, CD171 (L1CAM), CD19, CD47, pMHC[NY-ESO1], pMHC[MART1], pMHC[MAGEA1], pMHC[tyrosinase], pMHC[gp100], pMHC[MUC1], pMHC[tax], pMHC[WT-1], pMHC[EBNA-1], pMHC[LMP2], pMHC[hTERT], GPC3, CD80, CD23, and fibronectin extracellular domain B.

[0072] Methods for producing HDTVS antibodies of the present technology include engineered recombinant monoclonal antibodies with additional cysteine ​​residues to cross-link more strongly than common immunoglobulin isotypes. See, e.g., FitzGerald et al., Protein Eng. 10(10):1221-1225 (1997). HDTVS recombinant fusion proteins can be engineered by linking two or more different single-chain antibody or antibody fragment segments with the required dual specificity. See, e.g., Coloma et al., Nature Biotech. 15:159-163 (1997).

[0073] Recombinant methods can be used to create a variety of fusion proteins. In some embodiments, HDTVS antibodies according to the present technology include an immunoglobulin comprising two heavy chains, two light chains, and two scFvs, where each scFv is linked to the C-terminus of one of the two light chains of any immunoglobulin disclosed herein. In various embodiments, the scFv is linked to the light chain via a linker sequence. In some embodiments, the linker is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 amino acids in length, or more.

[0074] In some embodiments, the linker is characterized in that it does not adopt an inflexible three-dimensional structure, but rather tends to provide flexibility to the polypeptide (e.g., the first antigen-binding site and / or the second antigen-binding site). In some embodiments, linkers are utilized within the HDTVS antibodies described herein based on the specific properties they impart to the HDTVS antibody, such as, for example, increased stability. In some embodiments, the HDTVS antibodies of the present technology comprise a G4S linker. In certain embodiments, the HDTVS antibodies of the present technology comprise a (G4S) linker. nlinker, wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more. Exemplary V that may be utilized within the HDTVS antibodies of the present technology include: H Amino acid sequence and V L The amino acid sequences are presented in Table 1.

[0075] [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7] [Table 1-8] [Table 1-9] [Table 1-10] [Table 1-11] [Table 1-12] [Table 1-13] [Table 1-14] Table 1-15 Table 1-16 Table 1-17 Table 1-18 Table 1-19 Table 1-20 Table 1-21 Table 1-22 Table 1-23 Table 1-24 Table 1-25 Table 1-26 Table 1-27 Table 1-28 Table 1-29 Table 1-30 Table 1-31 Table 1-32 Table 1-33 Table 1-34 Table 1-35 Table 1-36 Table 1-37 Table 1-38 Table 1-39 Table 1-40 Table 1-41 Table 1-42 Table 1-43 Table 1-44 Table 1-45 Table 1-46 Table 1-47 Table 1-48 Table 1-49 Table 1-50 Table 1-51 Table 1-52 Table 1-53 Table 1-54 Table 1-55 Table 1-56 Table 1-57 Table 1-58 Table 1-59 Table 1-60 Table 1-61 Table 1-62 Table 1-63 Table 1-64 Table 1-65 Table 1-66 Table 1-67 Table 1-68 Table 1-69 Table 1-70 Table 1-71 Table 1-72 Table 1-73 Table 1-74 Table 1-75 Table 1-76 Table 1-77 Table 1-78 Table 1-79 Table 1-80 Table 1-81 Table 1-82

[0076] In one aspect, the disclosure provides a polypeptide comprising a first polypeptide chain, a second polypeptide chain, a third polypeptide chain, and a fourth polypeptide chain, wherein the first polypeptide chain and the second polypeptide chain are covalently coupled to each other, the second polypeptide chain and the third polypeptide chain are covalently coupled to each other, and the third polypeptide chain and the fourth polypeptide chain are covalently coupled to each other, and wherein (a) the first polypeptide chain comprises, in an N-terminal to C-terminal direction: (i) a light chain variable domain of a first immunoglobulin capable of specifically binding to a first epitope; (ii) a light chain constant domain (CL-1) of a first immunoglobulin; (iii) a flexible peptide linker comprising the amino acid sequence (GGGGS)3; and (iv) a light chain variable domain (VL-2) of a second immunoglobulin linked to a heavy chain variable domain (VH-2) of a complementary second immunoglobulin, or a heavy chain variable domain (VH-2) of a second immunoglobulin linked to a light chain variable domain (VL-2) of a complementary second immunoglobulin, wherein the VL-2 and VH-2 specifically bind to a second epitope. (b) a second polypeptide comprising, from N-terminal to C-terminal direction: (i) a heavy chain variable domain (VH-1) of a first immunoglobulin capable of specifically binding to a first epitope; (ii) a first CH1 domain (CH1-1) of the first immunoglobulin; and (iii) a first heterodimerization domain of the first immunoglobulin, the first heterodimerization domain being linked together via a flexible peptide linker comprising the amino acid sequence (GGGGS)6 to form a single-chain variable fragment. (c) a third polypeptide comprising, from N-terminal to C-terminal direction: (i) a heavy chain variable domain (VH-3) of a third immunoglobulin capable of specifically binding to a third epitope; (ii) a second CH1 domain (CH1-3) of the third immunoglobulin; and (iii) a second heterodimerization domain of the third immunoglobulin, which comprises an amino acid sequence or nucleic acid sequence distinct from the first heterodimerization domain of the first immunoglobulin;(d) a fourth polypeptide comprising, from N-terminal to C-terminal direction: (i) a light chain variable domain (VL-3) of a third immunoglobulin capable of specifically binding to a third epitope; (ii) a light chain constant domain (CL-3) of the third immunoglobulin; (iii) a flexible peptidic linker comprising the amino acid sequence (GGGGS)3; and (iv) a light chain variable domain (VL-4) of a fourth immunoglobulin linked to a heavy chain variable domain (VH-4) of a complementary fourth immunoglobulin, or a heavy chain variable domain (VH-4) of a fourth immunoglobulin linked to a light chain variable domain (VL-4) of a complementary fourth immunoglobulin, wherein the VL-4 and VH-4 are capable of specifically binding to a second epitope, and the VL-4 or VH-4 are linked together via a flexible peptide linker comprising the amino acid sequence (GGGGS)6 to form a single-chain variable fragment. , VL-1 and VL-3 are each independently selected from the group consisting of SEQ ID NOs: 1, 9, 17, 25, 33, 41, 49, 57, 65, 73, 81, 89, 97, 105, 113, 121, 129, 145, 153, 161, 169, 177, 193, 201, 233, 241, 257, 273, 281, 289, 297, 305, 313, 321, 329, 337, 345, 353, 361, 369, 377, 385, 393, 401, 409, 417, 425, 433, 441, 449, 457, 465, 481, 489, 497, 521, 529, 537, 545, 553 , 561, 609, 617, 681, 689, 697, 705, 713, 721, 729, 737, 745, 753, 761, 769, 777, 785, 793, 801, 809, 817, 825, 833, 841, 849, 857, 865, 873, 881, 889, 945, 953, 961, 977, 985, 993, 1001, 1009, 1017, 1025, 1033, 1041, 1049, 1065, 1073, 1081, 1089, 1097, 1105, 1113, 1121, 1129, 1137, 1145, 1153, 1161, 1169,1177, 1185, 1193, 1201, 1209, 1217, 1225, 1233, 1241, 1249, 1257, 1265, 1273, 1281, 1289, 1297, 1305, 1313, 1321, 1329, 1337, 1345, 1353, 1361, 1369, 1377, 1385, 1393, 1401, 1409, 1417, 1425, 1433, 1441, 1449, 1450 57, 1465, 1473, 1481, 1489, 1497, 1505, 1513, 1521, 1529, 1545, 1553, 1561, 1569, 1577, 1585, 1593, 1601, 1609, 1617, 1625, 1633, 1649, 1657, 1673, 1681, 1689, 1697, 1705, 1713, 1721, 1729, 1737, 1745, 1753, 1761, 1769, 1777, 1785, 1793, 1801, 1809, 1817, 1833, 1841, 1849, 1857, 1865, 1873, 1881, 1889, 1913, 1937, 1945, 1953, 1961, 1969, 1977, 1985, 1993, 2001, 2009, 2017, 2025, 2033, 2041, 2049, 2057, 2065, 2073, 2081, 2082, 2083, 2084, 2085, 2086, 2087, 2088, 2089, 2090, 2091, 2092, 2093, 2094, 2095, 2096, 2097, 2098, 2099, 2000, 2001, 2002, 2003, 2004, 2005, 2006, 2007, 2008, 2009, 2010, 2011, 2012, 2013, 2014, 2015, 2016, 2017, 2018, 2019, 2020, 2021, 2022, 2023, 2024, 2025, 2026, 2027, 2028, 2029, 2030, 2031, V selected from any one of 89, 2097, 2105, 2113, 2121, 2129, 2137, 2145, 2153, 2161, 2169, 2177, 2185, 2193, 2201, 2209, 2217, 2225, 2233, 2241, 2249, 2257, 2265, 2273, 2281, 2297, 2305, 2313, 2321, 2329, 2337, and 2345; L comprising CDR1, CDR2, and CDR3 amino acid sequences;

[0077] and / or each of VH-1 and VH-3 independently is selected from the group consisting of SEQ ID NOs: 5, 13, 21, 29, 37, 45, 53, 61, 69, 77, 85, 93, 101, 109, 117, 125, 133, 149, 157, 165, 173, 181, 197, 205, 237, 245, 261, 277, 285, 293, 301, 309, 317, 325, 333, 341, 349, 357, 365, 373, 381, 389, 397, 405, 413, 421, 429, 437, 445, 453, 461, 469, 485, 493, 501, 525, 533, 541, 552, 561, 571, 581, 591, 601, 612, 614, 616, 618, 620, 622, 624, 626, 628, 629, 630, 632, 634, 636, 638, 639, 640, 642, 643, 644, 645, 646, 647, 648, 650, 651, 652, 653, 654, 655, 656, 657, 658, 660, 662, 49, 557, 565, 613, 621, 685, 693, 701, 709, 717, 725, 733, 741, 749, 757, 765, 773, 781, 789, 797, 805, 813, 821, 829, 837, 845, 853, 861, 869, 877, 885, 89 3, 949, 957, 965, 981, 989, 997, 1005, 1013, 1021, 1029, 1037, 1045, 1053, 1069, 1077, 1085, 1093, 1101, 1109, 1117, 1125, 1133, 1141, 1149, 1157, 1165 , 1173, 1181, 1189, 1197, 1205, 1213, 1221, 1229, 1237, 1245, 1253, 1261, 1269, 1277, 1285, 1293, 1301, 1309, 1317, 1325, 1333, 1341, 1349, 1357, 1365 , 1373, 1381, 1389, 1397, 1405, 1413, 1421, 1429, 1437, 1445, 1453, 1461, 1469, 1477, 1485, 1493, 1501, 1509, 1517, 1525, 1533, 1549, 1557, 1565, 1573 , 1581, 1589, 1597, 1605, 1613, 1621, 1629, 1637, 1653, 1661, 1677, 1685, 1693, 1701, 1709, 1717, 1725, 1733, 1741, 1749, 1757, 1765, 1773, 1781, 1789 , 1797, 1805, 1813, 1821, 1837, 1845, 1853, 1861, 1869, 1877, 1885, 1893, 1917, 1941, 1949, 1957, 1965, 1973, 1981, 1989, 1997, 2005, 2013, 2021, 2029,V selected from any one of 2037, 2045, 2053, 2061, 2069, 2077, 2085, 2093, 2101, 2109, 2117, 2125, 2133, 2141, 2149, 2157, 2165, 2173, 2181, 2189, 2197, 2205, 2213, 2221, 2229, 2237, 2245, 2253, 2261, 2269, 2277, 2285, 2301, 2309, 2317, 2325, 2333, 2341, and 2349; H Heterodimeric multispecific antibodies are provided that comprise CDR1, CDR2, and CDR3 amino acid sequences.

[0078] In one aspect, the disclosure provides a polypeptide comprising a first polypeptide chain, a second polypeptide chain, a third polypeptide chain, and a fourth polypeptide chain, wherein the first polypeptide chain and the second polypeptide chain are covalently coupled to each other, the second polypeptide chain and the third polypeptide chain are covalently coupled to each other, and the third polypeptide chain and the fourth polypeptide chain are covalently coupled to each other, and wherein (a) the first polypeptide chain specifically binds, in an N-terminal to C-terminal direction: (i) to a first epitope; (ii) a light chain constant domain (CL-1) of the first immunoglobulin; (iii) a flexible peptide linker comprising the amino acid sequence (GGGGS)3; and (iv) a light chain variable domain (VL-2) of a second immunoglobulin linked to a heavy chain variable domain (VH-2) of a complementary second immunoglobulin, or a heavy chain variable domain (VL-2) of a second immunoglobulin linked to a light chain variable domain (VH-2) of a complementary second immunoglobulin. (b) a second polypeptide comprising, from N-terminal to C-terminal direction: (i) a heavy chain variable domain (VH-1) of a first immunoglobulin capable of specifically binding to a first epitope; (ii) a heavy chain variable domain (VH-2) of a first immunoglobulin capable of specifically binding to a second epitope; (iii) a heavy chain variable domain (VH-3) of a first immunoglobulin capable of specifically binding to a second epitope; (iv) a heavy chain variable domain (VH-4) of a first immunoglobulin capable of specifically binding to a second epitope; (v) a heavy chain variable domain (VH-5) of a first immunoglobulin capable of specifically binding to a second epitope; (v) a heavy chain variable domain (VH-6) of a first immunoglobulin capable of specifically binding to a second epitope; (v) a heavy chain variable domain (VH-7) of a first immunoglobulin capable of specifically binding to a second epitope; (v) a heavy chain variable domain (VH-8) of a first immunoglobulin capable of specifically binding to a second epitope; (v) a heavy chain variable domain (VH-9) of a first immunoglobulin capable of specifically binding to a second epitope; (v) a heavy chain variable domain (VH-1 ... first CH1 domain (VH-1) of a first immunoglobulin capable of specifically binding to a second epitope; (v) a first CH2 domain (VH-1) of a first immunoglobulin capable of specifically binding to a second epitope; (v) a first CH3 domain (VH-1) of a first immunoglobulin capable of specifically binding to a second epitope; (v) a first CH4 domain (VH-1) of a first immunoglobulin capable of specifically binding to a second epi (iii) a first heterodimerization domain of a first immunoglobulin, the first heterodimerization domain being incapable of forming a stable homodimer with another first heterodimerization domain; and (c) a third polypeptide comprising, from N-terminal to C-terminal direction: (i) a heavy chain variable domain (VH-3) of a third immunoglobulin capable of specifically binding to a first epitope; (ii) a second CH1 domain (CH1-3) of the third immunoglobulin;and (iii) a second heterodimerization domain of a third immunoglobulin, the second heterodimerization domain comprising an amino acid sequence or nucleic acid sequence distinct from the first heterodimerization domain of the first immunoglobulin, the second heterodimerization domain being incapable of forming stable homodimers with another second heterodimerization domain, and being configured to form a heterodimer with the first heterodimerization domain of the first immunoglobulin; (d) the fourth polypeptide comprising, from N-terminal to C-terminal direction: (i) a light chain variable domain (VL-3) of the third immunoglobulin capable of specifically binding to the first epitope; (ii) a light chain constant domain (CL-3) of the third immunoglobulin; (iii) a flexible peptide linker comprising the amino acid sequence (GGGGS)3;and (iv) a fourth immunoglobulin light chain variable domain (VL-4) linked to a complementary fourth immunoglobulin heavy chain variable domain (VH-4), or a fourth immunoglobulin heavy chain variable domain (VH-4) linked to a complementary fourth immunoglobulin light chain variable domain (VL-4), wherein the VL-4 and VH-4 are capable of specifically binding to a third epitope, and wherein the VL-4 and VH-4 are linked together via a flexible peptide linker comprising the amino acid sequence (GGGGS)6 to form a single-chain variable fragment, wherein each of VL-2 and VL-4 is independently selected from the group consisting of SEQ ID NOs: 17, 25, 33, 41, 121, 137, 169, 177, 185, 193, 201, 209, 217, 225, 233, 24 1, 249, 257, 265, 321, 329, 337, 393, 401, 409, 473, 481, 489, 497, 505, 513, 545, 553, 561, 569, 577, 585, 593, 601, 625, 633, 641, 649, 657, 665, 673, 681, 689, 697, 705, 713, 721, 729, 737, 745, 753, 76 V selected from any one of 1, 769, 785, 793, 801, 809, 817, 849, 857, 865, 873, 881, 889, 897, 905, 913, 921, 929, 937, 945, 969, 977, 1009, 1057, 1537, 1569, 1601, 1641, 1665, 1825, 1865, 1897, 1905, 1913, 1921, 1929, 2265, 2281 2289, 2329, and 2345; L comprising CDR1, CDR2, and CDR3 amino acid sequences;

[0079] and / or each of VH-2 and VH-4 independently selects from SEQ ID NOs: 21, 29, 37, 45, 125, 141, 173, 181, 189, 197, 205, 213, 221, 229, 237, 245, 253, 261, 269, 325, 333, 341, 397, 405, 413, 477, 485, 493, 501, 509, 517, 549, 557, 565, 573, 581, 589, 597, 605, 629, 637, 645, 653, 661, 669, 677, 685, 693, 701, 709, V selected from any one of 717, 725, 733, 741, 749, 757, 765, 773, 789, 797, 805, 813, 821, 853, 861, 869, 877, 885, 893, 901, 909, 917, 925, 933, 941, 949, 973, 981, 1013, 1061, 1541, 1573, 1605, 1645, 1669, 1829, 1869, 1901, 1909, 1917, 1925, 1933, 2269, 2285, 2293, 2333, and 2349 H Heterodimeric multispecific antibodies are provided that comprise CDR1, CDR2, and CDR3 amino acid sequences.

[0080] In another aspect, the disclosure provides a polypeptide comprising a first polypeptide chain, a second polypeptide chain, a third polypeptide chain, and a fourth polypeptide chain, wherein the first polypeptide chain and the second polypeptide chain are covalently linked to each other, the second polypeptide chain and the third polypeptide chain are covalently linked to each other, and the third polypeptide chain and the fourth polypeptide chain are covalently linked to each other, and wherein (a) the first polypeptide chain comprises, in an N-terminal to C-terminal direction: (i) a light chain variable region of a first immunoglobulin capable of specifically binding to a first epitope; (ii) a light chain constant domain (CL-1) of a first immunoglobulin; (iii) a flexible peptide linker comprising the amino acid sequence (GGGGS)3; and (iv) a light chain variable domain (VL-2) of a second immunoglobulin linked to a heavy chain variable domain (VH-2) of a complementary second immunoglobulin, or a heavy chain variable domain (VH-2) of a second immunoglobulin linked to a light chain variable domain (VL-2) of a complementary second immunoglobulin, wherein VL-2 and VH-2 specifically bind to a second epitope. (b) a second polypeptide comprising, from N-terminal to C-terminal direction: (i) a heavy chain variable domain (VH-1) of a first immunoglobulin capable of specifically binding to a first epitope; (ii) a first CH1 domain (CH1-1) of the first immunoglobulin; and (iii) a first heterodimerization domain of the first immunoglobulin, the first heterodimerization domain being capable of specifically binding to another first heterodimerization domain. (c) a third polypeptide comprising, from N-terminal to C-terminal direction: (i) a heavy chain variable domain (VH-3) of a third immunoglobulin capable of specifically binding to a third epitope; (ii) a second CH1 domain (CH1-3) of the third immunoglobulin; and (iii) a second heterodimerization domain of the third immunoglobulin, which comprises an amino acid sequence or nucleic acid sequence distinct from the first heterodimerization domain of the first immunoglobulin;(d) a fourth polypeptide comprising, from N-terminal to C-terminal direction: (i) a light chain variable domain (VL-3) of a third immunoglobulin capable of specifically binding to a third epitope; (ii) a light chain constant domain (CL-3) of the third immunoglobulin; (iii) a flexible peptidic linker comprising the amino acid sequence (GGGGS)3; and (iv) a light chain variable domain of a fourth immunoglobulin (VL-4) linked to a heavy chain variable domain (VH-4) of a complementary fourth immunoglobulin, or a heavy chain variable domain of a fourth immunoglobulin (VH-4) linked to a light chain variable domain of a fourth immunoglobulin (VL-4), wherein the VL-4 and VH-4 are capable of specifically binding to a fourth epitope, and the VL-4 or VH-4 are linked together via a flexible peptide linker comprising the amino acid sequence (GGGGS)6 to form a single-chain variable fragment. , VL-1 and VL-3 are each independently selected from the group consisting of SEQ ID NOs: 1, 9, 17, 25, 33, 41, 49, 57, 65, 73, 81, 89, 97, 105, 113, 121, 129, 145, 153, 161, 169, 177, 193, 201, 233, 241, 257, 273, 281, 289, 297, 305, 313, 321, 329, 337, 345, 353, 361, 369, 377, 385, 393, 401, 409, 417, 425, 433, 441, 449, 457, 465, 481, 489, 497, 521, 529, 537, 545, 553 , 561, 609, 617, 681, 689, 697, 705, 713, 721, 729, 737, 745, 753, 761, 769, 777, 785, 793, 801, 809, 817, 825, 833, 841, 849, 857, 865, 873, 881, 889, 945, 953, 961, 977, 985, 993, 1001, 1009, 1017, 1025, 1033, 1041, 1049, 1065, 1073, 1081, 1089, 1097, 1105, 1113, 1121, 1129, 1137, 1145, 1153, 1161, 1169,1177, 1185, 1193, 1201, 1209, 1217, 1225, 1233, 1241, 1249, 1257, 1265, 1273, 1281, 1289, 1297, 1305, 1313, 1321, 1329, 1337, 1345, 1353, 1361, 1369, 1377, 1385, 1393, 1401, 1409, 1417, 1425, 1433, 1441, 1449, 1450 57, 1465, 1473, 1481, 1489, 1497, 1505, 1513, 1521, 1529, 1545, 1553, 1561, 1569, 1577, 1585, 1593, 1601, 1609, 1617, 1625, 1633, 1649, 1657, 1673, 1681, 1689, 1697, 1705, 1713, 1721, 1729, 1737, 1745, 1753, 1761, 1769, 1777, 1785, 1793, 1801, 1809, 1817, 1833, 1841, 1849, 1857, 1865, 1873, 1881, 1889, 1913, 1937, 1945, 1953, 1961, 1969, 1977, 1985, 1993, 2001, 2009, 2017, 2025, 2033, 2041, 2049, 2057, 2065, 2073, 2081, 2082, 2083, 2084, 2085, 2086, 2087, 2088, 2089, 2090, 2091, 2092, 2093, 2094, 2095, 2096, 2097, 2098, 2099, 2000, 2001, 2002, 2003, 2004, 2005, 2006, 2007, 2008, 2009, 2010, 2011, 2012, 2013, 2014, 2015, 2016, 2017, 2018, 2019, 2020, 2021, 2022, 2023, 2024, 2025, 2026, 2027, 2028, 2029, 2030, 2031, V selected from any one of 89, 2097, 2105, 2113, 2121, 2129, 2137, 2145, 2153, 2161, 2169, 2177, 2185, 2193, 2201, 2209, 2217, 2225, 2233, 2241, 2249, 2257, 2265, 2273, 2281, 2297, 2305, 2313, 2321, 2329, 2337, and 2345; L comprising CDR1, CDR2, and CDR3 amino acid sequences;

[0081] and / or each of VH-1 and VH-3 independently is selected from the group consisting of SEQ ID NOs: 5, 13, 21, 29, 37, 45, 53, 61, 69, 77, 85, 93, 101, 109, 117, 125, 133, 149, 157, 165, 173, 181, 197, 205, 237, 245, 261, 277, 285, 293, 301, 309, 317, 325, 333, 341, 349, 357, 365, 373, 381, 389, 397, 405, 413, 421, 429, 437, 445, 453, 461, 469, 485, 493, 501, 525, 533, 541, 552, 561, 571, 581, 591, 601, 612, 614, 616, 618, 620, 622, 624, 626, 628, 629, 630, 632, 634, 636, 638, 639, 640, 642, 643, 644, 645, 646, 647, 648, 650, 651, 652, 653, 654, 655, 656, 657, 658, 660, 662, 49, 557, 565, 613, 621, 685, 693, 701, 709, 717, 725, 733, 741, 749, 757, 765, 773, 781, 789, 797, 805, 813, 821, 829, 837, 845, 853, 861, 869, 877, 885, 89 3, 949, 957, 965, 981, 989, 997, 1005, 1013, 1021, 1029, 1037, 1045, 1053, 1069, 1077, 1085, 1093, 1101, 1109, 1117, 1125, 1133, 1141, 1149, 1157, 1165 , 1173, 1181, 1189, 1197, 1205, 1213, 1221, 1229, 1237, 1245, 1253, 1261, 1269, 1277, 1285, 1293, 1301, 1309, 1317, 1325, 1333, 1341, 1349, 1357, 1365 , 1373, 1381, 1389, 1397, 1405, 1413, 1421, 1429, 1437, 1445, 1453, 1461, 1469, 1477, 1485, 1493, 1501, 1509, 1517, 1525, 1533, 1549, 1557, 1565, 1573 , 1581, 1589, 1597, 1605, 1613, 1621, 1629, 1637, 1653, 1661, 1677, 1685, 1693, 1701, 1709, 1717, 1725, 1733, 1741, 1749, 1757, 1765, 1773, 1781, 1789 , 1797, 1805, 1813, 1821, 1837, 1845, 1853, 1861, 1869, 1877, 1885, 1893, 1917, 1941, 1949, 1957, 1965, 1973, 1981, 1989, 1997, 2005, 2013, 2021, 2029,V selected from any one of 2037, 2045, 2053, 2061, 2069, 2077, 2085, 2093, 2101, 2109, 2117, 2125, 2133, 2141, 2149, 2157, 2165, 2173, 2181, 2189, 2197, 2205, 2213, 2221, 2229, 2237, 2245, 2253, 2261, 2269, 2277, 2285, 2301, 2309, 2317, 2325, 2333, 2341, and 2349; H and / or each of VL-2 and VL-4 independently comprises a CDR1 sequence, a CDR2 sequence, and a CDR3 sequence of the amino acid sequences SEQ ID NOs: 17, 25, 33, 41, 121, 137, 169, 177, 185, 193, 201, 209, 217, 225, 233, 241, 249, 257, 265, 321, 329, 337, 393, 401, 409, 473, 481, 489, 497, 505, 513, 545, 553, 561, 569, 577, 585, 593, 601, 625, 633, 640, 650, 660, 670, 680, 691, 702, 710, 720, 730, 740, 750, 760, 771, 782, 791, 802, 810, 825, 830, 840, 850, 860, 870, 882, 891, 902, 910, 920, 930, 940, 950, 960, 970, 982, 991, 1002, 1010, 1020, 1030, 1040, 1050, 1060, 1070, 1080, 1090, 1102, 1150, 1160, 1170, 41, 649, 657, 665, 673, 681, 689, 697, 705, 713, 721, 729, 737, 745, 753, 761, 769, 785, 793, 801, 809, 817, 849, 857, 865, 873, 881, 889, 897, 905, 913, 921, 929, 937, 945, 969, 977, 1009, 1057, 1537, 1569, 1601, 1641, 1665, 1825, 1865, 1897, 1905, 1913, 1921, 1929, 2265, 2281 V selected from any one of 2289, 2329, and 2345 Land / or each of VH-2 and VH-4 independently comprises the amino acid sequences of SEQ ID NOs: 21, 29, 37, 45, 125, 141, 173, 181, 189, 197, 205, 213, 221, 229, 237, 245, 253, 261, 269, 325, 333, 341, 397, 405, 413, 477, 485, 493, 501, 509, 517, 549, 557, 565, 573, 581, 589, 597, 605, 629, 637, 645, 653, 661, 669, 677 , 685, 693, 701, 709, 717, 725, 733, 741, 749, 757, 765, 773, 789, 797, 805, 813, 821, 853, 861, 869, 877, 885, 893, 901, 909, 917, 925, 933, 941, 949, 973, 981, 1013, 1061, 1541, 1573, 1605, 1645, 1669, 1829, 1869, 1901, 1909, 1917, 1925, 1933, 2269, 2285, 2293, 2333, and 2349. H Heterodimeric multispecific antibodies are provided that comprise CDR1, CDR2, and CDR3 amino acid sequences.

[0082] In another aspect, the disclosure provides a polypeptide comprising a first polypeptide chain, a second polypeptide chain, a third polypeptide chain, and a fourth polypeptide chain, wherein the first polypeptide chain and the second polypeptide chain are covalently coupled to each other, the second polypeptide chain and the third polypeptide chain are covalently coupled to each other, and the third polypeptide chain and the fourth polypeptide chain are covalently coupled to each other, and wherein (a) the first polypeptide chain, in an N-terminal to C-terminal direction: (i) specifically binds to a first epitope; (ii) a light chain constant domain (CL-1) of the first immunoglobulin; (iii) a flexible peptide linker comprising the amino acid sequence (GGGGS)3; and (iv) a light chain variable domain (VL-2) of a second immunoglobulin linked to a heavy chain variable domain (VH-2) of a complementary second immunoglobulin, or a heavy chain variable domain (VL-2) of a second immunoglobulin linked to a light chain variable domain (VH-2) of a complementary second immunoglobulin. (b) a second polypeptide comprising, from N-terminal to C-terminal direction: (i) a heavy chain variable domain (VH-1) of a first immunoglobulin capable of specifically binding to a first epitope; (ii) a heavy chain variable domain (VH-2) of a first immunoglobulin capable of specifically binding to a second epitope; (iii) a heavy chain variable domain (VH-3) of a first immunoglobulin capable of specifically binding to a second epitope; (iv) a heavy chain variable domain (VH-4) of a first immunoglobulin capable of specifically binding to a second epitope; (v) a heavy chain variable domain (VH-5) of a first immunoglobulin capable of specifically binding to a second epitope; (v) a heavy chain variable domain (VH-6) of a first immunoglobulin capable of specifically binding to a second epitope; (v) a heavy chain variable domain (VH-7) of a first immunoglobulin capable of specifically binding to a second epitope; (v) a heavy chain variable domain (VH-8) of a first immunoglobulin capable of specifically binding to a second epitope; (v) a heavy chain variable domain (VH-9) of a first immunoglobulin capable of specifically binding to a second epitope; (v) a heavy chain variable domain (VH-1 ... (iii) a first heterodimerization domain of a first immunoglobulin, the first heterodimerization domain being incapable of forming a stable homodimer with another first heterodimerization domain; and (c) a third polypeptide comprising, from N-terminal to C-terminal direction: (i) a heavy chain variable domain (VH-3) of a third immunoglobulin capable of specifically binding to a first epitope; (ii) a second CH1 domain (CH1-3) of the third immunoglobulin;and (iii) a second heterodimerization domain of a third immunoglobulin, the second heterodimerization domain comprising an amino acid sequence or nucleic acid sequence distinct from the first heterodimerization domain of the first immunoglobulin, the second heterodimerization domain being incapable of forming stable homodimers with another second heterodimerization domain, and being configured to form heterodimers with the first heterodimerization domain of the first immunoglobulin; (d) the fourth polypeptide comprising, from N-terminal to C-terminal direction: (i) a light chain variable domain (VL-3) of the third immunoglobulin capable of specifically binding to a first epitope; and (ii) a light chain constant domain (CL-3) of the third immunoglobulin, wherein VL-2 is selected from the group consisting of SEQ ID NOs: 17, 25, 33, 41, 121, 137, 169, 177, 185, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 335, 340, 350, 360, 370, 380, 390, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 93, 201, 209, 217, 225, 233, 241, 249, 257, 265, 321, 329, 337, 393, 401, 409, 473, 481, 489, 497, 505, 513, 545, 553, 561, 569, 577, 585, 593, 601, 625, 633, 641, 649, 657, 665, 673, 681, 689, 697, 705, 713, 721, 729, 73 V selected from any one of 7, 745, 753, 761, 769, 785, 793, 801, 809, 817, 849, 857, 865, 873, 881, 889, 897, 905, 913, 921, 929, 937, 945, 969, 977, 1009, 1057, 1537, 1569, 1601, 1641, 1665, 1825, 1865, 1897, 1905, 1913, 1921, 1929, 2265, 2281 2289, 2329, and 2345; Land / or VH-2 comprises the CDR1 sequence, CDR2 sequence, and CDR3 sequence of the amino acid sequences SEQ ID NOs: 21, 29, 37, 45, 125, 141, 173, 181, 189, 197, 205, 213, 221, 229, 237, 245, 253, 261, 269, 325, 333, 341, 397, 405, 413, 477, 485, 493, 501, 509, 517, 549, 557, 565, 573, 581, 589, 597, 605, 629, 637, 645, 653, 661, 669, 677, 685, 69 V selected from any one of 3, 701, 709, 717, 725, 733, 741, 749, 757, 765, 773, 789, 797, 805, 813, 821, 853, 861, 869, 877, 885, 893, 901, 909, 917, 925, 933, 941, 949, 973, 981, 1013, 1061, 1541, 1573, 1605, 1645, 1669, 1829, 1869, 1901, 1909, 1917, 1925, 1933, 2269, 2285, 2293, 2333, and 2349 H Heterodimeric multispecific antibodies are provided that comprise CDR1, CDR2, and CDR3 amino acid sequences.

[0083] In some embodiments, both the VH-1 and VH-3 are selected from the group consisting of SEQ ID NOs: 5, 13, 21, 29, 37, 45, 53, 61, 69, 77, 85, 93, 101, 109, 117, 125, 133, 149, 157, 165, 173, 181, 197, 205, 237, 245, 261, 277, 285, 293, 301, 309, 317, 325, 333, 341, 349, 357, 365, 373, 381, 389, 397, 405, 413, 421, 429, 437, 445, 453, 461, 469, 485, 493, 501, 525, 533, 541, 551, 561, 571, 581, 591, 601, 612, 614, 616, 618, 620, 622, 624, 626, 628, 630, 632, 634, 636, 638, 640, 642, 644, 645, 646, 648, 650, 652, 654, 656, 658, 660, 662, 664, 670, 672, 674, 676, 678, 681, 682, 49, 557, 565, 613, 621, 685, 693, 701, 709, 717, 725, 733, 741, 749, 757, 765, 773, 781, 789, 797, 805, 813, 821, 829, 837, 845, 853, 861, 869, 877, 885, 89 3, 949, 957, 965, 981, 989, 997, 1005, 1013, 1021, 1029, 1037, 1045, 1053, 1069, 1077, 1085, 1093, 1101, 1109, 1117, 1125, 1133, 1141, 1149, 1157, 1165 , 1173, 1181, 1189, 1197, 1205, 1213, 1221, 1229, 1237, 1245, 1253, 1261, 1269, 1277, 1285, 1293, 1301, 1309, 1317, 1325, 1333, 1341, 1349, 1357, 1365 , 1373, 1381, 1389, 1397, 1405, 1413, 1421, 1429, 1437, 1445, 1453, 1461, 1469, 1477, 1485, 1493, 1501, 1509, 1517, 1525, 1533, 1549, 1557, 1565, 1573 , 1581, 1589, 1597, 1605, 1613, 1621, 1629, 1637, 1653, 1661, 1677, 1685, 1693, 1701, 1709, 1717, 1725, 1733, 1741, 1749, 1757, 1765, 1773, 1781, 1789 , 1797, 1805, 1813, 1821, 1837, 1845, 1853, 1861, 1869, 1877, 1885, 1893, 1917, 1941, 1949, 1957, 1965, 1973, 1981, 1989, 1997, 2005, 2013, 2021, 2029,V selected from any one of 2037, 2045, 2053, 2061, 2069, 2077, 2085, 2093, 2101, 2109, 2117, 2125, 2133, 2141, 2149, 2157, 2165, 2173, 2181, 2189, 2197, 2205, 2213, 2221, 2229, 2237, 2245, 2253, 2261, 2269, 2277, 2285, 2301, 2309, 2317, 2325, 2333, 2341, and 2349; Hand / or both VL-1 and VL-3 comprise the amino acid sequences CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 1, 9, 17, 25, 33, 41, 49, 57, 65, 73, 81, 89, 97, 105, 113, 121, 129, 145, 153, 161, 169, 177, 193, 201, 233, 241, 257, 273, 281, 289, 297, 305, 313, 321, 329, 337, 345, 353, 361, 369, 377, 385, 393, 401, 409, 417, 425, 433, 441, 449, 457, 465, 481, 489, 497, 521, 529, 537, 545, 553, 561, 609, 617, 681, 689, 697, 705, 713, 721, 729, 737, 745, 753, 761, 769, 777, 785, 793, 801, 809, 817, 825, 8 33, 841, 849, 857, 865, 873, 881, 889, 945, 953, 961, 977, 985, 993, 1001, 1009, 1017, 1025, 1033, 1041, 1049, 1065, 1073, 1081, 1089, 1097, 1105, 1113, 1121, 1129, 1137, 1145, 1153, 1161, 1169, 1177, 1185, 1193, 1201, 1209, 1217, 1225, 1233, 1241, 1249, 1257, 1265, 1273, 1281, 1289, 1297, 1305, 1313, 1321, 1329, 1337, 1345, 1353, 1361, 1369, 1377, 1385, 1393, 1401, 1409, 1417, 1425, 1433, 1441, 1449, 1457, 1465, 1473, 1481, 1489, 1497, 1505, 1513, 1521, 1529, 1545, 1553, 1561, 1569, 1577, 1585, 1593, 1601, 1609, 1617, 1625, 1633, 1649, 1657, 1673, 1681, 1689, 1697, 1705, 1713, 1721, 1729, 1737, 1745, 1753, 1761, 1769, 1777, 1785, 1793, 1801, 1809, 1817, 1833, 1841, 1849, 1857, 1865, 1873, 1881, 1889, 1913, 1937, 1945, 1953, 1961, 1969, 1977,V selected from any one of 1985, 1993, 2001, 2009, 2017, 2025, 2033, 2041, 2049, 2057, 2065, 2073, 2081, 2089, 2097, 2105, 2113, 2121, 2129, 2137, 2145, 2153, 2161, 2169, 2177, 2185, 2193, 2201, 2209, 2217, 2225, 2233, 2241, 2249, 2257, 2265, 2273, 2281, 2297, 2305, 2313, 2321, 2329, 2337, and 2345; L It includes CDR1, CDR2, and CDR3 amino acid sequences.

[0084] In yet another aspect, the disclosure provides a polypeptide comprising a first polypeptide chain, a second polypeptide chain, a third polypeptide chain, and a fourth polypeptide chain, wherein the first polypeptide chain and the second polypeptide chain are covalently coupled to each other, the second polypeptide chain and the third polypeptide chain are covalently coupled to each other, and the third polypeptide chain and the fourth polypeptide chain are covalently coupled to each other, and wherein (a) the first polypeptide chain comprises, in an N-terminal to C-terminal direction: (i) a light chain of a first immunoglobulin capable of specifically binding to a first epitope; (ii) a light chain constant domain (CL-1) of a first immunoglobulin; (iii) a flexible peptide linker comprising the amino acid sequence (GGGGS)3; and (iv) a light chain variable domain (VL-2) of a second immunoglobulin linked to a heavy chain variable domain (VH-2) of a complementary second immunoglobulin, or a heavy chain variable domain (VH-2) of a second immunoglobulin linked to a light chain variable domain (VL-2) of a complementary second immunoglobulin, wherein VL-2 and VH-2 specifically bind to a second epitope. (b) a second polypeptide comprising, from N-terminal to C-terminal direction: (i) a heavy chain variable domain (VH-1) of a first immunoglobulin capable of specifically binding to a first epitope; (ii) a first CH1 domain (CH1-1) of the first immunoglobulin; and (iii) a first heterodimerization domain of the first immunoglobulin, the first heterodimerization domain being linked together via a flexible peptide linker comprising the amino acid sequence (GGGGS)6 to form a single-chain variable fragment; (c) a third polypeptide comprising, from N-terminal to C-terminal direction: (i) a heavy chain variable domain (VH-3) of a third immunoglobulin capable of specifically binding to a third epitope; (ii) a second CH1 domain (CH1-3) of the third immunoglobulin; and (iii) a second heterodimerization domain of the third immunoglobulin, which comprises an amino acid sequence or nucleic acid sequence distinct from the first heterodimerization domain of the first immunoglobulin;(d) a fourth polypeptide comprising, from N-terminal to C-terminal direction: (i) a light chain variable domain (VL-3) of a third immunoglobulin capable of specifically binding to a third epitope; and (ii) a light chain constant domain (CL-3) of the third immunoglobulin, wherein each of VL-1 and VL-3 independently has a sequence similar to SEQ ID NO: 1. , 9, 17, 25, 33, 41, 49, 57, 65, 73, 81, 89, 97, 105, 113, 121, 129, 145, 153, 161, 169, 177, 193, 201, 233, 241, 257, 273, 281, 289, 297, 305, 313, 321, 329, 337, 345, 353, 361, 369, 377, 385, 393, 401, 409, 417, 425, 433, 441, 449, 457, 465, 481, 489, 497, 521, 529, 537, 545, 553, 561, 609, 617, 681, 689, 697, 7 05, 713, 721, 729, 737, 745, 753, 761, 769, 777, 785, 793, 801, 809, 817, 825, 833, 841, 849, 857, 865, 873, 881, 889, 945, 953, 961, 977, 985, 993, 1001, 1009, 1017, 1025, 1033, 1041, 1049, 1065, 1073, 1081, 1089, 1097, 1105, 1113, 1121, 1129, 1137, 1145, 1153, 1161, 1169, 1177, 1185, 1193, 1201, 1209, 1217, 1225, 1233, 1241, 1249, 1257, 1265, 1273, 1281, 1289, 1297, 1305, 1313, 1321, 1329, 1337, 1345, 1353, 1361, 1369, 1377, 1385, 1393, 1401, 1409, 1417, 1425, 1433, 1441, 1449, 1457, 1465, 1473, 1481, 1489, 1497, 1505, 1513, 1521, 1529, 1545, 1553, 1561, 1569, 1577, 1585, 1593, 1601, 1609, 1617,1625, 1633, 1649, 1657, 1673, 1681, 1689, 1697, 1705, 1713, 1721, 1729, 1737, 1745, 1753, 1761, 1769, 1777, 1785, 1793, 1801, 1809, 1817, 1833, 1841, 1849, 1857, 1865, 1873, 1881, 1889, 1913, 1937, 1945, 1953, 1961, 1969, 1977, 1985, 1993, 2001, 2009, 2017, 2018 V selected from any one of 25, 2033, 2041, 2049, 2057, 2065, 2073, 2081, 2089, 2097, 2105, 2113, 2121, 2129, 2137, 2145, 2153, 2161, 2169, 2177, 2185, 2193, 2201, 2209, 2217, 2225, 2233, 2241, 2249, 2257, 2265, 2273, 2281, 2297, 2305, 2313, 2321, 2329, 2337, and 2345; L comprising CDR1, CDR2, and CDR3 amino acid sequences;

[0085] and / or each of VH-1 and VH-3 independently is selected from the group consisting of SEQ ID NOs: 5, 13, 21, 29, 37, 45, 53, 61, 69, 77, 85, 93, 101, 109, 117, 125, 133, 149, 157, 165, 173, 181, 197, 205, 237, 245, 261, 277, 285, 293, 301, 309, 317, 325, 333, 341, 349, 357, 365, 373, 381, 389, 397, 405, 413, 421, 429, 437, 445, 453, 461, 469, 485, 493, 501, 525, 533, 541, 552, 561, 571, 581, 591, 601, 612, 614, 616, 618, 620, 622, 624, 626, 628, 629, 630, 632, 634, 636, 638, 639, 640, 642, 643, 644, 645, 646, 647, 648, 650, 651, 652, 653, 654, 655, 656, 657, 658, 660, 662, 49, 557, 565, 613, 621, 685, 693, 701, 709, 717, 725, 733, 741, 749, 757, 765, 773, 781, 789, 797, 805, 813, 821, 829, 837, 845, 853, 861, 869, 877, 885, 89 3, 949, 957, 965, 981, 989, 997, 1005, 1013, 1021, 1029, 1037, 1045, 1053, 1069, 1077, 1085, 1093, 1101, 1109, 1117, 1125, 1133, 1141, 1149, 1157, 1165 , 1173, 1181, 1189, 1197, 1205, 1213, 1221, 1229, 1237, 1245, 1253, 1261, 1269, 1277, 1285, 1293, 1301, 1309, 1317, 1325, 1333, 1341, 1349, 1357, 1365 , 1373, 1381, 1389, 1397, 1405, 1413, 1421, 1429, 1437, 1445, 1453, 1461, 1469, 1477, 1485, 1493, 1501, 1509, 1517, 1525, 1533, 1549, 1557, 1565, 1573 , 1581, 1589, 1597, 1605, 1613, 1621, 1629, 1637, 1653, 1661, 1677, 1685, 1693, 1701, 1709, 1717, 1725, 1733, 1741, 1749, 1757, 1765, 1773, 1781, 1789 , 1797, 1805, 1813, 1821, 1837, 1845, 1853, 1861, 1869, 1877, 1885, 1893, 1917, 1941, 1949, 1957, 1965, 1973, 1981, 1989, 1997, 2005, 2013, 2021, 2029,V selected from any one of 2037, 2045, 2053, 2061, 2069, 2077, 2085, 2093, 2101, 2109, 2117, 2125, 2133, 2141, 2149, 2157, 2165, 2173, 2181, 2189, 2197, 2205, 2213, 2221, 2229, 2237, 2245, 2253, 2261, 2269, 2277, 2285, 2301, 2309, 2317, 2325, 2333, 2341, and 2349; H comprising CDR1, CDR2, and CDR3 amino acid sequences;

[0086] and / or VL-2 is selected from the group consisting of SEQ ID NOs: 17, 25, 33, 41, 121, 137, 169, 177, 185, 193, 201, 209, 217, 225, 233, 241, 249, 257, 265, 321, 329, 337, 393, 401, 409, 473, 481, 489, 497, 505, 513, 545, 553, 561, 569, 577, 585, 593, 601, 625, 633, 641, 649, 657, 665, 673, 681, 692, 701, 713, 721, 733, 741, 753, 761, 773, 781, 792, 801, 813, 825, 833, 841, 853, 861, 873, 881, 892, 901, 913, 925, 933, 941, 953, 961, 973, 981, 992, 1001, 1025, 1033, 1041, 1053, 1061, 1073, 1081, 1093, 1101, 1125, 1133, 1141, 1153, 1161, 1173, 1181, 11 V selected from any one of 89, 697, 705, 713, 721, 729, 737, 745, 753, 761, 769, 785, 793, 801, 809, 817, 849, 857, 865, 873, 881, 889, 897, 905, 913, 921, 929, 937, 945, 969, 977, 1009, 1057, 1537, 1569, 1601, 1641, 1665, 1825, 1865, 1897, 1905, 1913, 1921, 1929, 2265, 2281, 2289, 2329, and 2345 Land / or VH-2 comprises the CDR1 sequence, CDR2 sequence, and CDR3 sequence of the amino acid sequences SEQ ID NOs: 21, 29, 37, 45, 125, 141, 173, 181, 189, 197, 205, 213, 221, 229, 237, 245, 253, 261, 269, 325, 333, 341, 397, 405, 413, 477, 485, 493, 501, 509, 517, 549, 557, 565, 573, 581, 589, 597, 605, 629, 637, 645, 653, 661, 669, 677, 685, 69 V selected from any one of 3, 701, 709, 717, 725, 733, 741, 749, 757, 765, 773, 789, 797, 805, 813, 821, 853, 861, 869, 877, 885, 893, 901, 909, 917, 925, 933, 941, 949, 973, 981, 1013, 1061, 1541, 1573, 1605, 1645, 1669, 1829, 1869, 1901, 1909, 1917, 1925, 1933, 2269, 2285, 2293, 2333, and 2349 H Heterodimeric multispecific antibodies are provided that comprise CDR1, CDR2, and CDR3 amino acid sequences.

[0087] Additionally or alternatively, for the heterodimeric multispecific antibodies disclosed herein, in some embodiments, VH-1 or VH-3 is selected from the group consisting of SEQ ID NOs: 5, 13, 21, 29, 37, 45, 53, 61, 69, 77, 85, 93, 101, 109, 117, 125, 133, 149, 157, 165, 173, 181, 197, 205, 237, 245, 261, 277, 285, 293, 301, 309, 317, 325, 333, 341, 349, 357, 365, 373, 381, 389, 397, 405, 413, 421, 429, 437, 44 5, 453, 461, 469, 485, 493, 501, 525, 533, 541, 549, 557, 565, 613, 621, 685, 693, 701, 709, 717, 725, 733, 741, 749, 757, 765, 773, 781, 789, 797, 805, 813 , 821, 829, 837, 845, 853, 861, 869, 877, 885, 893, 949, 957, 965, 981, 989, 997, 1005, 1013, 1021, 1029, 1037, 1045, 1053, 1069, 1077, 1085, 1093, 1101, 1109, 1117, 1125, 1133, 1141, 1149, 1157, 1165, 1173, 1181, 1189, 1197, 1205, 1213, 1221, 1229, 1237, 1245, 1253, 1261, 1269, 1277, 1285, 1293, 1301, 1309, 1317, 1325, 1333, 1341, 1349, 1357, 1365, 1373, 1381, 1389, 1397, 1405, 1413, 1421, 1429, 1437, 1445, 1453, 1461, 1469, 1477, 1485, 1493, 1501, 1509, 1517, 1525, 1533, 1549, 1557, 1565, 1573, 1581, 1589, 1597, 1605, 1613, 1621, 1629, 1637, 1653, 1661, 1677, 1685, 1693, 1701, 1709, 1717, 1725, 1733, 1741, 1749, 1757, 1765, 1773, 1781, 1789, 1797, 1805, 1813, 1821, 1837, 1845, 1853, 1861, 1869, 1877, 1885, 1893, 1917, 1941, 1949, 1957, 1965,V selected from any one of 1973, 1981, 1989, 1997, 2005, 2013, 2021, 2029, 2037, 2045, 2053, 2061, 2069, 2077, 2085, 2093, 2101, 2109, 2117, 2125, 2133, 2141, 2149, 2157, 2165, 2173, 2181, 2189, 2197, 2205, 2213, 2221, 2229, 2237, 2245, 2253, 2261, 2269, 2277, 2285, 2301, 2309, 2317, 2325, 2333, 2341, and 2349; H comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to the amino acid sequence;

[0088] and / or VL-1 or VL-3 is selected from the group consisting of SEQ ID NOs: 1, 9, 17, 25, 33, 41, 49, 57, 65, 73, 81, 89, 97, 105, 113, 121, 129, 145, 153, 161, 169, 177, 193, 201, 233, 241, 257, 273, 281, 289, 297, 305, 313, 321, 329, 337, 345, 353, 361, 369, 377, 385, 393, 401, 409, 417, 425, 433, 441, 449, 457, 465, 481, 489, 497, 521, 529, 537, 545, 553, 561, 571, 581, 591, 602, 603, 604, 605, 606, 607, 608, 610, 611, 612, 613, 614, 615, 616, 617, 618, 619, 620, 621, 622, 623, 624, 625, 626, 627, 628, 629, 630, 631, 632, 633, 634, 635, 636, 637, 638, 61, 609, 617, 681, 689, 697, 705, 713, 721, 729, 737, 745, 753, 761, 769, 777, 785, 793, 801, 809, 817, 825, 833, 841, 849, 857, 865, 873, 881, 889, 945, 9 53, 961, 977, 985, 993, 1001, 1009, 1017, 1025, 1033, 1041, 1049, 1065, 1073, 1081, 1089, 1097, 1105, 1113, 1121, 1129, 1137, 1145, 1153, 1161, 1169, 1 177, 1185, 1193, 1201, 1209, 1217, 1225, 1233, 1241, 1249, 1257, 1265, 1273, 1281, 1289, 1297, 1305, 1313, 1321, 1329, 1337, 1345, 1353, 1361, 1369, 1377, 1385, 1393, 1401, 1409, 1417, 1425, 1433, 1441, 1449, 1457, 1465, 1473, 1481, 1489, 1497, 1505, 1513, 1521, 1529, 1545, 1553, 1561, 1569, 1577, 1585, 1593, 1601, 1609, 1617, 1625, 1633, 1649, 1657, 1673, 1681, 1689, 1697, 1705, 1713, 1721, 1729, 1737, 1745, 1753, 1761, 1769, 1777, 1785, 1793, 1801, 1809, 1817, 1833, 1841, 1849, 1857, 1865, 1873, 1881, 1889, 1913, 1937, 1945, 1953, 1961, 1969, 1977, 1985, 1993, 2001, 2009, 2017, 2025, 2033,V selected from any one of 2041, 2049, 2057, 2065, 2073, 2081, 2089, 2097, 2105, 2113, 2121, 2129, 2137, 2145, 2153, 2161, 2169, 2177, 2185, 2193, 2201, 2209, 2217, 2225, 2233, 2241, 2249, 2257, 2265, 2273, 2281, 2297, 2305, 2313, 2321, 2329, 2337, and 2345; L It includes an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to the amino acid sequence.

[0089] Additionally or alternatively, for the heterodimeric multispecific antibodies disclosed herein, in some embodiments, VH-2 or VH-4 is selected from the group consisting of SEQ ID NOs: 21, 29, 37, 45, 125, 141, 173, 181, 189, 197, 205, 213, 221, 229, 237, 245, 253, 261, 269, 325, 333, 341, 397, 405, 413, 477, 485, 493, 501, 509, 517, 549, 557, 565, 573, 581, 589, 597, 605, 629, 637, 645, 653, 661, 669, 670, 671, 672, 673, 674, 675, 676, 677, 678, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689, 690, 700, 701, 702, 703, 704, 705, 706, 707, 708, 710, 711, 712, 713, 714, 715, 716, 717, 718, 719, 720, 721, 722, 723, 724, 725, 726, 727, 728, 729, 73 V selected from any one of 77, 685, 693, 701, 709, 717, 725, 733, 741, 749, 757, 765, 773, 789, 797, 805, 813, 821, 853, 861, 869, 877, 885, 893, 901, 909, 917, 925, 933, 941, 949, 973, 981, 1013, 1061, 1541, 1573, 1605, 1645, 1669, 1829, 1869, 1901, 1909, 1917, 1925, 1933, 2269, 2285, 2293, 2333, and 2349 Hand / or VL-2 or VL-4 comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NOs: 17, 25, 33, 41, 121, 137, 169, 177, 185, 193, 201, 209, 217, 225, 233, 241, 249, 257, 265, 321, 329, 337, 393, 401, 409, 473, 481, 489, 497, 505, 513, 545, 553, 561, 569, 577, 585, 593, 601, 625, 633, 641, 649, 657, 665, 673, 681, 689, 697, 705, 713, 721, 729, 737, 745, 753, 761, 769, 785, 793, 801, 809, 817, 849, 857, 865, 873, 881, 889, 897, 905, 913, 921, 929, 937, 945, 969, 977, 1009, 1057, 1537, 1569, 1601, 1641, 1665, 1825, 1865, 1897, 1905, 1913, 1921, 1929, 2265, 2281 V selected from any one of 2289, 2329, and 2345 L It includes an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to the amino acid sequence.

[0090] Additionally or alternatively, for the heterodimeric multispecific antibodies disclosed herein, in some embodiments, each of the VL-1 and VH-1 is selected from the group consisting of SEQ ID NOs: 1 and 5, respectively; SEQ ID NOs: 9 and 13, respectively; SEQ ID NOs: 17 and 21, respectively; SEQ ID NOs: 25 and 29, respectively; SEQ ID NOs: 33 and 37, respectively; SEQ ID NOs: 41 and 45, respectively; SEQ ID NOs: 49 and 53, respectively; SEQ ID NOs: 57 and 61, respectively; SEQ ID NOs: 73 and 77, respectively; SEQ ID NOs: 89 and 93, respectively; SEQ ID NOs: 97 and 98, respectively. 101; SEQ ID NOs: 105 and 109, respectively; SEQ ID NOs: 113 and 117, respectively; SEQ ID NOs: 121 and 125, respectively; SEQ ID NOs: 129 and 133, respectively; SEQ ID NOs: 145 and 149, respectively; SEQ ID NOs: 161 and 165, respectively; SEQ ID NOs: 169 and 173, respectively; SEQ ID NOs: 177 and 181, respectively; SEQ ID NOs: 193 and 197, respectively; SEQ ID NOs: 201 and 205, respectively; SEQ ID NOs: 233 and 237, respectively; SEQ ID NOs: 241 and 245, respectively; SEQ ID NOs: 257 and 260, respectively. 61; SEQ ID NOs: 273 and 277, respectively; SEQ ID NOs: 281 and 285, respectively; SEQ ID NOs: 289 and 293, respectively; SEQ ID NOs: 297 and 301, respectively; SEQ ID NOs: 305 and 309, respectively; SEQ ID NOs: 313 and 317, respectively; SEQ ID NOs: 321 and 325, respectively; SEQ ID NOs: 329 and 333, respectively; SEQ ID NOs: 337 and 341, respectively; SEQ ID NOs: 345 and 349, respectively; SEQ ID NOs: 353 and 357, respectively; SEQ ID NOs: 361 and 365, respectively; SEQ ID NOs: 369 and 37, respectively 3; SEQ ID NOs:377 and 381, respectively; SEQ ID NOs:385 and 389, respectively; SEQ ID NOs:393 and 397, respectively; SEQ ID NOs:401 and 405, respectively; SEQ ID NOs:409 and 413, respectively; SEQ ID NOs:417 and 421, respectively; SEQ ID NOs:425 and 429, respectively; SEQ ID NOs:433 and 437, respectively; SEQ ID NOs:441 and 445, respectively; SEQ ID NOs:449 and 453, respectively; SEQ ID NOs:457 and 461, respectively; SEQ ID NOs:465 and 469, respectively; SEQ ID NOs:481 and 485, respectively;SEQ ID NOs: 489 and 493, respectively; SEQ ID NOs: 497 and 501, respectively; SEQ ID NOs: 521 and 525, respectively; SEQ ID NOs: 529 and 533, respectively; SEQ ID NOs: 537 and 541, respectively; SEQ ID NOs: 545 and 549, respectively; SEQ ID NOs: 553 and 557, respectively; SEQ ID NOs: 561 and 565, respectively; SEQ ID NOs: 609 and 613, respectively; SEQ ID NOs: 617 and 621, respectively; SEQ ID NOs: 681 and 685, respectively; SEQ ID NOs: 689 and 693, respectively; SEQ ID NOs: 697 and 701, respectively. SEQ ID NOs:705 and 709, respectively; SEQ ID NOs:713 and 717, respectively; SEQ ID NOs:721 and 725, respectively; SEQ ID NOs:729 and 733, respectively; SEQ ID NOs:737 and 741, respectively; SEQ ID NOs:745 and 749, respectively; SEQ ID NOs:753 and 757, respectively; SEQ ID NOs:761 and 765, respectively; SEQ ID NOs:769 and 773, respectively; SEQ ID NOs:785 and 789, respectively; SEQ ID NOs:793 and 797, respectively; SEQ ID NOs:801 and 805, respectively; SEQ ID NOs:809 and 813, respectively. SEQ ID NOs: 817 and 821, respectively; SEQ ID NOs: 825 and 829, respectively; SEQ ID NOs: 833 and 837, respectively; SEQ ID NOs: 841 and 845, respectively; SEQ ID NOs: 849 and 853, respectively; SEQ ID NOs: 857 and 861, respectively; SEQ ID NOs: 865 and 869, respectively; SEQ ID NOs: 873 and 877, respectively; SEQ ID NOs: 881 and 885, respectively; SEQ ID NOs: 889 and 893, respectively; SEQ ID NOs: 945 and 949, respectively; SEQ ID NOs: 953 and 957, respectively; SEQ ID NOs: 961 and 965, respectively. SEQ ID NOs: 977 and 981, respectively; SEQ ID NOs: 985 and 989, respectively; SEQ ID NOs: 993 and 997, respectively; SEQ ID NOs: 1001 and 1005, respectively; SEQ ID NOs: 1009 and 1013, respectively; SEQ ID NOs: 1017 and 1021, respectively; SEQ ID NOs: 1025 and 1029, respectively; SEQ ID NOs: 1033 and 1037, respectively; SEQ ID NOs: 1041 and 1045, respectively; SEQ ID NOs: 1065 and 1069, respectively; SEQ ID NOs: 1073 and 1077, respectively; SEQ ID NOs: 1081 and 1085, respectively;SEQ ID NOs: 1089 and 1093, respectively; SEQ ID NOs: 1097 and 1101, respectively; SEQ ID NOs: 1113 and 1117, respectively; SEQ ID NOs: 1121 and 1125, respectively; SEQ ID NOs: 1129 and 1133, respectively; SEQ ID NOs: 1137 and 1141, respectively; SEQ ID NOs: 1145 and 1149, respectively; SEQ ID NOs: 1153 and 1157, respectively; SEQ ID NOs: 1161 and 1165, respectively; SEQ ID NOs: 1169 and 1173, respectively; SEQ ID NOs: 1185 and 1189, respectively; SEQ ID NOs: 1193 and 1198, respectively. and 1197; SEQ ID NOs: 1201 and 1205, respectively; SEQ ID NOs: 1209 and 1213, respectively; SEQ ID NOs: 1217 and 1221, respectively; SEQ ID NOs: 1225 and 1229, respectively; SEQ ID NOs: 1233 and 1237, respectively; SEQ ID NOs: 1241 and 1245, respectively; SEQ ID NOs: 1249 and 1253, respectively; SEQ ID NOs: 1257 and 1261, respectively; SEQ ID NOs: 1265 and 1269, respectively; SEQ ID NOs: 1273 and 1277, respectively; SEQ ID NOs: 1281 and 1285, respectively; SEQ ID NOs: 1 SEQ ID NOs: 1329 and 1333, respectively; SEQ ID NOs: 1337 and 1341, respectively; SEQ ID NOs: 1345 and 1349, respectively; SEQ ID NOs: 1353 and 1357, respectively; SEQ ID NOs: 1361 and 1365, respectively; SEQ ID NOs: 1369 and 1373, respectively; SEQ ID NOs: 1377 and 1381, respectively; SEQ ID NOs: 1385 and 1389; SEQ ID NOs: 1393 and 1397, respectively; SEQ ID NOs: 1401 and 1405, respectively; SEQ ID NOs: 1409 and 1413, respectively; SEQ ID NOs: 1417 and 1421, respectively; SEQ ID NOs: 1433 and 1437, respectively; SEQ ID NOs: 1441 and 1445, respectively; SEQ ID NOs: 1457 and 1461, respectively; SEQ ID NOs: 1465 and 1469, respectively; SEQ ID NOs: 1473 and 1477, respectively; SEQ ID NOs: 1481 and 1485, respectively; SEQ ID NOs: 1489 and 1493, respectively;SEQ ID NOs: 1497 and 1501, respectively; SEQ ID NOs: 1505 and 1509, respectively; SEQ ID NOs: 1513 and 1517, respectively; SEQ ID NOs: 1521 and 1525, respectively; SEQ ID NOs: 1529 and 1533, respectively; SEQ ID NOs: 1545 and 1549, respectively; SEQ ID NOs: 1553 and 1557, respectively; SEQ ID NOs: 1561 and 1565, respectively; SEQ ID NOs: 1569 and 1573, respectively; SEQ ID NOs: 1577 and 1581, respectively; SEQ ID NOs: 1585 and 1589, respectively; SEQ ID NOs: 1593 and 1598, respectively. and 1597; SEQ ID NOs: 1601 and 1605, respectively; SEQ ID NOs: 1609 and 1613, respectively; SEQ ID NOs: 1617 and 1621, respectively; SEQ ID NOs: 1625 and 1629, respectively; SEQ ID NOs: 1633 and 1637, respectively; SEQ ID NOs: 1649 and 1653, respectively; SEQ ID NOs: 1657 and 1661, respectively; SEQ ID NOs: 1673 and 1677, respectively; SEQ ID NOs: 1681 and 1685, respectively; SEQ ID NOs: 1689 and 1693, respectively; SEQ ID NOs: 1697 and 1701, respectively; SEQ ID NOs: 1 SEQ ID NOs: 1705 and 1709; SEQ ID NOs: 1713 and 1717, respectively; SEQ ID NOs: 1721 and 1725, respectively; SEQ ID NOs: 1729 and 1733, respectively; SEQ ID NOs: 1737 and 1741, respectively; SEQ ID NOs: 1745 and 1749, respectively; SEQ ID NOs: 1753 and 1757, respectively; SEQ ID NOs: 1761 and 1765, respectively; SEQ ID NOs: 1769 and 1773, respectively; SEQ ID NOs: 1777 and 1781, respectively; SEQ ID NOs: 1785 and 1789, respectively; SEQ ID NOs: 1793 and 1797, respectively; SEQ ID NOs: 1801 and 1805; SEQ ID NOs: 1809 and 1813, respectively; SEQ ID NOs: 1817 and 1821, respectively; SEQ ID NOs: 1833 and 1837, respectively; SEQ ID NOs: 1841 and 1845, respectively; SEQ ID NOs: 1849 and 1853, respectively; SEQ ID NOs: 1857 and 1861, respectively; SEQ ID NOs: 1865 and 1869, respectively; SEQ ID NOs: 1873 and 1877, respectively; SEQ ID NOs: 1881 and 1885, respectively; SEQ ID NOs: 1889 and 1893, respectively; SEQ ID NOs: 1913 and 1917, respectively;SEQ ID NOs: 1937 and 1941, respectively; SEQ ID NOs: 1945 and 1949, respectively; SEQ ID NOs: 1953 and 1957, respectively; SEQ ID NOs: 1961 and 1965, respectively; SEQ ID NOs: 1969 and 1973, respectively; SEQ ID NOs: 1977 and 1981, respectively; SEQ ID NOs: 1985 and 1989, respectively; SEQ ID NOs: 1993 and 1997, respectively; SEQ ID NOs: 2001 and 2005, respectively; SEQ ID NOs: 2009 and 2013, respectively; SEQ ID NOs: 2017 and 2021, respectively; SEQ ID NOs: 2025 and 2026, respectively. and 2029; SEQ ID NOs: 2033 and 2037, respectively; SEQ ID NOs: 2041 and 2045, respectively; SEQ ID NOs: 2049 and 2053, respectively; SEQ ID NOs: 2057 and 2061, respectively; SEQ ID NOs: 2065 and 2069, respectively; SEQ ID NOs: 2073 and 2077, respectively; SEQ ID NOs: 2081 and 2085, respectively; SEQ ID NOs: 2089 and 2093, respectively; SEQ ID NOs: 2097 and 2101, respectively; SEQ ID NOs: 2105 and 2109, respectively; SEQ ID NOs: 2113 and 2117, respectively; SEQ ID NOs: 2114 and 2118, respectively. SEQ ID NOs: 2161 and 2165, respectively; SEQ ID NOs: 2169 and 2173, respectively; SEQ ID NOs: 2177 and 2181, respectively; SEQ ID NOs: 2185 and 2189, respectively; SEQ ID NOs: 2193 and 2197, respectively; SEQ ID NOs: 2201 and 2205, respectively; SEQ ID NOs: 2209 and 2213, respectively; SEQ ID NOs: 2217 and 2221; SEQ ID NOs: 2225 and 2229, respectively; SEQ ID NOs: 2233 and 2237, respectively; SEQ ID NOs: 2241 and 2245, respectively; SEQ ID NOs: 2249 and 2253, respectively; SEQ ID NOs: 2257 and 2261, respectively; SEQ ID NOs: 2273 and 2277, respectively; SEQ ID NOs: 2281 and 2285, respectively; SEQ ID NOs: 2305 and 2309, respectively; SEQ ID NOs: 2313 and 2317, respectively; SEQ ID NOs: 2321 and 2325, respectively; SEQ ID NOs: 2329 and 2333, respectively;V selected from the group consisting of SEQ ID NOs: 2337 and 2341, respectively; and SEQ ID NOs: 2345 and 2349, respectively; L Amino acid sequence and V H Contains the amino acid sequence.

[0091] Additionally or alternatively, for the heterodimeric multispecific antibodies disclosed herein, in some embodiments, each of the VL-3 and VH-3 is selected from SEQ ID NOs: 1 and 5, respectively; SEQ ID NOs: 9 and 13, respectively; SEQ ID NOs: 17 and 21, respectively; SEQ ID NOs: 25 and 29, respectively; SEQ ID NOs: 33 and 37, respectively; SEQ ID NOs: 41 and 45, respectively; SEQ ID NOs: 49 and 53, respectively; SEQ ID NOs: 57 and 61, respectively; SEQ ID NOs: 73 and 77, respectively; SEQ ID NOs: 89 and 93, respectively; SEQ ID NOs: 97 and 98, respectively. 101; SEQ ID NOs: 105 and 109, respectively; SEQ ID NOs: 113 and 117, respectively; SEQ ID NOs: 121 and 125, respectively; SEQ ID NOs: 129 and 133, respectively; SEQ ID NOs: 145 and 149, respectively; SEQ ID NOs: 161 and 165, respectively; SEQ ID NOs: 169 and 173, respectively; SEQ ID NOs: 177 and 181, respectively; SEQ ID NOs: 193 and 197, respectively; SEQ ID NOs: 201 and 205, respectively; SEQ ID NOs: 233 and 237, respectively; SEQ ID NOs: 241 and 245, respectively; SEQ ID NOs: 257 and 260, respectively. 61; SEQ ID NOs: 273 and 277, respectively; SEQ ID NOs: 281 and 285, respectively; SEQ ID NOs: 289 and 293, respectively; SEQ ID NOs: 297 and 301, respectively; SEQ ID NOs: 305 and 309, respectively; SEQ ID NOs: 313 and 317, respectively; SEQ ID NOs: 321 and 325, respectively; SEQ ID NOs: 329 and 333, respectively; SEQ ID NOs: 337 and 341, respectively; SEQ ID NOs: 345 and 349, respectively; SEQ ID NOs: 353 and 357, respectively; SEQ ID NOs: 361 and 365, respectively; SEQ ID NOs: 369 and 37, respectively 3; SEQ ID NOs:377 and 381, respectively; SEQ ID NOs:385 and 389, respectively; SEQ ID NOs:393 and 397, respectively; SEQ ID NOs:401 and 405, respectively; SEQ ID NOs:409 and 413, respectively; SEQ ID NOs:417 and 421, respectively; SEQ ID NOs:425 and 429, respectively; SEQ ID NOs:433 and 437, respectively; SEQ ID NOs:441 and 445, respectively; SEQ ID NOs:449 and 453, respectively; SEQ ID NOs:457 and 461, respectively; SEQ ID NOs:465 and 469, respectively; SEQ ID NOs:481 and 485, respectively;SEQ ID NOs: 489 and 493, respectively; SEQ ID NOs: 497 and 501, respectively; SEQ ID NOs: 521 and 525, respectively; SEQ ID NOs: 529 and 533, respectively; SEQ ID NOs: 537 and 541, respectively; SEQ ID NOs: 545 and 549, respectively; SEQ ID NOs: 553 and 557, respectively; SEQ ID NOs: 561 and 565, respectively; SEQ ID NOs: 609 and 613, respectively; SEQ ID NOs: 617 and 621, respectively; SEQ ID NOs: 681 and 685, respectively; SEQ ID NOs: 689 and 693, respectively; SEQ ID NOs: 697 and 701, respectively. SEQ ID NOs:705 and 709, respectively; SEQ ID NOs:713 and 717, respectively; SEQ ID NOs:721 and 725, respectively; SEQ ID NOs:729 and 733, respectively; SEQ ID NOs:737 and 741, respectively; SEQ ID NOs:745 and 749, respectively; SEQ ID NOs:753 and 757, respectively; SEQ ID NOs:761 and 765, respectively; SEQ ID NOs:769 and 773, respectively; SEQ ID NOs:785 and 789, respectively; SEQ ID NOs:793 and 797, respectively; SEQ ID NOs:801 and 805, respectively; SEQ ID NOs:809 and 813, respectively. SEQ ID NOs: 817 and 821, respectively; SEQ ID NOs: 825 and 829, respectively; SEQ ID NOs: 833 and 837, respectively; SEQ ID NOs: 841 and 845, respectively; SEQ ID NOs: 849 and 853, respectively; SEQ ID NOs: 857 and 861, respectively; SEQ ID NOs: 865 and 869, respectively; SEQ ID NOs: 873 and 877, respectively; SEQ ID NOs: 881 and 885, respectively; SEQ ID NOs: 889 and 893, respectively; SEQ ID NOs: 945 and 949, respectively; SEQ ID NOs: 953 and 957, respectively; SEQ ID NOs: 961 and 965, respectively. SEQ ID NOs: 977 and 981, respectively; SEQ ID NOs: 985 and 989, respectively; SEQ ID NOs: 993 and 997, respectively; SEQ ID NOs: 1001 and 1005, respectively; SEQ ID NOs: 1009 and 1013, respectively; SEQ ID NOs: 1017 and 1021, respectively; SEQ ID NOs: 1025 and 1029, respectively; SEQ ID NOs: 1033 and 1037, respectively; SEQ ID NOs: 1041 and 1045, respectively; SEQ ID NOs: 1065 and 1069, respectively; SEQ ID NOs: 1073 and 1077, respectively; SEQ ID NOs: 1081 and 1085, respectively;SEQ ID NOs: 1089 and 1093, respectively; SEQ ID NOs: 1097 and 1101, respectively; SEQ ID NOs: 1113 and 1117, respectively; SEQ ID NOs: 1121 and 1125, respectively; SEQ ID NOs: 1129 and 1133, respectively; SEQ ID NOs: 1137 and 1141, respectively; SEQ ID NOs: 1145 and 1149, respectively; SEQ ID NOs: 1153 and 1157, respectively; SEQ ID NOs: 1161 and 1165, respectively; SEQ ID NOs: 1169 and 1173, respectively; SEQ ID NOs: 1185 and 1189, respectively; SEQ ID NOs: 1193 and 1198, respectively. and 1197; SEQ ID NOs: 1201 and 1205, respectively; SEQ ID NOs: 1209 and 1213, respectively; SEQ ID NOs: 1217 and 1221, respectively; SEQ ID NOs: 1225 and 1229, respectively; SEQ ID NOs: 1233 and 1237, respectively; SEQ ID NOs: 1241 and 1245, respectively; SEQ ID NOs: 1249 and 1253, respectively; SEQ ID NOs: 1257 and 1261, respectively; SEQ ID NOs: 1265 and 1269, respectively; SEQ ID NOs: 1273 and 1277, respectively; SEQ ID NOs: 1281 and 1285, respectively; SEQ ID NOs: 1 SEQ ID NOs: 1329 and 1333, respectively; SEQ ID NOs: 1337 and 1341, respectively; SEQ ID NOs: 1345 and 1349, respectively; SEQ ID NOs: 1353 and 1357, respectively; SEQ ID NOs: 1361 and 1365, respectively; SEQ ID NOs: 1369 and 1373, respectively; SEQ ID NOs: 1377 and 1381, respectively; SEQ ID NOs: 1385 and 1389; SEQ ID NOs: 1393 and 1397, respectively; SEQ ID NOs: 1401 and 1405, respectively; SEQ ID NOs: 1409 and 1413, respectively; SEQ ID NOs: 1417 and 1421, respectively; SEQ ID NOs: 1433 and 1437, respectively; SEQ ID NOs: 1441 and 1445, respectively; SEQ ID NOs: 1457 and 1461, respectively; SEQ ID NOs: 1465 and 1469, respectively; SEQ ID NOs: 1473 and 1477, respectively; SEQ ID NOs: 1481 and 1485, respectively; SEQ ID NOs: 1489 and 1493, respectively;SEQ ID NOs: 1497 and 1501, respectively; SEQ ID NOs: 1505 and 1509, respectively; SEQ ID NOs: 1513 and 1517, respectively; SEQ ID NOs: 1521 and 1525, respectively; SEQ ID NOs: 1529 and 1533, respectively; SEQ ID NOs: 1545 and 1549, respectively; SEQ ID NOs: 1553 and 1557, respectively; SEQ ID NOs: 1561 and 1565, respectively; SEQ ID NOs: 1569 and 1573, respectively; SEQ ID NOs: 1577 and 1581, respectively; SEQ ID NOs: 1585 and 1589, respectively; SEQ ID NOs: 1593 and 1598, respectively. and 1597; SEQ ID NOs: 1601 and 1605, respectively; SEQ ID NOs: 1609 and 1613, respectively; SEQ ID NOs: 1617 and 1621, respectively; SEQ ID NOs: 1625 and 1629, respectively; SEQ ID NOs: 1633 and 1637, respectively; SEQ ID NOs: 1649 and 1653, respectively; SEQ ID NOs: 1657 and 1661, respectively; SEQ ID NOs: 1673 and 1677, respectively; SEQ ID NOs: 1681 and 1685, respectively; SEQ ID NOs: 1689 and 1693, respectively; SEQ ID NOs: 1697 and 1701, respectively; SEQ ID NOs: 1 SEQ ID NOs: 1705 and 1709; SEQ ID NOs: 1713 and 1717, respectively; SEQ ID NOs: 1721 and 1725, respectively; SEQ ID NOs: 1729 and 1733, respectively; SEQ ID NOs: 1737 and 1741, respectively; SEQ ID NOs: 1745 and 1749, respectively; SEQ ID NOs: 1753 and 1757, respectively; SEQ ID NOs: 1761 and 1765, respectively; SEQ ID NOs: 1769 and 1773, respectively; SEQ ID NOs: 1777 and 1781, respectively; SEQ ID NOs: 1785 and 1789, respectively; SEQ ID NOs: 1793 and 1797, respectively; SEQ ID NOs: 1801 and 1805; SEQ ID NOs: 1809 and 1813, respectively; SEQ ID NOs: 1817 and 1821, respectively; SEQ ID NOs: 1833 and 1837, respectively; SEQ ID NOs: 1841 and 1845, respectively; SEQ ID NOs: 1849 and 1853, respectively; SEQ ID NOs: 1857 and 1861, respectively; SEQ ID NOs: 1865 and 1869, respectively; SEQ ID NOs: 1873 and 1877, respectively; SEQ ID NOs: 1881 and 1885, respectively; SEQ ID NOs: 1889 and 1893, respectively; SEQ ID NOs: 1913 and 1917, respectively;SEQ ID NOs: 1937 and 1941, respectively; SEQ ID NOs: 1945 and 1949, respectively; SEQ ID NOs: 1953 and 1957, respectively; SEQ ID NOs: 1961 and 1965, respectively; SEQ ID NOs: 1969 and 1973, respectively; SEQ ID NOs: 1977 and 1981, respectively; SEQ ID NOs: 1985 and 1989, respectively; SEQ ID NOs: 1993 and 1997, respectively; SEQ ID NOs: 2001 and 2005, respectively; SEQ ID NOs: 2009 and 2013, respectively; SEQ ID NOs: 2017 and 2021, respectively; SEQ ID NOs: 2025 and 2026, respectively. and 2029; SEQ ID NOs: 2033 and 2037, respectively; SEQ ID NOs: 2041 and 2045, respectively; SEQ ID NOs: 2049 and 2053, respectively; SEQ ID NOs: 2057 and 2061, respectively; SEQ ID NOs: 2065 and 2069, respectively; SEQ ID NOs: 2073 and 2077, respectively; SEQ ID NOs: 2081 and 2085, respectively; SEQ ID NOs: 2089 and 2093, respectively; SEQ ID NOs: 2097 and 2101, respectively; SEQ ID NOs: 2105 and 2109, respectively; SEQ ID NOs: 2113 and 2117, respectively; SEQ ID NOs: 2114 and 2118, respectively. SEQ ID NOs: 2161 and 2165, respectively; SEQ ID NOs: 2169 and 2173, respectively; SEQ ID NOs: 2177 and 2181, respectively; SEQ ID NOs: 2185 and 2189, respectively; SEQ ID NOs: 2193 and 2197, respectively; SEQ ID NOs: 2201 and 2205, respectively; SEQ ID NOs: 2209 and 2213, respectively; SEQ ID NOs: 2217 and 2221; SEQ ID NOs: 2225 and 2229, respectively; SEQ ID NOs: 2233 and 2237, respectively; SEQ ID NOs: 2241 and 2245, respectively; SEQ ID NOs: 2249 and 2253, respectively; SEQ ID NOs: 2257 and 2261, respectively; SEQ ID NOs: 2273 and 2277, respectively; SEQ ID NOs: 2281 and 2285, respectively; SEQ ID NOs: 2305 and 2309, respectively; SEQ ID NOs: 2313 and 2317, respectively; SEQ ID NOs: 2321 and 2325, respectively; SEQ ID NOs: 2329 and 2333, respectively;V selected from the group consisting of SEQ ID NOs: 2337 and 2341, respectively; and SEQ ID NOs: 2345 and 2349, respectively; L Amino acid sequence and V H Contains the amino acid sequence.

[0092] Additionally or alternatively, for the heterodimeric multispecific antibodies disclosed herein, in some embodiments, each of the VL-1 and VH-1 is selected from the group consisting of SEQ ID NOs: 9 and 13, respectively; SEQ ID NOs: 49 and 53, respectively; SEQ ID NOs: 57 and 61, respectively; SEQ ID NOs: 65 and 69, respectively; SEQ ID NOs: 81 and 85, respectively; SEQ ID NOs: 153 and 157, respectively; SEQ ID NOs: 161 and 165, respectively; SEQ ID NOs: 193 and 197, respectively; SEQ ID NOs: 201 and 205, respectively; SEQ ID NOs: 273 and 277, respectively; SEQ ID NOs: 281 and 285, respectively; SEQ ID NOs: 289 and 293, respectively; SEQ ID NOs: 297 and 301, respectively; SEQ ID NOs: 305 and 309, respectively; SEQ ID NOs: 313 and 317, respectively; SEQ ID NOs: 361 and 365, respectively; SEQ ID NOs: 377 and 381, respectively; SEQ ID NOs: 393 and 397, respectively; SEQ ID NOs: 401 and 405, respectively; SEQ ID NOs: 409 and 413, respectively; SEQ ID NOs: 417 and 421, respectively; SEQ ID NOs: 425 and 429, respectively; SEQ ID NOs: 433 and 437, respectively. , SEQ ID NOs: 441 and 445, respectively; SEQ ID NOs: 449 and 453, respectively; SEQ ID NOs: 457 and 461, respectively; SEQ ID NOs: 465 and 469, respectively; SEQ ID NOs: 681 and 685, respectively; SEQ ID NOs: 689 and 693, respectively; SEQ ID NOs: 697 and 701, respectively; SEQ ID NOs: 705 and 709, respectively; SEQ ID NOs: 713 and 717, respectively; SEQ ID NOs: 721 and 725, respectively; SEQ ID NOs: 737 and 741, respectively; SEQ ID NOs: 745 and 749, respectively; SEQ ID NOs: 753 and 757, respectively; Sequence numbers 761 and 765; SEQ ID NOs: 777 and 781, respectively; SEQ ID NOs: 825 and 829, respectively; SEQ ID NOs: 833 and 837, respectively; SEQ ID NOs: 841 and 845, respectively; SEQ ID NOs: 953 and 957, respectively; SEQ ID NOs: 961 and 965, respectively; SEQ ID NOs: 977 and 981, respectively; SEQ ID NOs: 993 and 997, respectively; SEQ ID NOs: 1001 and 1005, respectively; SEQ ID NOs: 1009 and 1013, respectively; SEQ ID NOs: 1017 and 1021, respectively; SEQ ID NOs: 1033 and 1037, respectively;SEQ ID NOs: 1049 and 1053, respectively; SEQ ID NOs: 1073 and 1077, respectively; SEQ ID NOs: 1081 and 1085, respectively; SEQ ID NOs: 1089 and 1093, respectively; SEQ ID NOs: 1105 and 1109, respectively; SEQ ID NOs: 1129 and 1133, respectively; SEQ ID NOs: 1137 and 1141, respectively; SEQ ID NOs: 1153 and 1157, respectively; SEQ ID NOs: 1161 and 1165, respectively; SEQ ID NOs: 1177 and 1181, respectively; SEQ ID NOs: 1225 and 1229, respectively; SEQ ID NOs: 1241 and 1242, respectively. and 1245; SEQ ID NOs: 1257 and 1261, respectively; SEQ ID NOs: 1265 and 1269, respectively; SEQ ID NOs: 1297 and 1301, respectively; SEQ ID NOs: 1393 and 1397, respectively; SEQ ID NOs: 1409 and 1413, respectively; SEQ ID NOs: 1425 and 1429, respectively; SEQ ID NOs: 1441 and 1445, respectively; SEQ ID NOs: 1449 and 1453, respectively; SEQ ID NOs: 1457 and 1461, respectively; SEQ ID NOs: 1465 and 1469, respectively; SEQ ID NOs: 1473 and 1477, respectively; SEQ ID NOs: 1 SEQ ID NOs: 1545 and 1549, respectively; SEQ ID NOs: 1553 and 1557, respectively; SEQ ID NOs: 1561 and 1565, respectively; SEQ ID NOs: 1569 and 1573, respectively; SEQ ID NOs: 1577 and 1581, respectively; SEQ ID NOs: 1585 and 1589, respectively; SEQ ID NOs: 1609 and 1613; SEQ ID NOs: 1617 and 1621, respectively; SEQ ID NOs: 1649 and 1653, respectively; SEQ ID NOs: 1657 and 1661, respectively; SEQ ID NOs: 1673 and 1677, respectively; SEQ ID NOs: 1689 and 1693, respectively; SEQ ID NOs: 1697 and 1701, respectively; SEQ ID NOs: 1705 and 1709, respectively; SEQ ID NOs: 1713 and 1717, respectively; SEQ ID NOs: 1721 and 1725, respectively; SEQ ID NOs: 1729 and 1733, respectively; SEQ ID NOs: 1745 and 1749, respectively;SEQ ID NOs: 1753 and 1757, respectively; SEQ ID NOs: 1761 and 1765, respectively; SEQ ID NOs: 1769 and 1773, respectively; SEQ ID NOs: 1777 and 1781, respectively; SEQ ID NOs: 1785 and 1789, respectively; SEQ ID NOs: 1793 and 1797, respectively; SEQ ID NOs: 1817 and 1821, respectively; SEQ ID NOs: 1833 and 1837, respectively; SEQ ID NOs: 1841 and 1845, respectively; SEQ ID NOs: 1849 and 1853, respectively; SEQ ID NOs: 1857 and 1861, respectively; SEQ ID NOs: 1865 and 1869, respectively. V selected from the group consisting of SEQ ID NOs: 1889 and 1893, respectively; SEQ ID NOs: 2257 and 2261, respectively; SEQ ID NOs: 2265 and 2269, respectively; SEQ ID NOs: 2281 and 2285, respectively; SEQ ID NOs: 2297 and 2301, respectively; SEQ ID NOs: 2305 and 2309, respectively; SEQ ID NOs: 2313 and 2317, respectively; SEQ ID NOs: 2321 and 2325, respectively; SEQ ID NOs: 2329 and 2333, respectively; SEQ ID NOs: 2337 and 2341, respectively; and SEQ ID NOs: 2345 and 2349, respectively; L Amino acid sequence and V H Contains the amino acid sequence.

[0093] Additionally or alternatively, for the heterodimeric multispecific antibodies disclosed herein, in some embodiments, each of the VL-3 and VH-3 is selected from the group consisting of SEQ ID NOs: 9 and 13, respectively; SEQ ID NOs: 49 and 53, respectively; SEQ ID NOs: 57 and 61, respectively; SEQ ID NOs: 65 and 69, respectively; SEQ ID NOs: 81 and 85, respectively; SEQ ID NOs: 153 and 157, respectively; SEQ ID NOs: 161 and 165, respectively; SEQ ID NOs: 193 and 197, respectively; SEQ ID NOs: 201 and 205, respectively; SEQ ID NOs: 273 and 277, respectively; SEQ ID NOs: 281 and 285, respectively; SEQ ID NOs: 289 and 293, respectively; SEQ ID NOs: 297 and 301, respectively; SEQ ID NOs: 305 and 309, respectively; SEQ ID NOs: 313 and 317, respectively; SEQ ID NOs: 361 and 365, respectively; SEQ ID NOs: 377 and 381, respectively; SEQ ID NOs: 393 and 397, respectively; SEQ ID NOs: 401 and 405, respectively; SEQ ID NOs: 409 and 413, respectively; SEQ ID NOs: 417 and 421, respectively; SEQ ID NOs: 425 and 429, respectively; SEQ ID NOs: 433 and 437, respectively. , SEQ ID NOs: 441 and 445, respectively; SEQ ID NOs: 449 and 453, respectively; SEQ ID NOs: 457 and 461, respectively; SEQ ID NOs: 465 and 469, respectively; SEQ ID NOs: 681 and 685, respectively; SEQ ID NOs: 689 and 693, respectively; SEQ ID NOs: 697 and 701, respectively; SEQ ID NOs: 705 and 709, respectively; SEQ ID NOs: 713 and 717, respectively; SEQ ID NOs: 721 and 725, respectively; SEQ ID NOs: 737 and 741, respectively; SEQ ID NOs: 745 and 749, respectively; SEQ ID NOs: 753 and 757, respectively; Sequence numbers 761 and 765; SEQ ID NOs: 777 and 781, respectively; SEQ ID NOs: 825 and 829, respectively; SEQ ID NOs: 833 and 837, respectively; SEQ ID NOs: 841 and 845, respectively; SEQ ID NOs: 953 and 957, respectively; SEQ ID NOs: 961 and 965, respectively; SEQ ID NOs: 977 and 981, respectively; SEQ ID NOs: 993 and 997, respectively; SEQ ID NOs: 1001 and 1005, respectively; SEQ ID NOs: 1009 and 1013, respectively; SEQ ID NOs: 1017 and 1021, respectively; SEQ ID NOs: 1033 and 1037, respectively;SEQ ID NOs: 1049 and 1053, respectively; SEQ ID NOs: 1073 and 1077, respectively; SEQ ID NOs: 1081 and 1085, respectively; SEQ ID NOs: 1089 and 1093, respectively; SEQ ID NOs: 1105 and 1109, respectively; SEQ ID NOs: 1129 and 1133, respectively; SEQ ID NOs: 1137 and 1141, respectively; SEQ ID NOs: 1153 and 1157, respectively; SEQ ID NOs: 1161 and 1165, respectively; SEQ ID NOs: 1177 and 1181, respectively; SEQ ID NOs: 1225 and 1229, respectively; SEQ ID NOs: 1241 and 1242, respectively. and 1245; SEQ ID NOs: 1257 and 1261, respectively; SEQ ID NOs: 1265 and 1269, respectively; SEQ ID NOs: 1297 and 1301, respectively; SEQ ID NOs: 1393 and 1397, respectively; SEQ ID NOs: 1409 and 1413, respectively; SEQ ID NOs: 1425 and 1429, respectively; SEQ ID NOs: 1441 and 1445, respectively; SEQ ID NOs: 1449 and 1453, respectively; SEQ ID NOs: 1457 and 1461, respectively; SEQ ID NOs: 1465 and 1469, respectively; SEQ ID NOs: 1473 and 1477, respectively; SEQ ID NOs: 1 SEQ ID NOs: 1545 and 1549, respectively; SEQ ID NOs: 1553 and 1557, respectively; SEQ ID NOs: 1561 and 1565, respectively; SEQ ID NOs: 1569 and 1573, respectively; SEQ ID NOs: 1577 and 1581, respectively; SEQ ID NOs: 1585 and 1589, respectively; SEQ ID NOs: 1609 and 1613; SEQ ID NOs: 1617 and 1621, respectively; SEQ ID NOs: 1649 and 1653, respectively; SEQ ID NOs: 1657 and 1661, respectively; SEQ ID NOs: 1673 and 1677, respectively; SEQ ID NOs: 1689 and 1693, respectively; SEQ ID NOs: 1697 and 1701, respectively; SEQ ID NOs: 1705 and 1709, respectively; SEQ ID NOs: 1713 and 1717, respectively; SEQ ID NOs: 1721 and 1725, respectively; SEQ ID NOs: 1729 and 1733, respectively; SEQ ID NOs: 1745 and 1749, respectively;SEQ ID NOs: 1753 and 1757, respectively; SEQ ID NOs: 1761 and 1765, respectively; SEQ ID NOs: 1769 and 1773, respectively; SEQ ID NOs: 1777 and 1781, respectively; SEQ ID NOs: 1785 and 1789, respectively; SEQ ID NOs: 1793 and 1797, respectively; SEQ ID NOs: 1817 and 1821, respectively; SEQ ID NOs: 1833 and 1837, respectively; SEQ ID NOs: 1841 and 1845, respectively; SEQ ID NOs: 1849 and 1853, respectively; SEQ ID NOs: 1857 and 1861, respectively; SEQ ID NOs: 1865 and 1869, respectively. V selected from the group consisting of SEQ ID NOs: 1889 and 1893, respectively; SEQ ID NOs: 2257 and 2261, respectively; SEQ ID NOs: 2265 and 2269, respectively; SEQ ID NOs: 2281 and 2285, respectively; SEQ ID NOs: 2297 and 2301, respectively; SEQ ID NOs: 2305 and 2309, respectively; SEQ ID NOs: 2313 and 2317, respectively; SEQ ID NOs: 2321 and 2325, respectively; SEQ ID NOs: 2329 and 2333, respectively; SEQ ID NOs: 2337 and 2341, respectively; and SEQ ID NOs: 2345 and 2349, respectively; L Amino acid sequence and V H Contains the amino acid sequence.

[0094] Additionally or alternatively, for the heterodimeric multispecific antibodies disclosed herein, in some embodiments, each of the VL-2 and VH-2 is SEQ ID NOs: 17 and 21, respectively; SEQ ID NOs: 25 and 29, respectively; SEQ ID NOs: 33 and 37, respectively; SEQ ID NOs: 41 and 45, respectively; SEQ ID NOs: 121 and 125, respectively; SEQ ID NOs: 137 and 141, respectively; SEQ ID NOs: 169 and 173, respectively; SEQ ID NOs: 177 and 181, respectively; SEQ ID NOs: 185 and 189, respectively; SEQ ID NOs: 193 and 194, respectively. 7; SEQ ID NOs:201 and 205, respectively; SEQ ID NOs:209 and 213, respectively; SEQ ID NOs:217 and 221, respectively; SEQ ID NOs:225 and 229, respectively; SEQ ID NOs:233 and 237, respectively; SEQ ID NOs:241 and 245, respectively; SEQ ID NOs:249 and 253, respectively; SEQ ID NOs:257 and 261, respectively; SEQ ID NOs:265 and 269, respectively; SEQ ID NOs:321 and 325, respectively; SEQ ID NOs:329 and 333, respectively; SEQ ID NOs:337 and 341, respectively; SEQ ID NOs:393 and 397, respectively SEQ ID NOs: 401 and 405, respectively; SEQ ID NOs: 409 and 413, respectively; SEQ ID NOs: 473 and 477, respectively; SEQ ID NOs: 481 and 485, respectively; SEQ ID NOs: 489 and 493, respectively; SEQ ID NOs: 497 and 501, respectively; SEQ ID NOs: 505 and 509, respectively; SEQ ID NOs: 513 and 517, respectively; SEQ ID NOs: 545 and 549, respectively; SEQ ID NOs: 553 and 557, respectively; SEQ ID NOs: 561 and 565, respectively; SEQ ID NOs: 569 and 573, respectively; SEQ ID NOs: 577 and 581, respectively SEQ ID NOs: 585 and 589, respectively; SEQ ID NOs: 593 and 597, respectively; SEQ ID NOs: 601 and 605, respectively; SEQ ID NOs: 625 and 629, respectively; SEQ ID NOs: 633 and 637, respectively; SEQ ID NOs: 641 and 645, respectively; SEQ ID NOs: 649 and 653, respectively; SEQ ID NOs: 657 and 661, respectively; SEQ ID NOs: 665 and 669, respectively; SEQ ID NOs: 673 and 677, respectively; SEQ ID NOs: 681 and 685, respectively; SEQ ID NOs: 689 and 693, respectively; SEQ ID NOs: 697 and 701, respectively;SEQ ID NOs: 705 and 709, respectively; SEQ ID NOs: 713 and 717, respectively; SEQ ID NOs: 721 and 725, respectively; SEQ ID NOs: 729 and 733, respectively; SEQ ID NOs: 737 and 741, respectively; SEQ ID NOs: 745 and 749, respectively; SEQ ID NOs: 753 and 757, respectively; SEQ ID NOs: 761 and 765, respectively; SEQ ID NOs: 769 and 773, respectively; SEQ ID NOs: 785 and 789, respectively; SEQ ID NOs: 793 and 797, respectively; SEQ ID NOs: 801 and 805, respectively; SEQ ID NOs: 809 and 809, respectively. and 813; SEQ ID NOs: 817 and 821, respectively; SEQ ID NOs: 849 and 853, respectively; SEQ ID NOs: 857 and 861, respectively; SEQ ID NOs: 865 and 869, respectively; SEQ ID NOs: 873 and 877, respectively; SEQ ID NOs: 881 and 885, respectively; SEQ ID NOs: 889 and 893, respectively; SEQ ID NOs: 897 and 901, respectively; SEQ ID NOs: 905 and 909, respectively; SEQ ID NOs: 913 and 917, respectively; SEQ ID NOs: 921 and 925, respectively; SEQ ID NOs: 929 and 933, respectively; SEQ ID NOs: SEQ ID NOs: 937 and 941, respectively; SEQ ID NOs: 945 and 949, respectively; SEQ ID NOs: 969 and 973, respectively; SEQ ID NOs: 977 and 981, respectively; SEQ ID NOs: 1009 and 1013, respectively; SEQ ID NOs: 1057 and 1061, respectively; SEQ ID NOs: 1537 and 1541, respectively; SEQ ID NOs: 1569 and 1573, respectively; SEQ ID NOs: 1601 and 1605, respectively; SEQ ID NOs: 1641 and 1645, respectively; SEQ ID NOs: 1665 and 1669, respectively; SEQ ID NOs: 1825 and 1829, respectively; SEQ ID NOs: 1865 and 1869; SEQ ID NOs: 1897 and 1901, respectively; SEQ ID NOs: 1905 and 1909, respectively; SEQ ID NOs: 1913 and 1917, respectively; SEQ ID NOs: 1921 and 1925, respectively; SEQ ID NOs: 1929 and 1933, respectively; SEQ ID NOs: 2265 and 2269, respectively; SEQ ID NOs: 2281 and 2285, respectively; SEQ ID NOs: 2289 and 2293, respectively; SEQ ID NOs: 2329 and 2333, respectively; and SEQ ID NOs: 2345 and 2349, respectively; L Amino acid sequence and VH Contains the amino acid sequence.

[0095] Additionally or alternatively, for the heterodimeric multispecific antibodies disclosed herein, in some embodiments, each of the VL-4 and VH-4 is SEQ ID NOs: 17 and 21, respectively; SEQ ID NOs: 25 and 29, respectively; SEQ ID NOs: 33 and 37, respectively; SEQ ID NOs: 41 and 45, respectively; SEQ ID NOs: 121 and 125, respectively; SEQ ID NOs: 137 and 141, respectively; SEQ ID NOs: 169 and 173, respectively; SEQ ID NOs: 177 and 181, respectively; SEQ ID NOs: 185 and 189, respectively; SEQ ID NOs: 193 and 194, respectively. 7; SEQ ID NOs:201 and 205, respectively; SEQ ID NOs:209 and 213, respectively; SEQ ID NOs:217 and 221, respectively; SEQ ID NOs:225 and 229, respectively; SEQ ID NOs:233 and 237, respectively; SEQ ID NOs:241 and 245, respectively; SEQ ID NOs:249 and 253, respectively; SEQ ID NOs:257 and 261, respectively; SEQ ID NOs:265 and 269, respectively; SEQ ID NOs:321 and 325, respectively; SEQ ID NOs:329 and 333, respectively; SEQ ID NOs:337 and 341, respectively; SEQ ID NOs:393 and 397, respectively SEQ ID NOs: 401 and 405, respectively; SEQ ID NOs: 409 and 413, respectively; SEQ ID NOs: 473 and 477, respectively; SEQ ID NOs: 481 and 485, respectively; SEQ ID NOs: 489 and 493, respectively; SEQ ID NOs: 497 and 501, respectively; SEQ ID NOs: 505 and 509, respectively; SEQ ID NOs: 513 and 517, respectively; SEQ ID NOs: 545 and 549, respectively; SEQ ID NOs: 553 and 557, respectively; SEQ ID NOs: 561 and 565, respectively; SEQ ID NOs: 569 and 573, respectively; SEQ ID NOs: 577 and 581, respectively SEQ ID NOs: 585 and 589, respectively; SEQ ID NOs: 593 and 597, respectively; SEQ ID NOs: 601 and 605, respectively; SEQ ID NOs: 625 and 629, respectively; SEQ ID NOs: 633 and 637, respectively; SEQ ID NOs: 641 and 645, respectively; SEQ ID NOs: 649 and 653, respectively; SEQ ID NOs: 657 and 661, respectively; SEQ ID NOs: 665 and 669, respectively; SEQ ID NOs: 673 and 677, respectively; SEQ ID NOs: 681 and 685, respectively; SEQ ID NOs: 689 and 693, respectively; SEQ ID NOs: 697 and 701, respectively;SEQ ID NOs: 705 and 709, respectively; SEQ ID NOs: 713 and 717, respectively; SEQ ID NOs: 721 and 725, respectively; SEQ ID NOs: 729 and 733, respectively; SEQ ID NOs: 737 and 741, respectively; SEQ ID NOs: 745 and 749, respectively; SEQ ID NOs: 753 and 757, respectively; SEQ ID NOs: 761 and 765, respectively; SEQ ID NOs: 769 and 773, respectively; SEQ ID NOs: 785 and 789, respectively; SEQ ID NOs: 793 and 797, respectively; SEQ ID NOs: 801 and 805, respectively; SEQ ID NOs: 809 and 809, respectively. and 813; SEQ ID NOs: 817 and 821, respectively; SEQ ID NOs: 849 and 853, respectively; SEQ ID NOs: 857 and 861, respectively; SEQ ID NOs: 865 and 869, respectively; SEQ ID NOs: 873 and 877, respectively; SEQ ID NOs: 881 and 885, respectively; SEQ ID NOs: 889 and 893, respectively; SEQ ID NOs: 897 and 901, respectively; SEQ ID NOs: 905 and 909, respectively; SEQ ID NOs: 913 and 917, respectively; SEQ ID NOs: 921 and 925, respectively; SEQ ID NOs: 929 and 933, respectively; SEQ ID NOs: SEQ ID NOs: 937 and 941, respectively; SEQ ID NOs: 945 and 949, respectively; SEQ ID NOs: 969 and 973, respectively; SEQ ID NOs: 977 and 981, respectively; SEQ ID NOs: 1009 and 1013, respectively; SEQ ID NOs: 1057 and 1061, respectively; SEQ ID NOs: 1537 and 1541, respectively; SEQ ID NOs: 1569 and 1573, respectively; SEQ ID NOs: 1601 and 1605, respectively; SEQ ID NOs: 1641 and 1645, respectively; SEQ ID NOs: 1665 and 1669, respectively; SEQ ID NOs: 1825 and 1829, respectively; SEQ ID NOs: 1865 and 1869; SEQ ID NOs: 1897 and 1901, respectively; SEQ ID NOs: 1905 and 1909, respectively; SEQ ID NOs: 1913 and 1917, respectively; SEQ ID NOs: 1921 and 1925, respectively; SEQ ID NOs: 1929 and 1933, respectively; SEQ ID NOs: 2265 and 2269, respectively; SEQ ID NOs: 2281 and 2285, respectively; SEQ ID NOs: 2289 and 2293, respectively; SEQ ID NOs: 2329 and 2333, respectively; and SEQ ID NOs: 2345 and 2349, respectively; L Amino acid sequence and VH Contains the amino acid sequence.

[0096] Additionally or alternatively, in some embodiments of the heterodimeric multispecific antibodies disclosed herein, the first immunoglobulin or the third immunoglobulin is selected from the group consisting of a2b b3 (glycoprotein IIb / IIIa), a4, a4b7, a4b7+aEb7, a5, activin receptor type 2B, ALK1, alpha-synuclein, amyloid beta, APP, AXL, blood group A, CAIX, CCL-2, CD105 (endoglin), CD115 (CSF1R), CD116a (CSF2Ra), CD123, CD152 (CTLA4), CD184 (CXCR4), CD19, CD192 (CCR2), CD194 (CCR4), CD195 (CCR5), CD20, CD200, CD22 , CD221(IGF1R), CD248, CD25, CD257(BAFF), CD26, CD262(DR5), CD276(B7H3), CD3, CD30(TNFRSF8), CD319(SLAMF7), CD33, CD332(FGFR2), C D350(FZD10), CD37, CD371(CLEC12A), CD38, CD4, CD49b(a2), CD51(a5), CD52, CD56, CD61(a4b3), CD70, CD73(NT5E), CD74, CEA, claudin-18.2, cMET, CRLR, DLL3, DLL4, DNA / histone (H1) complex, EGFR, EpCAM, EGFR-HER3, EGFRvIII, EphA3, ERGT(GalNAc)Tn antigen, FLT1, FOLR1, frizzled family receptor (FZD), Lewis Y, Lewis X, GCGR, GD2, GD2 α-acetyl, GD3, GM1, GM1 fucosyl, GM2, GPA33, GPNMB, GUCY2C, HER2, HER3, HGFR (cMET), IgHe, IGLF2, kallikrein, LINGO1, LOXL2, Ly6 / PLAUR domain-containing protein 3, MADCAM1, MAG, mesothelin, MT1-MMP (MMP14), MUC1, mucin 5AC, NaPi2b, NeuGc-GM3, notch, NOTCH2 / NOTCH3 receptor, oxLDL, P-selectin, PCSK9, PDGFRA, PDGFRa, phosphatidylserine, polysialic acid, PSMA, PVRL4, RGMA, and CD240D, a blood antigen of type D The first immunoglobulin binds to a cell surface antigen selected from the group consisting of root plate-specific spondin 3, serum amyloid P component, STEAP-1, TACSTD2, TGFb, TWEAKR, TYRP1, VEGFR2, VSIR, CD171 (L1CAM), CD19, CD47, pMHC[NY-ESO1], pMHC[MART1], pMHC[MAGEA1], pMHC[tyrosinase], pMHC[gp100], pMHC[MUC1], pMHC[tax], pMHC[WT-1], pMHC[EBNA-1], pMHC[LMP2], pMHC[hTERT], GPC3, CD80, CD23, and fibronectin extracellular domain B. The first immunoglobulin and the third immunoglobulin may bind to the same epitope on the target cell, or may bind to two different epitopes on the target cell. In some embodiments, the target cells are cancer cells.

[0097] Additionally or alternatively, in some embodiments of the heterodimeric multispecific antibodies disclosed herein, the second immunoglobulin or fourth immunoglobulin binds to an epitope on a leukocyte, on a monocyte, on a lymphocyte, on a granulocyte, on a macrophage, on a T cell, on an NK cell, on a B cell, on an NKT cell, on an ILC, or on a neutrophil.

[0098] In any of the above embodiments of the heterodimeric multispecific antibody disclosed herein, the second immunoglobulin or the fourth immunoglobulin is selected from the group consisting of dabigatran, a4, a4b7, a4b7+aEb7, a5, AXL, BnDOTA, CD11a (LFA-1), CD3, CD4, CD8, CD16, CD19, CD22, CD23, CD25, CD28, CD30 (TNFRSF8), CD33, CD38, CD40, CD40L, CD47, CD49b(a2), CD54 (ICAM-1), CD56, CD74, CD80, CD115 (CSF1R), CD116a (CSF2Ra), CD123, CD134 (OX40), CD137 (41BB), CD1 52 (CTLA4), CD184 (CXCR4), CD192 (CCR2), CD194 (CCR4), CD195 (CCR5), CD223 (LAG-3), CD252 (OX40L), CD254 (RANKL), CD262 (DR5), CD27, CD200, CD221 (IGF1R), CD248, CD274 (PD-L1), CD275 (ICOS-L), CD278 (ICOS), CD279 (PD-1), CD319 (SLAMF7), CD371 (CLEC12A), MADCAM1, MT1-MMP (MMP14), NKG2A, NRP1, TIGIT, VSIR, KIRDL1 / 2 / 3, and KIR2DL2. The second immunoglobulin and the fourth immunoglobulin may bind to the same or different epitopes on leukocytes, monocytes, lymphocytes, granulocytes, macrophages, T cells, NK cells, B cells, NKT cells, ILCs, or neutrophils. In some embodiments, the second immunoglobulin binds to CD3 and the fourth immunoglobulin binds to an immune cell receptor selected from the group consisting of CD4, CD8, CD25, CD28, CTLA4, OX40, ICOS, PD-1, PD-L1, 41BB, CD2, CD69, and CD45. In other embodiments, the second immunoglobulin binds to CD16 and the fourth immunoglobulin binds to an immune cell receptor selected from the group consisting of CD56, NKG2D, and KIRDL1 / 2 / 3.In certain embodiments, the fourth immunoglobulin binds to an agent selected from the group consisting of a cytokine, a nucleic acid, a hapten, a small molecule, a radionuclide, an antitoxin, a vitamin, a peptide, a lipid, a carbohydrate, biotin, digoxin, or a conjugated variant of any of these.

[0099] Additionally or alternatively, in some embodiments of the heterodimeric multispecific antibodies disclosed herein, the first and third immunoglobulins bind to their respective epitopes with a monovalent or effective affinity of between about 100 nM and about 100 pM, hi certain embodiments, the first and third immunoglobulins bind to cell surface epitopes that are 60 to 120 angstroms apart.

[0100] Additionally or alternatively, in some embodiments of the heterodimeric multispecific antibodies disclosed herein, the first and third immunoglobulins bind to their respective epitopes with a monovalent or effective affinity of less than 100 pM, hi certain embodiments, the first and third immunoglobulins bind to cell surface epitopes that are up to 180 angstroms apart. Additionally or alternatively, in some embodiments of the heterodimeric multispecific antibodies disclosed herein, the first heterodimerization domain of the first immunoglobulin and / or the second heterodimerization domain of the third immunoglobulin is a CH2-CH3 domain and has an isotype selected from the group consisting of IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgM, IgD, and IgE. Non-limiting examples of constant region sequences include:

[0101] Constant region of human IgD; Uniprot: P01880 (SEQ ID NO: 2381) APTKAPDVFPIISGCRHPKDNSPVVLACLITGYHPTSVTVTWYMGTQSQPQRTFPEIQRRDSYYMTSSQLSTPLQQWRQGEYKCVVQHTASKSKKEIFRWPESPKAQASSVPTAQPQAEGSLAKATTAPATTRNTGRGGEEKKKEKEKEEQEERETKTPECPSHTQPLGVYLLTPAVQDLWLRDKATFTCFV VGSDLKDAHLTWEVAGKVPTGGVEEGLLERHSNGSQSQHSRLTLPRSLWNAGTSVTCTLNHPSLPPQRLMALREPAAQAPVKLSLNLLASSDPPEAASWLLCEVSGFSPPNILLMWLEDQREVNTSGFAPARPPPQPGSTTFWAWSVLRVPAPPSPQPATYTCVVSHEDSRTLLNASRSLEVSYVTDHGPMK Constant region of human IgG1; Uniprot: P01857 (SEQ ID NO: 2382) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0102] Constant region of human IgG2; Uniprot: P01859 (SEQ ID NO: 2383) ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKDTLMISRTPEVTCVVDVSHEDPEVQFNWYVDGVEV HNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDISVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Constant region of human IgG3; Uniprot: P01860 (SEQ ID NO: 2384) ASTKGPSVFPLAPCSRSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYTCNVNHKPSNTKVDKRVELKTPLGDTTHTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPAPELLGGPSVFLFPPKPKDTLMISRTPE VTCVVVDVSHEDPEVQFKWYVDGVEVHNAKTKPREEQYNSTFRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKTKGQPREPQVYTLP PSREEMTKNQVSLTCLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHEALHNRFTQKSLSLSPGK

[0103] Human IgM constant region; Uniprot: P01871 (SEQ ID NO: 2385) GSASAPTLFPLVSCENSPSDTSSVAVGCLAQDFLPDSITLSWKYKNNSDISSTRGFPSVLRGGKYAATSQVLLPSKDVMQGTDEHVVCKVQHPNGNKEKNVPLPVIAELPPKV SVFVPPRDGFFGNPRKSKLICQATGFSPRQIQVSWLREGKQVGSGVTTDQVQAEAKESGPTTYKVTSTLTIKESDWLGQSMFTCRVDHRGLTFQQNASSMCVPDQDTAIRVFA IPPSFASIFLTKSTKLTCLVTDLTTYDSVTISWTRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNSGERFTCTVTHTDLPSPLKQTISRPKGVALHRPDVYLLPPARE QLNLRESATITCLVTGFSPADVFVQWMQRGQPLSPEKYVTSAPMPEPQAPGRYFAHSILTVSEEEWNTGETYTCVAHEALPNRVTERTVDKSTGKPTLYNVSLVMSDTAGTCY

[0104] Constant region of human IgG4; Uniprot: P01861 (SEQ ID NO: 2386) ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVE VHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK Constant region of human IgA1; Uniprot: P01876 (SEQ ID NO: 2387) ASPTSPKVFPLSLCSTQPDGNVVIACLVQGFFPQEPLSVTWSESGQGVTARNFPPSQDASGDLYTTSSQLTLPATQCLAGKSVTCHVKHYTNPSQDVTVPCPVPSTPPTPSPSTPPTPSPSCCHPRLSLHRPALEDLLLGSEANLTCTLTGLRDASGVTFTWTPSSGKSAVQGPPE RDLCGCYSVSSVLPGCAEPWNHGKTFTCTAAYPESKTPLTATLSKSGNTFRPEVHLLPPPSEELALNELVTLTCLARGFSPKDVLVRWLQGSQELPREKYLTWASRQEPSQGTTTFAVTSILRVAAEDWKKGDTFSCMVGHEALPLAFTQKTIDRLAGKPTHVNVSVVMAEVDGTCY

[0105] Constant region of human IgA2; Uniprot: P01877 (SEQ ID NO: 2388) ASPTSPKVFPLSLDSTPQDGNVVVACLVQGFFPQEPLSVTWSESGQNVTARNFPPSQDASGDLYTTSSQLTLPATQCPDGKSVTCHVKHYTNPSQDVTVPCPVPPPPPCCHPRLSLHRPALEDLLLGSEANLTCTLTGLRDASGATFTWTPSSGKSAVQGPPERDLCGCY SVSSVLPGCAQPWNHGETFTCTAAHPELKTPLTANITKSGNTFRPEVHLLPPPSEELALNELVTLTCLARGFSPKDVLVRWLQGSQELPREKYLTWASRQEPSQGTTTFAVTSILRVAAEDWKKGDTFSCMVGHEALPLAFTQKTIDRMAGKPTHVNVSVVMAEVDGTCY Human Ig kappa constant region; Uniprot: P01834 (SEQ ID NO: 2389) TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0106] In some embodiments, the immunoglobulin analog constructs of the present technology comprise a heavy chain constant region that is at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NOs: 2381-2388. Additionally, or alternatively, in some embodiments, the immunoglobulin analog constructs of the present technology comprise a light chain constant region that is at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: 2389.

[0107] Additionally or alternatively, in some embodiments of the heterodimeric multispecific antibodies disclosed herein, the first heterodimerization domain of the first immunoglobulin and / or the second heterodimerization domain of the third immunoglobulin is an IgG1 constant region comprising one or more amino acid substitutions selected from the group consisting of N297A and K322A. Additionally or alternatively, in some embodiments of the heterodimeric multispecific antibodies disclosed herein, the first heterodimerization domain of the first immunoglobulin is a CH2-CH3 domain comprising a K409R mutation and the second heterodimerization domain of the third immunoglobulin is a CH2-CH3 domain comprising a F405L mutation. Also disclosed herein are recombinant nucleic acid sequences encoding any of the antibodies described herein. In another aspect, the technology provides host cells or vectors expressing any of the nucleic acid sequences encoding any of the immunoglobulin analog constructs described herein.

[0108] In some embodiments, the immunoglobulin analogue construct of the present technology is a chimeric construct, a humanized construct, or a monoclonal construct.The immunoglobulin analogue construct of the present technology can also be recombinantly fused to heterologous polypeptides at N-terminus or C-terminus, or can be chemically conjugated to polypeptides or other constructs (including covalent conjugation and non-covalent conjugation).For example, the immunoglobulin analogue construct of the present technology can be recombinantly fused to or conjugated with molecules useful as labels, and effector molecules such as heterologous polypeptides, drugs, or toxins in detection assays.For example, see WO92 / 08495; WO91 / 14438; WO89 / 12624; U.S. Patent No. 5,314,995; and EP0396387.

[0109] In any of the above-described embodiments of the immunoglobulin analog constructs of the present technology, the HDTVS antibody may be conjugated to an agent selected from the group consisting of an isotope, a dye, a chromogen, a contrast agent, a drug, a toxin, a cytokine, an enzyme, an enzyme inhibitor, a hormone, a hormone antagonist, a growth factor, a radionuclide, a metal, a liposome, a nanoparticle, RNA, DNA, or any combination thereof. For chemical or physical bonding, a functional group on the immunoglobulin analog construct typically binds to a functional group on the agent. Alternatively, a functional group on the agent binds to a functional group on the immunoglobulin analog construct. A functional group on an agent and a functional group on an immunoglobulin analog construct can be directly linked. For example, a functional group (e.g., a sulfhydryl group) on an agent can bond with a functional group (e.g., a sulfhydryl group) on an immunoglobulin analog construct to form a disulfide. Alternatively, functional groups can be linked via a cross-linking agent (i.e., a linker). Some examples of cross-linking agents are described below. The cross-linking agent can be conjugated to either the agent or the immunoglobulin analog construct. The number of agents or immunoglobulin analog constructs in a conjugate is also limited by the number of functional groups present on the other. For example, the maximum number of agents that can be associated with a conjugate depends on the number of functional groups present on the immunoglobulin analog construct. Alternatively, the maximum number of immunoglobulin analog constructs that can be conjugated to an agent depends on the number of functional groups present on the agent.

[0110] In yet another embodiment, the conjugate comprises one immunoglobulin analog construct linked to one agent. In one embodiment, the conjugate comprises at least one agent chemically linked (e.g., conjugated) to at least one immunoglobulin analog construct. The agent can be chemically linked to the immunoglobulin analog construct by any method known to those skilled in the art. For example, a functional group on the agent can be directly joined to a functional group on the immunoglobulin analog construct. Some examples of suitable functional groups include, for example, amino, carboxyl, sulfhydryl, maleimide, isocyanate, isothiocyanate, and hydroxyl. Agents can also be chemically coupled to immunoglobulin analog constructs by cross-linking agents such as dialdehydes, carbodiimides, dimaleimides, etc. Cross-linking agents can be obtained, for example, from Pierce Biotechnology, Inc., Rockford, Ill. The Pierce Biotechnology, Inc. website can be helpful. Additional cross-linking agents include platinum cross-linkers described in U.S. Patent Nos. 5,580,990; 5,985,566; and 6,133,038, all to Kreatech Biotechnology, BV, Amsterdam, Netherlands.

[0111] Alternatively, the functional groups on the agent and the immunoglobulin analog construct can be the same. Homobifunctional crosslinkers are typically used to crosslink identical functional groups. Examples of homobifunctional crosslinkers include EGS (i.e., ethylene glycol bis[succinimidyl succinate]), DSS (i.e., disuccinimidyl suberate), DMA (i.e., dimethyl adipimidate dihydrochloride), DTSSP (i.e., 3,3'-dithiobis[sulfosuccinimidyl propionate]), DPDPB (i.e., 1,4-di-[3'-(2'-pyridyldithio)-propionamido]butane), and BMH (i.e., bis-maleimidohexane). Such homobifunctional crosslinkers are also commercially available from Pierce Biotechnology, Inc.

[0112] In other cases, it may be beneficial to cleave the agent from the immunoglobulin analog construct. The Pierce Biotechnology, Inc. website mentioned above may also provide assistance to those skilled in the art in selecting appropriate cross-linkers that can be cleaved, for example, by intracellular enzymes. Thus, the agent can be separated from the immunoglobulin analog construct. Examples of cleavable linkers include SMPT (i.e., 4-succinimidyloxycarbonyl-methyl-a-[2-pyrimidyldithio]toluene), sulfo-LC-SPDP (i.e., sulfosuccinimidyl 6-(3-[2-pyrimidyldithio]-propionamido)hexanoate), LC-SPDP (i.e., succinimidyl 6-(3-[2-pyrimidyldithio]-propionamido)hexanoate), sulfo-LC-SPDP (i.e., sulfosuccinimidyl 6-(3-[2-pyrimidyldithio]-propionamido)hexanoate), SPDP (i.e., N-succinimidyl 3-[2-pyrimidyldithio]-propionamidohexanoate), and AEDP (i.e., 3-[(2-aminoethyl)dithio]propionic acid hydrochloride).

[0113] In another embodiment, the conjugate comprises at least one agent physically associated with at least one immunoglobulin analog construct. Any method known to those of skill in the art can be used to physically associate the agent with the immunoglobulin analog construct. For example, the immunoglobulin analog construct and agent can be mixed together by any method known to those of skill in the art. The order of mixing is not important. For example, the agent can be physically mixed with the immunoglobulin analog construct by any method known to those of skill in the art. For example, the immunoglobulin analog construct and agent can be placed in a container and agitated, for example, by shaking the container to mix the immunoglobulin analog construct and the agent.

[0114] The immunoglobulin analog constructs may be modified by any method known to those of skill in the art, for example, they may be modified with crosslinking agents or functional groups, as described above. Heterodimerization: This technology relies on heterodimerization of two IgG-scFv half molecules via mutations in the heterodimerization domains using techniques known in the art. Any heterodimerization method that keeps the hinge domain in place can be used, provided that sufficient antibody stability is achieved.

[0115] CH2-CH3 Domain Heterodimerization: The formation of heterodimeric trivalent / tetravalent multispecific antibody molecules of the present technology requires the interaction of four different polypeptide chains. Such interactions are difficult to achieve efficiently in a single-cell recombinant production system due to the many variations in potential chain mismatches. One solution to the increased probability of mismatching is to engineer "knob-into-hole" (KIH) mutations into the desired polypeptide chain pair. Such mutations favor heterodimerization over homodimerization. For example, with regard to Fc-Fc interactions, amino acid substitutions (preferably with amino acids containing a "knob"-forming bulky side chain group, e.g., tryptophan) can be introduced into the CH2 or CH3 domain so that steric hindrance prevents interaction with similarly mutated domains and directs the mutated domain to pair with a complementary or receptively mutated domain, i.e., a "hole" (e.g., glycine substitution). Such a set of mutations may be engineered into a pair of polypeptides (e.g., a pair of a second polypeptide chain and a third polypeptide chain) incorporated into a heterodimeric trivalent / tetravalent molecule, and may further be engineered into any portion of the polypeptide chains of the pair. Methods of protein engineering that favor heterodimerization over homodimerization, particularly with respect to the engineering of immunoglobulin-like molecules, are well known in the art and are encompassed herein (see, e.g., Ridgway et al., 1996, Protein Engr. 9:617-621; Atwell et al., 1997, J. Mol. Biol. 270: 26-35; and Xie et al., 2005, J. Immunol. Methods 296:95-101, each of which is incorporated by reference in its entirety).

[0116] The design of variant Fc heterodimers derived from wild-type homodimers is exemplified by the concepts of positive and negative design in the context of protein engineering through a balance of stability versus specificity, where mutations are introduced with the goal of driving heterodimer formation over homodimer formation when the polypeptide is expressed under cell culture conditions. Negative design strategies maximize interactions unfavorable for homodimer formation by introducing bulky side chains on one strand and small side chains on the opposite strand, e.g., by the knob-into-hole (KIH) strategy developed by Genentech (Ridgway JB, Presta LG, Carter P. Protein Eng. 1996 July; 9(7):617-21; Atwell S, Ridgway JB, Wells JA, Carter P. J Mol. Biol. 270(1):26-35 (1997)), or by electrostatic manipulation that results in repulsion for homodimer formation, e.g., the electrostatic steering strategy developed by Amgen (Gunaskekaran K, et al. JBC 285(25):19637-19646 (2010)). In two examples of these negative designs, asymmetric point mutations are introduced into the wild-type CH3 domain to drive heterodimer formation. Other heterodimerization methods are described in US20120149876 (e.g., Tables 1, 6, and 7) and US20140294836 (e.g., Figures 15A-15B, 16A-16B, and 17). Methods for engineering Fc heterodimers using electrostatic steering are described in detail in US8,592,562.

[0117] In some embodiments of the HDTVS antibodies disclosed herein, the second and third polypeptide chains comprise a first CH2-CH3 domain and a second CH2-CH3 domain, respectively, wherein the first CH2-CH3 domain and the second CH2-CH3 domain are: T366Y and Y407T, respectively; F405A and T394W, respectively; Y349C / T366S / L368A / Y407V and S354C / T366W, respectively; K409D / K392D and D399K, respectively; T366S / L368A / Y407V and T366W, respectively; K409D / K392D and D399K / E356K, respectively; L351Y / Y407A and T366A / K409F, respectively; L351Y / Y407A and T366V / K409F; Y407A and T366A / K409F, respectively; D399R / S400R / Y407A and T366A / K409F / K392E / T411E, respectively; L351Y / F405A / Y407V and T394W, respectively; L351Y / F405A / Y407V and T366L, respectively; F405 A / Y407V and T366I / K392M / T394W, respectively; F405A / Y407V and T366L / K392M / T394W, respectively; F405A / Y407V and T366L / T394W, respectively; F405A / Y407V and T366I / T394W, respectively; and K409R and F405L, respectively.

[0118] In some embodiments of the HDTVS antibodies disclosed herein, the second and third polypeptide chains comprise a first and second CH2-CH3 domain, respectively, wherein the first CH2-CH3 domain comprises an amino acid modification at position F405 and amino acid modifications L351Y and Y407V, and the second CH2-CH3 domain comprises amino acid modification T394W. In some embodiments, the amino acid modification at position F405 is F405A, F405I, F405M, F405T, F405S, F405V, or F405W.

[0119] In some embodiments of the HDTVS antibodies disclosed herein, the second and third polypeptide chains comprise a first and second CH2-CH3 domain, respectively, wherein the first CH2-CH3 domain comprises amino acid modifications at positions L351 and Y407, and the second CH2-CH3 domain comprises an amino acid modification at position T366 and an amino acid modification K409F. In some embodiments, the amino acid modification at position L351 is L351Y, L351I, L351D, L351R, or L351F. In some embodiments, the amino acid modification at position Y407 is Y407A, Y407V, or Y407S. In certain embodiments, the amino acid modification at position T366 is T366A, T366I, T366L, T366M, T366Y, T366S, T366C, T366V, or T366W.

[0120] In some embodiments of the HDTVS antibodies disclosed herein, the second and third polypeptide chains comprise a first and second CH2-CH3 domain, respectively, wherein the first or second CH2-CH3 domain comprises an amino acid modification at K392, T411, T366, L368, or S400. The amino acid modification at K392 can be K392V, K392M, K392R, K392L, K392F, or K392E. The amino acid modification at T411 can be T411N, T411R, T411Q, T411K, T411D, T411E, or T411W. The amino acid modification at S400 can be S400E, S400D, S400R, or S400K. The amino acid modification at position T366 can be T366A, T3661, T366L, T366M, T366Y, T366S, T366C, T366V, or T366W. The amino acid modification at position L368 can be L368D, L368R, L368T, L368M, L368V, L368F, L368S, and L368A.

[0121] In some embodiments of the HDTVS antibodies disclosed herein, the second and third polypeptide chains comprise a first and second CH2-CH3 domain, respectively, where the first CH2-CH3 domain comprises amino acid modifications L351Y and Y407A, the second CH2-CH3 domain comprises amino acid modifications T366A and K409F, and the first or second CH2-CH3 domain may comprise one or more amino acid modifications at positions T411, D399, S400, F405, N390, or K392. The amino acid modification at position T411 may be T411N, T411R, T411Q, T411K, T411D, T411E, or T411W. The amino acid modification at position D399 can be D399R, D399W, D399Y, or D399K. The amino acid modification at position S400 can be S400E, S400D, S400R, or S400K. The amino acid modification at position F405 can be F4051, F405M, F405T, F405S, F405V, or F405W. The amino acid modification at position N390 can be N390R, N390K, or N390D. The amino acid modification at position K392 can be K392V, K392M, K392R, K392L, K392F, or K392E.

[0122] In some embodiments of the HDTVS antibodies disclosed herein, the second and third polypeptide chains comprise a first and second CH2-CH3 domain, respectively, where the first and second CH2-CH3 domains comprise the set of amino acid modifications shown in Figure 11a. In some embodiments of the HDTVS antibodies disclosed herein, the second and third polypeptide chains comprise a first and second CH2-CH3 domain, respectively, where the first and second CH2-CH3 domains comprise the set of amino acid modifications shown in Figure 11b. In some embodiments of the HDTVS antibodies disclosed herein, the second and third polypeptide chains comprise a first and second CH2-CH3 domain, respectively, where the first and second CH2-CH3 domains comprise the set of amino acid modifications shown in Figure 11c. In some embodiments of the HDTVS antibodies disclosed herein, the second and third polypeptide chains comprise a first and second CH2-CH3 domain, respectively, where the first and second CH2-CH3 domains comprise the set of amino acid modifications shown in Figure 11d. In some embodiments of the HDTVS antibodies disclosed herein, the second and third polypeptide chains comprise a first and second CH2-CH3 domain, respectively, wherein the first and second CH2-CH3 domains comprise the set of amino acid modifications shown in Figure 11e.

[0123] Other Fc Modifications: In some embodiments, the heterodimeric trivalent / tetravalent multispecific antibodies of the present technology comprise a variant Fc region, wherein the variant Fc region comprises at least one amino acid modification compared to the wild-type Fc region (or parent Fc region) such that the molecule has altered affinity for an Fc receptor (e.g., FcγR), provided that the variant Fc region does not have a substitution at a position that directly contacts an Fc receptor, based on crystallographic and structural analysis of Fc-Fc receptor interactions, such as the analysis disclosed by Sondermann et al., Nature, 406:267-273 (2000). Examples of positions within the Fc region that directly contact an Fc receptor, such as FcγR, include amino acids 234-239 (hinge region), amino acids 265-269 (B / C loop), amino acids 297-299 (C7E loop), and amino acids 327-332 (F / G loop).

[0124] In some embodiments, the heterodimeric trivalent / tetravalent multispecific antibodies of the present technology have altered affinity for activating and / or inhibitory receptors and comprise a variant Fc Region with one or more amino acid modifications, wherein the one or more amino acid modifications are a substitution of N297 with alanine or a substitution of K322 with alanine. Glycosylation modifications: In some embodiments, the heterodimeric trivalent / tetravalent multispecific antibodies of the present technology have an Fc region with altered glycosylation compared to the parent Fc region. In some embodiments, the altered glycosylation comprises the absence of fucose; in some embodiments, the altered glycosylation results from expression in GnT1-deficient CHO cells.

[0125] In some embodiments, the antibodies of the present technology may have modified glycosylation sites compared to a suitable reference antibody that binds to an antigen of interest without altering the functionality of the antibody, e.g., its binding activity to the antigen. As used herein, a "glycosylation site" includes any specific amino acid sequence within an antibody to which an oligosaccharide (i.e., a carbohydrate containing two or more monosaccharides linked together) is specifically and covalently attached. Oligosaccharide side chains are typically linked to the antibody backbone via N- or O-linkages. N-linked glycosylation refers to the attachment of an oligosaccharide moiety to the side chain of an asparagine residue. O-linked glycosylation refers to the attachment of an oligosaccharide moiety to a hydroxyamino acid, e.g., serine or threonine. For example, Fc glycoforms lacking certain oligosaccharides, including fucose and terminal N-acetylglucosamine, can be produced in specialized CHO cells and exhibit enhanced ADCC effector function.

[0126] In some embodiments, the carbohydrate content of the immunoglobulin analog constructs disclosed herein is modified by adding or deleting glycosylation sites. Methods for modifying the carbohydrate content of antibodies are well known in the art and are within the scope of the present technology (see, e.g., U.S. Pat. No. 6,218,149; EP 0359096; U.S. Pat. Publication No. 2002 / 0028486; International Patent Application Publication No. WO 03 / 035835; U.S. Pat. Publication No. 2003 / 0115614; U.S. Pat. No. 6,218,149; U.S. Pat. No. 6,472,511, all of which are incorporated by reference in their entireties). In some embodiments, the carbohydrate content of an antibody (or of a relevant portion or component thereof) is modified by deleting one or more endogenous carbohydrate moieties of the antibody. In certain embodiments, the technology includes deleting the glycosylation site of the Fc region of an antibody by modifying position 297 from asparagine to alanine. Such antibodies lack Fc effector function. In some embodiments, FcR-dependent nonspecific binding in normal tissues is eliminated or reduced (for example, through the N297A mutation in the Fc region, which results in nonglycosylation).

[0127] Engineered glycoforms may be useful for a variety of purposes, including, but not limited to, enhancing or reducing effector function. Engineered glycoforms may be produced by any method known to those of skill in the art, for example, by using engineered or mutant expression strains, by co-expression with one or more enzymes, e.g., DI N-acetylglucosaminyltransferase III (GnTIII), by expressing molecules comprising an Fc region in different organisms or cell lines derived from different organisms, or by modifying the carbohydrates after expression of the molecules comprising an Fc region. Methods for producing engineered glycoforms are known in the art, each of which is incorporated herein by reference in its entirety: Umana et al., 1999, Nat. Biotechnol. 17: 176-180; Davies et al., 2001, Biotechnol. Bioeng. 74:288-294; Shields et al., 2002, J. Biol. Chem. 277:26733-26740; Shinkawa et al., 2003, J. Biol. Chem. 278:3466-3473; U.S. Patent No. 6,602,684; U.S. Patent Application No. 10 / 277,370; U.S. Patent Application No. 10 / 113,929; International Patent Application Publication Nos. WO 00 / 61739; 01 / 292246; 02 / 311140; 02 / 30954; POTILLEGENT™ technology (Biowa, Inc., Princeton, NJ); GLYCOMAB™ glycosylation engineering technology (GLYCART biotechnology AG, Zurich, Switzerland). See, for example, International Patent Application Publication No. WO 00 / 61739; US Patent Application Publication No. 2003 / 0115614; Okazaki et al., 2004, JMB, 336: 1239-49.

[0128] A. Methods of Preparing Heterodimeric Trivalent / Tetravalent Multispecific Antibodies of the Present Technology Overview: The heterodimeric trivalent / tetravalent multispecific antibodies of the present disclosure can be produced using a variety of methods known in the art, including de novo protein synthesis and expression of recombinant nucleic acids encoding the binding proteins. First, a target antigen is selected against which the antibodies of the present technology can be raised. For example, in some embodiments, antibodies can be raised against the full-length target protein or against a portion of the target protein. Techniques for generating antibodies directed against such target polypeptides are well known to those skilled in the art. Examples of such techniques include, but are not limited to, techniques involving display libraries, XenoMouse or human mice, hybridomas, etc.

[0129] Generally, antibody is obtained from original species.More specifically, antibody is obtained from the nucleic acid sequence or amino acid sequence of the variable part of light chain, heavy chain, or both of them of the original species antibody that has specificity for target antigen.Original species can be any species that has been useful for producing the antibody or antibody library of the present technology, such as rat, mouse, rabbit, chicken, monkey, human, etc.

[0130] Phage display technology or phagemid display technology is a useful technique for deriving the antibody of the present technology. Those skilled in the art are familiar with techniques for producing and cloning monoclonal antibodies. The expression of the sequence encoding the antibody of the present technology can be carried out in E. coli.

[0131] Due to the degeneracy of nucleic acid coding sequences, other sequences encoding substantially the same amino acid sequence as that of a naturally occurring protein can also be used in the practice of this technology. These include, but are not limited to, nucleic acid sequences comprising all or part of the nucleic acid sequences encoding the above polypeptides, altered by substitution of different codons that encode functionally equivalent amino acid residues within the sequence, thus resulting in a silent change. It is understood that the nucleotide sequences of immunoglobulins in accordance with this technology allow for variations in sequence homology of up to 25%, as calculated by standard methods ("Current Methods in Sequence Comparison and Analysis," Macromolecule Sequencing and Synthesis, Selected Methods and Applications, pp. 127-149, 1998, Alan R. Liss, Inc.), so long as such variations result in an effective antibody that recognizes the intended target. For example, one or more amino acid residues within a polypeptide sequence can be substituted with another amino acid of a similar polarity, which acts as a functional equivalent and results in a silent change. Substitutes for an amino acid within the sequence can be selected from other members of the class to which the amino acid belongs. For example, nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine. Polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine. Positively charged (basic) amino acids include arginine, lysine, and histidine. Negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Also included within the scope of the present technology are proteins, or fragments or derivatives thereof, that are differentially modified during or after translation, for example, by glycosylation, proteolytic cleavage, linkage to antibody molecules or other cellular ligands, etc.In addition, nucleic acid sequences encoding immunoglobulins can be mutated in vitro or in vivo to create and / or destroy translation sequences, initiation, and / or termination sequences, or to create variations in the coding region and / or to create new or destroy existing restriction endonuclease sites to facilitate further in vitro modifications. Any technique for mutagenesis known in the art can be used, including, but not limited to, in vitro site-directed mutagenesis, J. Biol. Chem. 253:6551, the use of Tab linkers (Pharmacia), etc.

[0132] Monoclonal antibody: In one embodiment of the present technology, the heterodimeric trivalent / tetravalent multispecific antibody is a monoclonal antibody. For example, in some embodiments, the heterodimeric trivalent / tetravalent multispecific monoclonal antibody may be a human heterodimeric trivalent / tetravalent multispecific monoclonal antibody or a murine heterodimeric trivalent / tetravalent multispecific monoclonal antibody. To prepare monoclonal antibodies directed against a target molecule of interest, any technique that provides for the production of antibody molecules by subculture of a cell line may be utilized. Such techniques include, but are not limited to, the hybridoma method (see, e.g., Kohler & Milstein, 1975. Nature 256: 495-497); the trioma method; the human B cell hybridoma method (see, e.g., Kozbor, et al., 1983. Immunol. Today 4: 72), and the EBV hybridoma method for producing human monoclonal antibodies (see, e.g., Cole, et al., 1985. In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96). Human monoclonal antibodies may be utilized in the practice of this technology and may be produced by using human hybridomas (see, e.g., Cote, et al., 1983. Proc. Natl. Acad. Sci. USA 80: 2026-2030) or by transforming human B cells in vitro with Epstein-Barr virus (see, e.g., Cole, et al., 1985. In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96). For example, a population of nucleic acids encoding a region of the antibody may be isolated.PCR using primers derived from sequences encoding conserved regions of the antibody amplifies sequences encoding portions of the antibody from a population and then uses the amplified sequences to reconstruct DNA encoding the polypeptide chains of the heterodimeric trivalent / tetravalent multispecific antibody, or fragments thereof, such as variable domains, from the amplified sequences. Such amplified sequences can also be fused to DNA encoding other proteins (e.g., bacteriophage coat or bacterial cell surface proteins) for expression and display of the fusion polypeptide on phage or bacteria. The amplified sequences can then be expressed and further selected or isolated based, for example, on the affinity of the expressed antibody or fragment thereof for an antigen or epitope present on a target molecule of interest. Alternatively, hybridomas expressing heterodimeric trivalent / tetravalent multispecific monoclonal antibodies can be prepared by immunizing a subject and then isolating hybridomas from the subject's spleen using standard methods. See, for example, Milstein et al. (Galfre and Milstein, Methods Enzymol (1981) 73: 3-46). Screening of hybridomas using standard methods will yield monoclonal antibodies with varying specificities (i.e., for different epitopes) and affinities. Selected monoclonal antibodies with desired properties, e.g., binding to a target antigen, can be used as expressed by the hybridoma, or they can be conjugated to molecules such as polyethylene glycol (PEG) to alter their properties, and the cDNA encoding them can be isolated, sequenced, and manipulated in various ways. Synthetic dendrimer trees can be added to reactive amino acid side chains, e.g., lysine, to enhance the immunogenic properties of target proteins. CPG dinucleotide methods can also be used to enhance the immunogenic properties of target proteins. Other manipulations include substitution or deletion of specific aminoacyl residues that contribute to antibody stability during storage or after administration to a subject, and affinity maturation methods that improve the affinity of the antibody for its target antigen.

[0133] Hybridoma method: In some embodiments, the antibody of the present technology is a heterodimeric trivalent / tetravalent multispecific monoclonal antibody produced by a hybridoma comprising B cells obtained from a transgenic non-human animal, such as a transgenic mouse, whose genome comprises a human heavy chain transgene and a human light chain transgene, fused with an immortalized cell.Hybridoma method includes the hybridoma method known in the art, and is taught in Harlow et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 349 (1988); Hammerling et al., Monoclonal Antibodies And T-Cell Hybridomas, 563-681 (1981).Other methods for producing hybridomas and monoclonal antibodies are also well known to those skilled in the art.

[0134] Phage display methods: As mentioned above, the antibodies of this technology can be produced through the application of recombinant DNA technology and phage display technology. For example, heterodimeric trivalent / tetravalent multispecific antibodies can be prepared using various phage display methods known in the art. In phage display methods, functional antibody domains are displayed on the surface of phage particles carrying the polynucleotide sequences encoding them. Phages with desired binding properties are selected from repertoire antibody libraries or combinatorial antibody libraries (e.g., human or murine) by direct selection with antigen, typically bound or captured to a solid surface or bead. The phages used in these methods are typically filamentous phages, including fd and M13, with Fab, Fv, or disulfide-stabilized Fv antibody domains recombinantly fused to phage gene III or phage gene VIII proteins. In addition, the methods can be adapted for the construction of Fab expression libraries (see, e.g., Huse, et al., Science 246: 1275-1281, 1989), which allow for the rapid and efficient identification of monoclonal Fab fragments, or derivatives, fragments, analogs, or homologs thereof, with desired specificity against a target antigen, e.g., a target polypeptide.Other examples of phage display methods that can be used to generate antibodies of the present technology include those described in Huston et al., Proc. Natl. Acad. Sci USA, 85: 5879-5883, 1988; Chaudhary et al., Proc. Natl. Acad. Sci USA, 87: 1066-1070, 1990; Brinkman et al., J. Immunol. Methods 182: 41-50, 1995; Ames et al., J. Immunol. Methods 184: 177-186, 1995; Kettleborough et al., Eur. J. Immunol. 24: 952-958, 1994; Persic et al., Gene 187: 9-18, 1997; Burton et al., Advances in Immunology 57: 191-280, 1994;PCT / GB91 / 01134;WO90 / 02809;WO91 / 10737;WO92 / 01047;WO92 / 18619;WO93 / 11236;WO95 / 15982;WO95 / 20401;WO96 / 06213;WO92 / 01047(Medical Research Council et al.); WO97 / 08320 (Morphosys); WO92 / 01047 (CAT / MRC); WO91 / 17271 (Affymax); and phage display methods disclosed in U.S. Patent Nos. 5,698,426; 5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753; 5,821,047; 5,571,698; 5,427,908; 5,516,637; 5,780,225; 5,658,727; and 5,733,743. A method useful for displaying polypeptides on the surface of bacteriophage particles by joining the polypeptides via disulfide bonds is described by Lohning in US Pat. No. 6,753,136.As described in the above references, after phage selection, antibody coding regions from the phage can be isolated and used to generate whole antibodies, including human antibodies, or any other desired antigen-binding fragments, and expressed in any desired host, including mammalian cells, insect cells, plant cells, yeast, and bacteria. Techniques for recombinantly producing Fab fragments, Fab' fragments, and F(ab')2 fragments can also be used, using methods known in the art, such as those disclosed in WO92 / 22324; Mullinax et al., BioTechniques 12: 864-869, 1992; and Sawai et al., AJRI 34: 26-34, 1995; and Better et al., Science 240: 1041-1043, 1988.

[0135] Generally, hybrid antibodies or hybrid antibody fragments cloned into a display vector can be selected against an appropriate antigen to identify mutants that maintain good binding activity, since the antibody or antibody fragment is present on the surface of a phage or phagemid particle. See, for example, Barbas III et al., Phage Display, A Laboratory Manual (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2001). However, other vector formats, such as cloning an antibody fragment library into a lytic phage vector (modified T7 system or modified Lambda Zap system) for selection and / or screening, could also be used for this step.

[0136] Single-chain Fv: The heterodimeric trivalent / tetravalent multispecific antibody of the present technology comprises two single-chain Fvs.According to the present technology, techniques can be adapted to produce target antigen-specific single-chain antibodies (see, for example, U.S. Patent No. 4,946,778).Examples of techniques that can be used to produce the single-chain Fvs and antibodies of the present technology include those described in U.S. Patent Nos. 4,946,778; and 5,258,498;Huston et al., Methods in Enzymology, 203: 46-88, 1991;Shu, L. et al., Proc. Natl. Acad. Sci. USA, 90: 7995-7999, 1993;and Skerra et al., Science 240: 1038-1040, 1988.

[0137] Chimeric antibodies and humanized antibodies: In one embodiment, the heterodimeric trivalent / tetravalent multispecific antibody of the present technology is a chimeric antibody. In one embodiment, the heterodimeric trivalent / tetravalent multispecific antibody of the present technology is a humanized antibody. In one embodiment of the present technology, the donor antibody and the acceptor antibody are monoclonal antibodies derived from different species. For example, the acceptor antibody is a human antibody (to minimize its antigenicity in humans), in which case the resulting CDR-grafted antibody is referred to as a "humanized" antibody.

[0138] Recombinant heterodimeric trivalent / tetravalent multispecific antibodies, such as chimeric monoclonal antibodies and humanized monoclonal antibodies, containing both human and non-human portions, may be produced using standard recombinant DNA methods and are within the scope of the present technology. For some uses, including the use of the heterodimeric trivalent / tetravalent multispecific antibodies of the present technology in humans in vivo, as well as the use of these agents in in vitro detection assays, it is possible to use chimeric heterodimeric trivalent / tetravalent multispecific antibodies or humanized heterodimeric trivalent / tetravalent multispecific antibodies. Such chimeric and humanized monoclonal antibodies may be produced by recombinant DNA methods known in the art.Such useful methods are described, for example, in International Application No. PCT / US86 / 02269; U.S. Patent No. 5,225,539; European Patent No. 184187; European Patent No. 171496; European Patent No. 173494; PCT International Application No. WO86 / 01533; U.S. Patent Nos. 4,816,567; 5,225,539; European Patent No. 125023; Better, et al., 1988. Science 240: 1041-1043; Liu, et al., 1987. Proc. Natl. Acad. Sci. USA 84: 3439-3443; Liu, et al., 1987. J. Immunol. 139: 3521-3526; Sun, et al., 1987. Proc. Natl. Acad. Sci. USA 84: 214-218;Nishimura, et al., 1987. Cancer Res. 47: 999-1005;Wood, et al., 1985. Nature 314: 446-449;Shaw, et al., 1988. J. Natl. Cancer Inst. 80: 1553-1559;Morrison (1985) Science 229: 1202-1207;Oi, et al. (1986) BioTechniques 4: 214;Jones, et al., 1986. Nature 321: 552-525;Verhoeyan, et al., 1988. Science 239: 1534;Morrison, Science 229: 1202, 1985; Oi et al., BioTechniques 4: 214, 1986; Gillies et al., J. Immunol. Methods, 125: 191-202, 1989; U.S. Patent No. 5,807,715; and Beidler, et al., 1988. J. Immunol. 141: 4053-4060.For example, antibodies can be humanized using a variety of techniques, including CDR grafting (EP 0239400; WO 91 / 09967; U.S. Patent Nos. 5,530,101; 5,585,089; 5,859,205; 6,248,516; EP 460167), veneering or resurfacing (EP 0592106; EP 0519596; Padlan EA, Molecular Immunology, 28: 48...

Claims

1. a first polypeptide chain, a second polypeptide chain, a third polypeptide chain, and a fourth polypeptide chain, wherein the first polypeptide chain and the second polypeptide chain are covalently bonded to each other, the second polypeptide chain and the third polypeptide chain are covalently bonded to each other, and the third polypeptide chain and the fourth polypeptide chain are covalently bonded to each other; a. a first polypeptide chain having, in the N-terminal to C-terminal direction: i. a first immunoglobulin light chain variable domain (VL-1) capable of specifically binding to a first epitope; ii. the light chain constant domain of the first immunoglobulin (CL-1); iii. Amino acid sequence (GGGGS) 3 a flexible peptide linker comprising: iv. a light chain variable domain (VL-2) of a second immunoglobulin linked to a heavy chain variable domain (VH-2) of a complementary second immunoglobulin, or a heavy chain variable domain (VH-2) of a second immunoglobulin linked to a light chain variable domain (VL-2) of a complementary second immunoglobulin, wherein the VL-2 and VH-2 are capable of specifically binding to a second epitope, and having the amino acid sequence (GGGGS). 6 VL-2 or VH-2 linked together to form a single chain variable fragment via a flexible peptide linker comprising Including, b. a second polypeptide comprising, in the N-terminal to C-terminal direction: i. a heavy chain variable domain (VH-1) of a first immunoglobulin capable of specifically binding to a first epitope; ii. a first CH1 domain (CH1-1) of a first immunoglobulin; and iii. a first heterodimerization domain of a first immunoglobulin, the first heterodimerization domain being incapable of forming a stable homodimer with another first heterodimerization domain; Including, c. a third polypeptide comprising, in the N-terminal to C-terminal direction: i. a third immunoglobulin heavy chain variable domain (VH-3) capable of specifically binding to a third epitope; ii. the second CH1 domain of a third immunoglobulin (CH1-3); and iii. a second heterodimerization domain of a third immunoglobulin, the second heterodimerization domain comprising an amino acid sequence or a nucleic acid sequence distinct from the first heterodimerization domain of the first immunoglobulin, incapable of forming stable homodimers with another second heterodimerization domain, and configured to form a heterodimer with the first heterodimerization domain of the first immunoglobulin. Including, d. a fourth polypeptide comprising, in the N-terminal to C-terminal direction: i. a third immunoglobulin light chain variable domain (VL-3) capable of specifically binding to a third epitope; ii. a third immunoglobulin light chain constant domain (CL-3); iii. Amino acid sequence (GGGGS) 3 a flexible peptide linker comprising: iv. a light chain variable domain of a fourth immunoglobulin (VL-4) linked to a heavy chain variable domain of a complementary fourth immunoglobulin (VH-4), or a heavy chain variable domain of a fourth immunoglobulin (VH-4) linked to a light chain variable domain of a complementary fourth immunoglobulin (VL-4), wherein the VL-4 and VH-4 are capable of specifically binding to a second epitope, the heavy chain variable domain having the amino acid sequence (GGGGS). 6 VL-4 and VH-4 linked together to form a single chain variable fragment via a flexible peptide linker comprising Including, Each of VL-1 and VL-3 is independently selected from SEQ ID NOs: 1, 9, 17, 25, 33, 41, 49, 57, 65, 73, 81, 89, 97, 105, 113, 121, 129, 145, 153, 161, 169, 177, 193, 201, 233, 241, 257, 273, 281, 289, 297, 305, 313, 321, 329, 337, 345, 353, 361, 369, 377, 385, 393, 401, 409, 417, 425, 433, 441, 449, 457, 465, 481, 489, 497, 521, 529, 537, 545, 553, 561, 609, 617, 681, 689, 697, 705, 713, 721, 729, 737, 745, 753, 761, 769, 777, 785, 793, 801, 809, 817, 825, 833, 841, 849, 857, 865, 873, 881, 889, 945, 9 53, 961, 977, 985, 993, 1001, 1009, 1017, 1025, 1033, 1041, 1049, 1065, 1073, 1081, 1089, 1097, 1105, 1113, 1121, 1129, 1137, 1145, 1153, 1161, 1169, 1 177, 1185, 1193, 1201, 1209, 1217, 1225, 1233, 1241, 1249, 1257, 1265, 1273, 1281, 1289, 1297, 1305, 1313, 1321, 1329, 1337, 1345, 1353, 1361, 1369, 1377, 1385, 1393, 1401, 1409, 1417, 1425, 1433, 1441, 1449, 1457, 1465, 1473, 1481, 1489, 1497, 1505, 1513, 1521, 1529, 1545, 1553, 1561, 1569, 1577, 1585, 1593, 1601, 1609, 1617, 1625, 1633, 1649, 1657, 1673, 1681, 1689, 1697, 1705, 1713, 1721, 1729, 1737, 1745, 1753, 1761, 1769, 1777, 1785, 1793, 1801, 1809, 1817, 1833, 1841, 1849, 1857, 1865, 1873, 1881, 1889, 1913, 1937, 1945, 1953, 1961, 1969, 1977, 1985, 1993, 2001, 2009, 2017, 2025, 2033,V selected from any one of 2041, 2049, 2057, 2065, 2073, 2081, 2089, 2097, 2105, 2113, 2121, 2129, 2137, 2145, 2153, 2161, 2169, 2177, 2185, 2193, 2201, 2209, 2217, 2225, 2233, 2241, 2249, 2257, 2265, 2273, 2281, 2297, 2305, 2313, 2321, 2329, 2337, and 2345; L and / or comprising the amino acid sequences CDR1, CDR2, and CDR3; Each of VH-1 and VH-3 is independently selected from SEQ ID NOs: 5, 13, 21, 29, 37, 45, 53, 61, 69, 77, 85, 93, 101, 109, 117, 125, 133, 149, 157, 165, 173, 181, 197, 205, 237, 245, 261, 277, 285, 293, 301, 309, 317, 325, 333, 341, 349, 357, 365, 373, 381, 389, 397, 405, 413, 421, 429, 437, 445, 453, 461, 469, 485, 493, 501, 525, 533, 541, 549, 557 , 565, 613, 621, 685, 693, 701, 709, 717, 725, 733, 741, 749, 757, 765, 773, 781, 789, 797, 805, 813, 821, 829, 837, 845, 853, 861, 869, 877, 885, 893, 949, 957, 965, 981, 989, 997, 1005, 1013, 1021, 1029, 1037, 1045, 1053, 1069, 1077, 1085, 1093, 1101, 1109, 1117, 1125, 1133, 1141, 1149, 1157, 1165, 1173, 1181, 1189, 1197, 1205, 1213, 1221, 1229, 1237, 1245, 1253, 1261, 1269, 1277, 1285, 1293, 1301, 1309, 1317, 1325, 1333, 1341, 1349, 1357, 1365, 1373, 1381, 1389, 1397, 1405, 1413, 1421, 1429, 1437, 1445, 1453, 1461, 1469, 1477, 1485, 1493, 1501, 1509, 1517, 1525, 1533, 1549, 1557, 1565, 1573, 1581, 1589, 1597, 1605, 1613, 1621, 1629, 1637, 1653, 1661, 1677, 1685, 1693, 1701, 1709, 1717, 1725, 1733, 1741, 1749, 1757, 1765, 1773, 1781, 1789, 1797, 1805, 1813, 1821, 1837, 1845, 1853, 1861, 1869, 1877, 1885, 1893, 1917, 1941, 1949, 1957, 1965, 1973, 1981, 1989, 1997, 2005, 2013, 2021, 2029, 2037,V selected from any one of 2045, 2053, 2061, 2069, 2077, 2085, 2093, 2101, 2109, 2117, 2125, 2133, 2141, 2149, 2157, 2165, 2173, 2181, 2189, 2197, 2205, 2213, 2221, 2229, 2237, 2245, 2253, 2261, 2269, 2277, 2285, 2301, 2309, 2317, 2325, 2333, 2341, and 2349; H The amino acid sequences include CDR1, CDR2, and CDR3 sequences, Heterodimeric multispecific antibodies.

2. a first polypeptide chain, a second polypeptide chain, a third polypeptide chain, and a fourth polypeptide chain, wherein the first polypeptide chain and the second polypeptide chain are covalently bonded to each other, the second polypeptide chain and the third polypeptide chain are covalently bonded to each other, and the third polypeptide chain and the fourth polypeptide chain are covalently bonded to each other; a. a first polypeptide chain having, in the N-terminal to C-terminal direction: i. a first immunoglobulin light chain variable domain (VL-1) capable of specifically binding to a first epitope; ii. the light chain constant domain of the first immunoglobulin (CL-1); iii. Amino acid sequence (GGGGS) 3 a flexible peptide linker comprising: iv. a light chain variable domain (VL-2) of a second immunoglobulin linked to a heavy chain variable domain (VH-2) of a complementary second immunoglobulin, or a heavy chain variable domain (VH-2) of a second immunoglobulin linked to a light chain variable domain (VL-2) of a complementary second immunoglobulin, wherein the VL-2 and VH-2 are capable of specifically binding to a second epitope, and having the amino acid sequence (GGGGS). 6 VL-2 or VH-2 linked together to form a single chain variable fragment via a flexible peptide linker comprising Including, b. a second polypeptide comprising, in the N-terminal to C-terminal direction: i. a first immunoglobulin heavy chain variable domain (VH-1) capable of specifically binding to a first epitope; ii. a first CH1 domain (CH1-1) of a first immunoglobulin; and iii. a first heterodimerization domain of a first immunoglobulin, the first heterodimerization domain being incapable of forming a stable homodimer with another first heterodimerization domain; Including, c. a third polypeptide comprising, in the N-terminal to C-terminal direction: i. a third immunoglobulin heavy chain variable domain (VH-3) capable of specifically binding to the first epitope; ii. the second CH1 domain of a third immunoglobulin (CH1-3); and iii. a second heterodimerization domain of a third immunoglobulin, the second heterodimerization domain comprising an amino acid sequence or a nucleic acid sequence distinct from the first heterodimerization domain of the first immunoglobulin, incapable of forming stable homodimers with another second heterodimerization domain, and configured to form a heterodimer with the first heterodimerization domain of the first immunoglobulin. Including, d. a fourth polypeptide comprising, in the N-terminal to C-terminal direction: i. a third immunoglobulin light chain variable domain (VL-3) capable of specifically binding to the first epitope; ii. a third immunoglobulin light chain constant domain (CL-3); iii. Amino acid sequence (GGGGS) 3 a flexible peptide linker comprising: iv. a light chain variable domain of a fourth immunoglobulin (VL-4) linked to a heavy chain variable domain of a complementary fourth immunoglobulin (VH-4), or a heavy chain variable domain of a fourth immunoglobulin (VH-4) linked to a light chain variable domain of a complementary fourth immunoglobulin (VL-4), wherein the VL-4 and VH-4 are capable of specifically binding to a third epitope, the heavy chain variable domain having the amino acid sequence (GGGGS). 6 VL-4 or VH-4 linked together to form a single chain variable fragment via a flexible peptide linker comprising Including, Each of VL-2 and VL-4 is independently selected from the group consisting of SEQ ID NOs: 17, 25, 33, 41, 121, 137, 169, 177, 185, 193, 201, 209, 217, 225, 233, 241, 249, 257, 265, 321, 329, 337, 393, 401, 409, 473, 481, 489, 497, 505, 513, 545, 553, 561, 569, 577, 585, 593, 601, 625, 633, 641, 649, 657, 665, 673, 681, 692, 701, 713, 725, 733, 741, 757, 765, 773, 785, 793, 801, 813, 825, 833, 841, 857, 865, 873, 885, 893, 901, 913, 925, 933, 941, 957, 965, 973, 985, 993, 1001, 1025, 1045, 1065, 1085, 1093, 1093, 1025, 1065, 1073, 1085, 1093, 1102, 1145, 1153, 1165, 117 81, 689, 697, 705, 713, 721, 729, 737, 745, 753, 761, 769, 785, 793, 801, 809, 817, 849, 857, 865, 873, 881, 889, 897, 905, 913, 921, 929, 937, 945, 969, 977, 1009, 1057, 1537, 1569, 1601, 1641, 1665, 1825, 1865, 1897, 1905, 1913, 1921, 1929, 2265, 2281, 2289, 2329, and 2345; L and / or comprising the amino acid sequences CDR1, CDR2, and CDR3; Each of VH-2 and VH-4 is independently selected from the group consisting of SEQ ID NOs: 21, 29, 37, 45, 125, 141, 173, 181, 189, 197, 205, 213, 221, 229, 237, 245, 253, 261, 269, 325, 333, 341, 397, 405, 413, 477, 485, 493, 501, 509, 517, 549, 557, 565, 573, 581, 589, 597, 605, 629, 637, 645, 653, 661, 669, 677, 685, 693, 701, 709, 717 , 725, 733, 741, 749, 757, 765, 773, 789, 797, 805, 813, 821, 853, 861, 869, 877, 885, 893, 901, 909, 917, 925, 933, 941, 949, 973, 981, 1013, 1061, 1541, 1573, 1605, 1645, 1669, 1829, 1869, 1901, 1909, 1917, 1925, 1933, 2269, 2285, 2293, 2333, and 2349. H The amino acid sequences include CDR1, CDR2, and CDR3 sequences, Heterodimeric multispecific antibodies.

3. a first polypeptide chain, a second polypeptide chain, a third polypeptide chain, and a fourth polypeptide chain, wherein the first polypeptide chain and the second polypeptide chain are covalently bonded to each other, the second polypeptide chain and the third polypeptide chain are covalently bonded to each other, and the third polypeptide chain and the fourth polypeptide chain are covalently bonded to each other; a. a first polypeptide chain having, in the N-terminal to C-terminal direction: i. a first immunoglobulin light chain variable domain (VL-1) capable of specifically binding to a first epitope; ii. the light chain constant domain of the first immunoglobulin (CL-1); iii. Amino acid sequence (GGGGS) 3 a flexible peptide linker comprising: iv. a light chain variable domain (VL-2) of a second immunoglobulin linked to a heavy chain variable domain (VH-2) of a complementary second immunoglobulin, or a heavy chain variable domain (VH-2) of a second immunoglobulin linked to a light chain variable domain (VL-2) of a complementary second immunoglobulin, wherein the VL-2 and VH-2 are capable of specifically binding to a second epitope, and having the amino acid sequence (GGGGS). 6 wherein the VL-2 or VH-2 are linked together to form a single chain variable fragment via a flexible peptide linker comprising b. a second polypeptide comprising, in the N-terminal to C-terminal direction: i. a heavy chain variable domain (VH-1) of a first immunoglobulin capable of specifically binding to a first epitope; ii. a first CH1 domain (CH1-1) of a first immunoglobulin; and iii. a first heterodimerization domain of a first immunoglobulin, the first heterodimerization domain being incapable of forming a stable homodimer with another first heterodimerization domain; Including, c. a third polypeptide comprising, in the N-terminal to C-terminal direction: i. a third immunoglobulin heavy chain variable domain (VH-3) capable of specifically binding to a third epitope; ii. the second CH1 domain of a third immunoglobulin (CH1-3); and iii. a second heterodimerization domain of a third immunoglobulin, the second heterodimerization domain comprising an amino acid sequence or a nucleic acid sequence distinct from the first heterodimerization domain of the first immunoglobulin, incapable of forming a stable homodimer with another second heterodimerization domain, and configured to form a heterodimer with the first heterodimerization domain of the first immunoglobulin. Including, d. a fourth polypeptide comprising, in the N-terminal to C-terminal direction: i. a third immunoglobulin light chain variable domain (VL-3) capable of specifically binding to a third epitope; ii. a third immunoglobulin light chain constant domain (CL-3); iii. Amino acid sequence (GGGGS) 3 a flexible peptide linker comprising: iv. a fourth immunoglobulin light chain variable domain (VL-4) linked to a complementary fourth immunoglobulin heavy chain variable domain (VH-4) or a fourth immunoglobulin heavy chain variable domain (VH-4) linked to a complementary fourth immunoglobulin light chain variable domain (VL-4), wherein the VL-4 and VH-4 are capable of specifically binding to a fourth epitope, the fourth immunoglobulin having the amino acid sequence (GGGGS). 6 VL-4 or VH-4 linked together to form a single chain variable fragment via a flexible peptide linker comprising Including, Each of VL-1 and VL-3 is independently selected from SEQ ID NOs: 1, 9, 17, 25, 33, 41, 49, 57, 65, 73, 81, 89, 97, 105, 113, 121, 129, 145, 153, 161, 169, 177, 193, 201, 233, 241, 257, 273, 281, 289, 297, 305, 313, 321, 329, 337, 345, 353, 361, 369, 377, 385, 393, 401, 409, 417, 425, 433, 441, 449, 457, 465, 481, 489, 497, 521, 529, 537, 545, 553, 561, 609, 617, 681, 689, 697, 705, 713, 721, 729, 737, 745, 753, 761, 769, 777, 785, 793, 801, 809, 817, 825, 833, 841, 849, 857, 865, 873, 881, 889, 945, 9 53, 961, 977, 985, 993, 1001, 1009, 1017, 1025, 1033, 1041, 1049, 1065, 1073, 1081, 1089, 1097, 1105, 1113, 1121, 1129, 1137, 1145, 1153, 1161, 1169, 1 177, 1185, 1193, 1201, 1209, 1217, 1225, 1233, 1241, 1249, 1257, 1265, 1273, 1281, 1289, 1297, 1305, 1313, 1321, 1329, 1337, 1345, 1353, 1361, 1369, 1377, 1385, 1393, 1401, 1409, 1417, 1425, 1433, 1441, 1449, 1457, 1465, 1473, 1481, 1489, 1497, 1505, 1513, 1521, 1529, 1545, 1553, 1561, 1569, 1577, 1585, 1593, 1601, 1609, 1617, 1625, 1633, 1649, 1657, 1673, 1681, 1689, 1697, 1705, 1713, 1721, 1729, 1737, 1745, 1753, 1761, 1769, 1777, 1785, 1793, 1801, 1809, 1817, 1833, 1841, 1849, 1857, 1865, 1873, 1881, 1889, 1913, 1937, 1945, 1953, 1961, 1969, 1977, 1985, 1993, 2001, 2009, 2017, 2025, 2033,V selected from any one of 2041, 2049, 2057, 2065, 2073, 2081, 2089, 2097, 2105, 2113, 2121, 2129, 2137, 2145, 2153, 2161, 2169, 2177, 2185, 2193, 2201, 2209, 2217, 2225, 2233, 2241, 2249, 2257, 2265, 2273, 2281, 2297, 2305, 2313, 2321, 2329, 2337, and 2345; L and / or comprising the amino acid sequences CDR1, CDR2, and CDR3; Each of VH-1 and VH-3 is independently selected from SEQ ID NOs: 5, 13, 21, 29, 37, 45, 53, 61, 69, 77, 85, 93, 101, 109, 117, 125, 133, 149, 157, 165, 173, 181, 197, 205, 237, 245, 261, 277, 285, 293, 301, 309, 317, 325, 333, 341, 349, 357, 365, 373, 381, 389, 397, 405, 413, 421, 429, 437, 445, 453, 461, 469, 485, 493, 501, 525, 533, 541, 549, 557 , 565, 613, 621, 685, 693, 701, 709, 717, 725, 733, 741, 749, 757, 765, 773, 781, 789, 797, 805, 813, 821, 829, 837, 845, 853, 861, 869, 877, 885, 893, 949, 957, 965, 981, 989, 997, 1005, 1013, 1021, 1029, 1037, 1045, 1053, 1069, 1077, 1085, 1093, 1101, 1109, 1117, 1125, 1133, 1141, 1149, 1157, 1165, 1173, 1181, 1189, 1197, 1205, 1213, 1221, 1229, 1237, 1245, 1253, 1261, 1269, 1277, 1285, 1293, 1301, 1309, 1317, 1325, 1333, 1341, 1349, 1357, 1365, 1373, 1381, 1389, 1397, 1405, 1413, 1421, 1429, 1437, 1445, 1453, 1461, 1469, 1477, 1485, 1493, 1501, 1509, 1517, 1525, 1533, 1549, 1557, 1565, 1573, 1581, 1589, 1597, 1605, 1613, 1621, 1629, 1637, 1653, 1661, 1677, 1685, 1693, 1701, 1709, 1717, 1725, 1733, 1741, 1749, 1757, 1765, 1773, 1781, 1789, 1797, 1805, 1813, 1821, 1837, 1845, 1853, 1861, 1869, 1877, 1885, 1893, 1917, 1941, 1949, 1957, 1965, 1973, 1981, 1989, 1997, 2005, 2013, 2021, 2029, 2037,V selected from any one of 2045, 2053, 2061, 2069, 2077, 2085, 2093, 2101, 2109, 2117, 2125, 2133, 2141, 2149, 2157, 2165, 2173, 2181, 2189, 2197, 2205, 2213, 2221, 2229, 2237, 2245, 2253, 2261, 2269, 2277, 2285, 2301, 2309, 2317, 2325, 2333, 2341, and 2349; H and / or comprising the amino acid sequences CDR1, CDR2, and CDR3; Each of VL-2 and VL-4 is independently selected from the group consisting of SEQ ID NOs: 17, 25, 33, 41, 121, 137, 169, 177, 185, 193, 201, 209, 217, 225, 233, 241, 249, 257, 265, 321, 329, 337, 393, 401, 409, 473, 481, 489, 497, 505, 513, 545, 553, 561, 569, 577, 585, 593, 601, 625, 633, 641, 649, 657, 665, 673, 681, 692, 701, 713, 725, 733, 741, 757, 765, 773, 785, 793, 801, 813, 825, 833, 841, 857, 865, 873, 885, 893, 901, 913, 925, 933, 941, 957, 965, 973, 985, 993, 1001, 1025, 1045, 1065, 1085, 1093, 1093, 1105, 1115, 1125, 1137, 1201, 1215, 1225, 1233, 1241, 125 V selected from any one of 81, 689, 697, 705, 713, 721, 729, 737, 745, 753, 761, 769, 785, 793, 801, 809, 817, 849, 857, 865, 873, 881, 889, 897, 905, 913, 921, 929, 937, 945, 969, 977, 1009, 1057, 1537, 1569, 1601, 1641, 1665, 1825, 1865, 1897, 1905, 1913, 1921, 1929, 2265, 2281, 2289, 2329, and 2345 L and / or comprising the amino acid sequences CDR1, CDR2, and CDR3; Each of VH-2 and VH-4 is independently selected from the group consisting of SEQ ID NOs: 21, 29, 37, 45, 125, 141, 173, 181, 189, 197, 205, 213, 221, 229, 237, 245, 253, 261, 269, 325, 333, 341, 397, 405, 413, 477, 485, 493, 501, 509, 517, 549, 557, 565, 573, 581, 589, 597, 605, 629, 637, 645, 653, 661, 669, 677, 685, 693, 701, 709, 717 , 725, 733, 741, 749, 757, 765, 773, 789, 797, 805, 813, 821, 853, 861, 869, 877, 885, 893, 901, 909, 917, 925, 933, 941, 949, 973, 981, 1013, 1061, 1541, 1573, 1605, 1645, 1669, 1829, 1869, 1901, 1909, 1917, 1925, 1933, 2269, 2285, 2293, 2333, and 2349. H The amino acid sequences include CDR1, CDR2, and CDR3 sequences, Heterodimeric multispecific antibodies.

4. a first polypeptide chain, a second polypeptide chain, a third polypeptide chain, and a fourth polypeptide chain, wherein the first polypeptide chain and the second polypeptide chain are covalently bonded to each other, the second polypeptide chain and the third polypeptide chain are covalently bonded to each other, and the third polypeptide chain and the fourth polypeptide chain are covalently bonded to each other; a. a first polypeptide chain having, in the N-terminal to C-terminal direction: i. a first immunoglobulin light chain variable domain (VL-1) capable of specifically binding to a first epitope; ii. the light chain constant domain of the first immunoglobulin (CL-1); iii. Amino acid sequence (GGGGS) 3 a flexible peptide linker comprising: iv. a light chain variable domain (VL-2) of a second immunoglobulin linked to a heavy chain variable domain (VH-2) of a complementary second immunoglobulin, or a heavy chain variable domain (VH-2) of a second immunoglobulin linked to a light chain variable domain (VL-2) of a complementary second immunoglobulin, wherein the VL-2 and VH-2 are capable of specifically binding to a second epitope, and having the amino acid sequence (GGGGS). 6 VL-2 or VH-2 linked together to form a single chain variable fragment via a flexible peptide linker comprising Including, b. a second polypeptide comprising, in the N-terminal to C-terminal direction: i. a first immunoglobulin heavy chain variable domain (VH-1) capable of specifically binding to a first epitope; ii. a first CH1 domain (CH1-1) of a first immunoglobulin; and iii. a first heterodimerization domain of a first immunoglobulin, the first heterodimerization domain being incapable of forming a stable homodimer with another first heterodimerization domain; Including, c. a third polypeptide comprising, in the N-terminal to C-terminal direction: i. a third immunoglobulin heavy chain variable domain (VH-3) capable of specifically binding to the first epitope; ii. the second CH1 domain of a third immunoglobulin (CH1-3); and iii. a second heterodimerization domain of a third immunoglobulin, the second heterodimerization domain comprising an amino acid sequence or a nucleic acid sequence distinct from the first heterodimerization domain of the first immunoglobulin, incapable of forming stable homodimers with another second heterodimerization domain, and configured to form a heterodimer with the first heterodimerization domain of the first immunoglobulin. Including, d. a fourth polypeptide comprising, in the N-terminal to C-terminal direction: i. a third immunoglobulin light chain variable domain (VL-3) capable of specifically binding to the first epitope; and ii. A third immunoglobulin light chain constant domain (CL-3) Including, VL-2 is selected from the group consisting of SEQ ID NOs: 17, 25, 33, 41, 121, 137, 169, 177, 185, 193, 201, 209, 217, 225, 233, 241, 249, 257, 265, 321, 329, 337, 393, 401, 409, 473, 481, 489, 497, 505, 513, 545, 553, 561, 569, 577, 585, 593, 601, 625, 633, 641, 649, 657, 665, 673, 681, 689, V selected from any one of 697, 705, 713, 721, 729, 737, 745, 753, 761, 769, 785, 793, 801, 809, 817, 849, 857, 865, 873, 881, 889, 897, 905, 913, 921, 929, 937, 945, 969, 977, 1009, 1057, 1537, 1569, 1601, 1641, 1665, 1825, 1865, 1897, 1905, 1913, 1921, 1929, 2265, 2281, 2289, 2329, and 2345 L and / or comprising the amino acid sequences CDR1, CDR2, and CDR3; VH-2 is selected from the group consisting of SEQ ID NOs: 21, 29, 37, 45, 125, 141, 173, 181, 189, 197, 205, 213, 221, 229, 237, 245, 253, 261, 269, 325, 333, 341, 397, 405, 413, 477, 485, 493, 501, 509, 517, 549, 557, 565, 573, 581, 589, 597, 605, 629, 637, 645, 653, 661, 669, 677, 685, 693, 701, 709, 717, 725, 73 3, 741, 749, 757, 765, 773, 789, 797, 805, 813, 821, 853, 861, 869, 877, 885, 893, 901, 909, 917, 925, 933, 941, 949, 973, 981, 1013, 1061, 1541, 1573, 1605, 1645, 1669, 1829, 1869, 1901, 1909, 1917, 1925, 1933, 2269, 2285, 2293, 2333, and 2349. H The amino acid sequences include CDR1, CDR2, and CDR3 sequences, Heterodimeric multispecific antibodies.

5. Both VH-1 and VH-3 are selected from the group consisting of SEQ ID NOs: 5, 13, 21, 29, 37, 45, 53, 61, 69, 77, 85, 93, 101, 109, 117, 125, 133, 149, 157, 165, 173, 181, 197, 205, 237, 245, 261, 277, 285, 293, 301, 309, 317, 325, 333, 341, 349, 357, 365, 373, 381, 389, 397, 405, 413, 421, 429, 437, 445, 453, 461, 469, 485, 493, 501, 525, 533, 541, 549, 557, 565 , 613, 621, 685, 693, 701, 709, 717, 725, 733, 741, 749, 757, 765, 773, 781, 789, 797, 805, 813, 821, 829, 837, 845, 853, 861, 869, 877, 885, 893, 949, 957, 965, 981, 989, 997, 1005, 1013, 1021, 1029, 1037, 1045, 1053, 1069, 1077, 1085, 1093, 1101, 1109, 1117, 1125, 1133, 1141, 1149, 1157, 1165, 1173, 1181 , 1189, 1197, 1205, 1213, 1221, 1229, 1237, 1245, 1253, 1261, 1269, 1277, 1285, 1293, 1301, 1309, 1317, 1325, 1333, 1341, 1349, 1357, 1365, 1373, 1381 , 1389, 1397, 1405, 1413, 1421, 1429, 1437, 1445, 1453, 1461, 1469, 1477, 1485, 1493, 1501, 1509, 1517, 1525, 1533, 1549, 1557, 1565, 1573, 1581, 1589 , 1597, 1605, 1613, 1621, 1629, 1637, 1653, 1661, 1677, 1685, 1693, 1701, 1709, 1717, 1725, 1733, 1741, 1749, 1757, 1765, 1773, 1781, 1789, 1797, 1805 , 1813, 1821, 1837, 1845, 1853, 1861, 1869, 1877, 1885, 1893, 1917, 1941, 1949, 1957, 1965, 1973, 1981, 1989, 1997, 2005, 2013, 2021, 2029, 2037, 2045,V selected from any one of 2053, 2061, 2069, 2077, 2085, 2093, 2101, 2109, 2117, 2125, 2133, 2141, 2149, 2157, 2165, 2173, 2181, 2189, 2197, 2205, 2213, 2221, 2229, 2237, 2245, 2253, 2261, 2269, 2277, 2285, 2301, 2309, 2317, 2325, 2333, 2341, and 2349; H and / or comprising the amino acid sequences CDR1, CDR2, and CDR3; Both VL-1 and VL-3 are selected from SEQ ID NOs: 1, 9, 17, 25, 33, 41, 49, 57, 65, 73, 81, 89, 97, 105, 113, 121, 129, 145, 153, 161, 169, 177, 193, 201, 233, 241, 257, 273, 281, 289, 297, 305, 313, 321, 329, 337, 345, 353, 361, 369, 377, 385, 393, 401, 409, 417, 425, 433, 441, 449, 457, 465, 481, 489, 497, 521, 529, 537, 545, 553, 561, 609, 617, 681, 689, 697, 705, 713, 721, 729, 737, 745, 753, 761, 769, 777, 785, 793, 801, 809, 817, 825, 833, 841, 849, 857, 865, 873, 881, 889, 945, 953, 9 61, 977, 985, 993, 1001, 1009, 1017, 1025, 1033, 1041, 1049, 1065, 1073, 1081, 1089, 1097, 1105, 1113, 1121, 1129, 1137, 1145, 1153, 1161, 1169, 1177, 1185, 1193, 1201, 1209, 1217, 1225, 1233, 1241, 1249, 1257, 1265, 1273, 1281, 1289, 1297, 1305, 1313, 1321, 1329, 1337, 1345, 1353, 1361, 1369, 1377, 1385, 1393, 1401, 1409, 1417, 1425, 1433, 1441, 1449, 1457, 1465, 1473, 1481, 1489, 1497, 1505, 1513, 1521, 1529, 1545, 1553, 1561, 1569, 1577, 1585, 1593, 1601, 1609, 1617, 1625, 1633, 1649, 1657, 1673, 1681, 1689, 1697, 1705, 1713, 1721, 1729, 1737, 1745, 1753, 1761, 1769, 1777, 1785, 1793, 1801, 1809, 1817, 1833, 1841, 1849, 1857, 1865, 1873, 1881, 1889, 1913, 1937, 1945, 1953, 1961, 1969, 1977, 1985, 1993, 2001, 2009, 2017, 2025, 2033, 2041,V selected from any one of 2049, 2057, 2065, 2073, 2081, 2089, 2097, 2105, 2113, 2121, 2129, 2137, 2145, 2153, 2161, 2169, 2177, 2185, 2193, 2201, 2209, 2217, 2225, 2233, 2241, 2249, 2257, 2265, 2273, 2281, 2297, 2305, 2313, 2321, 2329, 2337, and 2345; L The heterodimeric multispecific antibody of claim 4, comprising the amino acid sequences CDR1, CDR2, and CDR3.

6. a first polypeptide chain, a second polypeptide chain, a third polypeptide chain, and a fourth polypeptide chain, wherein the first polypeptide chain and the second polypeptide chain are covalently bonded to each other, the second polypeptide chain and the third polypeptide chain are covalently bonded to each other, and the third polypeptide chain and the fourth polypeptide chain are covalently bonded to each other; a. a first polypeptide chain having, in the N-terminal to C-terminal direction: i. a first immunoglobulin light chain variable domain (VL-1) capable of specifically binding to a first epitope; ii. the light chain constant domain of the first immunoglobulin (CL-1); iii. Amino acid sequence (GGGGS) 3 a flexible peptide linker comprising: iv. a light chain variable domain (VL-2) of a second immunoglobulin linked to a heavy chain variable domain (VH-2) of a complementary second immunoglobulin, or a heavy chain variable domain (VH-2) of a second immunoglobulin linked to a light chain variable domain (VL-2) of a complementary second immunoglobulin, wherein the VL-2 and VH-2 are capable of specifically binding to a second epitope, and having the amino acid sequence (GGGGS). 6 VL-2 or VH-2 linked together to form a single chain variable fragment via a flexible peptide linker comprising Including, b. a second polypeptide comprising, in the N-terminal to C-terminal direction: i. a first immunoglobulin heavy chain variable domain (VH-1) capable of specifically binding to a first epitope; ii. a first CH1 domain (CH1-1) of a first immunoglobulin; and iii. a first heterodimerization domain of a first immunoglobulin, the first heterodimerization domain being incapable of forming a stable homodimer with another first heterodimerization domain; Including, c. a third polypeptide comprising, in the N-terminal to C-terminal direction: i. a third immunoglobulin heavy chain variable domain (VH-3) capable of specifically binding to a third epitope; ii. the second CH1 domain of a third immunoglobulin (CH1-3); and iii. a second heterodimerization domain of a third immunoglobulin, the second heterodimerization domain comprising an amino acid sequence or a nucleic acid sequence distinct from the first heterodimerization domain of the first immunoglobulin, incapable of forming stable homodimers with another second heterodimerization domain, and configured to form a heterodimer with the first heterodimerization domain of the first immunoglobulin. Including, d. a fourth polypeptide comprising, in the N-terminal to C-terminal direction: i. a third immunoglobulin light chain variable domain (VL-3) capable of specifically binding to a third epitope; and ii. A third immunoglobulin light chain constant domain (CL-3) Including, Each of VL-1 and VL-3 is independently selected from SEQ ID NOs: 1, 9, 17, 25, 33, 41, 49, 57, 65, 73, 81, 89, 97, 105, 113, 121, 129, 145, 153, 161, 169, 177, 193, 201, 233, 241, 257, 273, 281, 289, 297, 305, 313, 321, 329, 337, 345, 353, 361, 369, 377, 385, 393, 401, 409, 417, 425, 433, 441, 449, 457, 465, 481, 489, 497, 521, 529, 537, 545, 553, 561, 609, 617, 681, 689, 697, 705, 713, 721, 729, 737, 745, 753, 761, 769, 777, 785, 793, 801, 809, 817, 825, 833, 841, 849, 857, 865, 873, 881, 889, 945, 9 53, 961, 977, 985, 993, 1001, 1009, 1017, 1025, 1033, 1041, 1049, 1065, 1073, 1081, 1089, 1097, 1105, 1113, 1121, 1129, 1137, 1145, 1153, 1161, 1169, 1 177, 1185, 1193, 1201, 1209, 1217, 1225, 1233, 1241, 1249, 1257, 1265, 1273, 1281, 1289, 1297, 1305, 1313, 1321, 1329, 1337, 1345, 1353, 1361, 1369, 1377, 1385, 1393, 1401, 1409, 1417, 1425, 1433, 1441, 1449, 1457, 1465, 1473, 1481, 1489, 1497, 1505, 1513, 1521, 1529, 1545, 1553, 1561, 1569, 1577, 1585, 1593, 1601, 1609, 1617, 1625, 1633, 1649, 1657, 1673, 1681, 1689, 1697, 1705, 1713, 1721, 1729, 1737, 1745, 1753, 1761, 1769, 1777, 1785, 1793, 1801, 1809, 1817, 1833, 1841, 1849, 1857, 1865, 1873, 1881, 1889, 1913, 1937, 1945, 1953, 1961, 1969, 1977, 1985, 1993, 2001, 2009, 2017, 2025, 2033,V selected from any one of 2041, 2049, 2057, 2065, 2073, 2081, 2089, 2097, 2105, 2113, 2121, 2129, 2137, 2145, 2153, 2161, 2169, 2177, 2185, 2193, 2201, 2209, 2217, 2225, 2233, 2241, 2249, 2257, 2265, 2273, 2281, 2297, 2305, 2313, 2321, 2329, 2337, and 2345; L and / or comprising the amino acid sequences CDR1, CDR2, and CDR3; Each of VH-1 and VH-3 is independently selected from SEQ ID NOs: 5, 13, 21, 29, 37, 45, 53, 61, 69, 77, 85, 93, 101, 109, 117, 125, 133, 149, 157, 165, 173, 181, 197, 205, 237, 245, 261, 277, 285, 293, 301, 309, 317, 325, 333, 341, 349, 357, 365, 373, 381, 389, 397, 405, 413, 421, 429, 437, 445, 453, 461, 469, 485, 493, 501, 525, 533, 541, 549, 557 , 565, 613, 621, 685, 693, 701, 709, 717, 725, 733, 741, 749, 757, 765, 773, 781, 789, 797, 805, 813, 821, 829, 837, 845, 853, 861, 869, 877, 885, 893, 949, 957, 965, 981, 989, 997, 1005, 1013, 1021, 1029, 1037, 1045, 1053, 1069, 1077, 1085, 1093, 1101, 1109, 1117, 1125, 1133, 1141, 1149, 1157, 1165, 1173, 1181, 1189, 1197, 1205, 1213, 1221, 1229, 1237, 1245, 1253, 1261, 1269, 1277, 1285, 1293, 1301, 1309, 1317, 1325, 1333, 1341, 1349, 1357, 1365, 1373, 1381, 1389, 1397, 1405, 1413, 1421, 1429, 1437, 1445, 1453, 1461, 1469, 1477, 1485, 1493, 1501, 1509, 1517, 1525, 1533, 1549, 1557, 1565, 1573, 1581, 1589, 1597, 1605, 1613, 1621, 1629, 1637, 1653, 1661, 1677, 1685, 1693, 1701, 1709, 1717, 1725, 1733, 1741, 1749, 1757, 1765, 1773, 1781, 1789, 1797, 1805, 1813, 1821, 1837, 1845, 1853, 1861, 1869, 1877, 1885, 1893, 1917, 1941, 1949, 1957, 1965, 1973, 1981, 1989, 1997, 2005, 2013, 2021, 2029, 2037,V selected from any one of 2045, 2053, 2061, 2069, 2077, 2085, 2093, 2101, 2109, 2117, 2125, 2133, 2141, 2149, 2157, 2165, 2173, 2181, 2189, 2197, 2205, 2213, 2221, 2229, 2237, 2245, 2253, 2261, 2269, 2277, 2285, 2301, 2309, 2317, 2325, 2333, 2341, and 2349; H and / or comprising the amino acid sequences CDR1, CDR2, and CDR3; VL-2 is selected from the group consisting of SEQ ID NOs: 17, 25, 33, 41, 121, 137, 169, 177, 185, 193, 201, 209, 217, 225, 233, 241, 249, 257, 265, 321, 329, 337, 393, 401, 409, 473, 481, 489, 497, 505, 513, 545, 553, 561, 569, 577, 585, 593, 601, 625, 633, 641, 649, 657, 665, 673, 681, 689, V selected from any one of 697, 705, 713, 721, 729, 737, 745, 753, 761, 769, 785, 793, 801, 809, 817, 849, 857, 865, 873, 881, 889, 897, 905, 913, 921, 929, 937, 945, 969, 977, 1009, 1057, 1537, 1569, 1601, 1641, 1665, 1825, 1865, 1897, 1905, 1913, 1921, 1929, 2265, 2281, 2289, 2329, and 2345 L and / or comprising the amino acid sequences CDR1, CDR2, and CDR3; VH-2 is selected from the group consisting of SEQ ID NOs: 21, 29, 37, 45, 125, 141, 173, 181, 189, 197, 205, 213, 221, 229, 237, 245, 253, 261, 269, 325, 333, 341, 397, 405, 413, 477, 485, 493, 501, 509, 517, 549, 557, 565, 573, 581, 589, 597, 605, 629, 637, 645, 653, 661, 669, 677, 685, 693, 701, 709, 717, 725, 73 3, 741, 749, 757, 765, 773, 789, 797, 805, 813, 821, 853, 861, 869, 877, 885, 893, 901, 909, 917, 925, 933, 941, 949, 973, 981, 1013, 1061, 1541, 1573, 1605, 1645, 1669, 1829, 1869, 1901, 1909, 1917, 1925, 1933, 2269, 2285, 2293, 2333, and 2349. H The amino acid sequences include CDR1, CDR2, and CDR3 sequences, Heterodimeric multispecific antibodies.

7. VH-1 or VH-3 is selected from the group consisting of SEQ ID NOs: 5, 13, 21, 29, 37, 45, 53, 61, 69, 77, 85, 93, 101, 109, 117, 125, 133, 149, 157, 165, 173, 181, 197, 205, 237, 245, 261, 277, 285, 293, 301, 309, 317, 325, 333, 341, 349, 357, 365, 373, 381, 389, 397, 405, 413, 421, 429, 437, 445, 453, 461, 469, 485, 493, 501, 525, 533, 541, 549, 557, 565, 6 13, 621, 685, 693, 701, 709, 717, 725, 733, 741, 749, 757, 765, 773, 781, 789, 797, 805, 813, 821, 829, 837, 845, 853, 861, 869, 877, 885, 893, 949, 957, 96 5, 981, 989, 997, 1005, 1013, 1021, 1029, 1037, 1045, 1053, 1069, 1077, 1085, 1093, 1101, 1109, 1117, 1125, 1133, 1141, 1149, 1157, 1165, 1173, 1181, 1 189, 1197, 1205, 1213, 1221, 1229, 1237, 1245, 1253, 1261, 1269, 1277, 1285, 1293, 1301, 1309, 1317, 1325, 1333, 1341, 1349, 1357, 1365, 1373, 1381, 1389, 1397, 1405, 1413, 1421, 1429, 1437, 1445, 1453, 1461, 1469, 1477, 1485, 1493, 1501, 1509, 1517, 1525, 1533, 1549, 1557, 1565, 1573, 1581, 1589, 1597, 1605, 1613, 1621, 1629, 1637, 1653, 1661, 1677, 1685, 1693, 1701, 1709, 1717, 1725, 1733, 1741, 1749, 1757, 1765, 1773, 1781, 1789, 1797, 1805, 1813, 1821, 1837, 1845, 1853, 1861, 1869, 1877, 1885, 1893, 1917, 1941, 1949, 1957, 1965, 1973, 1981, 1989, 1997, 2005, 2013, 2021, 2029, 2037, 2045,V selected from any one of 2053, 2061, 2069, 2077, 2085, 2093, 2101, 2109, 2117, 2125, 2133, 2141, 2149, 2157, 2165, 2173, 2181, 2189, 2197, 2205, 2213, 2221, 2229, 2237, 2245, 2253, 2261, 2269, 2277, 2285, 2301, 2309, 2317, 2325, 2333, 2341, and 2349; H and / or VL-1 or VL-3 is selected from the group consisting of SEQ ID NOs: 1, 9, 17, 25, 33, 41, 49, 57, 65, 73, 81, 89, 97, 105, 113, 121, 129, 145, 153, 161, 169, 177, 193, 201, 233, 241, 257, 273, 281, 289, 297 , 305, 313, 321, 329, 337, 345, 353, 361, 369, 377, 385, 393, 401, 409, 417, 425, 433, 441, 449, 457, 465, 481, 489, 497, 521, 529, 537, 545, 553, 561, 609, 617, 681, 689, 697, 705, 713, 721, 729, 737, 745, 753, 761, 769, 777, 785, 793, 801, 809, 817, 825, 833, 841, 849, 857, 865, 873, 881, 889, 945, 953, 961, 9 77, 985, 993, 1001, 1009, 1017, 1025, 1033, 1041, 1049, 1065, 1073, 1081, 1089, 1097, 1105, 1113, 1121, 1129, 1137, 1145, 1153, 1161, 1169, 1177, 1185 , 1193, 1201, 1209, 1217, 1225, 1233, 1241, 1249, 1257, 1265, 1273, 1281, 1289, 1297, 1305, 1313, 1321, 1329, 1337, 1345, 1353, 1361, 1369, 1377, 1385 , 1393, 1401, 1409, 1417, 1425, 1433, 1441, 1449, 1457, 1465, 1473, 1481, 1489, 1497, 1505, 1513, 1521, 1529, 1545, 1553, 1561, 1569, 1577, 1585, 1593 , 1601, 1609, 1617, 1625, 1633, 1649, 1657, 1673, 1681, 1689, 1697, 1705, 1713, 1721, 1729, 1737, 1745, 1753, 1761, 1769, 1777, 1785, 1793, 1801, 1809 , 1817, 1833, 1841, 1849, 1857, 1865, 1873, 1881, 1889, 1913, 1937, 1945, 1953, 1961, 1969, 1977, 1985, 1993, 2001, 2009, 2017, 2025, 2033, 2041, 2049,V selected from any one of 2057, 2065, 2073, 2081, 2089, 2097, 2105, 2113, 2121, 2129, 2137, 2145, 2153, 2161, 2169, 2177, 2185, 2193, 2201, 2209, 2217, 2225, 2233, 2241, 2249, 2257, 2265, 2273, 2281, 2297, 2305, 2313, 2321, 2329, 2337, and 2345; L The heterodimeric multispecific antibody of any one of claims 1 to 6, comprising the amino acid sequence

8. VH-2 or VH-4 is selected from the group consisting of SEQ ID NOs: 21, 29, 37, 45, 125, 141, 173, 181, 189, 197, 205, 213, 221, 229, 237, 245, 253, 261, 269, 325, 333, 341, 397, 405, 413, 477, 485, 493, 501, 509, 517, 549, 557, 565, 573, 581, 589, 597, 605, 629, 637, 645, 653, 661, 669, 677, 685, 693, 701, 709, 717, 725 , 733, 741, 749, 757, 765, 773, 789, 797, 805, 813, 821, 853, 861, 869, 877, 885, 893, 901, 909, 917, 925, 933, 941, 949, 973, 981, 1013, 1061, 1541, 1573, 1605, 1645, 1669, 1829, 1869, 1901, 1909, 1917, 1925, 1933, 2269, 2285, 2293, 2333, and 2349. H and / or VL-2 or VL-4 is selected from the group consisting of SEQ ID NOs: 17, 25, 33, 41, 121, 137, 169, 177, 185, 193, 201, 209, 217, 225, 233, 241, 249, 257, 265, 321, 329, 337, 393, 401, 409, 473, 481, 489, 497, 505, 513, 545, 553, 561, 569, 577, 585, 593, 601, 625, 633, 641, 649, 657, 665, 673, 681, 6 89, 697, 705, 713, 721, 729, 737, 745, 753, 761, 769, 785, 793, 801, 809, 817, 849, 857, 865, 873, 881, 889, 897, 905, 913, 921, 929, 937, 945, 969, 977, 1009, 1057, 1537, 1569, 1601, 1641, 1665, 1825, 1865, 1897, 1905, 1913, 1921, 1929, 2265, 2281, 2289, 2329, and 2345 L The heterodimeric multispecific antibody of any one of claims 1 to 7, comprising the amino acid sequence

9. Each of VL-1 and VH-1 is SEQ ID NO:1 and 5, respectively; SEQ ID NO:9 and 13, respectively; SEQ ID NO:17 and 21, respectively; SEQ ID NO:25 and 29, respectively; SEQ ID NO:33 and 37, respectively; SEQ ID NO:41 and 45, respectively; SEQ ID NO:49 and 53, respectively; SEQ ID NO:57 and 61, respectively; SEQ ID NO:73 and 77, respectively; SEQ ID NO:89 and 93, respectively; SEQ ID NO:97 and 101, respectively; SEQ ID NO:105 and 109, respectively; SEQ ID NO:113 and 117, respectively; SEQ ID NO:1, SEQ ID NOs: 21 and 125; SEQ ID NOs: 129 and 133, respectively; SEQ ID NOs: 145 and 149, respectively; SEQ ID NOs: 161 and 165, respectively; SEQ ID NOs: 169 and 173, respectively; SEQ ID NOs: 177 and 181, respectively; SEQ ID NOs: 193 and 197, respectively; SEQ ID NOs: 201 and 205, respectively; SEQ ID NOs: 233 and 237, respectively; SEQ ID NOs: 241 and 245, respectively; SEQ ID NOs: 257 and 261, respectively; SEQ ID NOs: 273 and 277, respectively; SEQ ID NOs: 281 and 285, respectively; SEQ ID NOs: 289 and 290, respectively. and 293; SEQ ID NOs:297 and 301, respectively; SEQ ID NOs:305 and 309, respectively; SEQ ID NOs:313 and 317, respectively; SEQ ID NOs:321 and 325, respectively; SEQ ID NOs:329 and 333, respectively; SEQ ID NOs:337 and 341, respectively; SEQ ID NOs:345 and 349, respectively; SEQ ID NOs:353 and 357, respectively; SEQ ID NOs:361 and 365, respectively; SEQ ID NOs:369 and 373, respectively; SEQ ID NOs:377 and 381, respectively; SEQ ID NOs:385 and 389, respectively; SEQ ID NOs:393 and 3 SEQ ID NOs: 401 and 405, respectively; SEQ ID NOs: 409 and 413, respectively; SEQ ID NOs: 417 and 421, respectively; SEQ ID NOs: 425 and 429, respectively; SEQ ID NOs: 433 and 437, respectively; SEQ ID NOs: 441 and 445, respectively; SEQ ID NOs: 449 and 453, respectively; SEQ ID NOs: 457 and 461, respectively; SEQ ID NOs: 465 and 469, respectively; SEQ ID NOs: 481 and 485, respectively; SEQ ID NOs: 489 and 493, respectively; SEQ ID NOs: 497 and 501, respectively; SEQ ID NOs: 521 and 525, respectively;SEQ ID NOs:529 and 533, respectively; SEQ ID NOs:537 and 541, respectively; SEQ ID NOs:545 and 549, respectively; SEQ ID NOs:553 and 557, respectively; SEQ ID NOs:561 and 565, respectively; SEQ ID NOs:609 and 613, respectively; SEQ ID NOs:617 and 621, respectively; SEQ ID NOs:681 and 685, respectively; SEQ ID NOs:689 and 693, respectively; SEQ ID NOs:697 and 701, respectively; SEQ ID NOs:705 and 709, respectively; SEQ ID NOs:713 and 717, respectively; SEQ ID NOs:721 and 725, respectively; SEQ ID NOs:729 and 733, respectively; SEQ ID NOs:737 and 741, respectively; SEQ ID NOs:745 and 749, respectively; SEQ ID NOs:753 and 757, respectively; SEQ ID NOs:761 and 765, respectively; SEQ ID NOs:769 and 773, respectively; SEQ ID NOs:785 and 789, respectively; SEQ ID NOs:793 and 797, respectively; SEQ ID NOs:801 and 805, respectively; SEQ ID NOs:809 and 813, respectively; SEQ ID NOs:817 and 821, respectively; SEQ ID NOs:825 and 829, respectively; SEQ ID NOs:833 and 837, respectively; SEQ ID NOs:841 and 845, respectively; SEQ ID NOs:849 and 853, respectively; SEQ ID NOs:857 and 861, respectively; SEQ ID NOs:865 and 869, respectively; SEQ ID NOs:873 and 877, respectively; SEQ ID NOs:881 and 885, respectively; SEQ ID NOs:889 and 893, respectively; SEQ ID NOs:945 and 949, respectively; SEQ ID NOs:953 and 957, respectively; SEQ ID NOs:961 and 965, respectively; SEQ ID NOs:977 and 981, respectively; SEQ ID NOs:985 and 989, respectively; SEQ ID NOs:993 and 997, respectively; SEQ ID NOs: 1001 and 1005, respectively; SEQ ID NOs: 1009 and 1013, respectively; SEQ ID NOs: 1017 and 1021, respectively; SEQ ID NOs: 1025 and 1029, respectively; SEQ ID NOs: 1033 and 1037, respectively; SEQ ID NOs: 1041 and 1045, respectively; SEQ ID NOs: 1065 and 1069, respectively; SEQ ID NOs: 1073 and 1077, respectively; SEQ ID NOs: 1081 and 1085, respectively; SEQ ID NOs: 1089 and 1093, respectively; SEQ ID NOs: 1097 and 1101, respectively; SEQ ID NOs: 1113 and 1117, respectively;SEQ ID NOs:1121 and 1125, respectively; SEQ ID NOs:1129 and 1133, respectively; SEQ ID NOs:1137 and 1141, respectively; SEQ ID NOs:1145 and 1149, respectively; SEQ ID NOs:1153 and 1157, respectively; SEQ ID NOs:1161 and 1165, respectively; SEQ ID NOs:1169 and 1173, respectively; SEQ ID NOs:1185 and 1189, respectively; SEQ ID NOs:1193 and 1197, respectively; SEQ ID NOs:1201 and 1205, respectively; SEQ ID NOs:1209 and 1213, respectively; SEQ ID NOs:1217 and and 1221; SEQ ID NOs: 1225 and 1229, respectively; SEQ ID NOs: 1233 and 1237, respectively; SEQ ID NOs: 1241 and 1245, respectively; SEQ ID NOs: 1249 and 1253, respectively; SEQ ID NOs: 1257 and 1261, respectively; SEQ ID NOs: 1265 and 1269, respectively; SEQ ID NOs: 1273 and 1277, respectively; SEQ ID NOs: 1281 and 1285, respectively; SEQ ID NOs: 1289 and 1293, respectively; SEQ ID NOs: 1297 and 1301, respectively; SEQ ID NOs: 1305 and 1309, respectively; SEQ ID NOs: 1306 and 1309, respectively; SEQ ID NOs: 1371 and 1365, respectively; SEQ ID NOs: 1369 and 1373, respectively; SEQ ID NOs: 1377 and 1381, respectively; SEQ ID NOs: 1385 and 1389, respectively; SEQ ID NOs: 1393 and 1397, respectively; SEQ ID NOs: 1401 and 1405, respectively; SEQ ID NOs:1409 and 1413; SEQ ID NOs:1417 and 1421, respectively; SEQ ID NOs:1433 and 1437, respectively; SEQ ID NOs:1441 and 1445, respectively; SEQ ID NOs:1457 and 1461, respectively; SEQ ID NOs:1465 and 1469, respectively; SEQ ID NOs:1473 and 1477, respectively; SEQ ID NOs:1481 and 1485, respectively; SEQ ID NOs:1489 and 1493, respectively; SEQ ID NOs:1497 and 1501, respectively; SEQ ID NOs:1505 and 1509, respectively; SEQ ID NOs:1513 and 1517, respectively;SEQ ID NOs: 1521 and 1525, respectively; SEQ ID NOs: 1529 and 1533, respectively; SEQ ID NOs: 1545 and 1549, respectively; SEQ ID NOs: 1553 and 1557, respectively; SEQ ID NOs: 1561 and 1565, respectively; SEQ ID NOs: 1569 and 1573, respectively; SEQ ID NOs: 1577 and 1581, respectively; SEQ ID NOs: 1585 and 1589, respectively; SEQ ID NOs: 1593 and 1597, respectively; SEQ ID NOs: 1601 and 1605, respectively; SEQ ID NOs: 1609 and 1613, respectively; SEQ ID NOs: 1617 and SEQ ID NOs:1673 and 1677, respectively; SEQ ID NOs:1681 and 1685, respectively; SEQ ID NOs:1689 and 1693, respectively; SEQ ID NOs:1697 and 1701, respectively; SEQ ID NOs:1705 and 1709, respectively; SEQ ID NOs:1713 and 1717, respectively; SEQ ID NOs:1721 and 1725, respectively; SEQ ID NOs:1722 and 1725, respectively; SEQ ID NOs: 1729 and 1733, respectively; SEQ ID NOs: 1737 and 1741, respectively; SEQ ID NOs: 1745 and 1749, respectively; SEQ ID NOs: 1753 and 1757, respectively; SEQ ID NOs: 1761 and 1765, respectively; SEQ ID NOs: 1769 and 1773, respectively; SEQ ID NOs: 1777 and 1781, respectively; SEQ ID NOs: 1785 and 1789, respectively; SEQ ID NOs: 1793 and 1797, respectively; SEQ ID NOs: 1801 and 1805, respectively; SEQ ID NOs: 1809 and 1813, respectively; SEQ ID NOs: 1817 and 1821, respectively; SEQ ID NOs:1833 and 1837; SEQ ID NOs:1841 and 1845, respectively; SEQ ID NOs:1849 and 1853, respectively; SEQ ID NOs:1857 and 1861, respectively; SEQ ID NOs:1865 and 1869, respectively; SEQ ID NOs:1873 and 1877, respectively; SEQ ID NOs:1881 and 1885, respectively; SEQ ID NOs:1889 and 1893, respectively; SEQ ID NOs:1913 and 1917, respectively; SEQ ID NOs:1937 and 1941, respectively; SEQ ID NOs:1945 and 1949, respectively; SEQ ID NOs:1953 and 1957, respectively;SEQ ID NOs: 1961 and 1965, respectively; SEQ ID NOs: 1969 and 1973, respectively; SEQ ID NOs: 1977 and 1981, respectively; SEQ ID NOs: 1985 and 1989, respectively; SEQ ID NOs: 1993 and 1997, respectively; SEQ ID NOs: 2001 and 2005, respectively; SEQ ID NOs: 2009 and 2013, respectively; SEQ ID NOs: 2017 and 2021, respectively; SEQ ID NOs: 2025 and 2029, respectively; SEQ ID NOs: 2033 and 2037, respectively; SEQ ID NOs: 2041 and 2045, respectively; SEQ ID NOs: 2049 and and 2053; SEQ ID NOs:2057 and 2061, respectively; SEQ ID NOs:2065 and 2069, respectively; SEQ ID NOs:2073 and 2077, respectively; SEQ ID NOs:2081 and 2085, respectively; SEQ ID NOs:2089 and 2093, respectively; SEQ ID NOs:2097 and 2101, respectively; SEQ ID NOs:2105 and 2109, respectively; SEQ ID NOs:2113 and 2117, respectively; SEQ ID NOs:2121 and 2125, respectively; SEQ ID NOs:2129 and 2133, respectively; SEQ ID NOs:2137 and 2141, respectively; SEQ ID NOs:2138 and 2142, respectively. SEQ ID NOs: 2145 and 2149; SEQ ID NOs: 2153 and 2157, respectively; SEQ ID NOs: 2161 and 2165, respectively; SEQ ID NOs: 2169 and 2173, respectively; SEQ ID NOs: 2177 and 2181, respectively; SEQ ID NOs: 2185 and 2189, respectively; SEQ ID NOs: 2193 and 2197, respectively; SEQ ID NOs: 2201 and 2205, respectively; SEQ ID NOs: 2209 and 2213, respectively; SEQ ID NOs: 2217 and 2221, respectively; SEQ ID NOs: 2225 and 2229, respectively; SEQ ID NOs: 2233 and 2237, respectively; V selected from the group consisting of SEQ ID NOs:2241 and 2245, respectively; SEQ ID NOs:2249 and 2253, respectively; SEQ ID NOs:2257 and 2261, respectively; SEQ ID NOs:2273 and 2277, respectively; SEQ ID NOs:2281 and 2285, respectively; SEQ ID NOs:2305 and 2309, respectively; SEQ ID NOs:2313 and 2317, respectively; SEQ ID NOs:2321 and 2325, respectively; SEQ ID NOs:2329 and 2333, respectively; SEQ ID NOs:2337 and 2341, respectively; and SEQ ID NOs:2345 and 2349, respectively; L Amino acid sequence and V H The heterodimeric multispecific antibody of any one of claims 1 to 8, comprising the amino acid sequence

10. Each of the VL-3 and VH-3 is SEQ ID NO:1 and 5, respectively; SEQ ID NO:9 and 13, respectively; SEQ ID NO:17 and 21, respectively; SEQ ID NO:25 and 29, respectively; SEQ ID NO:33 and 37, respectively; SEQ ID NO:41 and 45, respectively; SEQ ID NO:49 and 53, respectively; SEQ ID NO:57 and 61, respectively; SEQ ID NO:73 and 77, respectively; SEQ ID NO:89 and 93, respectively; SEQ ID NO:97 and 101, respectively; SEQ ID NO:105 and 109, respectively; SEQ ID NO:113 and 117, respectively; SEQ ID NO:1, SEQ ID NOs: 21 and 125; SEQ ID NOs: 129 and 133, respectively; SEQ ID NOs: 145 and 149, respectively; SEQ ID NOs: 161 and 165, respectively; SEQ ID NOs: 169 and 173, respectively; SEQ ID NOs: 177 and 181, respectively; SEQ ID NOs: 193 and 197, respectively; SEQ ID NOs: 201 and 205, respectively; SEQ ID NOs: 233 and 237, respectively; SEQ ID NOs: 241 and 245, respectively; SEQ ID NOs: 257 and 261, respectively; SEQ ID NOs: 273 and 277, respectively; SEQ ID NOs: 281 and 285, respectively; SEQ ID NOs: 289 and 290, respectively. and 293; SEQ ID NOs:297 and 301, respectively; SEQ ID NOs:305 and 309, respectively; SEQ ID NOs:313 and 317, respectively; SEQ ID NOs:321 and 325, respectively; SEQ ID NOs:329 and 333, respectively; SEQ ID NOs:337 and 341, respectively; SEQ ID NOs:345 and 349, respectively; SEQ ID NOs:353 and 357, respectively; SEQ ID NOs:361 and 365, respectively; SEQ ID NOs:369 and 373, respectively; SEQ ID NOs:377 and 381, respectively; SEQ ID NOs:385 and 389, respectively; SEQ ID NOs:393 and 3 SEQ ID NOs: 401 and 405, respectively; SEQ ID NOs: 409 and 413, respectively; SEQ ID NOs: 417 and 421, respectively; SEQ ID NOs: 425 and 429, respectively; SEQ ID NOs: 433 and 437, respectively; SEQ ID NOs: 441 and 445, respectively; SEQ ID NOs: 449 and 453, respectively; SEQ ID NOs: 457 and 461, respectively; SEQ ID NOs: 465 and 469, respectively; SEQ ID NOs: 481 and 485, respectively; SEQ ID NOs: 489 and 493, respectively; SEQ ID NOs: 497 and 501, respectively; SEQ ID NOs: 521 and 525, respectively;SEQ ID NOs:529 and 533, respectively; SEQ ID NOs:537 and 541, respectively; SEQ ID NOs:545 and 549, respectively; SEQ ID NOs:553 and 557, respectively; SEQ ID NOs:561 and 565, respectively; SEQ ID NOs:609 and 613, respectively; SEQ ID NOs:617 and 621, respectively; SEQ ID NOs:681 and 685, respectively; SEQ ID NOs:689 and 693, respectively; SEQ ID NOs:697 and 701, respectively; SEQ ID NOs:705 and 709, respectively; SEQ ID NOs:713 and 717, respectively; SEQ ID NOs:721 and 725, respectively; SEQ ID NOs:729 and 733, respectively; SEQ ID NOs:737 and 741, respectively; SEQ ID NOs:745 and 749, respectively; SEQ ID NOs:753 and 757, respectively; SEQ ID NOs:761 and 765, respectively; SEQ ID NOs:769 and 773, respectively; SEQ ID NOs:785 and 789, respectively; SEQ ID NOs:793 and 797, respectively; SEQ ID NOs:801 and 805, respectively; SEQ ID NOs:809 and 813, respectively; SEQ ID NOs:817 and 821, respectively; SEQ ID NOs:825 and 829, respectively; SEQ ID NOs:833 and 837, respectively; SEQ ID NOs:841 and 845, respectively; SEQ ID NOs:849 and 853, respectively; SEQ ID NOs:857 and 861, respectively; SEQ ID NOs:865 and 869, respectively; SEQ ID NOs:873 and 877, respectively; SEQ ID NOs:881 and 885, respectively; SEQ ID NOs:889 and 893, respectively; SEQ ID NOs:945 and 949, respectively; SEQ ID NOs:953 and 957, respectively; SEQ ID NOs:961 and 965, respectively; SEQ ID NOs:977 and 981, respectively; SEQ ID NOs:985 and 989, respectively; SEQ ID NOs:993 and 997, respectively; SEQ ID NOs: 1001 and 1005, respectively; SEQ ID NOs: 1009 and 1013, respectively; SEQ ID NOs: 1017 and 1021, respectively; SEQ ID NOs: 1025 and 1029, respectively; SEQ ID NOs: 1033 and 1037, respectively; SEQ ID NOs: 1041 and 1045, respectively; SEQ ID NOs: 1065 and 1069, respectively; SEQ ID NOs: 1073 and 1077, respectively; SEQ ID NOs: 1081 and 1085, respectively; SEQ ID NOs: 1089 and 1093, respectively; SEQ ID NOs: 1097 and 1101, respectively; SEQ ID NOs: 1113 and 1117, respectively;SEQ ID NOs:1121 and 1125, respectively; SEQ ID NOs:1129 and 1133, respectively; SEQ ID NOs:1137 and 1141, respectively; SEQ ID NOs:1145 and 1149, respectively; SEQ ID NOs:1153 and 1157, respectively; SEQ ID NOs:1161 and 1165, respectively; SEQ ID NOs:1169 and 1173, respectively; SEQ ID NOs:1185 and 1189, respectively; SEQ ID NOs:1193 and 1197, respectively; SEQ ID NOs:1201 and 1205, respectively; SEQ ID NOs:1209 and 1213, respectively; SEQ ID NOs:1217 and and 1221; SEQ ID NOs: 1225 and 1229, respectively; SEQ ID NOs: 1233 and 1237, respectively; SEQ ID NOs: 1241 and 1245, respectively; SEQ ID NOs: 1249 and 1253, respectively; SEQ ID NOs: 1257 and 1261, respectively; SEQ ID NOs: 1265 and 1269, respectively; SEQ ID NOs: 1273 and 1277, respectively; SEQ ID NOs: 1281 and 1285, respectively; SEQ ID NOs: 1289 and 1293, respectively; SEQ ID NOs: 1297 and 1301, respectively; SEQ ID NOs: 1305 and 1309, respectively; SEQ ID NOs: 1306 and 1309, respectively; SEQ ID NOs: 1371 and 1365, respectively; SEQ ID NOs: 1369 and 1373, respectively; SEQ ID NOs: 1377 and 1381, respectively; SEQ ID NOs: 1385 and 1389, respectively; SEQ ID NOs: 1393 and 1397, respectively; SEQ ID NOs: 1401 and 1405, respectively; SEQ ID NOs:1409 and 1413; SEQ ID NOs:1417 and 1421, respectively; SEQ ID NOs:1433 and 1437, respectively; SEQ ID NOs:1441 and 1445, respectively; SEQ ID NOs:1457 and 1461, respectively; SEQ ID NOs:1465 and 1469, respectively; SEQ ID NOs:1473 and 1477, respectively; SEQ ID NOs:1481 and 1485, respectively; SEQ ID NOs:1489 and 1493, respectively; SEQ ID NOs:1497 and 1501, respectively; SEQ ID NOs:1505 and 1509, respectively; SEQ ID NOs:1513 and 1517, respectively;SEQ ID NOs: 1521 and 1525, respectively; SEQ ID NOs: 1529 and 1533, respectively; SEQ ID NOs: 1545 and 1549, respectively; SEQ ID NOs: 1553 and 1557, respectively; SEQ ID NOs: 1561 and 1565, respectively; SEQ ID NOs: 1569 and 1573, respectively; SEQ ID NOs: 1577 and 1581, respectively; SEQ ID NOs: 1585 and 1589, respectively; SEQ ID NOs: 1593 and 1597, respectively; SEQ ID NOs: 1601 and 1605, respectively; SEQ ID NOs: 1609 and 1613, respectively; SEQ ID NOs: 1617 and SEQ ID NOs:1673 and 1677, respectively; SEQ ID NOs:1681 and 1685, respectively; SEQ ID NOs:1689 and 1693, respectively; SEQ ID NOs:1697 and 1701, respectively; SEQ ID NOs:1705 and 1709, respectively; SEQ ID NOs:1713 and 1717, respectively; SEQ ID NOs:1721 and 1725, respectively; SEQ ID NOs:1722 and 1725, respectively; SEQ ID NOs: 1729 and 1733, respectively; SEQ ID NOs: 1737 and 1741, respectively; SEQ ID NOs: 1745 and 1749, respectively; SEQ ID NOs: 1753 and 1757, respectively; SEQ ID NOs: 1761 and 1765, respectively; SEQ ID NOs: 1769 and 1773, respectively; SEQ ID NOs: 1777 and 1781, respectively; SEQ ID NOs: 1785 and 1789, respectively; SEQ ID NOs: 1793 and 1797, respectively; SEQ ID NOs: 1801 and 1805, respectively; SEQ ID NOs: 1809 and 1813, respectively; SEQ ID NOs: 1817 and 1821, respectively; SEQ ID NOs:1833 and 1837; SEQ ID NOs:1841 and 1845, respectively; SEQ ID NOs:1849 and 1853, respectively; SEQ ID NOs:1857 and 1861, respectively; SEQ ID NOs:1865 and 1869, respectively; SEQ ID NOs:1873 and 1877, respectively; SEQ ID NOs:1881 and 1885, respectively; SEQ ID NOs:1889 and 1893, respectively; SEQ ID NOs:1913 and 1917, respectively; SEQ ID NOs:1937 and 1941, respectively; SEQ ID NOs:1945 and 1949, respectively; SEQ ID NOs:1953 and 1957, respectively;SEQ ID NOs: 1961 and 1965, respectively; SEQ ID NOs: 1969 and 1973, respectively; SEQ ID NOs: 1977 and 1981, respectively; SEQ ID NOs: 1985 and 1989, respectively; SEQ ID NOs: 1993 and 1997, respectively; SEQ ID NOs: 2001 and 2005, respectively; SEQ ID NOs: 2009 and 2013, respectively; SEQ ID NOs: 2017 and 2021, respectively; SEQ ID NOs: 2025 and 2029, respectively; SEQ ID NOs: 2033 and 2037, respectively; SEQ ID NOs: 2041 and 2045, respectively; SEQ ID NOs: 2049 and and 2053; SEQ ID NOs:2057 and 2061, respectively; SEQ ID NOs:2065 and 2069, respectively; SEQ ID NOs:2073 and 2077, respectively; SEQ ID NOs:2081 and 2085, respectively; SEQ ID NOs:2089 and 2093, respectively; SEQ ID NOs:2097 and 2101, respectively; SEQ ID NOs:2105 and 2109, respectively; SEQ ID NOs:2113 and 2117, respectively; SEQ ID NOs:2121 and 2125, respectively; SEQ ID NOs:2129 and 2133, respectively; SEQ ID NOs:2137 and 2141, respectively; SEQ ID NOs:2138 and 2142, respectively. SEQ ID NOs: 2145 and 2149; SEQ ID NOs: 2153 and 2157, respectively; SEQ ID NOs: 2161 and 2165, respectively; SEQ ID NOs: 2169 and 2173, respectively; SEQ ID NOs: 2177 and 2181, respectively; SEQ ID NOs: 2185 and 2189, respectively; SEQ ID NOs: 2193 and 2197, respectively; SEQ ID NOs: 2201 and 2205, respectively; SEQ ID NOs: 2209 and 2213, respectively; SEQ ID NOs: 2217 and 2221, respectively; SEQ ID NOs: 2225 and 2229, respectively; SEQ ID NOs: 2233 and 2237, respectively; V selected from the group consisting of SEQ ID NOs:2241 and 2245, respectively; SEQ ID NOs:2249 and 2253, respectively; SEQ ID NOs:2257 and 2261, respectively; SEQ ID NOs:2273 and 2277, respectively; SEQ ID NOs:2281 and 2285, respectively; SEQ ID NOs:2305 and 2309, respectively; SEQ ID NOs:2313 and 2317, respectively; SEQ ID NOs:2321 and 2325, respectively; SEQ ID NOs:2329 and 2333, respectively; SEQ ID NOs:2337 and 2341, respectively; and SEQ ID NOs:2345 and 2349, respectively; L Amino acid sequence and V H The heterodimeric multispecific antibody of any one of claims 1 to 9, comprising the amino acid sequence

11. Each of the VL-1 and VH-1 are SEQ ID NOs:9 and 13, respectively; SEQ ID NOs:49 and 53, respectively; SEQ ID NOs:57 and 61, respectively; SEQ ID NOs:65 and 69, respectively; SEQ ID NOs:81 and 85, respectively; SEQ ID NOs:153 and 157, respectively; SEQ ID NOs:161 and 165, respectively; SEQ ID NOs:193 and 197, respectively; SEQ ID NOs:201 and 205, respectively; SEQ ID NOs:273 and 277, respectively; SEQ ID NOs:281 and 285, respectively; SEQ ID NOs:289 and 293, respectively; SEQ ID NOs:297 and 298, respectively. and 301; SEQ ID NOs:305 and 309, respectively; SEQ ID NOs:313 and 317, respectively; SEQ ID NOs:361 and 365, respectively; SEQ ID NOs:377 and 381, respectively; SEQ ID NOs:393 and 397, respectively; SEQ ID NOs:401 and 405, respectively; SEQ ID NOs:409 and 413, respectively; SEQ ID NOs:417 and 421, respectively; SEQ ID NOs:425 and 429, respectively; SEQ ID NOs:433 and 437, respectively; SEQ ID NOs:441 and 445, respectively; SEQ ID NOs:449 and 453, respectively; SEQ ID NOs:457 and 460, respectively. and 461; SEQ ID NOs:465 and 469, respectively; SEQ ID NOs:681 and 685, respectively; SEQ ID NOs:689 and 693, respectively; SEQ ID NOs:697 and 701, respectively; SEQ ID NOs:705 and 709, respectively; SEQ ID NOs:713 and 717, respectively; SEQ ID NOs:721 and 725, respectively; SEQ ID NOs:737 and 741, respectively; SEQ ID NOs:745 and 749, respectively; SEQ ID NOs:753 and 757, respectively; SEQ ID NOs:761 and 765, respectively; SEQ ID NOs:777 and 781, respectively; SEQ ID NO:825, respectively. and 829; SEQ ID NOs:833 and 837, respectively; SEQ ID NOs:841 and 845, respectively; SEQ ID NOs:953 and 957, respectively; SEQ ID NOs:961 and 965, respectively; SEQ ID NOs:977 and 981, respectively; SEQ ID NOs:993 and 997, respectively; SEQ ID NOs:1001 and 1005, respectively; SEQ ID NOs:1009 and 1013, respectively; SEQ ID NOs:1017 and 1021, respectively; SEQ ID NOs:1033 and 1037, respectively; SEQ ID NOs:1049 and 1053, respectively; SEQ ID NOs:1073 and 1077, respectively;SEQ ID NOs: 1081 and 1085, respectively; SEQ ID NOs: 1089 and 1093, respectively; SEQ ID NOs: 1105 and 1109, respectively; SEQ ID NOs: 1129 and 1133, respectively; SEQ ID NOs: 1137 and 1141, respectively; SEQ ID NOs: 1153 and 1157, respectively; SEQ ID NOs: 1161 and 1165, respectively; SEQ ID NOs: 1177 and 1181, respectively; SEQ ID NOs: 1225 and 1229, respectively; SEQ ID NOs: 1241 and 1245, respectively; SEQ ID NOs: 1257 and 1261, respectively; SEQ ID NOs: 1265 and and 1269; SEQ ID NOs: 1297 and 1301, respectively; SEQ ID NOs: 1393 and 1397, respectively; SEQ ID NOs: 1409 and 1413, respectively; SEQ ID NOs: 1425 and 1429, respectively; SEQ ID NOs: 1441 and 1445, respectively; SEQ ID NOs: 1449 and 1453, respectively; SEQ ID NOs: 1457 and 1461, respectively; SEQ ID NOs: 1465 and 1469, respectively; SEQ ID NOs: 1473 and 1477, respectively; SEQ ID NOs: 1481 and 1485, respectively; SEQ ID NOs: 1497 and 1501, respectively; SEQ ID NOs: 1 SEQ ID NOs: 1505 and 1509; SEQ ID NOs: 1513 and 1517, respectively; SEQ ID NOs: 1521 and 1525, respectively; SEQ ID NOs: 1529 and 1533, respectively; SEQ ID NOs: 1545 and 1549, respectively; SEQ ID NOs: 1553 and 1557, respectively; SEQ ID NOs: 1561 and 1565, respectively; SEQ ID NOs: 1569 and 1573, respectively; SEQ ID NOs: 1577 and 1581, respectively; SEQ ID NOs: 1585 and 1589, respectively; SEQ ID NOs: 1609 and 1613, respectively; SEQ ID NOs: 1617 and 1621, respectively; SEQ ID NOs:1649 and 1653, respectively; SEQ ID NOs:1657 and 1661, respectively; SEQ ID NOs:1673 and 1677, respectively; SEQ ID NOs:1689 and 1693, respectively; SEQ ID NOs:1697 and 1701, respectively; SEQ ID NOs:1705 and 1709, respectively; SEQ ID NOs:1713 and 1717, respectively; SEQ ID NOs:1721 and 1725, respectively; SEQ ID NOs:1729 and 1733, respectively; SEQ ID NOs:1745 and 1749, respectively; SEQ ID NOs:1753 and 1757, respectively; SEQ ID NOs:1761 and 1765, respectively;SEQ ID NOs:1769 and 1773, respectively; SEQ ID NOs:1777 and 1781, respectively; SEQ ID NOs:1785 and 1789, respectively; SEQ ID NOs:1793 and 1797, respectively; SEQ ID NOs:1817 and 1821, respectively; SEQ ID NOs:1833 and 1837, respectively; SEQ ID NOs:1841 and 1845, respectively; SEQ ID NOs:1849 and 1853, respectively; SEQ ID NOs:1857 and 1861, respectively; SEQ ID NOs:1865 and 1869, respectively; SEQ ID NOs:1889 and 1893, respectively. V selected from the group consisting of SEQ ID NOs:2257 and 2261, respectively; SEQ ID NOs:2265 and 2269, respectively; SEQ ID NOs:2281 and 2285, respectively; SEQ ID NOs:2297 and 2301, respectively; SEQ ID NOs:2305 and 2309, respectively; SEQ ID NOs:2313 and 2317, respectively; SEQ ID NOs:2321 and 2325, respectively; SEQ ID NOs:2329 and 2333, respectively; SEQ ID NOs:2337 and 2341, respectively; and SEQ ID NOs:2345 and 2349, respectively; L Amino acid sequence and V H The heterodimeric multispecific antibody of any one of claims 1 to 8, comprising the amino acid sequence

12. Each of the VL-3 and VH-3 is SEQ ID NO:9 and 13, respectively; SEQ ID NO:49 and 53, respectively; SEQ ID NO:57 and 61, respectively; SEQ ID NO:65 and 69, respectively; SEQ ID NO:81 and 85, respectively; SEQ ID NO:153 and 157, respectively; SEQ ID NO:161 and 165, respectively; SEQ ID NO:193 and 197, respectively; SEQ ID NO:201 and 205, respectively; SEQ ID NO:273 and 277, respectively; SEQ ID NO:281 and 285, respectively; SEQ ID NO:289 and 293, respectively; SEQ ID NO:297 and and 301; SEQ ID NOs:305 and 309, respectively; SEQ ID NOs:313 and 317, respectively; SEQ ID NOs:361 and 365, respectively; SEQ ID NOs:377 and 381, respectively; SEQ ID NOs:393 and 397, respectively; SEQ ID NOs:401 and 405, respectively; SEQ ID NOs:409 and 413, respectively; SEQ ID NOs:417 and 421, respectively; SEQ ID NOs:425 and 429, respectively; SEQ ID NOs:433 and 437, respectively; SEQ ID NOs:441 and 445, respectively; SEQ ID NOs:449 and 453, respectively; SEQ ID NOs:457 and 460, respectively. and 461; SEQ ID NOs:465 and 469, respectively; SEQ ID NOs:681 and 685, respectively; SEQ ID NOs:689 and 693, respectively; SEQ ID NOs:697 and 701, respectively; SEQ ID NOs:705 and 709, respectively; SEQ ID NOs:713 and 717, respectively; SEQ ID NOs:721 and 725, respectively; SEQ ID NOs:737 and 741, respectively; SEQ ID NOs:745 and 749, respectively; SEQ ID NOs:753 and 757, respectively; SEQ ID NOs:761 and 765, respectively; SEQ ID NOs:777 and 781, respectively; SEQ ID NO:825, respectively. and 829; SEQ ID NOs:833 and 837, respectively; SEQ ID NOs:841 and 845, respectively; SEQ ID NOs:953 and 957, respectively; SEQ ID NOs:961 and 965, respectively; SEQ ID NOs:977 and 981, respectively; SEQ ID NOs:993 and 997, respectively; SEQ ID NOs:1001 and 1005, respectively; SEQ ID NOs:1009 and 1013, respectively; SEQ ID NOs:1017 and 1021, respectively; SEQ ID NOs:1033 and 1037, respectively; SEQ ID NOs:1049 and 1053, respectively; SEQ ID NOs:1073 and 1077, respectively;SEQ ID NOs: 1081 and 1085, respectively; SEQ ID NOs: 1089 and 1093, respectively; SEQ ID NOs: 1105 and 1109, respectively; SEQ ID NOs: 1129 and 1133, respectively; SEQ ID NOs: 1137 and 1141, respectively; SEQ ID NOs: 1153 and 1157, respectively; SEQ ID NOs: 1161 and 1165, respectively; SEQ ID NOs: 1177 and 1181, respectively; SEQ ID NOs: 1225 and 1229, respectively; SEQ ID NOs: 1241 and 1245, respectively; SEQ ID NOs: 1257 and 1261, respectively; SEQ ID NOs: 1265 and and 1269; SEQ ID NOs: 1297 and 1301, respectively; SEQ ID NOs: 1393 and 1397, respectively; SEQ ID NOs: 1409 and 1413, respectively; SEQ ID NOs: 1425 and 1429, respectively; SEQ ID NOs: 1441 and 1445, respectively; SEQ ID NOs: 1449 and 1453, respectively; SEQ ID NOs: 1457 and 1461, respectively; SEQ ID NOs: 1465 and 1469, respectively; SEQ ID NOs: 1473 and 1477, respectively; SEQ ID NOs: 1481 and 1485, respectively; SEQ ID NOs: 1497 and 1501, respectively; SEQ ID NOs: 1 SEQ ID NOs: 1505 and 1509; SEQ ID NOs: 1513 and 1517, respectively; SEQ ID NOs: 1521 and 1525, respectively; SEQ ID NOs: 1529 and 1533, respectively; SEQ ID NOs: 1545 and 1549, respectively; SEQ ID NOs: 1553 and 1557, respectively; SEQ ID NOs: 1561 and 1565, respectively; SEQ ID NOs: 1569 and 1573, respectively; SEQ ID NOs: 1577 and 1581, respectively; SEQ ID NOs: 1585 and 1589, respectively; SEQ ID NOs: 1609 and 1613, respectively; SEQ ID NOs: 1617 and 1621, respectively; SEQ ID NOs:1649 and 1653, respectively; SEQ ID NOs:1657 and 1661, respectively; SEQ ID NOs:1673 and 1677, respectively; SEQ ID NOs:1689 and 1693, respectively; SEQ ID NOs:1697 and 1701, respectively; SEQ ID NOs:1705 and 1709, respectively; SEQ ID NOs:1713 and 1717, respectively; SEQ ID NOs:1721 and 1725, respectively; SEQ ID NOs:1729 and 1733, respectively; SEQ ID NOs:1745 and 1749, respectively; SEQ ID NOs:1753 and 1757, respectively; SEQ ID NOs:1761 and 1765, respectively;SEQ ID NOs:1769 and 1773, respectively; SEQ ID NOs:1777 and 1781, respectively; SEQ ID NOs:1785 and 1789, respectively; SEQ ID NOs:1793 and 1797, respectively; SEQ ID NOs:1817 and 1821, respectively; SEQ ID NOs:1833 and 1837, respectively; SEQ ID NOs:1841 and 1845, respectively; SEQ ID NOs:1849 and 1853, respectively; SEQ ID NOs:1857 and 1861, respectively; SEQ ID NOs:1865 and 1869, respectively; SEQ ID NOs:1889 and 1893, respectively. V selected from the group consisting of SEQ ID NOs:2257 and 2261, respectively; SEQ ID NOs:2265 and 2269, respectively; SEQ ID NOs:2281 and 2285, respectively; SEQ ID NOs:2297 and 2301, respectively; SEQ ID NOs:2305 and 2309, respectively; SEQ ID NOs:2313 and 2317, respectively; SEQ ID NOs:2321 and 2325, respectively; SEQ ID NOs:2329 and 2333, respectively; SEQ ID NOs:2337 and 2341, respectively; and SEQ ID NOs:2345 and 2349, respectively; L Amino acid sequence and V H 12. The heterodimeric multispecific antibody of any one of claims 1 to 8 or 11, comprising the amino acid sequence

13. Each of VL-2 and VH-2 is SEQ ID NO:17 and 21, respectively; SEQ ID NO:25 and 29, respectively; SEQ ID NO:33 and 37, respectively; SEQ ID NO:41 and 45, respectively; SEQ ID NO:121 and 125, respectively; SEQ ID NO:137 and 141, respectively; SEQ ID NO:169 and 173, respectively; SEQ ID NO:177 and 181, respectively; SEQ ID NO:185 and 189, respectively; SEQ ID NO:193 and 197, respectively; SEQ ID NO:201 and 205, respectively; SEQ ID NO:209 and 213, respectively; SEQ ID NO:217 and 220, respectively. and 221; SEQ ID NOs:225 and 229, respectively; SEQ ID NOs:233 and 237, respectively; SEQ ID NOs:241 and 245, respectively; SEQ ID NOs:249 and 253, respectively; SEQ ID NOs:257 and 261, respectively; SEQ ID NOs:265 and 269, respectively; SEQ ID NOs:321 and 325, respectively; SEQ ID NOs:329 and 333, respectively; SEQ ID NOs:337 and 341, respectively; SEQ ID NOs:393 and 397, respectively; SEQ ID NOs:401 and 405, respectively; SEQ ID NOs:409 and 413, respectively; SEQ ID NOs:473 and SEQ ID NOs: 481 and 485, respectively; SEQ ID NOs: 489 and 493, respectively; SEQ ID NOs: 497 and 501, respectively; SEQ ID NOs: 505 and 509, respectively; SEQ ID NOs: 513 and 517, respectively; SEQ ID NOs: 545 and 549, respectively; SEQ ID NOs: 553 and 557, respectively; SEQ ID NOs: 561 and 565, respectively; SEQ ID NOs: 569 and 573, respectively; SEQ ID NOs: 577 and 581, respectively; SEQ ID NOs: 585 and 589, respectively; SEQ ID NOs: 593 and 597, respectively; SEQ ID NOs: 601 and 60 SEQ ID NOs:625 and 629, respectively; SEQ ID NOs:633 and 637, respectively; SEQ ID NOs:641 and 645, respectively; SEQ ID NOs:649 and 653, respectively; SEQ ID NOs:657 and 661, respectively; SEQ ID NOs:665 and 669, respectively; SEQ ID NOs:673 and 677, respectively; SEQ ID NOs:681 and 685, respectively; SEQ ID NOs:689 and 693, respectively; SEQ ID NOs:697 and 701, respectively; SEQ ID NOs:705 and 709, respectively; SEQ ID NOs:713 and 717, respectively; SEQ ID NOs:721 and 725, respectively;SEQ ID NOs:729 and 733, respectively; SEQ ID NOs:737 and 741, respectively; SEQ ID NOs:745 and 749, respectively; SEQ ID NOs:753 and 757, respectively; SEQ ID NOs:761 and 765, respectively; SEQ ID NOs:769 and 773, respectively; SEQ ID NOs:785 and 789, respectively; SEQ ID NOs:793 and 797, respectively; SEQ ID NOs:801 and 805, respectively; SEQ ID NOs:809 and 813, respectively; SEQ ID NOs:817 and 821, respectively; SEQ ID NOs:849 and 853, respectively; SEQ ID NOs:857 and 861, respectively; SEQ ID NOs:865 and 869, respectively; SEQ ID NOs:873 and 877, respectively; SEQ ID NOs:881 and 885, respectively; SEQ ID NOs:889 and 893, respectively; SEQ ID NOs:897 and 901, respectively; SEQ ID NOs:905 and 909, respectively; SEQ ID NOs:913 and 917, respectively; SEQ ID NOs:921 and 925, respectively; SEQ ID NOs:929 and 933, respectively; SEQ ID NOs:937 and 941, respectively; SEQ ID NOs:945 and 949, respectively; SEQ ID NOs: 969 and 973, respectively; SEQ ID NOs: 977 and 981, respectively; SEQ ID NOs: 1009 and 1013, respectively; SEQ ID NOs: 1057 and 1061, respectively; SEQ ID NOs: 1537 and 1541, respectively; SEQ ID NOs: 1569 and 1573, respectively; SEQ ID NOs: 1601 and 1605, respectively; SEQ ID NOs: 1641 and 1645, respectively; SEQ ID NOs: 1665 and 1669, respectively; SEQ ID NOs: 1825 and 1829, respectively; SEQ ID NOs: 1865 and 1869, respectively; V selected from the group consisting of SEQ ID NOs: 1897 and 1901, respectively; SEQ ID NOs: 1905 and 1909, respectively; SEQ ID NOs: 1913 and 1917, respectively; SEQ ID NOs: 1921 and 1925, respectively; SEQ ID NOs: 1929 and 1933, respectively; SEQ ID NOs: 2265 and 2269, respectively; SEQ ID NOs: 2281 and 2285, respectively; SEQ ID NOs: 2289 and 2293, respectively; SEQ ID NOs: 2329 and 2333, respectively; and SEQ ID NOs: 2345 and 2349, respectively; L Amino acid sequence and V H The heterodimeric multispecific antibody of any one of claims 1 to 12, comprising the amino acid sequence

14. Each of VL-4 and VH-4 is set forth as SEQ ID NOs:17 and 21, respectively; SEQ ID NOs:25 and 29, respectively; SEQ ID NOs:33 and 37, respectively; SEQ ID NOs:41 and 45, respectively; SEQ ID NOs:121 and 125, respectively; SEQ ID NOs:137 and 141, respectively; SEQ ID NOs:169 and 173, respectively; SEQ ID NOs:177 and 181, respectively; SEQ ID NOs:185 and 189, respectively; SEQ ID NOs:193 and 197, respectively; SEQ ID NOs:201 and 205, respectively; SEQ ID NOs:209 and 213, respectively; SEQ ID NOs:217 and 220, respectively. and 221; SEQ ID NOs:225 and 229, respectively; SEQ ID NOs:233 and 237, respectively; SEQ ID NOs:241 and 245, respectively; SEQ ID NOs:249 and 253, respectively; SEQ ID NOs:257 and 261, respectively; SEQ ID NOs:265 and 269, respectively; SEQ ID NOs:321 and 325, respectively; SEQ ID NOs:329 and 333, respectively; SEQ ID NOs:337 and 341, respectively; SEQ ID NOs:393 and 397, respectively; SEQ ID NOs:401 and 405, respectively; SEQ ID NOs:409 and 413, respectively; SEQ ID NOs:473 and SEQ ID NOs: 481 and 485, respectively; SEQ ID NOs: 489 and 493, respectively; SEQ ID NOs: 497 and 501, respectively; SEQ ID NOs: 505 and 509, respectively; SEQ ID NOs: 513 and 517, respectively; SEQ ID NOs: 545 and 549, respectively; SEQ ID NOs: 553 and 557, respectively; SEQ ID NOs: 561 and 565, respectively; SEQ ID NOs: 569 and 573, respectively; SEQ ID NOs: 577 and 581, respectively; SEQ ID NOs: 585 and 589, respectively; SEQ ID NOs: 593 and 597, respectively; SEQ ID NOs: 601 and 60 SEQ ID NOs:625 and 629, respectively; SEQ ID NOs:633 and 637, respectively; SEQ ID NOs:641 and 645, respectively; SEQ ID NOs:649 and 653, respectively; SEQ ID NOs:657 and 661, respectively; SEQ ID NOs:665 and 669, respectively; SEQ ID NOs:673 and 677, respectively; SEQ ID NOs:681 and 685, respectively; SEQ ID NOs:689 and 693, respectively; SEQ ID NOs:697 and 701, respectively; SEQ ID NOs:705 and 709, respectively; SEQ ID NOs:713 and 717, respectively; SEQ ID NOs:721 and 725, respectively;SEQ ID NOs:729 and 733, respectively; SEQ ID NOs:737 and 741, respectively; SEQ ID NOs:745 and 749, respectively; SEQ ID NOs:753 and 757, respectively; SEQ ID NOs:761 and 765, respectively; SEQ ID NOs:769 and 773, respectively; SEQ ID NOs:785 and 789, respectively; SEQ ID NOs:793 and 797, respectively; SEQ ID NOs:801 and 805, respectively; SEQ ID NOs:809 and 813, respectively; SEQ ID NOs:817 and 821, respectively; SEQ ID NOs:849 and 853, respectively; SEQ ID NOs:857 and 861, respectively; SEQ ID NOs:865 and 869, respectively; SEQ ID NOs:873 and 877, respectively; SEQ ID NOs:881 and 885, respectively; SEQ ID NOs:889 and 893, respectively; SEQ ID NOs:897 and 901, respectively; SEQ ID NOs:905 and 909, respectively; SEQ ID NOs:913 and 917, respectively; SEQ ID NOs:921 and 925, respectively; SEQ ID NOs:929 and 933, respectively; SEQ ID NOs:937 and 941, respectively; SEQ ID NOs:945 and 949, respectively; SEQ ID NOs: 969 and 973, respectively; SEQ ID NOs: 977 and 981, respectively; SEQ ID NOs: 1009 and 1013, respectively; SEQ ID NOs: 1057 and 1061, respectively; SEQ ID NOs: 1537 and 1541, respectively; SEQ ID NOs: 1569 and 1573, respectively; SEQ ID NOs: 1601 and 1605, respectively; SEQ ID NOs: 1641 and 1645, respectively; SEQ ID NOs: 1665 and 1669, respectively; SEQ ID NOs: 1825 and 1829, respectively; SEQ ID NOs: 1865 and 1869, respectively; V selected from the group consisting of SEQ ID NOs: 1897 and 1901, respectively; SEQ ID NOs: 1905 and 1909, respectively; SEQ ID NOs: 1913 and 1917, respectively; SEQ ID NOs: 1921 and 1925, respectively; SEQ ID NOs: 1929 and 1933, respectively; SEQ ID NOs: 2265 and 2269, respectively; SEQ ID NOs: 2281 and 2285, respectively; SEQ ID NOs: 2289 and 2293, respectively; SEQ ID NOs: 2329 and 2333, respectively; and SEQ ID NOs: 2345 and 2349, respectively; L Amino acid sequence and V H The heterodimeric multispecific antibody of any one of claims 1 to 3 or 7 to 12, comprising the amino acid sequence

15. The first immunoglobulin or the third immunoglobulin is b3 (glycoprotein IIb / IIIa), a4, a4b7, a4b7+aEb7, a5, activin receptor type 2B, ALK1, alpha-synuclein, amyloid beta, APP, AXL, blood type A, CAIX, CCL-2, CD105 (endoglin), CD115 (CSF1R), CD116a (CSF2Ra), CD123, CD152 (CTLA4), CD184 (CXCR4), CD19, CD192 (CCR2), CD194 (CCR4), CD195 (CCR5), CD20, CD200, CD22, CD221 (IGF1R), CD248, CD25, CD257 (BAFF), CD26, CD262 (DR5), CD276 (B7H3) , CD3, CD30 (TNFRSF8), CD319 (SLAMF7), CD33, CD332 (FGFR2), CD350 (FZD10), CD37, CD371 (CLEC12A), CD38, CD4, CD49b (a2), CD51 (a5), CD52, CD56, CD61 (a4b3), CD70, CD73 (NT5E), CD74, CEA, claudin-18.2, cMET, CRLR, DLL3, DLL4, DNA / histone (H1) complex, EGFR, EpCAM, EGFR-HER3, EGFRvIII, EphA3, ERGT(GalNAc)Tn antigen, FLT1, FOLR1, frizzled family receptor (FZD), Lewis Y, Lewis X, GCGR, GD2, GD2α-acetyl, GD3, GM1, GM1 fucosyl, GM2, GPA33, GPNMB, GUCY2C, HER2, HER3, HGFR (cMET), IgHe, IGLF2, kallikrein, LINGO1, LOXL2, Ly6 / PLAUR domain-containing protein 3, MADCAM1, MAG, mesothelin, MT1-MMP (MMP14), MUC1, mucin 5AC, NaPi2b, NeuGc-GM3, notch, NOTCH2 / NOTCH3 receptor, oxLDL, P-selectin, PCSK9, PDGFRA, PDGFRa, phosphatidylserine, polysialic acid, PSMA, PVRL4, RGMA, CD240D, a blood antigen of type D, and root plate specific spondin 3, serum amyloid P component, STEAP-1, TACSTD2, TGFb, TWEAKR, TYRP1, VEGFR2, VSIR, CD171 (L1CAM), CD19, CD47, pMHC[NY-ESO1], pMHC[MART1], pMHC[MAGEA1], pMHC[tyrosinase], pMHC[gp100], pMHC[MUC1], pMHC[tax], pMHC[WT-1], pMHC[EBNA-1], pMHC[LMP2], pMHC[hTERT], GPC3, CD80, CD23, and fibronectin extracellular domain B.

16. The heterodimeric multispecific antibody of any one of claims 1 to 15, wherein the first immunoglobulin and the third immunoglobulin bind to two different epitopes on a target cell.

17. The heterodimeric multispecific antibody of claim 16, wherein the target cell is a cancer cell.

18. The heterodimeric multispecific antibody according to any one of claims 1 to 17, wherein the second immunoglobulin or the fourth immunoglobulin binds to an epitope on a leukocyte, on a monocyte, on a lymphocyte, on a granulocyte, on a macrophage, on a T cell, on a NK cell, on a B cell, on a NKT cell, on an ILC or on a neutrophil.

19. The second or fourth immunoglobulin is selected from the group consisting of dabigatran, a4, a4b7, a4b7+aEb7, a5, AXL, BnDOTA, CD11a (LFA-1), CD3, CD4, CD8, CD16, CD19, CD22, CD23, CD25, CD28, CD30 (TNFRSF8), CD33, CD38, CD40, CD 40L, CD47, CD49b (a2), CD54 (ICAM-1), CD56, CD74, CD80, CD115 (CSF1R), CD116a (CSF2Ra), CD123, CD134 (OX40), CD137 (41BB), CD152 (CTLA4), CD184 (CXCR4), CD192 (CCR2), CD194 (C 19. The heterodimeric multispecific antibody of any one of claims 1 to 3 or 7 to 18, which binds to an antigen selected from the group consisting of CD195 (CCR4), CD223 (LAG-3), CD252 (OX40L), CD254 (RANKL), CD262 (DR5), CD27, CD200, CD221 (IGF1R), CD248, CD274 (PD-L1), CD275 (ICOS-L), CD278 (ICOS), CD279 (PD-1), CD319 (SLAMF7), CD371 (CLEC12A), MADCAM1, MT1-MMP (MMP14), NKG2A, NRP1, TIGIT, VSIR, KIRDL1 / 2 / 3, and KIR2DL2.

20. 20. The heterodimeric multispecific antibody of any one of claims 1 to 3 or 7 to 19, wherein the second and fourth immunoglobulins bind to two different epitopes on leukocytes, on monocytes, on lymphocytes, on granulocytes, on macrophages, on T cells, on NK cells, on B cells, on NKT cells, on ILCs, or on neutrophils.

21. 20. The heterodimeric multispecific antibody of any one of claims 1 to 3 or 7 to 19, wherein the second immunoglobulin binds to CD3 and the fourth immunoglobulin binds to an immune cell receptor selected from the group consisting of CD4, CD8, CD25, CD28, CTLA4, OX40, ICOS, PD-1, PD-L1, 41BB, CD2, CD69, and CD45.

22. 20. The heterodimeric multispecific antibody of any one of claims 1 to 3 or 7 to 19, wherein the second immunoglobulin binds to CD16 and the fourth immunoglobulin binds to an immune cell receptor selected from the group consisting of CD56, NKG2D, and KIRDL1 / 2 / 3.

23. 23. The heterodimeric multispecific antibody of any one of claims 1 to 22, wherein the fourth immunoglobulin binds to an agent selected from the group consisting of a cytokine, a nucleic acid, a hapten, a small molecule, a radionuclide, an antitoxin, a vitamin, a peptide, a lipid, a carbohydrate, biotin, digoxin, or a conjugated variant of any of these.

24. 24. The heterodimeric multispecific antibody of any one of claims 1 to 23, wherein the first and third immunoglobulins bind to their respective epitopes with a monovalent or effective affinity of between about 100 nM and about 100 pM.

25. The heterodimeric multispecific antibody of claim 24, wherein the first and third immunoglobulins bind to cell surface epitopes that are 60-120 angstroms apart.

26. 26. The heterodimeric multispecific antibody of any one of claims 1 to 25, wherein the first and third immunoglobulins bind to their respective epitopes with a monovalent or effective affinity of less than 100 pM.

27. 27. The heterodimeric multispecific antibody of claim 26, wherein the first and third immunoglobulins bind to cell surface epitopes that are up to 180 angstroms apart.

28. 28. The heterodimeric multispecific antibody according to any one of claims 1 to 27, wherein the first heterodimerization domain and / or the second heterodimerization domain is a CH2-CH3 domain and has an isotype selected from the group consisting of IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgM, IgD and IgE.

29. 29. The heterodimeric multispecific antibody of claim 28, wherein the first heterodimerization domain and / or the second heterodimerization domain is a constant region of IgG1 comprising one or more amino acid substitutions selected from the group consisting of N297A and K322A.

30. 30. The heterodimeric multispecific antibody of claim 28 or 29, wherein the first heterodimerization domain is a CH2-CH3 domain comprising a K409R mutation and the second heterodimerization domain is a CH2-CH3 domain comprising a F405L mutation.

31. The heterodimeric multispecific antibody of any one of claims 1 to 30, wherein the antibody is a monoclonal antibody, a chimeric antibody or a humanized antibody.

32. A recombinant nucleic acid sequence encoding the heterodimeric multispecific antibody according to any one of claims 1 to 31.

33. A host cell or vector comprising the recombinant nucleic acid sequence of claim 32.

34. 32. A composition comprising the heterodimeric multispecific antibody of any one of claims 1 to 31 and a pharma- ceutically acceptable carrier, wherein the antibody is optionally conjugated to an agent selected from the group consisting of an isotope, a dye, a chromogen, an imaging agent, a drug, a toxin, a cytokine, an enzyme, an enzyme inhibitor, a hormone, a hormone antagonist, a growth factor, a radionuclide, a metal, a liposome, a nanoparticle, RNA, DNA, or any combination thereof.

35. 32. A method for treating cancer in a subject in need thereof, comprising administering to the subject an effective amount of a heterodimeric multispecific antibody according to any one of claims 1 to 31.

36. 36. The method of claim 35, wherein the cancer is selected from the group consisting of lung cancer, colorectal cancer, skin cancer, breast cancer, ovarian cancer, leukemia, pancreatic cancer, and gastric cancer.

37. 37. The method of claim 35 or 36, wherein the heterodimeric multispecific antibody is administered to a subject separately, sequentially or simultaneously with a further therapeutic agent.

38. A kit comprising the heterodimeric multispecific antibody of any one of claims 1 to 31 and instructions for use.

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