HETERODIMERIC TETRAVALENCY AND SPECIFICITY ANTIBODY COMPOSITIONS AND USES THEREOF

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Dec. 23, 2019, is named 115872-0497 SL.txt and is 1,200,059 bytes in size.

TECHNICAL FIELD

The present technology relates generally to the preparation of heterodimeric trivalent/tetravalent multispecific antibodies that specifically bind three or four distinct target antigens, and their uses. The heterodimeric trivalent/tetravalent multispecific antibodies described herein are useful in methods for detecting and treating cancer in a subject in need thereof.

BACKGROUND

The following description of the background of the present technology is provided simply as an aid in 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 BiTE. 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 to date has shown a clear and significant functional advantage over others within the clinic.

In the case of multispecific antibodies that engage immune cells, such as BiTEs, the ideal structure that maximizes anti-tumor activity has not been defined, and likely varies based on the target antigens or the parental antibodies (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). Even less is understood about the spatial orientation imparted by the antibody on the cell-to-cell interface, the strength of each individual specificity interaction, or the number of interactions. Moreover, the size of the antibody format, the flexibility of each binding domain, and their relative orientations to one another may influence the capacity to properly or effectively engage multiple antigens at once. Given these different complexities, it is of paramount importance to understand if a given platform design is properly optimized for therapeutic function.

Summary of the Present Technology

In one aspect, the present disclosure provides a heterodimeric multispecific antibody comprising 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, and wherein: (a) the first polypeptide chain comprises in the N-terminal to C-terminal direction: (i) a light chain variable domain of a first immunoglobulin (VL-1) that is capable of specifically binding to a first epitope; (ii) a light chain constant domain of the first immunoglobulin (CL-1); (iii) a flexible peptide linker comprising the amino acid sequence (GGGGS)₃(SEQ ID NO: 2506); and (iv) a light chain variable domain of a second immunoglobulin (VL-2) that is linked to a complementary heavy chain variable domain of the second immunoglobulin (VH-2), or a heavy chain variable domain of a second immunoglobulin (VH-2) that is linked to a complementary light chain variable domain of the second immunoglobulin (VL-2), wherein VL-2 and VH-2 are capable of specifically binding to a second epitope, and are linked together via a flexible peptide linker comprising the amino acid sequence (GGGGS)₆(SEQ ID NO: 2507) to form a single-chain variable fragment; (b) the second polypeptide comprises in the N-terminal to C-terminal direction: (i) a heavy chain variable domain of the first immunoglobulin (VH-1) that is capable of specifically binding to the first epitope; (ii) a first CH1 domain of the first immunoglobulin (CH1-1); and (iii) a first heterodimerization domain of the first immunoglobulin, wherein the first heterodimerization domain is incapable of forming a stable homodimer with another first heterodimerization domain; (c) the third polypeptide comprises in the N-terminal to C-terminal direction: (i) a heavy chain variable domain of a third immunoglobulin (VH-3) that is capable of specifically binding to a third epitope; (ii) a second CH1 domain of the third immunoglobulin (CH1-3); and (iii) a second heterodimerization domain of the third immunoglobulin, wherein the second heterodimerization domain comprises an amino acid sequence or a nucleic acid sequence that is distinct from the first heterodimerization domain of the first immunoglobulin, wherein the second heterodimerization domain is incapable of forming a stable homodimer with another second heterodimerization domain, and wherein the second heterodimerization domain of the third immunoglobulin is configured to form a heterodimer with the first heterodimerization domain of the first immunoglobulin; (d) the fourth polypeptide comprises in the N-terminal to C-terminal direction: (i) a light chain variable domain of the third immunoglobulin (VL-3) that is capable of specifically binding to the third epitope; (ii) a light chain constant domain of the third immunoglobulin (CL-3); (iii) a flexible peptide linker comprising the amino acid sequence (GGGGS)₃(SEQ ID NO: 2506); and (iv) a light chain variable domain of a fourth immunoglobulin (VL-4) that is linked to a complementary heavy chain variable domain of the fourth immunoglobulin (VH-4), or a heavy chain variable domain of a fourth immunoglobulin (VH-4) that is linked to a complementary light chain variable domain of the fourth immunoglobulin (VL-4), wherein VL-4 and VH-4 are capable of specifically binding to the second epitope, and are linked together via a flexible peptide linker comprising the amino acid sequence (GGGGS)₆(SEQ ID NO: 2507) to form a single-chain variable fragment, and wherein each of VL-1 and VL-3 independently comprises the CDR1 sequence, the CDR2 sequence and the CDR3 sequence of a V_Lamino acid sequence selected from any one 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, 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, 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; and/or wherein each of VH-1 and VH-3 independently comprises the CDR1 sequence, the CDR2 sequence and the CDR3 sequence of a V_Hamino acid sequence selected from any one 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, 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.

In one aspect, the present disclosure provides a heterodimeric multispecific antibody comprising 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, and wherein: (a) the first polypeptide chain comprises in the N-terminal to C-terminal direction: (i) a light chain variable domain of a first immunoglobulin (VL-1) that is capable of specifically binding to a first epitope; (ii) a light chain constant domain of the first immunoglobulin (CL-1); (iii) a flexible peptide linker comprising the amino acid sequence (GGGGS)₃(SEQ ID NO: 2506); and (iv) a light chain variable domain of a second immunoglobulin (VL-2) that is linked to a complementary heavy chain variable domain of the second immunoglobulin (VH-2), or a heavy chain variable domain of a second immunoglobulin (VH-2) that is linked to a complementary light chain variable domain of the second immunoglobulin (VL-2), wherein the VL-2 and VH-2 are capable of specifically binding to a second epitope, and are linked together via a flexible peptide linker comprising the amino acid sequence (GGGGS)₆(SEQ ID NO: 2507) to form a single-chain variable fragment; (b) the second polypeptide comprises in the N-terminal to C-terminal direction: (i) a heavy chain variable domain of the first immunoglobulin (VH-1) that is capable of specifically binding to the first epitope; (ii) a first CH1 domain of the first immunoglobulin (CH1-1); and (iii) a first heterodimerization domain of the first immunoglobulin, wherein the first heterodimerization domain is incapable of forming a stable homodimer with another first heterodimerization domain; (c) the third polypeptide comprises in the N-terminal to C-terminal direction: (i) a heavy chain variable domain of a third immunoglobulin (VH-3) that is capable of specifically binding to the first epitope; (ii) a second CH1 domain of the third immunoglobulin (CH1-3); and (iii) a second heterodimerization domain of the third immunoglobulin, wherein the second heterodimerization domain comprises an amino acid sequence or a nucleic acid sequence that is distinct from the first heterodimerization domain of the first immunoglobulin, wherein the second heterodimerization domain is incapable of forming a stable homodimer with another second heterodimerization domain, and wherein the second heterodimerization domain of the third immunoglobulin is configured to form a heterodimer with the first heterodimerization domain of the first immunoglobulin; (d) the fourth polypeptide comprises in the N-terminal to C-terminal direction: (i) a light chain variable domain of the third immunoglobulin (VL-3) that is capable of specifically binding to the first epitope; (ii) a light chain constant domain of the third immunoglobulin (CL-3); (iii) a flexible peptide linker comprising the amino acid sequence (GGGGS)₃(SEQ ID NO: 2506); and (iv) a light chain variable domain of a fourth immunoglobulin (VL-4) that is linked to a complementary heavy chain variable domain of the fourth immunoglobulin (VH-4), or a heavy chain variable domain of a fourth immunoglobulin (VH-4) that is linked to a complementary light chain variable domain of the fourth immunoglobulin (VL-4), wherein the VL-4 and VH-4 are capable of specifically binding to a third epitope, and are linked together via a flexible peptide linker comprising the amino acid sequence (GGGGS)₆(SEQ ID NO: 2507) to form a single-chain variable fragment, and wherein each of VL-2 and VL-4 independently comprises the CDR1 sequence, the CDR2 sequence and the CDR3 sequence of a V_Lamino acid sequence selected from any one 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 2289, 2329, and 2345; and/or wherein each of VH-2 and VH-4 independently comprises the CDR1 sequence, the CDR2 sequence and the CDR3 sequence of a V_Hamino acid sequence selected from any one 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.

In another aspect, the present disclosure provides a heterodimeric multispecific antibody comprising 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, and wherein: (a) the first polypeptide chain comprises in the N-terminal to C-terminal direction: (i) a light chain variable domain of a first immunoglobulin (VL-1) that is capable of specifically binding to a first epitope; (ii) a light chain constant domain of the first immunoglobulin (CL-1); (iii) a flexible peptide linker comprising the amino acid sequence (GGGGS)₃(SEQ ID NO: 2506); and (iv) a light chain variable domain of a second immunoglobulin (VL-2) that is linked to a complementary heavy chain variable domain of the second immunoglobulin (VH-2), or a heavy chain variable domain of a second immunoglobulin (VH-2) that is linked to a complementary light chain variable domain of the second immunoglobulin (VL-2), wherein VL-2 and VH-2 are capable of specifically binding to a second epitope, and are linked together via a flexible peptide linker comprising the amino acid sequence (GGGGS)₆(SEQ ID NO: 2507) to form a single-chain variable fragment; (b) the second polypeptide comprises in the N-terminal to C-terminal direction: (i) a heavy chain variable domain of the first immunoglobulin (VH-1) that is capable of specifically binding to the first epitope; (ii) a first CH1 domain of the first immunoglobulin (CH1-1); and (iii) a first heterodimerization domain of the first immunoglobulin, wherein the first heterodimerization domain is incapable of forming a stable homodimer with another first heterodimerization domain; (c) the third polypeptide comprises in the N-terminal to C-terminal direction: (i) a heavy chain variable domain of a third immunoglobulin (VH-3) that is capable of specifically binding to a third epitope; (ii) a second CH1 domain of the third immunoglobulin (CH1-3); and (iii) a second heterodimerization domain of the third immunoglobulin, wherein the second heterodimerization domain comprises an amino acid sequence or a nucleic acid sequence that is distinct from the first heterodimerization domain of the first immunoglobulin, wherein the second heterodimerization domain is incapable of forming a stable homodimer with another second heterodimerization domain, and wherein the second heterodimerization domain of the third immunoglobulin is configured to form a heterodimer with the first heterodimerization domain of the first immunoglobulin; (d) the fourth polypeptide comprises in the N-terminal to C-terminal direction: (i) a light chain variable domain of the third immunoglobulin (VL-3) that is capable of specifically binding to the third epitope; (ii) a light chain constant domain of the third immunoglobulin (CL-3); (iii) a flexible peptide linker comprising the amino acid sequence (GGGGS)₃(SEQ ID NO: 2506); and (iv) a light chain variable domain of a fourth immunoglobulin (VL-4) that is linked to a complementary heavy chain variable domain of the fourth immunoglobulin (VH-4), or a heavy chain variable domain of a fourth immunoglobulin (VH-4) that is linked to a complementary light chain variable domain of the fourth immunoglobulin (VL-4), wherein VL-4 and VH-4 are capable of specifically binding to the fourth epitope, and are linked together via a flexible peptide linker comprising the amino acid sequence (GGGGS)₆(SEQ ID NO: 2507) to form a single-chain variable fragment; and wherein each of VL-1 and VL-3 independently comprises the CDR1 sequence, the CDR2 sequence and the CDR3 sequence of a V_Lamino acid sequence selected from any one 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, 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, 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; and/or wherein each of VH-1 and VH-3 independently comprises the CDR1 sequence, the CDR2 sequence and the CDR3 sequence of a V_Hamino acid sequence selected from any one 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, 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; and/or wherein each of VL-2 and VL-4 independently comprises the CDR1 sequence, the CDR2 sequence and the CDR3 sequence of a V_Lamino acid sequence selected from any one 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 2289, 2329, and 2345; and/or wherein each of VH-2 and VH-4 independently comprises the CDR1 sequence, the CDR2 sequence and the CDR3 sequence of a V_Hamino acid sequence selected from any one 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.

In another aspect, the present disclosure provides a heterodimeric multispecific antibody comprising 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, and wherein: (a) the first polypeptide chain comprises in the N-terminal to C-terminal direction: (i) a light chain variable domain of a first immunoglobulin (VL-1) that is capable of specifically binding to a first epitope; (ii) a light chain constant domain of the first immunoglobulin (CL-1); (iii) a flexible peptide linker comprising the amino acid sequence (GGGGS)₃(SEQ ID NO: 2506); and (iv) a light chain variable domain of a second immunoglobulin (VL-2) that is linked to a complementary heavy chain variable domain of the second immunoglobulin (VH-2), or a heavy chain variable domain of a second immunoglobulin (VH-2) that is linked to a complementary light chain variable domain of the second immunoglobulin (VL-2), wherein VL-2 and VH-2 are capable of specifically binding to a second epitope, and are linked together via a flexible peptide linker comprising the amino acid sequence (GGGGS)₆(SEQ ID NO: 2507) to form a single-chain variable fragment; (b) the second polypeptide comprises in the N-terminal to C-terminal direction: (i) a heavy chain variable domain of the first immunoglobulin (VH-1) that is capable of specifically binding to the first epitope; (ii) a first CH1 domain of the first immunoglobulin (CH1-1); and (iii) a first heterodimerization domain of the first immunoglobulin, wherein the first heterodimerization domain is incapable of forming a stable homodimer with another first heterodimerization domain; (c) the third polypeptide comprises in the N-terminal to C-terminal direction: (i) a heavy chain variable domain of a third immunoglobulin (VH-3) that is capable of specifically binding to the first epitope; (ii) a second CH1 domain of the third immunoglobulin (CH1-3); and (iii) a second heterodimerization domain of the third immunoglobulin, wherein the second heterodimerization domain comprises an amino acid sequence or a nucleic acid sequence that is distinct from the first heterodimerization domain of the first immunoglobulin, wherein the second heterodimerization domain is incapable of forming a stable homodimer with another second heterodimerization domain, and wherein the second heterodimerization domain of the third immunoglobulin is configured to form a heterodimer with the first heterodimerization domain of the first immunoglobulin; (d) the fourth polypeptide comprises in the N-terminal to C-terminal direction: (i) a light chain variable domain of the third immunoglobulin (VL-3) that is capable of specifically binding to the first epitope; and (ii) a light chain constant domain of the third immunoglobulin (CL-3); and wherein VL-2 comprises the CDR1 sequence, the CDR2 sequence and the CDR3 sequence of a V_Lamino acid sequence selected from any one 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 2289, 2329, and 2345; and/or wherein VH-2 comprises the CDR1 sequence, the CDR2 sequence and the CDR3 sequence of a V_Hamino acid sequence selected from any one 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. In some embodiments, both VH-1 and VH-3 comprise the CDR1 sequence, the CDR2 sequence and the CDR3 sequence of a V_Hamino acid sequence selected from any one 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, 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; and/or both VL-1 and VL-3 comprise the CDR1 sequence, the CDR2 sequence and the CDR3 sequence of a V_Lamino acid sequence selected from any one 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, 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, 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.

In yet another aspect, the present disclosure provides a heterodimeric multispecific antibody comprising 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, and wherein: (a) the first polypeptide chain comprises in the N-terminal to C-terminal direction: (i) a light chain variable domain of a first immunoglobulin (VL-1) that is capable of specifically binding to a first epitope; (ii) a light chain constant domain of the first immunoglobulin (CL-1); (iii) a flexible peptide linker comprising the amino acid sequence (GGGGS)₃(SEQ ID NO: 2506); and (iv) a light chain variable domain of a second immunoglobulin (VL-2) that is linked to a complementary heavy chain variable domain of the second immunoglobulin (VH-2), or a heavy chain variable domain of a second immunoglobulin (VH-2) that is linked to a complementary light chain variable domain of the second immunoglobulin (VL-2), wherein VL-2 and VH-2 are capable of specifically binding to a second epitope, and are linked together via a flexible peptide linker comprising the amino acid sequence (GGGGS)₆(SEQ ID NO: 2507) to form a single-chain variable fragment; (b) the second polypeptide comprises in the N-terminal to C-terminal direction: (i) a heavy chain variable domain of the first immunoglobulin (VH-1) that is capable of specifically binding to the first epitope; (ii) a first CH1 domain of the first immunoglobulin (CH1-1); and (iii) a first heterodimerization domain of the first immunoglobulin, wherein the first heterodimerization domain is incapable of forming a stable homodimer with another first heterodimerization domain; (c) the third polypeptide comprises in the N-terminal to C-terminal direction: (i) a heavy chain variable domain of a third immunoglobulin (VH-3) that is capable of specifically binding to a third epitope; (ii) a second CH1 domain of the third immunoglobulin (CH1-3); and (iii) a second heterodimerization domain of the third immunoglobulin, wherein the second heterodimerization domain comprises an amino acid sequence or a nucleic acid sequence that is distinct from the first heterodimerization domain of the first immunoglobulin, wherein the second heterodimerization domain is incapable of forming a stable homodimer with another second heterodimerization domain, and wherein the second heterodimerization domain of the third immunoglobulin is configured to form a heterodimer with the first heterodimerization domain of the first immunoglobulin; (d) the fourth polypeptide comprises in the N-terminal to C-terminal direction: (i) a light chain variable domain of the third immunoglobulin (VL-3) that is capable of specifically binding to the third epitope; and (ii) a light chain constant domain of the third immunoglobulin (CL-3); and wherein each of VL-1 and VL-3 independently comprises the CDR1 sequence, the CDR2 sequence and the CDR3 sequence of a V_Lamino acid sequence selected from any one 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, 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, 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; and/or wherein each of VH-1 and VH-3 independently comprises the CDR1 sequence, the CDR2 sequence and the CDR3 sequence of a V_Hamino acid sequence selected from any one 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, 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; and/or wherein VL-2 comprises the CDR1 sequence, the CDR2 sequence and the CDR3 sequence of a V_Lamino acid sequence selected from any one 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 2289, 2329, and 2345; and/or wherein VH-2 comprises the CDR1 sequence, the CDR2 sequence and the CDR3 sequence of a V_Hamino acid sequence selected from any one 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.

Additionally or alternatively, in some embodiments of the heterodimeric multispecific antibodies disclosed herein, VH-1 or VH-3 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 a V_Hamino acid sequence selected from any one 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, 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; and/or the VL-1 or VL-3 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 a V_Lamino acid sequence selected from any one 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, 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, 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.

Additionally or alternatively, in some embodiments of the heterodimeric multispecific antibodies disclosed herein, VH-2 or VH-4 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 a V_Hamino acid sequence selected from any one 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; and/or VL-2 or VL-4 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 a VL amino acid sequence selected from any one 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 2289, 2329, and 2345.

Additionally or alternatively, in some embodiments of the heterodimeric multispecific antibodies disclosed herein, each of VL-1 and VH-1 comprise a V_Lamino acid sequence and a V_Hamino acid sequence 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 101 respectively; 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 261 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: 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 1197 respectively; 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: 1289 and 1293 respectively; SEQ ID NOs: 1297 and 1301 respectively; SEQ ID NOs: 1305 and 1309 respectively; SEQ ID NOs: 1313 and 1317 respectively; SEQ ID NOs: 1321 and 1325 respectively; 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 respectively; 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 1597 respectively; 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: 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: 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 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 2029 respectively; 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: 2121 and 2125 respectively; SEQ ID NOs: 2129 and 2133 respectively; SEQ ID NOs: 2137 and 2141 respectively; SEQ ID NOs: 2145 and 2149 respectively; 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; 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.

Additionally or alternatively, in some embodiments of the heterodimeric multispecific antibodies disclosed herein, each of VL-3 and VH-3 comprise a V_Lamino acid sequence and a V_Hamino acid sequence 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 101 respectively; 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 261 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: 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 1197 respectively; 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: 1289 and 1293 respectively; SEQ ID NOs: 1297 and 1301 respectively; SEQ ID NOs: 1305 and 1309 respectively; SEQ ID NOs: 1313 and 1317 respectively; SEQ ID NOs: 1321 and 1325 respectively; 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 respectively; 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 1597 respectively; 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: 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: 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 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 2029 respectively; 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: 2121 and 2125 respectively; SEQ ID NOs: 2129 and 2133 respectively; SEQ ID NOs: 2137 and 2141 respectively; SEQ ID NOs: 2145 and 2149 respectively; 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; 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.

Additionally or alternatively, in some embodiments of the heterodimeric multispecific antibodies disclosed herein, each of VL-2 and VH-2 comprise a V_Lamino acid sequence and a V_Hamino acid sequence selected from the group consisting of 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 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 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; 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; 2289 and 2293 respectively; 2329 and 2333 respectively; and SEQ ID NOs: 2345 and 2349, respectively.

Additionally or alternatively, in some embodiments of the heterodimeric multispecific antibodies disclosed herein, each of VL-4 and VH-4 comprise a V_Lamino acid sequence and a V_Hamino acid sequence selected from the group consisting of 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 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 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; 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; 2289 and 2293 respectively; 2329 and 2333 respectively; and SEQ ID NOs: 2345 and 2349, respectively.

Additionally or alternatively, in some embodiments of the heterodimeric multispecific antibodies disclosed herein, the first immunoglobulin or the third immunoglobulin binds to a cell surface antigen 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), CD350 (FZD10), CD37, CD371 (CLEC12A), CD38, CD4, CD49b (a2), CD51 (a5), CD52, CD56, CD61 (a4b3), CD70, CD73 (NTSE), 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, Kallikreins, LINGO1, LOXL2, Ly6/PLAUR domain-containing protein 3, MADCAM1, MAG, Mesothelin, MT1-MMP (MMP14), MUC1, Mucin SAC, NaPi2b, NeuGc-GM3, notch, NOTCH2/NOTCH3 receptors, oxLDL, P-selectin, PCSK9, PDGFRA, PDGFRa, phosphatidylserine, polysialic acid, PSMA, PVRL4, RGMA, CD240D Blood group D antigen, 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 extra domain-B. The first immunoglobulin and the third immunoglobulin may bind to the same epitope on a target cell or two different epitopes on a target cell. In some embodiments, the target cell is a cancer cell.

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

In any of the above embodiments of the heterodimeric multispecific antibodies disclosed herein, the second immunoglobulin or the fourth immunoglobulin bind to an antigen 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), 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 epitope or different epitopes on a white blood cell, a monocyte, a lymphocyte, a granulocyte, a macrophage, a T cell, a NK cell, a B cell, a NKT cell, an ILC, or neutrophil. In some embodiments, the second immunoglobulin binds CD3 and the fourth immunoglobulin binds 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 CD16 and the fourth immunoglobulin binds 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 immunotoxin, a vitamin, a peptide, a lipid, a carbohydrate, biotin, digoxin, or any conjugated variants thereof.

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

Additionally or alternatively, in some embodiments of the heterodimeric multispecific antibodies disclosed herein, the first immunoglobulin and the third immunoglobulin bind to their respective epitopes with a monovalent affinity or an effective affinity that is less than 100 pM. In certain embodiments, the first immunoglobulin and the third immunoglobulin 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 comprises 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 present technology provides a host cell or vector expressing any nucleic acid sequence encoding any of the antibodies described herein.

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 may 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 detection and/or treatment of a disease (e.g., cancers), comprising at least one heterodimeric trivalent/tetravalent multispecific antibody of the present technology and instructions for use.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1a shows the basic design strategy of each HeteroDimeric TetraValency and Specificity (HDTVS) variant compared with the parental 2+2 IgG-[L]-scFv. The 5 heterodimeric IgG-L-scFv designs display novel biological activities. Each construct uses heterodimerization to achieve tri- or tetraspecificity.

FIG. 1b shows a schematic of the 1+1+2 Low affinity design and how it can be used to distinguish single-antigen positive healthy cells from dual-antigen positive target cells. Single antigen positivity would result in inferior immune cell activation over dual antigen positivity.

FIG. 1c shows a schematic of the 1+1+2 High affinity design and how it can be used to target either (or both) of two different cellular antigens.

FIG. 1d shows a schematic of the 2+1+1 design and how it can be used to improve immune cell activation. Targeting of two different immune cell receptors can be used to more specifically recruit an immune cell population or provide greater immune cell activation or inhibition through cross linking of multiple receptors.

FIG. 1e shows a schematic of the 2+1+1 design and how it can be used to broaden immune cell recruitment or combine payload delivery with immunotherapy. Each HDTVS antibody needs only one immune cell receptor for recruitment and activation. The additional domain can then be used to bind payloads (for diagnostics, therapy, recruitment, etc.) or additional effector cells.

FIG. 1f shows a schematic of the 1+1+1+1 design and how it can be used to combine the benefits of 1+1+2 with 2+1+1. In this embodiment, tetraspecificity can bring better specificity or a broader range of targets, as well and improved immune cell activation or payload delivery.

FIG. 2a shows the superior cytotoxicity, binding and in vivo potency of the IgG-[L]-scFv design over the IgG-Het and BiTE formats. A 4 hr Cr⁵¹′ release assay was used to evaluate cytotoxicity of activated T-cells against M14 melanoma tumor cells. Flow cytometry was used to evaluate differences in antigen binding of each bispecific antibody to huCD3 or GD2 on activated T cells or M14 melanoma tumor cells, respectively. Affinities were measured using SPR on GD2 coated streptavidin chips. Two mouse models were used for assessing in vivo potency, a syngeneic transgenic model which has huCD3 expressing murine T cells, and a humanized xenograft model using activated human T-cells engrafted into immunodeficient IL2-re^−/− Rag2^−/− BALB/c mice. Mice were implanted subcutaneously with GD2(+) tumors and treated intravenously with a particular test bispecific antibody.

FIG. 2b shows the superior cytotoxicity of the IgG-[L]-scFv design over the IgG-het using two additional anti-GD2 sequences.

FIG. 3 shows the schematics of 4 IgG-[L]-scFv heterodimeric variants along with the parental format and the IgG-Het format. Designs are ranked by their relative potency.

FIG. 4 shows the in vitro binding activity of the various IgG-[L]-scFv variants. GD2 and CD3 affinities were measured using SPR with GD2 or huCD3de coated chips, respectively. Cell binding was assayed by flow cytometry using activated human T cells or M14 melanoma cells. T-cell: tumor cell conjugate formation was measured by flow cytometry using differentially labeled activated human T cells and M14 melanoma tumor cells.

FIG. 5 shows the in vitro cytotoxicity of each IgG-[L]-scFv variant against two cell lines: M14 melanoma and IMR32 neuroblastoma. Cytotoxicity was measured using a 4 hr Cr⁵¹release assay and activated human T-cells.

FIG. 6 shows the in vitro immune cell activation of each IgG-[L]-scFv variant. Activation was measured by flow cytometry. Naïve purified T cells and M14 melanoma cells were co-cultured for 24 or 96 hrs, harvested and stained for CD69 or CD25, respectively. T cells for the 96 hr time points were also labeled with Cell Trace Violet (CTV). Culture supernatant was also collected at the 24 hr time point for cytokine measurements.

FIG. 7 shows the in vivo activity of each IgG-[L]-scFv variant. Two mouse models were used for assessing in vivo potency, a syngeneic transgenic model which has huCD3 expressing murine T cells, 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 a particular test bispecific antibody.

FIG. 8 shows various dual bivalent bispecific antibody formats compared to the IgG-[L]-scFv design. Cytotoxicity was evaluated using a 4 hr Cr⁵¹release assay using activated human T cells and M14 melanoma cells. Conjugation activity was measured using flow cytometry. Cell binding was evaluated by flow cytometry using activated human T cells and M14 melanoma cells.

FIG. 9 shows IgG-[L]-scFv variants which bind CD33 or HER2. Cell binding activities were measured by flow cytometry using Molm13, SKMEL28, or MCF7 cells. Cytotoxicity was assessed using Molm13 cells and activated human T cells in a 4 hr Cr⁵¹release assay.

FIG. 10a shows two 1+1+2 designs (high and low affinity variants). Cell binding and cytotoxicity assays used the GD2(+)HER2(+) cell line U2OS. Cytotoxicity was measured using 4 hr Cr⁵¹release, and cell binding was evaluated using flow cytometry.

FIG. 10b shows two 1+1+2 designs (high and low affinity variants). Cell binding and cytotoxicity assays used the GD2(+) IMR32 neuroblastoma cells or HER2(+) HCC1954 breast cancer cells. Cytotoxicity was measured using 4 hr Cr⁵¹release, and cell binding was evaluated using flow cytometry.

FIGS. 11a-11e show exemplary Fc variants that are capable of heterodimerization.

FIG. 12a shows various dual bivalent bispecific antibody formats compared in vivo to the IgG-[L]-scFv design. Schematics show all four dual bivalent bispecific antibodies expressed.

FIG. 12b shows the mean tumor growth for in vivo huDKO arming model. Tumor responses were evaluated using a T-cell arming model, where T-cells were preincubated with each BsAb for 20 min at a concentration to achieve equal anti-GD2 binding domains (as verified by flow cytometry). These prelabeled or “armed” T-cells were injected intravenously into tumor bearing DKO mice. Each line represents one BsAb. Solid black triangles represent a dose of BsAb armed human activated T-cells (huATC) and IL-2. The dotted black line represents no measurable tumor and the star represents the tumor implantation. Error bars represent standard deviation.

FIG. 12c shows tumor growth from individual mice. Each figure represents one treatment group, with schematics (see above) for reference. Each solid line represents a single mouse, and the dotted lines represents the group average.

FIG. 13 demonstrates the combined binding effect of L1CAM/GD2 1+1+2 Lo, a heterodimeric 1+1+2Lo format antibody that can bind ganglioside GD2 and adhesion protein L1CAM simultaneously. Design of the 1+1+2 Lo format antibody is shown on the left side. Homodimeric formats against GD2 and L1CAM were 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, thus showing improved targeting specificity for tumors expressing both GD2 and L1CAM.

FIG. 14 demonstrates the combined binding effect of HER2/EGFR 1+1+2 Hi, a heterodimeric 1+1+2Hi format antibody that can bind both HER2 and EGFR, either simultaneously or separately. Design of the 1+1+2 Hi format antibody is shown on the right side. Homodimeric formats against HER2 and EGFR were included for reference. For this binding assay, Desmoplastic Small Cell Round 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 that of either anti-HER2 or anti-EGFR homodimeric antibodies, while maintaining specificity for both antigens, demonstrating cooperative binding.

FIG. 15 demonstrates the combined binding effect of GD2/B7H3 1+1+2 Lo, a heterodimeric 1+1+2Lo format antibody that can bind both GD2 and B7H3 simultaneously. Design of the 1+1+2 Lo format antibody is shown on the left hand side. Homodimeric formats against GD2 and B7H3, and monovalent control antibodies against GD2 or B7H3 (GD2 or B7H3 ctrl, 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 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 similar to the anti-B7H3 homodimeric antibody, but weaker than the anti-GD2 homodimeric antibody. Importantly, GD2/B7H3 1+1+2 Lo also showed improved binding over monovalent control antibodies, demonstrating cooperative binding.

FIG. 16 demonstrates the cytotoxic selectivity of HER2/GD2 1+1+2 Lo, a heterodimeric 1+1+2Lo format that can bind both GD2 and HER2 simultaneously. In this format, a low affinity HER2 sequence was used. Design of the 1+1+2 Lo format antibody is shown below the line graph. Homodimeric formats against GD2 and HER2, and monovalent control antibodies against GD2 or HER2 (GD2 and HER2 ctrl, respectively) were included for reference. For this cytotoxicity assay, Osteosarcoma cells (U2OS) were first incubated with ⁵¹Cr for one hour. After the incubation, the ⁵¹Cr labeled target cells were mixed with serial dilutions of the indicated antibody and activated human T-cells for four hours at 37° C. After four hours, supernatant was harvested and analyzed on a gamma counter to quantify the released ⁵¹Cr. Cytotoxicity was measured as the % of released ⁵¹Cr from maximum release. In this example, the low affinity 1+1+2 heterodimer antibody killed the target cells as effectively as the anti-GD2 and anti-HER2 homodimeric antibodies yet showing clear superiority over the monovalent control formats. This demonstrates the selectivity possible with the 1+1+2Lo design: target cells expressing each individual antigen will be targeted with 10-100-fold lower cytotoxic potency than targets expressing both antigens simultaneously. Using a homodimeric design for either GD2 or HER2 would lose such selectivity.

FIG. 17a demonstrates the cytotoxic dual specificity of HER2/GPA33 1+1+2 Hi, a heterodimeric 1+1+2Hi format that can bind both GPA33 and HER2 simultaneously. Design of the 1+1+2 Hi format antibody is shown below the line graph. Homodimeric formats against GPA33 and HER2, and monovalent control antibodies against GPA33 or HER2 were included for reference. For this cytotoxicity assay, Colon cancer cells (Colo205) were first incubated with ⁵¹Cr for one hour. After the incubation, the ⁵¹Cr labeled target cells were mixed with serial dilutions of the indicated antibody and activated human T-cells for four hours at 37° C. After four hours, the supernatant was harvested and read on a gamma counter to quantify the released ⁵¹Cr. Cytotoxicity was measured as the % of released ⁵¹Cr from maximum release. In this example, the high affinity 1+1+2 heterodimer antibody killed target cells as effectively as the anti-GPA33 homodimeric antibody, but with greater potency than the anti-HER2 homodimeric antibody and monovalent control antibodies. These data demonstrate functional cooperativity between the HER2 and GPA33 antigen-binding domains and illustrate that the dual specificity of a 1+1+2Hi format does not significantly compromise its cytotoxicity against either antigen individually.

FIG. 17b demonstrates the combined binding effect of HER2/GPA33 1+1+2 Hi, a heterodimeric 1+1+2Hi format that can bind both HER2 and GPA33, either simultaneously or separately. Design of the 1+1+2 Hi format antibody is shown on the right hand side. 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 the final wash, the cells were analyzed using flow cytometry. In this example, the affinity binding of the 1+1+2 heterodimer antibody was stronger than either anti-HER2 or anti-GPA33 homodimeric and monovalent control antibodies, while maintaining specificity for both antigens, demonstrating cooperative binding.

FIG. 18 demonstrates the utility of CD3/CD28 2+1+1, a heterodimeric 2+1+1 design that can bind both CD3 and CD28 on T-cells. Design of the heterodimeric 1+1+2 format antibody is shown below the line graph. Homodimeric formats against CD3 and CD28 were included for reference. For this cytokine assay, naïve human T-cells and Melanoma tumor cells (M14) were co-cultured along with the indicated BsAb for 20 hours before culture supernatants were harvested and analyzed for secreted cytokine IL-2 by flow cytometry. Data was normalized to T-cell cytokine release after 20 hours without target cells or antibody. The CD3/CD28 2+1+1 design showed clearly more potent cytokine release activity than either CD3 or CD28 engagement alone, illustrating cooperative activity from dual CD3/CD28 engagement.

FIG. 19 demonstrates the combined binding effect of CD3/CD4 2+1+1, a heterodimeric 2+1+1 format antibody that can bind both CD3 and CD4 simultaneously. Design of the heterodimeric 2+1+1 format antibody is shown on the right side. For this binding assay, active 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 the final wash, the cells were analyzed using flow cytometry. In this example, the 2+1+1 heterodimer shows enhanced binding compared to the bivalent CD4 and monomeric CD3 binder (2+1) demonstrating cooperative binding.

FIG. 20 demonstrates the combined binding effect of CD3/PD-1 2+1+1, a heterodimeric 2+1+1 format antibody that can bind both CD3 and PD-1 simultaneously. Design of the heterodimeric 2+1+1 format antibody is shown on the right side. For this binding assay active 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 the final wash, the cells were analyzed using flow cytometry. In this example, the binding of the 2+1+1 heterodimer was better than either anti-PD-1 homodimeric or anti-CD3 monomeric (2+1) binder, demonstrating cooperative binding.

FIGS. 21a-21c show the unique characteristics of the IgG-L-scFv design, compared to two other common dual bivalent design strategies: the BiTE-Fc and the IgG-H-scFv. FIG. 21a demonstrates the potent T-cell functional activity of the IgG-L-scFv design compared to other dual bivalent T-cell bispecific antibody formats. Designs of the IgG-L-scFv, BiTE-Fc and the IgG-H-scFv format antibodies are shown below the line graph. For this cytokine assay, naïve T-cells and melanoma tumor cells (M14) were co-cultured along with each BsAb for 20 hours before culture supernatants were harvested and analyzed for secreted cytokine IL-2 by flow cytometry. Data were normalized to T-cell cytokine release after 20 hours without target cells or antibody. In contrast to the IgG-H-scFv (2+2HC) and the BiTE-Fc (2+2B) designs, the IgG-L-scFv format (2+2) demonstrated significant cytokine IL-2 responses in vitro, which correlated with stronger in vivo activity (shown in FIG. 21c). FIG. 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 separately 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. Shown is CD3-specific (FIG. 21b, upper panel), and GD2-specific binding (FIG. 21b, middle panel). Designs of the IgG-L-scFv, BiTE-Fc and the IgG-H-scFv format antibodies are shown in FIG. 21b (lower panel). In contrast to their GD2 binding activity, each BsAb demonstrated quite different T-cell binding activities. These data demonstrated how the IgG-L-scFv design is uniquely different than other dual-bivalent designs, with each scFv showing incomplete bivalent binding. Although the inclusion of two scFv domains in the IgG-L-scFv does show improvement over monovalent designs, it still does not compare to the binding activity of the 2+2HC or 2+2B designs, illustrating the sterically hindered binding of this format. FIG. 21c illustrates the in vivo superiority of the IgG-L-scFv design. In contrast to other dual bivalent designs, the IgG-L-scFv format was the only one capable of controlling tumor growth in mice. Here, immunodeficient mice (Balb/c IL-2Rgc−/−, Rag2−/−) were implanted with neuroblastoma cells (IMR32) subcutaneously, before being treated with intravenous activated T-cells and antibody (2-times per week). Tumors sizes were measured by caliper.

FIG. 22 demonstrates the in vitro properties and design of anti-CD33/CD3 IgG-[L]-scFv panel. The in vitro cytotoxicity EC₅₀, fold-difference in EC₅₀, antigen valency, heterodimer design and protein purity by SEC-HPLC for anti-CD33/CD3 IgG-[L]-scFv panel are summarized. Fold change is based on the EC₅₀of 2+2. Purity was calculated as the fraction of protein at correct elution time out of the total protein by area under the curve of the SEC-HPLC chromatogram. For the cytotoxicity assays, CD33-transfected cells (Nalm6) were first incubated with ⁵¹Cr for one hour. Afterwards, ⁵¹Cr labeled target cells were mixed with serial titrations of the indicated antibody and activated human T-cells for four hours at 37° C. The supernatant was harvested and analyzed on a gamma counter to quantify the released ⁵¹Cr. Cytotoxicity was measured as the % of released ⁵¹Cr from maximum release. These results confirm the relative importance of Cis-oriented binding domains in an additional antigen system (CD33) which is much more membrane distal than GD2 (see FIG. 5).

FIG. 23 provides a summary of the various HDTVS antibodies tested in the Examples disclosed herein. The table summarizes all successfully produced HDTVS formatted multi-specific antibodies across a variety of antigen models. All clones were expressed in Expi293 cells and heterodimerized using the controlled Fab Arm Exchange method. HDTVS type displays the category of each clone. Fab 1 and scFv 1 (and corresponding Ag1 and Ag3) are attached in a cis-orientation on one heavy chain (linked by the light chain of Fab) while Fab 2 and scFv 2 (and corresponding Ag2 and Ag4) are on a separate heavy chain molecule in a cis-orientation (linked by the light chain of Fab).

DETAILED DESCRIPTION

It is to be appreciated that certain aspects, modes, embodiments, variations and features of the present methods are described below in various levels of detail in order to provide a substantial understanding of the present technology.

In practicing the present methods, many conventional techniques in molecular biology, protein biochemistry, cell biology, immunology, microbiology and recombinant DNA are used. See, e.g., 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., N.Y.); 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; U.S. Pat. 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 to detect and measure levels of polypeptide gene expression products (i.e., gene translation level) are well-known in the art and include the use of polypeptide detection methods such as antibody detection and quantification techniques. (See also, Strachan & Read, Human Molecular Genetics, Second Edition. (John Wiley and Sons, Inc., NY, 1999)).

Advances in protein engineering can enhance the functional output of proteins by linking different peptides in sequences, or by arranging them in complexes that do not exist naturally. Antibodies have served as a platform for such enhancements, where antigen binding can be modulated through antigen affinity maturation (Boder et al., Proc Natl Acad Sci USA 97:10701-10705 (2000)) or increases in valency (Cuesta et al., Trends Biotechnol 28:355-362 (2010)). Fc receptor binding can be modulated through point mutations (Leabman et al., MAbs 5:896-903 (2013)) or changes in glycosylation (Xu et al., Cancer Immun Res 4: 631-638 (2016)) whereas pharmacokinetics can be influenced through ablation of FcR(n) binding (Suzuki et al., J Immunol 184:1968-1976 (2010)) or removal of entire antibody domains. However, no single antibody platform to date has shown a clear and significant functional advantage over others within the clinic.

The present disclosure provides an antibody platform in which up to 4 different antigen binding domains can be used to simultaneously engage up to 4 different cellular targets, thereby increasing avidity and modulating specificity of the therapeutic antibodies. This platform is based on the heterodimerization of two IgG half molecules, in which each IgG half molecule comprises a heavy chain and a light chain, wherein a scFv is linked to the C-terminus of at least one light chain (i.e., IgG-[L]-scFv platform). The resulting heterodimers are both trivalent/tetravalent and multispecific and are collectively referred to as HDTVS antibodies.

The native form of the IgG-[L]-scFv platform has bivalent binding to two different targets (2+2) (each integer represents a different specificity, while its value represents the valency). The present disclosure provides 5 HDTVS platform variants which vary the 4 functional domains (2 Fabs and 2 scFv) in the IgG(L)-scFv format: (1) the Lo1+1+2 HDTVS variant to achieve improved tumor cell specificity, (2) the Hi1+1+2 HDTVS variant to achieve broader tumor cell selectivity, (3) the 2+1+1 HDTVS variant to achieve improved immune cell activation, (4) the 2+1+1 HDTVS variant which allows recruitment of different cells and/or payloads and (5) the 1+1+1+1 HDTVS variant which combines designs from (1) or (2) with (3) or (4) to achieve more effective immune activation or payload delivery with finer specificity or broader selectivity. (FIGS. 1a-1f). In order to test the functional output of these HDTVS variants, one of the 2 Fab domains can be neutralized by using an irrelevant Fab that has no binding to either tumor cells or effector immune cells (e.g., T cells), creating monovalency for tumor. Alternatively, one of the scFv domains can be removed to create monovalency towards effector immune cells (e.g., T cells).

As described herein, the biological potency of each design is dependent on the biophysical characteristics of the antigen binding domains of the HDTVS variants. Unexpectedly, the changes in valency did not entirely correlate with changes in functional output. As shown in Examples described herein, the biological activity of the tri- and tetra-specific variants of the HDTVS platform is dependent on the antigen/epitope combinations, as well as the relative binding affinities to each target antigen (up to 4 targets total). The Lo1+1+2 HDTVS variant requires its Fab domains to bind to two distinct tumor antigens that are within a proximity of 60-120 angstroms from each other (thus allowing simultaneous binding), and (b) have monovalent and/or effective binding affinities (K_D) that range from about 100 nM to about 100 pM to reduce bystander reactivity with healthy cells. The Hi1+1+2 HDTVS variant on the other hand exploits the high monovalent and/or effective binding affinity (K_D<100 pM) of its Fab domains such that monovalency is nearly as effective as bivalency. Moreover, the 2+1+1 HDTVS variant exhibited in vivo tumor clearance activity that was comparable to that observed with the 2+2 native form of the IgG-[L]-scFv platform. These results were unexpected given that the binding activities of the 2+1+1 HDTVS variant were about 6-fold lower than the 2+2 native form of the IgG-[L]-scFv platform.

Accordingly, biophysical properties such as orientation (cis vs trans), valency (mono- vs bi-valent) and target affinity (K_D˜nM or <pM) had an unpredictable impact on the functionality of the HDTVS variants (e.g., log-fold enhancement of therapeutic efficacy). Moreover, the HDTVS antibodies of the present technology show superior therapeutic potency compared to other conventional antibody platforms, such as BiTE or heterodimeric IgG (IgG-Het). These results also demonstrate that different multispecific antibody platforms yield antibodies that possess substantially different biological properties. Without wishing to be bound by theory, it is believed that spatial distances between the antigen binding domains of multispecific antibodies, as well as the relative flexibility and orientation of the individual antigen binding domains may determine their ability to drive cell-to-cell interactions.

Definitions

Unless defined otherwise, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this technology belongs. As used in this specification and the appended claims, the singular forms “a”, “an” and “the” include plural referents unless the content clearly dictates otherwise. For example, reference to “a cell” includes a combination of two or more cells, and the like. Generally, the nomenclature used herein and the laboratory procedures in cell culture, molecular genetics, organic chemistry, analytical chemistry and nucleic acid chemistry and hybridization described below are those well-known and commonly employed in the art.

As used herein, a “2+1+1” design refers to a HDTVS antibody in which the two Fab domains recognize and bind to the same target antigen, and the two scFvs recognize and bind to two distinct target antigens. In some embodiments, the two scFvs of the 2+1+1 HDTVS antibody binds to two distinct target antigens that are up to 180 angstroms apart from each other in order to engage two separate molecules on the same cell.

As used herein, the term “about” in reference to a number is generally taken to include numbers that fall within a range of 1%, 5%, or 10% in either direction (greater than or less than) of the number unless otherwise stated or otherwise evident from the context (except where such number would be less than 0% or exceed 100% of a possible value).

As used herein, the “administration” of an agent or drug to a subject includes any route of introducing or delivering to a subject a compound to perform its intended function. Administration can be carried out by any suitable route, including but not limited to, orally, intranasally, parenterally (intravenously, intramuscularly, intraperitoneally, or subcutaneously), rectally, intrathecally, intratumorally or topically. Administration includes self-administration and the administration by another.

As used herein, the term “antibody” collectively refers to immunoglobulins or immunoglobulin-like molecules including by way of example and without limitation, IgA, IgD, IgE, IgG and IgM, combinations thereof, and similar molecules produced during an immune response in any vertebrate, for example, in mammals such as humans, goats, rabbits and mice, as well as non-mammalian species, such as shark immunoglobulins. As used herein, “antibodies” (includes intact immunoglobulins) and “antigen binding fragments” 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 (for example, antibodies and antibody fragments that have a binding constant for the molecule of interest that is at least 10³greater, at least 10⁴M⁻¹greater or at least 10⁵greater than a binding constant for other molecules in a biological sample). The term “antibody” also includes genetically engineered forms such as chimeric antibodies (for example, humanized murine antibodies), heteroconjugate antibodies (such as, bispecific antibodies). See also, Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, Ill.); Kuby, J., Immunology, 3^rdEd., W.H. Freeman & Co., New York, 1997.

More particularly, antibody refers to a polypeptide ligand comprising at least a light chain immunoglobulin variable region or heavy chain immunoglobulin variable region which specifically recognizes and binds an epitope of an antigen. Antibodies are composed of a heavy and a light chain, each of which has a variable region, termed the variable heavy (V_H) region and the variable light (V_L) region. Together, the VH region and the VL region are responsible for binding the antigen recognized by the antibody. Typically, an immunoglobulin has heavy (H) chains and light (L) chains interconnected by disulfide bonds. There are two types of light chain, lambda (λ) and kappa (κ). There are five main heavy chain classes (or isotypes) which determine the functional activity of an antibody molecule: IgM, IgD, IgG, IgA and IgE. Each heavy and light chain contains a constant region and a variable region, (the regions are also known as “domains”). In combination, the heavy and the light chain variable regions specifically bind the antigen. Light and heavy chain variable regions contain a “framework” region interrupted by three hypervariable regions, also called “complementarity-determining regions” or “CDRs”. The extent of the framework region and CDRs have been defined (see, Kabat et al., Sequences of Proteins of Immunological Interest, U.S. Department of Health and Human Services, 1991, which is hereby incorporated by reference). The Kabat database is now maintained online. The sequences of the framework regions of different light or heavy chains are relatively conserved within a species. The framework region of an antibody, that is the combined framework regions of the constituent light and heavy chains, largely adopt a β-sheet conformation and the CDRs form loops which connect, and in some cases form part of, the β-sheet structure. Thus, framework regions act to form a scaffold that provides for positioning the CDRs in correct orientation by inter-chain, non-covalent interactions.

The 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, a V_HCDR3 is located in the variable domain of the heavy chain of the antibody in which it is found, whereas a V_LCDR1 is the CDR1 from the variable domain of the light chain of the antibody in which it is found. An antibody that binds a target antigen will have a specific V_Hregion and the V_Lregion sequence, and thus specific CDR sequences. Antibodies with different specificities (i.e. different combining sites for different antigens) have different CDRs. Although it is the CDRs that vary from antibody to antibody, only a limited number of amino acid positions within the CDRs are directly involved in antigen binding. These positions within the CDRs are called specificity determining residues (SDRs). “Immunoglobulin-related compositions” as used herein, refers 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 antigen binding fragment thereof specifically binds to an antigen.

As used herein, the term “antibody-related polypeptide” means antigen binding antibody fragments, including single-chain antibodies, that can comprise the variable region(s) alone, or in combination, with all or part of the following polypeptide elements: hinge region, CH₁, CH₂, and CH₃domains of an antibody molecule. Also included in the technology are any combinations of variable region(s) and hinge region, CH₁, CH₂, and CH₃domains. Antibody-related molecules useful in the present methods, e.g., but are not limited to, Fab, Fab′ and F(ab′)₂, Fd, single-chain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs (sdFv) and fragments comprising either a V_Lor V_Hdomain. Examples include: (i) a Fab fragment, a monovalent fragment consisting of the V_L, V_H, C_Land CH₁domains; (ii) a F(ab′)₂fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the V_Hand CH₁domains; (iv) a Fv fragment consisting of the V_Land V_Hdomains of a single arm of an antibody, (v) a dAb fragment (Ward et al., Nature 341: 544-546, 1989), which consists of a V_Hdomain; and (vi) an isolated complementarity determining region (CDR). As such “antibody fragments” or “antigen binding fragments” can comprise a portion of a full-length antibody, generally the antigen binding or variable region thereof. Examples of antibody fragments or antigen binding fragments include Fab, Fab′, F(ab′)₂, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.

“Bispecific antibody” or “BsAb”, as used herein, refers to an antibody that can bind simultaneously to two targets that have a distinct structure, e.g., two different target antigens, two different epitopes on the same target antigen, or a hapten and a target antigen or 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 includes V_Hand/or V_Lregions; in some such embodiments, the V_Hand/or V_Lregions are those found in a particular monoclonal antibody. In some embodiments, the bispecific antibody contains two antigen binding moieties, each including VH and/or VL regions from different monoclonal antibodies. In some embodiments, the bispecific antibody contains two antigen binding moieties, wherein one of the two antigen binding moieties includes an immunoglobulin molecule having VH and/or VL regions that contain CDRs from a first monoclonal antibody, and the other antigen binding moiety includes an antibody fragment (e.g., Fab, F(ab′), F(ab′)₂, Fd, Fv, dAB, scFv, etc.) having VH and/or VL regions that contain CDRs from a second monoclonal antibody.

As used herein, the term “diabodies” refers to small antibody fragments with two antigen binding sites, which fragments comprise a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) in the same polypeptide chain (VH VL). 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 the complementary domains of another chain and create two antigen binding sites. Diabodies are described more fully in, e.g., EP 404,097; WO 93/11161; and 30 Hollinger et al., Proc. Natl. Acad. Sci. USA, 90: 6444-6448 (1993).

As used herein, the terms “single-chain antibodies” or “single-chain Fv (scFv)” refer to an antibody fusion molecule of the two domains of the Fv fragment, VL and VH. Single-chain antibody molecules may comprise a polymer with a number of individual molecules, for example, dimer, trimer or other polymers. Furthermore, although the two domains of the F_vfragment, V_Land V_H, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the V_Land V_Hregions pair to form monovalent molecules (known as single-chain F_v(scFv)). 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 enzymatic or chemical cleavage of intact antibodies.

Any of the above-noted antibody fragments are obtained using conventional techniques known to those of skill in the art, and the fragments are screened for binding specificity and neutralization activity in the same manner as are intact antibodies.

As used herein, an “antigen” refers to a molecule to which an antibody (or antigen binding fragment thereof) can selectively bind. The target antigen may be a protein, carbohydrate, nucleic acid, lipid, hapten, or other naturally occurring or synthetic compound. In some embodiments, the target antigen may be a polypeptide. An antigen may also be administered to an animal to generate an immune response in the animal.

The term “antigen binding fragment” refers to a fragment of the whole immunoglobulin structure which possesses a part of a polypeptide responsible for binding to antigen. Examples of the antigen binding fragment useful in the present technology include scFv, (scFv)₂, scFvFc, Fab, Fab′ and F(ab′)₂, but are not limited thereto.

By “binding affinity” is meant 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 can generally be represented by the dissociation constant (Ku). Affinity can be measured by standard methods known in the art, including those described herein. A low-affinity complex contains an antibody that generally tends to dissociate readily from the antigen, whereas a high-affinity complex contains an antibody that generally tends to remain bound to the antigen for a longer duration.

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

As used herein, the term “cancer” refers to a neoplasm or tumor resulting from abnormal uncontrolled growth of cells. In some embodiments, cancer refers to a benign tumor or a malignant tumor. In some embodiments, the cancer is associated with a specific cancer antigen.

As used herein, the term “CDR-grafted antibody” means an antibody in which at least one CDR of an “acceptor” antibody is replaced by a CDR “graft” from a “donor” antibody possessing a desirable antigen specificity.

As used herein, the term “chimeric antibody” means an antibody in which the Fc constant region of a monoclonal antibody from one species (e.g., a mouse Fc constant region) is replaced, using recombinant DNA techniques, with an Fc constant region 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 184,187; Taniguchi, European Patent Application 171,496; Morrison et al., European Patent Application 173,494; Neuberger et al., WO 86/01533; Cabilly et al. U.S. Pat. No. 4,816,567; Cabilly et al., European Patent Application 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. Sci. USA 84: 214-218, 1987; Nishimura et al., Cancer Res 47: 999-1005, 1987; Wood et al., Nature 314: 446-449, 1885; and Shaw et al., J. Natl. Cancer Inst. 80: 1553-1559, 1988.

As used herein, the term “consensus FR” means a framework (FR) antibody region in a consensus immunoglobulin sequence. The FR regions of an antibody do not contact the antigen.

As used herein, a “control” is an alternative sample used in an experiment for comparison purpose. A control can be “positive” or “negative.” For example, where the purpose of the experiment is to determine a correlation of the efficacy of a therapeutic agent for the 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 a sample that does not receive the therapy or receives a placebo) are typically employed.

As used herein, the term “effective affinity” refers to the binding constant derived from measuring the overall binding kinetics of a compound with two or more simultaneous binding interactions (e.g., with an IgG, IgM, IgA, IgD, or IgE molecule instead of a Fab domain).

As used herein, the term “effective amount” refers to a quantity sufficient to achieve a desired therapeutic and/or prophylactic effect, e.g., an amount which results in the prevention of, or a decrease in 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 the subject will vary depending on the composition, the degree, type, and severity of the disease and on the characteristics of the individual, such as general health, age, sex, body weight and tolerance to drugs. The skilled artisan will be able to determine appropriate dosages depending on these and other factors. The compositions can also be administered in combination with one or more additional therapeutic compounds. In the methods described herein, the therapeutic compositions may be administered to a subject having 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 composition levels in which the physiological effects of a disease or condition are ameliorated or eliminated. A therapeutically effective amount can be given in one or more administrations.

As used herein, the term “effector cell” means an immune cell which is involved in the effector phase of an immune response, as opposed to the cognitive and activation phases of an immune response. Exemplary immune cells include a cell of a myeloid or lymphoid origin, e.g., 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 carry out specific immune functions. An effector cell can induce antibody-dependent cell-mediated cytotoxicity (ADCC), e.g., a neutrophil capable of inducing ADCC. For example, monocytes, macrophages, neutrophils, eosinophils, and lymphocytes which express FcaR are involved in specific killing of target cells and presenting antigens to other components of the immune system, or binding to cells that present antigens.

“Effector function” as used herein refers to a biochemical event that results from the interaction of an antibody Fc region 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 those that operate after the binding of an antigen and those that operate independent of antigen binding.

As used herein, the term “epitope” means an antigenic determinant (site on an antigen) capable of specific binding to an antibody. Epitopes usually 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 non-conformational epitopes are distinguished in that the binding to the former but not the latter is lost in the presence of denaturing solvents. Thus, in some embodiments, the heterodimeric trivalent/tetravalent multispecific antibodies disclosed herein may bind a non-conformational epitope and/or a conformational epitope. To screen for antibodies which bind to an epitope, a routine cross-blocking assay such as that described in Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, Ed Harlow and David Lane (1988), can be performed. This assay can be used to determine if an antibody binds the same site or epitope as a heterodimeric trivalent/tetravalent multispecific antibody of the present technology. Alternatively, or additionally, 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 a target protein antigen can be used in competition assays with the test antibodies or with a test antibody and an antibody with a characterized or known epitope.

As used herein, “expression” includes one or more of the following: transcription of the gene into precursor mRNA; splicing and other processing of the precursor mRNA to produce mature mRNA; mRNA stability; translation of the mature mRNA into protein (including codon usage and tRNA availability); and glycosylation and/or other modifications of the translation product, if required for proper expression and function.

As used herein, the term “gene” means a segment of DNA that contains all the information for the regulated biosynthesis of an RNA product, including promoters, exons, introns, and other untranslated regions that control expression.

As used herein, a “heterodimerization domain that is incapable of forming a stable homodimer” 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) which either exclusively self-assembles as a heterodimer with the second complementary member of the pair, or shows at least a 10⁴fold preference for assembling into a heterodimer with the second complementary member of the pair, or forms a homodimer with an identical member that is not stable under reducing conditions such as >2 mM 2-MEA at room temperature for 90 minutes (see e.g., Labrijn, A. F. et al., Proc. Natl. Acad. Sci. 110, 5145-50 (2013). Examples of such heterodimerization domains include, but are not limited to CH2-CH3 that include any of the Fc variants/mutations described herein, WinZip-A1B1, a pair of complementary oligonucleotides, and a CH-1 and CL pair.

As used herein, “Hi1+1+2” refers to a heterodimeric tetravalent multispecific antibody in which the Fab domains (a) bind to two distinct target epitopes and (b) have monovalent binding affinities or effective affinities (K_D) that are <100 pM.

As used herein, the term “humanized” forms of non-human (e.g., murine) antibodies are chimeric antibodies which contain minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins in which hypervariable region residues of the recipient are replaced by hypervariable region residues from a non-human species (donor antibody) such as mouse, rat, rabbit or nonhuman 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 which are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance such as binding affinity. Generally, the 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 all or substantially all of the FR regions are those of a human immunoglobulin consensus FR sequence although the FR regions may include one or more amino acid substitutions that improve binding affinity. The number of these amino acid substitutions in the FR is typically no more than 6 in the H chain, and in the L chain, no more than 3. The humanized antibody optionally 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).

As used herein, the term “hypervariable region” refers to the amino acid residues of an antibody which are responsible for antigen binding. The hypervariable region generally comprises amino acid residues from a “complementarity determining region” or “CDR” (e.g., around about residues 24-34 (L1), 50-56 (L2) and 89-97 (L3) in the V_L, and around about 31-35B (H1), 50-65 (H2) and 95-102 (H3) in the V_H(Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)) and/or those residues from a “hypervariable loop” (e.g., residues 26-32 (L1), 50-52 (L2) and 91-96 (L3) in the V_L, and 26-32 (H1), 52A-55 (H2) and 96-101 (H3) in the V_H(Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)).

As used herein, the term “intact antibody” or “intact immunoglobulin” means an antibody that has at least two heavy (H) chain polypeptides and two light (L) chain polypeptides interconnected by disulfide bonds. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as HCVR or V_H) and a heavy chain constant region. The heavy chain constant region is comprised of three domains, CH₁, CH₂and CH₃. Each light chain is comprised of a light chain variable region (abbreviated herein as LCVR or V_L) and a light chain constant region. The light chain constant region is comprised of one domain, CL. The V_Hand V_Lregions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each V_Hand V_Lis composed of three CDRs and four FRs, arranged from amino-terminus to carboxyl-terminus in the following order: FR₁, CDR₁, FR₂, CDR₂, FR₃, CDR₃, FR₄. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant regions of the antibodies can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.

As used herein, the terms “individual”, “patient”, or “subject” can be an individual organism, a vertebrate, a mammal, or a human. In some embodiments, the individual, patient or subject is a human.

As used herein, “Lo1+1+2” refers to a heterodimeric tetravalent multispecific antibody in which the Fab domains (a) bind to two distinct target epitopes that are within a proximity of 60-120 angstroms from each other (thus allowing simultaneous binding), and (b) have monovalent binding affinities or effective affinities (K_D) that range from about 100 nM to about 100 pM.

The term “monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. For example, a monoclonal antibody can be an antibody that is 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 as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. Monoclonal antibodies can be prepared using a wide variety of techniques known in the art including, e.g., but not limited to, hybridoma, recombinant, and phage display technologies. For example, the monoclonal antibodies to be 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). The “monoclonal antibodies” may also be isolated from phage antibody libraries using the techniques described in Clackson et al., Nature 352:624-628 (1991) and Marks et al., J. Mol. Biol. 222:581-597 (1991), for example.

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, and the like, compatible with pharmaceutical administration. Pharmaceutically-acceptable carriers and their formulations are known to one skilled in the art and are described, for example, in Remington's Pharmaceutical Sciences (20^thedition, ed. A. Gennaro, 2000, Lippincott, Williams & Wilkins, Philadelphia, Pa.).

As used herein, the term “polyclonal antibody” means a preparation of antibodies derived from at least two (2) different antibody-producing cell lines. The use of this term includes preparations of at least two (2) antibodies that contain antibodies that specifically bind to different epitopes or regions of an antigen.

As used herein, the term “polynucleotide” or “nucleic acid” means any RNA or DNA, which may be unmodified or modified RNA or DNA. Polynucleotides include, without limitation, single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, RNA that is mixture of single- and double-stranded regions, and hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions. In addition, polynucleotide refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA. The term polynucleotide also includes DNAs or RNAs containing one or more modified bases and DNAs or RNAs with backbones modified for stability or for other reasons.

As used herein, the terms “polypeptide”, “peptide” and “protein” are used interchangeably herein to mean a polymer comprising two or more amino acids joined 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, generally referred to as proteins. Polypeptides may contain amino acids other than the 20 gene-encoded amino acids. Polypeptides include amino acid sequences modified either 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 in more detailed monographs, as well as in a voluminous research literature.

As used herein, the term “recombinant” when used with reference, e.g., to a cell, or 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 native nucleic acid or protein, or that the material is derived from a cell so modified. Thus, for example, recombinant cells express genes that are not found within the native (non-recombinant) form of the cell or express native genes that are otherwise abnormally expressed, under expressed or not expressed at all.

As used herein, the term “separate” therapeutic use refers to an administration of at least two active ingredients at the same time or at substantially the same time by different routes.

As used herein, the term “sequential” therapeutic use refers to administration of at least two active ingredients at different times, the administration route being identical or different. More particularly, sequential use refers to the whole administration of one of the active ingredients before administration of the other or others commences. It is thus possible to administer one of the active ingredients over several minutes, hours, or days before administering the other active ingredient or ingredients. There is no simultaneous treatment in this case.

As used herein, “specifically binds” refers to a molecule (e.g., an antibody or antigen binding fragment thereof) which recognizes and binds another molecule (e.g., an antigen), but that does not substantially recognize and bind other molecules. The terms “specific binding,” “specifically binds to,” or is “specific for” a particular molecule (e.g., a polypeptide, or an epitope on a polypeptide), as used herein, can be exhibited, for example, by a molecule having a K_Dfor the molecule to which it binds to of about 10⁻⁴M, 10⁻⁵M, 10⁻⁶M, 10⁻⁷M, 10⁻⁸M, 10⁻⁹M, 10⁻¹⁰M, 10⁻¹¹M, or 10⁻¹²M. The term “specifically binds” may also refer to binding where 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 polypeptide, or polypeptide epitope.

As used herein, the term “simultaneous” therapeutic use refers to the administration of at least two active ingredients by the same route and at the same time or at substantially the same time.

As used herein, the term “therapeutic agent” is intended to mean a compound that, when present in an effective amount, produces a desired therapeutic effect on a subject in need thereof.

“Treating” or “treatment” as used herein covers the treatment of a disease or disorder described herein, in a subject, such as a human, and includes: (i) inhibiting a disease or disorder, i.e., arresting its development; (ii) relieving a disease or disorder, i.e., causing regression of the disorder; (iii) slowing progression of the disorder; and/or (iv) inhibiting, relieving, or slowing progression of one or more symptoms of the disease or disorder. In some embodiments, treatment means that the symptoms associated with the disease are, e.g., alleviated, reduced, cured, or placed in a state of remission.

It is also to be appreciated that the various modes of treatment of disorders as described herein are intended to mean “substantial,” which includes total but also less than total treatment, and wherein some biologically or medically relevant result is achieved. The treatment may be a continuous prolonged treatment for a chronic disease or a single, or few time administrations for the treatment of an acute condition.

Heterodimeric Trivalent/Tetravalent Multispecific Antibodies of the Present Technology

The heterodimeric trivalent/tetravalent multispecific antibodies of the present technology can bind simultaneously to three or four targets that have a distinct structure, e.g., 3-4 different target antigens, 3-4 different epitopes on the same target antigen, or a combination of haptens and target antigens or epitopes on a target antigen. A variety of HDTVS antibodies can be produced using molecular engineering. For example, the HDTVS antibodies disclosed herein utilize combinations of the full immunoglobulin framework (e.g., IgG), and single chain variable fragments (scFvs).

HDTVS antibodies can be made, for example, by combining and/or engineering heavy chains and/or light chains that recognize different epitopes of the same or different antigen. In some embodiments, the HDTVS protein is trivalent and tri-specific, comprising, for example, an immunoglobulin (e.g., IgG) with a binding site for a first antigen (one V_H/V_Lpair) and a binding site for a second antigen (a different V_H/V_Lpair) and an scFv for a third antigen. In some embodiments, the HDTVS protein is trivalent and bispecific, comprising, for example, an immunoglobulin (e.g., IgG) with two binding sites (two V_H/V_Lpairs) for a first antigen, and a scFv for a second antigen. In some embodiments, the HDTVS protein is tetravalent and tri-specific, comprising, for example, an immunoglobulin (e.g., IgG) with a binding site for a first antigen (one V_H/V_Lpair) and a binding site for a second antigen (a different V_H/V_Lpair) and two identical scFvs for a third antigen. In some embodiments, the HDTVS protein is tetravalent and tri-specific, comprising, for example, an immunoglobulin (e.g., IgG) with two binding sites (two V_H/V_Lpairs) for a first antigen, an scFv for a second antigen and an scFv for a third antigen. In some embodiments, the HDTVS protein is tetravalent and tetra-specific, comprising, for example, an immunoglobulin (e.g., IgG) with a binding site for a first antigen (one V_H/V_Lpair) and a binding site for a second antigen (different V_H/V_Lpair), an scFv for a third antigen and an scFv for a fourth antigen.

In some embodiments, at least one scFv of the HDTVS antibodies 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 antibodies of the present technology binds to an antigen 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), 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.

Additionally or alternatively, in certain embodiments, the HDTVS antibodies disclosed herein are capable of binding to cells (e.g., tumor cells) that express a cell surface antigen 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, CD25? (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 (NTSE), 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, Kallikreins, LINGO1, LOXL2, Ly6/PLAUR domain-containing protein 3, MADCAM1, MAG, Mesothelin, MT1-MMP (MMP14), MUC1, Mucin SAC, NaPi2b, NeuGc-GM3, notch, NOTCH2/NOTCH3 receptors, oxLDL, P-selectin, PCSK9, PDGFRA, PDGFRa, phosphatidylserine, polysialic acid, PSMA, PVRL4, RGMA, CD240D Blood group D antigen, 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 extra domain-B.

Methods for producing the HDTVS antibodies of the present technology include engineered recombinant monoclonal antibodies which have additional cysteine residues so that they crosslink more strongly than the more 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 needed dual specificities. See, e.g., Coloma et al., Nature Biotech. 15:159-163 (1997).

Recombinant methods can be used to produce a variety of fusion proteins. In some embodiments, a HDTVS antibody according to the present technology comprises an immunoglobulin, which immunoglobulin comprises two heavy chains and two light chains, and two scFvs, wherein each scFv is linked to the C-terminal end of one of the two light chains of any immunoglobulin disclosed herein. In various embodiments, scFvs are linked to the light chains via a linker sequence. In some embodiments, a 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 or more amino acids in length.

In some embodiments, a linker is characterized in that it tends not to adopt a rigid three-dimensional structure, but rather provides flexibility to the polypeptide (e.g., first and/or second antigen binding sites). In some embodiments, a linker is employed in a HDTVS antibody described herein based on specific properties imparted to the HDTVS antibody such as, for example, an increase in stability. In some embodiments, a HDTVS antibody of the present technology comprises a G₄S linker (SEQ ID NO: 2508). In certain embodiments, a HDTVS antibody of the present technology comprises a (G₄S)_nlinker, wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15 or more (SEQ ID NO: 2509).

Exemplary V_Hand V_Lamino acid sequences that may be employed in the HDTVS antibodies of the present technology are provided in Table 1.

TABLE 1

SEQ

SEQ

SEQ

SEQ

SEQ

SEQ

SEQ

SEQ

ID
V_L
ID
V_L
ID
V_L
ID

ID
V_H
ID
V_H
ID
V_H
ID

Antigen
V_L
NO
CDR1
NO
CDR2
NO
CDR3
NO
V_H
NO
CDR1
NO
CDR2
NO
CDR3
NO

a2b b3
DILMTQSPSSM
1
QGIS
2
YGT
3
VQY
4
EVQLQQSGAELV
5
GFNI
6
IDPA
7
VRPL
8

(Glyco-
SVSLGDTVSIT

SN

AQLP

KPGASVKLSCTA

KDT

NGYT

YDYY

protein
CHASQGISSNI

YT

SGFNIKDTYVHW

Y

AMDY

IIb/
GWLQQKPGKS

VKQRPEQGLEWI

IIIa)
FMGLIYYGTN

GRIDPANGYTKY

LVDGVPSRFS

DPKFQGKATITA

GSGSGADYSL

DTSSNTAYLQLS

TISSLDSEDFA

SLTSEDTAVYYC

DYYCVQYAQ

VRPLYDYYAMD

LPYTFGGGTK

YWGQGTSVTVSS

LEIK

a2b b3
DIQMTQTPSTL
9
QDIN
10
YTS
11
QQG
12
QVQLVQSGAEV
13
GYA
14
IYPG
15
ARRD
16

(Glyco-
SASVGDRVTIS

NY

NTLP

KKPGSSVKVSCK

FTNY

SGGT

GNYG

protein
CRASQDINNY

WT

ASGYAFTNYLIE

L

WFAY

IIb/
LNWYQQKPG

WVRQAPGQGLE

IIIa)
KAPKLLIYYTS

WIGVIYPGSGGT

TLHSGVPSRFS

NYNEKFKGRVTL

GSGSGTDYTL

TVDESTNTAYME

TISSLQPDDFA

LSSLRSEDTAVY

TYFCQQGNTL

FCARRDGNYGWF

PWTFGQGTKV

AYWGQGTLVTV

EVK

SS

a4
DIQMTQSPSSL
17
QDIN
18
YTS
19
LQY
20
QVQLVQSGAEV
21
GFNI
22
IDPA
23
AREG
24

SASVGDRVTIT

KY

DNL

KKPGASVKVSCK

KDT

NGYT

YYGN

CKTSQDINKY

WT

ASGFNIKDTYIH

Y

YGVY

MAWYQQTPG

WVRQAPGQRLE

AMDY

KAPRLLIHYTS

WMGRIDPANGY

ALQPGIPSRFS

TKYDPKFQGRVT

GSGSGRDYTF

ITADTSASTAYM

TISSLQPEDIA

ELSSLRSEDEAV

TYYCLQYDNL

YYCAREGYYGN

WTFGQGTKVE

YGVYAMDYWG

IK

QGTLVTVSS

a4b7
DVVMTQSPLS
25
QSLA
26
GIS
27
LQGT
28
QVQLVQSGAEV
29
GYTF
30
IDPS
31
ARGG
32

LPVTPGEPASI

KSYG

HQP

KKPGASVKVSCK

TSY

ESNT

YDGW

SCRSSQSLAKS

NTY

YT

GSGYTFTSYWM

W

DYAI

YGNTYLSWYL

HWVRQAPGQRL

DY

QKPGQSPQLLI

EWIGEIDPSESN

YGISNRFSGVP

TNYNQKFKGRVT

DRFSGSGSGT

LTVDISASTAYM

DFTLKISRVEA

ELSSLRSEDTAV

EDVGVYYCLQ

YYCARGGYDGWD

GTHQPYTFGQ

YAIDYWGQGTL

GTKVEIK

VTVSS

a4b7 +
DIQMTQSPSSL
33
ESVD
34
YAS
35
QQG
36
EVQLVESGGGLV
37
GFFI
38
ISYS
39
ARTG
40

aEb7
SASVGDRVTIT

DL

Q

NSLP

QPGGSLRLSCAA

TNN

GST

SSGY

CRASESVDDL

NT

SGFFITNNYWGW

Y

FDF

LHWYQQKPG

VRQAPGKGLEW

KAPKLLIKYAS

VGYISYSGSTSY

QSISGVPSRFS

NPSLKSRFTISR

GSGSGTDFTLT

DTSKNTFYLQMN

ISSLQPEDFAT

SLRAEDTAVYYC

YYCQQGNSLP

ARTGSSGYFDFW

NTFGQGTKVE

GQGTLVTVSS

IK

a5
EIVLTQSPATL
41
QSVS
42
DAS
43
QQRS
44
QVQLVESGGGV
45
GFTF
46
ISFD
47
AREA
48

SLSPGERATLS

SY

NWP

VQPGRSRRLSCA

SRYT

GSNK

RGSY

CRASQSVSSY

PFT

ASGFTFSRYTMH

AFDI

LAWYQQKPG

WVRQAPGKGLE

QAPRLLIYDAS

WVAVISFDGSNK

NRATGIPARFS

YYVDSVKGRFTI

GSGSGTDFTLT

SRDNSENTLYLQ

ISSLEPEDFAV

VNILRAEDTAVY

YYCQQRSNWP

YCAREARGSYAF

PFTFGPGTKV

DIWGQGTMVTV

DIK

SS

Activin
QSALTQPASV
49
SSDV
50
GVS
51
GTFA
52
QVQLVQSGAEV
53
GYTF
54
INPV
55
ARGG
56

recep-
SGSPGQSITIS

GSYN

GGS

KKPGASVKVSCK

TSSY

SGST

WFDY

tor
CTGTSSDVGSY

Y

YYG

ASGYTFTSSYIN

type-2B
NYVNWYQQH

V

WVRQAPGQGLE

PGKAPKLMIY

WMGTINPVSGST

GVSKRPSGVS

SYAQKFQGRVT

NRFSGSKSGN

MTRDTSISTAYM

TASLTISGLQA

ELSRLRSDDTAV

EDEADYYCGT

YYCARGGWFDY

FAGGSYYGVF

WGQGTLVTVSS

GGGTKLTVL

ALK1
EIVLTQSPGTL
57
QSVS
58
GTS
59
QQY
60
QVQLQESGPGLV
61
GGSI
62
IYYS
63
ARES
64

SLSPGERATLS

SSY

GSSP

KPSQTLSLTCTV

SSGE

GST

VAGF

CRASQSVSSSY

IT

SGGSISSGEYYW

YY

DY

LAWYQQKPG

NWIRQHPGKGLE

QAPRLLIYGTS

WIGYIYYSGSTY

SRATGIPDRFS

YNPSLKSRVTIS

GSGSGTDFTLT

VDTSKNQFSLKL

ISRLEPEDFAV

SSVTAADTAVYY

YYCQQYGSSPI

CARESVAGFDYW

TFGQGTRLEIK

GQGTLVTVSS

Alpha-
DIQMTQSPSSL
65
QTLL
66
WAS
67
QQY
68
EVQLVESGGGLV
69
GFTF
70
ISSG
71
ARGG
72

synu-
SASVGDRVTIT

YSSN

YSYP

QPGGSLRLSCAA

SNY

GGST

AGID

clein
CKSIQTLLYSS

QKNY

LT

SGFTFSNYGMSW

G

YW

NQKNYLAWF

VRQAPGKGLEW

QQKPGKAPKL

VASISSGGGSTY

LIYWASIRKSG

YPDNVKGRFTIS

VPSRFSGSGSG

RDDAKNSLYLQM

TDFTLTISSLQ

NSLRAEDTAVYY

PEDLATYYCQ

CARGGAGIDYW

QYYSYPLTFG

GQGTLVTVSS

GGTKLEIK

amyloid
DVVMTQSPLS
73
QSLL
74
LYS
75
WQG
76
EVQLLESGGGLV
77
GFTF
78
IRSG
79
VRYD
80

beta
LPVTPGEPASI

DSDG

THFP

QPGGSLRLSCAA

SNY

GGRT

HYSG

SCKSSQSLLDS

KTY

RT

SGFTFSNYGMSW

G

SSDY

DGKTYLNWLL

VRQAPGKGLEW

QKPGQSPQRLI

VASIRSGGGRTY

YLVSKLDSGV

YSDNVKGRFTIS

PDRFSGSGSGT

RDNSKNTLYLQ

DFTLKISRVEA

MNSLRAEDTAV

EDVGVYYCW

YYCVRYDHYSGS

QGTHFPRTFG

SDYWGQGTLVT

QGTKVEIK

VSS

amyloid
DVVMTQSPLS
81
qSLI
82
KVS
83
SQST
84
EVQLVESGGGLV
85
GFTF
86
INSV
87
ASGD
88

beta
LPVTLGQPASI

YSDG

HVP

QPGGSLRLSCAA

SRYS

GNST

Y

SCRSSQSLIYS

NAY

WT

SGFTFSRYSMSW

DGNAYLHWF

VRQAPGKGLELV

LQKPGQSPRL

AQINSVGNSTYY

LIYKVSNRFSG

PDTVKGRFTISR

VPDRFSGSGS

DNAKNTLYLQMN

GTDFTLKISRV

SLRAEDTAVYYC

EAEDVGVYYC

ASGDYWGQGTL

SQSTHVPWTF

VTVSS

GQGTKVEIK

amyloid
DIVMTQSPLSL
89
QSLV
90
KVS
91
SQST
92
EVQLVESGGGLV
93
GFTF
94
INSN
95
ASGD
96

beta
PVTPGEPASIS

YSNG

HVP

QPGGSLRLSCAA

SSYG

GGST

YW

CRSSQSLVYS

DTY

WT

SGFTFSSYGMSW

NGDTYLHWY

VRQAPGKGLELV

LQKPGQSPQL

ASINSNGGSTYY

LIYKVSNRFSG

PDSVKGRFTISR

VPDRFSGSGS

DNAKNSLYLQMN

GTDFTLKISRV

SLRAEDTAVYYC

EAEDVGVYYC

ASGDYWGQGTT

SQSTHVPWTF

VTVSS

GQGTKVEIK

amyloid
DVVMTQSPLS
97
QSLL
98
QIS
99
LQGT
100
QVQLVQSGAEV
101
GYY
102
IDPA
103
ASLY
104

beta
LPVTLGQPASI

YSD

HYP

KKPGASVKVSCK

TEA

TGNT

SLPV

SCKSSQSLLYS

AKTY

VL

ASGYYTEAYYIH

YY

Y

DAKTYLNWF

WVRQAPGQGLE

QQRPGQSPRR

WMGRIDPATGNT

LIYQISRLDPG

KYAPRLQDRVT

VPDRFSGSGS

MTRDTSTSTVYM

GTDFTLKISRV

ELSSLRSEDTAV

EAEDVGVYYC

YYCASLYSLPVY

LQGTHYPVLF

WGQGTTVTVSS

GQGTRLEIK

amyloid
DIQMTQSPSSL
105
QSIS
106
AAS
107
QQS
108
QVQLVESGGGV
109
GFAF
110
IWFD
111
ARDRG
112

beta
SASVGDRVTIT

SY

YSTP

VQPGRSLRLSCA

SSYG

GTKK

IGARR

CRASQSISSYL

LT

ASGFAFSSYGMH

GPYYM

NWYQQKPGK

WVRQAPGKGLE

DV

APKLLIYAASS

WVAVIWFDGTK

LQSGVPSRFSG

KYYTDSVKGRFT

SGSGTDFTLTI

ISRDNSKNTLYL

SSLQPEDFATY

QMNTLRAEDTA

YCQQSYSTPL

VYYCARDRGIGA

TFGGGTKVEI

RRGPYYMDVWG

K

KGTTVTVSS

APP
DIVLTQSPATL
113
QSVS
114
GAS
115
LQIY
116
QVELVESGGGLV
117
GFTF
118
INAS
119
ARGKG
120

SLSPGERATLS

SSY

NMPI

QPGGSLRLSCAA

SSYA

TRT

GNTH

CRASQSVSSSY

T

SGFTFSSYAMSW

KPYG

LAWYQQKPG

VRQAPGKGLEW

YVRY

QAPRLLIYGAS

VSAINASGTRTY

FDV

SRATGVPARF

YADSVKGRFTIS

SGSGSGTDFTL

RDNSKNTLYLQ

TISSLEPEDFA

MNSLRAEDTAV

TYYCLQIYNM

YYCARGKGNTH

PITFGQGTKVE

KPYGYVRYFDV

IK

WGQGTLVTVSS

AXL
EIVLTQSPGTL
121
QSVS
122
GAS
123
QQY
124
EVQLLESGGGLV
125
GFTF
126
TSGS
127
AKIWI
128

SLSPGERATLS

SSY

GSSP

QPGGSLRLSCAA

SSYA

GAST

AFDI

CRASQSVSSSY

YT

SGFTFSSYAMNW

LAWYQQKPG

VRQAPGKGLEW

QAPRLLIYGAS

VSTTSGSGASTY

SRATGIPDRFS

YADSVKGRFTIS

GSGSGTDFTLT

RDNSKNTLYLQ

ISRLEPEDFAV

MNSLRAEDTAV

YYCQQYGSSP

YYCAKIWIAFDI

YTFGQGTKLEI

WGQGTMVTVSS

K

Blood
DIQMTQTTSSL
129
QDIN
130
YTS
131
QQG
132
QVQLQQPGAELV
133
GYN
134
IYPG
135
AGQY
136

group A
SASLGDRVTIS

NY

NTLP

KPGTSVKLSCKA

FTSY

SGIT

GNLW

CRASQDINNY

WT

SGYNFTSYWINW

W

FAY

LNWYQQKPD

VKLRPGQGLEWI

GTVKLLIHYTS

GDIYPGSGITNY

RLHSGVPSRFS

NEKFKSKATLTV

GSGSGTDYSL

DTSSSTAYMQLS

TISNLEQEDIA

SLASEDSALYYC

TYFCQQGNTL

AGQYGNLWFAYW

PWTFGGGTKL

GQGTLVTVSS

EIK

BnDOTA
QAVVIQESAL
137
TGAV
138
GHN
139
ALW
140
HVKLQESGPGLV
141
GFSL
142
IWSG
143
ARRG
144

TTPPGETVTLT

TASN

YSD

QPSQSLSLTCTV

TDY

GGT

SYPY

CGSSTGAVTA

Y

HWV

SGFSLTDYGVHW

G

NYFD

SNYANWVQE

IGGG

VRQSPGKGLEWL

A

KPDHCFTGLIG

GVIWSGGGTAYN

GHNNRPPGVP

TALISRLNIYRD

ARFSGSLIGDK

NSKNQVFLEMNS

AALTIAGTQTE

LQAEDTAMYYCA

DEAIYFCALW

RRGSYPYNYFDA

YSDHWVIGGG

WGCGTTVTVSS

TRLTVL

CAIX
DIVMTQSQRF
145
QNVV
146
SAS
147
QQY
148
DVKLVESGGGLV
149
GFTF
150
INSD
151
ARHR
152

MSTTVGDRVS

SA

SNYP

KLGGSLKLSCAA

SNY

GGIT

SGYF

ITCKASQNVV

WT

SGFTFSNYYMSW

Y

SMDY

SAVAWYQQK

VRQTPEKRLELV

PGQSPKLLIYS

AAINSDGGITYY

ASNRYTGVPD

LDTVKGRFTISR

RFTGSGSGTDF

DNAKNTLYLQMS

TLTISNMQSED

SLKSEDTALFYC

LADFFCQQYS

ARHRSGYFSMDY

NYPWTFGGGT

WGQGTSVTVSS

KLEIK

CCL-2
EIVLTQSPATL
153
QSVS
154
DAS
155
HQYI
156
QVQLVQSGAEV
157
GGTF
158
IIPI
159
ARYD
160

SLSPGERATLS

DAY

QLHS

KKPGSSVKVSCK

SSYG

FGTA

GIYG

CRASQSVSDA

FT

ASGGTFSSYGIS

ELDF

YLAWYQQKP

WVRQAPGQGLE

GQAPRLLIYD

WMGGIIPIFGTA

ASSRATGVPA

NYAQKFQGRVTI

RFSGSGSGTDF

TADESTSTAYME

TLTISSLEPED

LSSLRSEDTAVY

FAVYYCHQYIQ

YCARYDGIYGEL

LHSFTFGQGT

DFWGQGTLVTVS

KVEIK

S

CD105
QIVLSQSPAIL
161
SSVS
162
ATS
163
QQW
164
EVKLEESGGGLV
165
GFTF
166
IRSK
167
TRWR
168

(endo-
SASPGEKVTMT

Y

SSNP

QPGGSMKLSCAA

SDA

ASNH

RFFD

glin)
CRASSSVSYM

LT

SGFTFSDAWMD

W

AT

S

HWYQQKPGSS

WVRQSPEKGLE

PKPWIYATSN

WVAEIRSKASNH

LASGVPVRFS

ATYYAESVKGRF

GSGSGTSYSLT

TISRDDSKSSVY

ISRVEAEDAAT

LQMNSLRAEDTG

YYCQQWSSNP

IYYCTRWRRFFD

LTFGAGTKLE

SWGQGTTLTVSS

LK

CD115
EIVLTQSPATL
169
QSVD
170
AAS
171
HLSN
172
QVQLVQSGAEV
173
GYTF
174
INPY
175
ARES
176

(CSF1R)
SLSPGERATLS

YDGD

EDLS

KKPGSSVKVSCK

TDN

NGGT

PYFS

CKASQSVDYD

NY

T

ASGYTFTDNYMI

Y

NLYV

GDNYMNWYQ

WVRQAPGQGLE

MDYW

QKPGQAPRLLI

WMGDINPYNGG

YAASNLESGIP

TTFNQKFKGRVT

ARFSGSGSGT

ITADKSTSTAYM

DFTLTISSLEP

ELSSLRSEDTAV

EDFAVYYCHLS

YYCARESPYFSN

NEDLSTFGGG

LYVMDYWGQGT

TKVEIK

LVTVSS

CD116a
QSVLTQPPSVS
177
GSNI
178
HNN
179
ATVE
180
QVQLVQSGAEV
181
GYT
182
FDPE
183
AIVG
184

(CSF2
GAPGQRVTISC

GAPY

AGLS

KKPGASVKVSCK

LTEL

ENEI

SFSP

Ra)
TGSGSNIGAPY

D

GSV

VSGYTLTELSIH

S

LTLG

DVSWYQQLPG

WVRQAPGKGLE

L

TAPKLLIYHN

WMGGFDPEENEI

NKRPSGVPDR

VYAQRFQGRVT

FSGSKSGTSAS

MTEDTSTDTAY

LAITGLQAEDE

MELSSLRSEDTA

ADYYCATVEA

VYYCAIVGSFSP

GLSGSVFGGG

LTLGLWGQGTMV

TKLTVL

TVSS

CD11a
DIQMTQSPSSL
185
KTISKY
186
SGS
187
QQH
188
EVQLVESGGGLV
189
GYSF
190
IHPS
191
ARGI
192

(LFA-1)
SASVGDRVTIT

NEYP

QPGGSLRLSCAA

TGH

DSET

YFYG

CRASKTISKYL

LT

SGYSFTGHWMN

W

TTYF

AWYQQKPGK

WVRQAPGKGLE

DYW

APKLLIYSGST

WVGMIHPSDSET

LQSGVPSRFSG

RYNQKFKDRFTI

SGSGTDFTLTI

SVDKSKNTLYLQ

SSLQPEDFATY

MNSLRAEDTAV

YCQQHNEYPL

YYCARGIYFYGT

TFGQGTKVEI

TYFDYWGQGTL

K

VTVSS

CD123
DFVMTQSPSS
193
QSLL
194
WAS
195
QND
196
EVQLQQSGPELV
197
GYTF
198
IIPS
199
TRSH
200

LTVTAGEKVT

NSGN

YSYP

KPGASVKMSCK

TDY

N

LLRA

MSCKSSQSLL

QKNY

YT

ASGYTFTDYYM

Y

GAT

SWFA

NSGNQKNYLT

KWVKQSHGKSL

Y

WYLQKPGQPP

EWIGDIIPSNGA

KLLIYWASTR

TFYNQKFKGKAT

ESGVPDRFTGS

LTVDRSSSTAYM

GSGTDFTLTIS

HLNSLTSEDSAV

SVQAEDLAVY

YYCTRSHLLRAS

YCQNDYSYPY

WFAYWGQGTLVT

TFGGGTKLEIK

VSA

CD123
QAVVTQEPSL
201
TGAV
202
GTN
203
ALW
204
EVQLVESGGGLV
205
GFTF
206
IRSK
207
VRHG
208

TVSPGGTVTL

TTSN

YSNL

QPGGSLRLSCAA

STYA

YNNY

NFGN

TCRSSTGAVT

Y

WV

SGFTFSTYAMNW

AT

SYVS

TSNYANWVQ

VRQAPGKGLEW

WFAY

QKPGQAPRGL

VGRIRSKYNNYA

IGGTNKRAPW

TYYADSVKDRFT

TPARFSGSLLG

ISRDDSKNSLYL

GKAALTITGA

QMNSLKTEDTAV

QAEDEADYYC

YYCVRHGNFGNS

ALWYSNLWV

YVSWFAYWGQGT

FGGGTKLTVL

LVTVSS

CD134
DIQMTQSPSSL
209
QDIS
210
YTS
211
QQG
212
EVQLVQSGAEVK
213
GYTF
214
MYPD
215
VLAP
216

(OX40)
SASVGDRVTIT

NY

HTLP

KPGASVKVSCKA

TDSY

NGDS

RWYF

CRASQDISNYL

PT

SGYTFTDSYMSW

SVW

NWYQQKPGK

VRQAPGQGLEWI

APKLLIYYTSR

GDMYPDNGDSS

LRSGVPSRFSG

YNQKFRERVTIT

SGSGTDFTLTI

RDTSTSTAYLEL

SSLQPEDFATY

SSLRSEDTAVYY

YCQQGHTLPP

CVLAPRWYFSVW

TFGQGTKVEI

GQGTLVTVSS

K

CD137
SYELTQPPSVS
217
NIGD
218
QDK
219
ATYT
220
EVQLVQSGAEVK
221
GYSF
222
IYPG
223
ARGY
224

(41BB)
VSPGQTASITC

QY

GFGS

KPGESLRISCKG

STY

DSYT

GIFD

SGDNIGDQYA

LAV

SGYSFSTYWISW

W

Y

HWYQQKPGQ

VRQMPGKGLEWM

SPVLVIYQDK

GKIYPGDSYTNY

NRPSGIPERFS

SPSFQGQVTISA

GSNSGNTATL

DKSISTAYLQWS

TISGTQAMDE

SLKASDTAMYYC

ADYYCATYTG

ARGYGIFDYWGQ

FGSLAVFGGG

GTLVTVSS

TKLTVL

CD137
EIVLTQSPATL
225
QSVS
226
DAS
227
QQRS
228
QVQLQQWGAGL
229
GGSF
230
INHG
231
ARDY
232

(41BB)
SLSPGERATLS

SY

NWP

LKPSETLSLTCA

SGY

GYV

GPGN

CRASQSVSSY

PALT

VYGGSFSGYYWS

Y

YDWY

LAWYQQKPG

WIRQSPEKGLEW

FDL

QAPRLLIYDAS

IGEINHGGYVTY

NRATGIPARFS

NPSLESRVTISV

GSGSGTDFTLT

DTSKNQFSLKLS

ISSLEPEDFAV

SVTAADTAVYYC

YYCQQRSNWP

ARDYGPGNYDWY

PALTFCGGTK

FDLWGRGTLVTV

VEIK

SS

CD152
DIQMTQSPSSL
233
QSIN
234
AAS
235
QQY
236
QVQLVESGGGV
237
GFTF
238
IWYD
239
ARDP
240

(CTLA4)
SASVGDRVTIT

SY

YSTP

VQPGRSLRLSCA

SSYG

GSNK

RGAT

CRASQSINSYL

FT

ASGFTFSSYGMH

LYYY

DWYQQKPGK

WVRQAPGKGLE

YYGM

APKLLIYAASS

WVAVIWYDGSN

DV

LQSGVPSRFSG

KYYADSVKGRFT

SGSGTDFTLTI

ISRDNSKNTLYL

SSLQPEDFATY

QMNSLRAEDTA

YCQQYYSTPF

VYYCARDPRGAT

TFGPGTKVEIK

LYYYYYGMDVW

GQGTTVTVSS

CD152
EIVLTQSPGTL
241
QSVGS
242
GAF
243
QQY
244
QVQLVESGGGV
245
GFTF
246
ISYD
247
ARTG
248

(CTLA4)
SLSPGERATLS

SY

GSSP

VQPGRSLRLSCA

SSYT

GNNK

WLGP

CRASQSVGSS

WT

ASGFTFSSYTMH

FDY

YLAWYQQKP

WVRQAPGKGLE

GQAPRLLIYG

WVTFISYDGNNK

AFSRATGIPDR

YYADSVKGRFTI

FSGSGSGTDFT

SRDNSKNTLYLQ

LTISRLEPEDF

MNSLRAEDTAIY

AVYYCQQYGS

YCARTGWLGPFD

SPWTFGQGTK

YWGQGTLVTVSS

VEIK

CD16
DTVLTQSPASL
249
QSVD
250
TTS
251
QQS
252
QVTLKESGPGIL
253
GFSL
254
IWWD
255
AQIN
256

AVSLGQRATIS

FDGD

NEDP

QPSQTLSLTCSF

RTSG

DDK

PAWF

CKASQSVDFD

SF

YT

SGFSLRTSGMGV

MG

AY

GDSFMNWYQ

GWIRQPSGKGLE

QKPGQPPKLLI

WLAHIWWDDDKR

YTTSNLESGIP

YNPALKSRLTIS

ARFSASGSGT

KDTSSNQVFLKI

DFTLNIHPVEE

ASVDTADTATYY

EDTATYYCQQ

CAQINPAWFAYW

SNEDPYTFGG

GQGTLVTVSA

GTKLEIK

CD184
DIQMTQSPSSL
257
QGIS
258
AAS
259
QQY
260
EVQLVESGGGLV
261
GFTF
262
ISSR
263
ARDY
264

(CXCR4)
SASVGDRVTIT

SW

NSYP

QPGGSLRLSCAA

SSYS

SRTI

GGQP

CRASQGISSW

RT

AGFTFSSYSMNW

PYYY

LAWYQQKPE

VRQAPGKGLEW

YYGM

KAPKSLIYAAS

VSYISSRSRTIY

DV

SLQSGVPSRFS

YADSVKGRFTIS

GSGSGTDFTLT

RDNAKNSLYLQM

ISSLQPEDFVT

NSLRDEDTAVYY

YYCQQYNSYP

CARDYGGQPPYY

RTFGQGTKVEI

YYYGMDVWGQ

K

GTTVTVSS

CD19
DIQMTQTTSSL
265
QDISK
266
HTS
267
QQG
268
EVKLQESGPGLV
269
GVSL
270
IWGS
271
AKHY
272

SASLGDRVTIS

Y

NTLP

APSQSLSVTCTV

PDY

ETT

YYGG

CRASQDISKYL

YT

SGVSLPDYGVSW

G

SYAM

NWYQQKPDG

IRQPPRKGLEWL

DY

TVKLLIYHTSR

GVIWGSETTYYN

LHSGVPSRFSG

SALKSRLTIIKD

SGSGTDYSLTI

NSKSQVFLK

SNLEQE

MNSLQTDDTAIY

DIATYFCQQG

YCAKHYYYGGS

NTLPYTFGGG

YAMDYWGQGTS

TKLEIK

VTVSS

CD19
EIVLTQSPDFQ
273
ESVDT
274
EAS
275
QQS
276
EVQLVESGGGLV
277
GFTF
278
IYPG
279
ARSG
280

SVTPKEKVTIT

FGISF

KEVP

QPGGSLRLSCAA

SSSW

DGDT

FITT

CRASESVDTF

FT

SGFTFSSSWMNW

VRDF

GISFMNWFQQ

VRQAPGKGLEW

DY

KPDQSPKLLIH

VGRIYPGDGDTN

EASNQGSGVP

YNVKFKGRFTIS

SRFSGSGSGTD

RDDSKNSLYLQM

FTLTINSLEAE

NSLKTEDTAVYY

DAATYYCQQS

CARSGFITTVRD

KEVPFTFGGG

FDYWGQGTLVTV

TKVEIK

SS

CD19
DIQMTQSPSSL
281
TDIS
282
YGS
283
GQG
284
QVQLQESGPGLV
285
GHSI
286
ISYS
287
ARSL
288

SASVGDSVTIT

SH

NRLP

KPSETLSLTCAV

SHD

GIT

ARTT

CQASTDISSHL

YT

SGHSISHDHAWS

HA

AMDY

NWYQQKPGK

WVRQPPGEGLEW

APELLIYYGSH

IGFISYSGITNY

LLSGVPSRFSG

NPSLQGRVTISR

SGSGTDFTFTI

DNSKNTLYLQMN

SSLEAEDAAT

SLRAEDTAVYYC

YYCGQGNRLP

ARSLARTTAMDY

YTFGQGTKVE

WGEGTLVTVSS

IE

CD19
EIVLTQSPATL
289
SSVS
290
DTS
291
FQGS
292
QVQLQESGPGLV
293
GGSI
294
IWWD
295
ARME
296

SLSPGERATLS

Y

VYPF

KPSQTLSLTCTV

STSG

DDK

LWSY

CSASSSVSYM

T

SGGSISTSGMGV

MG

YFDY

HWYQQKPGQ

GWIRQHPGKGLE

APRLLIYDTSK

WIGHIWWDDDK

LASGIPARFSG

RYNPALKSRVTI

SGSGTDFTLTI

SVDTSKNQFSLK

SSLEPEDVAV

LSSVTAADTAVY

YYCFQGSVYP

YCARMELWSYYF

FTFGQGTKLEI

DYWGQGTLVTV

K

SS

CD19
DIQLTQSPASL
297
QSVD
298
DAS
299
QQST
300
QVQLQQSGAELV
301
GYA
302
IWPG
303
ARRE
304

AVSLGQRATIS

YDGD

EDP

RPGSSVKISCKA

FSSY

DGDT

TTTV

CKASQSVDYD

SY

WT

SGYAFSSYWMNW

W

GRYY

GDSYLNWYQ

VKQRPGQGLEWI

YAMD

QIPGQPPKLLI

GQIWPGDGDTNY

Y

YDASNLVSGIP

NGKFKGKATLTA

PRFSGSGSGTD

DESSSTAYMQLS

FTLNIHPVEKV

SLASEDSAVYFC

DAATYHCQQS

ARRETTTVGRYY

TEDPWTFGGG

YAMDYWGQGTT

TKLEIK

VTVSS

CD19
DIVMTQAAPSI
305
KSLL
306
RMS
307
MQH
308
QVQLQQSGPELI
309
GYTF
310
INPY
311
ARGT
312

PVTPGESVSIS

NSNG

LEYP

KPGASVKMSCK

TSYV

NDGT

YYYG

CRSSKSLLNSN

NTY

LT

ASGYTFTSYVMH

SRVF

GNTYLYWFLQ

WVKQKPGQGLE

DY

RPGQSPQLLIY

QIGYINPYNDGT

RMSNLASGVP

KYNEKFKGKATL

DRFSGSGSGT

TSDKSSTAYMEL

AFTLRISRVEA

SSLTSEDSAVYY

EDVGVYYCM

CARGTYYYGSRV

QHLEYPLTFG

FDYWGQGTTLT

AGTKLEIK

VTVSS

CD19
EIVLTQSPAIM
313
SGVN
314
DTS
315
HQR
316
QVQLVQPGAEV
317
GYTF
318
IDPS
319
ARGS
320

SASPGERVTM

Y

GSYT

VKPGASVKLSCK

TSN

DSYT

NPYY

TCSASSGVNY

TSGYTFTSNWMH

W

YAMD

MHWYQQKPG

WVKQAPGQGLE

Y

TSPRRWIYDTS

WIGEIDPSDSYT

KLASGVPARF

NYNQNFQGKAKL

SGSGSGTDYS

TVDKSTSTAYME

LTISSMEPEDA

VSSLRSDDTAVY

ATYYCHQRGS

YCARGSNPYYYA

YTFGGGTKLEI

MDYWGQGTSVT

K

VSS

CD192
DVVMTQSPLS
321
QSLL
322
LVS
323
WQG
324
EVQLVESGGGLV
325
GFTF
326
IRTK
327
TTFY
328

(CCR2)
LPVTLGQPASI

DSDG

THFP

KPGGSLRLSCAA

SAY

NNNY

GNGV

SCKSSQSLLDS

KTF

YT

SGFTFSAYAMN

A

AT

W

DGKTFLNWFQ

WVRQAPGKGLE

QRPGQSPRRLI

WVGRIRTKNNN

YLVSKLDSGV

YATYYADSVKD

PDRFSGSGSGT

RFTISRDDSKNT

DFTLKISRVEA

LYLQMNSLKTED

EDVGVYYCW

TAVYYCTTFYGN

QGTHFPYTFG

GVWGQGTLVTVS

QGTRLEIK

S

CD194
DVLMTQSPLS
329
RNIV
330
KVS
331
FQGS
332
EVQLVESGGDLV
333
GFIF
334
ISSA
335
GRHS
336

(CCR4)
LPVTPGEPASI

HING

LLP

QPGRSLRLSCAA

SNY

STYS

DGNF

SCRSSRNIVHI

DTY

WT

SGFIFSNYGMSW

G

AFGY

NGDTYLEWYL

VRQAPGKGLEW

QKPGQSPQLLI

VATISSASTYSYY

YKVSNRFSGV

PDSVKGRFTISRD

PDRFSGSGSGT

NAKNSLYLQMN

DFTLKISRVEA

SLRVEDTALYYC

EDVGVYYCFQ

GRHSDGNFAFGY

GSLLPWTFGQ

WGQGTLVTVSS

GTKVEIK

CD195
DIVMTQSPLSL
337
QRLL
338
EVS
339
SQST
340
EVQLVESGGGLV
341
GYTF
342
IYPG
343
GSSF
344

(CCR5)
PVTPGEPASIS

SSYG

HVPL

KPGGSLRLSCAA

SNY

GNYI

GSN

CRSSQRLLSSY

HTY

T

SGYTFSNYWIGW

W

YVFA

GHTYLHWYL

VRQAPGKGLEWI

WFTY

QKPGQSPQLLI

GDIYPGGNYIRN

W

YEVSNRFSGV

NEKFKDKTTLSA

PDRFSGSGSGT

DTSKNTAYLQM

DFTLKISRVEA

NSLKTEDTAVYY

EDVGVYYCSQ

CGSSFGSNYVFA

STHVPLTFGQ

WFTYWGQGTLV

GTKVEIK

TVSS

CD20
EIVLTQSPATL
345
QSVS
346
DAS
347
QQRS
348
EVQLVESGGGLV
349
GFTF
350
ISWN
351
AKDI
352

SLSPGERATLS

SY

NWPI

QPGRSLRLSCAA

NDY

SGSI

QYGN

CRASQSVSSY

T

SGFTFNDYAMH

A

YYYG

LAWYQQKPG

WVRQAPGKGLE

MDV

QAPRLLIYDAS

WVSTISWNSGSI

NRATGIPARFS

GYADSVKGRFTI

GSGSGTDFTLT

SRDNAKKSLYLQ

ISSLEPEDFAV

MNSLRAEDTALY

YYCQQRSNWP

YCAKDIQYGNYY

ITFGQGTRLEI

YGMDVWGQGTT

K

VTVSS

CD20
QIVLSQSPAILS
353
SSVS
354
APS
355
QQW
356
QAYLQQSGAELV
357
GYTF
358
IYPG
359
ARVV
360

ASPGEKVTMT

Y

SFNP

RPGASVKMSCKA

TSYN

NGDT

YYSN

CRASSSVSYM

PT

SGYTFTSYNMH

SYWY

HWYQQKPGSS

WVKQTPRQGLE

FDV

PKPWIYAPSNL

WIGAIYPGNGDT

ASGVPARFSG

SYNQKFKGKATL

SGSGTSYSLTI

TVDKSSSTAYMQ

SRVEAEDAAT

LSSLTSEDSAVYF

YYCQQWSFNP

CARVVYYSNSY

PTFGAGTKLE

WYFDVWGTGTT

LK

VTVSGPSVFPLAP

SS

CD20
QIVLSQSPAILS
361
SSVS
362
ATS
363
QQW
364
QAYLQQSGAELV
365
GYTF
366
IYPG
367
ARYD
368

ASPGEKVTMT

Y

TFNP

RPGASVKMSCKA

TSYN

NGDT

YNYA

CRASSSVSYM

PT

SGYTFTSYNMH

MDY

HWYQQKPGSS

WVKQTPRQGLE

PKPWIYATSN

WIGGIYPGNGDT

LASGVPARFS

SYNQKFKGKATL

GSGSGTSYSFT

TVGKSSSTAYMQ

ISRVEAEDAAT

LSSLTSEDSAVYF

YYCQQWTFNP

CARYDYNYAMD

PTFGGGTRLEI

YWGQGTSVTVSS

K

CD20
QIVLSQSPAILS
369
SSVS
370
ATS
371
QQW
372
QVQLQQPGAELV
373
GYTF
374
IYPG
375
ARST
376

ASPGEKVTMT

Y

TSNP

KPGASVKMSCK

TSYN

NGDT

YYGG

CRASSSVSYIH

PT

ASGYTFTSYNMH

DWYF

WFQQKPGSSP

WVKQTPGRGLE

NV

KPWIYATSNL

WIGAIYPGNGDT

ASGVPVRFSG

SYNQKFKGKATL

SGSGTSYSLTI

TADKSSSTAYMQ

SRVEAEDAAT

LSSLTSEDSAVY

YYCQQWTSNP

YCARSTYYGGD

PTFGGGTKLEI

WYFNVWGAGTT

K

VTVSA

CD20
DIQLTQSPSSL
377
SSVS
378
ATS
379
QQW
380
QVQLQQSGAEV
381
GYTF
382
IYPG
383
ARST
384

SASVGDRVTM

Y

TSNP

KKPGSSVKVSCK

TSYN

NGDT

YYGG

TCRASSSVSYI

PT

ASGYTFTSYNMH

DWYF

HWFQQKPGK

WVKQAPGQGLE

DV

APKPWIYATS

WIGAIYPGNGDT

NLASGVPVRF

SYNQKFKGKATL

SGSGSGTDYT

TADESTNTAYME

FTISSLQPEDIA

LSSLRSEDTAFYY

TYYCQQWTSN

CARSTYYGGDW

PPTFGGGTKLE

YFDVWGQGTTV

IK

TVSS

CD20
DIVMTQTPLSL
385
KSLL
386
QMS
387
AQN
388
QVQLVQSGAEV
389
GYA
390
IFPG
391
ARNV
392

PVTPGEPASIS

HSNG

LELP

KKPGSSVKVSCK

FSYS

DGDT

FDGY

CRSSKSLLHSN

ITY

YT

ASGYAFSYSWIN

W

WLVY

GITYLYWYLQ

WVRQAPGQGLE

KPGQSPQLLIY

WMGRIFPGDGDT

QMSNLVSGVP

DYNGKFKGRVTI

DRFSGSGSGT

TADKSTSTAYME

DFTLKISRVEA

LSSLRSEDTAVY

EDVGVYYCA

YCARNVFDGYW

QNLELPYTFG

LVYWGQGTLVT

GGTKVEIK

VSS

CD200
DIQMTQSPSSL
393
QDIN
394
RAN
395
LQY
396
QVQLQQSGSELK
397
GYSF
398
IDPY
399
GRSK
400

SASIGDRVTIT

SY

DEFP

KPGASVKISCKA

TDYI

YGSS

RDYF

CKASQDINSY

YT

SGYSFTDYIILWV

DYW

LSWFQQKPGK

RQNPGKGLEWIG

APKLLIYRAN

HIDPYYGSSNYN

RLVDGVPSRF

LKFKGRVTITAD

SGSGSGTDYT

QSTTTAYMELSS

LTISSLQPEDF

LRSEDTAVYYCG

AVYYCLQYDE

RSKRDYFDYWG

FPYTFGGGTK

QGTTLTVSS

LEIK

CD22
DIQLTQSPSSL
401
QSVL
402
WAS
403
HQY
404
QVQLQESGAELS
405
GYTF
406
INPR
407
ARRD
408

AVSAGENVTM

YSAN

LSSW

KPGASVKMSCK

TSY

NDYT

ITTF

SCKSSQSVLYS

HKNY

T

ASGYTFTSYWLH

W

Y

ANHKNYLAW

WIKQRPGQGLE

YQQKPGQSPK

WIGYINPRNDYT

LLIYWASTRES

EYNQNFKDKATL

GVPDRFTGSG

TADKSSSTAYMQ

SGTDFTLTISR

LSSLTSEDSAVY

VQVEDLAIYY

YCARRDITTFYW

CHQYLSSWTF

GQGTTLTVSS

GGGTKLEIK

CD221
DIQMTQFPSSL
409
QGIR
410
AAS
411
LQH
412
EVQLLESGGGLV
413
GFTF
414
ISGS
415
AKDL
416

(IGF1R)
SASVGDRVTIT

ND

NSYP

QPGGSLRLSCTA

SSYA

GGTT

GWSD

CRASQGIRND

CS

SGFTFSSYAMNW

SYYY

LGWYQQKPG

VRQAPGKGLEW

YYGM

KAPKRLIYAA

VSAISGSGGTTFY

DV

SRLHRGVPSRF

ADSVKGRFTISR

SGSGSGTEFTL

DNSRTTLYLQMN

TISSLQPEDFA

SLRAEDTAVYYC

TYYCLQHNSY

AKDLGWSDSYY

PCSFGQGTKL

YYYGMDVWGQ

EIK

GTTVTVSS

CD221
DIQMTQSPSSL
417
QGIS
418
AKS
419
QQY
420
EVQLLQSGGGLV
421
GFM
422
ISGS
423
AKDF
424

(IGF1R)
SASLGDRVTIT

SY

WTFP

QPGGSLRLSCAA

FSRY

GGAT

YQIL

CRASQGISSYL

LT

SGFMFSRYPMH

P

TGNA

AWYQQKPGK

WVRQAPGKGLE

FDY

APKLLIYAKST

WVGSISGSGGAT

LQSGVPSRFSG

PYADSVKGRFTIS

SGSGTDFTLTI

RDNSKNTLYLQ

SSLQPEDSATY

MNSLRAEDTAV

YCQQYWTFPL

YYCAKDFYQILT

TFGGGTKVEI

GNAFDYWGQGT

K

TVTVSS

CD221
QIVLTQSPAIM
425
SSVS
426
GTS
427
QQRS
428
EVQLQQSGPELV
429
GYSF
430
RINP
431
CAKS
432

(IGF1R)
SASPGEKVTIT

Y

SYPF

KPGSSVKISCKAS

TAY

D

TSYD

CSASSSVSYIH

T

GYSFTAYYMHW

Y

NGG

YDGY

WFQQKPGTSP

VKQSHGKSLEQI

WFDV

KVWIYGTSNL

SGRINPDNGGNS

ASGVPARFTG

YNQFKFGKAILT

SGSGTSYSLTI

VDKSSNTAYMEL

SRMEAEDAAT

RSLTSEDSAVYY

YYCQQRSSYP

CAKSTSYDYDGY

FTFGSGTKLEI

WFDVWGAGTTV

K

TVSS

CD221
SSELTQDPAVS
433
SLRS
434
GEN
435
KSRD
436
EVQLVQSGAEVK
437
GGTF
438
IIPI
439
ARAP
440

(IGF1R)
VALGQTVRIT

YY

GSG

KPGSSVKVSCKA

SSYA

FGTA

LRFL

CQGDSLRSYY

QHL

SGGTFSSYAISW

EWST

ATWYQQKPG

V

VRQAPGQGLEW

QDHY

QAPILVIYGEN

MGGIIPIFGTANY

YYYY

KRPSGIPDRFS

AQKFQGRVTITA

MDV

GSSSGNTASLT

DKSTSTAYMELS

ITGAQAEDEA

SLRSEDTAVYYC

DYYCKSRDGS

ARAPLRFLEWST

GQHLVFGGGT

QDHYYYYYMDV

KLTVL

WGKGTTVTVSS

CD221
EIVLTQSPGTL
441
QSIG
442
YAS
443
HQSS
444
EVQLVQSGGGLV
445
GFTF
446
IDTR
447
ARLG
448

(IGF1R)
SVSPGERATLS

SS

RLPH

KPGGSLRLSCAA

SSFA

GAT

NFYY

CRASQSIGSSL

T

SGFTFSSFAMHW

GMDV

HWYQQKPGQ

VRQAPGKGLEWI

APRLLIKYASQ

SVIDTRGATYYA

SLSGIPDRFSG

DSVKGRFTISRD

SGSGTDFTLTI

NAKNSLYLQMN

SRLEPEDFAV

SLRAEDTAVYYC

YYCHQSSRLP

ARLGNFYYGMD

HTFGQGTKVE

VWGQGTTVTVSS

IK

CD221
EIVLTQSPATL
449
QSVS
450
DAS
451
QQRS
452
QVELVESGGGVV
453
GFTF
454
IWFD
455
AREL
456

(IGF1R)
SLSPGERATLS

SY

KWP

QPGRSQRLSCAA

SSYG

GSST

GRRY

CRASQSVSSY

PWT

SGFTFSSYGMHW

FDL

LAWYQQKPG

VRQAPGKGLEW

QAPRLLIYDAS

VAIIWFDGSSTYY

KRATGIPARFS

ADSVRGRFTISRD

GSGSGTDFTLT

NSKNTLYLQMNS

ISSLEPEDFAV

LRAEDTAVYFCA

YYCQQRSKWP

RELGRRYFDLWG

PWTFGQGTKV

RGTLVSVSS

ESK

CD221
DIVMTQSPLSL
457
QSIV
458
KVS
459
FQGS
460
QVQLQESGPGLV
461
GYSI
462
ISYD
463
ARYG
464

(IGF1R)
PVTPGEPASIS

HSNG

HVP

KPSETLSLTCTVS

TGG

GTN

RVFF

CRSSQSIVHSN

NTY

WT

GYSITGGYLWN

YL

DY

GNTYLQWYL

WIRQPPGKGLEW

QKPGQSPQLLI

IGYISYDGTNNY

YKVSNRLYGV

KPSLKDRVTISRD

PDRFSGSGSGT

TSKNQFSLKLSSV

DFTLKISRVEA

TAADTAVYYCA

EDVGVYYCFQ

RYGRVFFDYWG

GSHVPWTFGQ

QGTLVTVSS

GTKVEIK

CD221
DVVMTQSPLS
465
QSLL
466
LGS
467
MQG
468
QVQLQESGPGLV
469
GGSI
470
IYHS
471
ARWT
472

(IGF1R)
LPVTPGEPASI

HSNG

THW

KPSGTLSLTCAVS

SSSN

GST

GRTD

SCRSSQSLLHS

YNY

PLT

GGSISSSNWWSW

W

AFDI

NGYNYLDWY

VRQPPGKGLEWI

LQKPGQSPQL

GEIYHSGSTNYN

LIYLGSNRASG

PSLKSRVTISVDK

VPDRFSGSGS

SKNQFSLKLSSVT

GTDFTLKISRV

AADTAVYYCAR

EAEDVGVYYC

WTGRTDAFDIW

MQGTHWPLTF

GQGTMVTVSS

GQGTKVEIK

CD223
EIVLTQSPATL
473
QSIS
474
DAS
475
QQRS
476
QVQLQQWGAGL
477
GGSF
478
INHR
479
AFGY
480

(L4G-3)
SLSPGERATLS

SY

NWP

LKPSETLSLTCAV

SDY

GST

SDYE

CRASQSISSYL

LT

YGGSFSDYYWN

Y

YNWF

AWYQQKPGQ

WIRQPPGKGLEW

DP

APRLLIYDASN

IGEINHRGSTNSN

RATGIPARFSG

PSLKSRVTLSLDT

SGSGTDFTLTI

SKNQFSLKLRSV

SSLEPEDFAVY

TAADTAVYYCAF

YCQQRSNWPL

GYSDYEYNWFD

TFGQGTNLEIK

PWGQGTLVTVSS

CD248
DIQMTQSPSSL
481
QNVG
482
SAS
483
QQY
484
QVQLQESGPGLV
485
GYTF
486
INPY
487
ARRG
488

SASVGDRVTIT

TA

TNYP

RPSQTLSLTCTAS

TDY

DDDT

NSYD

CRASQNVGTA

MYT

GYTFTDYVIHWV

V

GYFD

VAWLQQTPG

KQPPGRGLEWIG

YSMD

KAPKLLIYSAS

YINPYDDDTTYN

Y

NRYTGVPSRF

QKFKGRVTMLV

SGSGSGTDYT

DTSSNTAYLRLSS

FTISSLQPEDIA

VTAEDTAVYYC

TYYCQQYTNY

ARRGNSYDGYFD

PMYTFGQGTK

YSMDYWGSGTP

VQIK

VTVSS

CD25
QIVSTQSPAIM
489
SSRS
490
DTS
491
HQRS
492
QLQQSGTVLARP
493
GYSF
494
IYPG
495
SRDY
496

SASPGEKVTM

Y

SYT

GASVKMSCKAS

TRY

NSDT

GYYF

TCSASSSRSY

GYSFTRYWMHW

W

DF

MQWYQQKPG

IKQRPGQGLEWI

TSPKRWIYDTS

GAIYPGNSDTSY

KLASGVPARF

NQKFEGKAKLTA

SGSGSGTSYSL

VTSASTAYMELS

TISSMEAEDA

SLTHEDSAVYYC

ATYYCHQRSS

SRDYGYYFDFW

YTFGGGTKLEI

GQGTTLTVSS

K

CD25
DIQMTQSPSTL
497
SSIS
498
TTS
499
HQRS
500
QVQLVQSGAEV
501
GYTF
502
INPS
503
ARGG
504

SASVGDRVTIT

Y

TYPL

KKPGSSVKVSCK

TSYR

TGYT

GVFD

CSASSSISYMH

T

ASGYTFTSYRMH

YW

WYQQKPGKA

WVRQAPGQGLE

PKLLIYTTSNL

WIGYINPSTGYTE

ASGVPARFSG

YNQKFKDKATIT

SGSGTEFTLTI

ADESTNTAYMEL

SSLQPDDFAT

SSLRSEDTAVYY

YYCHQRSTYP

CARGGGVFDYW

LTFGQGTKVE

GQGTLVTVSS

VK

CD252
DIQMTQSPSSL
505
QDIS
506
YTS
507
QQG
508
QVQLQESGPGLV
509
GGSF
510
ISYN
511
ARYK
512

(OX40L)
SASVGDRVTIT

NY

SALP

KPSQTLSLTCAV

SSGY

GIT

YDYD

CRASQDISNYL

WT

YGGSFSSGYWN

GGHA

NWYQQKPGK

WIRKHPGKGLEY

MDY

APKLLIYYTSK

IGYISYNGITYHN

LHSGVPSRFSG

PSLKSRITINRDTS

SGSGTDYTLTI

KNQYSLQLNSVT

SSLQPEDFATY

PEDTAVYYCARY

YCQQGSALPW

KYDYDGGHAMD

TFGQGTKVEI

YWGQGTLVTVSS

K

CD254
EIVLTQSPGTL
513
QSVR
514
GAS
515
QQY
516
EVQLLESGGGLV
517
GFTF
518
ITGS
519
AKDP
520

(RANKL)
SLSPGERATLS

GRY

GSSP

QPGGSLRLSCAA

SSYA

GGST

GTTV

CRASQSVRGR

RT

SGFTFSSYAMSW

IMSW

YLAWYQQKP

VRQAPGKGLEW

FDP

GQAPRLLIYG

VSGITGSGGSTY

ASSRATGIPDR

YADSVKGRFTIS

FSGSGSGTDFT

RDNSKNTLYLQ

LTISRLEPEDF

MNSLRAEDTAV

AVFYCQQYGS

YYCAKDPGTTVI

SPRTFGQGTK

MSWFDPWGQGT

VEIK

LVTVSS

CD257
EIVLTQSPATL
521
QSVS
522
DAS
523
QQRS
524
QVQLQQWGAGL
525
GGSF
526
INHS
527
ARGY
528

(BAFF)
SLSPGERATLS

RY

NWP

LKPSETLSLTCAV

SGY

GST

YDIL

CRASQSVSRY

RT

YGGSFSGYYWS

Y

TGYY

LAWYQQKPG

WIRQPPGKGLEW

YYFD

QAPRLLIYDAS

IGEINHSGSTNYN

Y

NRATGIPARFS

PSLKSRVTISVDT

GSGSGTDSTLT

SKNQFSLKLSSVT

ISSLEPEDFAV

AADTAVYYCAR

YYCQQRSNWP

GYYDILTGYYYY

RTFGQGTKVEI

FDYWGQGTLVT

K

VSS

CD257
SSELTQDPAVS
529
SLRS
530
GKN
531
SSRD
532
QVQLQQSGAEV
533
GGTF
534
IIPM
535
ARSR
536

(BAFF)
VALGQTVRVT

YY

SSGN

KKPGSSVRVSCK

NNN

FGTA

DLLL

CQGDSLRSYY

HWV

ASGGTFNNNAIN

A

FPHH

ASQYQQKPGQ

WVRQAPGQGLE

ALSP

APVLVIYGKN

WMGGIIPMFGTA

NRPSGIPDRFS

KYSQNFQGRVAI

GSSSGNTASLT

TADESTGTASME

ITGAQAEDEA

LSSLRSEDTAVY

DYYCSSRDSS

YCARSRDLLLFP

GNHWVFGGG

HHALSPWGRGT

TEL

MVTVSS

CD26
QIVLTQSPAIM
537
SSVS
538
STS
539
QQRS
540
QVQLQQSGAELV
541
GYTF
542
IFPG
543
ARWT
544

SASPGEKVTIT

Y

SYPN

KPGASVKLSCKA

RSY

DGST

VVGP

CSASSSVSYM

T

SGYTFRSYDINW

D

GYFD

NWFQQKPGTS

VRQRPEQGLEWI

V

PKLWIYSTSNL

GWIFPGDGSTKY

ASGVPARFSG

NEKFKGKATLTT

SGSGTSYSLTI

DKSSSTAYMQLS

SRMEAEDAAT

RLTSEDSAVYFC

YYCQQRSSYP

ARWTVVGPGYF

NTFGGGTKLEI

DVWGAGTTVTV

K

SS

CD262
DIQMTQSPSSL
545
QDVG
546
WAS
547
QQY
548
EVQLVESGGGLV
549
GFTF
550
ISSG
551
ARRG
552

(DR5)
SASVGDRVTIT

TA

SSYR

QPGGSLRLSCAA

SSYV

GSYT

DSMI

CKASQDVGTA

T

SGFTFSSYVMSW

TTDY

VAWYQQKPG

VRQAPGKGLEW

W

KAPKLLIYWA

VATISSGGSYTY

STRHTGVPSRF

YPDSVKGRFTISR

SGSGSGTDFTL

DNAKNTLYLQM

TISSLQPEDFA

NSLRAEDTAVYY

TYYCQQYSSY

CARRGDSMITTD

RTFGQGTKVEI

YWGQGTLVTVSS

K

CD262
SSELTQDPAVS
553
SLRS
554
GKN
555
NSRD
556
EVQLVQSGGGVE
557
GFTF
558
INWN
559
AKIL
560

(DR5)
VALGQTVRIT

YY

SSGN

RPGGSLRLSCAA

DDY

GGST

GAGR

CQGDSLRSYY

HVV

SGFTFDDYGMS

G

GWYF

ASWYQQKPG

WVRQAPGKGLE

DL

QAPVLVIYGK

WVSGINWNGGS

NNRPSGIPDRF

TGYADSVKGRVT

SGSSSGNTASL

ISRDNAKNSLYL

TITGAQAEDE

QMNSLRAEDTA

ADYYCNSRDS

VYYCAKILGAGR

SGNHVVFGGG

GWYFDLWGKGT

TKLTVL

TVTVSS

CD262
EIVLTQSPGTL
561
QGIS
562
GAS
563
QQF
564
QVQLQESGPGLV
565
GGSI
566
IHNS
567
ARDR
568

(DR5)
SLSPGERATLS

RSY

GSSP

KPSQTLSLTCTVS

SSGD

GTT

GGDY

CRASQGISRSY

WT

GGSISSGDYFWS

YF

YYGM

LAWYQQKPG

WIRQLPGKGLEW

DV

QAPSLLIYGAS

IGHIHNSGTTYYN

SRATGIPDRFS

PSLKSRVTISVDT

GSGSGTDFTLT

SKKQFSLRLSSVT

ISRLEPEDFAV

AADTAVYYCAR

YYCQQFGSSP

DRGGDYYYGMD

WTFGQGTKVE

VWGQGTTVTVSS

IK

CD27
DIQMTQSPSSL
569
QGIS
570
AAS
571
QQY
572
QVQLVESGGGV
573
GFTF
574
IWYD
575
ARGSG
576

SASVGDRVTIT

RW

NTYP

VQPGRSLRLSCA

SSYD

GSNK

NWGFF

CRASQGISRW

RT

ASGFTFSSYDMH

DY

LAWYQQKPE

WVRQAPGKGLE

KAPKSLIYAAS

WVAVIWYDGSN

SLQSGVPSRFS

KYYADSVKGRFT

GSGSGTDFTLT

ISRDNSKNTLYL

ISSLQPEDFAT

QMNSLRAEDTA

YYCQQYNTYP

VYYCARGSGNW

RTFGQGTKVEI

GFFDYWGQGTL

K

VTVSS

CD274
QSALTQPASV
577
SSDV
578
DVS
579
SSYT
580
EVQLLESGGGLV
581
GFTF
582
IYPS
583
ARIK
584

(PD-L1)
SGSPGQSITISC

GGYN

SSST

QPGGSLRLSCAA

SSYI

GGIT

LGTV

TGTSSDVGGY

Y

RV

SGFTFSSYIMMW

TTVD

NYVSWYQQH

VRQAPGKGLEW

Y

PGKAPKLMIY

VSSIYPSGGITFY

DVSNRPSGVS

ADTVKGRFTISR

NRFSGSKSGN

DNSKNTLYLQM

TASLTISGLQA

NSLRAEDTAVYY

EDEADYYCSS

CARIKLGTVTTV

YTSSSTRVFGT

DYWGQGTLVTV

GTKVTVL

SS

CD274
DIQMTQSPSSL
585
QDVST
586
SAS
587
QQY
588
EVQLVESGGGLV
589
GFTF
590
ISPY
591
ARRH
592

(PD-L1)
SASVGDRVTIT

A

LYHP

QPGGSLRLSCAA

SDS

GGST

WPGG

CRASQDVSTA

AT

SGFTFSDSWIHW

W

FDY

VAWYQQKPG

VRQAPGKGLEW

KAPKLLIYSAS

VAWISPYGGSTY

FLYSGVPSRFS

YADSVKGRFTIS

GSGSGTDFTLT

ADTSKNTAYLQ

ISSLQPEDFAT

MNSLRAEDTAV

YYCQQYLYHP

YYCARRHWPGG

ATFGQGTKVE

FDYWGQGTLVT

IK

VSS

CD274
EIVLTQSPGTL
593
QRVS
594
DAS
595
QQY
596
EVQLVESGGGLV
597
GFTF
598
IKQD
599
AREG
600

(PD-L1)
SLSPGERATLS

SSY

GSLP

QPGGSLRLSCAA

SRY

GSEK

GWFG

CRASQRVSSS

WT

SGFTFSRYWMS

W

ELAF

YLAWYQQKP

WVRQAPGKGLE

DY

GQAPRLLIYD

WVANIKQDGSEK

ASSRATGIPDR

YYVDSVKGRFTI

FSGSGSGTDFT

SRDNAKNSLYLQ

LTISRLEPEDF

MNSLRAEDTAV

AVYYCQQYGS

YYCAREGGWFG

LPWTFGQGTK

ELAFDYWGQGT

VEIK

LVTVSS

CD275
DIQMTQSPSSL
601
QGIS
602
AAS
603
QQY
604
EVQLVESGGGLV
605
GFTF
606
IKQD
607
AREG
608

(ICOS-L)
SASVGDRVTIT

NW

DSYP

QPGGSLRLSCAA

SSY

GNEK

ILWF

CRASQGISNW

RT

SGFTFSSYWMSW

W

GDLP

LAWYQQKPE

VRQAPGKGLEW

TF

KAPKSLIYAAS

VAYIKQDGNEKY

SLQSGVPSRFS

YVDSVKGRFTIS

GSGSGTDFTLT

RDNAKNSLYLQ

ISSLQPEDFAT

MNSLRAEDTAV

YYCQQYDSYP

YYCAREGILWFG

RTFGQGTKVEI

DLPTFWGQGTLV

K

TVSS

CD276
DIQLTQSPSFL
609
QNVD
610
SAS
611
QQY
612
EVQLVESGGGLV
613
GFTF
614
ISSD
615
GRGR
616

(B7H3)
SASVGDRVTIT

TN

NNY

QPGGSLRLSCAA

SSFG

SSAI

ENIY

CKASQNVDTN

PFT

SGFTFSSFGMHW

YGSR

VAWYQQKPG

VRQAPGKGLEW

LDY

KAPKALIYSAS

VAYISSDSSAIYY

YRYSGVPSRFS

ADTVKGRFTISR

GSGSGTDFTLT

DNAKNSLYLQM

ISSLQPEDFAT

NSLRDEDTAVYY

YYCQQYNNYP

CGRGRENIYYGS

FTFGQGTKLEI

RLDYWGQGTTV

K

TVSS

CD276
DIVMTQSPAT
617
QSIS
618
YAS
619
QNG
620
QVQLQQSGAELV
621
GYTF
622
IFPG
623
ARQT
624

(B7H3)
LSVTPGDRVS

DY

HSFP

KPGASVKLSCKA

TNY

DGST

TATW

LSCRASQSISD

LT

SGYTFTNYDINW

D

FAY

YLHWYQQKS

VRQRPEQGLEWI

HESPRLLIKYA

GWIFPGDGSTQY

SQSISGIPSRFS

NEKFKGKATLTT

GSGSGSDFTLS

DTSSSTAYMQLS

INSVEPEDVGV

RLTSEDSAVYFC

YYCQNGHSFP

ARQTTATWFAY

LTFGAGTKLE

WGQGTLVTVSA

LK

CD279
DIQMTQSPSSL
625
LSIN
626
AAS
627
QQSS
628
EVQLLESGGVLV
629
GFTF
630
ISGG
631
VKWG
632

(PD-1)
SASVGDSITIT

TF

NTPF

QPGGSLRLSCAA

GSNF

RDT

NIYF

CRASLSINTFL

T

SGFTFSNFGMTW

G

DY

NWYQQKPGK

VRQAPGKGLEW

APNLLIYAASS

VSGISGGGRDTY

LHGGVPSRFS

FADSVKGRFTISR

GSGSGTDFTLT

DNSKNTLYLQM

IRTLQPEDFAT

NSLKGEDTAVYY

YYCQQSSNTP

CVKWGNIYFDY

FTFGPGTVVD

WGQGTLVTVSS

FR

CD279
DIQMTQSPSSL
633
QTIG
634
TAT
635
QQV
636
EVQLVESGGGLV
637
GFTF
638
ISGG
639
ARQL
640

(PD-1)
SASVGDRVTIT

TW

YSIP

QPGGSLRLSCAA

SSY

GANT

YYFD

CLASQTIGTW

WT

SGFTFSSYMMSW

M

Y

LTWYQQKPG

VRQAPGKGLEW

KAPKLLIYTAT

VATISGGGANTY

SLADGVPSRFS

YPDSVKGRFTISR

GSGSGTDFTLT

DNAKNSLYLQM

ISSLQPEDFAT

NSLRAEDTAVYY

YYCQQVYSIP

CARQLYYFDYW

WTFGGGTKVE

GQGTTVTVSS

IK

CD279
EIVLTQSPATL
641
QSVS
642
DAS
643
QQSS
644
QVQLVESGGGV
645
GITF
646
IWYD
647
ATND
648

(PD-1)
SLSPGERATLS

SY

NWP

VQPGRSLRLDCK

SNSG

GSKR

DY

CRASQSVSSY

RT

ASGITFSNSGMH

LAWYQQKPG

WVRQAPGKGLE

QAPRLLIYDAS

WVAVIWYDGSK

NRATGIPARFS

RYYADSVKGRFT

GSGSGTDFTLT

ISRDNSKNTLFLQ

ISSLEPEDFAV

MNSLRAEDTAV

YYCQQSSNWP

YYCATNDDYWG

RTFGQGTKVEI

QGTLVTVSS

K

CD279
DIQMTQSPSSV
649
QGIS
650
AAS
651
QQA
652
QVQLVQSGAEV
653
GGTF
654
IIPM
655
ARAE
656

(PD-1)
SASVGDRVTIT

SW

NHLP

KKPGSSVKVSCK

SSYA

FDTA

HSST

CRASQGISSW

FT

ASGGTFSSYAIS

GTFD

LAWYQQKPG

WVRQAPGQGLE

Y

KAPKLLISAAS

WMGLIIPMFDTA

SLQSGVPSRFS

GYAQKFQGRVAI

GSGSGTDFTLT

TVDESTSTAYME

ISSLQPEDFAT

LSSLRSEDTAVY

YYCQQANHLP

YCARAEHSSTGT

FTFGGGTKVEI

FDYWGQGTLVT

K

VSS

CD279
QPVLTQPLSVS
657
NIGS
658
RDS
659
QVW
660
QVQLVQSGGGL
661
GFTF
662
IDTG
663
ARDE
664

(PD-1)
VALGQTARIT

KN

DSST

VQPGGSLRLSCA

SSY

GGRT

GGGT

CGGNNIGSKN

AV

ASGFTFSSYWMY

W

GWGV

VHWYQQKPG

WVRQVPGKGLE

LKDW

QAPVLVIYRD

WVSAIDTGGGRT

PYGL

SNRPSGIPERF

YYADSVKGRFAI

DA

SGSNSGNTAT

SRVNAKNTMYL

LTISRAQAGDE

QMNSLRAEDTA

ADYYCQVWD

VYYCARDEGGG

SSTAVFGTGT

TGWGVLKDWPY

KLTVL

GLDAWGQGTLV

TVSS

CD279
EIVLTQSPATL
665
KGVS
666
LAS
667
QHSR
668
QVQLVQSGVEV
669
GYTF
670
INPS
671
ARRD
672

(PD-1)
SLSPGERATLS

TSGY

DLPL

KKPGASVKVSCK

TNY

NGGT

YRFD

CRASKGVSTS

SY

T

ASGYTFTNYYM

Y

MGFD

GYSYLHWYQ

YWVRQAPGQGL

YW

QKPGQAPRLLI

EWMGGINPSNGG

YLASYLESGV

TNFNEKFKNRVT

PARFSGSGSGT

LTTDSSTTTAYM

DFTLTISSLEPE

ELKSLQFDDTAV

DFAVYYCQHS

YYCARRDYRFD

RDLPLTFGGG

MGFDYWGQGTT

TKVEIK

VTVSS

CD279
EIVLTQSPATL
673
QSVR
674
DAS
675
QQR
676
VQLVQSGAEVK
677
GGTF
678
IIPI
679
ARPG
680

(PD-1)
SLSPGERATLS

SY

NYW

KPGSSVKVSCKA

SSYA

FDTA

LAAA

CRASQSVRSY

PLT

SGGTFSSYAISW

YDTG

LAWYQQKPG

VRQAPGQGLEW

SLDY

QAPRLLIYDAS

MGGIIPIFDTANY

NRATGIPARFS

AQKFQGRVTITA

GSGSGTDFTLT

DESTSTAYMELS

ISSLEPEDFAV

SLRSEDTAVYYC

YYCQQRNYW

ARPGLAAAYDTG

PLTFGQGTKV

SLDYWGQGTLV

EIK

TVSS

CD3
DIQLTQSPAIM
681
SSVS
682
DTS
683
QQW
684
DIKLQQSGAELA
685
GYTF
686
INPS
687
ARYY
688

SASPGEKVTM

Y

SSNP

RPGASVKMSCKT

TRYT

RGYT

DDHY

TCRASSSVSY

LT

SGYTFTRYTMH

CLDY

MNWYQQKSG

WVKQRPGQGLE

TSPKRWIYDTS

WIGYINPSRGYT

KVASGVPYRF

NYNQKFKDKAT

SGSGSGTSYSL

LTTDKSSSTAYM

TISSMEAEDA

QLSSLTSEDSAV

ATYYCQQWSS

YYCARYYDDHY

NPLTFGAGTK

CLDYWGQGTTL

LELK

TVSS

CD3
DIQLTQPNSVS
689
SGNI
690
DDD
691
HSY
692
EVQLLESGGGLV
693
GFTF
694
ISTS
695
AKFR
696

TSLGSTVKLSC

ENNY

VSSF

QPGGSLRLSCAA

SSFP

GGRT

QYSG

TLSSGNIENNY

NV

SGFTFSSFPMAW

GFDY

VHWYQLYEG

VRQAPGKGLEW

RSPTTMIYDD

VSTISTSGGRTYY

DKRPDGVPDR

RDSVKGRFTISRD

FSGSIDRSSNS

NSKNTLYLQMNS

AFLTIHNVAIE

LRAEDTAVYYCA

DEAIYFCHSY

KFRQYSGGFDY

VSSFNVFGGG

WGQGTLVTVSS

TKLTVL

CD3
DIQMTQTTSSL
697
QDIR
698
YTS
699
QQG
700
EVQLQQSGPELV
701
GYSF
702
INPY
703
ARSG
704

SASLGDRVTIS

NY

NTLP

KPGASMKISCKA

TGY

KGVS

YYGD

CRASQDIRNY

WTF

SGYSFTGYTMN

T

SDWY

LNWYQQKPD

AGG

WVKQSHGKNLE

FDV

GTVKLLIYYTS

WMGLINPYKGVS

RLHSGVPSKFS

TYNQKFKDKATL

GSGSGTDYSL

TVDKSSSTAYME

TISNLEQEDIA

LLSLTSEDSAVY

TYFCQQGNTL

YCARSGYYGDSD

PWTFAGGTKL

WYFDVWGQGTT

EIK

LTVFS

CD3
QTVVTQEPSL
705
TGAVT
706
GTK
707
VLW
708
EVQLVESGGGLV
709
GFTF
710
IRSK
711
VRHG
712

TVSPGGTVTL

SGNY

YSNR

QPGGSLKLSCAA

NKY

YNNY

NFGN

TCGSSTGAVT

WV

SGFTFNKYAMN

A

AT

SYIS

SGNYPNWVQ

WVRQAPGKGLE

YWAY

QKPGQAPRGL

WVARIRSKYNNY

IGGTKFLAPGT

ATYYADSVKDRF

PARFSGSLLGG

TISRDDSKNTAY

KAALTLSGVQ

LQMNNLKTEDT

PEDEAEYYCV

AVYYCVRHGNF

LWYSNRWVF

GNSYISYWAYW

GGGTKLTVL

GQGTLVTVSS

CD3
DFVMTQSPDS
713
QSLL
714
WAS
715
QND
716
EVQLVQSGAELK
717
GYTF
718
IIPS
719
ARSH
720

LAVSLGERVT

NSGN

YSYP

KPGASVKVSCKA

TDY

NGAT

LLRA

MSCKSSQSLL

QKNY

YT

SGYTFTDYYMK

Y

SWFA

NSGNQKNYLT

WVRQAPGQGLE

YW

WYQQKPGQPP

WIGDIIPSNGATF

KLLIYWASTR

YNQKFKGRVTIT

ESGVPDRFSGS

VDKSTSTAYMEL

GSGTDFTLTIS

SSLRSEDTAVYY

SLQAEDVAVY

CARSHLLRASWF

YCQNDYSYPY

AYWGQGTLVTV

TFGQGTKLEIK

SS

CD3
QIVLTQSPAIM
721
SSVS
722
DTS
723
QQW
724
QVQLQQSGAELA
725
GYTF
726
INPS
727
ARYY
728

SASPGEKVTM

Y

SSNP

RPGASVKMSCKA

TRYT

RGYT

DDHY

TCSASSSVSY

FT

SGYTFTRYTMH

CLDY

MNWYQQKSG

WVKQRPGQGLE

TSPKRWIYDTS

WIGYINPSRGYT

KLASGVPAHF

NYNQKFKDKAT

RGSGSGTSYSL

LTTDKSSSTAYM

TISGMEAEDA

QLSSLTSEDSAV

ATYYCQQWSS

YYCARYYDDHY

NPFTFGSGTKL

CLDYWGQGTTL

EIN

TVSS

CD3
EIVLTQSPATL
729
QSVS
730
DAS
731
QQRS
732
QVQLVESGGGV
733
GFKF
734
IWYD
735
ARQM
736

SLSPGERATLS

SY

NWP

VQPGRSLRLSCA

SGY

GSKK

GYWH

CRASQSVSSY

PLT

ASGFKFSGYGMH

G

FDL

LAWYQQKPG

WVRQAPGKGLE

QAPRLLIYDAS

WVAVIWYDGSK

NRATGIPARFS

KYYVDSVKGRFT

GSGSGTDFTLT

ISRDNSKNTLYL

ISSLEPEDFAV

QMNSLRAEDTA

YYCQQRSNWP

VYYCARQMGYW

PLTFGGGTKV

HFDLWGRGTLVT

EIK

VSS

CD3
DIQMTQSPSSL
737
QSIS
738
AAS
739
QQS
740
QVQLVQSGAEV
741
GYTF
742
INPS
743
AKGT
744

SASVGDRVTIT

SY

YSTP

KKPGASVKVSCK

TSYY

GGST

TGDW

CRASQSISSYL

PT

ASGYTFTSYYMH

FDY

NWYQQKPGK

WVRQAPGQGLE

APKLLIYAASS

WMGIINPSGGSTS

LQSGVPSRFSG

YAQKFQGRVTM

SGSGTDFTLTI

TRDTSTSTVYME

SSLQPEDFATY

LSSLRSEDTAVY

YCQQSYSTPPT

YCAKGTTGDWF

FGQGTKVEIK

DYWGQGTLVTV

SS

CD30
DIVLTQSPASL
745
QSVD
746
AAS
747
QQS
748
QIQLQQSGPEVV
749
GYTF
750
IYPG
751
ANYG
752

(TNFRS
AVSLGQRATIS

FDGD

NEDP

KPGASVKISCKA

TDY

SGNT

NYWF

F8)
CKASQSVDFD

SY

WT

SGYTFTDYYITW

Y

AY

GDSYMNWYQ

VKQKPGQGLEWI

QKPGQPPKVLI

GWIYPGSGNTKY

YAASNLESGIP

NEKFKGKATLTV

ARFSGSGSGT

DTSSSTAFMQLSS

DFTLNIHPVEE

LTSEDTAVYFCA

EDAATYYCQQ

NYGNYWFAYWG

SNEDPWTFGG

QGTQVTVSA

GTKLEIK

CD30
DIQMTQSPTSL
753
QGIS
754
AAS
755
QQY
756
QVQLQQWGAGL
757
GGSF
758
INHG
759
ASLT
760

(TNFRS
SASVGDRVTIT

SW

DSYP

LKPSETLSLTCAV

SAY

GGT

AY

F8)
CRASQGISSW

IT

YGGSFSAYYWS

Y

LTWYQQKPEK

WIRQPPGKGLEW

APKSLIYAASS

IGDINHGGGTNY

LQSGVPSRFSG

NPSLKSRVTISVD

SGSGTDFTLTI

TSKNQFSLKLNS

SSLQPEDFATY

VTAADTAVYYC

YCQQYDSYPI

ASLTAYWGQGSL

TFGQGTRLEIK

VTVSS

CD319
DIQMTQSPSSL
761
QDVG
762
WAS
763
QQY
764
EVQLVESGGGLV
765
GFDF
766
INPD
767
ARPD
768

(SLAMF
SASVGDRVTIT

IA

SSYP

QPGGSLRLSCAA

SRY

SSTI

GNYW

7)
CKASQDVGIA

YT

SGFDFSRYWMS

W

YFDV

VAWYQQKPG

WVRQAPGKGLE

KVPKLLIYWA

WIGEINPDSSTIN

STRHTGVPDR

YAPSLKDKFIISR

FSGSGSGTDFT

DNAKNSLYLQM

LTISSLQPEDV

NSLRAEDTAVYY

ATYYCQQYSS

CARPDGNYWYF

YPYTFGQGTK

DVWGQGTLVTV

VEIK

SS

CD33
DIVLTQSPTIM
769
SSVN
770
DTS
771
QQW
772
EVKLQESGPELV
773
GYK
774
INPY
775
ARDY
776

SASPGERVTM

Y

RSYP

KPGASVKMSCK

FTDY

NDGT

RYEV

TCTASSSVNYI

LT

ASGYKFTDYVVH

V

YGMD

HWYQQKSGD

WLKQKPGQGLE

Y

SPLRWIFDTSK

WIGYINPYNDGT

VASGVPARFS

KYNEKFKGKATL

GSGSGTSYSLT

TSDKSSSTAYME

ISTMEAEDAA

VSSLTSEDSAVY

TYYCQQWRS

YCARDYRYEVY

YPLTFGDGTR

GMDYWGQGTSV

LELK

TVSS

CD33
DIVMTQSPSSL
777
QDIN
778
YTS
779
LQY
780
EVKLQQSGPELV
781
GYSF
782
IDPY
783
AREM
784

SASLGGKVTIT

KY

DNLL

KPGTSVKVSCKA

TDY

KGGT

ITAY

CKASQDINKYI

T

SGYSFTDYNMY

N

YFDY

AWYQHKPGK

WVKQSHGKSLE

GPRLLIHYTST

WIGYIDPYKGGTI

LQPGIPSRFSG

YNQKFKGKATLT

SGSGRDYSFSI

VDKSSSTAFMHL

SNLEPEDIATY

NSLTSEDSAVYY

YCLQYDNLLT

CAREMITAYYFD

FGAGTKLELK

YWGQGSSVTVSS

CD33
DIVLTQSPASL
785
ESVD
786
AAS
787
QQS
788
EVQLQQSGPELV
789
GYTF
790
IYPY
791
ARGR
792

AVSLGQRATIS

NYGI

KEVP

KPGASVKISCKA

TDY

NGGT

PAMD

CRASESVDNY

SF

WT

SGYTFTDYNMH

N

Y

GISFMNWFQQ

WVKQSHGKSLE

KPGQPPKLLIY

WIGYIYPYNGGT

AASNQGSGVP

GYNQKFKSKATL

ARFSGSGSGT

TVDNSSSTAYMD

DFSLNIHPMEE

VRSLTSEDSAVY

DDTAMYFCQ

YCARGRPAMDY

QSKEVPWTFG

WGQGTSVTVSS

GGTKLEIK

CD33
DIQLTQSPSTL
793
ESLD
794
AAS
795
QQT
796
EVQLVQSGAEVK
797
GYTI
798
IYPYN
799
VNGNP
800

SASVGDRVTIT

NYGI

KEVP

KPGSSVKVSCKA

TDSN

GGT

WLAY

CRASESLDNY

RF

WS

SGYTITDSNIHW

GIRFLTWFQQ

VRQAPGQSLEWI

KPGKAPKLLM

GYIYPYNGGTDY

YAASNQGSGV

NQKFKNRATLTV

PSRFSGSGSGT

DNPTNTAYMELS

EFTLTISSLQP

SLRSEDTAFYYC

DDFATYYCQQ

VNGNPWLAYWG

TKEVPWSFGQ

QGTLVTVSS

GTKVEVK

CD33
NIMLTQSPSSL
801
QSVF
802
WAS
803
HQY
804
QVQLQQPGAEV
805
GYTF
806
IYPG
807
AREV
808

AVSAGEKVTM

FSSS

LSSR

VKPGASVKMSC

TSYY

NDDI

RLRY

SCKSSQSVFFS

QKNY

T

KASGYTFTSYYI

FDV

SSQKNYLAWY

HWIKQTPGQGLE

QQIPGQSPKLL

WVGVIYPGNDDI

IYWASTRESG

SYNQKFKGKATL

VPDRFTGSGS

TADKSSTTAYMQ

GTDFTLTISSV

LSSLTSEDSAVY

QSEDLAIYYC

YCAREVRLRYFD

HQYLSSRTFG

VWGAGTTVTVSS

GGTKLEIK

CD33
DIKMTQSPSS
809
QDIN
810
RAN
811
LQY
812
QVQLQQSGPELV
813
GYTF
814
IYPG
815
ASGY
816

MYASLGERVII

SY

DEFP

RPGTFVKISCKAS

TNY

DGST

EDAM

NCKASQDINS

LT

GYTFTNYDINWV

D

DY

YLSWFQQKPG

NQRPGQGLEWIG

KSPKTLIYRAN

WIYPGDGSTKYN

RLVDGVPSRF

EKFKAKATLTAD

SGSGSGQDYS

KSSSTAYLQLNN

LTISSLEYEDM

LTSENSAVYFCA

GIYYCLQYDE

SGYEDAMDYWG

FPLTFGAGTKL

QGTSVTVSS

ELK

CD33
DIQMTQSPSSL
817
ESVD
818
AAS
819
QQS
820
QVQLVQSGAEV
821
GYTF
822
IYPY
823
ARGR
824

SASVGDRVTIT

NYGI

KEVP

KKPGSSVKVSCK

TDY

NGGT

PAMD

CRASESVDNY

SF

WT

ASGYTFTDYNM

N

YW

GISFMNWFQQ

HWVRQAPGQGL

KPGKAPKLLL

EWLGYIYPYNGG

YAASNQGSGV

TGYNQKFKSKAT

PSRFSGSGSGT

ITADESTNTAYM

DFTLTISSLQP

ELSSLRSEDTAV

DDFATYYCQQ

YYCARGRPAMD

SKEVPWTFGQ

YWGQGTLVTVSS

GTKVELK

CD332
QSVLTQPPSAS
825
SSNI
826
ENY
827
SSW
828
EVQLLESGGGLV
829
GFTF
830
ISGS
831
ARVR
832

(FGFR2)
GTPGQRVTISC

GNNY

N

DDSL

QPGGSLRLSCAA

SSYA

GTST

YNWN

SGSSSNIGNNY

NYW

SGFTFSSYAMSW

HGDW

VSWYQQLPGT

V

VRQAPGKGLEW

FDP

APKLLIYENY

VSAISGSGTSTYY

NRPAGVPDRF

ADSVKGRFTISR

SGSKSGTSASL

DNSKNTLYLQM

AISGLRSEDEA

NSLRAEDTAVYY

DYYCSSWDDS

CARVRYNWNHG

LNYWVFGGG

DWFDPWGQGTL

TKLTVL

VTVSS

CD350
DIQMTQSPASL
833
ENIY
834
VAT
835
QHF
836
EVQLQQSGAELV
837
GFNI
838
IDPA
839
ARGA
840

(FZD
SVSVGETVTIT

SN

WGT

KPGASVKLSCTA

NDT

NGNT

RGSR

10)
CRASENIYSNL

PYT

SGFNINDTYMHW

Y

FAY

AWYQQKQGK

VKQRPEQGLEWI

SPQLLVYVAT

GRIDPANGNTKY

NLADGVPSRF

DPKFQGKATITA

SGSGSGTQYS

DTSSNTAYLQLS

LKINSLQSEDF

SLTSEDTAVYYC

GSYYCQHFW

ARGARGSRFAY

GTPYTFGGGT

WGQGTLVTVSA

KLEIK

CD37
DIQMTQSPSSL
841
ENIR
842
VAT
843
QHY
844
QVQVQESGPGLV
845
GFSL
846
IWGD
847
AKGG
848

SVSVGERVTIT

SN

WGT

APSQTLSITCTVS

TTSG

GST

YSLA

CRASENIRSNL

TWT

GFSLTTSGVSWV

H

AWYQQKPGK

RQPPGKGLEWLG

SPKLLVNVAT

VIWGDGSTNYHP

NLADGVPSRF

SLKSRLSIKKDHS

SGSGSGTDYS

KSQVFLKLNSLT

LKINSLQPEDF

AADTATYYCAK

GTYYCQHYW

GGYSLAHWGQG

GTTWTFGQGT

TLVTVSS

KLEIK

CD371
DIQMTQSPSSL
849
QSIS
850
AAS
851
QQS
852
QVQLVQSGGGV
853
GFTF
854
IWYN
855
TRGT
856

(CLEC1
SASVGDRVTIT

SY

YSTP

VQPGRSLRLSCV

SSYG

ARKQ

GYNW

2A)
CRASQSISSYL

PT

ASGFTFSSYGMH

FDP

NWYQQKPGK

WVRQAPGKGLE

APKLLIYAASS

WVAAIWYNARK

LQSGVPSRFSG

QDYADSVKGRFT

SGSGTDFTLTI

ISRDNSKNTLYL

SSLQPEDFATY

QMNSLRAEDTA

YCQQSYSTPPT

VYYCTRGTGYN

FGQGTKVEIK

WFDPWGQGTLV

TVSS

CD38
DIVMAQSHKF
857
ASQD
858
ITSA
859
TCQ
860
QVKLVESGGGLV
861
GFTF
862
ISIG
863
TRDF
864

MSTSVGDRVS

VS

QHY

KPGGSLKLSCEA

SSYT

GRYT

NGTS

ITCKASQDVST

SPYT

SGFTFSSYTLSW

DF

VVAWYQQKP

VRQTPETRLEWV

GQSPKRLITSA

ATISIGGRYTTTP

SYRYIGVPDRF

DSVEGRFTISRDN

TGSGSGTDFTF

AKNTLYLQMNSL

TISSVQAEDLA

KSEDTAMYYCTR

VTTCQQHYSP

DFNGTSDFWGQ

YTFGGGTKLEI

GTTLTVSS

K

CD38
EIVLTQSPATL
865
QSVS
866
DAS
867
QQRS
868
EVQLLESGGGLV
869
GFTF
870
ISGS
871
AKDK
872

SLSPGERATLS

SY

NWP

QPGGSLRLSCAV

NSFA

GGGT

ILWF

CRASQSVSSY

PT

SGFTFNSFAMSW

GEPV

LAWYQQKPG

VRQAPGKGLEW

FDY

QAPRLLIYDAS

VSAISGSGGGTY

NRATGIPARFS

YADSVKGRFTIS

GSGSGTDFTLT

RDNSKNTLYLQ

ISSLEPEDFAV

MNSLRAEDTAV

YYCQQRSNWP

YFCAKDKILWFG

PTFGQGTKVEI

EPVFDYWGQGTL

K

VTVSS

CD38
DIVMTQSHLS
873
QDVS
874
SAS
875
QQH
876
QVQLVQSGAEV
877
GYTF
878
IYPG
879
ARGDY
880

MSTSLGDPVSI

TV

YSPP

AKPGTSVKLSCK

TDY

GDT

DYGS

TCKASQDVST

YT

ASGYTFTDYWM

W

NSLD

VVAWYQQKP

QWVKQRPGQGL

Y

GQSPRRLIYSA

EWIGTIYPGDGD

SYRYIGVPDRF

TGYAQKFQGKA

TGSGAGTDFT

TLTADKSSKTVY

FTISSVQAEDL

MHLSSLASEDSA

AVYYCQQHYS

VYYCARGDYYG

PPYTFGGGTK

SNSLDYWGQGTS

LEIK

VTVSS

CD4
DIVMTQSPDSL
881
KSVS
882
LAS
883
QHSR
884
EEQLVESGGGLV
885
GFSF
886
ISVK
887
SASY
888

AVSLGERATIN

TSGY

ELP

KPGGSLRLSCAA

SDCR

SENY

YRYD

CRASKSVSTS

SY

WT

SGFSFSDCRMYW

GA

VGAW

GYSYIYWYQQ

LRQAPGKGLEWI

FAYW

KPGQPPKLLIY

GVISVKSENYGA

LASILESGVPD

NYAESVRGRFTIS

RFSGSGSGTDF

RDDSKNTVYLQ

TLTISSLQAED

MNSLKTEDTAVY

VAVYYCQHSR

YCSASYYRYDVG

ELPWTFGQGT

AWFAYWGQGTL

KVEIK

VTVSS

CD4
DIVMTQSPDSL
889
QSLL
890
WAS
891
QQY
892
QVQLQQSGPEVV
893
GYTF
894
INPY
895
AREK
896

AVSLGERVTM

YSTN

YSYR

KPGASVKMSCK

TSYV

NDGT

DNYA

NCKSSQSLLYS

QKNY

T

ASGYTFTSYVIH

TGAW

TNQKNYLAW

WVRQKPGQGLD

FAY

YQQKPGQSPK

WIGYINPYNDGT

LLIYWASTRES

DYDEKFKGKATL

GVPDRFSGSG

TSDTSTSTAYME

SGTDFTLTISS

LSSLRSEDTAVY

VQAEDVAVY

YCAREKDNYAT

YCQQYYSYRT

GAWFAYWGQGT

FGGGTKLEIK

LVTVSS

CD40
DIQMTQSPSSL
897
QSLV
898
TVS
899
SQTT
900
EVQLVESGGGLV
901
GYSF
902
VIPN
903
AREG
904

SASVGDRVTIT

HSNG

HVP

QPGGSLRLSCAA

TGY

AGGT

IYW

CRSSQSLVHS

NTF

WT

SGYSFTGYYIHW

Y

NGNTFLHWY

VRQAPGKGLEW

QQKPGKAPKL

VARVIPNAGGTS

LIYTVSNRFSG

YNQKFKGRFTLS

VPSRFSGSGSG

VDNSKNTAYLQ

TDFTLTISSLQ

MNSLRAEDTAV

PEDFATYFCSQ

YYCAREGIYWW

TTHVPWTFGQ

GQGTLVTVSS

GTKVEIK

CD40
AIQLTQSPSSL
905
QGIS
906
DAS
907
QQF
908
QLQLQESGPGLL
909
GGSI
910
IYKS
911
TRPV
912

SASVGDRVTIT

SA

NSYP

KPSETLSLTCTVS

SSPG

GST

VRYF

CRASQGISSAL

T

GGSISSPGYYGG

YY

GWFD

AWYQQKPGK

WIRQPPGKGLEW

P

APKLLIYDASN

IGSIYKSGSTYHN

LESGVPSRFSG

PSLKSRVTISVDT

SGSGTDFTLTI

SKNQFSLKLSSVT

SSLQPEDFATY

AADTAVYYCTRP

YCQQFNSYPT

VVRYFGWFDPW

FGQGTKVEIK

GQGTLVTVSS

CD40
DIVMTQSPLSL
913
QSLL
914
LGS
915
MQA
916
QVQLVESGGGV
917
GFTF
918
ISYE
919
ARDG
920

TVTPGEPASIS

YSNG

RQTP

VQPGRSLRLSCA

SSYG

ESNR

GIAA

CRSSQSLLYSN

YNY

FT

ASGFTFSSYGMH

PGPD

GYNYLDWYL

WVRQAPGKGLE

Y

QKPGQSPQVLI

WVAVISYEESNR

SLGSNRASGV

YHADSVKGRFTI

PDRFSGSGSGT

SRDNSKITLYLQ

DFTLKISRVEA

MNSLRTEDTAVY

EDVGVYYCM

YCARDGGIAAPG

QARQTPFTFGP

PDYWGQGTLVT

GTKVDIR

VSS

CD40
DIQMTQSPSSV
921
QGIY
922
TAS
923
QQA
924
QVQLVQSGAEV
925
GYTF
926
INPD
927
ARDQ
928

SASVGDRVTIT

SW

NIFP

KKPGASVKVSCK

TGY

SGGT

PLGY

CRASQGIYSW

LT

ASGYTFTGYYM

Y

CTNG

LAWYQQKPG

HWVRQAPGQGL

VCSY

KAPNLLIYTAS

EWMGWINPDSG

FDY

TLQSGVPSRFS

GTNYAQKFQGR

GSGSGTDFTLT

VTMTRDTSISTA

ISSLQPEDFAT

YMELNRLRSDDT

YYCQQANIFP

AVYYCARDQPL

LTFGGGTKVEI

GYCTNGVCSYFD

K

YWGQGTLVTVSS

CD40L
DIQMTQSPSSL
929
EDLY
930
DTY
931
QQY
932
EVQLVESGGGLV
933
GFSS
934
IWGD
935
ARQL
936

SASVGDRVTIT

YN

YKFP

QPGGSLRLSCAV

TNY

GDT

THYY

CRASEDLYYN

FT

SGFSSTNYHVHW

H

VLAA

LAWYQRKPG

VRQAPGKGLEW

KAPKLLIYDT

MGVIWGDGDTS

YRLADGVPSR

YNSVLKSRFTISR

FSGSGSGTDY

DTSKNTVYLQM

TLTISSLQPED

NSLRAEDTAVYY

FASYYCQQYY

CARQLTHYYVLA

KFPFTFGQGT

AWGQGTLVTVSS

KVEIK

CD40L
DIVLTQSPATL
937
QRVS
938
YAS
939
QHS
940
QVQLVQSGAEV
941
GYIF
942
INPS
943
TRSD
944

SVSPGERATIS

SSTY

WEIP

VKPGASVKLSCK

TSYY

NGDT

GRND

CRASQRVSSST

SY

PT

ASGYIFTSYYMY

MDS

YSYMHWYQQ

WVKQAPGQGLE

KPGQPPKLLIK

WIGEINPSNGDT

YASNLESGVP

NFNEKFKSKATL

ARFSGSGSGT

TVDKSASTAYME

DFTLTISSVEP

LSSLRSEDTAVY

EDFATYYCQH

YCTRSDGRNDM

SWEIPPTFGGG

DSWGQGTLVTVS

TKLEIK

S

DFVMTQSPAF

CD49b
LSVTPGEKVTI
945
SSVN
946
DTS
947
QQW
948
QVQLQESGPGLV
949
GFSL
950
IWAR
951
ARAN
952

(a2)
TCSAQSSVNYI

Y

TTNP

KPSETLSLTCTVS

TNY

GFT

DGVY

HWYQQKPDQ

LT

GFSLTNYGIHWIR

G

YAMD

APKKLIYDTSK

QPPGKGLEWLGV

Y

LASGVPSRFSG

IWARGFTNYNSA

SGSGTDYTFTI

LMSRLTISKDNS

SSLEAEDAAT

KNQVSLKLSSVT

YYCQQWTTNP

AADTAVYYCAR

LTFGQGTKVEI

ANDGVYYAMDY

K

WGQGTLVTVSS

CD51
DIQMTQSPSSL
953
QDIS
954
YTS
955
QQG
956
QVQLQQSGGELA
957
GYTF
958
INPRS
959
ASFL
960

(a5)
SASVGDRVTIT

NY

NTFP

KPGASVKVSCKA

SSFW

GYT

GRGA

CRASQDISNYL

YT

SGYTFSSFWMH

MDY

AWYQQKPGK

WVRQAPGQGLE

APKLLIYYTSK

WIGYINPRSGYTE

IHSGVPSRFSG

YNEIFRDKATMT

SGSGTDYTFTI

TDTSTSTAYMEL

SSLQPEDIATY

SSLRSEDTAVYY

YCQQGNTFPY

CASFLGRGAMD

TFGQGTKVEI

YWGQGTTVTVSS

K

CD52
DIQMTQSPSSL
961
QNID
962
NTN
963
LQHI
964
QVQLQESGPGLV
965
GFTF
966
IRDK
967
AREG
968

SASVGDRVTIT

KY

SRPR

RPSQTLSLTCTVS

TDFY

AKGY

HTAA

CKASQNIDKY

T

GFTFTDFYMNW

TT

PFDY

LNWYQQKPG

VRQPPGRGLEWI

KAPKLLIYNT

GFIRDKAKGYTT

NNLQTGVPSR

EYNPSVKGRVTM

FSGSGSGTDFT

LVDTSKNQFSLR

FTISSLQPEDIA

LSSVTAADTAVY

TYYCLQHISRP

YCAREGHTAAPF

RTFGQGTKVEI

DYWGQGSLVTV

K

SS

CD54(IC
QSVLTQPPSAS
969
SSNI
970
DNN
971
QSY
972
EVQLLESGGGLV
973
GFTF
974
IWYD
975
ARYS
976

AM-1)
GTPGQRVTISC

GAGY

DSSL

QPGGSLRLSCAA

SNA

GSNK

GWYF

TGSSSNIGAGY

D

SAW

SGFTFSNAWMS

W

Y

DVHWYQQLP

L

WVRQAPGKGLE

GTAPKLLIYD

WVAFIWYDGSN

NNNRPSGVPD

KYYADSVKGRFT

RFSGSKSGTSA

ISRDNSKNTLYL

SLAISGLRSED

QMNSLRAEDTA

EADYYCQSYD

VYYCARYSGWY

SSLSAWLFGG

FDYWGQGTLVT

GTKLTVL

VSS

CD56
DVVMTQSPLS
977
QIII
978
KVS
979
FQGS
980
QVQLVESGGGV
981
GFTF
982
ISSG
983
ARMR
984

LPVTLGQPASI

HSDG

HVP

VQPGRSLRLSCA

SSFG

SFTI

KGYA

SCRSSQIIIHSD

NTY

HT

ASGFTFSSFGMH

MDY

GNTYLEWFQQ

WVRQAPGKGLE

RPGQSPRRLIY

WVAYISSGSFTIY

KVSNRFSGVP

YADSVKGRFTIS

DRFSGSGSGT

RDNSKNTLYLQ

DFTLKISRVEA

MNSLRAEDTAV

EDVGVYYCFQ

YYCARMRKGYA

GSHVPHTFGQ

MDYWGQGTLVT

GTKVEIK

VSS

CD61
EIVLTQSPATL
985
QSIS
986
YRS
987
QQS
988
QVQLVESGGGV
989
GFTF
990
VSSG
991
ARHL
992

(a4b3)
SLSPGERATLS

NF

GSW

VQPGRSLRLSCA

SSYD

GGST

HGSF

CQASQSISNFL

PLT

ASGFTFSSYDMS

AS

HWYQQRPGQ

WVRQAPGKGLE

APRLLIRYRSQ

WVAKVSSGGGS

SISGIPARFSGS

TYYLDTVQGRFT

GSGTDFTLTIS

ISRDNSKNTLYL

SLEPEDFAVY

QMNSLRAEDTA

YCQQSGSWPL

VYYCARHLHGSF

TFGGGTKVEI

ASWGQGTTVTVS

K

S

CD70
QAVVTQEPSL
993
SGSV
994
NTN
995
ALFI
996
EVQLVESGGGLV
997
GFTF
998
INNE
999
ARDA
1000

TVSPGGTVTL

TSDN

SNPS

QPGGSLRLSCAA

SVY

GGTT

GYSN

TCGLKSGSVT

F

VEFG

SGFTFSVYYMN

Y

HVPI

SDNFPTWYQQ

G

WVRQAPGKGLE

FDS

TPGQAPRLLIY

WVSDINNEGGTT

NTNTRHSGVP

YYADSVKGRFTI

DRFSGSILGNK

SRDNSKNSLYLQ

AALTITGAQA

MNSLRAEDTAV

DDEAEYFCAL

YYCARDAGYSN

FISNPSVEFGG

HVPIFDSWGQGT

GTQLTVL

LVTVSS

CD73
QSVLTQPPSAS
1001
LSNI
1002
LDN
1003
ATW
1004
EVQLLESGGGLV
1005
GFTF
1006
ISGS
1007
ARLG
1008

(NT5E)
GTPGQRVTISC

GRNP

DDS

QPGGSLRLSCAA

SSYA

GGRT

YGRV

SGSLSNIGRNP

HPG

SGFTFSSYAYSW

DE

VNWYQQLPG

WT

VRQAPGKGLEW

TAPKLLIYLDN

VSAISGSGGRTY

LRLSGVPDRFS

YADSVKGRFTIS

GSKSGTSASL

RDNSKNTLYLQ

AISGLQSEDEA

MNSLRAEDTAV

DYYCATWDD

YYCARLGYGRV

SHPGWTFGGG

DEWGRGTLVTVS

TKLTVL

S

CD74
DIQLTQSPLSL
1009
QSLV
1010
TVS
1011
SQSS
1012
QVQLQQSGSELK
1013
GYTF
1014
INPN
1015
SRSR
1016

PVTLGQPASIS

HRNG

HVPP

KPGASVKVSCKA

TNY

TGEP

GKNE

CRSSQSLVHR

NTY

T

SGYTFTNYGVN

G

AWFA

NGNTYLHWF

WIKQAPGQGLQ

Y

QQRPGQSPRL

WMGWINPNTGE

LIYTVSNRFSG

PTFDDDFKGRFA

VPDRFSGSGS

FSLDTSVSTAYL

GTDFTLKISRV

QISSLKADDTAV

EAEDVGVYFC

YFCSRSRGKNEA

SQSSHVPPTFG

WFAYWGQGTLV

AGTRLEIK

TVSS

CEA
QTVLSQSPAIL
1017
SSVT
1018
ATS
1019
QHW
1020
EVKLVESGGGLV
1021
GFTF
1022
IGNK
1023
TRDR
1024

SASPGEKVTM

Y

SSKP

QPGGSLRLSCAT

TDY

ANGY

GLRF

TCRASSSVTYI

PT

SGFTFTDYYMN

Y

TT

YFDY

HWYQQKPGSS

WVRQPPGKALE

PKSWIYATSN

WLGFIGNKANGY

LASGVPARFS

TTEYSASVKGRF

GSGSGTSYSLT

TISRDKSQSILYL

ISRVEAEDAAT

QMNTLRAEDSAT

YYCQHWSSKP

YYCTRDRGLRFY

PTFGGGTKLEI

FDYWGQGTTLT

K

VSS

CEA
DIQLTQSPSSL
1025
QDVG
1026
WTS
1027
QQY
1028
EVQLVESGGGVV
1029
GFDF
1030
IHPD
1031
ASLY
1032

SASVGDRVTIT

TS

SLYR

QPGRSLRLSCSAS

TTY

SSTI

FGFP

CKASQDVGTS

S

GFDFTTYWMSW

W

WFAY

VAWYQQKPG

VRQAPGKGLEWI

KAPKLLIYWT

GEIHPDSSTINYA

STRHTGVPSRF

PSLKDRFTISRDN

SGSGSGTDFTF

AKNTLFLQMDSL

TISSLQPEDIAT

RPEDTGVYFCAS

YYCQQYSLYR

LYFGFPWFAYW

SFGQGTKVEIK

GQGTPVTVSS

Clau-
DIVMTQSPSSL
1033
QSLL
1034
WAS
1035
QND
1036
QVQLQQPGAELV
1037
GYTF
1038
IYPS
1039
TRSW
1040

din-
TVTAGEKVTM

NSGN

YSYP

RPGASVKLSCKA

TSY

DSYT

RGNS

18.2
SCKSSQSLLNS

QKNY

FT

SGYTFTSYWINW

GNQKNYLTW

VKQRPGQGLEWI

W

FDY

YQQKPGQPPK

GNIYPSDSYTNY

LLIYWASTRES

NQKFKDKATLTV

GVPDRFTGSG

DKSSSTAYMQLS

SGTDFTLTISS

SPTSEDSAVYYC

VQAEDLAVYY

TRSWRGNSFDY

CQNDYSYPFT

WGQGTTLTVSS

FGSGTKLEIK

cMET
DIQMTQSPSSL
1041
QSLL
1042
WAS
1043
QQY
1044
EVQLVESGGGLV
1045
GYTF
1046
IDPS
1047
ATYR
1048

SASVGDRVTIT

YTSS

YAY

QPGGSLRLSCAA

TSY

NSDT

SYVT

CKSSQSLLYTS

QKNY

PWT

SGYTFTSYWLH

W

PLDY

SQKNYLAWY

WVRQAPGKGLE

QQKPGKAPKL

WVGMIDPSNSDT

LIYWASTRES

RFNPNFKDRFTIS

GVPSRFSGSGS

ADTSKNTAYLQ

GTDFTLTISSL

MNSLRAEDTAV

QPEDFATYYC

YYCATYRSYVTP

QQYYAYPWTF

LDYWGQGTLVT

GQGTKVEIK

VSS

CRLR
QSVLTQPPSVS
1049
SSNI
1050
DNN
1051
GTW
1052
QVQLVESGGGV
1053
GFTF
1054
ISFD
1055
ARDR
1056

AAPGQKVTIS

GNNY

DSRL

VQPGRSLRLSCA

SSFG

GSIK

LNYY

CSGSSSNIGNN

SAV

ASGFTFSSFGMH

DSSG

YVSWYQQLPG

V

WVRQAPGKGLE

YYHY

TAPKLLIYDN

WVAVISFDGSIK

KYYG

NKRPSGIPDRF

YSVDSVKGRFTIS

MAV

SGSKSGTSTTL

RDNSKNTLFLQM

GITGLQTGDE

NSLRAEDTAVYY

ADYYCGTWD

CARDRLNYYDSS

SRLSAVVFGG

GYYHYKYYGMA

GTKLTVL

VWGQGTTVTVSS

Dabiga-
DVVMTQSPLS
1057
QSLL
1058
LVS
1059
LQST
1060
QVQLQESGPGLV
1061
GFSL
1062
IWAG
1063
ASAA
1064

tran
LPVTLGQPASI

YTDG

HFPH

KPSETLSLTCTVS

TSYI

GST

YYSY

SCKSSQSLLYT

KTY

T

GFSLTSYIVDWIR

YNYD

DGKTYLYWFL

QPPGKGLEWIGV

GFAY

QRPGQSPRRLI

IWAGGSTGYNSA

YLVSKLDSGV

LRSRVSITKDTSK

PDRFSGSGSGT

NQFSLKLSSVTA

DFTLKISRVEA

ADTAVYYCASA

EDVGVYYCLQ

AYYSYYNYDGF

STHFPHTFGG

AYWGQGTLVTV

GTKVEIK

SS

DLL3
EIVMTQSPATL
1065
QSVS
1066
YAS
1067
QQD
1068
QVQLVQSGAEV
1069
GYTF
1070
INTY
1071
ARIG
1072

SVSPGERATLS

ND

YTSP

KKPGASVKVSCK

TNY

TGEP

DSSP

CKASQSVSND

WT

ASGYTFTNYGM

G

SDY

VVWYQQKPG

NWVRQAPGQGL

QAPRLLIYYAS

EWMGWINTYTG

NRYTGIPARFS

EPTYADDFKGRV

GSGSGTEFTLT

TMTTDTSTSTAY

ISSLQSEDFAV

MELRSLRSDDTA

YYCQQDYTSP

VYYCARIGDSSPS

WTFGQGTKLE

DYWGQGTLVTV

IK

SS

DLL4
EIVLTQSPATL
1073
QSVS
1074
DAS
1075
QHRS
1076
QVQLVESGGGV
1077
GFTF
1078
LWYD
1079
ARDH
1080

SLSPGERATLS

SY

NWP

VQPGRSLRLSCA

SSYG

GTNK

DFRS

CRASQSVSSY

PT

ASGFTFSSYGMH

GYEG

LAWYQQKPG

WVRQAPGKGLE

WFDP

QAPRLLIYDAS

WVSFLWYDGTN

NRATGIPARFS

KNYVESVKGRFT

GSGSGTDFTLT

ISRDNSKNMLYL

ISSLEPEDFAV

EMNSLRAEDTAV

YYCQHRSNWP

YYCARDHDFRSG

PTFGGGTKVEI

YEGWFDPWGQG

K

TLVTVSS

DLL4
DIVMTQSPDSL
1081
ESVD
1082
AAS
1083
QQS
1084
QVQLVQSGAEV
1085
GYSF
1086
ISSY
1087
ARDY
1088

AVSLGERATIS

NYGI

KEVP

KKPGASVKISCK

TAY

NGAT

DYDV

CRASESVDNY

SF

WT

ASGYSFTAYYIH

Y

GMDY

GISFMKWFQQ

WVKQAPGQGLE

KPGQPPKLLIY

WIGYISSYNGAT

AASNQGSGVP

NYNQKFKGRVTF

DRFSGSGSGT

TTDTSTSTAYME

DFTLTISSLQA

LRSLRSDDTAVY

EDVAVYYCQ

YCARDYDYDVG

QSKEVPWTFG

MDYWGQGTLVT

GGTKVEIK

VSS

DNA/his
ENVLTQSPAI
1089
SSVS
1090
STS
1091
QQY
1092
QVQLKESGPGLV
1093
GFSL
1094
IWGG
1095
AKEK
1096

tone
MSASPGEKVT

SSY

SGYP

APSQSLSITCTVS

TDY

GST

RRGY

(H1)
MTCRASSSVS

LT

GFSLTDYGVRWI

G

YYAM

complex
SSYLHWYQQK

RQPPGKGLEWLG

DY

SGASPKLWIYS

VIWGGGSTYYNS

TSNLASGVPA

ALKSRLSISKDNS

RFSGSGSGTSY

KSQVFLKMNSLQ

SLTISSVEAED

TDDTAMYYCAK

AATYYCQQYS

EKRRGYYYAMD

GYPLTFGGGT

YWGQGTSVTVSS

KLEIK

EGFR
DILLTQSPVILS
1097
QSIG
1098
YAS
1099
QQN
1100
QVQLKQSGPGLV
1101
GFSL
1102
IWSG
1103
ARAL
1104

VSPGERVSFSC

TN

NNW

QPSQSLSITCTVS

TNY

GNT

TYYD

RASQSIGTNIH

PTT

GFSLTNYGVHW

G

YEFA

WYQQRTNGSP

VRQSPGKGLEWL

Y

RLLIKYASESIS

GVIWSGGNTDYN

GIPSRFSGSGS

TPFTSRLSINKDN

GTDFTLSINSV

SKSQVFFKMNSL

ESEDIADYYC

QSNDTAIYYCAR

QQNNNWPTTF

ALTYYDYEFAY

GAGTKLELK

WGQGTLVTVSA

EGFR
DIQMTQSPSSL
1105
QDIS
1106
DAS
1107
QHF
1108
QVQLQESGPGLV
1109
GGS
1110
IYYS
1111
VRDR
1112

SASVGDRVTIT

NY

DHLP

KPSETLSLTCTVS

VSSG

GNT

VTGA

CQASQDISNY

LA

GGSVSSGDYYW

DYY

FDI

LNWYQQKPG

TWIRQSPGKGLE

KAPKLLIYDAS

WIGHIYYSGNTN

NLETGVPSRFS

YNPSLKSRLTISI

GSGSGTDFTFT

DTSKTQFSLKLSS

ISSLQPEDIAT

VTAADTAIYYCV

YFCQHFDHLP

RDRVTGAFDIWG

LAFGGGTKVE

QGTMVTVSS

IK

EGFR
EIVMTQSPATL
1113
QSVS
1114
DAS
1115
HQY
1116
QVQLQESGPGLV
1117
GGSI
1118
IYYS
1119
ARVS
1120

SLSPGERATLS

SY

GSTP

KPSQTLSLTCTVS

SSGD

GST

IFGV

CRASQSVSSY

LT

GGSISSGDYYWS

YY

GTFD

LAWYQQKPG

WIRQPPGKGLEW

Y

QAPRLLIYDAS

IGYIYYSGSTDYN

NRATGIPARFS

PSLKSRVTMSVD

GSGSGTDFTLT

TSKNQFSLKVNS

ISSLEPEDFAV

VTAADTAVYYC

YYCHQYGSTP

ARVSIFGVGTFD

LTFGGGTKAEI

YWGQGTLVTVSS

KR

EGFR
DIQMTQSPSSL
1121
QNIV
1122
KVS
1123
FQYS
1124
QVQLQQSGAEV
1125
GYTF
1126
INPT
1127
ARQG
1128

SASVGDRVTIT

HSNG

HVP

KKPGSSVKVSCK

TNY

SGGS

LWFD

CRSSQNIVHSN

NTY

WT

ASGYTFTNYYIY

Y

SDGR

GNTYLDWYQ

WVRQAPGQGLE

GFDF

QTPGKAPKLLI

WIGGINPTSGGSN

W

YKVSNRFSGV

FNEKFKTRVTITV

PSRFSGSGSGT

DESTNTAYMELS

DFTFTISSLQPE

SLRSEDTAFYFC

DIATYYCFQY

ARQGLWFDSDG

SHVPWTFGQG

RGFDFWGQGSTV

TKLQIT

TVSS

EGFR
IQLTQSPSSLS
1129
QDIS
1130
DAS
1131
QQF
1132
QVQLVESGGGV
1133
GFTF
1134
IWDD
1135
ARDG
1136

ASVGDRVTIT

SA

NSYP

VQPGRSLRLSCA

STYG

GSYK

ITMV

CRASQDISSAL

LT

ASGFTFSTYGMH

RGVM

VWYQQKPGK

WVRQAPGKGLE

KDYF

APKLLIYDASS

WVAVIWDDGSY

DY

LESGVPSARFS

KYYGDSVKGRFT

GSESGTDFTLT

ISRDNSKNTLYL

ISSLQPEDFAT

QMNSLRAEDTA

YYCQQFNSYP

VYYCARDGITMV

LTFGGGTKVEI

RGVMKDYFDYW

K

GQGTLVTVSS

EGFR
DIQMTQSPSSL
1137
QDIN
1138
YTS
1139
LQY
1140
QVQLVQSGAEV
1141
GYTF
1142
IYPG
1143
ARYD
1144

SASVGDRVTIT

NY

DNLL

AKPGASVKLSCK

TSY

DGDT

APGY

CRASQDINNY

YT

ASGYTFTSYWM

W

AMDY

LAWYQHKPG

QWVKQRPGQGL

KGPKLLIHYTS

ECIGTIYPGDGDT

TLHPGIPSRFS

TYTQKFQGKATL

GSGSGRDYSF

TADKSSSTAYMQ

SISSLEPEDIAT

LSSLRSEDSAVY

YYCLQYDNLL

YCARYDAPGYA

YTFGQGTKLEI

MDYWGQGTLVT

K

VSS

EGFR
DIQMTQSPSSL
1145
SSVT
1146
DTS
1147
QQW
1148
QVQLVQSGAEV
1149
GYTF
1150
FNPS
1151
ASRD
1152

SASVGDRVTIT

Y

SSHI

KKPGASVKVSCK

TSH

NGRT

YDYD

CSASSSVIYM

FT

ASGYTFTSHWM

W

GRYF

YWYQQKPGK

HWVRQAPGQGL

DY

APKLLIYDTSN

EWIGEFNPSNGR

LASGVPSRFSG

TNYNEKFKSKAT

SGSGTDYTFTI

MTVDTSTNTAY

SSLQPEDIATY

MELSSLRSEDTA

YCQQWSSHIF

VYYCASRDYDY

TFGQGTKVEI

DGRYFDYWGQG

K

TLVTVSS

EGFR
DIQMTQSPSSL
1153
QGIN
1154
NTN
1155
LQH
1156
QVQLVQSGAEV
1157
GFTF
1158
FNPN
1159
ARLS
1160

SASVGDRVTIT

NY

NSFP

KKPGSSVKVSCK

TDY

SGYS

PGGY

CRASQGINNY

T

ASGFTFTDYKIH

K

YVMD

LNWYQQKPG

WVRQAPGQGLE

AW

KAPKRLIYNT

WMGYFNPNSGY

NNLQTGVPSR

STYAQKFQGRVT

FSGSGSGTEFT

ITADKSTSTAYM

LTISSLQPEDF

ELSSLRSEDTAV

ATYYCLQHNS

YYCARLSPGGYY

FPTFGQGTKLE

VMDAWGQGTTV

IK

TVSS

EGFR
DIQMTQSPSSL
1161
QNIA
1162
SAS
1163
QQSE
1164
EVQLVESGGGLV
1165
GFTL
1166
ISAA
1167
ARES
1168

HER3
SASVGDRVTIT

TD

PEPY

QPGGSLRLSCAA

SGD

GGYT

RVSF

CRASQNIATD

T

SGFTLSGDWIHW

W

EAAM

VAWYQQKPG

VRQAPGKGLEW

DY

KAPKLLIYSAS

VGEISAAGGYTD

FLYSGVPSRFS

YADSVKGRFTIS

GSGSGTDFTLT

ADTSKNTAYLQ

ISSLQPEDFAT

MNSLRAEDTAV

YYCQQSEPEP

YYCARESRVSFE

YTFGQGTKVE

AAMDYWGQGTL

IK

VTVSS

EGFRvII
DILMTQSPSSM
1169
QDIN
1170
HGT
1171
VQY
1172
DVQLQESGPSLV
1173
GYSI
1174
ISYS
1175
VTAG
1176

I
SVSLGDTVSIT

SN

AQFP

KPSQSLSLTCTVT

TSDF

GNT

RGFP

CHSSQDINSNI

WT

GYSITSDFAWNW

A

Y

GWLQQRPGKS

IRQFPGNKLEWM

FKGLIYHGTN

GYISYSGNTRYN

LDDEVPSRFSG

PSLKSRISITRDTS

SGSGADYSLTI

KNQFFLQLNSVTI

SSLESEDFADY

EDTATYYCVTAG

YCVQYAQFP

RGFPYWGQGTL

WTFGGGTKLE

VTVSS

IKA

EGFRvII
DIQMTQSPSS
1177
QDIN
1178
HGT
1179
VQY
1180
QVQLQESGPGLV
1181
GYSI
1182
ISYS
1183
VTAG
1184

I
MSVSVGDRVT

SN

AQFP

KPSQTLSLTCTVS

SSDF

GNT

RGFP

ITCHSSQDINS

WT

GYSISSDFAWNW

A

Y

NIGWLQQKPG

IRQPPGKGLEWM

KSFKGLIYHGT

GYISYSGNTRYQ

NLDDGVPSRF

PSLKSRITISRDTS

SGSGSGTDYT

KNQFFLKLNSVT

LTISSLQPEDF

AADTATYYCVT

ATYYCVQYA

AGRGFPYWGQG

QFPWTFGGGT

TLVTVSS

KLEIK

EpCAM
ELVMTQSPSSL
1185
QSLL
1186
WAS
1187
QND
1188
EVQLLEQSGAEL
1189
ASG
1190
GDIH
1191
FCAR
1192

TVTAGEKVTM

NSGN

YSYP

VRPGTSVKISCK

YAFT

FPGS

LRNW

SCKSSQSLLNS

QKNY

LT

ASGYAFTNYWL

N

G

DEPM

GNQKNYLTW

GWVKQRPGHGL

DY

YQQKPGQPPK

EWIGDIHFPGSGN

LLIYWASTRES

IHYNEKFKGKAT

GVPDRFTGSG

LTADKSSSTAYM

SGTDFTLTISS

QLSSLTFEDSAV

VQAEDLAVYY

YFCARLRNWDEP

CQNDYSYPLT

MDYWGQGTTVT

FGAGTKLEIK

VSS

EpCAM
ELQMTQSPSSL
1193
QSIS
1194
WAS
1195
QQS
1196
EVQLLESGGGVV
1197
GFTF
1198
ISYD
1199
AKDM
1200

SASVGDRVTIT

SY

YDIP

QPGRSLRLSCAA

SSYG

GSNK

GWGS

CRTSQSISSYL

YT

SGFTFSSYGMHW

GWRP

NWYQQKPGQ

VRQAPGKGLEW

YYYY

PPKLLIYWAST

VAVISYDGSNKY

GMDV

RESGVPDRFS

YADSVKGRFTIS

GSGSGTDFTLT

RDNSKNTLYLQ

ISSLQPEDSAT

MNSLRAEDTAV

YYCQQSYDIP

YYCAKDMGWGS

YTFGQGTKLEI

GWRPYYYYGMD

K

VWGQGTTVTVSS

EpCAM
DIVLTQSPFSN
1201
KSLL
1202
QMS
1203
AQN
1204
QVKLQQSGPELK
1205
GYTF
1206
INTY
1207
ARFA
1208

PVTLGTSASIS

HSNG

LEIP

KPGETVKISCKAS

TNY

TGES

IKGD

CRSTKSLLHSN

ITY

RT

GYTFTNYGMNW

G

Y

GITYLYWYLQ

VKQAPGKGLKW

KPGQSPQLLIY

MGWINTYTGEST

QMSNLASGVP

YADDFKGRFAFS

DRFSSSGSGTD

LETSASAAYLQIN

FTLRISRVEAE

NLKNEDTATYFC

DVGVYYCAQ

ARFAIKGDYWG

NLEIPRTFGGG

QGTTVTVSS

TKLEIK

EpCAM
NIVMTQSPKS
1209
ENVV
1210
GAS
1211
GQG
1212
QVQLQQSGAELV
1213
GYA
1214
INPG
1215
ARDG
1216

MSMSVGERVT

TY

YSYP

RPGTSVKVSCKA

FTNY

SGGT

PWFA

LTCKASENVV

YT

SGYAFTNYLIEW

L

Y

TYVSWYQQKP

VKQRPGQGLEWI

EQSPKLLIYGA

GVINPGSGGTNY

SNRYTGVPDR

NEKFKGKATLTA

FTGSGSATDFT

DKSSSTAYMQLS

LTISSVQAEDL

SLTSDDSAVYFC

ADYHCGQGYS

ARDGPWFAYWG

YPYTFGGGTK

QGTLVTVSA

LEIK

EpCAM
EIVMTQSPATL
1217
QSVS
1218
GAS
1219
QQY
1220
QVQLVQSGAEV
1221
SGGT
1222
GIIP
1223
CARG
1224

SVSPGERATLS

SN

NNW

KKPGSSVKVSCIC

FSSY

IFGT

LLWN

CRASQSVSSN

PPAY

ASGGTFSSYAIS

Y

LAWYQQKPG

T

WVRQAPGQGLE

QAPRLITYGAS

WMGGIIPIFGTAN

TTASGIPARFS

YAQKFQGRVTIT

ASGSGTDFTLT

ADESTSTAYMEL

ISSLQSEDFAV

SSLRSEDTAVYY

YYCQQYNNW

CARGLLWNYWG

PPAYTFGQGT

QGTLVTVSS

KLEIK

EphA3
DIQMTQSPSFL
1225
QGII
1226
AAS
1227
GQY
1228
QVQLVQSGAEV
1229
GYTF
1230
IYPG
1231
ARGG
1232

SASVGDRVTIT

SY

ANY

KKPGASVKVSCK

TGY

SGNT

YYED

CRASQGIISYL

PYT

ASGYTFTGYWM

W

FDS

AWYQQKPEK

NWVRQAPGQGL

APKRLIYAASS

EWMGDIYPGSGN

LQSGVPSRFSG

TNYDEKFQGRVT

SGSGTEFTLTI

MTRDTSISTAYM

SSLQPEDFATY

ELSRLRSDDTAV

YCGQYANYPY

YYCARGGYYED

TFGQGTKLEIK

FDSWGQGTTVTV

SS

ERGT(G
DIQLTQTPLSL
1233
QSLV
1234
KVS
1235
SQST
1236
QVQLQQSGGGL
1237
GFTF
1238
IRNK
1239
SGGK
1240

alNAc)
PVSLGDQASIS

HSNG

HVPT

VQPGGSMKIFCA

SDA

ANNH

VRNA

Tn
CRSSQSLVHS

NTY

ASGFTFSDAWM

W

ET

Y

Antigen
NGNTYLHWY

DWVRQSPEKGLE

LQKPGQSPKL

WVAEIRNKANN

LIYKVSNRFSG

HETYYAESVKGR

VPDRFSGSGS

FTITRDDSKSRMS

GTDFTLKISSV

LQMNSLRAEDTG

EAEDLGVYFC

IYYCSGGKVRNA

SQSTHVPTFG

YWGQGTTVTVSS

GGTKLEIK

FLT1
EIVLTQSPGTL
1241
QSVS
1242
GAS
1243
QQY
1244
QAQVVESGGGV
1245
GFAF
1246
IWYD
1247
ARDH
1248

SLSPGERATLS

SSY

GSSP

VQSGRSLRLSCA

SSYG

GSNK

YGSG

CRASQSVSSSY

LT

ASGFAFSSYGMH

VHHY

LAWYQQKPG

WVRQAPGKGLE

FYYG

QAPRLLIYGAS

WVAVIWYDGSN

LDV

SRATGIPDRFS

KYYADSVRGRFT

GSGSGTDFTLT

ISRDNSENTLYLQ

ISRLEPEDFAV

MNSLRAEDTAV

YYCQQYGSSP

YYCARDHYGSG

LTFGGGTKVEI

VHHYFYYGLDV

K

WGQGTTVTVSS

FOLR1
DIQLTQSPSSL
1249
SSIS
1250
GTS
1251
QQW
1252
EVQLVESGGGVV
1253
GFTF
1254
ISSG
1255
ARHG
1256

SASVGDRVTIT

SNN

SSYP

QPGRSLRLSCSAS

SGY

GSYT

DDPA

CSVSSSISSNN

YMY

GFTFSGYGLSWV

G

WFAY

LHWYQQKPG

T

RQAPGKGLEWV

W

KAPKPWIYGT

AMISSGGSYTYY

SNLASGVPSRF

ADSVKGRFAISR

SGSGSGTDYT

DNAKNTLFLQM

FTISSLQPEDIA

DSLRPEDTGVYF

TYYCQQWSSY

CARHGDDPAWF

PYMYTFGQGT

AYWGQGTPVTV

KVEIK

SS

FOLR1
DIVLTQSPLSL
1257
QSVS
1258
RAS
1259
QQSR
1260
QVQLVQSGAEV
1261
GYTF
1262
IHPY
1263
TRYD
1264

AVSLGQPAIIS

FAGT

EYPY

VKPGASVKISCK

TGYF

DGDT

GSRA

CKASQSVSFA

SL

T

ASGYTFTGYFMN

MDY

GTSLMHWYH

WVKQSPGQSLE

QKPGQQPRLLI

WIGRIHPYDGDT

YRASNLEAGV

FYNQKFQGKATL

PDRFSGSGSKT

TVDKSSNTAHME

DFTLTISPVEA

LLSLTSEDFAVY

EDAATYYCQQ

YCTRYDGSRAM

SREYPYTFGG

DYWGQGTTVTV

GTKLEIK

SS

frizzled
DIELTQPPSVS
1265
NIGS
1266
DKS
1267
QSY
1268
EVQLVESGGGLV
1269
GFTF
1270
ISGD
1271
ARNF
1272

family
VAPGQTARISC

FY

ANTL

QPGGSLRLSCAA

SHYT

GSYT

IKYV

receptor
SGDNIGSFYV

SLV

SGFTFSHYTLSW

FAN

(FZD)
HWYQQKPGQ

VRQAPGKGLEW

APVLVIYDKS

VSVISGDGSYTY

NRPSGIPERFS

YADSVKGRFTISS

GSNSGNTATL

DNSKNTLYLQM

TISGTQAEDEA

NSLRAEDTAVYY

DYYCQSYANT

CARNFIKYVFAN

LSLVFGGGTK

WGQGTLVTVSS

LTVLG

Lewis Y
DVLMTQIPVS
1273
QIIV
1274
KVS
1275
FQGS
1276
EVNLVESGGGLV
1277
GFTF
1278
ISQG
1279
ARGL
1280

LPVSLGDQASI

HNNG

HVPF

QPGGSLKVSCVT

SDY

GDIT

DDGA

SCRSSQIIVHN

NTY

T

SGFTFSDYYMY

Y

WFAY

NGNTYLEWYL

WVRQTPEKRLE

QKPGQSPQLLI

WVAYISQGGDIT

YKVSNRFSGV

DYPDTVKGRFTIS

PDRFSGSGSGT

RDNAKNSLYLQ

DFTLKISRVEA

MSRLKSEDTAM

EDLGVYYCFQ

YYCARGLDDGA

GSHVPFTFGSG

WFAYWGQGTLV

TKLEIK

TVSV

Lewis Y
DIQMTQSPSSL
1281
QRIV
1282
KVS
1283
FQGS
1284
EVQLVESGGGVV
1285
GFTF
1286
MSNV
1287
ARGT
1288

SASVGDRVTIT

HSNG

HVPF

QPGRSLRLSCSTS

SDY

GAIT

RDGS

CRSSQRIVHSN

NTY

T

GFTFSDYYMYW

Y

WFAY

GNTYLEWYQ

VRQAPGKGLEW

QTPGKAPKLLI

VAYMSNVGAITD

YKVSNRFSGV

YPDTVKGRFTISR

PSRFSGSGSGT

DNSKNTLFLQMD

DFTFTISSLQPE

SLRPEDTGVYFC

DIATYYCFQG

ARGTRDGSWFA

SHVPFTFGQG

YWGQGTPVTVSS

TKLQIT

Lewis X
DIVMTQAAFS
1289
KSLL
1290
QMS
1291
AQN
1292
EVKLLESGGGLV
1293
SGFD
1294
EINP
1295
CARE
1296

NPVTLGTSASI

YSNG

LEVP

QPGGSQKLSCAA

FSGY

DSST

TGTR

SCRSSKSLLYS

ITY

WT

SGFDFSGYWMS

FDY

NGITYLYWYL

WVRQAPGKGLE

QKPGQSPQLLI

WIGEINPDSSTIN

YQMSNLASGV

YTPSLKDKFIISR

PDRFSSSGSGT

DNAKNTLYLQM

DFTLRISRVEA

SKVRSEDTALYY

EDVGVYYCA

CARETGTRFDYW

QNLEVPWTFG

GQGTTLTVSS

GGTKLEIK

GCGR
DIQMTQSPSSL
1297
QGIR
1298
AAS
1299
LQY
1300
EVQLVESGGGLV
1301
GFTF
1302
IQED
1303
AREP
1304

SASVGDRVTIT

ND

NSNP

QPGGSLRLSCAA

SNYL

GIEK

SHYD

CRASQGIRND

FT

SGFTFSNYLMNW

ILTG

LGWYQQKPG

VRQAPGKGLEW

YDYY

KAPKRLIYAA

LANIQEDGIEKY

YGMD

SSLQSGVPSRF

YVDSVKGRFTIS

V

SGSGSGTEFIL

RDNAKNSLYLQ

TVSSLQPEDFA

MNSLRAEDTAV

TYYCLQYNSN

YYCAREPSHYDI

PFTFGPGTKV

LTGYDYYYGMD

DIK

VWGQGTTVTVSS

GD2
EIVMTQSPATL
1305
QSLV
1306
KVS
1307
SQST
1308
EVQLLQSGPELE
1309
GSSF
1310
IDPY
1311
VSGM
1312

SVSPGERATLS

HRNG

HVPP

KPGASVMISCKA

TGY

YGGT

EY

CRSSQSLVHR

NTY

LT

SGSSFTGYNMN

N

NGNTYLHWY

WVRQNIGKSLE

LQKPGQSPKL

WIGAIDPYYGGT

LIHKVSNRFSG

SYNQKFKGRATL

VPDRFSGSGS

TVDKSSSTAYMH

GTDFTLKISRV

LKSLTSEDSAVY

EAEDLGVYFC

YCVSGMEYWGQ

SQSTHVPPLTF

GTSVTVSS

GAGTKLELK

GD2
SIVMTQTPKFL
1313
QSVS
1314
SAS
1315
QQD
1316
QVQLKESGPGLV
1317
GFSV
1318
IWAG
1319
ASRG
1320

LVSAGDRVTIT

ND

YSS

APSQSLSITCTVS

TNY

GIT

GHYG

CKASQSVSND

GFSVTNYGVHW

G

YALD

VTWYQQKAG

VRQPPGKGLEWL

Y

QSPKLLIYSAS

GVIWAGGITNYN

NRYSGVPDRF

SAFMSRLSISKDN

TGSGYGTAFT

SKSQVFLKMNSL

FTISTVQAEDL

QIDDTAMYYCAS

AVYFCQQDYS

RGGHYGYALDY

SFGGGTKLEIK

WGQGTSVTVSS

GD2
EIVMTQTPATL
1321
QSVS
1322
SAS
1323
QQD
1324
QVQLVESGPGVV
1325
GFSV
1326
IWAG
1327
ASRG
1328

SVSAGERVTIT

ND

YSS

QPGRSLRISCAVS

TNY

GIT

GHYG

CKASQSVSND

GFSVTNYGVHW

G

YALD

VTWYQQKPG

VRQPPGKGLEWL

Y

QAPRLLIYSAS

GVIWAGGITNYN

NRYSGVPARF

SAFMSRLTISKDN

SGSGYGTEFTF

SKNTVYLQMNSL

TISSVQSEDFA

RAEDTAMYYCA

VYFCQQDYSS

SRGGHYGYALD

FGQGTKLEIK

YWGQGTLVTVSS

GD2 o-
DVVMTQTPLS
1329
QSLL
1330
KVS
1331
SQST
1332
EVKLVESGGGLV
1333
KFTF
1334
IRNR
1335
ARVS
1336

acetyl
LPVSLGDQASI

KNNG

HIPY

LPGDSLRLSCATS

TDY

ANGY

NWAF

SCRSSQSLLKN

NTF

T

KFTFTDYYMTW

Y

TT

DY

NGNTFLHWYL

VRQPPRKALEQL

QKSGQSPKLLI

GFIRNRANGYTT

YKVSNRLSGV

EYNPSVKGRFTIS

PDRFSGSGSGT

RDNSQSILYLQM

YFTLKISRVEA

NTLRTEDSATYY

EDLGVYFCSQ

CARVSNWAFDY

STHIPYTFGGG

WGQGTTLTVSS

TKLELK

GD3
DIQMTQITSSL
1337
GFTF
1338
ISSG
1339
TRG
1340
DVQLVESGGGLV
1341
QDIG
1342
YTS
1343
QQGK
1344

SVSLGDRVIIS

SNFG

GSSI

GTGT

QPGGSRKLSCAA

NF

TLP

CRASQDIGNFL

RSLY

SGFTFSNFGMHW

NWYQQKPDG

YFD

VRQAPEKGLEW

SLKLLIYYTSR

Y

VAYISSGGSSINY

LQSGVPSRFSG

ADTVKGRFTISR

WGSGTDYSLT

DNPKNTLFLQMT

ISNLEEEDIATF

SLRSEDTAIYYCT

FCQQGKTLPY

RGGTGTRSLYYF

TFGGGTKLEIK

DYWGQGATLIVS

S

GD3
DIQMTQTASS
1345
QDIS
1346
YSS
1347
HQY
1348
EVTLVESGGDFV
1349
GFAF
1350
ISSG
1351
TRVK
1352

LPASLGDRVTI

NY

SKLP

KPGGSLKVSCAA

SHY

GSGT

LGTY

SCSASQDISNY

WT

SGFAFSHYAMSW

A

YFDS

LNWYQQKPD

VRQTPAKRLEW

GTVKLLIFYSS

VAYISSGGSGTY

NLHSGVPSRFS

YSDSVKGRFTISR

GGGSGTDYSL

DNAKNTLYLQM

TISNLEPEDIAT

RSLRSEDSAMYF

YFCHQYSKLP

CTRVKLGTYYFD

WTFGGGTKLE

SWGQGTTLTVSS

IK

GM1
DIQMTQSPSSL
1353
QGIS
1354
AAS
1355
QQY
1356
EVQLVESGGGLV
1357
GFTF
1358
ISRS
1359
AGTV
1360

SASVGDRVTIT

SW

NSYP

QPGESLRLSCVA

SRY

GRDI

TTYY

CRASQGISSW

PT

SGFTFSRYKMNW

K

YYFG

LAWYQQKPE

VRQAPGKGLEW

MDV

KAPKSLIYAAS

VSYISRSGRDIYY

SLQSGVPSRFS

ADSVKGRFTISR

GSGSGTDFTLT

DNAKNSLYLQM

ISSLQPEDFAT

NSLRDEDTAVYY

YYCQQYNSYP

CAGTVTTYYYYF

PTFGGGTKVEI

GMDVWGHGTTV

K

TVSS

GM1
DIQMTQSPSSL
1361
QGIS
1362
AAS
1363
QQY
1364
EVQLVESGGGLY
1365
GFTF
1366
YISR
1367
CAGT
1368

fucosyl
SASVGDRVTIT

W

NSYP

QPGESLRLSCVA

SRYK

SGRD

VTTY

CRASQGISWL

PT

SGFTFSRYKMNW

YYYF

AWYQQKPEK

VRQAPGKGLLE

GMDV

APKSLIYAASS

WVSYISRSGRDIY

LQSGVPSRFSG

YADSVKGRFTIS

SGSGTDFTLTI

RDNAKNSLYLQ

SSLQPEDFATY

MNSLRDEDTAV

YCQQYNSYPP

YYCAGTVTTYY

TFGGGTKVEI

YYFGMDVWGHG

K

TTVTVSS

GM1
DIQMTQSPSSL
1369
QGIS
1370
AAS
1371
QQY
1372
EVQLVESGGGLV
1373
GFTF
1374
ISRS
1375
AGTV
1376

fucosyl
ASVGDRVTIT

SW

NSYP

QPGESLRLSCVV

SRY

GRDI

TTYY

CRASQGISSW

PT

SGFTFSRYKMNW

K

YYFG

LAWYQQKPE

VRQAPGKGLEWI

MDVW

KAPKSLIYAAS

SYISRSGRDIYYA

G

SLQSGVPSRFS

DSVKGRFTISRD

GSGSGTDFTLT

NAKNSLYLQMSS

ISCLQPEDFAT

LRDEDTAVYYCA

YYCQQYNSYP

GTVTTYYYYFG

PTFGGGTKVEI

MDVWGLGITVT

K

VSS

GM1
DIQMTQSPSSL
1377
QGIS
1378
AA
1379
QQY
1380
EVQLVESGGGSV
1381
GFTF
1382
ISRS
1383
AGTV
1384

fucosyl
SASVGDRVTIT

SW

NSYP

QPGESLRLSCVA

SRY

GRDI

TTYY

CRASQGISSW

PT

SGFTFSRYKMNW

K

YDFG

LAWYQQKPE

VRQAPGKGLEW

MDV

KAPKSLIYAAS

VSYISRSGRDIYY

LQSGVPSRFSG

ADSVKGRFTISR

SGSGTDFTLTI

DNAKNSLYLQM

SSLQPEDFATY

NSLRDEDTAVYY

YCQQYNSYPP

CAGTVTTYYYDF

TFGGGTKVEI

GMDVWGQGTTV

K

TVSS

GM2
QIVLTQSPAIM
1385
SSVS
1386
STS
1387
QQRS
1388
EVQLQQSGPELV
1389
GYTF
1390
IYPN
1391
ATYG
1392

SASPGEKVTIT

Y

SYPY

KPGASVKISCKA

TDY

NGGT

HYYG

CSASSSVSYM

T

SGYTFTDYNMD

N

YMFA

HWFQQKPGTS

WVKQSHGKSLE

Y

PKLWIYSTSNL

WIGYIYPNNGGT

ASGVPARFSG

GYNQKFKSKATL

SGSGTSYSLTI

TVDKSSSTAYME

SRMEAEDAAT

LHSLTSEDSAVY

YYCQQRSSYP

YCATYGHYYGY

YTFGGGTKLEI

MFAYWGQGTLV

K

TVSA

GPA33
DIVMTQSQKF
1393
QNVR
1394
LAS
1395
LQH
1396
EVKLVESGGGLV
1397
GFAF
1398
ISSG
1399
APTT
1400

MSTSVGDRVS

TV

WSY

KPGGSLKLSCAA

STYD

GSYT

VVPF

ITCKASQNVR

PLT

SGFAFSTYDMSW

AY

TVVAWYQQK

VRQTPEKRLEWV

PGQSPKTLIYL

ATISSGGSYTYYL

ASNRHTGVPD

DSVKGRFTISRDS

RFTGSGSGTDF

ARNTLYLQMSSL

TLTISNVQSED

RSEDTALYYCAP

LADYFCLQHW

TTVVPFAYWGQ

SYPLTFGSGTK

GTLVTVSA

LEVK

GPNMB
EIVMTQSPATL
1401
QSVD
1402
GAS
1403
QQY
1404
QVQLQESGPGLV
1405
GGSI
1406
IYYS
1407
ARGY
1408

SVSPGERATLS

NN

NNW

KPSQTLSLTCTVS

SSFN

GST

NWNY

CRASQSVDNN

PPWT

GGSISSFNYYWS

YY

FDY

LVWYQQKPG

WIRHHPGKGLE

QAPRLLIYGAS

WIGYIYYSGSTYS

TRATGIPARFS

NPSLKSRVTISVD

GSGSGTEFTLT

TSKNQFSLTLSSV

ISSLQSEDFAV

TAADTAVYYCA

YYCQQYNNW

RGYNWNYFDYW

PPWTFGQGTK

GQGTLVTVSS

VEIK

GUCY2
EIVMTQSPATL
1409
QSVS
1410
GAS
1411
QQY
1412
QVQLQQWGAGL
1413
GGSF
1414
INHR
1415
ARER
1416

C
SVSPGERATLS

RN

KTW

LKPSETLSLTCAV

SGY

GNT

GYTY

CRASQSVSRN

PRT

FGGSFSGYYWS

Y

GNFD

LAWYQQKPG

WIRQPPGKGLEW

H

QAPRLLIYGAS

IGEINHRGNTND

TRATGIPARFS

NPSLKSRVTISVD

GSGSGTEFTLT

TSKNQFALKLSS

IGSLQSEDFAV

VTAADTAVYYC

YYCQQYKTW

ARERGYTYGNFD

PRTFGQGTNV

HWGQGTLVTVSS

EIK

HER2
DIQMTQSPSSL
1417
QDVN
1418
SAS
1419
QQA
1420
EVQLVESGGGLV
1421
GFNI
1422
IYPT
1423
SRWG
1424

SASVGDRVTIT

TA

YTTP

QPGGSLRLSCAA

KDT

NGYT

GDGF

CRASQDVNTA

PT

SGFNIKDTYIHW

Y

YAMD

VAWYQQKPG

VRQAPGKGLEW

Y

KAPKLLIYSAS

VARIYPTNGYTR

FLYSGVPSRFS

YADSVKGRFTIS

GSRSGTDFTLT

ADTSKNTAYLQ

ISSLQPEDFAT

MNSLRAEDTAV

YYCQQAYTTP

YYCSRWGGDGF

PTFGQGTKVEI

YAMDYWGQGTL

K

VTVSS

HER2
DIQMTQSPSSL
1425
QDVN
1426
SAS
1427
QQH
1428
EVQLVESGGGLV
1429
GFNI
1430
IYPT
1431
SRWG
1432

SASVGDRVTIT

TA

YTTP

QPGGSLRLSCAA
KDT

NGYT

GDGF

CRASQDVNTA

PT

SGFNIKDTYIHW

Y

YAMD

VAWYQQKPG

VRQAPGKGLEW

Y

KAPKLLIYSAS

VARIYPTNGYTR

FLYSGVPSRFS

YADSVKGRFTIS

GSRSGTDFTLT

ADTSKNTAYLQ

ISSLQPEDFAT

MNSLRAEDTAV

YYCQQHYTTP

YYCSRWGGDGF

PTFGQGTKVEI

YAMDYWGQGTL

K

VTVSS

HER2
DIQMTQSPSSL
1433
QDVS
1434
SAS
1435
QQY
1436
EVQLVESGGGLV
1437
GFTF
1438
VNPN
1439
ARNL
1440

SASVGDRVTIT

IG

YIYP

QPGGSLRLSCAA

TDY

SGGS

GPSF

CKASQDVSIG

YT

SGFTFTDYTMDW

T

YFDY

VAWYQQKPG

VRQAPGKGLEW

W

KAPKLLIYSAS

VADVNPNSGGSI

YRYTGVPSRF

YNQRFKGRFTLS

SGSGSGTDFTL

VDRSKNTLYLQ

TISSLQPEDFA

MNSLRAEDTAV

TYYCQQYYIY

YYCARNLGPSFY

PYTFGQGTKV

FDYWGQGTLVT

EIK

VSS

HER2
QSVLTQPPSVS
1441
SSNI
1442
GNT
1443
QSY
1444
QVQLVESGGGLV
1445
GFTF
1446
ISGR
1447
AKMT
1448

GAPGQRVTISC

GAGY

DSSL

QPGGSLRLSCAA

RSY

GDNT

SNAF

TGSSSNIGAGY

G

SGW

SGFTFRSYAMSW

A

AFDY

GVHWYQQLP

V

VRQAPGKGLEW

GTAPKLLIYG

VSAISGRGDNTY

NTNRPSGVPD

YADSVKGRFTIS

RFSGFKSGTSA

RDNSKNTLYLQ

SLAITGLQAED

MNSLRAEDTAV

EADYYCQSYD

YYCAKMTSNAF

SSLSGWVFGG

AFDYWGQGTLV

GTKLTVL

TVSS

HER3
QSALTQPASV
1449
SSDV
1450
EVS
1451
CSYA
1452
EVQLLESGGGLV
1453
GFTF
1454
ISSS
1455
TRGL
1456

SGSPGQSITISC

GSYN

GSSI

QPGGSLRLSCAA

SHY

GGWT

KMAT

TGTSSDVGSY

V

FVI

SGFTFSHYVMA

V

IFDY

NVVSWYQQH

WVRQAPGKGLE

PGKAPKLIIYE

WVSSISSSGGWT

VSQRPSGVSN

LYADSVKGRFTIS

RFSGSKSGNT

RDNSKNTLYLQ

ASLTISGLQTE

MNSLRAEDTAV

DEADYYCCSY

YYCTRGLKMATI

AGSSIFVIFGG

FDYWGQGTLVT

GTKVTVL

VSS

HER3
DIQMTQSPSSL
1457
QGIS
1458
GAS
1459
QQY
1460
EVQLLESGGGLV
1461
GFTF
1462
INSQ
1463
ARWG
1464

SASVGDRVTIT

NW

SSFP

QPGGSLRLSCAA

SSYA

GKST

DEGF

CRASQGISNW

TT

SGFTFSSYAMSW

DI

LAWYQQKPG

VRQAPGKGLEW

KAPKLLIYGAS

VSAINSQGKSTY

SLQSGVPSRFS

YADSVKGRFTIS

GSGSGTDFTLT

RDNSKNTLYLQ

ISSLQPEDFAT

MNSLRAEDTAV

YYCQQYSSFP

YYCARWGDEGF

TTFGQGTKVEI

DIWGQGTLVTVS

K

S

HER3
DIEMTQSPDSL
1465
QSVL
1466
WAS
1467
QQY
1468
QVQLQQWGAGL
1469
GGSF
1470
INHS
1471
ARDK
1472

AVSLGERATIN

YSSS

YSTP

LKPSETLSLTCAV

SGY

GST

WTWY

CRSSQSVLYSS

NRNY

RT

YGGSFSGYYWS

Y

FDL

SNRNYLAWY

WIRQPPGKGLEW

QQNPGQPPKL

IGEINHSGSTNYN

LIYWASTRES

PSLKSRVTISVET

GVPDRFSGSG

SKNQFSLKLSSVT

SGTDFTLTISS

AADTAVYYCAR

LQAEDVAVYY

DKWTWYFDLWG

CQQYYSTPRT

RGTLVTVSS

FGQGTKVEIK

HER3
DIVMTQSPDSL
1473
QSVL
1474
WAS
1475
QSD
1476
QVQLVQSGAEV
1477
GYTF
1478
IYAG
1479
ARHR
1480

AVSLGERATIN

NSGN

YSYP

KKPGASVKVSCK

RSSY

TGSP

DYYS

CKSSQSVLNS

QKNY

YT

ASGYTFRSSYISW

NSLT

GNQKNYLTW

VRQAPGQGLEW

Y

YQQKPGQPPK

MGWIYAGTGSPS

LLIYWASTRES

YNQKLQGRVTM

GVPDRFSGSG

TTDTSTSTAYME

SGTDFTLTISS

LRSLRSDDTAVY

LQAEDVAVYY

YCARHRDYYSNS

CQSDYSYPYT

LTYWGQGTLVT

FGQGTKLEIK

VSS

HER3
YELTQDPAVS
1481
SLRS
1482
GKN
1483
NSRD
1484
QVQLVQSGGGL
1485
GFTF
1486
ISWD
1487
ARDL
1488

VALGQTVRIT

YY

SPGN

VQPGGSLRLSCA

DDY

SGST

GAYQ

CQGDSLRSYY

QWV

ASGFTFDDYAMH

A

WVEG

ASWYQQKPG

WVRQAPGKGLE

FDY

QAPVLVIYGK

WVAGISWDSGST

NNRPSGIPDRF

GYADSVKGRFTI

SGSTSGNSASL

SRDNAKNSLYLQ

TITGAQAEDE

MNSLRAEDTALY

ADYYCNSRDS

YCARDLGAYQW

PGNQWVFGG

VEGFDYWGQGT

GTKVTVL

LVTVSS

HGFR
DIVMTQSPDSL
1489
ESVD
1490
RAS
1491
QQS
1492
QVQLVQSGAEV
1493
GYIF
1494
IKPN
1495
ARSE
1496

(cMET)
AVSLGERATIN

SYAN

KEDP

KKPGASVKVSCK

TAY

NGLA

ITTE

CKSSESVDSY

SF

LT

ASGYIFTAYTMH

T

FDY

ANSFLHWYQQ

WVRQAPGQGLE

KPGQPPKLLIY

WMGWIKPNNGL

RASTRESGVP

ANYAQKFQGRV

DRFSGSGSGT

TMTRDTSISTAY

DFTLTISSLQA

MELSRLRSDDTA

EDVAVYYCQ

VYYCARSEITTEF

QSKEDPLTFG

DYWGQGTLVTV

GGTKVEIK

SS

HGFR
DIQMTQSPSSL
1497
SSVS
1498
STS
1499
QVY
1500
QVQLVQSGAEV
1501
GYTF
1502
VNPN
1503
ARAN
1504

(cMET)
SASVGDRVTIT

SIY

SGYP

KKPGASVKVSCK

TDY

RRGT

WLDY

CSVSSSVSSIY

LT

ASGYTFTDYYM

Y

LHWYQQKPG

HWVRQAPGQGL

KAPKLLIYSTS

EWMGRVNPNRR

NLASGVPSRFS

GTTYNQKFEGRV

GSGSGTDFTLT

TMTTDTSTSTAY

ISSLQPEDFAT

MELRSLRSDDTA

YYCQVYSGYP

VYYCARANWLD

LTFGGGTKVEI

YWGQGTTVTVSS

K

IgHe
DIQLTQSPSSL
1505
QSVD
1506
AAS
1507
QQS
1508
EVQLVESGGGLV
1509
GYSI
1510
ITYD
1511
ARGS
1512

SASVGDRVTIT

YDGD

HEDP

QPGGSLRLSCAV

TSGY

GST

HYFG

CRASQSVDYD

SY

YT

SGYSITSGYSWN

S

HWHF

GDSYMNWYQ

WIRQAPGKGLE

AV

QKPGKAPKLLI

WVASITYDGSTN

YAASYLESGV

YNPSVKGRITISR

PSRFSGSGSGT

DDSKNTFYLQM

DFTLTISSLQP

NSLRAEDTAVYY

EDFATYYCQQ

CARGSHYFGHW

SHEDPYTFGQ

HFAVWGQGTLV

GTKVEIK

TVSS

IgHe
EIVMTQSPATL
1513
QSIG
1514
YAS
1515
QQS
1516
QVQLVQSGAEV
1517
GYTF
1518
IDPG
1519
ARFS
1520

SVSPGERATLS

TN

WSW

MKPGSSVKVSCK

SWY

TFTT

HFSG

CRASQSIGTNI

PTT

ASGYTFSWYWL

W

SNYD

HWYQQKPGQ

EWVRQAPGHGL

YFDY

APRLLIYYASE

EWMGEIDPGTFT

W

SISGIPARFSGS

TNYNEKFKARVT

GSGTEFTLTIS

FTADTSTSTAYM

SLQSEDFAVY

ELSSLRSEDTAV

YCQQSWSWPT

YYCARFSHFSGS

TFGGGTKVEI

NYDYFDYWGQG

K

TLVTVSS

IGLF2
QSVLTQPPSVS
1521
SSNI
1522
DNN
1523
ETW
1524
QVQLVQSGAEV
1525
GYTF
1526
MNPN
1527
ARDP
1528

AAPGQKVTIS

ENNH

DTSL

KKPGASVKVSCK

TSYD

SGNT

YYYY

CSGSSSNIENN

SAGR

ASGYTFTSYDIN

YGMD

HVSWYQQLPG

V

WVRQATGQGLE

V

TAPKLLIYDN

WMGWMNPNSG

NKRPSGIPDRF

NTGYAQKFQGR

SGSKSGTSATL

VTMTRNTSISTA

GITGLQTGDE

YMELSSLRSEDT

ADYYCETWD

AVYYCARDPYY

TSLSAGRVFG

YYYGMDVWGQ

GGTKLTVL

GTTVTVSS

Kalli-
DIQMTQSPSTL
1529
QSIS
1530
KAS
1531
QQY
1532
EVQLLESGGGLV
1533
GFTF
1534
IYSS
1535
AYRR
1536

kreins
SASVGDRVTIT

SW

NTY

QPGGSLRLSCAA

SHYI

GGIT

IGVP

CRASQSISSWL

WT

SGFTFSHYIMMW

RRDE

AWYQQKPGK

VRQAPGKGLEW

FDI

APKLLIYKAST

VSGIYSSGGITVY

LESGVPSRFSG

ADSVKGRFTISR

SGSGTEFTLTI

DNSKNTLYLQM

SSLQPDDFAT

NSLRAEDTAVYY

YYCQQYNTY

CAYRRIGVPRRD

WTFGQGTKVE

EFDIWGQGTMVT

IK

VSS

KIRDL1/
EIVLTQSPVTL
1537
QSVS
1538
DAS
1539
QQRS
1540
QVQLVQSGAEV
1541
GGTF
1542
FIPI
1543
ARIP
1544

2/3
SLSPGERATLS

SY

NWM

KKPGSSVKVSCK

SFYA

FGAA

SGSY

CRASQSVSSY

YT

ASGGTFSFYAIS

YYDY

LAWYQQKPG

WVRQAPGQGLE

DMDV

QAPRLLIYDAS

WMGGFIPIFGAA

NRATGIPARFS

NYAQKFQGRVTI

GSGSGTDFTLT

TADESTSTAYME

ISSLEPEDFAV

LSSLRSDDTAVY

YYCQQRSNW

YCARIPSGSYYY

MYTFGQGTKL

DYDMDVWGQGT

EIK

TVTVSS

LINGO1
DIQMTQSPAT
1545
QSVS
1546
DAS
1547
QQRS
1548
EVQLLESGGGLV
1549
GFTF
1550
IGPS
1551
ATEG
1552

LSLSPGERATL

SY

NWP

QPGGSLRLSCAA

SAYE

GGFT

DNDA

SCRASQSVSSY

MYT

SGFTFSAYEMKW

FDI

LAWYQQKPG

VRQAPGKGLEW

QAPRLLIYDAS

VSVIGPSGGFTFY

NRATGIPARFS

ADSVKGRFTISR

GSGSGTDFTLT

DNSKNTLYLQM

ISSLEPEDFAV

NSLRAEDTAVYY

YYCQQRSNWP

CATEGDNDAFDI

MYTFGQGTKL

WGQGTTVTVSS

EIK

LOXL2
DIVMTQTPLSL
1553
KSLL
1554
RMS
1555
MQH
1556
QVQLVQSGAEV
1557
GYA
1558
INPG
1559
ARNW
1560

SVTPGQPASIS

HSNG

LEYP

KKPGASVKVSCK

FTYY

SGGT

MNFD

CRSSKSLLHSN

NTY

YT

ASGYAFTYYLIE

L

Y

GNTYLYWFLQ

WVRQAPGQGLE

KPGQSPQFLIY

WIGVINPGSGGT

RMSNLASGVP

NYNEKFKGRATI

DRFSGSGSGT

TADKSTSTAYME

DFTLKISRVEA

LSSLRSEDTAVYF

EDVGVYYCM

CARNWMNFDY

QHLEYPYTFG

WGQGTTVTVSS

GGTKVEIK

Ly6/PLA
ESVLTQPPSVS
1561
SSNI
1562
DNN
1563
AAW
1564
EVQLLESGGGLV
1565
GFTF
1566
ISSS
1567
AREG
1568

UR
GAPGQRVTISC

GAGY

DDR

QPGGSLRLSCAA

SNA

GSTI

LWAF

domain-
TGSSSNIGAGY

V

LNGP

SGFTFSNAWMS

W

DY

con-
VVHWYQQLP

V

WVRQAPGKGLE

taining
GTAPKLLIYD

WVSYISSSGSTIY

protein
NNKRPSGVPD

YADSVKGRFTIS

3
RFSGSKSGTSA

RDNSKNTLYLQ

SLAISGLRSED

MNSLRAEDTAV

EADYYCAAW

YYCAREGLWAF

DDRLNGPVFG

DYWGQGTLVTV

GGTKLTVL

SS

MADCA
DIVMTQTPLSL
1569
QSLL
1570
EVS
1571
MQNI
1572
QVQLVQSGAEV
1573
GYTF
1574
ISVY
1575
AREG
1576

M1
SVTPGQPASIS

HTDG

QLP

KKPGASVKVSCK

TSYG

SGNT

SSSS

CKSSQSLLHT

TTY

WT

ASGYTFTSYGIN

GDYY

DGTTYLYWYL

WVRQAPGQGLE

YGMD

QKPGQPPQLLI

WMGWISVYSGN

V

YEVSNRFSGV

TNYAQKVQGRV

PDRFSGSGSGT

TMTADTSTSTAY

DFTLKISRVEA

MDLRSLRSDDTA

EDVGIYYCMQ

VYYCAREGSSSS

NIQLPWTFGQ

GDYYYGMDVW

GTKVEIK

GQGTTVTVSS

MAG
DIVMTQSPDSL
1577
HSVL
1578
WAS
1579
HQY
1580
QVQLVQSGSELK
1581
GYTF
1582
INTY
1583
ARNP
1584

AVSLGERATIN

YSSN

LSSL

KPGASVKVSCKA

TNY

TGEP

INYY

CKSSHSVLYSS

QKNY

T

SGYTFTNYGMN

G

GINY

NQKNYLAWY

WVRQAPGQGLE

EGYV

QQKPGQPPKL

WMGWINTYTGE

MDY

LIYWASTRES

PTYADDFTGRFV

GVPDRFSGSG

FSLDTSVSTAYL

SGTDFTLTISS

QISSLKAEDTAV

LQAEDVAVYY

YYCARNPINYYG

CHQYLSSLTF

INYEGYVMDYW

GQGTKLEIK

GQGTLVTVSS

Meso-
DIALTQPASVS
1585
SSDI
1586
GVN
1587
SSYD
1588
QVELVQSGAEVK
1589
GYSF
1590
IDPG
1591
ARGQ
1592

thelin
GSPGQSITISCT

GGYN

IESA

KPGESLKISCKGS

TSY

DSRT

LYGG

GTSSDIGGYNS

S

TPV

GYSFTSYWIGWV

W

TYMD

VSWYQQHPG

RQAPGKGLEWM

G

KAPKLMIYGV

GIIDPGDSRTRYS

NNRPSGVSNR

PSFQGQVTISADK

FSGSKSGNTAS

SISTAYLQWSSLK

LTISGLQAEDE

ASDTAMYYCAR

ADYYCSSYDI

GQLYGGTYMDG

ESATPVFGGG

WGQGTLVTVSS

TKLTVL

Meso-
DIELTQSPAIM
1593
SSVS
1594
DTS
1595
QQW
1596
QVQLQQSGPELE
1597
GYSF
1598
ITPY
1599
ARGG
1600

thelin
SASPGEKVTM

Y

SKHP

KPGASVKISCKA

TGY

NGAS

YDGR

TCSASSSVSY

LT

SGYSFTGYTMN

T

GFDY

MHWYQQKSG

WVKQSHGKSLE

TSPKRWIYDTS

WIGLITPYNGASS

KLASGVPGRF

YNQKFRGKATLT

SGSGSGNSYSL

VDKSSSTAYMDL

TISSVEAEDDA

LSLTSEDSAVYFC

TYYCQQWSK

ARGGYDGRGFD

HPLTFGSGTK

YWGSGTPVTVSS

VEIK

MT1-
DIQMTQSPSSL
1601
QDVR
1602
SSS
1603
QQH
1604
QVQLQESGPGLV
1605
GFSL
1606
IWTG
1607
ARYY
1608

MMP
SASVGDRVTIT

NT

YITP

KPSETLSLTCTVS

LSYG

GTT

YGMD

(MMP14)
CKASQDVRNT

YT

GFSLLSYGVHWV

Y

VAWYQQKPG

RQPPGKGLEWLG

KAPKLLIYSSS

VIWTGGTTNYNS

YRNTGVPDRF

ALMSRFTISKDDS

SGSGSGTDFTL

KNTVYLKMNSL

TISSLQAEDVA

KTEDTAIYYCAR

VYYCQQHYIT

YYYGMDYWGQ

PYTFGGGTKV

GTLVTVSS

EIK

MUC1
DIQLTQSPSSL
1609
SSVS
1610
STS
1611
HQW
1612
QVQLQQSGAEV
1613
GYTF
1614
INPY
1615
ARGF
1616

SASVGDRVTM

SSY

NRYP

KKPGASVKVSCE

PSYV

NDGT

GGSY

TCSASSSVSSS

YT

ASGYTFPSYVLH

GFAY

YLYWYQQKP

WVKQAPGQGLE

GKAPKLWIYS

WIGYINPYNDGT

TSNLASGVPA

QYNEKFKGKATL

RFSGSGSGTDF

TRDTSINTAYME

TLTISSLQPED

LSRLRSDDTAVY

SASYFCHQWN

YCARGFGGSYGF

RYPYTFGGGT

AYWGQGTLVTV

RLEIK

SS

Mucin
QVVLTQSPVI
1617
SSIS
1618
DTS
1619
HQR
1620
QVQLKESGPDLV
1621
GFSL
1622
IWGD
1623
VKPG
1624

5AC
MSASPGEKVT

Y

DSYP

APSQSLSITCTVS

SKFG

GST

GDY

MTCSASSSISY

WT

GFSLSKFGVNWV

MYWYQQKPG

RQPPGKGLEWLG

TSPKRWIYDTS

VIWGDGSTSYNS

KLASGVPARF

GLISRLSISKENS

SGSGSGTSYSL

KSQVFLKLNSLQ

TISNMEAGDA

ADDTATYYCVKP

ATYYCHQRDS

GGDYWGHGTSV

YPWTFGGGIN

TVSS

LEIK

NaPi2b
DIQMTQSPSSL
1625
ETLV
1626
RVS
1627
FQGS
1628
EVQLVESGGGLV
1629
GFSF
1630
IGRV
1631
ARHR
1632

SASVGDRVTIT

HSSG

FNPL

QPGGSLRLSCAA

SDFA

AFHT

GFDV

CRSSETLVHSS

NTY

T

SGFSFSDFAMSW

GHFD

GNTYLEWYQ

VRQAPGKGLEW

F

QKPGKAPKLLI

VATIGRVAFHTY

YRVSNRFSGV

YPDSMKGRFTIS

PSRFSGSGSGT

RDNSKNTLYLQ

DFTLTISSLQP

MNSLRAEDTAV

EDFATYYCFQ

YYCARHRGFDV

GSFNPLTFGQ

GHFDFWGQGTL

GTKVEIK

VTVSS

NeuGc-
DIVMTQSHKF
1633
QDVS
1634
SAS
1635
QQH
1636
EVQLKESGPGLV
1637
GFSL
1638
IWGG
1639
ARSG
1640

GM3
MSTSVGDRVS

TA

YSTP

APSQSLSITCTVS

SRYS

GST

VREG

ITCKASQDVST

WT

GFSLSRYSVHWV

RAQA

AVAWYQQKP

RQPPGKGLEWLG

WFAY

GQSPKLLIYSA

MIWGGGSTDYNS

SYRYTGVPDR

ALKSRLSISKDNS

FTGSGSGTDFT

KSQVFLKMNSLQ

FTISSVQAEDL

TDDTAMYYCAR

AVYYCQQHYS

SGVREGRAQAW

TPWTFGGGTK

FAYWGQGTLVT

LELK

VSA

NKG2A
DIQMTQSPSSL
1641
ENIY
1642
NAK
1643
QHH
1644
QVQLVQSGAEV
1645
GYTF
1646
IDPY
1647
ARGG
1648

SASVGDRVTIT

SY

YGTP

KKPGASVKVSCK

TSY

DSET

YDFD

CRASENIYSYL

RT

ASGYTFTSYWM

W

VGTL

AWYQQKPGK

NWVRQAPGQGL

YWFF

APKLLIYNAK

EWMGRIDPYDSE

DV

TLAEGVPSRFS

THYAQKLQGRV

GSGSGTDFTLT

TMTTDTSTSTAY

ISSLQPEDFAT

MELRSLRSDDTA

YYCQHHYGTP

VYYCARGGYDF

RTFGGGTKVEI

DVGTLYWFFDV

K

WGQGTTVTVSS

notch
QAVVTQEPSL
1649
TGAV
1650
GTN
1651
ALW
1652
QVQLVQSGAEV
1653
GAS
1654
ILPG
1655
ARFD
1656

TVSPGGTVTL

TTSN

YSN

KKPGASVKISCK

VKIS

TGRT

GNYG

TCRSSTGAVT

Y

HWV

VSGYTLRGYWIE

CKV

YYAM

TSNYANWFQQ

WVRQAPGKGLE

S

DYW

KPGQAPRTLIG

WIGQILPGTGRT

GTNNRAPGVP

NYNEKFKGRVT

ARFSGSLLGG

MTADTSTDTAY

KAALTLSGAQ

MELSSLRSEDTA

PEDEAEYYCA

VYYCARFDGNY

LWYSNHWVF

GYYAMDYWGQ

GGGTKLTV

GTTVTVSS

NOTCH2/
DIVLTQSPATL
1657
QSVR
1658
GAS
1659
QQY
1660
EVQLVESGGGLV
1661
GFTF
1662
IASS
1663
ARSI
1664

NOTCH3
SLSPGERATLS

SNY

SNFP

QPGGSLRLSCAA

SSSG

GSNT

FYTT

recep-
CRASQSVRSN

IT

SGFTFSSSGMSW

tors
YLAWYQQKP

VRQAPGKGLEW

GQAPRLLIYG

VSVIASSGSNTYY

ASSRATGVPA

ADSVKGRFTISR

RFSGSGSGTDF

DNSKNTLYLQM

TLTISSLEPEDF

NSLRAEDTAVYY

AVYYCQQYSN

CARSIFYTTWGQ

FPITFGQGTKV

GTLVTVSS

EIK

NRP1
DIQMTQSPSSL
1665
QYFS
1666
GAS
1667
QQY
1668
EVQLVESGGGLV
1669
GFTF
1670
ISPA
1671
ARGE
1672

SASVGDRVTIT

SY

LGSP

QPGGSLRLSCAA

SSYA

GGYT

LPYY

CRASQYFSSY

PT

SGFTFSSYAMSW

RMSK

LAWYQQKPG

VRQAPGKGLEW

VMDV

KAPKLLIYGAS

VSQISPAGGYTN

SRASGVPSRFS

YADSVKGRFTIS

GSGSGTDFTLT

ADTSKNTAYLQ

ISSLQPEDFAT

MNSLRAEDTAV

YYCQQYLGSP

YYCARGELPYYR

PTFGQGTKVEI

MSKVMDVWGQ

K

GTLVTVSS

oxLDL
QSVLTQPPSAS
1673
NTNI
1674
ANS
1675
ASW
1676
EVQLLESGGGLV
1677
GFTF
1678
ISVG
1679
ARIR
1680

GTPGQRVTISC

GKNY

DASL

QPGGSLRLSCAA

SNA

GHRT

VGPS

SGSNTNIGKN

NGW

SGFTFSNAWMS

W

GGAF

YVSWYQQLPG

V

WVRQAPGKGLE

DY

TAPKLLIYANS

WVSSISVGGHRT

NRPSGVPDRFS

YYADSVKGRSTI

GSKSGTSASL

SRDNSKNTLYLQ

AISGLRSEDEA

MNSLRAEDTAV

DYYCASWDA

YYCARIRVGPSG

SLNGWVFGGG

GAFDYWGQGTL

TKLTVL

VTVSS

P-
EIVLTQSPATL
1681
QSVS
1682
DAS
1683
QQRS
1684
EVQLVESGGGLV
1685
GFTF
1686
ITAA
1687
ARGR
1688

selectin
SLSPGERATLS

SY

NWP

RPGGSLRLSCAA

SNY

GDI

YSGS

CRASQSVSSY

LT

SGFTFSNYDMH

D

GSYY

LAWYQQKPG

WVRQATGKGLE

NDWF

QAPRLLIYDAS

WVSAITAAGDIY

DP

NRATGIPARFS

YPGSVKGRFTISR

GSGSGTDFTLT

ENAKNSLYLQM

ISSLEPEDFAV

NSLRAGDTAVYY

YYCQQRSNWP

CARGRYSGSGSY

LTFGGGTKVEI

YNDWFDPWGQG

K

TLVTVSS

PCSK9
DIVMTQSPDSL
1689
QSVL
1690
WAS
1691
QQY
1692
EVQLVESGGGLV
1693
GFTF
1694
ISGS
1695
AKDS
1696

AVSLGERATIN

YRSN

YTTP

QPGGSLRLSCAA

NNY

GGTT

NWGN

CKSSQSVLYR

NRNF

YT

SGFTFNNYAMN

A

FDL

SNNRNFLGWY

WVRQAPGKGLD

QQKPGQPPNL

WVSTISGSGGTT

LIYWASTRES

NYADSVKGRFIIS

GVPDRFSGSG

RDSSKHTLYLQM

SGTDFTLTISS

NSLRAEDTAVYY

LQAEDVAVYY

CAKDSNWGNFD

CQQYYTTPYT

LWGRGTLVTVSS

FGQGTKLEIK

PCSK9
ESALTQPASVS
1697
SSDV
1698
EVS
1699
NSYT
1700
EVQLVQSGAEVK
1701
GYT
1702
VSFY
1703
ARGY
1704

GSPGQSITISCT

GGYN

STSM

KPGASVKVSCKA

LTSY

NGNT

GMDV

GTSSDVGGYN

S

V

SGYTLTSYGISW

G

SVSWYQQHPG

VRQAPGQGLEW

KAPKLMIYEV

MGWVSFYNGNT

SNRPSGVSNRF

NYAQKLQGRGT

SGSKSGNTAS

MTTDPSTSTAYM

LTISGLQAEDE

ELRSLRSDDTAV

ADYYCNSYTS

YYCARGYGMDV

TSMVFGGGTK

WGQGTTVTVSS

LTVL

PCSK9
DIQMTQSPSSL
1705
QGIS
1706
SAS
1707
QQR
1708
QVQLVQSGAEV
1709
GYTF
1710
ISPF
1711
ARER
1712

SASVGDRVTIT

SA

YSL

KKPGASVKVSCK

TSYY

GGRT

PLYA

CRASQGISSAL

WRT

ASGYTFTSYYMH

SDL

AWYQQKPGK

WVRQAPGQGLE

APKLLIYSASY

WMGEISPFGGRT

RYTGVPSRFS

NYNEKFKSRVTM

GSGSGTDFTFT

TRDTSTSTVYME

ISSLQPEDIAT

LSSLRSEDTAVY

YYCQQRYSL

YCARERPLYASD

WRTFGQGTKL

LWGQGTTVTVSS

EIK

PDGFR
EIVLTQSPATL
1713
QSVS
1714
DAS
1715
QQRS
1716
QLQLQESGPGLV
1717
GGSI
1718
FFYT
1719
ARQS
1720

A
SLSPGERATLS

SY

NWP

KPSETLSLTCTVS

NSSS

GST

TYYY

CRASQSVSSY

PA

GGSINSSSYYWG

YY

GSGN

LAWYQQKPG

WLRQSPGKGLE

YYGW

QAPRLLIYDAS

WIGSFFYTGSTY

FDR

NRATGIPARFS

YNPSLRSRLTISV

GSGSGTDFTLT

DTSKNQFSLMLS

ISSLEPEDFAV

SVTAADTAVYYC

YYCQQRSNWP

ARQSTYYYGSGN

PAFGQGTKVEI

YYGWFDRWDQG

K

TLVTVSS

PDGFRa
DIQMTQSPSSL
1721
QSFS
1722
AAS
1723
QQT
1724
QVQLVESGGGLV
1725
GFTF
1726
ISSS
1727
AREG
1728

SASVGDRVSIT

RY

YSNP

KPGGSLRLSCAA

SDY

GSII

RIAA

CRPSQSFSRYI

PIT

SGFTFSDYYMN

Y

RGMD

NWYQQKPGK

WIRQAPGKGLE

V

APKLLIHAASS

WVSYISSSGSIIY

LVGGVPSRFS

YADSVKGRFTIS

GSGSGTDFTLT

RDNAKNSLYLQ

ISSLQPEDFAT

MNSLRAEDTAV

YYCQQTYSNP

YYCAREGRIAAR

PITFGQGTRLE

GMDVWGQGTTV

MK

TVSS

phospha-
DIQMTQSPSSL
1729
QDIG
1730
ATS
1731
LQY
1732
EVQLQQSGPELE
1733
GYSF
1734
IDPY 1735
VKGG
1736

tidyl-
SASLGERVSLT

SS

VSSP

KPGASVKLSCKA

TGY

Y
GDT

YYGH

serine
CRASQDIGSSL

PT

SGYSFTGYNMN

N

WYFD

NWLQQGPDG

WVKQSHGKSLE

V

TIKRLIYATSS

WIGHIDPYYGDT

LDSGVPKRFS

SYNQKFRGKATL

GSRSGSDYSLT

TVDKSSSTAYMQ

ISSLESEDFVD

LKSLTSEDSAVY

YYCLQYVSSP

YCVKGGYYGHW

PTFGAGTKLE

YFDVWGAGTTV

LK

TVSS

polysi-
DVVMTQTPLS
1737
QSLV
1738
RVS
1739
FQGT
1740
QIQLQQSGPELV
1741
GYTF
1742
IYPG
1743
ARGG
1744

alic
LPVSLGDQASI

HSNG

HVP

RPGASVKISCKAS

TDY

SGNT

KFAM

acid
SCRSSQSLVHS

NTY

YT

GYTFTDYYIHWV

Y

DY

NGNTYLYWY

KQRPGEGLEWIG

LQKPGQSPKP

WIYPGSGNTKYN

LIYRVSNRFSG

EKFKGKATLTVD

VPDRFSGSGS

TSSSTAYMQLSS

GTDFTLKISRV

LTSEDSAVYFCA

EAEDLGVYFC

RGGKFAMDYWG

FQGTHVPYTF

QGTSVTVSS

GGGTRLEIK

PSMA
DIVMTQSHKF
1745
QDVG
1746
WAS
1747
QQY
1748
EVQLQQSGPELV
1749
GYTF
1750
INPN
1751
AAGW
1752

MSTSVGDRVS

TA

NSYP

KPGTSVRISCKTS

TEYT

NGGT

NFDY

IICKASQDVGT

LTFG

GYTFTEYTIHWV

AVDWYQQKP

AGT

KQSHGKSLEWIG

GQSPKLLIYW

M

NINPNNGGTTYN

ASTRHTGVPD

QKFEDKATLTVD

RFTGSGSGTDF

KSSSTAYMELRS

TLTITNVQSED

LTSEDSAVYYCA

LADYFCQQYN

AGWNFDYWGQG

SYPLTFGAGT

TTLTVSS

MLDLK

PSMA
DIQMTQSPSSL
1753
QNVD
1754
SAS
1755
QQY
1756
QVQLVESGGGLV
1757
GFTF
1758
ISDG
1759
ARGF
1760

SASVGDRVTIT

TN

DSYP

KPGESLRLSCAA

SDY

GYYT

PLLR

CKASQNVDTN

YT

SGFTFSDYYMY

Y

HGAM

VAWYQQKPG

WVRQAPGKGLE

DY

QAPKSLIYSAS

WVAIISDGGYYT

YRYSDVPSRFS

YYSDIIKGRFTISR

GSASGTDFTLT

DNAKNSLYLQM

ISSVQSEDFAT

NSLKAEDTAVYY

YYCQQYDSYP

CARGFPLLRHGA

YTFGGGTKLEI

MDYWGQGTLVT

K

VSS

PVRL4
DIQMTQSPSSV
1761
QGIS
1762
AAS
1763
QQA
1764
EVQLVESGGGLV
1765
GFTF
1766
ISSS
1767
ARAY
1768

SASVGDRVTIT

GW

NSFP

QPGGSLRLSCAA

SSYN

SSTI

YYGM

CRASQGISGW

PT

SGFTFSSYNMNW

DV

LAWYQQKPG

VRQAPGKGLEW

KAPKFLIYAAS

VSYISSSSSTIYYA

TLQSGVPSRFS

DSVKGRFTISRD

GSGSGTDFTLT

NAKNSLSLQMNS

ISSLQPEDFAT

LRDEDTAVYYCA

YYCQQANSFP

RAYYYGMDVW

PTFGGGTKVEI

GQGTTVTVSS

K

RGMA
QSALTQPRSVS
1769
SSSV
1770
DVT
1771
YSY
1772
EVQLVQSGAEVK
1773
GYTF
1774
ISPY
1775
ARVG
1776

GSPGQSVTISC

GDSI

AGT

KPGASVKVSCKA

TSHG

SGNT

SGPY

TGTSSSVGDSI

Y

DTL

SGYTFTSHGISW

YYMD

YVSWYQQHP

VRQAPGQGLDW

V

GKAPKLMLYD

MGWISPYSGNTN

VTKRPSGVPD

YAQKLQGRVTM

RFSGSKSGNT

TTDTSTSTAYME

ASLTISGLQAE

LSSLRSEDTAVY

DEADYYCYSY

YCARVGSGPYYY

AGTDTLFGGG

MDVWGQGTLVT

TKVTVL

VSS

CD240D
AIRMTQSPSSF
1777
QDIR
1778
AAS
1779
QQY
1780
QVQLVESGGGV
1781
GFTF
1782
ISYD
1783
ARPV
1784

Blood
SASTGDRVTIT

NY

YNSP

VQPGRSLRLSCT

KNY

GRNI

RSRW

group D
CRASQDIRNY

PT

ASGFTFKNYAMEI

A

LQLG

antigen
VAWYQQKSG

WVRQAPAKGLE

LEDA

KAPKFLIYAAS

WVATISYDGRNI

FHI

TLQSGVPSRFS

QYADSVKGRFTF

GSGSGTDFTLT

SRDNSQDTLYLQ

INSLQSEDFAT

LNSLRPEDTAVY

YYCQQYYNSP

YCARPVRSRWLQ

PTFGQGTRVEI

LGLEDAFHIWGQ

T

GTMVTVSS

root
DIQMTQSPSSL
1785
QSVD
1786
AAS
1787
QQS
1788
QVQLVQSGAEV
1789
GYTF
1790
IYPS
1791
ATYF
1792

plate-
SASVGDRVTIT

YDGD

NEDP

KKPGASVKVSCK

TDYS

NGDS

ANNF

speci-
CKASQSVDYD

SY

LT

ASGYTFTDYSIH

DYW

fic
GDSYMNWYQ

WVRQAPGQGLE

spondin
QKPGKAPKLLI

WIGYIYPSNGDS

3
YAASNLESGV

GYNQKFKNRVT

PSRFSGSGSGT

MTRDTSTSTAYM

DFTLTISPVQA

ELSRLRSEDTAV

EDFATYYCQQ

YYCATYFANNFD

SNEDPLTFGA

YWGQGTTLTVSS

GTKLELK

serum
DIQMTQSPSSL
1793
ENIY
1794
NAK
1795
QHH
1796
QVQLVQSGAEV
1797
GFTF
1798
IYPG
1799
ARGD
1800

amyloid
SASVGDRVTIT

SY

YGA

KKPGSSVKVSCK

ATY

DGNA

FDYD

P
CRASENIYSYL

PLT

ASGFTFATYNMH

N

GGYY

com-
AWYQQKPGK

WVRQAPGQGLE

FDS

ponent
APKLLIHNAK

WMGYIYPGDGN

TLAEGVPSRFS

ANYNQQFKGRV

GSGSGTDFTLT

TITADKSTSTAY

ISSLQPEDFAT

MELSSLRSEDTA

YYCQHHYGAP

VYYCARGDFDY

LTFGQGTKLEI

DGGYYFDSWGQ

K

GTLVTVSS

STEAP-
DIQMTQSPSSL
1801
QSLL
1802
WAS
1803
QQY
1804
EVQLVESGGGLV
1805
GYSI
1806
ISNS
1807
ARER
1808

1
SASVGDRVTIT

YRSN

YNY

QPGGSLRLSCAV

TSDY

GST

NYDY

CKSSQSLLYRS

QKNY

PRT

SGYSITSDYAWN

A

DDYY

NQKNYLAWY

WVRQAPGKGLE

YAMD

QQKPGKAPKL

WVGYISNSGSTS

Y

LIYWASTRES

YNPSLKSRFTISR

GVPSRFSGSGS

DTSKNTLYLQMN

GTDFTLTISSL

SLRAEDTAVYYC

QPEDFATYYC

ARERNYDYDDY

QQYYNYPRTF

YYAMDYWGQGT

GQGTKVEIK

LVTVSS

TACST
DIQLTQSPSSL
1809
QDVS
1810
SAS
1811
QQH
1812
QVQLQQSGSELK
1813
GYTF
1814
INTY
1815
ARGG
1816

D2
SASVGDRVSIT

IA

YITP

KPGASVKVSCKA

TNY

TGEP

FGSS

CKASQDVSIA

LT

SGYTFTNYGMN

G

YWYF

VAWYQQKPG

WVKQAPGQGLK

DV

KAPKLLIYSAS

WMGWINTYTGE

YRYTGVPDRF

PTYTDDFKGRFA

SGSGSGTDFTL

FSLDTSVSTAYL

TISSLQPEDFA

QISSLKADDTAV

VYYCQQHYIT

YFCARGGFGSSY

PLTFGAGTKV

WYFDVWGQGSL

EIK

VTVSS

TGFb
ETVLTQSPGTL
1817
QSLG
1818
GAS
1819
QQY
1820
QVQLVQSGAEV
1821
GYTF
1822
VIPI
1823
ASTL
1824

SLSPGERATLS

SSY

ADSP

KKPGSSVKVSCK

SSNV

VDIA

GLVL

CRASQSLGSS

IT

ASGYTFSSNVIS

DAMD

YLAWYQQKP

WVRQAPGQGLE

Y

GQAPRLLIYG

WMGGVIPIVDIA

ASSRAPGIPDR

NYAQRFKGRVTI

FSGSGSGTDFT

TADESTSTTYME

LTISRLEPEDF

LSSLRSEDTAVY

AVYYCQQYA

YCASTLGLVLDA

DSPITFGQGTR

MDYWGQGTLVT

LEIK

VSS

TIGIT
DIVMTQSPDSL
1825
QTVL
1826
WAS
1827
QQY
1828
EVQLQQSGPGLV
1829
GDS
1830
TYYR
1831
TRES
1832

AVSLGERATIN

YSSN

YSTP

KPSQTLSLTCAIS

VSSN

FKWY

TTYD

CKSSQTVLYSS

NKKY

FT

GDSVSSNSAAWN

SAA

S

LLAG

NNKKYLAWY

WIRQSPSRGLEW

PFDY

QQKPGQPPNL

LGKTYYRFKWY

LIYWASTRES

SDYAVSVKGRITI

GVPDRFSGSG

NPDTSKNQFSLQ

SGTDFTLTISS

LNSVTPEDTAVF

LQAEDVAVYY

YCTRESTTYDLL

CQQYYSTPFTF

AGPFDYWGQGT

GPGTKVEIK

LVTVSS

TWEAK
DIQMTQSPSSL
1833
QSVS
1834
YAS
1835
QHS
1836
EVQLVESGGGLV
1837
GFTF
1838
IRLK
1839
TGYY
1840

R
SASVGDRVTIT

TSSY

WEIP

QPGGSLRLSCAA

SSY

SDNY

ADAM

CRASQSVSTSS

SY

YT

SGFTFSSYWMSW

W

AT

DY

YSYMHWYQQ

VRQAPGKGLEW

KPGKAPKLLIK

VAEIRLKSDNYA

YASNLESGVP

THYAESVKGRFT

SRFSGSGSGTD

ISRDDSKNSLYLQ

FTLTISSLQPE

MNSLRAEDTAV

DFATYYCQHS

YYCTGYYADAM

WEIPYTFGGG

DYWGQGTLVTV

TKVEIK

SS

TYRP1
EIVLTQSPATL
1841
QSVS
1842
DAS
1843
QQRS
1844
QVQLVQSGSELK
1845
GYTF
1846
INTN
1847
APRY
1848

SLSPGERATLS

SY

NWL

KPGASVKISCKA

TSYA

TGNP

SSSW

CRASQSVSSY

MYT

SGYTFTSYAMN

YLDY

LAWYQQKPG

WVRQAPGQGLE

QAPRLLIYDAS

SMGWINTNTGNP

NRATGIPARFS

TYAQGFTGRFVF

GSGSGTDFTLT

SMDTSVSTAYLQ

ISSLEPEDFAV

ISSLKAEDTAIYY

YYCQQRSNW

CAPRYSSSWYLD

LMYTFGQGTK

YWGQGTLVTVSS

LEIK

VEGFR2
DIQMTQSPSSL
1849
QDIA
1850
ATS
1851
LQY
1852
EVQLVESGGGLV
1853
GFTF
1854
ITSG
1855
VRIG
1856

SASVGDRVTIT

GS

GSFP

QPGGSLRLSCAA

SSYG

GSYT

EDAL

CRASQDIAGSL

PT

SGFTFSSYGMSW

DY

NWLQQKPGK

VRQAPGKGLEW

AIKRLIYATSS

VATITSGGSYTY

LDSGVPKRFS

YVDSVKGRFTIS

GSRSGSDYTL

RDNAKNTLYLQ

TISSLQPEDFA

MNSLRAEDTAV

TYYCLQYGSF

YYCVRIGEDALD

PPTFGQGTKV

YWGQGTLVTVSS

EIK

VEGFR2
DIQMTQSPSSV
1857
QGID
1858
DAS
1859
QQA
1860
EVQLVQSGGGLV
1861
GFTF
1862
ISSS
1863
ARVT
1864

SASIGDRVTIT

NW

KAFP

KPGGSLRLSCAA

SSYS

SSYI

DAFD

CRASQGIDNW

PT

SGFTFSSYSMNW

I

LGWYQQKPG

VRQAPGKGLEW

KAPKLLIYDAS

VSSISSSSSYIYY

NLDTGVPSRFS

ADSVKGRFTISR

GSGSGTYFTLT

DNAKNSLYLQM

ISSLQAEDFAV

NSLRAEDTAVYY

YFCQQAKAFP

CARVTDAFDIWG

PTFGGGTKVDI

QGTMVTVSS

K

VSIR
DIQMTQSPSSL
1865
QSID
1866
SAS
1867
QQS
1868
QVQLVQSGAEV
1869
GGTF
1870
IIPI
1871
ARSS
1872

SASVGDRVTIT

TR

AYN

KKPGSSVKVSCK

SSYA

FGTA

YGWS

CRASQSIDTRL

PIT

ASGGTFSSYAIS

YEFD

NWYQQKPGK

WVRQAPGQGLE

Y

APKLLIYSASS

WMGGIIPIFGTAN

LQSGVPSRFSG

YAQKFQGRVTIT

SGSGTDFTLTI

ADESTSTAYMEL

SSLQPEDFATY

SSLRSEDTAVYY

YCQQSAYNPI

CARSSYGWSYEF

TFGQGTKVEI

DYWGQGTLVTV

K

SS

CD171
DIVMSQSPSSL
1873
QSLL
1874
WAS
1875
QQY
1876
QVQLQQPGDELV
1877
GYTF
1878
INPS
1879
ALYD
1880

(L1CAM)
AVSVGEKVTM

YSSN

HSYP

KPGASVKLSCKA

TSY

NGRT

GYYA

SCKSSQSLLYS

QKNY

FT

SGYTFTSYWMQ

W

MDY

SNQKNYLAW

WVKQRPGQGLE

YQQKPGQSPK

WIGEINPSNGRTN

LLIYWASTRES

YNEMFKSKATLT

GVPDRFTGSG

VDKSSSTAYMQL

SGTDFTLTISS

SSLTSEDSAVYY

VKAEDLALYY

CALYDGYYAMD

CQQYHSYPFT

YWGQGTSVTVSS

FGSGTKLEIK

CD171
DIQMTQSSSSF
1881
EDIN
1882
GAT
1883
QQY
1884
QVQLQQPGAELV
1885
GYTF
1886
INPS
1887
ARDY
1888

(L1CAM)
SVSLGDRVTIT

NR

WSTP

KPGASVKLSCKA

TGY

NGRT

YGTS

CKANEDINNR

FT

SGYTFTGYWMH

W

YNFD

LAWYQQTPG

WVKQRPGHGLE

Y

NSPRLLISGAT

WIGEINPSNGRTN

NLVTGVPSRFS

YNERFKSKATLT

GSGSGKDYTL

VDKSSTTAFMQL

TITSLQAEDFA

SGLTSEDSAVYF

TYYCQQYWST

CARDYYGTSYNF

PFTFGSGTELE

DYWGQGTTLTV

IK

SS

CD19
EIVLTQSPAIM
1889
SGVN
1890
DTS
1891
HQR
1892
QVQLVQPGAEV
1893
GYTF
1894
IDPS
1895
ARGS
1896

SASPGERVTM

Y

GSYT

VKPGASVKLSCK

TSN

DSYT

NPYY

TCSASSGVNY

TSGYTFTSNWMH

W

YAMD

MHWYQQKPG

WVKQAPGQGLE

Y

TSPRRWIYDTS

WIGEIDPSDSYTN

KLASGVPARF

YNQNFQGKAKL

SGSGSGTSYSL

TVDKSTSTAYME

TISSMEPEDAA

VSSLRSDDTAVY

TYYCHQRGSY

YCARGSNPYYYA

TFGGGTKLEIK

MDYWGQGTSVT

VSS

CD28
DIQMTQSPSSL
1897
QNIY
1898
KAS
1899
QQG
1900
QVQLVQSGAEV
1901
GYTF
1902
IYPG
1903
TRSH
1904

SASVGDRVTIT

VW

QTYP

KKPGASVKVSCK

TSYY

NVNT

YGLD

CHASQNIYVW

YT

ASGYTFTSYYIH

WNFD

LNWYQQKPG

WVRQAPGQGLE

V

KAPKLLIYKAS

WIGCIYPGNVNT

NLHTGVPSRFS

NYNEKFKDRATL

GSGSGTDFTLT

TVDTSISTAYME

ISSLQPEDFAT

LSRLRSDDTAVY

YYCQQGQTYP

FCTRSHYGLDWN

YTFGGGTKVE

FDVWGQGTTVT

IK

VSS

CD4
DIQMTQSPSSL
1905
QDIN
1906
HTS
1907
IQYN
1908
EVILVESGGAIVE
1909
GFTF
1910
ISDH
1911
ARKY
1912

SASLGGKVTIA

NY

DLFL

PGGSLKLSCSAS

SNY

STNT

GGDY

CKASQDINNYI

TT

GFTFSNYAMSW

A

DPED

AWYQHKPGK

VRQTPEKRLEWV

Y

GPRLLIYHTST

AAISDHSTNTYY

LQPGIPSRFSG

PDSVKGRFTISRD

SGSGRDYSFSI

NAKNTLYLQMN

SNLEPEDIATY

SLRSEDTAIYYCA

YCIQYNDLFLT

RKYGGDYDPED

TFGGGTKLEIK

YWGQGTTLTVSS

CD47
DVLMTQTPLS
1913
QSIV
1914
KVS
1915
FQGS
1916
QVQLQQPGAELV
1917
GYTF
1918
IYPG
1919
ARGG
1920

LPVSLGDQASI

YSNG

HVP

KPGASVMMSCK

TNY

NDDT

YRAM

SCRSSQSIVYS

NTY

YT

ASGYTFTNYNM

DYN

NGNTYLGWY

HWVKQTPGQGL

LQKPGQSPKL

EWIGTIYPGNDD

LIYKVSNRFSG

TSYNQKFKDKAT

VPDRFSGSGS

LTADKSSSAAYM

GTDFTLKISRV

QLSSLTSEDSAV

EAEDLGVYHC

YYCARGGYRAM

FQGSHVPYTF

DYWGQGTSVTV

GGGTKVEIK

SS

CD8
DVQINQSPSFL
1921
RSIS
1922
SGS
1923
QQH
1924
QVQLQQSGAELV
1925
GFNI
1926
IDPA
1927
GRGY
1928

AASPGETITNC

QY

NENP

KPGASVKLSCTA

KDT

NDNT

GYYV

RTSRSISQYLA

LT

SGFNIKDTYIHFV

Y

FDH

WYQEKPGKT

RQRPEQGLEWIG

NKLLIYSGSTL

RIDPANDNTLYA

QSGIPSRFSGS

SKFQGKATITAD

GSGTDFTLTIS

TSSNTAYMHLCS

GLEPEDFAMY

LTSGDTAVYYCG

YCQQHNENPL

RGYGYYVFDHW

TFGAGTKLEL

GQGTTLTVSS

K

KIR2DL
EIVLTQSPVTL
1929
QSVS
1930
DAS
1931
QQRS
1932
QVQLVQSGAEV
1933
GGTF
1934
FIPI
1935
ARIP
1936

2
SLSPGERATLS

SY

NWM

KKPGSSVKVSCK

SFYA

FGAA

SGSY

CRASQSVSSY

YT

ASGGTFSFYAIS

YYDY

LAWYQQKPG

WVRQAPGQGLE

DMDV

QAPRLLIYDAS

WMGGFIPIFGAA

NRATGIPARFS

NYAQKFQGRVTI

GSGSGTDFTLT

TADESTSTAYME

ISSLEPEDFAV

LSSLRSDDTAVY

YYCQQRSNW

YCARIPSGSYYY

MYTFGQGTKL

DYDMDVWGQGT

EIK

TVTVSS

pMHC
QSELTQPRSVS
1937
SRDV
1938
DVI
1939
WSF
1940
EVQLLESGGGLV
1941
GFTF
1942
IVSS
1943
AGEL
1944

[NY-
GSPGQSVTISC

GGYN

AGS

QPGGSLRLSCAA

STYQ

GGST

LPYY

ESO1]
TGTSRDVGGY

Y

YYV

SGFTFSTYQMSW

GMDV

NYVSWYQQH

VRQAPGKGLEW

PGKAPKLIIHD

VSGIVSSGGSTAY

VIERSSGVPDR

ADSVKGRFTISR

FSGSKSGNTAS

DNSKNTLYLQM

LTISGLQAEDE

NSLRAEDTAVYY

ADYYCWSFA

CAGELLPYYGM

GSYYVFGTGT

DVWGQGTTVTV

DVTVL

SS

pMHC
QSVLTQPPSVS
1945
SSNI
1946
GNS
1947
QSY
1948
EVQLQQSGAEVK
1949
GGTF
1950
IIPI
1951
ARDV
1952

[MART1]
GAPGQRVTISC

GAGY

DNSL

KPGSSVKVSCKA

SSYA

LGIA

GSGS

TGSSSNIGAGY

D

SSW

SGGTFSSYAISW

YSLD

DVHWYQQLP

V

VRQAPGQGLEW

Y

GTAPKLLIYG

MGRIIPILGIANY

NSNRPSGVPD

AQKFQGRVTITA

RFSGSKSGTSA

DKSTSAYMELSS

SLAITGLQAED

LRSEDTAVYYCA

EADYYCQSYD

RDVGSGSYSLDY

NSLSSWVFGG

WGQGTLVTVSS

GTKLTVL

pMHC
SYVLTQPPSVS
1953
NIGS
1954
DDS
1955
QVW
1956
EVQLVESGGGLV
1957
GFTF
1958
ISWN
1959
ARGR
1960

[MAGEA1]
VAPGQTARIT

RS

DSRT

QPGRSLRLSCAA

DDY

SGSI

GFHY

CGGNNIGSRS

DHW

SGFTFDDYAMH

A

YYYG

VHWYQQKPG

V

WVRQAPGKGLE

MDI

QAPVLVVYDD

WVSGISWNSGSI

SDRPSGIPERF

GYADSVKGRFTI

SGSNSGNMAT

SRDNAKNSLYLQ

LTISRVEAGDE

MNSLRAEDTAV

ADYYCQVWD

YYCARGRGFHY

SRTDHWVFGG

YYYGMDIWGQG

GTDLTVL

TTVTVSS

pMHC
EVILTQSPLSL
1961
QSLL
1962
LGS
1963
MQA
1964
EVQLVESGGGVV
1965
GFTF
1966
ISYD
1967
ARGG
1968

[Tyrosi-
PVTPGEPASIS

HSIG

LQTP

QPGRSLRLSCAA

RSY

G

GYYE

nase]
CRSSQSLLHSI

YN

LT

SGFTFRSYGMHW

G

SNK

TSGP

GYNYLDWYL

VRQAPGKGLEW

DY

QKPGQSPQLLI

VAVISYDGSNKY

YLGSNRASGV

YTDSVNGRFTISR

PDRFSGSGSGT

DNSKNTLYQMN

DFTLKISRVEA

SLRAEDTAVYYC

EDVGVYYCM

ARGGGYYETSGP

QALQTPLTFG

DYWGQGTLVTV

GGTKVEIK

SS

pMHC
DVVMTQSPLS
1969
QSLL
1970
LGS
1971
MQT
1972
EVQLVETGGGVV
1973
GFTF
1974
ISYD
1975
AKDR
1976

[Tyrosi-
PVTPGEPASIS

HSNG

LQTP

QPGRSLRLSCAA

SSYG

GSNK

YGWG

nase]
CRSSQSLLHSN

HNY

LT

SGFTFSSYGMHW

SSFG

GHNYLDWYL

VRQAPGKGLEW

HDY

QKPGQSQLLIY

VAVISYDGSNKY

LGSNRSGVPD

YADSVKGRFTIS

RFSGSGSGTDF

DNSKNTLYLQM

TLKISRVEAED

NSLRAEDTAVYY

VGVYYCMQT

CAKDRYGWGSS

LQTPLTFGPGT

FGHDYWGQGTL

KVDIK

TVSS

pMHC
QSVLTQPPSVS
1977
SSNI
1978
DNN
1979
GTW
1980
EVQLVQSGAEVK
1981
GYTF
1982
INPS
1983
ARDG
1984

[gp100]
AAPGQTVTISC

GRNY

DSTL

KPGASVKVSCKA

TSYY

GGST

TYGS

SGSSSNIGRNY

DLY

SGYTFTSYYIHW

GSYP

VSWFQQVPGR

V

VRQAPGQGLEW

YYYY

APKLLIYDNN

MGAINPSGGSTP

YGMD

QRPSGIPGRFS

YAQKFQGRVTM

V

ASKSDTSATL

TRDTSTSTVYME

DITGLQSGDE

LSSLRSEDTAVY

AVYYCGTWD

YCARDGTYGSGS

STLDLYVFGG

YPYYYYYGMDV

GTHVPVL

WGQGTTVTVSS

pMHC
ETTLTQSPGTL
1985
ASQS
1986
IYGA
1987
YCQ
1988
EVQLVQSGAEVK
1989
GGTF
1990
IIPI
1991
ARGP
1992

[MUC1]
SLSLSPGERAT

VSS

QYG

KPGSSVKVSCKA

SSYA

FGTA

EYCI

LSCRASQSVSS

SSPR

SGGTFSSYAISW

NGVC

SYLAWYQQKP

T

VRQAPGQGLEW

SLDV

GQAPRLLIYG

MGGIIPIFGTANY

ASSRATGIPDR

AQKFQGRVTITA

FSGSGSGTDFT

DESTSTAYMELS

LTISRLEPEDF

SLRSEDTAVYYC

AVYYCQQYGS

ARGPEYCINGVC

SPRTFGQQGT

SLDVWGQGTTV

KVEIK

TVSS

pMHC
EIVMTQSPATL
1993
QSVS
1994
DAS
1995
HQY
1996
EVQLVQSGAEVK
1997
GGTF
1998
IIPI
1999
AVHY
2000

[MUC1]
SLSPGERATLS

SY

GSSP

KPGSSVKVSCKA

SSYA

FGTA

GDYV

CRASQSVSSY

QT

SGGTFSSYAISW

FSSM

LAWYQQKPG

VRQAPGQGLEW

DV

QAPRLLIYDAS

MGGIIPIFGTANY

NRATGIPARFS

AQKFQGRVTITA

GSGSGTDFTLT

DESTSTAYMELS

ISSLEPEDFAV

SLRSEDTAVYYC

YYCHQYGSSP

AVHYGDYVFSS

QTFGQGTKVE

MDVWGQGTTVT

IK

VSS

pMHC
EIVLTQSPATL
2001
QSVG
2002
DAS
2003
QQRS
2004
EVQLVQSGAEVK
2005
GGTF
2006
IIPI
2007
YCAG
2008

[tax]
SLSPGERATLS

SY

NWP

KPGSSVKVSCKA

SSYT

FGTA

DTDS

CRASQSVGSY

PMY

SGGTFSSYTISWV

SGYY

LAWYQQKPG

T

RQAPGQGLEWM

GAVD

XAPRLLIYDAS

GGIIPIFGTANYA

Y

HRATGIPARFS

QKFQGRVTITAD

GSGSGTDFTLT

KSTSTSTAYMEL

ISSLEPEDFAV

SSLRSEDTAVYY

YYCQQRSNWP

CAGDTDSSGYYG

PMYTFGQGTK

AVDYWGQGTLV

LEIK

TVSS

pMHC
NFMLTQPHSV
2009
GGSI
2010
EDN
2011
QSSD
2012
EVQLVQSGGGV
2013
GFTF
2014
ISYD
2015
AKTL
2016

[gp100]
SESPGKTVTIS

DNNY

GSK

VQPGRSLTLSCA

SSYG

GSNK

SAGE

CTGSGGSIDN

VV

ASGFTFSSYGMH

WIGG

NYVHWYQQR

WVRQAPGKGLE

GAFD

PGSAPTTVMF

WVSVISYDGSNK

I

EDNQRPSGVP

YYADSVKGRFTI

DRFSGSIDSSS

SRDNSKNTLYLM

NSASLVISGLK

NSLRTEDTAVYY

TEDEGDYYCQ

CAKTLSAGEWIG

SSDGSKVVFG

GGAFDIWGHGT

GGTKLTVL

MVTVSS

pMHC
DIVMTQSPDSL
2017
QSLL
2018
WAS
2019
QQY
2020
QVQLQESGPGLV
2021
GGSI
2022
ISDS
2023
ARVR
2024

[NY-
AVSLGERVTIN

YTSN

YKSP

KPSQTLALTCSVI

SSGD

GST

IQGA

ESO1]
CKSSQSLLYTS

NRNY

L

GGSISSGDYYWS

YY

SWGF

NNRNYLAWY

WIRQPPGKGLEW

FDL

QLKPGQPPKL

VGYISDSGSTYN

LIYWASTRES

EPSLNSRVTISVD

GVPDRFSGSG

TSKNQFSLKLFS

SGTDFTLTISG

MTAADTAVYYC

LQAEDVAVYY

ARVRIQGASWGF

CQQYYKSPLF

FDLWGRGTLVSV

GQGTKLEIK

SS

pMHC
EIVMTQSPATL
2025
QSFS
2026
AAS
2027
QQY
2028
QVQLVQSGVEV
2029
GYTF
2030
ISVY
2031
AREG
2032

[NY-
SVSPGERATLS

DD

NNW

KKPGASVKVSCK

ASY

NGKT

GFYG

ESO1]
CRASQSFSDD

PQT

ASGYTFASYGIS

G

SGSH

LAWYQQKPG

WVRQAPGQGLE

YRYF

QAPRLLIYAAS

WMGWISVYNGK

AMDV

TRATGIPARFS

TNPAERHLGRVT

GRGSGTEFTLT

MTTDTSTNTAY

ISSLQSEDSAV

MELRNLKSDDTA

YYCQQYNNW

VYYCAREGGFY

PQTFGQGTKV

GSGSHYRYFAM

EIK

DVWGQGTTVIVS

S

pMHC
DIVMTQTPLSL
2033
QSLV
2034
KVS
2035
MQG
2036
QVQLVQSGGGV
2037
GFSF
2038
MNWS
2039
ARGE
2040

[NY-
PVTLGQPASLS

FTDG

THW

VRPGGSLRLSCA

IDYG

GDKK

YSNR

ESO1]
CRSSQSLVFTD

NTY

PPI

ASGFSFIDYGMS

GNTYLNWFQ

WVRQVPGKGLE

QRPGQSPRRLI

WVAGMNWSGD

YKVSSRDPGV

KKGHAESVKGRF

PDRFSGTGSGT

IISRDNAKNTLYL

DFTLEISRVEA

EMSSLRVEDTAL

EDIGVYYCMQ

YFCARGEYSNRF

GTHWPPIFGQ

DPRGRGTLVTVS

GTKVEIK

S

pMHC
EIVLTQSPGTL
2041
QSVS
2042
GAS
2043
QHY
2044
EVQLQESGPGLV
2045
GGSI
2046
IYPR
2047
AREY
2048

[NY-
SLSPGERATLS

SSY

DNSL

KPSETLSLTCTVS

SSDY

GTS

YYVT

ESO1]
CRASQSVSSSY

ITFG

GGSISSDYWTWI

NGYF

LGWYQQKPG

HGT

RQPAGKGLEWIG

SPGF

QAPRLLIYGAS

R

RIYPRGTSNYNPS

DY

IRATGIPDRFS

LKSRVTMSVDTS

GSGSGTDFTLT

KNQISLRLSSVTA

ISRLEPDDFAV

ADTAVYYCARE

YYCQHYDNSL

YYYVTNGYFSPG

ITFGHGTRLDI

FDYWGQGTLVT

K

VSS

pMHC
DIVMTQSPLSL
2049
QSLH
2050
LVS
2051
MQA
2052
EVQLVESGGGVV
2053
GFIF
2054
ISSD
2055
GTGH
2056

[NY-
PVTPGEPASIS

SNGY

VQTP

QPGKSLRLSCAA

SSFA

GSNE

STEY

ESO1]
CRSSQSLHSN

NY

FT

SGFIFSSFAVHWV

YDGL

GYNYLDWYL

RQAPGKGLEWV

LGV

QKPGQSPQLLI

ATISSDGSNEDY

YLVSNRASGV

VDSVKGRFIISRD

PDRFSGTGSGT

NSKNTLYLQMNS

DFTLKISRVEA

LRRDDTAVYYC

EDVGVYYCM

GTGHSTEYYDGL

QAVQTPFTFG

LGVWGHGTTVS

PGTKVDIK

VSS

pMHC
QSVVTQPPSVS
2057
SSNI
2058
EDD
2059
ATW
2060
QLQLQESGPGLV
2061
GGSI
2062
IYYS
2063
ARHV
2064

[MARTI]
AAPGRKVTISC

GSNY

DRT

KPSETLSLTCTVS

SSSS

GT

GHEL

SGSSSNIGSNY

VNV

GGSISSSSYYWG

YY

DY

VSWYQQVPGT

VR

WIRQPPGKGLEW

APKLLIYEDD

GSIYYSGTYYNPS

KRPSGIPDRFS

LKSRVTISVDTSK

GSKGTSATLGI

NQFSLKLSSTAA

TGLQTGDEAD

DTAVYYCARHV

YFCATWDRTV

GHELDYWGQGT

NVVRFGGGTR

LVTVSS

LTV

pMHC
DVVMTQSPLS
2065
QSLL
2066
LGS
2067
MQA
2068
QLQLQESGPGLV
2069
GGSI
2070
IYHS
2071
VGSP
2072

[Tyrosi-
LPVTPGEPASI

HSIG

LQTP

KPSGTLSLTCAVS

SSSN

GST

YGDY

nase]
SCRSSQSLLHS

YNY

PT

GGSISSSNWWSW

W

VLDY

IGYNYLHWFL

VRQPPGKGLEWI

QKGQSPQLLIY

GEIYHSGSTNYN

LGSNRASGVP

PSLKSRVTISDKS

DRFSGSGSGT

KNQFSLKLSSVT

DFTLKISRVEA

AADTAVYYCVG

EDVGVYYCM

SPYGDYVLDYW

QALQTPPTFG

GQGTLVTVSS

QGTRLEIK

pMHC
QAVVTQPPSA
2073
SSNI
2074
SNN
2075
AAW
2076
QMQLVQSGAEV
2077
GYSF
2078
VDPG
2079
ARVQ
2080

[WT-1]
SGTPGQRVTIS

GSNT

DDSL

KEPGESLRISCKG

TNF

YSYS

YSGY

CSGSSSNIGSN

NGW

SGYSFTNFWISW

W

YDWF

TVNWYQQVP

V

VRQMPGKGLEW

DP

GTAPKLLIYSN

MGRVDPGYSYST

NQRPSGVPDR

YSPSFQGHVTISA

FSGSKSGTSAS

DKSTSTAYLQWN

LAISGLQSEDE

SLKASDTAMYYC

ADYYCAAWD

ARVQYSGYYDW

DSLNGWVFGG

FDPWGQGTLVTV

GTKLTVL

SS

pMHC
DIVMTQSQKF
2081
QNVH
2082
LAS
2083
LQH
2084
QVQLKESGPGLV
2085
GFSL
2086
IWGD
2087
ARDP
2088

[EBNA-1]
MSTSVGDRVS

TA

WNN

APSQSLSITCTVS

TGY

GST

YGYI

ITCKASQNVH

PLT

GFSLTGYGVNW

G

FDY

TAVAWYQQK

VRQPPGKGLEWL

AGQSPKALIYL

GMIWGDGSTDY

ASNRHTGVPD

NSALKSRLSISKD

RFTGSGSGTDF

NSKSQVFLKMNS

TLTISNVQSED

LQTDDTARYYCA

LADYFCLQHW

RDPYGYIFDYWG

NNPLTFGAGT

QGTTLTVSS

KLELK

pMHC
DIVMTQSQKF
2089
QNVF
2090
STS
2091
QQYI
2092
QVQLKQSGPGLV
2093
GFSL
2094
IWSG
2095
ARNW
2096

[LMP2]
MSTSVGDRVS

TN

SYPL

QPSQSLSITCTVS

TNY

GST

VPYY

VTCRASQNVF

T

GFSLTNYGVHW

G

FDY

TNVAWYQQK

VRQSPGKGLEwl

PGQAPKALIYS

GVIWSGGSTDYN

TSYRYSGVPD

AAFISRLSISKDN

RFTGSGSGTDF

SKQVFFKMNSLQ

TLTISNVQSED

ANDTAIYYCARN

LAEYFCQQYIS

WVPYYFDYWGQ

YPLTFGAGTK

GTTLTVSS

LELK

pMHC
ETTLTQSPGTL
2097
QSVS
2098
AAS
2099
QQY
2100
QVQLQESGGGLV
2101
GFTF
2102
ISSS
2103
VRGD
2104

[gp100]
SLSPGERATLS

SNY

GSSR

KPGGSLRLSCAA

SSYS

GSTI

PYFF

CRASQSVSSN

S

SGFTFSSYSMNW

YYYG

YLAWYQQKP

VRQAPGKGLEW

MDI

GQAPRLLIYA

VSYISSSGSTIYY

ASSRATGIPDR

ADSVRGRFTISRD

FSGSGSGTDFT

NAKNTLYLQMN

LTISRLEPEDF

SLRAEDTAVYYC

AVYYCQQYGS

VRGDPYFFYYYG

SRSFGQGTKL

MDIWGQGTTVT

EIK

VSS

pMHC
DIQLTQSPSSL
2105
QSIS
2106
SAS
2107
QQS
2108
QVQLQESGPGLV
2109
GGSI
2110
IDYS
2111
ARES
2112

[gp100]
SASVGDRVIIT

TH

YSSP

KPSETLSLTCTVS

SSN

GST

GSPY

CRATQSISTHL

PIT

GGSISSNMYYWG

MYY

YFDY

NWYQQKPGK

WVRQPPGKGLE

APKLLIYSASS

WIGSIDYSGSTYY

LQSGVPSRFSG

NPSLRSRVTMSV

SGSGSTDFTLT

DTSKKQFSLKMT

ISSLQPEDFAT

SVTAADTAVYYC

YYCQQSYSSP

ARESGSPYYFDY

PITFGQGTRLE

WGQGTLVTVSS

IK

pMHC
ETTLTQSPGTL
2113
QSVS
2114
GAS
2115
QQY
2116
QVQLQESGPGLV
2117
GGSI
2118
WINH
2119
ARVV
2120

[hTERT]
SLSPGERATLS

SSY

GTSL

KPSETLSLTCTVS

SSSS

SGST

AAAG

CRASQSVSSSY

TWY

GGSISSSSYYWA

YY

HYYY

LAWYQQKPG

WIRQPPGKLEWI

YYMD

QAPRLLIYGAS

GEWINHSGSTNY

V

TRATGVPDRF

NPSLKSRVTISVD

SGSGSGTDFTL

TSKNQFSLNLNS

ISRLEPEDFAV

VTAADTAVYYC

YYCQQYGTSL

ARVVAAAGHYY

TWYFGQGTK

YYYMDVWGKGT

VEIK

TVTVSS

pMHC
ETTLTQSPGTL
2121
QSVS
2122
GAS
2123
QQY
2124
QVQLQESGPGLV
2125
GGSI
2126
IYYS
2127
ARSR
2128

[hTERT]
SLSPGERATLS

SRY

GSSN

KPSETLSLTCTVS

SSSY

GST

SGSY

CRASQSVSSR

T

GGSISSSYYWGW

Y

LNDA

YLAWYQQKP

IRQPPGKGLEWIG

FDI

GQAPRLLIYG

SIYYSGSTYYNPS

ASSRATGIPDR

LKSRVTISVDTSK

FSGSGSGTDFT

NQFSLKLSSVTA

LTISRLEPEDF

ADTAVYYCARSR

AVYYCQQYGS

SGSYLNDAFDIW

SNTFGQGTKL

GQGTMVTVSS

EIK

pMHC
ETTLTQSPGTL
2129
QSVS
2130
GAS
2131
QQY
2132
QVQLQQSGAEV
2133
GGTF
2134
IIPI
2135
ARGF
2136

[hTERT]
SLSPGERATLS

SSY

GSSS

KKPGSSVKVSCK

SSYA

LGIA

RPYY

CRASQSVSSSY

GT

ASGGTFSSYAIS

YYGM

LAWYQQKPG

WVRQAPGQGLE

DV

QAPRLLIYGAS

WMGRIIPILGIAN

SRATGIPDRFS

YAQKFQGRVTIT

GSGSGTDFTLT

ADKSTSTAYMEL

ISRLEPEDFAV

SSLRSEDTAVYY

YYCQQYGSSS

CARGFRPYYYYG

GTFGQGTKVE

MDVWGQGTTVT

IK

VSS

pMHC
QSVVTQPPSVS
2137
SSNI
2138
GNS
2139
QSY
2140
QVQLQQSGPGLV
2141
GGSI
2142
MYYS
2143
ARIP
2144

[gp100]
GAPGQRVTISC

GAGY

DSSL

KPSETLSLTCTVS

RNY

GGA

NYYD

TGSSSNIGAGY

D

SAL

GGSIRNYYWSWI

Y

RSGY

DVHWYQQLP

RQPPGKGLEWIG

YPGY

GTAPKLLIYG

YMYYSGGANYN

WYFD

NSNRPSGVPD

PSLNSRVTISLDT

L

RFSGSKSGTSA

SKNQFSLKLTSV

SLAITGLQAED

TAADTAVYYCA

EADYYCQSYD

RIPNYYDRSGYY

SSLSALFGGGT

PGYWYFDLWGR

KLTVL

GTLVTVSS

pMHC
DIQLTQSPSSL
2145
QSIS
2146
SAS
2147
QQS
2148
QVQLQQSGPGLV
2149
GDSI
2150
TYYR
2151
ARAS
2152

[gp100]
SASVGDRVTIT

TY

DIIP

KPSQTLSLTCAIS

SSNS

SKWY

FGTS

CRASQSISTYL

LT

GDSISSNSVVWN

VV

N

GKFD

NWYQHRPGK

WIRQSPSRGLEW

D

APKLLIYSASS

LGRTYYRSKWY

LQSGVPSRFSG

NDYAVSVKSRITI

SGSGTDFTLTI

NPDTSKNQFSLQ

SSLQPEDFATY

LNSVTPDDTALY

YCQQSDIIPLT

YCARASFGTSGK

FGGGTKVEIN

FDDWGQGTLVT

VSS

pMHC
SYVLTQPPSVS
2153
TIGR
2154
DDT
2155
QVW
2156
QVQLQQSGPGLV
2157
GDS
2158
TYYR
2159
CVRG
2160

[hTERT]
EAPGKTARITC

KS

DSST

KPSQTLSLTCAIS

VSSK

SKWY

SIFD

EGITIGRKSVH

DPQ

GDSVSSKNSSWN

NSS

Y

V

WYQQKPGQA

VV

WIRQSPSRGLEW

PVLVVYDDTV

LGRTYYRSKWY

RPSGVPERFSG

YDYAVSVKGRIT

SNSGNTATLII

FTFPDTSKNQVSL

SGVEAGDEAD

HLNAVTPEDTAM

YCQVWDSSTD

YYCVRGSIFDVW

PQVVFGGGTK

GQGTMVTVSS

TVL

pMHC
NFMLTQPHSV
2161
GGSI
2162
EDD
2163
QSY
2164
QVQLQQWGAGL
2165
GGSF
2166
INHS
2167
ARMV
2168

[hTERT]
SESPGKTVTIS

ATNY

DSSN

LKPSETLSLTCAV

SGY

GST

RYYY

CTGSGGSIATN

QV

YGGSFSGYYWS

Y

GMDV

YVQWYQQRP

WIRQPPGKGLEW

GSAPATVIYED

IGEINHSGSTNYN

DQRPSGVPDR

PSLKSRVTISVDT

FSGSIDSSSNS

SKNQFSLKLSSVT

ASLTISGLKTE

AADTAVYYCAR

DEADYYCQSY

MVRYYYGMDV

DSSNQVFGGG

WGQGTTVTVSS

TKLTVL

pMHC
ETTLTQSPGTL
2169
QSVG
2170
GAS
2171
QQY
2172
QVQLQQWGAGL
2173
GGSF
2174
INHS
2175
ARVA
2176

[hTERT]
SLSPGERATLS

SN

GDSP

LKPSETLSLTCAV

SGY

GST

YYDS

CRASQSVGSN

RLYT

YGGSFSGYYWS

Y

SGYY

LAWYQQRPG

WIRQPPGKGLEW

PYDA

QAPSLLIYGAS

IGEINHSGSTNYN

FDI

SRATGVPDRF

PSLKSRVTISVDT

SGSGSGTDFTL

SKNQFSLKLSSVT

TISRLEPEDFA

AADTAVYYCAR

VYYCQQYGDS

VAYYDSSGYYPY

PRLYTFGQGT

DAFDIWGQGTM

KLEIK

VTVSS

pMHC
DVVMTQSPGT
2177
QLSD
2178
SAS
2179
HQY
2180
QVQLVQSGAEV
2181
GYTF
2182
ISSS
2183
ARYD
2184

[gp100]
LSVSPGDSATL

SY

GFLP

KKPGASVKVSCK

TRY

NGYT

ISGL

SCWASQLSDS

WT

ASGYTFTRYGIS

G

DGFD

YVSWYQQKP

WVRQAPGQGLE

I

GQAPRLLIHSA

WMGWISSSNGY

SIRAPGIPDRFS

TKYAQNLQGRLT

GSVSGTEFTLT

LTTDTSTGTAYM

ISGLEPEDFAV

ELRSLRSEDTAL

YSCHQYGFLP

YYCARYDISGLD

WTFGQGTKVE

GFDIWGQGTMV

IR

TVSS

pMHC
ETTLTQSPGTL
2185
RYIN
2186
DAS
2187
QQY
2188
QVQLVQSGAEV
2189
GGTF
2190
IIPI
2191
CARD
2192

[gp100]
SLSPGERATLS

ANF

GSSP

KKPGSSVKVSCK

SSYA

FGTA

SSGW

CRASRYINAN

RT

ASGGTFSSYAIS

LYDA

FLAWYQQKPG

WVRQAPGQGLE

FDI

QAPRLLIYDAS

WMGGIIPIFGTAT

TRATGIPDRFS

NYAQKFQGRVTI

GSGSGTDFTLT

TADESTSTAYME

ISRLEPEDFAV

LSSLRSEDTAVY

YYCQQYGSSP

YCARDSSGWLY

RTFGQGTKVEI

DAFDIWGQGTM

K

VTVSS

pMHC
DIQMTQSPSIL
2193
QRFG
2194
GAS
2195
QQA
2196
QVQLVQSGAEV
2197
GGTF
2198
INVG
2199
ARDG
2200

[hTERT]
SASVGDRVTIT

DY

NSFP

KKPGSSVKVSCK

SSYA

NGNA

ERAW

CRASQRFGDY

ITFG

ASGGTFSSYAIS

DLDY

LAWYQQKPG

KGT

WVRQAPGQGLE

QAPKLLIYGAS

R

WMGWINVGNGN

TLQSGVPSRFS

AIYSQKFQGRVTI

GSGSGTEFTLT

TRDTSATTAYME

ISGLQPEDFAT

LSSLRSEDTAVY

YYCQQANSFPI

YCARDGERAWD

TFGKGTRLDIR

LDYWGQGTLVT

VSS

pMHC
ETTLTQSPGTL
2201
QSVS
2202
GAS
2203
QQY
2204
QVQLVQSGGGV
2205
GFTF
2206
ISYD
2207
AREL
2208

[hTERT]
SLSPGERATLS

SSY

GSSP

VQPGRSLRLSCA

SSYA

GSNK

RFLE

CRASQSVSSSY

YT

ASGFTFSSYAMH

WSSD

LAWYQQKPG

WVRQAPGKGLE

AFDI

QAPRLLIYGAS

WVAVISYDGSNK

SRATGIPDRFS

YYADSVKGRFTI

GSGSGTDFTLT

SRDNSKNTLYLQ

ISRLEPEDFAV

MNSLRAEDTAV

YYCQQYGSSP

YYCARELRFLEW

YTFGQGTKLEI

SSDAFDIWGQGT

K

MVTVSS

pMHC
ETTLTQSPGTL
2209
QSVS
2210
GAS
2211
QQH
2212
QVQLVQSGGGV
2213
GFTF
2214
ISYD
2215
AKDS
2216

[gp100]
SLSPGERATLS

SSY

DSSP

VQPGRSLRLSCA

SSYG

GSDK

YYDN

CRASQSVSSSY

RT

ASGFTFSSYGMH

SAFQ

LAWYQQKPG

WVRQAPGKGLE

AD

QAPRLLIYGAS

WVAFISYDGSDK

SRATGIPDRFS

NFADSVKGRFTIS

GSGSGTDFTLT

RDNSKNTLYLQ

ISRLEPEDFAV

MNSLRAEDTAV

YYCQQHDSSP

YYCAKDSYYDN

RTFGQGTKVEI

SAFQADWGQGT

K

LVTVSS

pMHC
EIVLTQSPLSL
2217
QSLL
2218
LGS
2219
MQA
2220
QVQLVQSGGGV
2221
GFTF
2222
ISYD
2223
ARDF
2224

[tax]
PVTPGEPASIS

HSNG

LQTP

VQPGRSLRLSCA

SSYG

GSNK

DYGD

CRSSQSLLHSN

YNY

RT

ASGFTFSSYGMH

SYYY

GYNYLDWYL

WVRQAPGKGLE

YGMD

QKPGQSPQLLI

WVAVISYDGSNK

V

YLGSNRASGV

YYADSVKGRFTI

PDRFSGSGSGT

SRDNSKNTLYLQ

DFTLKISRVEA

MNSLRAEDTAV

EDVGVYYCM

YYCARDFDYGDS

QALQTPRTFG

YYYYGMDVWG

QGTKVEIK

QGTTVTVSS

pMHC
DVMTQSPLSL
2225
QSLL
2226
FGS
2227
MQA
2228
QVQLVQSGGGV
2229
GFTF
2230
ISYD
2231
ARDY
2232

[gp100]
PVTPGEPASIS

HSNG

THW

VQPGRSLRLSCA

SSYG

GSNK

YGDY

CRSSQSLLHSN

YKY

PYT

ASGFTFSSYGMH

ALLD

GYKYVNWYL

WVRQAPGKGLE

Y

QKPGQSPQLLI

WVAVISYDGSNK

YFGSYRASGV

YYADSVKGRFTI

PDRFSGSGSGT

SRDNSKNTLYLQ

DFTLKISRVEA

MNSLRAEDTAV

EDVGIYYCMQ

YYCARDYYGDY

ATHWPYTFGQ

ALLDYWGQGTL

GTRLEIK

VTVSS

pMHC
EIVLTQSPDTL
2233
SQSV
2234
YDT
2235
CQQ
2236
QVQLVQSGGGV
2237
GFTF
2238
ISYD
2239
AKTV
2240

[gp100]
SLSPGEREATL

SHS

YVSS

VQPGRSLRLSCA

STYG

GSNK

GVTF

SCRASQSVSHS

PLT

ASGFTFSTYGLH

VSDA

YLAQYQQKPG

WVRQAPGKGLE

FDI

QAPRLLIYDTS

WVAFISYDGSNK

SRATDIPDRFS

YYADSVKGRFTI

GSGSGTDFTLT

SRDNSKNTLYLQ

ISRLEPEDSAV

MNGLRAEDTAV

YYCQQYVSSP

YYCAKTVGVTFV

LTFGQGTKLEI

SDAFDIWGQGTM

K

VTVSS

pMHC
QSELTQPRSVS
2241
SRDV
2242
DVI
2243
WSF
2244
QVQLLESGGGLV
2245
GFTF
2246
IGSS
2247
AGEL
2248

[NY-
GSPGQSVTISC

GGYN

AGS

QPGGSLRLSCAA

SAY

GGGT

LPYY

ESO1]
TGTSRDVGGY

Y

YYV

SGFTFSAYGMG

G

GMDV

NYVSWYQQH

WVRQAPGKGLE

PGKAPKLIIHD

WVSSIGSSGGGT

VIIRPSGVPDR

AYADSVKGRFTI

FSGSKSGNTAS

SRDNSKNTLYLQ

LTISGLQAEDE

MNSLRAEDTAV

AHYYCWSFA

YYCAGELLPYYG

GSYYVFGTGT

MDVWGQGTTVT

DVTVL

VSS

GD2
ENVLTQSPAI
2249
SSVS
2250
STS
2251
QQY
2252
QVQLKESGPVLV
2253
GFSL
2254
IWAG
2255
AKRS
2256

MSASPGEKVT

SSL

SGYP

APSQTLSITCTVS

ASY

GST

DDYS

MTCRASSSVS

IT

GFSLASYNIHWV

N

WFAY

SSLYHWYQQK

RQPPGKGLEWLG

SGASPKVWIY

VIWAGGSTNYNS

STSNLASGVP

ALMSRLSISKDNS

GRFSGSGSGTS

KSQVFLQMNSLQ

YSLTISSVEAE

TDDTAMYYCAK

DAATYYCQQ

RSDDYSWFAYW

YSGYPITFGAG

GCQTLVTVSA

TKVEVK

STEAP_
DIVMSQSPSSL
2257
QSLL
2258
WAS
2259
QQY
2260
DVQVQESGPGLV
2261
GYSI
2262
ISNS
2263
ARER
2264

1
AVSVGEKVTM

YRSN

YNY

KPSQSLSLTCTVT

TSDY

GST

NYDY

SCKSSQSLLYR

QKNY

PRT

GYSITSDYAWN

A

DDYY

SNQKNYLAW

WIRQFPGNKLEW

YAMD

YQQKPGQSPK

MGYISNSGSTSY

Y

LLIYWASTRES

NPSLKSRISITRDT

GVPDRFTGSG

SKNQFFLQLISVT

SGTDFTLTISS

TEDTATYYCARE

VKAEDLAVYY

RNYDYDDYYYA

CQQYYNYPRT

MDYWGQGTTLT

FGGGTKLEIK

VSA

a4b7
DIQMTQSPSSV
2265
QGIS
2266
GAS
2267
QQA
2268
QVQLVQSGAEV
2269
GYT
2270
FDPQ
2271
ATGS
2272

SASVGDRVTIT

SW

NSFP

KKPGASVKVSCK

LSDL

DGET

SSSW

CRASQGISSW

WT

VSGYTLSDLSIH

S

FDP

LAWYQQKPG

WVRQAPGKGLE

KAPKLLIYGAS

WMGGFDPQDGE

NLESGVPSRFS

TIYAQKFQGRVT

GSGSGTDFTLT

MTEDTSTDTAY

ISSLQPEDFAN

MELSSLKSEDTA

YYCQQANSFP

VYYCATGSSSSW

WTFGQGTKVE

FDPWGQGTLVTV

IK

SS

GPC3
DVVMTQSPLS
2273
QSLV
2274
KVS
2275
SQNT
2276
QVQLVQSGAEV
2277
GYTF
2278
LDPK
2279
TRFY
2280

LPVTPGEPASI

HSNR

HVPP

KKPGASVKVSCK

TDY

TGDT

SYTY

SCRSSQSLVHS

NTY

T

ASGYTFTDYEMH

E

W

NRNTYLHWY

WVRQAPGQGLE

LQKPGQSPQL

WMGALDPKTGD

LIYKVSNRFSG

TAYSQKFKGRVT

VPDRFSGSGS

LTADKSTSTAYM

GTDFTLKISRV

ELSSLTSEDTAVY

EAEDVGVYYC

YCTRFYSYTYWG

SQNTHVPPTF

QGTLVTVSS

GQGTKLEIK

CD262
SELTQDPAVS
2281
SLRS
2282
GAN
2283
NSA
2284
EVQLVQSGGGVE
2285
GFTF
2286
INWQ
2287
AKIL
2288

(DR5)
VALGQTVRIT

YY

DSSG

RPGGSLRLSCAA

DDY

GGST

GAGR

CSGDSLRSYY

NHV

SGFTFDDYAMS

A

GWYF

ASWYQQKPG

V

WVRQAPGKGLE

DY

QAPVLVIYGA

WVSGINWQGGS

NNRPSGIPDRF

TGYADSVKGRVT

SGSSSGNTASL

ISRDNAKNSLYL

TITGAQAEDE

QMNSLRAEDTA

ADYYCNSADS

VYYCAKILGAGR

SGNHVVFGGG

GWYFDYWGKGT

TKLTVL

TVTVSS

CD80
ESALTQPPSVS
2289
TSNI
2290
DIN
2291
QSY
2292
QVQLQESGPGLV
2293
GGSI
2294
FYSS
2295
VRDR
2296

GAPGQKVTIS

GGYD

DSSL

KPSETLSLTCAVS

SGG

SGNT

LFSV

CTGSTSNIGGY

NAQ

GGSISGGYGWG

YG

VGMV

DLHWYQQLP

VFG

WIRQPPGKGLEW

YNNW

GTAPKLLIYDI

G

IGSFYSSSGNTYY

FDVW

NKRPSGISDRF

NPSLKSQVTISTD

SGSKSGTAAS

TSKNQFSLKLNS

LAITGLQTEDE

MTAADTAVYYC

ADYYCQSYDS

VRDRLFSVVGM

SLNAQVFGGG

VYNNWFDVWGP

TRLTVL

GVLVTVSS

CD22
DVQVTQSPSS
2297
QSLA
2298
GIS
2299
LQGT
2300
EVQLVQSGAEVK
2301
GYR
2302
INPG
2303
TREG
2304

LSASVGDRVTI

NSYG

HQP

KPGASVKVSCKA

FTNY

NNYA

YGNY

TCRSSQSLANS

NTF

YT

SGYRFTNYWIHW

W

GAWF

YGNTFLSWYL

VRQAPGQGLEWI

AY

HKPGKAPQLLI

GGINPGNNYATY

YGISNRFSGVP

RRKFQGRVTMT

DRFSGSGSGT

ADTSTSTVYMEL

DFTLTISSLQP

SSLRSEDTAVYY

EDFATYYCLQ

CTREGYGNYGA

GTHQPYTFGQ

WFAYWGQGTLV

GTKVEIK

TVSS

CD23
DIQMTQSPSSL
2305
QDIR
2306
VAS
2307
LQV
2308
EVQLVESGGGLA
2309
GFRF
2310
ISSS
2311
ASLT
2312

SASVGDRVTIT

YY

YSTP

KPGGSLRLSCAA

TFNN

GDPT

TGSD

CRASQDIRYY

RT

SGFRFTFNNYYM

YY

SW

LNWYQQKPG

DWVRQAPGQGL

KAPKLLIYVAS

EWVSRISSSGDPT

SLQSGVPSRFS

WYADSVKGRFTI

GSGSGTEFTLT

SRENANNTLFLQ

VSSLQPEDFAT

MNSLRAEDTAV

YYCLQVYSTP

YYCASLTTGSDS

RTFGQGTKVEI

WGQGVLVTVSS

K

CD20
DIQMTQSPSSL
2313
SSVS
2314
APS
2315
QQW
2316
EVQLVESGGGLV
2317
GYTF
2318
IYPG
2319
ARVV
2320

SASVGDRVTIT

Y

SFNP

QPGGSLRLSCAA

TSYN

NGDT

YYSN

CRASSSVSYM

PT

SGYTFTSYNMH

SYWY

HWYQQKPGK

WVRQAPGKGLE

FDV

APKPLIYAPSN

WVGAIYPGNGDT

LASGVPSRFSG

SYNQKFKGRFTIS

SGSGTDFTLTI

VDKSKNTLYLQ

SSLQPEDFATY

MNSLRAEDTAV

YCQQWSFNPP

YYCARVVYYSNS

TFGQGTKVEI

YWYFDVWGQGT

K

LVTVSS

CD37
EIVLTQSPATL
2321
ENVY
2322
FAK
2323
QHH
2324
EVQLVQSGAEVK
2325
GYSF
2326
IDPY
2327
ARSV
2328

SLSPGERATLS

SY

SDNP

KPGESLKISCKGS

TGY

YGGT

GPFD

CRASENVYSY

WT

GYSFTGYNMNW

N

S

LAWYQQKPG

VRQMPGKGLEW

QAPRLLIYFAK

MGNIDPYYGGTT

TLAEGIPARFS

YNRKFKGQVTIS

GSGSGTDFTLT

ADKSISTAYLQW

ISSLEPEDFAV

SSLKASDTAMYY

YYCQHHSDNP

CARSVGPFDSWG

WTFGQGTKVE

QGTLVTVSS

IK

CD22
DIQMTQSPSSL
2329
QSIV
2330
KVS
2331
FQGS
2332
EVQLVESGGGLV
2333
GYEF
2334
IYPGD
2335
ARDGS
2336

SASVGDRVTIT

HSVG

QFPY

QPGGSLRLSCAA

SRS

GDT

SWDW

CRSSQSIVHSV

NTF

T

SGYEFSRSWMN

W

YFDV

GNTFLEWYQQ

WVRQAPGKGLE

KPGKAPKLLIY

WVGRIYPGDGDT

KVSNRFSGVP

NYSGKFKGRFTIS

SRFSGSGSGTD

ADTSKNTAYLQ

FTLTISSLQPE

MNSLRAEDTAV

DFATYYCFQG

YYCARDGSSWD

SQFPYTFGQG

WYFDVWGQGTL

TKVEIK

VTVSS

fibro-
EIVLTQSPGTL
2337
QSVS
2338
YAS
2339
QQT
2340
EVQLLESGGGLV
2341
GFTF
2342
ISGS
2343
AKPF
2344

nectin
SLSPGERATLS

SSF

GRIP

QPGGSLRLSCAA

SSFS

SGTT

PYFD

extra
CRASQSVSSSF

PT

SGFTFSSFSMSW

Y

domain-
LAWYQQKPG

VRQAPGKGLEW

B
QAPRLLIYYAS

VSSISGSSGTTYY

SRATGIPDRFS

ADSVKGRFTISR

GSGSGTDFTLT

DNSKNTLYLQM

ISRLEPEDFAV

NSLRAEDTAVYY

YYCQQTGRIPP

CAKPFPYFDYWG

TFGQGTKVEI

QGTLVTVSS

K

CD3
DIQMTQSPSSL
2345
SSVS
2346
DTS
2347
QQW
2348
QVQLVQSGAEV
2349
GYTF
2350
INPR
2351
ARSAY
2352

SASVGDRVTIT

Y

SSNP

KKPGASVKVSCK

ISYT

SGYT

YDYDG

CSASSSVSYM

PT

ASGYTFISYTMH

FAY

NWYQQKPGK

WVRQAPGQGLE

APKRLIYDTSK

WMGYINPRSGYT

LASGVPSRFSG

HYNQKLKDKAT

SGSGTDFTLTI

LTADKSASTAYM

SSLQPEDFATY

ELSSLRSEDTAV

YCQQWSSNPP

YYCARSAYYDY

TFGGGTKVEI

DGFAYWGQGTL

K

VTVSS

*Italics means immune cell target/payload (scFv arm)

In one aspect, the present disclosure provides a heterodimeric multispecific antibody comprising 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, and wherein: (a) the first polypeptide chain comprises in the N-terminal to C-terminal direction: (i) a light chain variable domain of a first immunoglobulin (VL-1) that is capable of specifically binding to a first epitope; (ii) a light chain constant domain of the first immunoglobulin (CL-1); (iii) a flexible peptide linker comprising the amino acid sequence (GGGGS)₃(SEQ ID NO: 2506); and (iv) a light chain variable domain of a second immunoglobulin (VL-2) that is linked to a complementary heavy chain variable domain of the second immunoglobulin (VH-2), or a heavy chain variable domain of a second immunoglobulin (VH-2) that is linked to a complementary light chain variable domain of the second immunoglobulin (VL-2), wherein the VL-2 and VH-2 are capable of specifically binding to a second epitope, and are linked together via a flexible peptide linker comprising the amino acid sequence (GGGGS)₆(SEQ ID NO: 2507) to form a single-chain variable fragment; (b) the second polypeptide comprises in the N-terminal to C-terminal direction: (i) a heavy chain variable domain of the first immunoglobulin (VH-1) that is capable of specifically binding to the first epitope; (ii) a first CH1 domain of the first immunoglobulin (CH1-1); and (iii) a first heterodimerization domain of the first immunoglobulin, wherein the first heterodimerization domain is incapable of forming a stable homodimer with another first heterodimerization domain; (c) the third polypeptide comprises in the N-terminal to C-terminal direction: (i) a heavy chain variable domain of a third immunoglobulin (VH-3) that is capable of specifically binding to the first epitope; (ii) a second CH1 domain of the third immunoglobulin (CH1-3); and (iii) a second heterodimerization domain of the third immunoglobulin, wherein the second heterodimerization domain comprises an amino acid sequence or a nucleic acid sequence that is distinct from the first heterodimerization domain of the first immunoglobulin, wherein the second heterodimerization domain is incapable of forming a stable homodimer with another second heterodimerization domain, and wherein the second heterodimerization domain of the third immunoglobulin is configured to form a heterodimer with the first heterodimerization domain of the first immunoglobulin; (d) the fourth polypeptide comprises in the N-terminal to C-terminal direction: (i) a light chain variable domain of the third immunoglobulin (VL-3) that is capable of specifically binding to the first epitope; (ii) a light chain constant domain of the third immunoglobulin (CL-3); (iii) a flexible peptide linker comprising the amino acid sequence (GGGGS)₃(SEQ ID NO: 2506); and (iv) a light chain variable domain of a fourth immunoglobulin (VL-4) that is linked to a complementary heavy chain variable domain of the fourth immunoglobulin (VH-4), or a heavy chain variable domain of a fourth immunoglobulin (VH-4) that is linked to a complementary light chain variable domain of the fourth immunoglobulin (VL-4), wherein the VL-4 and VH-4 are capable of specifically binding to a third epitope, and are linked together via a flexible peptide linker comprising the amino acid sequence (GGGGS)₆(SEQ ID NO: 2507) to form a single-chain variable fragment, and wherein each of VL-2 and VL-4 independently comprises the CDR1 sequence, the CDR2 sequence and the CDR3 sequence of a V_Lamino acid sequence selected from any one 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 2289, 2329, and 2345; and/or wherein each of VH-2 and VH-4 independently comprises the CDR1 sequence, the CDR2 sequence and the CDR3 sequence of a V_Hamino acid sequence selected from any one 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.

In another aspect, the present disclosure provides a heterodimeric multispecific antibody comprising 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, and wherein: (a) the first polypeptide chain comprises in the N-terminal to C-terminal direction: (i) a light chain variable domain of a first immunoglobulin (VL-1) that is capable of specifically binding to a first epitope; (ii) a light chain constant domain of the first immunoglobulin (CL-1); (iii) a flexible peptide linker comprising the amino acid sequence (GGGGS)₃(SEQ ID NO: 2506); and (iv) a light chain variable domain of a second immunoglobulin (VL-2) that is linked to a complementary heavy chain variable domain of the second immunoglobulin (VH-2), or a heavy chain variable domain of a second immunoglobulin (VH-2) that is linked to a complementary light chain variable domain of the second immunoglobulin (VL-2), wherein VL-2 and VH-2 are capable of specifically binding to a second epitope, and are linked together via a flexible peptide linker comprising the amino acid sequence (GGGGS)₆(SEQ ID NO: 2507) to form a single-chain variable fragment; (b) the second polypeptide comprises in the N-terminal to C-terminal direction: (i) a heavy chain variable domain of the first immunoglobulin (VH-1) that is capable of specifically binding to the first epitope; (ii) a first CH1 domain of the first immunoglobulin (CH1-1); and (iii) a first heterodimerization domain of the first immunoglobulin, wherein the first heterodimerization domain is incapable of forming a stable homodimer with another first heterodimerization domain; (c) the third polypeptide comprises in the N-terminal to C-terminal direction: (i) a heavy chain variable domain of a third immunoglobulin (VH-3) that is capable of specifically binding to the first epitope; (ii) a second CH1 domain of the third immunoglobulin (CH1-3); and (iii) a second heterodimerization domain of the third immunoglobulin, wherein the second heterodimerization domain comprises an amino acid sequence or a nucleic acid sequence that is distinct from the first heterodimerization domain of the first immunoglobulin, wherein the second heterodimerization domain is incapable of forming a stable homodimer with another second heterodimerization domain, and wherein the second heterodimerization domain of the third immunoglobulin is configured to form a heterodimer with the first heterodimerization domain of the first immunoglobulin; (d) the fourth polypeptide comprises in the N-terminal to C-terminal direction: (i) a light chain variable domain of the third immunoglobulin (VL-3) that is capable of specifically binding to the first epitope; and (ii) a light chain constant domain of the third immunoglobulin (CL-3); and wherein VL-2 comprises the CDR1 sequence, the CDR2 sequence and the CDR3 sequence of a V_Lamino acid sequence selected from any one 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 2289, 2329, and 2345; and/or wherein VH-2 comprises the CDR1 sequence, the CDR2 sequence and the CDR3 sequence of a V_Hamino acid sequence selected from any one 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. In some embodiments, both VH-1 and VH-3 comprise the CDR1 sequence, the CDR2 sequence and the CDR3 sequence of a V_Hamino acid sequence selected from any one 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, 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; and/or both VL-1 and VL-3 comprise the CDR1 sequence, the CDR2 sequence and the CDR3 sequence of a V_Lamino acid sequence selected from any one 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, 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, 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.

Additionally or alternatively, in some embodiments of the heterodimeric multispecific antibodies disclosed herein, VH-2 or VH-4 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 a V_Hamino acid sequence selected from any one 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; and/or VL-2 or VL-4 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 a V_Lamino acid sequence selected from any one 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 2289, 2329, and 2345.

Additionally or alternatively, in some embodiments of the heterodimeric multispecific antibodies disclosed herein, the first immunoglobulin or the third immunoglobulin binds to a cell surface antigen 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), CD350 (FZD10), CD37, CD371 (CLEC12A), CD38, CD4, CD49b (a2), CD51 (a5), CD52, CD56, CD61 (a4b3), CD70, CD73 (NTSE), 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, Kallikreins, LINGO1, LOXL2, Ly6/PLAUR domain-containing protein 3, MADCAM1, MAG, Mesothelin, MT1-MMP (MMP14), MUC1, Mucin SAC, NaPi2b, NeuGc-GM3, notch, NOTCH2/NOTCH3 receptors, oxLDL, P-selectin, PCSK9, PDGFRA, PDGFRa, phosphatidylserine, polysialic acid, PSMA, PVRL4, RGMA, CD240D Blood group D antigen, 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 extra domain-B. The first immunoglobulin and the third immunoglobulin may bind to the same epitope on a target cell or two different epitopes on a target cell. In some embodiments, the target cell is a cancer cell.

Additionally or alternatively, in some embodiments of the heterodimeric multispecific antibodies disclosed herein, the first immunoglobulin and the third immunoglobulin bind to their respective epitopes with a monovalent affinity or an effective affinity that is less than 100 pM. In certain embodiments, the first immunoglobulin and the third immunoglobulin bind to cell surface epitopes that are up to 180 angstroms apart.

Human IgD constant region, Uniprot: P01880

(SEQ ID NO: 2381)

APTKAPDVFPIISGCRHPKDNSPVVLACLITGYHPTSVTVTWYMGTQSQP

QRTFPEIQRRDSYYMTSSQLSTPLQQWRQGEYKCVVQHTASKSKKEIFRW

PESPKAQASSVPTAQPQAEGSLAKATTAPATTRNTGRGGEEKKKEKEKEE

QEERETKTPECPSHTQPLGVYLLTPAVQDLWLRDKATFTCFVVGSDLKDA

HLTWEVAGKVPTGGVEEGLLERHSNGSQSQHSRLTLPRSLWNAGTSVTCT

LNHPSLPPQRLMALREPAAQAPVKLSLNLLASSDPPEAASWLLCEVSGFS

PPNILLMWLEDQREVNTSGFAPARPPPQPGSTTFWAWSVLRVPAPPSPQP

ATYTCVVSHEDSRTLLNASRSLEVSYVTDHGPMK

Human IgG1 constant region, Uniprot: P01857

(SEQ ID NO: 2382)

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV

HTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEP

KSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS

HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGK

EYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTC

LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW

QQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Human IgG2 constant region, Uniprot: P01859

(SEQ ID NO: 2383)

ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGV

HTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVER

KCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDP

EVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKC

KVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKG

FYPSDISVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGN

VFSCSVMHEALHNHYTQKSLSLSPGK

Human IgG3 constant region, Uniprot: P01860

(SEQ ID NO: 2384)

ASTKGPSVFPLAPCSRSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV

HTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYTCNVNHKPSNTKVDKRVEL

KTPLGDTTHTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPEPKSC

DTPPPCPRCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHED

PEVQFKWYVDGVEVHNAKTKPREEQYNSTFRVVSVLTVLHQDWLNGKEYK

CKVSNKALPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVK

GFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQQG

NIFSCSVMHEALHNRFTQKSLSLSPGK

Human IgM constant region, Uniprot: P01871

(SEQ ID NO: 2385)

GSASAPTLFPLVSCENSPSDTSSVAVGCLAQDFLPDSITLSWKYKNNSDI

SSTRGFPSVLRGGKYAATSQVLLPSKDVMQGTDEHVVCKVQHPNGNKEKN

VPLPVIAELPPKVSVFVPPRDGFFGNPRKSKLICQATGFSPRQIQVSWLR

EGKQVGSGVTTDQVQAEAKESGPTTYKVTSTLTIKESDWLGQSMFTCRVD

HRGLTFQQNASSMCVPDQDTAIRVFAIPPSFASIFLTKSTKLTCLVTDLT

TYDSVTISWTRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNSGER

FTCTVTHTDLPSPLKQTISRPKGVALHRPDVYLLPPAREQLNLRESATIT

CLVTGFSPADVFVQWMQRGQPLSPEKYVTSAPMPEPQAPGRYFAHSILTV

SEEEWNTGETYTCVAHEALPNRVTERTVDKSTGKPTLYNVSLVMSDTAGT

CY

Human IgG4 constant region, Uniprot: P01861

(SEQ ID NO: 2386)

ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGV

HTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVES

KYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQED

PEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYK

CKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVK

GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEG

NVFSCSVMHEALHNHYTQKSLSLSLGK

Human IgA1 constant region, Uniprot: P01876

(SEQ ID NO: 2387)

ASPTSPKVFPLSLCSTQPDGNVVIACLVQGFFPQEPLSVTWSESGQGVTA

RNFPPSQDASGDLYTTSSQLTLPATQCLAGKSVTCHVKHYTNPSQDVTVP

CPVPSTPPTPSPSTPPTPSPSCCHPRLSLHRPALEDLLLGSEANLTCTLT

GLRDASGVTFTWTPSSGKSAVQGPPERDLCGCYSVSSVLPGCAEPWNHGK

TFTCTAAYPESKTPLTATLSKSGNTFRPEVHLLPPPSEELALNELVTLTC

LARGFSPKDVLVRWLQGSQELPREKYLTWASRQEPSQGTTTFAVTSILRV

AAEDWKKGDTFSCMVGHEALPLAFTQKTIDRLAGKPTHVNVSVVMAEVDG

TCY

Human IgA2 constant region, Uniprot: P01877

(SEQ ID NO: 2388)

ASPTSPKVFPLSLDSTPQDGNVVVACLVQGFFPQEPLSVTWSESGQNVTA

RNFPPSQDASGDLYTTSSQLTLPATQCPDGKSVTCHVKHYTNPSQDVTVP

CPVPPPPPCCHPRLSLHRPALEDLLLGSEANLTCTLTGLRDASGATFTWT

PSSGKSAVQGPPERDLCGCYSVSSVLPGCAQPWNHGETFTCTAAHPELKT

PLTANITKSGNTFRPEVHLLPPPSEELALNELVTLTCLARGFSPKDVLVR

WLQGSQELPREKYLTWASRQEPSQGTTTFAVTSILRVAAEDWKKGDTFSC

MVGHEALPLAFTQKTIDRMAGKPTHVNVSVVMAEVDGTCY

Human Ig kappa constant region, Uniprot: P01834

(SEQ ID NO: 2389)

TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGN

SQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS

FNRGEC

In some embodiments, the immunoglobulin-related compositions 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 is 100% identical to SEQ ID NOS: 2381-2388. Additionally or alternatively, in some embodiments, the immunoglobulin-related compositions 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 is 100% identical to SEQ ID NO: 2389.

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.

In some embodiments, the immunoglobulin-related compositions of the present technology are chimeric, humanized, or monoclonal. The immunoglobulin-related compositions of the present technology can further be recombinantly fused to a heterologous polypeptide at the N or C terminus or chemically conjugated (including covalently and non-covalently conjugations) to polypeptides or other compositions. For example, the immunoglobulin-related compositions of the present technology can be recombinantly fused or conjugated to molecules useful as labels in detection assays and effector molecules such as heterologous polypeptides, drugs, or toxins. See, e.g., WO 92/08495; WO 91/14438; WO 89/12624; U.S. Pat. No. 5,314,995; and EP 0 396 387.

In any of the above embodiments of the immunoglobulin-related compositions of the present technology, the HDTVS antibody may be optionally conjugated to an agent selected from the group consisting of isotopes, dyes, chromagens, contrast agents, drugs, toxins, cytokines, enzymes, enzyme inhibitors, hormones, hormone antagonists, growth factors, radionuclides, metals, liposomes, nanoparticles, RNA, DNA or any combination thereof. For a chemical bond or physical bond, a functional group on the immunoglobulin-related composition typically associates with a functional group on the agent. Alternatively, a functional group on the agent associates with a functional group on the immunoglobulin-related composition.

The functional groups on the agent and immunoglobulin-related composition can associate directly. For example, a functional group (e.g., a sulfhydryl group) on an agent can associate with a functional group (e.g., sulfhydryl group) on an immunoglobulin-related composition to form a disulfide. Alternatively, the functional groups can associate through a cross-linking agent (i.e., linker). Some examples of cross-linking agents are described below. The cross-linker can be attached to either the agent or the immunoglobulin-related composition. The number of agents or immunoglobulin-related compositions in a conjugate is also limited by the number of functional groups present on the other. For example, the maximum number of agents associated with a conjugate depends on the number of functional groups present on the immunoglobulin-related composition. Alternatively, the maximum number of immunoglobulin-related compositions associated with an agent depends on the number of functional groups present on the agent.

In yet another embodiment, the conjugate comprises one immunoglobulin-related composition associated to one agent. In one embodiment, a conjugate comprises at least one agent chemically bonded (e.g., conjugated) to at least one immunoglobulin-related composition. The agent can be chemically bonded to an immunoglobulin-related composition by any method known to those in the art. For example, a functional group on the agent may be directly attached to a functional group on the immunoglobulin-related composition. Some examples of suitable functional groups include, for example, amino, carboxyl, sulfhydryl, maleimide, isocyanate, isothiocyanate and hydroxyl.

The agent may also be chemically bonded to the immunoglobulin-related composition by means of cross-linking agents, such as dialdehydes, carbodiimides, dimaleimides, and the like. Cross-linking agents can, for example, be obtained from Pierce Biotechnology, Inc., Rockford, Ill. The Pierce Biotechnology, Inc. web-site can provide assistance. Additional cross-linking agents include the platinum cross-linking agents described in U.S. Pat. Nos. 5,580,990; 5,985,566; and 6,133,038 of Kreatech Biotechnology, B.V., Amsterdam, The Netherlands.

Alternatively, the functional group on the agent and immunoglobulin-related composition can be the same. Homobifunctional cross-linkers are typically used to cross-link identical functional groups. Examples of homobifunctional cross-linkers include EGS (i.e., ethylene glycol bis[succinimidylsuccinate]), DSS (i.e., disuccinimidyl suberate), DMA (i.e., dimethyl adipimidate.2HCl), DTSSP (i.e., 3,3′-dithiobis[sulfosuccinimidylpropionate])), DPDPB (i.e., 1,4-di-[3′-(2′-pyridyldithio)-propionamido]butane), and BMH (i.e., bis-maleimidohexane). Such homobifunctional cross-linkers are also available from Pierce Biotechnology, Inc.

In other instances, it may be beneficial to cleave the agent from the immunoglobulin-related composition. The web-site of Pierce Biotechnology, Inc. described above can also provide assistance to one skilled in the art in choosing suitable cross-linkers which can be cleaved by, for example, enzymes in the cell. Thus the agent can be separated from the immunoglobulin-related composition. Examples of cleavable linkers include SMPT (i.e., 4-succinimidyloxycarbonyl-methyl-a-[2-pyridyldithio]toluene), Sulfo-LC-SPDP (i.e., sulfosuccinimidyl 6-(3-[2-pyridyldithio]-propionamido)hexanoate), LC-SPDP (i.e., succinimidyl 6-(3-[2-pyridyldithio]-propionamido)hexanoate), Sulfo-LC-SPDP (i.e., sulfosuccinimidyl 6-(3-[2-pyridyldithio]-propionamido)hexanoate), SPDP (i.e., N-succinimidyl 3-[2-pyridyldithio]-propionamidohexanoate), and AEDP (i.e., 3-[(2-aminoethyl)dithio]propionic acid HCl).

In another embodiment, a conjugate comprises at least one agent physically bonded with at least one immunoglobulin-related composition. Any method known to those in the art can be employed to physically bond the agents with the immunoglobulin-related compositions. For example, the immunoglobulin-related compositions and agents can be mixed together by any method known to those in the art. The order of mixing is not important. For instance, agents can be physically mixed with immunoglobulin-related compositions by any method known to those in the art. For example, the immunoglobulin-related compositions and agents can be placed in a container and agitated, by for example, shaking the container, to mix the immunoglobulin-related compositions and agents.

The immunoglobulin-related compositions can be modified by any method known to those in the art. For instance, the immunoglobulin-related composition may be modified by means of cross-linking agents or functional groups, as described above.

Heterodimerization. The present technology is dependent on heterodimerization of two IgG-scFv half-molecules through mutations in the heterodimerization domains using techniques known in the art. Any heterodimerization approach where the hinge domain is kept in place may be employed, provided that sufficient antibody stability is achieved.

Heterodimerization of CH2-CH3 domains. Formation of a heterodimeric trivalent/tetravalent multispecific antibody molecule of the present technology requires the interaction of four different polypeptide chains. Such interactions are difficult to achieve with efficiency within a single cell recombinant production system, due to the many variants of potential chain mispairings. One solution to increase the probability of mispairings, is to engineer “knobs-into-holes” type mutations into the desired polypeptide chain pairs. Such mutations favor heterodimerization over homodimerization. For example, with respect to Fc-Fc-interactions, an amino acid substitution (preferably a substitution with an amino acid comprising a bulky side group forming a ‘knob’, e.g., tryptophan) can be introduced into the CH2 or CH3 domain such that steric interference will prevent interaction with a similarly mutated domain and will obligate the mutated domain to pair with a domain into which a complementary, or accommodating mutation has been engineered, i.e., ‘the hole’ (e.g., a substitution with glycine). Such sets of mutations can be engineered into a pair of polypeptides that are included within the heterodimeric trivalent/tetravalent molecule (e.g., the second polypeptide chain and the third polypeptide chain), and further, engineered into any portion of the polypeptides chains of said pair. Methods of protein engineering to favor heterodimerization over homodimerization are well known in the art, in particular with respect to the engineering of immunoglobulin-like molecules, 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 hereby incorporated herein by reference in its entirety).

The design of variant Fc heterodimers from wildtype homodimers is illustrated by the concept of positive and negative design in the context of protein engineering by balancing stability vs. specificity, where mutations are introduced with the goal of driving heterodimer formation over homodimer formation when the polypeptides are expressed in cell culture conditions. Negative design strategies maximize unfavorable interactions for the formation of homodimers, by either introducing bulky sidechains on one chain and small sidechains on the opposite, for example the knobs-into-holes strategy developed by Genentech (Ridgway J B, Presta L G, Carter P. Protein Eng. 1996 July; 9(7):617-21; Atwell S, Ridgway J B, Wells J A, Carter P. J Mol. Biol. 270(1):26-35 (1997))), or by electrostatic engineering that leads to repulsion of homodimer formation, for example the electrostatic steering strategy developed by Amgen (Gunaskekaran K, et al. JBC 285 (25): 19637-19646 (2010)). In these two examples, negative design asymmetric point mutations are introduced into the wild-type CH3 domain to drive heterodimer formation. Other heterodimerization approaches are described in US 20120149876 (e.g., at Tables 1, 6 and 7), and US 20140294836 (e.g., at FIGS. 15A-B, 16A-B, and 17). Methods for engineering Fc heterodimers using electrostatic steering are described in detail in U.S. Pat. No. 8,592,562.

In some embodiments of the HDTVS antibodies disclosed herein, the second polypeptide chain and the third polypeptide chain comprise a first CH2-CH3 domain and a 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 amino acid modification K409F. In some embodiments, the amino acid modification at position L351 is L351Y, L3511, L351D, L351R or L351F. In some embodiments, the amino acid modification at position Y407 is Y407A, Y407V or Y4075. In certain embodiments, the amino acid modification at position T366 is T366A, T366I, T366L, T366M, T366Y, T366S, T366C, T366V or T366W.

In some embodiments of the HDTVS antibodies disclosed herein, the second polypeptide chain and the third polypeptide chain comprise a first CH2-CH3 domain and a second CH2-CH3 domain respectively, wherein the first CH2-CH3 domain comprises amino acid modifications L351Y and Y407A and the second CH2-CH3 domain comprises amino acid modifications T366A and K409F, and optionally wherein the first CH2-CH3 domain or the second CH2-CH3 domain comprises one or more amino acid modifications at position T411, D399, 5400, 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 may be D399R, D399W, D399Y or D399K. The amino acid modification at position 5400 may be S400E, 5400D, 5400R, or S400K. The amino acid modification at position F405 may be F4051, F405M, F405T, F4055, F405V or F405W. The amino acid modification at position N390 may be N390R, N390K or N390D. The amino acid modification at position K392 may be K392V, K392M, K392R, K392L, K392F or K392E.

In some embodiments of the HDTVS antibodies disclosed herein, the second polypeptide chain and the third polypeptide chain 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 comprise a set of amino acid modifications as shown in FIG. 11a. In some embodiments of the HDTVS antibodies disclosed herein, the second polypeptide chain and the third polypeptide chain 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 comprise a set of amino acid modifications as shown in FIG. 11b. In some embodiments of the HDTVS antibodies disclosed herein, the second polypeptide chain and the third polypeptide chain 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 comprise a set of amino acid modifications as shown in FIG. 11c. In some embodiments of the HDTVS antibodies disclosed herein, the second polypeptide chain and the third polypeptide chain 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 comprise a set of amino acid modifications as shown in FIG. 11d. In some embodiments of the HDTVS antibodies disclosed herein, the second polypeptide chain and the third polypeptide chain 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 comprise a set of amino acid modifications as shown in FIG. 11e.

Other Fc Modifications. In some embodiments, the heterodimeric trivalent/tetravalent multispecific antibodies of the present technology comprise a variant Fc region, wherein said variant Fc region comprises at least one amino acid modification relative to a wild-type Fc region (or the parental Fc region), such that said molecule has an altered affinity for an Fc receptor (e.g., an FcγR), provided that said variant Fc region does not have a substitution at positions that make a direct contact with Fc receptor based on crystallographic and structural analysis of Fc-Fc receptor interactions such as those disclosed by Sondermann et al., Nature, 406:267-273 (2000). Examples of positions within the Fc region that make a direct contact with an Fc receptor such as an 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.

In some embodiments, a heterodimeric trivalent/tetravalent multispecific antibody of the present technology has an altered affinity for activating and/or inhibitory receptors, and includes a variant Fc region with one or more amino acid modifications, wherein said one or more amino acid modification is a N297 substitution with alanine, or a K322 substitution with alanine.

Glycosylation Modifications. In some embodiments, heterodimeric trivalent/tetravalent multispecific antibodies of the present technology have an Fc region with variant glycosylation as compared to a parent Fc region. In some embodiments, variant glycosylation includes the absence of fucose; in some embodiments, variant glycosylation results from expression in GnT1-deficient CHO cells.

In some embodiments, the antibodies of the present technology, may have a modified glycosylation site relative to an appropriate reference antibody that binds to an antigen of interest, without altering the functionality of the antibody, e.g., binding activity to the antigen. As used herein, “glycosylation sites” include any specific amino acid sequence in an antibody to which an oligosaccharide (i.e., carbohydrates containing two or more simple sugars linked together) will specifically and covalently attach.

Oligosaccharide side chains are typically linked to the backbone of an antibody via either 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, threonine. For example, an Fc-glycoform that lacks certain oligosaccharides including fucose and terminal N-acetylglucosamine may be produced in special CHO cells and exhibit enhanced ADCC effector function.

In some embodiments, the carbohydrate content of an immunoglobulin-related composition disclosed herein is modified by adding or deleting a glycosylation site. Methods for modifying the carbohydrate content of antibodies are well known in the art and are included within the present technology, see, e.g., U.S. Pat. No. 6,218,149; EP 0359096B1; U.S. Patent Publication No. US 2002/0028486; International Patent Application Publication WO 03/035835; U.S. Patent Publication No. 2003/0115614; U.S. Pat. Nos. 6,218,149; 6,472,511; all of which are incorporated herein by reference in their entirety. In some embodiments, the carbohydrate content of an antibody (or relevant portion or component thereof) is modified by deleting one or more endogenous carbohydrate moieties of the antibody. In certain embodiments, the present 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, nonspecific FcR-dependent binding in normal tissues is eliminated or reduced (e.g., via N297A mutation in Fc region, which results in aglycosylation).

Engineered glycoforms may be useful for a variety of purposes, including but not limited to enhancing or reducing effector function. Engineered glycoforms may be generated by any method known to one skilled in the art, for example by using engineered or variant expression strains, by co-expression with one or more enzymes, for example DI N-acetylglucosaminyltransferase III (GnTIII), by expressing a molecule comprising an Fc region in various organisms or cell lines from various organisms, or by modifying carbohydrate(s) after the molecule comprising Fc region has been expressed. Methods for generating engineered glycoforms are known in the art, and include but are not limited to those described in Umana et al., 1999, Nat. Biotechnol. 17: 176-180; Davies et al., 2001, Biotechnol. Bioeng. 74:288-294; Shields et al., 2002, 1 Biol. Chem. 277:26733-26740; Shinkawa et al., 2003, J Biol. Chem. 278:3466-3473; U.S. Pat. No. 6,602,684; U.S. patent application Ser. No. 10/277,370; U.S. patent application Ser. No. 10/113,929; International Patent Application Publications WO 00/61739A1; WO 01/292246A1; WO 02/311140A1; WO 02/30954A1; POTILLEGENT™ technology (Biowa, Inc. Princeton, N.J.); GLYCOMAB™ glycosylation engineering technology (GLYCART biotechnology AG, Zurich, Switzerland); each of which is incorporated herein by reference in its entirety. See, e.g., International Patent Application Publication WO 00/061739; U.S. Patent Application Publication No. 2003/0115614; Okazaki et al., 2004, JMB, 336: 1239-49.

A. Methods of Preparing Heterodimeric Trivalent/Tetravalent Multispecific Antibodies of the Present Technology

General Overview. The heterodimeric trivalent/tetravalent multispecific antibodies of the present disclosure can be produced using a variety of methods well known in the art, including de novo protein synthesis and recombinant expression of nucleic acids encoding the binding proteins. Initially, a target antigen is chosen to which an antibody of the present technology can be raised. For example, in some embodiments, an antibody may be raised against a full-length target protein, or to a portion of the target protein. Techniques for generating antibodies directed to such target polypeptides are well known to those skilled in the art. Examples of such techniques include, for example, but are not limited to, those involving display libraries, xeno or human mice, hybridomas, and the like.

Generally, an antibody is obtained from an originating species. More particularly, the nucleic acid or amino acid sequence of the variable portion of the light chain, heavy chain or both, of an originating species antibody having specificity for a target antigen is obtained. An originating species is any species which was useful to generate the antibody of the present technology or library of antibodies, e.g., rat, mouse, rabbit, chicken, monkey, human, and the like.

Phage or phagemid display technologies are useful techniques to derive the antibodies of the present technology. Techniques for generating and cloning monoclonal antibodies are well known to those skilled in the art. Expression of sequences encoding antibodies of the present technology, can be carried out in E. coli.

Due to the degeneracy of nucleic acid coding sequences, other sequences which encode substantially the same amino acid sequences as those of the naturally occurring proteins may be used in the practice of the present technology. These include, but are not limited to, nucleic acid sequences including all or portions of the nucleic acid sequences encoding the above polypeptides, which are altered by the substitution of different codons that encode a functionally equivalent amino acid residue within the sequence, thus producing a silent change. It is appreciated that the nucleotide sequence of an immunoglobulin according to the present technology tolerates sequence homology variations 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 a variant yields an operative antibody which recognizes a target of interest. For example, one or more amino acid residues within a polypeptide sequence can be substituted by another amino acid of a similar polarity which acts as a functional equivalent, resulting in a silent alteration. Substitutes for an amino acid within the sequence may be selected from other members of the class to which the amino acid belongs. For example, the nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine. The polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine. The positively charged (basic) amino acids include arginine, lysine and histidine. The 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 which are differentially modified during or after translation, e.g., by glycosylation, proteolytic cleavage, linkage to an antibody molecule or other cellular ligands, etc. Additionally, an immunoglobulin encoding nucleic acid sequence can be mutated in vitro or in vivo to create and/or destroy translation, initiation, and/or termination sequences or to create variations in coding regions and/or form new restriction endonuclease sites or destroy pre-existing ones, to facilitate further in vitro modification. 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, use of Tab linkers (Pharmacia), and the like.

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 or a mouse heterodimeric trivalent/tetravalent multispecific monoclonal antibody. For preparation of monoclonal antibodies directed towards a target molecule of interest, any technique that provides for the production of antibody molecules by continuous cell line culture can be utilized. Such techniques include, but are not limited to, the hybridoma technique (See, e.g., Kohler & Milstein, 1975. Nature 256: 495-497); the trioma technique; the human B-cell hybridoma technique (See, e.g., Kozbor, et al., 1983. Immunol. Today 4: 72) and the EBV hybridoma technique to produce 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 can be utilized in the practice of the present technology and can 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 with Epstein Barr Virus in vitro (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 that encode regions of antibodies can be isolated. PCR utilizing primers derived from sequences encoding conserved regions of antibodies is used to amplify sequences encoding portions of antibodies from the population and then DNAs encoding polypeptide chains of the heterodimeric trivalent/tetravalent multispecific antibodies or fragments thereof, such as variable domains, are reconstructed from the amplified sequences. Such amplified sequences also can be fused to DNAs encoding other proteins—e.g., a bacteriophage coat, or a bacterial cell surface protein—for expression and display of the fusion polypeptides on phage or bacteria. Amplified sequences can then be expressed and further selected or isolated based, e.g., on the affinity of the expressed antibody or fragment thereof for an antigen or epitope present on the 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 routine methods. See, e.g., Milstein et al., (Galfre and Milstein, Methods Enzymol (1981) 73: 3-46). Screening the hybridomas using standard methods will produce monoclonal antibodies of varying specificity (i.e., for different epitopes) and affinity. A selected monoclonal antibody with the desired properties, e.g., binding to a target antigen, can be used as expressed by the hybridoma, it can be bound to a molecule such as polyethylene glycol (PEG) to alter its properties, or a cDNA encoding it can be isolated, sequenced and manipulated in various ways. Synthetic dendromeric trees can be added to reactive amino acid side chains, e.g., lysine, to enhance the immunogenic properties of a target protein. Also, CPG-dinucleotide techniques can be used to enhance the immunogenic properties of the target protein. Other manipulations include substituting or deleting particular amino acyl residues that contribute to instability of the antibody during storage or after administration to a subject, and affinity maturation techniques to improve affinity of the antibody towards its target antigen.

Hybridoma Technique. In some embodiments, the antibody of the present technology is a heterodimeric trivalent/tetravalent multispecific monoclonal antibody produced by a hybridoma which includes a B cell obtained from a transgenic non-human animal, e.g., a transgenic mouse, having a genome comprising a human heavy chain transgene and a light chain transgene fused to an immortalized cell. Hybridoma techniques include those known in the art and taught in Harlow et al., Antibodies: A Laboratory Manual Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 349 (1988); Hammerling et al., Monoclonal Antibodies And T-Cell Hybridomas, 563-681 (1981). Other methods for producing hybridomas and monoclonal antibodies are well known to those of skill in the art.

Phage Display Technique. As noted above, the antibodies of the present technology can be produced through the application of recombinant DNA and phage display technology. For example, heterodimeric trivalent/tetravalent multi specific 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 a phage particle which carries polynucleotide sequences encoding them. Phages with a desired binding property are selected from a repertoire or combinatorial antibody library (e.g., human or murine) by selecting directly with an antigen, typically an antigen bound or captured to a solid surface or bead. Phages used in these methods are typically filamentous phage including fd and M13 with Fab, Fv or disulfide stabilized Fv antibody domains that are recombinantly fused to either the phage gene III or gene VIII protein. In addition, methods can be adapted for the construction of Fab expression libraries (See, e.g., Huse, et al., Science 246: 1275-1281, 1989) to allow rapid and effective identification of monoclonal Fab fragments with the desired specificity for a target antigen, e.g., a target polypeptide or derivatives, fragments, analogs or homologs thereof. Other examples of phage display methods that can be used to make the antibodies of the present technology include those disclosed in Huston et al., Proc. Natl. Acad. Sci U.S.A., 85: 5879-5883, 1988; Chaudhary et al., Proc. Natl. Acad. Sci U.S.A., 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; WO 90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO 95/15982; WO 95/20401; WO 96/06213; WO 92/01047 (Medical Research Council et al.); WO 97/08320 (Morphosys); WO 92/01047 (CAT/MRC); WO 91/17271 (Affymax); and U.S. Pat. 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. Methods useful for displaying polypeptides on the surface of bacteriophage particles by attaching the polypeptides via disulfide bonds have been described by Lohning, U.S. Pat. No. 6,753,136. As described in the above references, after phage selection, the antibody coding regions from the phage can be isolated and used to generate whole antibodies, including human antibodies, or any other desired antigen binding fragment, and expressed in any desired host including mammalian cells, insect cells, plant cells, yeast, and bacteria. For example, techniques to recombinantly produce Fab, Fab′ and F(ab′)₂fragments can also be employed using methods known in the art such as those disclosed in WO 92/22324; 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.

Generally, hybrid antibodies or hybrid antibody fragments that are cloned into a display vector can be selected against the appropriate antigen in order to identify variants that maintain good binding activity, because the antibody or antibody fragment will be present on the surface of the phage or phagemid particle. See, e.g., Barbas III et al., Phage Display, A Laboratory Manual (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 2001). However, other vector formats could be used for this process, such as cloning the antibody fragment library into a lytic phage vector (modified T7 or Lambda Zap systems) for selection and/or screening.

Single-Chain Fvs. The heterodimeric trivalent/tetravalent multispecific antibody of the present technology comprises two single-chain Fvs. According to the present technology, techniques can be adapted for the production of single-chain antibodies specific to a target antigen (See, e.g., U.S. Pat. No. 4,946,778). Examples of techniques which can be used to produce single-chain Fvs and antibodies of the present technology include those described in U.S. Pat. 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.

Chimeric and Humanized Antibodies. In one embodiment, the heterodimeric trivalent/tetravalent multispecific antibody of the present technology is chimeric. In one embodiment, the heterodimeric trivalent/tetravalent multispecific antibody of the present technology is humanized. In one embodiment of the present technology, the donor and acceptor antibodies are monoclonal antibodies from different species. For example, the acceptor antibody is a human antibody (to minimize its antigenicity in a human), in which case the resulting CDR-grafted antibody is termed a “humanized” antibody.

Recombinant heterodimeric trivalent/tetravalent multispecific antibodies, such as chimeric and humanized monoclonal antibodies, comprising both human and non-human portions, can be made using standard recombinant DNA techniques, and are within the scope of the present technology. For some uses, including in vivo use of the heterodimeric trivalent/tetravalent multispecific antibody of the present technology in humans as well as use of these agents in in vitro detection assays, it is possible to use chimeric or humanized heterodimeric trivalent/tetravalent multispecific antibodies. Such chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art. Such useful methods include, e.g., but are not limited to, methods described in International Application No. PCT/US86/02269; U.S. Pat. No. 5,225,539; European Patent No. 184187; European Patent No. 171496; European Patent No. 173494; PCT International Publication No. WO 86/01533; U.S. Pat. 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. Pat. 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 0 239 400; WO 91/09967; U.S. Pat. Nos. 5,530,101; 5,585,089; 5,859,205; 6,248,516; EP460167), veneering or resurfacing (EP 0 592 106; EP 0 519 596; Padlan E. A., Molecular Immunology, 28: 489-498, 1991; Studnicka et al., Protein Engineering 7: 805-814, 1994; Roguska et al., PNAS 91: 969-973, 1994), and chain shuffling (U.S. Pat. No. 5,565,332). In one embodiment, a cDNA encoding a murine heterodimeric trivalent/tetravalent multispecific monoclonal antibody is digested with a restriction enzyme selected specifically to remove the sequence encoding the Fc constant region, and the equivalent portion of a cDNA encoding a human Fc constant region is substituted (See Robinson et al., PCT/US86/02269; Akira et al., European Patent Application 184,187; Taniguchi, European Patent Application 171,496; Morrison et al., European Patent Application 173,494; Neuberger et al., WO 86/01533; Cabilly et al. U.S. Pat. No. 4,816,567; Cabilly et al., European Patent Application 125,023; 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; and Shaw et al. (1988) J. Natl. Cancer Inst. 80: 1553-1559; U.S. Pat. Nos. 6,180,370; 6,300,064; 6,696,248; 6,706,484; 6,828,422.

In one embodiment, the present technology provides the construction of humanized heterodimeric trivalent/tetravalent multispecific antibodies that are unlikely to induce a human anti-mouse antibody (hereinafter referred to as “HAMA”) response, while still having an effective antibody effector function. As used herein, the terms “human” and “humanized”, in relation to antibodies, relate to any antibody which is expected to elicit a therapeutically tolerable weak immunogenic response in a human subject. In one embodiment, the present technology provides for a humanized heterodimeric trivalent/tetravalent multispecific antibody comprising both heavy chain and light chain polypeptides.

CDR Antibodies. In some embodiments, the heterodimeric trivalent/tetravalent multispecific antibody of the present technology is a CDR antibody. Generally the donor and acceptor antibodies used to generate the heterodimeric trivalent/tetravalent multispecific CDR antibody are monoclonal antibodies from different species; typically the acceptor antibody is a human antibody (to minimize its antigenicity in a human), in which case the resulting CDR-grafted antibody is termed a “humanized” antibody. The graft may be of a single CDR (or even a portion of a single CDR) within a single V_Hor V_Lof the acceptor antibody, or can be of multiple CDRs (or portions thereof) within one or both of the V_Hand V_L. Frequently, all three CDRs in all variable domains of the acceptor antibody will be replaced with the corresponding donor CDRs, though one need replace only as many as necessary to permit adequate binding of the resulting CDR-grafted antibody to the target antigen. Methods for generating CDR-grafted and humanized antibodies are taught by Queen et al. U.S. Pat. Nos. 5,585,089; 5,693,761; 5,693,762; and Winter U.S. Pat. No. 5,225,539; and EP 0682040. Methods useful to prepare V_Hand V_Lpolypeptides are taught by Winter et al., U.S. Pat. Nos. 4,816,397; 6,291,158; 6,291,159; 6,291,161; 6,545,142; EP 0368684; EP0451216; and EP0120694.

After selecting suitable framework region candidates from the same family and/or the same family member, either or both the heavy and light chain variable regions are produced by grafting the CDRs from the originating species into the hybrid framework regions. Assembly of hybrid antibodies or hybrid antibody fragments having hybrid variable chain regions with regard to either of the above aspects can be accomplished using conventional methods known to those skilled in the art. For example, DNA sequences encoding the hybrid variable domains described herein (i.e., frameworks based on the target species and CDRs from the originating species) can be produced by oligonucleotide synthesis and/or PCR. The nucleic acid encoding CDR regions can also be isolated from the originating species antibodies using suitable restriction enzymes and ligated into the target species framework by ligating with suitable ligation enzymes. Alternatively, the framework regions of the variable chains of the originating species antibody can be changed by site-directed mutagenesis.

Since the hybrids are constructed from choices among multiple candidates corresponding to each framework region, there exist many combinations of sequences which are amenable to construction in accordance with the principles described herein. Accordingly, libraries of hybrids can be assembled having members with different combinations of individual framework regions. Such libraries can be electronic database collections of sequences or physical collections of hybrids.

This process typically does not alter the acceptor antibody's FRs flanking the grafted CDRs. However, one skilled in the art can sometimes improve antigen binding affinity of the resulting heterodimeric trivalent/tetravalent multispecific CDR-grafted antibody by replacing certain residues of a given FR to make the FR more similar to the corresponding FR of the donor antibody. Suitable locations of the substitutions include amino acid residues adjacent to the CDR, or which are capable of interacting with a CDR (See, e.g., U.S. Pat. No. 5,585,089, especially columns 12-16). Or one skilled in the art can start with the donor FR and modify it to be more similar to the acceptor FR or a human consensus FR. Techniques for making these modifications are known in the art. Particularly if the resulting FR fits a human consensus FR for that position, or is at least 90% or more identical to such a consensus FR, doing so may not increase the antigenicity of the resulting modified heterodimeric trivalent/tetravalent multispecific CDR-grafted antibody significantly compared to the same antibody with a fully human FR.

Expression of Recombinant Heterodimeric Trivalent/Tetravalent Multispecific Antibodies. The desired nucleic acid sequences can be produced by recombinant methods (e.g., PCR mutagenesis of an earlier prepared variant of the desired polynucleotide) or by solid-phase DNA synthesis. Because of the degeneracy of the genetic code, a variety of nucleic acid sequences encode each immunoglobulin amino acid sequence, and the present disclosure includes all nucleic acids encoding the binding proteins described herein, which are suitable for use in accordance with the present disclosure.

Once the nucleotide sequence of the heterodimeric trivalent/tetravalent multispecific antibodies are determined, the nucleotide sequence may be manipulated using methods well known in the art, e.g., recombinant DNA techniques, site directed mutagenesis, PCR, etc. (see, for example, the techniques described in Sambrook et al., 2001, Molecular Cloning, A Laboratory Manual, 3rd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.; and Ausubel et al., eds., 1998, Current Protocols in Molecular Biology, John Wiley & Sons, NY, which are both incorporated by reference herein in their entireties), to generate, for example, antibodies having a different amino acid sequence, for example by generating amino acid substitutions, deletions, and/or insertions. In one embodiment, human libraries or any other libraries available in the art, can be screened by standard techniques known in the art, to clone the nucleic acids encoding the heterodimeric trivalent/tetravalent multispecific antibodies of the present disclosure.

As noted above, the antibodies of the present technology can be produced through the application of recombinant DNA technology. Recombinant polynucleotide constructs encoding a heterodimeric trivalent/tetravalent multispecific antibody of the present technology typically include an expression control sequence operably-linked to the coding sequences of heterodimeric trivalent/tetravalent multispecific antibody chains, including naturally-associated or heterologous promoter regions. As such, another aspect of the technology includes vectors containing one or more nucleic acid sequences encoding a heterodimeric trivalent/tetravalent multispecific antibody of the present technology. Methods which are well known to those skilled in the art can be used to construct expression vectors containing the coding sequences for the molecules of the present disclosure and appropriate transcriptional and translational control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. See, for example, the techniques described in Sambrook et al., 1990, Molecular Cloning, A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. and Ausubel et al. eds., 1998, Current Protocols in Molecular Biology, John Wiley & Sons, NY. For recombinant expression of one or more of the polypeptides of the present technology, the nucleic acid containing all or a portion of the nucleotide sequence encoding the heterodimeric trivalent/tetravalent multispecific antibody is inserted into an appropriate cloning vector, or an expression vector (i.e., a vector that contains the necessary elements for the transcription and translation of the inserted polypeptide coding sequence) by recombinant DNA techniques well known in the art and as detailed below. Methods for producing diverse populations of vectors have been described by Lerner et al., U.S. Pat. Nos. 6,291,160 and 6,680,192.

In general, expression vectors useful in recombinant DNA techniques are often in the form of plasmids. In the present disclosure, “plasmid” and “vector” can be used interchangeably as the plasmid is the most commonly used form of vector. However, the present technology is intended to include such other forms of expression vectors that are not technically plasmids, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions. Such viral vectors permit infection of a subject and expression of a construct in that subject. In some embodiments, the expression control sequences are eukaryotic promoter systems in vectors capable of transforming or transfecting eukaryotic host cells. Once the vector has been incorporated into the appropriate host, the host is maintained under conditions suitable for high level expression of the nucleotide sequences encoding the heterodimeric trivalent/tetravalent multispecific antibody, and the collection and purification of the heterodimeric trivalent/tetravalent multispecific antibody, e.g., cross-reacting heterodimeric trivalent/tetravalent multispecific antibodies. See generally, U.S. 2002/0199213. These expression vectors are typically replicable in the host organisms either as episomes or as an integral part of the host chromosomal DNA. Commonly, expression vectors contain selection markers, e.g., ampicillin-resistance or hygromycin-resistance, to permit detection of those cells transformed with the desired DNA sequences. Vectors can also encode signal peptide, e.g., pectate lyase, useful to direct the secretion of extracellular antibody fragments. See U.S. Pat. No. 5,576,195.

The recombinant expression vectors of the present technology comprise a nucleic acid encoding a protein having binding properties to a molecule of interest and in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vectors include one or more regulatory sequences, selected on the basis of the host cells to be used for expression that is operably-linked to the nucleic acid sequence to be expressed. Within a recombinant expression vector, “operably-linked” is intended to mean that the nucleotide sequence of interest is linked to the regulatory sequence(s) in a manner that allows for expression of the nucleotide sequence (e.g., in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell). The term “regulatory sequence” is intended to include promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are described, e.g., in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990). Regulatory sequences include those that direct constitutive expression of a nucleotide sequence in many types of host cell and those that direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences) or under certain environmental conditions (e.g., inducible regulatory sequences). It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of polypeptide desired, etc. Typical regulatory sequences useful as promoters of recombinant polypeptide expression (e.g., a heterodimeric trivalent/tetravalent multispecific antibody), include, e.g., but are not limited to, promoters of 3-phosphoglycerate kinase and other glycolytic enzymes. Inducible yeast promoters include, among others, promoters from alcohol dehydrogenase, isocytochrome C, and enzymes responsible for maltose and galactose utilization. In one embodiment, a polynucleotide encoding a heterodimeric trivalent/tetravalent multispecific antibody of the present technology is operably-linked to an ara B promoter and expressible in a host cell. See U.S. Pat. No. 5,028,530. The expression vectors of the present technology can be introduced into host cells to thereby produce polypeptides or peptides, including fusion polypeptides, encoded by nucleic acids as described herein (e.g., heterodimeric trivalent/tetravalent multispecific antibody, etc.).

Another aspect of the present technology pertains to heterodimeric trivalent/tetravalent multispecific antibody-expressing host cells, which contain a nucleic acid encoding one or more heterodimeric trivalent/tetravalent multispecific antibodies. A variety of host-expression vector systems may be utilized to express the heterodimeric trivalent/tetravalent multispecific antibodies of the present disclosure. Such host-expression systems represent vehicles by which the coding sequences of the heterodimeric trivalent/tetravalent multispecific antibodies of the present disclosure may be produced and subsequently purified, but also represent cells which may, when transformed or transfected with the appropriate nucleotide coding sequences, express the molecules of the present disclosure in situ. These include, but are not limited to, microorganisms such as bacteria (e.g., E. coli and B. subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA, expression vectors containing coding sequences for the heterodimeric trivalent/tetravalent multispecific antibodies of the present disclosure; yeast (e.g., Saccharomyces Pichia) transformed with recombinant yeast expression vectors containing sequences encoding the heterodimeric trivalent/tetravalent multispecific antibodies of the present disclosure; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing the sequences encoding the heterodimeric trivalent/tetravalent multispecific antibodies of the present disclosure; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus (CaMV) and tobacco mosaic virus (TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing sequences encoding the heterodimeric trivalent/tetravalent multispecific antibodies of the present disclosure; or mammalian cell systems (e.g., COS, CHO, BHK, 293, 293T, 3T3 cells, lymphotic cells (see U.S. Pat. No. 5,807,715), Per C.6 cells (human retinal cells developed by Crucell) harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5K promoter).

The recombinant expression vectors of the present technology can be designed for expression of a heterodimeric trivalent/tetravalent multispecific antibody in prokaryotic or eukaryotic cells. For example, a heterodimeric trivalent/tetravalent multispecific antibody can be expressed in bacterial cells such as Escherichia coli, insect cells (using baculovirus expression vectors), fungal cells, e.g., yeast, yeast cells or mammalian cells. Suitable host cells are discussed further in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990). Alternatively, the recombinant expression vector can be transcribed and translated in vitro, e.g., using T7 promoter regulatory sequences and T7 polymerase. Methods useful for the preparation and screening of polypeptides having a predetermined property, e.g., heterodimeric trivalent/tetravalent multispecific antibody, via expression of stochastically generated polynucleotide sequences have been previously described. See U.S. Pat. Nos. 5,763,192; 5,723,323; 5,814,476; 5,817,483; 5,824,514; 5,976,862; 6,492,107; 6,569,641.

Expression of polypeptides in prokaryotes is most often carried out in E. coli with vectors containing constitutive or inducible promoters directing the expression of either fusion or non-fusion polypeptides. Fusion vectors add a number of amino acids to a polypeptide encoded therein, usually to the amino terminus of the recombinant polypeptide. Such fusion vectors typically serve three purposes: (i) to increase expression of recombinant polypeptide; (ii) to increase the solubility of the recombinant polypeptide; and (iii) to aid in the purification of the recombinant polypeptide by acting as a ligand in affinity purification. Often, in fusion expression vectors, a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant polypeptide to enable separation of the recombinant polypeptide from the fusion moiety subsequent to purification of the fusion polypeptide. Such enzymes, and their cognate recognition sequences, include Factor Xa, thrombin and enterokinase. Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith and Johnson, 1988. Gene 67: 31-40), pMAL (New England Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) that fuse glutathione S-transferase (GST), maltose E binding polypeptide, or polypeptide A, respectively, to the target recombinant polypeptide.

Examples of suitable inducible non-fusion E. coli expression vectors include pTrc (Amrann et al., (1988) Gene 69: 301-315) and pET 11d (Studier et al., GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990) 60-89). Methods for targeted assembly of distinct active peptide or protein domains to yield multifunctional polypeptides via polypeptide fusion have been described by Pack et al., U.S. Pat. Nos. 6,294,353; 6,692,935. One strategy to maximize recombinant polypeptide expression, e.g., a heterodimeric trivalent/tetravalent multispecific antibody, in E. coli is to express the polypeptide in host bacteria with an impaired capacity to proteolytically cleave the recombinant polypeptide. See, e.g., Gottesman, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990) 119-128. Another strategy is to alter the nucleic acid sequence of the nucleic acid to be inserted into an expression vector so that the individual codons for each amino acid are those preferentially utilized in the expression host, e.g., E. coli (See, e.g., Wada, et al., 1992. Nucl. Acids Res. 20: 2111-2118). Such alteration of nucleic acid sequences of the present technology can be carried out by standard DNA synthesis techniques.

In another embodiment, the heterodimeric trivalent/tetravalent multispecific antibody expression vector is a yeast expression vector. Examples of vectors for expression in yeast Saccharomyces cerevisiae include pYepSec1 (Baldari, et al., 1987. EMBO J. 6: 229-234), pMFa (Kurjan and Herskowitz, Cell 30: 933-943, 1982), pJRY88 (Schultz et al., Gene 54: 113-123, 1987), pYES2 (Invitrogen Corporation, San Diego, Calif.), and picZ (Invitrogen Corp, San Diego, Calif.). Alternatively, a heterodimeric trivalent/tetravalent multispecific antibody can be expressed in insect cells using baculovirus expression vectors. Baculovirus vectors available for expression of polypeptides, e.g., heterodimeric trivalent/tetravalent multispecific antibody, in cultured insect cells (e.g., SF9 cells) include the pAc series (Smith, et al., Mol. Cell. Biol. 3: 2156-2165, 1983) and the pVL series (Lucklow and Summers, 1989. Virology 170: 31-39).

In yet another embodiment, a nucleic acid encoding a heterodimeric trivalent/tetravalent multispecific antibody of the present technology is expressed in mammalian cells using a mammalian expression vector. Examples of mammalian expression vectors include, e.g., but are not limited to, pCDM8 (Seed, Nature 329: 840, 1987) and pMT2PC (Kaufman, et al., EMBO J. 6: 187-195, 1987). When used in mammalian cells, the expression vector's control functions are often provided by viral regulatory elements. For example, commonly used promoters are derived from polyoma, adenovirus 2, cytomegalovirus, and simian virus 40. For other suitable expression systems for both prokaryotic and eukaryotic cells that are useful for expression of the heterodimeric trivalent/tetravalent multispecific antibody of the present technology, see, e.g., Chapters 16 and 17 of Sambrook, et al., MOLECULAR CLONING: A LABORATORY MANUAL. 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989.

In another embodiment, the recombinant mammalian expression vector is capable of directing expression of the nucleic acid in a particular cell type (e.g., tissue-specific regulatory elements). Tissue-specific regulatory elements are known in the art. Non-limiting examples of suitable tissue-specific promoters include the albumin promoter (liver-specific; Pinkert, et al., Genes Dev. 1: 268-277, 1987), lymphoid-specific promoters (Calame and Eaton, Adv. Immunol. 43: 235-275, 1988), promoters of T cell receptors (Winoto and Baltimore, EMBO J. 8: 729-733, 1989) and immunoglobulins (Banerji, et al., 1983. Cell 33: 729-740; Queen and Baltimore, Cell 33: 741-748, 1983.), neuron-specific promoters (e.g., the neurofilament promoter; Byrne and Ruddle, Proc. Natl. Acad. Sci. USA 86: 5473-5477, 1989), pancreas-specific promoters (Edlund, et al., 1985. Science 230: 912-916), and mammary gland-specific promoters (e.g., milk whey promoter; U.S. Pat. No. 4,873,316 and European Application Publication No. 264,166). Developmentally-regulated promoters are also encompassed, e.g., the murine hox promoters (Kessel and Gruss, Science 249: 374-379, 1990) and the α-fetoprotein promoter (Campes and Tilghman, Genes Dev. 3: 537-546, 1989).

Another aspect of the present methods pertains to host cells into which a recombinant expression vector of the present technology has been introduced. The terms “host cell” and “recombinant host cell” are used interchangeably herein. It is understood that such terms refer not only to the particular subject cell but also to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.

A host cell can be any prokaryotic or eukaryotic cell. For example, a heterodimeric trivalent/tetravalent multispecific antibody can be expressed in bacterial cells such as E. coli, insect cells, yeast or mammalian cells. Mammalian cells are a suitable host for expressing nucleotide segments encoding immunoglobulins or fragments thereof. See Winnacker, From Genes To Clones, (VCH Publishers, N Y, 1987). A number of suitable host cell lines capable of secreting intact heterologous proteins have been developed in the art, and include Chinese hamster ovary (CHO) cell lines, various COS cell lines, HeLa cells, L cells and myeloma cell lines. In some embodiments, the cells are non-human. For example, mammalian cells such as Chinese hamster ovary cells (CHO), in conjunction with a vector such as the major intermediate early gene promoter element from human cytomegalovirus can be an effective expression system for immunoglobulins (Foecking et al., 1998, Gene 45:101; Cockett et al., 1990, BioTechnology 8:2).

Expression vectors for these cells can include expression control sequences, such as an origin of replication, a promoter, an enhancer, and necessary processing information sites, such as ribosome binding sites, RNA splice sites, polyadenylation sites, and transcriptional terminator sequences. Queen et al., Immunol. Rev. 89: 49, 1986. Illustrative expression control sequences are promoters derived from endogenous genes, cytomegalovirus, SV40, adenovirus, bovine papillomavirus, and the like. Co et al., J Immunol. 148: 1149, 1992. Other suitable host cells are known to those skilled in the art.

Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. As used herein, the terms “transformation” and “transfection” are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, electroporation, biolistics or viral-based transfection. Other methods used to transform mammalian cells include the use of polybrene, protoplast fusion, liposomes, electroporation, and microinjection (See generally, Sambrook et al., Molecular Cloning). Suitable methods for transforming or transfecting host cells can be found in Sambrook, et al. (MOLECULAR CLONING: A LABORATORY MANUAL. 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989), and other laboratory manuals. The vectors containing the DNA segments of interest can be transferred into the host cell by well-known methods, depending on the type of cellular host.

For stable transfection of mammalian cells, it is known that, depending upon the expression vector and transfection technique used, only a small fraction of cells may integrate the foreign DNA into their genome. In order to identify and select these integrants, a gene that encodes a selectable marker (e.g., resistance to antibiotics) is generally introduced into the host cells along with the gene of interest. Various selectable markers include those that confer resistance to drugs, such as G418, hygromycin and methotrexate. Nucleic acid encoding a selectable marker can be introduced into a host cell on the same vector as that encoding the heterodimeric trivalent/tetravalent multispecific antibody or can be introduced on a separate vector. Cells stably transfected with the introduced nucleic acid can be identified by drug selection (e.g., cells that have incorporated the selectable marker gene will survive, while the other cells die).

A host cell that includes a heterodimeric trivalent/tetravalent multispecific antibody of the present technology, such as a prokaryotic or eukaryotic host cell in culture, can be used to produce (i.e., express) a recombinant heterodimeric trivalent/tetravalent multispecific antibody. In one embodiment, the method comprises culturing the host cell (into which a recombinant expression vector encoding the heterodimeric trivalent/tetravalent multispecific antibody has been introduced) in a suitable medium such that the heterodimeric trivalent/tetravalent multispecific antibody is produced. In another embodiment, the method further comprises the step of isolating the heterodimeric trivalent/tetravalent multispecific antibody from the medium or the host cell. Once expressed, collections of the heterodimeric trivalent/tetravalent multispecific antibody, e.g., the heterodimeric trivalent/tetravalent multispecific antibodies or the heterodimeric trivalent/tetravalent multispecific antibody-related polypeptides are purified from culture media and host cells. The heterodimeric trivalent/tetravalent multispecific antibody can be purified according to standard procedures of the art, including HPLC purification, column chromatography, gel electrophoresis and the like. In one embodiment, the heterodimeric trivalent/tetravalent multispecific antibody is produced in a host organism by the method of Boss et al., U.S. Pat. No. 4,816,397. Usually, heterodimeric trivalent/tetravalent multispecific antibody chains are expressed with signal sequences and are thus released to the culture media. However, if the heterodimeric trivalent/tetravalent multispecific antibody chains are not naturally secreted by host cells, the heterodimeric trivalent/tetravalent multispecific antibody chains can be released by treatment with mild detergent. Purification of recombinant polypeptides is well known in the art and includes ammonium sulfate precipitation, affinity chromatography purification technique, column chromatography, ion exchange purification technique, gel electrophoresis and the like (See generally Scopes, Protein Purification (Springer-Verlag, N.Y., 1982).

Polynucleotides encoding heterodimeric trivalent/tetravalent multispecific antibodies, e.g., the heterodimeric trivalent/tetravalent multispecific antibody coding sequences, can be incorporated in transgenes for introduction into the genome of a transgenic animal and subsequent expression in the milk of the transgenic animal. See, e.g., U.S. Pat. Nos. 5,741,957, 5,304,489, and 5,849,992. Suitable transgenes include coding sequences for light and/or heavy chains in operable linkage with a promoter and enhancer from a mammary gland specific gene, such as casein or β-lactoglobulin. For production of transgenic animals, transgenes can be microinjected into fertilized oocytes, or can be incorporated into the genome of embryonic stem cells, and the nuclei of such cells transferred into enucleated oocytes.

In bacterial systems, a number of expression vectors may be advantageously selected depending upon the use intended for the molecule being expressed. For example, when a large quantity of such a protein is to be produced, for the generation of pharmaceutical compositions of an antibody, vectors which direct the expression of high levels of fusion protein products that are readily purified may be desirable. Such vectors include, but are not limited, to the E. coli expression vector pUR278 (Ruther et al., 1983, EMBO J. 2:1791), in which the antibody coding sequence may be ligated individually into the vector in frame with the lac Z coding region so that a fusion protein is produced; pIN vectors (Inouye & Inouye, 1985, Nucleic Acids Res. 13:3101-3109; Van Heeke & Schuster, 1989, J. Biol. Chem. 24:5503-5509); and the like. pGEX vectors may also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST). In general, such fusion proteins are soluble and can easily be purified from lysed cells by adsorption and binding to a matrix glutathione-agarose beads followed by elution in the presence of free glutathione. The pGEX vectors are designed to include thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety.

In an insect system, Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes. The virus grows in Spodoptera frugiperda cells. The antibody coding sequence may be cloned individually into non-essential regions (e.g., the polyhedrin gene) of the virus and placed under control of an AcNPV promoter (e.g., the polyhedrin promoter).

In mammalian host cells, a number of viral-based expression systems may be utilized. In cases where an adenovirus is used as an expression vector, the antibody coding sequence of interest may be ligated to an adenovirus transcription/translation control complex, e.g., the late promoter and tripartite leader sequence. This chimeric gene may then be inserted in the adenovirus genome by in vitro or in vivo recombination. Insertion in a non-essential region of the viral genome (e.g., region E1 or E3) will result in a recombinant virus that is viable and capable of expressing the immunoglobulin molecule in infected hosts (e.g., see Logan & Shenk, 1984, Proc. Natl. Acad. Sci. USA 81:355-359). Specific initiation signals may also be required for efficient translation of inserted antibody coding sequences. These signals include the ATG initiation codon and adjacent sequences. Furthermore, the initiation codon must be in phase with the reading frame of the desired coding sequence to ensure translation of the entire insert. These exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, etc. (see Bittner et al., 1987, Methods in Enzymol. 153:51-544).

In addition, a host cell strain may be chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products may be important for the function of the protein. For example, in certain embodiments, the polypeptides of a heterodimeric trivalent/tetravalent multispecific antibody of the present disclosure may be expressed as a single gene product (e.g., as a single polypeptide chain, i.e., as a polyprotein precursor), requiring proteolytic cleavage by native or recombinant cellular mechanisms to form the separate polypeptides of the heterodimeric trivalent/tetravalent multispecific antibodies of the present disclosure. The present disclosure thus encompasses engineering a nucleic acid sequence to encode a polyprotein precursor molecule comprising the polypeptides of the heterodimeric trivalent/tetravalent multispecific antibodies of the present disclosure, which includes coding sequences capable of directing post translational cleavage of said polyprotein precursor. Post-translational cleavage of the polyprotein precursor results in the polypeptides of the heterodimeric trivalent/tetravalent multispecific antibodies of the present disclosure.

The post translational cleavage of the precursor molecule comprising the polypeptides of a heterodimeric trivalent/tetravalent multispecific antibody of the present disclosure may occur in vivo (i.e., within the host cell by native or recombinant cell systems/mechanisms, e.g. furin cleavage at an appropriate site) or may occur in vitro (e.g., incubation of said polypeptide chain in a composition comprising proteases or peptidases of known activity and/or in a composition comprising conditions or reagents known to foster the desired proteolytic action). Purification and modification of recombinant proteins are well known in the art such that the design of the polyprotein precursor could include a number of embodiments readily appreciated by a skilled artisan. Any known proteases or peptidases known in the art can be used for the described modification of the precursor molecule, e.g., thrombin (which recognizes the amino acid sequence LVPR{circumflex over ( )}GS (SEQ ID NO: 2500)), or factor Xa (which recognizes the amino acid sequence I(E/D)GR{circumflex over ( )} (SEQ ID NO: 2501) (Nagani et al., 1985, PNAS USA 82:7252-7255, and reviewed in Jenny et al., 2003, Protein Expr. Purif. 31:1-11, each of which is incorporated by reference herein in its entirety)), enterokinase (which recognizes the amino acid sequence DDDDK{circumflex over ( )} (SEQ ID NO: 2502) (Collins-Racie et al., 1995, Biotechnol. 13:982-987 hereby incorporated by reference herein in its entirety)), furin (which recognizes the amino acid sequence RXXR{circumflex over ( )}, with a preference for RX(K/R)R{circumflex over ( )} (SEQ ID NO: 2503 and SEQ ID NO: 2504, respectively) (additional R at P6 position appears to enhance cleavage)), and AcTEV (which recognizes the amino acid sequence ENLYFQ{circumflex over ( )}G (SEQ ID NO: 2505) (Parks et al., 1994, Anal. Biochem. 216:413 hereby incorporated by reference herein in its entirety)) and the Foot and Mouth Disease Virus Protease C3.

Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the foreign protein expressed. To this end, eukaryotic host cells which possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product may be used. Such mammalian host cells include but are not limited to CHO, VERY, BHK, Hela, COS, MDCK, 293, 293T, 3T3, WI38, BT483, Hs578T, HTB2, BT20 and T47D, CRL7030 and Hs578Bst.

For long-term, high-yield production of recombinant proteins, stable expression is desirable. For example, cell lines which stably express an antibody of the present disclosure may be engineered. Rather than using expression vectors which contain viral origins of replication, host cells can be transformed with DNA controlled by appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker. Following the introduction of the foreign DNA, engineered cells may be allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media. The selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci which in turn can be cloned and expanded into cell lines. This method may advantageously be used to engineer cell lines which express the antibodies of the present disclosure. Such engineered cell lines may be particularly useful in screening and evaluation of compounds that interact directly or indirectly with the heterodimeric trivalent/tetravalent multi specific antibodies of the present disclosure.

A number of selection systems may be used, including but not limited to the herpes simplex virus thymidine kinase (Wigler et al., 1977, Cell 11: 223), hypoxanthine-guanine phosphoribosyltransferase (Szybalska & Szybalski, 1992, Proc. Natl. Acad. Sci. USA 48: 202), and adenine phosphoribosyltransferase (Lowy et al., 1980, Cell 22: 817) genes can be employed in tk-, hgprt- or aprt-cells, respectively. Also, antimetabolite resistance can be used as the basis of selection for the following genes: dhfr, which confers resistance to methotrexate (Wigler et al., 1980, Proc. Natl. Acad. Sci. USA 77:357; O'Hare et al., 1981, Proc. Natl. Acad. Sci. USA 78: 1527); gpt, which confers resistance to mycophenolic acid (Mulligan & Berg, 1981, Proc. Natl. Acad. Sci. USA 78: 2072); neo, which confers resistance to the aminoglycoside G-418 Clinical Pharmacy 12: 488-505; Wu and Wu, 1991, 3:87-95; Tolstoshev, 1993, Ann. Rev. Pharmacol. Toxicol. 32:573-596; Mulligan, 1993, Science 260:926-932; and Morgan and Anderson, 1993, Ann. Rev. Biochem. 62:191-217; May, 1993, TIB TECH 11(5):155-215). Methods commonly known in the art of recombinant DNA technology which can be used are described in Ausubel et al. (eds.), 1993, Current Protocols in Molecular Biology, John Wiley & Sons, NY; Kriegler, 1990, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY; and in Chapters 12 and 13, Dracopoli et al. (eds), 1994, Current Protocols in Human Genetics, John Wiley & Sons, NY.; Colberre-Garapin et al., 1981, J. Mol. Biol. 150:1; and hygro, which confers resistance to hygromycin (Santerre et al., 1984, Gene 30:147).

The expression levels of a heterodimeric trivalent/tetravalent multispecific antibody of the present disclosure can be increased by vector amplification (for a review, see Bebbington and Hentschel, The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning, Vol. 3 (Academic Press, New York, 1987). When a marker in the vector system expressing an antibody is amplifiable, increase in the level of inhibitor present in culture of host cell will increase the number of copies of the selection marker gene. Since the amplified region is associated with the nucleotide sequence of a polypeptide of the heterodimeric trivalent/tetravalent multispecific antibody molecule, production of the polypeptide will also increase (Crouse et al., 1983, Mol. Cell. Biol. 3:257).

The host cell may be co-transfected with a plurality of expression vectors of the present disclosure, wherein each expression vector encodes at least one and no more than three of the first, second, third, or fourth polypeptide chains of the heterodimeric trivalent/tetravalent multispecific antibody. Alternatively, a single vector may be used which encodes the first, second, third, and fourth polypeptide chains of the heterodimeric trivalent/tetravalent multispecific antibody. The coding sequences for the polypeptides of the heterodimeric trivalent/tetravalent multispecific antibodies of the present disclosure may comprise cDNA or genomic DNA.

Once a molecule of the present disclosure (i.e., heterodimeric trivalent/tetravalent multispecific antibodies) has been recombinantly expressed, it may be purified by any method known in the art for purification of polypeptides, polyproteins or heterodimeric trivalent/tetravalent multispecific antibodies (e.g., analogous to antibody purification schemes based on antigen selectivity) for example, by chromatography (e.g., ion exchange, affinity, particularly by affinity for the specific antigen (optionally after Protein A selection where the heterodimeric trivalent/tetravalent multispecific antibodies molecule comprises an Fc domain (or portion thereof)), and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of polypeptides, polyproteins or heterodimeric trivalent/tetravalent multispecific antibodies.

Labeled Heterodimeric trivalent/tetravalent multispecific antibodies. In one embodiment, the heterodimeric trivalent/tetravalent multispecific antibody of the present technology is coupled with a label moiety, i.e., detectable group. The particular label or detectable group conjugated to the heterodimeric trivalent/tetravalent multispecific antibody is not a critical aspect of the technology, so long as it does not significantly interfere with the specific binding of the heterodimeric trivalent/tetravalent multispecific antibody of the present technology to its target antigens. The detectable group can be any material having a detectable physical or chemical property. Such detectable labels have been well-developed in the field of immunoassays and imaging. In general, almost any label useful in such methods can be applied to the present technology. Thus, a label is any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means. Labels useful in the practice of the present technology include magnetic beads (e.g., Dynabeads™), fluorescent dyes (e.g., fluorescein isothiocyanate, Texas red, rhodamine, and the like), radiolabels (e.g., ³H, ¹⁴C, ³⁵S ¹²⁵I, ¹²¹I, ¹³¹I, ¹¹²In, ⁹⁹mTc), other imaging agents such as microbubbles (for ultrasound imaging), ¹⁸F, 1^1C, ¹⁵O, (for Positron emission tomography), ^99mTc, ¹¹¹In (for Single photon emission tomography), enzymes (e.g., horse radish peroxidase, alkaline phosphatase and others commonly used in an ELISA), and calorimetric labels such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, and the like) beads. Patents that describe the use of such labels include U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241, each incorporated herein by reference in their entirety and for all purposes. See also Handbook of Fluorescent Probes and Research Chemicals (6^thEd., Molecular Probes, Inc., Eugene Oreg.).

The label can be coupled directly or indirectly to the desired component of an assay according to methods well known in the art. As indicated above, a wide variety of labels can be used, with the choice of label depending on factors such as required sensitivity, ease of conjugation with the compound, stability requirements, available instrumentation, and disposal provisions.

Non-radioactive labels are often attached by indirect means. Generally, a ligand molecule (e.g., biotin) is covalently bound to the molecule. The ligand then binds to an anti-ligand (e.g., streptavidin) molecule which is either inherently detectable or covalently bound to a signal system, such as a detectable enzyme, a fluorescent compound, or a chemiluminescent compound. A number of ligands and anti-ligands can be used. Where a ligand has a natural anti-ligand, e.g., biotin, thyroxine, and cortisol, it can be used in conjunction with the labeled, naturally-occurring anti-ligands. Alternatively, any haptenic or antigenic compound can be used in combination with an antibody, e.g., a heterodimeric trivalent/tetravalent multispecific antibody.

The molecules can also be conjugated directly to signal generating compounds, e.g., by conjugation with an enzyme or fluorophore. Enzymes of interest as labels will primarily be hydrolases, particularly phosphatases, esterases and glycosidases, or oxidoreductases, particularly peroxidases. Fluorescent compounds useful as labeling moieties, include, but are not limited to, e.g., fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, and the like. Chemiluminescent compounds useful as labeling moieties, include, but are not limited to, e.g., luciferin, and 2,3-dihydrophthalazinediones, e.g., luminol. For a review of various labeling or signal-producing systems which can be used, see U.S. Pat. No. 4,391,904.

Means of detecting labels are well known to those of skill in the art. Thus, for example, where the label is a radioactive label, means for detection include a scintillation counter or photographic film as in autoradiography. Where the label is a fluorescent label, it can be detected by exciting the fluorochrome with the appropriate wavelength of light and detecting the resulting fluorescence. The fluorescence can be detected visually, by means of photographic film, by the use of electronic detectors such as charge coupled devices (CCDs) or photomultipliers and the like. Similarly, enzymatic labels can be detected by providing the appropriate substrates for the enzyme and detecting the resulting reaction product. Finally simple colorimetric labels can be detected simply by observing the color associated with the label. Thus, in various dipstick assays, conjugated gold often appears pink, while various conjugated beads appear the color of the bead.

Some assay formats do not require the use of labeled components. For instance, agglutination assays can be used to detect the presence of the target antibodies, e.g., the heterodimeric trivalent/tetravalent multispecific antibodies. In this case, antigen-coated particles are agglutinated by samples comprising the target antibodies. In this format, none of the components need be labeled and the presence of the target antibody is detected by simple visual inspection.

Fusion Proteins. In one embodiment, the heterodimeric trivalent/tetravalent multispecific antibody of the present technology is a fusion protein. In some embodiments, the heterodimeric trivalent/tetravalent multispecific antibodies of the present technology, when fused to a second protein, can be used as an antigenic tag. Examples of domains that can be fused to polypeptides include not only heterologous signal sequences, but also other heterologous functional regions. The fusion does not necessarily need to be direct, but can occur through linker sequences. Moreover, fusion proteins of the present technology can also be engineered to improve characteristics of the heterodimeric trivalent/tetravalent multispecific antibodies. For instance, a region of additional amino acids, particularly charged amino acids, can be added to the N-terminus of the heterodimeric trivalent/tetravalent multispecific antibody to improve stability and persistence during purification from the host cell or subsequent handling and storage. Also, peptide moieties can be added to a heterodimeric trivalent/tetravalent multispecific antibody to facilitate purification. Such regions can be removed prior to final preparation of the heterodimeric trivalent/tetravalent multispecific antibody. The addition of peptide moieties to facilitate handling of polypeptides may be accomplished using familiar and routine techniques in the art. The heterodimeric trivalent/tetravalent multispecific antibody of the present technology can be fused to marker sequences, such as a peptide which facilitates purification of the fused polypeptide. In select embodiments, the marker amino acid sequence is a hexa-histidine peptide (SEQ ID NO: 2510), such as the tag provided in a pQE vector (QIAGEN, Inc., Chatsworth, Calif.), among others, many of which are commercially available. As described in Gentz et al., Proc. Natl. Acad. Sci. USA 86: 821-824, 1989, for instance, hexa-histidine (SEQ ID NO: 2510) provides for convenient purification of the fusion protein. Another peptide tag useful for purification, the “HA” tag, corresponds to an epitope derived from the influenza hemagglutinin protein. Wilson et al., Cell 37: 767, 1984.

Thus, any of these above fusion proteins can be engineered using the polynucleotides or the polypeptides of the present technology. Also, in some embodiments, the fusion proteins described herein show an increased half-life in vivo.

Fusion proteins having disulfide-linked dimeric structures (due to the IgG) can be more efficient in binding and neutralizing other molecules compared to the monomeric secreted protein or protein fragment alone. Fountoulakis et al., J. Biochem. 270: 3958-3964, 1995.

Similarly, EP-A-O 464 533 (Canadian counterpart 2045869) discloses fusion proteins comprising various portions of constant region of immunoglobulin molecules together with another human protein or a fragment thereof. In many cases, the Fc part in a fusion protein is beneficial in therapy and diagnosis, and thus can result in, e.g., improved pharmacokinetic properties. See EP-A 0232 262. Alternatively, deleting or modifying the Fc part after the fusion protein has been expressed, detected, and purified, may be desired. For example, the Fc portion can hinder therapy and diagnosis if the fusion protein is used as an antigen for immunizations. In drug discovery, e.g., human proteins, such as hIL-5, have been fused with Fc portions for the purpose of high-throughput screening assays to identify antagonists of hIL-5. Bennett et al., J. Molecular Recognition 8: 52-58, 1995; Johanson et al., J. Biol. Chem., 270: 9459-9471, 1995.

In some embodiments, the heterodimeric trivalent/tetravalent multispecific antibody of the present technology may be conjugated to a therapeutic agent or a payload. Examples of a payload include a toxin, a protein such as tumor necrosis factor, interferons including, but not limited to, α-interferon (IFN-α), β-interferon (IFN-β), nerve growth factor (NGF), platelet derived growth factor (PDGF), tissue plasminogen activator (TPA), an apoptotic agent (e.g., TNF-α, TNF-β, AIM I as disclosed in PCT Publication No. WO 97/33899), AIM II (see, PCT Publication No. WO 97/34911), Fas ligand (Takahashi et al., J. Immunol., 6:1567-1574, 1994), and VEGI (PCT Publication No. WO 99/23105), a thrombotic agent or an anti-angiogenic agent (e.g., angiostatin or endostatin), or a biological response modifier such as, for example, a lymphokine (e.g., interleukin-1 (“IL-1”), interleukin-2 (“IL-2”), interleukin-6 (“IL-6”), granulocyte macrophage colony stimulating factor (“GM-CSF”), and granulocyte colony stimulating factor (“G-CSF”), macrophage colony stimulating factor, (“M-CSF”), or a growth factor (e.g., growth hormone (“GH”); proteases, or ribonucleases. Examples of therapeutic agents include paclitaxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs thereof. Other examples of therapeutic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cisdichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC), and anti-mitotic agents (e.g., vincristine and vinblastine).

B. Identifying and Characterizing the Heterodimeric Trivalent/Tetravalent Multispecific Antibodies of the Present Technology

Methods for identifying and/or screening the heterodimeric trivalent/tetravalent multispecific antibodies of the present technology. Methods useful to identify and screen antibodies that possess the desired specificity to a target antigen include any immunologically-mediated techniques known within the art. Components of an immune response can be detected in vitro by various methods that are well known to those of ordinary skill in the art. For example, (1) cytotoxic T lymphocytes can be incubated with radioactively labeled target cells and the lysis of these target cells detected by the release of radioactivity; (2) helper T lymphocytes can be incubated with antigens and antigen presenting cells and the synthesis and secretion of cytokines measured by standard methods (Windhagen A et al., Immunity, 2: 373-80, 1995); (3) antigen presenting cells can be incubated with whole protein antigen and the presentation of that antigen on MHC detected by either T lymphocyte activation assays or biophysical methods (Harding et al., Proc. Natl. Acad. Sci., 86: 4230-4, 1989); (4) mast cells can be incubated with reagents that cross-link their Fc-epsilon receptors and histamine release measured by enzyme immunoassay (Siraganian et al., TIPS, 4: 432-437, 1983); and (5) enzyme-linked immunosorbent assay (ELISA).

Similarly, products of an immune response in either a model organism (e.g., mouse) or a human subject can also be detected by various methods that are well known to those of ordinary skill in the art. For example, (1) the production of antibodies in response to vaccination can be readily detected by standard methods currently used in clinical laboratories, e.g., an ELISA; (2) the migration of immune cells to sites of inflammation can be detected by scratching the surface of skin and placing a sterile container to capture the migrating cells over scratch site (Peters et al., Blood, 72: 1310-5, 1988); (3) the proliferation of peripheral blood mononuclear cells (PBMCs) in response to mitogens or mixed lymphocyte reaction can be measured using ³H-thymidine; (4) the phagocytic capacity of granulocytes, macrophages, and other phagocytes in PBMCs can be measured by placing PBMCs in wells together with labeled particles (Peters et al., Blood, 72: 1310-5, 1988); and (5) the differentiation of immune system cells can be measured by labeling PBMCs with antibodies to CD molecules such as CD4 and CD8 and measuring the fraction of the PBMCs expressing these markers.

In one embodiment, heterodimeric trivalent/tetravalent multispecific antibodies of the present technology are selected using display of target antigen peptides on the surface of replicable genetic packages. See, e.g., U.S. Pat. Nos. 5,514,548; 5,837,500; 5,871,907; 5,885,793; 5,969,108; 6,225,447; 6,291,650; 6,492,160; EP 585 287; EP 605522; EP 616640; EP 1024191; EP 589 877; EP 774 511; EP 844 306. Methods useful for producing/selecting a filamentous bacteriophage particle containing a phagemid genome encoding for a binding molecule with a desired specificity has been described. See, e.g., EP 774 511; U.S. Pat. Nos. 5,871,907; 5,969,108; 6,225,447; 6,291,650; 6,492,160.

In some embodiments, heterodimeric trivalent/tetravalent multispecific antibodies of the present technology are selected using display of target antigen peptides on the surface of a yeast host cell. Methods useful for the isolation of scFv polypeptides by yeast surface display have been described by Kieke et al., Protein Eng. 1997 November; 10(11): 1303-10.

In some embodiments, heterodimeric trivalent/tetravalent multispecific antibodies of the present technology are selected using ribosome display. Methods useful for identifying ligands in peptide libraries using ribosome display have been described by Mattheakis et al., Proc. Natl. Acad. Sci. USA 91: 9022-26, 1994; and Hanes et al., Proc. Natl. Acad. Sci. USA 94: 4937-42, 1997.

In certain embodiments, heterodimeric trivalent/tetravalent multispecific antibodies of the present technology are selected using tRNA display of target antigen peptides. Methods useful for in vitro selection of ligands using tRNA display have been described by Merryman et al., Chem. Biol., 9: 741-46, 2002.

In one embodiment, heterodimeric trivalent/tetravalent multispecific antibodies of the present technology are selected using RNA display. Methods useful for selecting peptides and proteins using RNA display libraries have been described by Roberts et al. Proc. Natl. Acad. Sci. USA, 94: 12297-302, 1997; and Nemoto et al., FEBS Lett., 414: 405-8, 1997. Methods useful for selecting peptides and proteins using unnatural RNA display libraries have been described by Frankel et al., Curr. Opin. Struct. Biol., 13: 506-12, 2003.

In some embodiments, heterodimeric trivalent/tetravalent multispecific antibodies of the present technology are expressed in the periplasm of gram negative bacteria and mixed with labeled target antigen. See WO 02/34886. In clones expressing recombinant polypeptides with affinity for a target antigen, the concentration of the labeled target antigen bound to the heterodimeric trivalent/tetravalent multispecific antibodies is increased and allows the cells to be isolated from the rest of the library as described in Harvey et al., Proc. Natl. Acad. Sci. 22: 9193-98 2004 and U.S. Pat. Publication No. 2004/0058403.

After selection of the desired heterodimeric trivalent/tetravalent multispecific antibodies, it is contemplated that said antibodies can be produced in large volume by any technique known to those skilled in the art, e.g., prokaryotic or eukaryotic cell expression and the like. For example, the heterodimeric trivalent/tetravalent multispecific antibodies can be produced by using conventional techniques to construct an expression vector that encodes an antibody heavy chain and/or light chain in which the CDRs and, if necessary, a minimal portion of the variable region framework, that are required to retain original species antibody binding specificity (as engineered according to the techniques described herein) are derived from the originating species antibody and the remainder of the antibody is derived from a target species immunoglobulin which can be manipulated as described herein, thereby producing a vector for the expression of a hybrid antibody heavy chain.

Measurement of Antigen Binding. In some embodiments, an antigen binding assay refers to an assay format wherein a target antigen and a heterodimeric trivalent/tetravalent multispecific antibody are mixed under conditions suitable for binding between the target antigen and the heterodimeric trivalent/tetravalent multispecific antibody and assessing the amount of binding between the target antigen and the heterodimeric trivalent/tetravalent multispecific antibody. The amount of binding is compared with a suitable control, which can be the amount of binding in the absence of the target antigen, the amount of the binding in the presence of a non-specific immunoglobulin composition, or both. The amount of binding can be assessed by any suitable method. Binding assay methods include, e.g., ELISA, radioimmunoassays, scintillation proximity assays, fluorescence energy transfer assays, liquid chromatography, membrane filtration assays, and the like. Biophysical assays for the direct measurement of target antigen binding to a heterodimeric trivalent/tetravalent multispecific antibody are, e.g., nuclear magnetic resonance, fluorescence, fluorescence polarization, surface plasmon resonance (BIACORE chips) and the like. Specific binding is determined by standard assays known in the art, e.g., radioligand binding assays, ELISA, FRET, immunoprecipitation, SPR, NMR (2D-NMR), mass spectroscopy and the like. If the specific binding of a candidate heterodimeric trivalent/tetravalent multispecific antibody is at least 1 percent greater than the binding observed in the absence of the candidate heterodimeric trivalent/tetravalent multispecific antibody, the candidate heterodimeric trivalent/tetravalent multi specific antibody is useful as a heterodimeric trivalent/tetravalent multispecific antibody of the present technology.

Measurement of Target Antigen Neutralization. As used here, “target antigen neutralization” refers to reduction of the activity and/or expression of a target antigen through the binding of a heterodimeric trivalent/tetravalent multispecific antibody disclosed herein. The capacity of heterodimeric trivalent/tetravalent multispecific antibodies of the present technology to neutralize activity/expression of a target antigen may be assessed in vitro or in vivo using methods known in the art.

Uses of the Heterodimeric Trivalent/Tetravalent Multispecific Antibodies of the Present Technology

General. The heterodimeric trivalent/tetravalent multispecific antibodies of the present technology are useful in methods known in the art relating to the localization and/or quantitation of a target antigen (e.g., for use in measuring levels of the target antigen within appropriate physiological samples, for use in diagnostic methods, for use in imaging the target antigen, and the like). Antibodies of the present technology are useful to isolate a target antigen by standard techniques, such as affinity chromatography or immunoprecipitation. A heterodimeric trivalent/tetravalent multispecific antibody of the present technology can facilitate the purification of natural immunoreactive target antigens from biological samples, e.g., mammalian sera or cells as well as recombinantly-produced immunoreactive target antigens expressed in a host system. Moreover, heterodimeric trivalent/tetravalent multispecific antibodies can be used to detect an immunoreactive target antigen (e.g., in plasma, a cellular lysate or cell supernatant) in order to evaluate the abundance and pattern of expression of the immunoreactive molecule. The heterodimeric trivalent/tetravalent multispecific antibodies of the present technology can be used diagnostically to monitor immunoreactive target antigen levels in tissue as part of a clinical testing procedure, e.g., to determine the efficacy of a given treatment regimen. As noted above, the detection can be facilitated by coupling (i.e., physically linking) the heterodimeric trivalent/tetravalent multispecific antibodies of the present technology to a detectable sub stance.

Detection of target antigen. An exemplary method for detecting the presence or absence of an immunoreactive target antigen in a biological sample involves obtaining a biological sample from a test subject and contacting the biological sample with a heterodimeric trivalent/tetravalent multispecific antibody of the present technology capable of detecting an immunoreactive target antigen such that the presence of an immunoreactive target antigen is detected in the biological sample. Detection may be accomplished by means of a detectable label attached to the antibody.

The term “labeled” with regard to the heterodimeric trivalent/tetravalent multispecific antibody is intended to encompass direct labeling of the antibody by coupling (i.e., physically linking) a detectable substance to the antibody, as well as indirect labeling of the antibody by reactivity with another compound that is directly labeled, such as a secondary antibody. Examples of indirect labeling include detection of a primary antibody using a fluorescently-labeled secondary antibody and end-labeling of a DNA probe with biotin such that it can be detected with fluorescently-labeled streptavidin.

In some embodiments, the heterodimeric trivalent/tetravalent multispecific antibodies disclosed herein are conjugated to one or more detectable labels. For such uses, heterodimeric trivalent/tetravalent multispecific antibodies may be detectably labeled by covalent or non-covalent attachment of a chromogenic, enzymatic, radioisotopic, isotopic, fluorescent, toxic, chemiluminescent, nuclear magnetic resonance contrast agent or other label.

Examples of suitable chromogenic labels include diaminobenzidine and 4-hydroxyazo-benzene-2-carboxylic acid. Examples of suitable enzyme labels include malate dehydrogenase, staphylococcal nuclease, Δ-5-steroid isomerase, yeast-alcohol dehydrogenase, α-glycerol phosphate dehydrogenase, triose phosphate isomerase, peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, β-galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase, and acetylcholine esterase.

Examples of suitable radioisotopic labels include ³H, ¹¹¹In, ¹²⁵I, ¹³¹I, ³²P, ³⁵S, ¹⁴C, ⁵¹Cr, ⁵⁷To, ⁵⁸Co, ⁵⁹Fe, ⁷⁵Se, ¹⁵²Eu, ⁹⁰Y, ⁶⁷Cu, ²¹⁷Ci, ²¹¹At, ²¹²Pb, ⁴⁷Sc, ¹⁰⁹Pd, etc. ¹¹¹In is an exemplary isotope where in vivo imaging is used since it avoids the problem of dehalogenation of the ¹²⁵I or ¹³¹I-labeled heterodimeric trivalent/tetravalent multispecific antibodies by the liver. In addition, this isotope has a more favorable gamma emission energy for imaging (Perkins et al, Eur. J. Nucl. Med. 70:296-301 (1985); Carasquillo et al., J. Nucl. Med. 25:281-287 (1987)). For example, ¹¹¹In coupled to monoclonal antibodies with 1-(P-isothiocyanatobenzyl)-DPTA exhibits little uptake in non-tumorous tissues, particularly the liver, and enhances specificity of tumor localization (Esteban et al., J. Nucl. Med. 28:861-870 (1987)). Examples of suitable non-radioactive isotopic labels include ¹⁵⁷Gd, ⁵⁵Mn, ¹⁶²Dy, ⁵²Tr, and ⁵⁶Fe.

Examples of suitable fluorescent labels include an ¹⁵²Eu label, a fluorescein label, an isothiocyanate label, a rhodamine label, a phycoerythrin label, a phycocyanin label, an allophycocyanin label, a Green Fluorescent Protein (GFP) label, an o-phthaldehyde label, and a fluorescamine label. Examples of suitable toxin labels include diphtheria toxin, ricin, and cholera toxin.

Examples of chemiluminescent labels include a luminol label, an isoluminol label, an aromatic acridinium ester label, an imidazole label, an acridinium salt label, an oxalate ester label, a luciferin label, a luciferase label, and an aequorin label. Examples of nuclear magnetic resonance contrasting agents include heavy metal nuclei such as Gd, Mn, and iron.

The detection method of the present technology can be used to detect an immunoreactive target antigen in a biological sample in vitro as well as in vivo. In vitro techniques for detection of an immunoreactive target antigen include enzyme linked immunosorbent assays (ELISAs), Western blots, immunoprecipitations, radioimmunoassay, and immunofluorescence. Furthermore, in vivo techniques for detection of an immunoreactive target antigen include introducing into a subject a labeled heterodimeric trivalent/tetravalent multispecific antibody. For example, the heterodimeric trivalent/tetravalent multispecific antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques. In one embodiment, the biological sample contains target antigen molecules from the test subject.

Immunoassay and Imaging. A heterodimeric trivalent/tetravalent multispecific antibody of the present technology can be used to assay immunoreactive target antigen levels in a biological sample (e.g., human plasma) using antibody-based techniques. For example, protein expression in tissues can be studied with classical immunohistological methods. Jalkanen, M. et al., J. Cell. Biol. 101: 976-985, 1985; Jalkanen, M. et al., J. Cell. Biol. 105: 3087-3096, 1987. Other antibody-based methods useful for detecting protein gene expression include immunoassays, such as the enzyme linked immunosorbent assay (ELISA) and the radioimmunoassay (RIA). Suitable antibody assay labels are known in the art and include enzyme labels, such as, glucose oxidase, and radioisotopes or other radioactive agent, such as iodine (¹²⁵I, ¹²¹I, ¹³¹I), and carbon (¹⁴C), sulfur (³⁵S), tritium (³H), indium (¹¹²In), and technetium (⁹⁹mTc), and fluorescent labels, such as fluorescein, rhodamine, and green fluorescent protein (GFP), as well as biotin.

In addition to assaying immunoreactive target antigen levels in a biological sample, heterodimeric trivalent/tetravalent multispecific antibodies of the present technology may be used for in vivo imaging of the target antigen. Antibodies useful for this method include those detectable by X-radiography, NMR or ESR. For X-radiography, suitable labels include radioisotopes such as barium or cesium, which emit detectable radiation but are not overtly harmful to the subject. Suitable markers for NMR and ESR include those with a detectable characteristic spin, such as deuterium, which can be incorporated into the heterodimeric trivalent/tetravalent multispecific antibodies by labeling of nutrients for the relevant scFv clone.

A heterodimeric trivalent/tetravalent multispecific antibody which has been labeled with an appropriate detectable imaging moiety, such as a radioisotope (e.g., ¹³¹I, ¹¹²In, ⁹⁹mTc), a radio-opaque substance, or a material detectable by nuclear magnetic resonance, is introduced (e.g., parenterally, subcutaneously, or intraperitoneally) into the subject. It will be understood in the art that the size of the subject and the imaging system used will determine the quantity of imaging moiety needed to produce diagnostic images. In the case of a radioisotope moiety, for a human subject, the quantity of radioactivity injected will normally range from about 5 to 20 millicuries of ⁹⁹mTc. The labeled heterodimeric trivalent/tetravalent multispecific antibody will then accumulate at the location of cells which contain the specific target antigen. For example, labeled heterodimeric trivalent/tetravalent multispecific antibodies of the present technology will accumulate within the subject in cells and tissues in which the target antigen has localized.

Thus, the present technology provides a diagnostic method of a medical condition, which involves: (a) assaying the expression of immunoreactive target antigen by measuring binding of a heterodimeric trivalent/tetravalent multispecific antibody of the present technology in cells or body fluid of an individual; (b) comparing the amount of immunoreactive target antigen present in the sample with a standard reference, wherein an increase or decrease in immunoreactive target antigen levels compared to the standard is indicative of a medical condition.

Affinity Purification. The heterodimeric trivalent/tetravalent multispecific antibodies of the present technology may be used to purify immunoreactive target antigen from a sample. In some embodiments, the antibodies are immobilized on a solid support. Examples of such solid supports include plastics such as polycarbonate, complex carbohydrates such as agarose and sepharose, acrylic resins and such as polyacrylamide and latex beads. Techniques for coupling antibodies to such solid supports are well known in the art (Weir et al., “Handbook of Experimental Immunology” 4th Ed., Blackwell Scientific Publications, Oxford, England, Chapter 10 (1986); Jacoby et al., Meth. Enzym. 34 Academic Press, N.Y. (1974)).

The simplest method to bind the antigen to the antibody-support matrix is to collect the beads in a column and pass the antigen solution down the column. The efficiency of this method depends on the contact time between the immobilized antibody and the antigen, which can be extended by using low flow rates. The immobilized antibody captures the antigen as it flows past. Alternatively, an antigen can be contacted with the antibody-support matrix by mixing the antigen solution with the support (e.g., beads) and rotating or rocking the slurry, allowing maximum contact between the antigen and the immobilized antibody. After the binding reaction has been completed, the slurry is passed into a column for collection of the beads. The beads are washed using a suitable washing buffer and then the pure or substantially pure antigen is eluted.

An antibody or target antigen of interest can be conjugated to a solid support, such as a bead. In addition, a first solid support such as a bead can also be conjugated, if desired, to a second solid support, which can be a second bead or other support, by any suitable means, including those disclosed herein for conjugation of a molecule to a support. Accordingly, any of the conjugation methods and means disclosed herein with reference to conjugation of a molecule to a solid support can also be applied for conjugation of a first support to a second support, where the first and second solid support can be the same or different.

Appropriate linkers, which can be cross-linking agents, for use for conjugating a molecule to a solid support include a variety of agents that can react with a functional group present on a surface of the support, or with the molecule, or both. Reagents useful as cross-linking agents include homo-bi-functional and, in particular, hetero-bi-functional reagents. Useful bi-functional cross-linking agents include, but are not limited to, N-SIAB, dimaleimide, DTNB, N-SATA, N-SPDP, SMCC and 6-HYNIC. In one exemplary embodiment, a cross-linking agent can be selected to provide a selectively cleavable bond between a target polypeptide and the solid support. For example, a photolabile cross-linker, such as 3-amino-(2-nitrophenyl)propionic acid can be employed as a means for cleaving a target polypeptide from a solid support. (Brown et al., Mol. Divers, pp, 4-12 (1995); Rothschild et al., Nucl. Acids Res., 24:351-66 (1996); and U.S. Pat. No. 5,643,722). Other cross-linking reagents are well-known in the art. (See, e.g., Wong (1991), supra; and Hermanson (1996), supra).

An antibody or target polypeptide can be immobilized on a solid support, such as a bead, through a covalent amide bond formed between a carboxyl group functionalized bead and the amino terminus of the target polypeptide or, conversely, through a covalent amide bond formed between an amino group functionalized bead and the carboxyl terminus of the target polypeptide. In addition, a bi-functional trityl linker can be attached to the support, e.g., to the 4-nitrophenyl active ester on a resin, such as a Wang resin, through an amino group or a carboxyl group on the resin via an amino resin. Using a bi-functional trityl approach, the solid support can require treatment with a volatile acid, such as formic acid or trifluoroacetic acid to ensure that the target polypeptide is cleaved and can be removed. In such a case, the target polypeptide can be deposited as a beadless patch at the bottom of a well of a solid support or on the flat surface of a solid support. After addition of a matrix solution, the target polypeptide can be desorbed into a MS.

Hydrophobic trityl linkers can also be exploited as acid-labile linkers by using a volatile acid or an appropriate matrix solution, e.g., a matrix solution containing 3-HPA, to cleave an amino linked trityl group from the target polypeptide. Acid lability can also be changed. For example, trityl, monomethoxytrityl, dimethoxytrityl or trimethoxytrityl can be changed to the appropriate p-substituted, or more acid-labile tritylamine derivatives, of the target polypeptide, i.e., trityl ether and tritylamine bonds can be made to the target polypeptide. Accordingly, a target polypeptide can be removed from a hydrophobic linker, e.g., by disrupting the hydrophobic attraction or by cleaving tritylether or tritylamine bonds under acidic conditions, including, if desired, under typical MS conditions, where a matrix, such as 3-HPA acts as an acid.

Orthogonally cleavable linkers can also be useful for binding a first solid support, e.g., a bead to a second solid support, or for binding a molecule of interest to a solid support. Using such linkers, a first solid support, e.g., a bead, can be selectively cleaved from a second solid support, without cleaving the target antigen from the support; the target antigen then can be cleaved from the bead at a later time. For example, a disulfide linker, which can be cleaved using a reducing agent, such as DTT, can be employed to bind a bead to a second solid support, and an acid cleavable bi-functional trityl group could be used to immobilize a target antigen to the support. As desired, the linkage of the target antigen to the solid support can be cleaved first, e.g., leaving the linkage between the first and second support intact. Trityl linkers can provide a covalent or hydrophobic conjugation and, regardless of the nature of the conjugation, the trityl group is readily cleaved in acidic conditions.

For example, a bead can be bound to a second support through a linking group which can be selected to have a length and a chemical nature such that high density binding of the beads to the solid support, or high density binding of the target antigens to the beads, is promoted. Such a linking group can have, e.g., “tree-like” structure, thereby providing a multiplicity of functional groups per attachment site on a solid support. Examples of such linking group; include polylysine, polyglutamic acid, penta-erythrole and tris-hydroxy-aminomethane.

Noncovalent Binding Association. An antibody or target antigen can be conjugated to a solid support, or a first solid support can also be conjugated to a second solid support, through a noncovalent interaction. For example, a magnetic bead made of a ferromagnetic material, which is capable of being magnetized, can be attracted to a magnetic solid support, and can be released from the support by removal of the magnetic field. Alternatively, the solid support can be provided with an ionic or hydrophobic moiety, which can allow the interaction of an ionic or hydrophobic moiety, respectively, with a target antigen, e.g., a polypeptide containing an attached trityl group or with a second solid support having hydrophobic character.

A solid support can also be provided with a member of a specific binding pair and, therefore, can be conjugated to a target antigen or a second solid support containing a complementary binding moiety. For example, a bead coated with avidin or with streptavidin can be bound to a target antigen (e.g., a polypeptide) having a biotin moiety incorporated therein, or to a second solid support coated with biotin or derivative of biotin, such as iminobiotin.

It should be recognized that any of the binding members disclosed herein or otherwise known in the art can be reversed. Thus, biotin, e.g., can be incorporated into either a target antigen or a solid support and, conversely, avidin or other biotin binding moiety would be incorporated into the support or the target antigen, respectively. Other specific binding pairs contemplated for use herein include, but are not limited to, hormones and their receptors, enzyme, and their substrates, a nucleotide sequence and its complementary sequence, an antibody and the antigen to which it interacts specifically, and other such pairs known to those skilled in the art.

A. Diagnostic Uses

General. The heterodimeric trivalent/tetravalent multispecific antibodies of the present technology are useful in diagnostic methods. As such, the present technology provides methods using the antibodies in the diagnosis of activity of a molecule of interest in a subject. Heterodimeric trivalent/tetravalent multispecific antibodies of the present technology may be selected such that they have any level of epitope binding specificity and binding affinity to a target antigen. In general, the higher the binding affinity of an antibody, the more stringent wash conditions can be performed in an immunoassay to remove nonspecifically bound material without removing the molecule of interest. Accordingly, heterodimeric trivalent/tetravalent multispecific antibodies of the present technology useful in diagnostic assays usually have binding affinities of about 10⁸M⁻¹, 10⁹M⁻¹, 10¹⁰M⁻¹, 10¹¹M⁻¹or 10¹²M⁻¹. Further, it is desirable that heterodimeric trivalent/tetravalent multispecific antibodies used as diagnostic reagents have a sufficient kinetic on-rate to reach equilibrium under standard conditions in at least 12 h, at least five (5) h, or at least one (1) hour.

Heterodimeric trivalent/tetravalent multispecific antibodies can be used to detect an immunoreactive target antigen in a variety of standard assay formats. Such formats include immunoprecipitation, Western blotting, ELISA, radioimmunoassay, and immunometric assays. See Harlow & Lane, Antibodies, A Laboratory Manual (Cold Spring Harbor Publications, New York, 1988); U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,879,262; 4,034,074, 3,791,932; 3,817,837; 3,839,153; 3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533; 3,996,345; 4,034,074; and 4,098,876. Biological samples can be obtained from any tissue or body fluid of a subject. In certain embodiments, the subject is at an early stage of cancer. In one embodiment, the early stage of cancer is determined by the level or expression pattern of a target antigen in a sample obtained from the subject. In certain embodiments, the sample is selected from the group consisting of urine, blood, serum, plasma, saliva, amniotic fluid, cerebrospinal fluid (CSF), and biopsied body tissue.

Immunometric or sandwich assays are one format for the diagnostic methods of the present technology. See U.S. Pat. Nos. 4,376,110, 4,486,530, 5,914,241, and 5,965,375. Such assays use one antibody, e.g., a heterodimeric trivalent/tetravalent multispecific antibody or a population of heterodimeric trivalent/tetravalent multispecific antibodies immobilized to a solid phase, and another heterodimeric trivalent/tetravalent multispecific antibody or a population of heterodimeric trivalent/tetravalent multispecific antibodies in solution. Typically, the solution heterodimeric trivalent/tetravalent multispecific antibody or population of heterodimeric trivalent/tetravalent multispecific antibodies is labeled. If an antibody population is used, the population can contain antibodies binding to different epitope specificities within the target antigen. Accordingly, the same population can be used for both solid phase and solution antibody. If heterodimeric trivalent/tetravalent multispecific monoclonal antibodies are used, first and second monoclonal heterodimeric trivalent/tetravalent multispecific antibodies having different binding specificities are used for the solid and solution phase. Solid phase (also referred to as “capture”) and solution (also referred to as “detection”) antibodies can be contacted with target antigen in either order or simultaneously. If the solid phase antibody is contacted first, the assay is referred to as being a forward assay. Conversely, if the solution antibody is contacted first, the assay is referred to as being a reverse assay. If the target is contacted with both antibodies simultaneously, the assay is referred to as a simultaneous assay. After contacting the target antigen with the heterodimeric trivalent/tetravalent multispecific antibody, a sample is incubated for a period that usually varies from about 10 min to about 24 hr and is usually about 1 hr. A wash step is then performed to remove components of the sample not specifically bound to the heterodimeric trivalent/tetravalent multispecific antibody being used as a diagnostic reagent. When solid phase and solution antibodies are bound in separate steps, a wash can be performed after either or both binding steps. After washing, binding is quantified, typically by detecting a label linked to the solid phase through binding of labeled solution antibody. Usually for a given pair of antibodies or populations of antibodies and given reaction conditions, a calibration curve is prepared from samples containing known concentrations of target antigen. Concentrations of the immunoreactive target antigen in samples being tested are then read by interpolation from the calibration curve (i.e., standard curve). Analyte can be measured either from the amount of labeled solution antibody bound at equilibrium or by kinetic measurements of bound labeled solution antibody at a series of time points before equilibrium is reached. The slope of such a curve is a measure of the concentration of the target antigen in a sample.

Suitable supports for use in the above methods include, e.g., nitrocellulose membranes, nylon membranes, and derivatized nylon membranes, and also particles, such as agarose, a dextran-based gel, dipsticks, particulates, microspheres, magnetic particles, test tubes, microtiter wells, SEPHADEX™ (Amersham Pharmacia Biotech, Piscataway N.J.), and the like. Immobilization can be by absorption or by covalent attachment. Optionally, heterodimeric trivalent/tetravalent multispecific antibodies can be joined to a linker molecule, such as biotin for attachment to a surface bound linker, such as avidin.

In some embodiments, the present disclosure provides a heterodimeric trivalent/tetravalent multispecific antibody of the present technology conjugated to a diagnostic agent. The diagnostic agent may comprise a radioactive or non-radioactive label, a contrast agent (such as for magnetic resonance imaging, computed tomography or ultrasound), and the radioactive label can be a gamma-, beta-, alpha-, Auger electron-, or positron-emitting isotope. A diagnostic agent is a molecule which is administered conjugated to an antibody moiety, i.e., antibody or antibody fragment, or subfragment, and is useful in diagnosing or detecting a disease by locating the cells containing the antigen. Radioactive levels emitted by the antibody may be detected using positron emission tomography or single photon emission computed tomography.

Useful diagnostic agents include, but are not limited to, radioisotopes, dyes (such as with the biotin-streptavidin complex), contrast agents, fluorescent compounds or molecules and enhancing agents (e.g., paramagnetic ions) for magnetic resonance imaging (MRI). U.S. Pat. No. 6,331,175 describes MRI technique and the preparation of antibodies conjugated to a MRI enhancing agent and is incorporated in its entirety by reference. In some embodiments, the diagnostic agents are selected from the group consisting of radioisotopes, enhancing agents for use in magnetic resonance imaging, and fluorescent compounds. In order to load an antibody component with radioactive metals or paramagnetic ions, it may be necessary to react it with a reagent having a long tail to which are attached a multiplicity of chelating groups for binding the ions. Such a tail can be a polymer such as a polylysine, polysaccharide, or other derivatized or derivatizable chain having pendant groups to which can be bound chelating groups such as, e.g., ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), porphyrins, polyamines, crown ethers, bis-thiosemicarbazones, polyoximes, and like groups known to be useful for this purpose. Chelates may be coupled to the antibodies of the present technology using standard chemistries. The chelate is normally linked to the antibody by a group which enables formation of a bond to the molecule with minimal loss of immunoreactivity and minimal aggregation and/or internal cross-linking. Other methods and reagents for conjugating chelates to antibodies are disclosed in U.S. Pat. No. 4,824,659. Particularly useful metal-chelate combinations include 2-benzyl-DTPA and its monomethyl and cyclohexyl analogs, used with diagnostic isotopes for radio-imaging. The same chelates, when complexed with non-radioactive metals, such as manganese, iron and gadolinium are useful for MM, when used along with the heterodimeric trivalent/tetravalent multispecific antibodies of the present technology.

B. Therapeutic Uses

The immunoglobulin-related compositions (e.g., heterodimeric trivalent/tetravalent multispecific antibodies) of the present technology are useful for the treatment of a disease or condition. Exemplary diseases or conditions include, but are not limited to cardiovascular disease, diabetes, autoimmune disease, dementia, Parkinson's disease, cancer or Alzheimer's disease. Such treatment can be used in patients identified as having pathological levels of a molecule of interest (e.g., those diagnosed by the methods described herein) or in patients diagnosed with a disease known to be associated with such pathological levels. 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 trivalent/tetravalent multispecific antibody of the present technology. Examples of cancers that can be treated by the antibodies of the present technology include, but are not limited to: lung cancer, colorectal cancer, skin cancer, breast cancer, ovarian cancer, leukemia, pancreatic cancer, and gastric cancer.

The compositions of the present technology may be employed in conjunction with other therapeutic agents useful in the treatment of cancer. For example, the antibodies of the present technology may be separately, sequentially or simultaneously administered with at least one additional therapeutic agent-selected from the group consisting of alkylating agents, platinum agents, taxanes, vinca agents, anti-estrogen drugs, aromatase inhibitors, ovarian suppression agents, VEGF/VEGFR inhibitors, EGF/EGFR inhibitors, PARP inhibitors, cytostatic alkaloids, cytotoxic antibiotics, antimetabolites, endocrine/hormonal agents, bisphosphonate therapy agents and targeted biological therapy agents (e.g., therapeutic peptides described in U.S. Pat. No. 6,306,832, WO 2012007137, WO 2005000889, WO 2010096603 etc.). In some embodiments, the at least one additional therapeutic agent is a chemotherapeutic agent. Specific chemotherapeutic agents include, but are not limited to, cyclophosphamide, fluorouracil (or 5-fluorouracil or 5-FU), methotrexate, edatrexate (10-ethyl-10-deaza-aminopterin), thiotepa, carboplatin, cisplatin, taxanes, paclitaxel, protein-bound paclitaxel, docetaxel, vinorelbine, tamoxifen, raloxifene, toremifene, fulvestrant, gemcitabine, irinotecan, ixabepilone, temozolmide, topotecan, vincristine, vinblastine, eribulin, mutamycin, capecitabine, anastrozole, exemestane, letrozole, leuprolide, abarelix, buserlin, goserelin, megestrol acetate, risedronate, pamidronate, ibandronate, alendronate, denosumab, zoledronate, trastuzumab, tykerb, anthracyclines (e.g., daunorubicin and doxorubicin), bevacizumab, oxaliplatin, melphalan, etoposide, mechlorethamine, bleomycin, microtubule poisons, annonaceous acetogenins, or combinations thereof.

In another aspect, the antibodies of the present technology may be separately, sequentially or simultaneously administered with one or more therapeutic agents useful in the treatment of Alzheimer's disease. Examples of such therapeutic agents include acetylcholine esterase inhibitors such as tacrine (tetrahydroaminoacridine), donepezil hydrochloride, and rivastigmine; gamma-secretase inhibitors; anti-inflammatory agents such as cyclooxygenase II inhibitors; antioxidants such as Vitamin E and ginkolides; immunological approaches, such as, for example, immunization with A beta peptide or administration of anti-A beta peptide antibodies; statins; and direct or indirect neurotropic agents such as Cerebrolysin®, AIT-082 (Emilieu, 2000, Arch. Neurol. 57:454).

The compositions of the present technology may optionally be administered as a single bolus to a subject in need thereof. Alternatively, the dosing regimen may comprise multiple administrations performed at various times after the appearance of tumors or amyloid plaques.

Administration can be carried out by any suitable route, including orally, intranasally, parenterally (intravenously, intramuscularly, intraperitoneally, or subcutaneously), rectally, intracranially, intrathecally, or topically. Administration includes self-administration and the administration by another. It is also to be appreciated that the various modes of treatment of medical conditions as described are intended to mean “substantial”, which includes total but also less than total treatment, and wherein some biologically or medically relevant result is achieved.

In some embodiments, the antibodies of the present technology comprise pharmaceutical formulations which may be administered to subjects in need thereof in one or more doses. Dosage regimens can be adjusted to provide the desired response (e.g., a therapeutic response).

Typically, an effective amount of the antibody compositions of the present technology, sufficient for achieving a therapeutic effect, range from about 0.000001 mg per kilogram body weight per day to about 10,000 mg per kilogram body weight per day. Typically, the dosage ranges are from about 0.0001 mg per kilogram body weight per day to about 100 mg per kilogram body weight per day. For administration of heterodimeric trivalent/tetravalent multispecific antibodies, the dosage ranges from about 0.0001 to 100 mg/kg, and more usually 0.01 to 5 mg/kg every week, every two weeks or every three weeks, of the subject body weight. For example, dosages can be 1 mg/kg body weight or 10 mg/kg body weight every week, every two weeks or every three weeks or within the range of 1-10 mg/kg every week, every two weeks or every three weeks. In one embodiment, a single dosage of antibody ranges from 0.1-10,000 micrograms per kg body weight. In one embodiment, antibody concentrations in a carrier range from 0.2 to 2000 micrograms per delivered milliliter. An exemplary treatment regime entails administration once per every two weeks or once a month or once every 3 to 6 months. Heterodimeric trivalent/tetravalent multispecific antibodies may be administered on multiple occasions. Intervals between single dosages can be hourly, daily, weekly, monthly or yearly. Intervals can also be irregular as indicated by measuring blood levels of the antibody in the subject. In some methods, dosage is adjusted to achieve a serum antibody concentration in the subject of from about 75 μg/mL to about 125 μg/mL, 100 μg/mL to about 150 μg/mL, from about 125 μg/mL to about 175 μg/mL, or from about 150 μg/mL to about 200 μg/mL. Alternatively, heterodimeric trivalent/tetravalent multispecific antibodies can be administered as a sustained release formulation, in which case less frequent administration is required. Dosage and frequency vary depending on the half-life of the antibody in the subject. The dosage and frequency of administration can vary depending on whether the treatment is prophylactic or therapeutic. In prophylactic applications, a relatively low dosage is administered at relatively infrequent intervals over a long period of time. In therapeutic applications, a relatively high dosage at relatively short intervals is sometimes required until progression of the disease is reduced or terminated, or until the subject shows partial or complete amelioration of symptoms of disease. Thereafter, the patient can be administered a prophylactic regime.

Toxicity. Optimally, an effective amount (e.g., dose) of heterodimeric trivalent/tetravalent multispecific antibody described herein will provide therapeutic benefit without causing substantial toxicity to the subject. Toxicity of the heterodimeric trivalent/tetravalent multispecific antibody described herein can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., by determining the LD₅₀(the dose lethal to 50% of the population) or the LD₁₀₀(the dose lethal to 100% of the population). The dose ratio between toxic and therapeutic effect is the therapeutic index. The data obtained from these cell culture assays and animal studies can be used in formulating a dosage range that is not toxic for use in human. The dosage of the heterodimeric trivalent/tetravalent multispecific antibody described herein lies within a range of circulating concentrations that include the effective dose with little or no toxicity. The dosage can vary within this range depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the subject's condition. See, e.g., Fingl et al., In: The Pharmacological Basis of Therapeutics, Ch. 1 (1975).

Formulations of Pharmaceutical Compositions

Formulations of Pharmaceutical Compositions. According to the methods of the present technology, the heterodimeric trivalent/tetravalent multispecific antibodies can be incorporated into pharmaceutical compositions suitable for administration. The pharmaceutical compositions generally comprise recombinant or substantially purified antibody and a pharmaceutically-acceptable carrier in a form suitable for administration to a subject. Pharmaceutically-acceptable carriers are determined in part by the particular composition being administered, as well as by the particular method used to administer the composition. Accordingly, there is a wide variety of suitable formulations of pharmaceutical compositions for administering the antibody compositions (See, e.g., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa. 18^thed., 1990). The pharmaceutical compositions are generally formulated as sterile, substantially isotonic and in full compliance with all Good Manufacturing Practice (GMP) regulations of the U.S. Food and Drug Administration.

The terms “pharmaceutically-acceptable,” “physiologically-tolerable,” and grammatical variations thereof, as they refer to compositions, carriers, diluents and reagents, are used interchangeably and represent that the materials are capable of administration to a subject without the production of undesirable physiological effects to a degree that would prohibit administration of the composition. For example, “pharmaceutically-acceptable excipient” means an excipient that is useful in preparing a pharmaceutical composition that is generally safe, non-toxic, and desirable, and includes excipients that are acceptable for veterinary use as well as for human pharmaceutical use. Such excipients can be solid, liquid, semisolid, or, in the case of an aerosol composition, gaseous. “Pharmaceutically-acceptable salts and esters” means salts and esters that are pharmaceutically-acceptable and have the desired pharmacological properties. Such salts include salts that can be formed where acidic protons present in the composition are capable of reacting with inorganic or organic bases. Suitable inorganic salts include those formed with the alkali metals, e.g., sodium and potassium, magnesium, calcium, and aluminum. Suitable organic salts include those formed with organic bases such as the amine bases, e.g., ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine, and the like. Such salts also include acid addition salts formed with inorganic acids (e.g., hydrochloric and hydrobromic acids) and organic acids (e.g., acetic acid, citric acid, maleic acid, and the alkane- and arene-sulfonic acids such as methanesulfonic acid and benzenesulfonic acid). Pharmaceutically-acceptable esters include esters formed from carboxy, sulfonyloxy, and phosphonoxy groups present in the heterodimeric trivalent/tetravalent multispecific antibody, e.g., C1-6 alkyl esters. When there are two acidic groups present, a pharmaceutically-acceptable salt or ester can be a mono-acid-mono-salt or ester or a di-salt or ester; and similarly where there are more than two acidic groups present, some or all of such groups can be salified or esterified. A heterodimeric trivalent/tetravalent multispecific antibody named in this technology can be present in unsalified or unesterified form, or in salified and/or esterified form, and the naming of such heterodimeric trivalent/tetravalent multispecific antibody is intended to include both the original (unsalified and unesterified) compound and its pharmaceutically-acceptable salts and esters. Also, certain embodiments of the present technology can be present in more than one stereoisomeric form, and the naming of such heterodimeric trivalent/tetravalent multispecific antibody is intended to include all single stereoisomers and all mixtures (whether racemic or otherwise) of such stereoisomers. A person of ordinary skill in the art, would have no difficulty determining the appropriate timing, sequence and dosages of administration for particular drugs and compositions of the present technology.

Examples of such carriers or diluents include, but are not limited to, water, saline, Ringer's solutions, dextrose solution, and 5% human serum albumin. Liposomes and non-aqueous vehicles such as fixed oils may also be used. The use of such media and compounds for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or compound is incompatible with the heterodimeric trivalent/tetravalent multispecific antibody, use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.

A pharmaceutical composition of the present technology is formulated to be compatible with its intended route of administration. The heterodimeric trivalent/tetravalent multispecific antibody compositions of the present technology can be administered by parenteral, topical, intravenous, oral, subcutaneous, intraarterial, intradermal, transdermal, rectal, intracranial, intrathecal, intraperitoneal, intranasal; or intramuscular routes, or as inhalants. The heterodimeric trivalent/tetravalent multispecific antibody can optionally be administered in combination with other agents that are at least partly effective in treating a disease or medical condition described herein.

Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial compounds such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating compounds such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates or phosphates, and compounds for the adjustment of tonicity such as sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringeability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, e.g., water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, e.g., by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal compounds, e.g., parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be desirable to include isotonic compounds, e.g., sugars, polyalcohols such as manitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition a compound which delays absorption, e.g., aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating a heterodimeric trivalent/tetravalent multispecific antibody of the present technology in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the heterodimeric trivalent/tetravalent multispecific antibody into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, methods of preparation are vacuum drying and freeze-drying that yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. The antibodies of the present technology can be administered in the form of a depot injection or implant preparation which can be formulated in such a manner as to permit a sustained or pulsatile release of the active ingredient.

Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the heterodimeric trivalent/tetravalent multispecific antibody can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed. Pharmaceutically compatible binding compounds, and/or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating compound such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening compound such as sucrose or saccharin; or a flavoring compound such as peppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the heterodimeric trivalent/tetravalent multispecific antibody is delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.

Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, e.g., for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the heterodimeric trivalent/tetravalent multispecific antibody is formulated into ointments, salves, gels, or creams as generally known in the art.

The heterodimeric trivalent/tetravalent multispecific antibody can also be prepared as pharmaceutical compositions in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

In one embodiment, the heterodimeric trivalent/tetravalent multispecific antibody is prepared with carriers that will protect the heterodimeric trivalent/tetravalent multispecific antibody against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically-acceptable carriers. These can be prepared according to methods known to those skilled in the art, e.g., as described in U.S. Pat. No. 4,522,811.

Kits

The present technology provides kits for the detection and/or treatment of cancer, comprising at least one heterodimeric trivalent/tetravalent multispecific antibody composition described herein, or a functional variant (e.g., substitutional variant) thereof. Optionally, the above described components of the kits of the present technology are packed in suitable containers and labeled for diagnosis and/or treatment of cancer. The above-mentioned components may be stored in unit or multi-dose containers, for example, sealed ampoules, vials, bottles, syringes, and test tubes, as an aqueous, preferably sterile, solution or as a lyophilized, preferably sterile, formulation for reconstitution. The kit may further comprise a second container which holds a diluent suitable for diluting the pharmaceutical composition towards a higher volume. Suitable diluents include, but are not limited to, the pharmaceutically acceptable excipient of the pharmaceutical composition and a saline solution. Furthermore, the kit may comprise instructions for diluting the pharmaceutical composition and/or instructions for administering the pharmaceutical composition, whether diluted or not. The containers may be formed from a variety of materials such as glass or plastic and may have a sterile access port (for example, the container may be an intravenous solution bag or a vial having a stopper which may be pierced by a hypodermic injection needle). The kit may further comprise more containers comprising a pharmaceutically acceptable buffer, such as phosphate-buffered saline, Ringer's solution and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, culture medium for one or more of the suitable hosts. The kits may optionally include instructions customarily included in commercial packages of therapeutic or diagnostic products, that contain information about, for example, the indications, usage, dosage, manufacture, administration, contraindications and/or warnings concerning the use of such therapeutic or diagnostic products.

The kits are useful for detecting the presence of a target antigen in a biological sample, e.g., any body fluid including, but not limited to, e.g., serum, plasma, lymph, cystic fluid, urine, stool, cerebrospinal fluid, ascitic fluid or blood and including biopsy samples of body tissue. For example, the kit can comprise: one or more heterodimeric trivalent/tetravalent multispecific antibodies of the present technology capable of binding a target antigen in a biological sample; means for determining the amount of the target antigen in the sample; and means for comparing the amount of the immunoreactive target antigen in the sample with a standard. One or more of the heterodimeric trivalent/tetravalent multispecific antibodies may be labeled. The kit components, (e.g., reagents) can be packaged in a suitable container. The kit can further comprise instructions for using the kit to detect the immunoreactive target antigen.

For antibody-based kits, the kit can comprise, e.g., 1) a first antibody, e.g. a humanized, or chimeric heterodimeric trivalent/tetravalent multispecific antibody of the present technology, attached to a solid support, which binds to a target antigen; and, optionally; 2) a second, different antibody which binds to either the target antigen or to the first antibody, and is conjugated to a detectable label.

The kit can also comprise, e.g., a buffering agent, a preservative or a protein-stabilizing agent. The kit can further comprise components necessary for detecting the detectable-label, e.g., an enzyme or a substrate. The kit can also contain a control sample or a series of control samples, which can be assayed and compared to the test sample. Each component of the kit can be enclosed within an individual container and all of the various containers can be within a single package, along with instructions for interpreting the results of the assays performed using the kit. The kits of the present technology may contain a written product on or in the kit container. The written product describes how to use the reagents contained in the kit, e.g., for detection of a target antigen in vitro or in vivo, or for treatment of cancer in a subject in need thereof. In certain embodiments, the use of the reagents can be according to the methods of the present technology.

EXAMPLES

The present technology is further illustrated by the following Examples, which should not be construed as limiting in any way.

Example 1: Materials and Methods

Protein production. All proteins were expressed using the expi293 expression system (Thermo Fisher Scientific, Waltham Mass.) according to manufacturer's instructions. Briefly, maxiprepped plasmids containing each antibody were diluted and incubated with expifectamine for 20 min before being added to expi293s in shaker flasks. Cells were incubated for 4 days or until cell viability dropped <70%, whichever came first. IgG-based proteins were purified over a protein A column using a GE P920 AKTA FPLC and eluted using 50 mM Citric acid. The BiTE was purified using prepacked Ni²⁺NTA columns (GE) and eluted using a 250 mM imidazole buffer. All proteins were run on SEC-HPLC to validate their size and quantify their purity.

Heterodimerization. Heterodimerization was achieved using Fab Arm Exchange (FAE). Briefly, K409R and F405L mutations were placed in the Fc regions of each reciprocal pair of IgG or IgG-[L]-scFv bispecific antibodies to be heterodimerized. Paired homodimers were then mixed at 3 different molar rations (1:1, 1.2:1 and 1:1.2) and incubated in reducing conditions for 5 hrs at 30° C. before being dialyzed overnight at room temperature in sodium citrate buffer (pH 8.2). After an initial overnight dialysis, samples were moved to 4° C. for another 24 hrs before being analyzed by SEC-HPLC and CZE chromatography to assess heterodimerization yields. In all experiments the 1:1 ratio was used, after validating its purity was optimal.

Cell lines. EL.4 cells were obtained from ATCC. M14 cells were obtained from ATCC and transfected with luciferase prior to use in all assays. IMR32 cells were obtained from ATCC and transfected with luciferase prior to use in all assays. Molm13-fluc cells were a gift from the Brentjens lab. Naïve T-cells were purified from PBMCs using the Dynabeads™ Untouched™ human T cells kit, according to manufacturer's protocol. Activated T cells were generated by using CD3/CD28 dynabeads and 30U/ml of human IL-2. T-cells were stimulated twice, at day 0 and day 7, and used in cytotoxicity, cell binding or conjugate assays day 15-18 of culture.

Cell binding FACS. For cell binding assays, 1M cells were incubated with 5 pmol of antibody for 30 min at 4° C., followed by either an anti-human Fc secondary or an anti-3F8 or anti-OKT3 idiotype antibody (5 pmol) and the corresponding anti-Fc secondary (anti-rat APC or anti-mouse PE, respectively). Samples were acquired using a FACSCalibur and analyzed by FlowJo.

Affinity Measurements. Binding kinetics were evaluated using SPR (GE, Biacore T200). Briefly, chips were coated with GD2, CD33 or huCD3de antigen and a titration series of each bispecific antibody were flowed over them. Binding affinities were calculated using a two-state reaction model.

Cytotoxicity measurements. Cytotoxicity was evaluated using a 4 hr ⁵¹Cr release assay. Briefly, 1M target cells were incubated with 100 μCi of activity and incubated with activated human T cells (10:1 E:T) and serially titrated bispecific antibody. Released ⁵¹Cr was measured using a gamma counter.

Animal Models. All experiments have been conducted in accordance with and approved by the Institutional Animal Care and Use Committee in MSKCC. Two mouse models were used: (1) a humanized immunodeficient xenograft model (huDKO) and (2) a transgenic huCD3e-expressing syngeneic model (huCD3e-tg). Briefly, huDKO (Balb/C IL2rg^−/−, Rag2^−/−) mice were implanted subcutaneously with 2M M14 melanoma cells. After 5-15 days, mice were treated with intravenous activated human T cells (20-40M/dose), intravenous bispecific antibody (25 pmol/dose) and subcutaneous IL-2 (100U/dose) for three weeks. For huCD3e-tg (C57BL/6) mice were implanted subcutaneously with EL.4 lymphoma cells. After 7 days, mice were treated intravenous bispecific antibody (25 pmol/dose) for three weeks. For BiTEs, either 7 pmol or 350 pmol were administered daily for 3 weeks. Weights and tumor volumes were measured once per week and overall mouse health was evaluated at least 3-times per week. Mice were sacrificed if tumor volumes reached 1.5-2.0 cm³volumes. No toxicities were seen during treatment of any mice.

Conjugate formation. For conjugate assays, T cells were labeled with CFSE (2.5 μM) and M14 melanoma cells were labeled with CTV (2.5 μM). 50 M/ml cells were incubated with dye for 5 min at room temperature, followed by the addition of 30 ml of complete RPMI (supplemented with 10% fetal calf serum (heat inactivated), 2 mM glutamine and 1% P/S) and incubated at 37° C. for 20 min. Cells were pelleted and washed with complete medium twice before being added antibodies or cells. Labeled cells were mixed at a 1:5 ratio (E:T) along with serially titrated bispecific antibody, in duplicate. After 30 min, cells were fixed with a final concentration of 2% PFA (10 min, RT) and washed in 5 ml of PBS. Cells were acquired using a BD LSR Fortessa and analyzed using Flowjo.

Activation assay. Purified naïve T cells were incubated with M14 melanoma cells (10:1 E:T) and serially titrated bispecific antibody, in duplicate. After 24 hrs supernatant was collected and frozen at −80° C. Cells were then stained with antibodies against CD4, CD8, CD45, and CD69 to assess the CD69 upregulation. For the 96 hr assay, T cells were first labeled with 2.504 of CTV. After 96 hrs cells were stained with antibodies against CD4, CD8, CD45 and CD25 to assess CD25 upregulation and CTV dilution.

Cytokine Assay. Frozen supernatant from the activation assay (24 hr) was used to quantify cytokine production after 24 hrs of coculture. IL-2, IFNγ, IL-10, IL-6 and TNFα were measured with the 5-plex legend plex system according to manufacturer guidelines.

Sequences. The amino acid sequences utilized in the Examples are provided below:

Anti-HER2

LC(VL-CL-scFv)

(SEQ ID NO: 2353)

DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYS

ASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQ

GTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV

DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG

LSSPVTKSFNRGECTSGGGGSGGGGSGGGGSQVQLVQSGGGVVQPGRSLR

LSCKASGYTFTRYTIVIRWVRQAPGKCLEWIGYINPSRGYTNYNQKFKDR

FTISRDNSKNTAFLQMDSLRPEDTGVYFCARYYDDHYSLDYWGQGTPVTV

SSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDR

VTITCSASSSVSYMNWYQQTPGKAPKRWIYDTSKLASGVPSRFSGSGSGT

DYTFTISSLQPEDIATYYCQQWSSNPFTFGCGTKLQITR

HC(VH-CH1-CH2-CH3, N297A, K322A):

(SEQ ID NO: 2354)

EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVAR

TYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWG

GDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVK

DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT

YICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKP

KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYA

STYRVVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTISKAKGQPREPQ

VYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV

LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

HC(VH-CH1-CH2-CH3, N297A, K322A, F405L):

(SEQ ID NO: 2355)

EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVAR

TYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWG

GDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVK

DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT

YICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKP

KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYA

STYRVVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTISKAKGQPREPQ

VYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV

LDSDGSFLLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

HC(VH-CH1-CH2-CH3, N297A, K322A, K409R):

(SEQ ID NO: 2356)

EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVAR

TYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWG

GDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVK

DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT

YICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKP

KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYA

STYRVVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTISKAKGQPREPQ

VYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV

LDSDGSFFLYSRLTVDKSRWQQGNVFScSVMHEALHNHYTQKSLSLSPGK

Anti-GD2

LC(VL-CL-scFv):

(SEQ ID NO: 2357)

EIVMTQTPATLSVSAGERVTITCKASQSVSNDVTWYQQKPGQAPRLLIYS

ASNRYSGVPARFSGSGYGTEFTFTISSVQSEDFAVYFCQQDYSSFGQGTK

LEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNA

LQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSS

PVTKSFNRGECTSGGGGSGGGGSGGGGSQVQLVQSGGGVVQPGRSLRLSC

KASGYTFTRYTMHWVRQAPGKCLEWIGYINPSRGYTNYNQKFKDRFTISR

DNSKNTAFLQMDSLRPEDTGVYFCARYYDDHYSLDYWGQGTPVTVSSGGG

GSGGGGSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITC

SASSSVSYMNWYQQTPGKAPKRWIYDTSKLASGVPSRFSGSGSGTDYTFT

ISSLQPEDIATYYCQQWSSNPFTFGCGTKLQITR

LC(VL-CL):

(SEQ ID NO: 2358)

EIVMTQTPATLSVSAGERVTITCKASQSVSNDVTWYQQKPGQAPRLLIYS

ASNRYSGVPARFSGSGYGTEFTFTISSVQSEDFAVYFCQQDYSSFGQGTK

LEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNA

LQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSS

PVTKSFNRGEC

HC(VH-CH1-CH2-CH3, N297A, K322A):

(SEQ ID NO: 2359)

QVQLVESGPGVVQPGRSLRISCAVSGFSVTNYGVHWVRQPPGKGLEWLGV

IWAGGITNYNSAFMSRLTISKDNSKNTVYLQMNSLRAEDTAMYYCASRGG

HYGYALDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKD

YFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTY

ICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPK

DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYAS

TYRVVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTISKAKGQPREPQV

YTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL

DSDGSFFLYSKLTVDKSRWQQGNVFScSVMHEALHNHYTQKSLSLSPGK

HC(VH-CH1-CH2-CH3, N297A, K322A, F405L):

(SEQ ID NO: 2360)

QVQLVESGPGVVQPGRSLRISCAVSGFSVTNYGVHWVRQPPGKGLEWLGV

IWAGGITNYNSAFMSRLTISKDNSKNTVYLQMNSLRAEDTAMYYCASRGG

HYGYALDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKD

YFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTY

ICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPK

DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYAS

TYRVVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTISKAKGQPREPQV

YTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL

DSDGSFLLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

HC(VH-CH1-CH2-CH3, N297A, K322A, K409R):

(SEQ ID NO: 2361)

QVQLVESGPGVVQPGRSLRISCAVSGFSVTNYGVHWVRQPPGKGLEWLGV

IWAGGITNYNSAFMSRLTISKDNSKNTVYLQMNSLRAEDTAMYYCASRGG

HYGYALDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKD

YFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTY

ICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPK

DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYAS

TYRVVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTISKAKGQPREPQV

YTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL

DSDGSFFLYSRLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Anti-GD2(2)

LC(VL-CL-scFv):

(SEQ ID NO: 2362)

KIVMTQTPATLSVSAGERVTITCKASQSVSNHVTWYQQKPGQAPRLLIYS

ASNRYSGVPARFSGSGYGTEFTFTISSVQSEDFAVYFCQQDYSSFGQGTK

LEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNA

LQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSS

PVTKSFNRGECTSGGGGSGGGGSGGGGSQVQLVQSGGGVVQPGRSLRLSC

KASGYTFTRYTMHWVRQAPGKCLEWIGYINPSRGYTNYNQKFKDRFTISR

DNSKNTAFLQMDSLRPEDTGVYFCARYYDDHYSLDYWGQGTPVTVSSGGG

GSGGGGSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITC

SASSSVSYMNWYQQTPGKAPKRWIYDTSKLASGVPSRFSGSGSGTDYTFT

ISSLQPEDIATYYCQQWSSNPFTFGCGTKLQITR

LC(VL-CL):

(SEQ ID NO: 2363)

KIVMTQTPATLSVSAGERVTITCKASQSVSNHVTWYQQKPGQAPRLLIYS

ASNRYSGVPARFSGSGYGTEFTFTISSVQSEDFAVYFCQQDYSSFGQGTK

LEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNA

LQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSS

PVTKSFNRGEC

HC(VH-CH1-CH2-CH3, N297A, K322A):

(SEQ ID NO: 2364)

QVQLVESGPGVVQPGRSLRISCAVSGFSVTNYGVHWVRQPPGKGLEWLGV

IWAGGITNYNSAFMSRLTISKDNSKNTVYLQMNSLRAEDTAMYYCASRGG

HYGYALDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKD

YFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTY

ICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPK

DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYAS

TYRVVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTISKAKGQPREPQV

YTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL

DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

HC(VH-CH1-CH2-CH3, N297A, K322A, F405L):

(SEQ ID NO: 2365)

QVQLVESGPGVVQPGRSLRISCAVSGFSVTNYGVHWVRQPPGKGLEWLGV

IWAGGITNYNSAFMSRLTISKDNSKNTVYLQMNSLRAEDTAMYYCASRGG

HYGYALDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKD

YFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTY

ICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPK

DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYAS

TYRVVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTISKAKGQPREPQV

YTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL

DSDGSFLLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

HC(VH-CH1-CH2-CH3, N297A, K322A, K409R):

(SEQ ID NO: 2366)

QVQLVESGPGVVQPGRSLRISCAVSGFSVTNYGVHWVRQPPGKGLEWLGV

IWAGGITNYNSAFMSRLTISKDNSKNTVYLQMNSLRAEDTAMYYCASRGG

HYGYALDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKD

YFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTY

ICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPK

DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYAS

TYRVVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTISKAKGQPREPQV

YTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL

DSDGSFFLYSRLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Anti-GD2(3)

LC(VL-CL-scFv):

(SEQ ID NO: 2367)

EIVMTQSPATLSVSPGERATLSCRSSQSLVHRNGNTYLHWYLQKPGQSPK

LLIHKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVP

PLTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREA

KVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYAC

EVTHQGLSSPVTKSFNRGECTSGGGGSGGGGSGGGGSQVQLVQSGGGVVQ

PGRSLRLSCKASGYTFTRYTMHWVRQAPGKCLEWIGYINPSRGYTNYNQK

FKDRFTISRDNSKNTAFLQMDSLRPEDTGVYFCARYYDDHYSLDYWGQGT

PVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSAS

VGDRVTITCSASSSVSYMNWYQQTPGKAPKRWIYDTSKLASGVPSRFSGS

GSGTDYTFTISSLQPEDIATYYCQQWSSNPFTFGCGTKLQITR

LC(VL-CL):

(SEQ ID NO: 2368)

EIVMTQSPATLSVSPGERATLSCRSSQSLVHRNGNTYLHWYLQKPGQSPK

LLIHKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVP

PLTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREA

KVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYAC

EVTHQGLSSPVTKSFNRGEC

HC(VH-CH1-CH2-CH3, N297A, K322A):

(SEQ ID NO: 2369)

EVQLLQSGPELEKPGASVMISCKASGSSFTGYNMNWVRQNIGKSLEWIGA

IDPYYGGTSYNQKFKGRATLTVDKSSSTAYMHLKSLTSEDSAVYYCVSGM

EYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPV

TVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNH

KPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMIS

RTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVS

VLTVLHQDWLNGKEYKCAVSNKALPAPIEKTISKAKGQPREPQVYTLPPS

RDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF

FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

HC(VH-CH1-CH2-CH3, N297A, K322A, F405L):

(SEQ ID NO: 2370)

EVQLLQSGPELEKPGASVMISCKASGSSFTGYNMNWVRQNIGKSLEWIGA

IDPYYGGTSYNQKFKGRATLTVDKSSSTAYMHLKSLTSEDSAVYYCVSGM

EYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPV

TVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNH

KPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMIS

RTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVS

VLTVLHQDWLNGKEYKCAVSNKALPAPIEKTISKAKGQPREPQVYTLPPS

RDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF

LLYSKLTVDKSRWQQGNVFScSVMHEALHNHYTQKSLSLSPGK

HC(VH-CH1-CH2-CH3, N297A, K322A, K409R):

(SEQ ID NO: 2371)

EVQLLQSGPELEKPGASVMISCKASGSSFTGYNMNWVRQNIGKSLEWIGA

IDPYYGGTSYNQKFKGRATLTVDKSSSTAYMHLKSLTSEDSAVYYCVSGM

EYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPV

TVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNH

KPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMIS

RTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVS

VLTVLHQDWLNGKEYKCAVSNKALPAPIEKTISKAKGQPREPQVYTLPPS

RDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF

FLYSRLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Anti-CD33

LC(VL-CL-scFv):

(SEQ ID NO: 2372)

EIVLTQSPATLSVSLGERATISCRASESVDNYGISFMNWFQQKPGQPPRL

LIYAASNQGSGVPARFSGSGPGTDFTLTISSMEPEDFAMYFCQQSKEVPW

TFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKV

QWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV

THQGLSSPVTKSFNRGECTSGGGGSGGGGSGGGGSQVQLVQSGGGVVQPG

RSLRLSCKASGYTFTRYTMHWVRQAPGKCLEWIGYINPSRGYTNYNQKFK

DRFTISRDNSKNTAFLQMDSLRPEDTGVYFCARYYDDHYSLDYWGQGTPV

TVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVG

DRVTITCSASSSVSYMNWYQQTPGKAPKRWIYDTSKLASGVPSRFSGSGS

GTDYTFTISSLQPEDIATYYCQQWSSNPFTFGCGTKLQITR

LC(VL-CL):

(SEQ ID NO: 2373)

EIVLTQSPATLSVSLGERATISCRASESVDNYGISFMNWFQQKPGQPPRL

LIYAASNQGSGVPARFSGSGPGTDFTLTISSMEPEDFAMYFCQQSKEVPW

TFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKV

QWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV

THQGLSSPVTKSFNRGEC

HC(VH-CH1-CH2-CH3, N297A, K322A):

(SEQ ID NO: 2374)

EVQLVQSGPEVVKPGASVKISCKASGYTFTDYNMHWVRQAHGQSLEWIGY

IYPYNGGTGYNQKFKSRATLTVDNSASTAYMEVSSLRSEDTAVYYCARGR

PAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFP

EPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICN

VNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTL

MISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYR

VVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTISKAKGQPREPQVYTL

PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD

GSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

HC(VH-CH1-CH2-CH3, N297A, K322A, F405L):

(SEQ ID NO: 2375)

EVQLVQSGPEVVKPGASVKISCKASGYTFTDYNMHWVRQAHGQSLEWIGY

IYPYNGGTGYNQKFKSRATLTVDNSASTAYMEVSSLRSEDTAVYYCARGR

PAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFP

EPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICN

VNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTL

MISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYR

VVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTISKAKGQPREPQVYTL

PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD

GSFLLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

HC(VH-CH1-CH2-CH3, N297A, K322A, K409R):

(SEQ ID NO: 2376)

EVQLVQSGPEVVKPGASVKISCKASGYTFTDYNMHWVRQAHGQSLEWIGY

IYPYNGGTGYNQKFKSRATLTVDNSASTAYMEVSSLRSEDTAVYYCARGR

PAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFP

EPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICN

VNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTL

MISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYR

VVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTISKAKGQPREPQVYTL

PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD

GSFFLYSRLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Anti-CD3

LC(VL-CL):

(SEQ ID NO: 2377)

DIQMTQSPSSLSASVGDRVTITCSASSSVSYMNWYQQTPGKAPKRWIYDT

SKLASGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQWSSNPFTFGQG

TKLQITRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVD

NALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGL

SSPVTKSFNRGEC

HC(VH-CH1-CH2-CH3, N297A, K322A):

(SEQ ID NO: 2378)

QVQLVQSGGGVVQPGRSLRLSCKASGYTFTRYTMHWVRQAPGKGLEWIGY

INPSRGYTNYNQKFKDRFTISRDNSKNTAFLQMDSLRPEDTGVYFCARYY

DDHYSLDYWGQGTPVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKD

YFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTY

ICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPK

DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYAS

TYRVVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTISKAKGQPREPQV

YTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL

DSDGSFFLYSKLTVDKSRWQQGNVFScSVMHEALHNHYTQKSLSLSPGK

HC(VH-CH1-CH2-CH3, N297A, K322A, F405L):

(SEQ ID NO: 2379)

QVQLVQSGGGVVQPGRSLRLSCKASGYTFTRYTMHWVRQAPGKGLEWIGY

INPSRGYTNYNQKFKDRFTISRDNSKNTAFLQMDSLRPEDTGVYFCARYY

DDHYSLDYWGQGTPVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKD

YFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTY

ICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPK

DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYAS

TYRVVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTISKAKGQPREPQV

YTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL

DSDGSFLLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

HC(VH-CH1-CH2-CH3, N297A, K322A, K409R):

(SEQ ID NO: 2380)

QVQLVQSGGGVVQPGRSLRLSCKASGYTFTRYTMHWVRQAPGKGLEWIGY

INPSRGYTNYNQKFKDRFTISRDNSKNTAFLQMDSLRPEDTGVYFCARYY

DDHYSLDYWGQGTPVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKD

YFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTY

ICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPK

DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYAS

TYRVVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTISKAKGQPREPQV

YTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL

DSDGSFFLYSRLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

huOKT3-VL

(SEQ ID NO: 2390)

DIQMTQSPSSLSASVGDRVTITCSASSSVSYMNWYQQTPGKAPKRWIYDT

SKLASGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQWSSNPFTFGCG

TKLQIT

huOKT3-VH

(SEQ ID NO: 2391)

QVQLVQSGGGVVQPGRSLRLSCKASGYTFTRYTMHWVRQAPGKCLEWIGY

INPSRGYTNYNQKFKDRFTISRDNSKNTAFLQMDSLRPEDTGVYFCARYY

DDHYSLDYWGQGTPVTVSS

huA33-VL

(SEQ ID NO: 2392)

DIQMTQSQSSLSTSVGDRVTITCKASQNVRTVVAWYQQKPGKSPKTLIYL

ASNRHTGVPSRFSGSGSGTEFTLTISNVQPEDFADYFCLQHWSYPLTFGS

GTKLEIK

huA33-VH

(SEQ ID NO: 2393)

EVQLVESGGGLVKPGGSLRLSCAASGFAFSTYDMSWVRQAPGKRLEWVAT

ISSGGSYTYYLDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAPTT

VVPFAYWGQGTLVTVSS

huM195-VL

(SEQ ID NO: 2394)

EIVLTQSPATLSVSLGERATISCRASESVDNYGISFMNWFQQKPGQPPRL

LIYAASNQGSGVPARFSGSGPGTDFTLTISSMEPEDFAMYFCQQSKEVPW

TFGGGTKLEIK

huM195-VH

(SEQ ID NO: 2395)

EVQLVQSGPEVVKPGASVKISCKASGYTFTDYNMHWVRQAHGQSLEWIGY

IYPYNGGTGYNQKFKSRATLTVDNSASTAYMEVSSLRSEDTAVYYCARGR

PAMDYWGQGTLVTVSS

Example 2: Functionality of Lo1+1+2, Hi1+1+1 and 2+1+1 HDTVS Variants

FIG. 1a shows the basic design strategy of each HDTVS variant compared with the parental 2+2 IgG-[L]-scFv. FIGS. 1b-1g describe each of the three designs in more detail.

The Lo1+1+2 utilizes two different Fab domains that (a) target two distinct antigens within a tumor and (b) have moderate to low binding affinities (e.g. K_D100 nM-100 pM), and two identical scFvs that target an immune cell so as to improve tumor cell specificity. As illustrated in FIG. 1b, this design targets tumors more specifically due to its unexpectedly poor activity when only one of the two Fab domains is engaged with the tumor target (such as when only one of the two Fab domain-specific antigens is expressed). Importantly, when both Fab domains bind their respective tumor targets, normal cytotoxic potency is restored. This allows for improved therapeutic index (or safety) when the target antigens are not unique to the tumor, where each target antigen (but never both) is shared to some extent by normal cells. While a standard BsAb or 2+2 design would harm normal tissues, this Lo1+1+2 design should spare normal tissues that express only one of the two targeted antigens, while maintaining the full potency against a tumor cell that expresses both antigens.

As illustrated in FIG. 1c, the Hi1+1+2 design is capable of recognizing two distinct antigens with equal potency, regardless of simultaneous binding. Since Fab domains of appropriately high affinity (e.g., K_D<100 pM) are sufficient to induce potent cytotoxicity even monovalently, two different Fab domains can be used to broaden the tumor cell selectivity and permits targeting of heterogeneous tumors with a single drug.

The 2+1+1 design is capable of improved immune cell interactions by virtue of its dual specificity toward the immune cell, either improving activation or providing more selective activation. As demonstrated herein, the second scFv domain is somewhat dispensable due to the biophysical properties of the IgG-[L]-scFv platform. Thus, using two different scFv domains can provide a greater diversity of interactions than a normal bivalent approach. As illustrated in FIG. 1d, the 2+1+1 design can be used to both improve signaling in a more selective population of immune cells (B1(+)B2(+)) or to enhance activation through colocalization of complementary pairs of receptors. Importantly, the 2+1+1 design can be used to interact with activating receptors and/or inhibitory receptors or antagonistic antibodies that specifically inhibit signaling of certain immune cell pathways, such as blocking PD-1 on T cells while activating through CD3.

The 2+1+1 design takes advantage of the two anti-immune cell binding domains to recruit a broader selection of immune cells (e.g., anti-CD3 for T cells+anti-CD16 for NK cells) or for combinatorial recruitment of payloads with immune cells as theranostics (e.g., anti-CD3 for T cells and anti-BnDOTA for imaging). As illustrated in FIG. 1e, the 2+1+1 design takes advantage of the minimal differences in therapeutic activity between a 2+1 design and a 2+2 design to add a new function, thus broadening the selection of delivered anti-tumor activity to multiple types of immune cells or to chemical or radiological payloads.

The 1+1+1+1 format combines the previous 4 designs to take advantage of all possible combinations. As shown in Figure if, this allows for the combinatorial properties of the 2+1+1 design to be combined with the specificity or selectivity improvements from the Hi1+1+2 and Lo1+1+2 designs.

Example 3: —Superiority of 2+2 IgG-[L]-scFv Design over BITE and IgG-Het

FIG. 2a-2b show the unexpected benefits of the IgG-[L]-scFv (2+2 BsAb) over other common designs such as IgG-Het and BiTE, highlighting both the benefit of having a valency >1 and the structural properties imparted by a Fab/scFv combination. As shown in FIG. 2a, the top panels compare cytotoxicity, cell binding and antigen affinity properties between the IgG-[L]-scFv, IgG-Het and BiTE formats.

The left most panel shows that the 2+2 BsAb achieved nearly 1,000-fold improved cytotoxicity over the 1+1 IgG-Het and >20-fold than the 1+1 BiTE. Measurements were made using a standard four hour ⁵¹Cr release assay using activated human T cells and GD2(+) M14-luciferase cells, with each antibody diluted over 7-logs. The center panel shows the varying levels of antigen binding (GD2 or CD3) between these three formats using GD2(+) M14-luciferase cells or CD3(+) activated human T cells. Cells were stained with each of the three formats and detected using either anti-hu3F8 or anti-huOKT3 idiotypic antibodies. As with the cytotoxicity, the cell binding to both antigens was superior for the 2+2 BsAb due to increased valency. The right panel displays the binding kinetics against the antigen GD2 for each of the three platforms. The 2+2 BsAb exhibited stronger antigen binding over either 1+1 design (BITE or IgG-Het). The bottom panels compare these three constructs in two separate animal models: a huCD3(+) transgenic syngeneic mouse model (left panel) or a humanized immunodeficient xenograft mouse model (right panel). Both models had antibodies injected twice per week and began approximately one week after tumor implantation. Only the 2+2 BsAb was capable of delaying subcutaneous GD2(+) EL.4 tumor growth in the syngeneic model. The 1+1 IgG-Het and the 1+1 BiTE were just as ineffective as the inactive negative control BsAb. Administering the BiTE format daily or at a 10× higher dose level (“hi dose” group, syngeneic mice, FIG. 2a) did not result in any anti-tumor effect. In the xenograft model, where human ATCs and IL-2 were added to support T cell survival in all groups, the 1+1 IgG-Het still failed to show any benefit compared to the control, while the 2+2 BsAb strongly inhibited subcutaneous GD2(+) M14Luc tumors. As show in FIG. 2b, these striking differences in cytotoxicity between the IgG-[L]-scFv and IgG-Het formats were reproducible using two additional anti-GD2 antibodies, suggesting that the effects were not specific to any one GD2 epitope.

These results demonstrate that the HDTVS antibodies disclosed herein are useful in methods for treating a disease or condition, such as cancer.

Example 4: Characterization of IgG-[L]-scFv HDTVS Variants

FIG. 3 describes the characterization of the IgG-[L]-scFv platform to identify the necessity and sufficiency of each binding domain as well as their relative impact on overall functional activity. Unexpectedly, the changes in valency did not entirely correlate with changes in functional output, suggesting a preference for tumor binding by the Fab domain over immune cell binding by the scFv domain, as well as a preference for cis-oriented domains over trans-oriented domains.

As illustrated in FIG. 3, the four IgG-[L]-scFv variants display potencies somewhere between the parental 2+2 IgG-[L]-scFv (top left) and the IgG-Het (bottom right). The 2+1 BsAb (second from left) used heterodimerization to remove one of the two immune cell binding scFv domains yet functioned quite similarly to the parental 2+2 BsAb. Neutralization of the second tumor cell binding Fab domain to create a 1+2 BsAb (third from right) reduced the potency further, but unexpectedly additional removal of an scFv domain did not significantly change the potency, as long as the two remaining domains were in a Cis orientation (1+1C, third from left). Neutralization of the second tumor cell binding Fab was achieved by replacing it with a Fab that binds CD33, an antigen not found on tumor cells or T cells. Neutralization/removal of both the tumor binding Fab domain and the T cell engaging scFv domain in a Trans orientation (1+1T, second from right) caused the biggest drop in potency (equivalent to the IgG-Het), even lower than the 1+1C despite equivalent valency. These results demonstrate that orientation or spatial arrangements of the antigen binding domains are important determinants of therapeutic potency.

These results demonstrate that the HDTVS antibodies disclosed herein are useful in methods for treating a disease or condition, such as cancer.

Example 5: Modifications of the 2+2 IgG-[L]-scFv and Their Relative Binding Activities

FIG. 4 describes the binding activities of each IgG-[L]-scFv variant, compared to the parental 2(GD2)+2(CD3) BsAb and the IgG-Het. Monovalency towards tumor (e.g. 1+2), was created by changing one of the 2 Fab domains to an irrelevant binder (i.e., a huCD33 targeting Fab). Monovalency (e.g. 2+1) towards T cells is created by removing one of the two scFv domains. As illustrated in FIG. 4, bivalency improves antigen binding over monovalency (upper panels). Surface Plasmon Resonance was used to measure antigen binding kinetics against both GD2 coated chips (upper left) and CD3 coated chips (upper right). Briefly, each BsAb was serially titrated and flowed against each chip. Against GD2, the 2+2 BsAb and 2(GD2)+1(CD3) BsAb showed equivalent binding activities whereas the 1+1C, 1+1T, 1+2 and 1+1 IgG-Het all displayed inferior GD2 binding. Against CD3, the pattern was similar, with bivalency being superior over monovalency, but to a lesser extent (which may be attributable in part to the spatial restrictions of bivalent scFv binding compared to Fab binding). The 2+2 and 1+2 BsAb showed the strongest binding, while the 2+1, 1+1T and 1+1C exhibited inferior binding kinetics. The Fab binding domain of the IgG-Het appeared to show some benefit over a monovalent scFv, but this may result from the more stable sequence of a Fab domain compared with an scFv domain, where CH1/CL interactions are lacking. Compared to SPR, cell binding (measured as described in FIG. 2 but using a standard anti-Fc secondary antibody instead of using anti-idiotypic antibodies) showed similar results (bottom left). GD2 binding (left Y-axis) was the strongest in constructs with bivalency (2+2, 2+1), and less for constructs with monovalency (1+1T, 1+1C, 1+2 and IgG-Het). The same pattern was observed with CD3-specific cell binding (right Y-axis), with 2+2 and 1+2 binding being more effective than 2+1, 1+1T and 1+1C.

Similar to the CD3-specific SPR readings, the IgG-Het showed stronger Fab binding than scFv binding. Conjugate formation between targets and effector cells when mixed together with titrated BsAb (bottom right), showed much smaller differences between IgG-[L]-scFv variants. The 2+2 BsAb showed the most efficient conjugate formation activity, followed by the 2+1 BsAb and then all others (except control). These results demonstrate that after the removal of the second anti-effector cell scFv, all other changes to the IgG-[L]-scFv do not markedly reduce its capacity to conjugate effector target cells together, or that the small differences in cell binding activities do not impact conjugate formation or the stability of conjugate formation.

These results demonstrate that the HDTVS antibodies disclosed herein are useful in methods for treating a disease or condition, such as cancer.

Example 6: Modifications of the 2+2 IgG-[L]-scFv and their Relative Cytotoxicity

FIG. 5 describes the anti-tumor cytotoxicity of each IgG-[L]-scFv variant in vitro, across two GD2(+) cell lines. As illustrated in FIG. 5 and summarized in TABLE 2, the variants showed a wide range of cytotoxic potency (assays were performed as described in FIG. 2).

TABLE 2

K_D
Cytotoxic EC50

Fold

Fold

Fold

GD2
Change
CD3
Change
EC50
Change

2 + 2
2.8 nM
—
10 nM
—
17 fM
—

2 + 1
2.5 nM
0.9
310 nM
30.1
106 fM
6.2

1 + 1C
30 nM
10.9
110 nM
11.0
292 fM
17.2

1 + 2
31 nM
11.3
11 nM
1.0
454 fM
26.7

1 + 1H
31 nM
11.4
70 nM
6.8
14 pM
823.5

1 + 1T
21 nM
7.7
88 nM
8.5
13 pM
764.7

Against both tumor cell lines, the 2+2 BsAb displayed the highest cytotoxic effect, followed by the 2+1 and then both 1+1C and 1+2. Interestingly, the 1+1T and IgG-Het (nearly 1,000-fold worse than 2+2) were nearly identical to each other, suggesting that: the cis-oriented binding domains provide superior killing activity compared to trans-oriented binding domains, and that a 2+1 interaction is superior to a 1+2 interaction. Despite the similarities of both the trans and cis oriented 1+1 variants having identical tumor cell binding, effector cell binding capacities, antigen binding kinetics, and conjugate formation activity, the cis-trans orientations of these two constructs differ substantially in the functional output (50-fold) as measured by in vitro cytotoxicity. This unexpected observation may account for why the 1+2 fails to kill as potently as the 2+1. Without wishing to be bound by theory, it is believed that the 1+2 interaction may be caught between a cis and trans interaction at all times, while the 2+1 is more often in a cis interaction. An alternative possibility is that the tumor-binding Fab domains may be more critical for driving anti-tumor potency.

Additionally, the value of each domain and its orientation was quantified. While the 2+2 was about 1,000-fold more potent than the IgG-Het (or 1+1T), it was only 6-fold more potent than the 2+1, and 20-25 fold more potent than the 1+2 or 1+1C. These data demonstrate that the second scFv imparts about 6-fold change in activity (2+2 is 6-fold better than 2+1), the bivalent Fab imparts about 25-fold change (2+2 is up to 25-fold better than 1+2 domain) and the Cis/Trans orientation imparts another 50-fold change (1+1C is 50-fold better than 1+1T).

These results demonstrate that the HDTVS antibodies disclosed herein are useful in methods for treating a disease or condition, such as cancer.

Example 7: Modifications of the 2+2 IgG-[L]-scFv and their Relative Immune Cell Activation

FIG. 6 describes the cell activation properties of each IgG-[L]-scFv variant in vitro. As illustrated in FIG. 6, the variations made to the IgG-[L]-scFv variants significantly influence their capacity to activate immune cells. The upper panels show upregulation of CD69 expression on T cells after 24 hours of in vitro coculture with varying concentrations of each BsAb and GD2(+) M14Luc tumor cells. As in FIG. 5, valency and cis/trans orientation appear to play an important role, suggesting that the activation potency and cytotoxicity are correlated. The 2+2 BsAb again displayed its superiority over all other variants tested, at both the level of expression level of CD69 (left) and the frequency of CD69(+) cells (right). Removal of a single domain (2+1 or 1+2) markedly lowered activation, and was made worse with the transition to 1+1C, 1+1T and finally IgG-Het. A similar pattern emerged after 96 hr of coculture (bottom panel). CD25 expression remained the highest for the 2+2, both in terms of expression level (left) and frequency of CD25(+) (center) cells. All other variants showed reduced activation of effector T cells. Proliferation was also measured using Cell Trace Violet (CTV) dilution. T cells were labeled with the cell penetrating dye CTV and incubated with target cells (M14Luc) and titrated with BsAb for 96 hrs. The frequency of cells fluorescing with less remaining CTV than an unstimulated control was considered to have divided at least once. As such, proliferation was the greatest for the 2+2 and reduced for all other IgG-[L]-scFv variants (right). No activation or proliferation was observed with any construct in the absence of tumor cells (data not shown) indicating that there is minimal activation without target antigen. These results demonstrate that a cis interaction is considerably more potent than a trans interaction (1+1C vs 1+1T) and furthermore that two cis interactions are more potent than one (2+2 vs 1+1C or 1+2 or 2+1) (two cis interactions are only possible in a dual bivalent approach, such as the 2+2 IgG-[L]-scFv).

These results demonstrate that the HDTVS antibodies disclosed herein are useful in methods for treating a disease or condition, such as cancer.

Example 8: Modifications of the 2+2 IgG-[L]-scFv and their Relative In Vivo Tumor Clearance

FIG. 7 describes the in vivo anti-tumor activity of each IgG-[L]-scFv variant in two different tumor models. As illustrated in FIG. 7, the in vivo anti-tumor activity of each variant largely correlated with in vitro cytotoxicity. In the xenograft model (right) the strongest anti-tumor activity was imparted by the 2+2 BsAb. Surprisingly, the 2+1 was very similar, with only a slight difference in tumor recurrence (5/5 CR for both). As with the cytotoxicity data, the next most effective were the 1+1C and 1+2, validating both in vitro findings that the cis orientation is superior to the trans and the 2+1 was superior to the 1+2. All other variants (1+1T, IgG-Het, control BsAb) failed to show any effect on tumor growth. In the more aggressive syngeneic model using EL.4 tumors (as done in FIG. 1), no IgG-[L]-scFv variant aside from the 2+2 showed an anti-tumor effect. As opposed to the xenograft model where activated T-cells are directly administered to the mouse, the syngeneic model requires activation in situ, suggesting that the in vitro cell activation differences may manifest in vivo leading to diminished capacity to shrink tumors. Taken together, these results suggest that the optimal BsAb platform is capable of strong cell activation in the presence of antigen, and that bivalency toward both cell populations, target cells and effector cells, is critical. In addition, these results confirm the importance of two cis-interactions in a bispecific antibody (2+2) over all single cis-interacting variants (2+1, 1+1C, 1+2) or non-cis interacting variants (1+1T, 1+1H).

These results demonstrate that the HDTVS antibodies disclosed herein are useful in methods for treating a disease or condition, such as cancer.

Example 9: 2+2 IgG-[L]-scFv is Superior to Other Bivalent Antibody Designs

FIG. 8 shows cytotoxicity and conjugate formation activity from 3 additional 2+2 designs, thus demonstrating the overall superiority of the IgG-[L]-scFv format. The 2+2 IgG-[L]-scFv format was more demonstrably more potent than other conventional 2+2 formats. The IgG-chemical conjugate (Yankelevich et al., Pediatr Blood Cancer 59:1198-1205 (2012)) the IgG-[H]-scFv (with scFv attached at the C-terminus of the HC instead of the LC of the IgG; Coloma & Morrison, Nat Biotechnol 15:159-163 (1997)) and the BITE-Fc, all failed to kill cells as potently in vitro, compared with the IgG-[L]-scFv design. The poor cytotoxic effects were observed despite apparently improved conjugate formation activity (bottom left) and cell binding activity (bottom right). These results demonstrate that the structural features of the IgG-[L]-scFv format (unique flexibility, orientations and arrangements of the four antigen binding domains) may be correlated with effects on T-cell recruitment, activation and cytotoxicity. FIGS. 12a-12c show the in vivo anti-tumor activity from two additional 2+2 designs, thus confirming the overall superiority of the IgG-[L]-scFv format (2+2). Using an in vivo T-cell arming model, only the IgG-[L]-scFv format (2+2) of the present technology was able to inhibit tumor growth. Strikingly, despite the dual bivalency of the dimeric BiTE-Fc and the IgG-[H]-scFv, both failed to display any anti-tumor activity compared to the control BsAb. These results confirm the in vitro findings, that the superiority of the IgG-[L]-scFv design is not strictly due to decreased distance between binding domains, but instead suggests that the potency of the IgG-[L]-scFv is not simply a function of minimization of intermembrane distance. Rather, the exceptional in vitro and in vivo potency of the IgG-[L]-scFv may be attributed at least in part to the properties of cis-configured Fab and scFv domains, spaced apart with a single Ig domain (CL), such as stiffness or flexibility.

These results demonstrate that the HDTVS antibodies disclosed herein are useful in methods for treating a disease or condition, such as cancer.

Example 10: 2+2 IgG-[L]-scFv and Subset of Variants Against Alternative Antigens

FIG. 9 describes some of the differences in activity observed with different tumor antigens. As illustrated in FIG. 9, the IgG-[L]-scFv platform does depend in part on the tumor antigen. When targeted to CD33 (top panels) a similar pattern of cell binding and cytotoxicity was found. CD33(+) MOLM13-fluc cells were assayed as described in FIG. 4 (left). As with GD2, reduction in valency (1+1T, 1+1C, or 1+2) significantly decreased binding activity. In terms of cytotoxicity, the Cis/Trans orientation appeared to play less of a role (both 1+1T and 1+C are most inferior, and equivalent to IgG-Het), and therefore the difference between the 2+1 and 1+2 was diminished. The lack of cis/trans difference may also explain the overall worse EC₅₀against CD33(+) MOLM-13fluc as compared to GD2(+) M14Luc or IMR32Luc. When the tumor antigen was changed to HER2 (lower panels), and the antigen binding domains possessed significantly higher binding affinity, a different pattern was observed. 2+2 and 1+2 variants appeared identical, with similar tumor binding levels despite the monovalency. This suggests that with sufficiently high affinities, the second tumor binding domain is dispensable, as predicated in the Hi1+1+2 HDTVS design.

These results demonstrate that the HDTVS antibodies disclosed herein are useful in methods for treating a disease or condition, such as cancer.

Example 11: Hi1+1+2 and Lo1+1+2 Proof of Concept Studies

As depicted in FIG. 10a (left side), the 2(HER2)+2(CD3) functions similarly to the 1(HER2)+2(CD3), where only one Fab domain binds the tumor and the second Fab recognizes an irrelevant antigen, due to the very high affinity interaction between HER2 and the anti-HER2 Fab used (Herceptin). In both FACS binding (top) and an in vitro cytotoxicity assay (bottom) with U2OS cells, the 2(HER2)+2(CD3) and the 1(HER2)+2(CD3) were indistinguishable, highlighting the possibility of using the second Fab arm to target a separate antigen. Conversely, the Lo1(GD2)+1(GD2)+2(CD3) (right side), shows the utility of two separate tumor antigen specificities when binding affinities are sufficiently low. Here the 2(GD2)+2(CD3), the 1(GD2)+2(CD3) and Lo1(GD2)+1(GD2)+2(CD3) showed major differences that are explained by the differences in valency between constructs. In both FACS binding (top) and in vitro cytotoxicity (bottom) with U2OS cells, the 2(GD2)+2(CD3) displayed superior activity over a 1(GD2)+2(CD3) format having an irrelevant second specificity (thus limiting binding to monovalency). However, adding a second relevant Fab binding specificity (e.g. HER2) in Lo1(GD2)+1(HER2)+2(CD3) was able to rescue this defect and even improve binding and killing. These results highlight the utility of targeting two separate antigens on the same cell when the Fab affinity for each individual antigen is sufficiently low (e.g., 100 pM to 100 nM K_D). Additionally, the approximately 100-fold difference in EC₅₀between the Lo1(GD2)+1(HER2)+2(CD3) and 1(GD2)+2(CD3) validates the improved therapeutic index between monovalent and bivalent binding of a Lo1(GD2)+1(HER2)+2(CD3) construct. Had the second specificity (i.e. HER2) of the Lo1+1+2(GD2) been irrelevant (no binding to tumor or T cells), it would have functioned as the 1(GD2)+2(CD3) with 100-fold less activity. This is in contrast to the 2+2 which would not be able to distinguish a dual-antigen positive tumor from a GD2(+) normal tissue (such as peripheral nerves).

As shown in FIG. 10b, when these two sets of constructs were presented to tumor cells expressing high levels of only one antigen (HER2 and GD2, left and right sides respectively), the same patterns were observed. With the 2(HER2)+2(CD3) and 1(HER2)+2(CD3), similar FACS binding and cytotoxicity were observed against the HCC1954 cell line which shows high expression of HER2(+). However, stronger binding and cytotoxicity was observed with the 2(GD2)+2(CD3) compared to the 1(GD2)+2(CD3) and a Lo1(GD2)+1(HER2)+2(CD3) having an irrelevant second specificity (second Fab domain did not recognize the tumor cell line IMR32Luc).

Taken together, with a sufficiently high effective affinity interaction a 1+2 IgG-[L]-scFv functions identically to a 2+2, suggesting the Hi1+1+2 can be used to target two separate antigens instead of just one. However, with a sufficiently low effective affinity interaction, a Lo1+1+2 can provide an improved therapeutic index to distinguish between single antigen positive normal tissue and double antigen positive tumor cells.

These results demonstrate that the HDTVS antibodies disclosed herein are useful in methods for treating a disease or condition, such as cancer.

Example 12: Binding Affinity and Cytotoxic Selectivity of the Low Affinity 1+1+2 Format Antibodies of the Present Technology

The binding affinity of L1CAM/GD2 1+1+2 Lo, a heterodimeric 1+1+2Lo format antibody, which can bind ganglioside GD2 and adhesion protein L1CAM simultaneously, was compared with homodimeric formats against GD2 and L1CAM. 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. As shown in FIG. 13, the binding of the low affinity 1+1+2 HDTVS antibody was stronger than that of the anti-L1CAM homodimeric antibody, but weaker than the anti-GD2 homodimeric antibody, thus showing improved targeting specificity for tumors expressing both GD2 and L1CAM.

The combined binding effect of GD2/B7H3 1+1+2 Lo, a heterodimeric 1+1+2Lo format antibody, which can bind both GD2 and B7H3 simultaneously was also compared with the homodimeric format antibodies against GD2 and B7H3, and monovalent control antibodies against GD2 or B7H3. 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 the final wash, the cells were analyzed using flow cytometry. As shown in FIG. 15, the binding of the low affinity 1+1+2 heterodimer antibody was similar to the anti-B7H3 homodimeric antibody, but weaker than the anti-GD2 homodimeric antibody. Importantly, the GD2/B7H3 1+1+2 Lo HDTVS antibody also shows improved binding over monovalent control antibodies, thus demonstrating cooperative binding of the heterodimeric GD2/B7H3 1+1+2 Lo antibody.

To assess the cytotoxic selectivity of the low affinity 1+1+2Lo format antibodies of the present technology, HER2/GD2 1+1+2 Lo, a heterodimeric 1+1+2Lo format antibody, which can bind both GD2 and HER2 simultaneously, was studied. In this format, a low affinity HER2 sequence was used. Homodimeric formats against GD2 and HER2, and monovalent control antibodies against GD2 or HER2 were included for reference. Osteosarcoma cells (U2OS) were first incubated with ⁵¹Cr for one hour. After the incubation, the ⁵¹Cr labeled target cells were mixed with serial dilutions of the antibodies and activated human T-cells for four hours at 37° C. After four hours, supernatant was harvested and analyzed on a gamma counter to quantify the released ⁵¹Cr. As shown in FIG. 16, the low affinity 1+1+2 heterodimer antibody killed U2OS cells as effectively as the anti-GD2 and anti-HER2 homodimeric antibodies and showed clear superiority over the monovalent control formats. Therefore, the 1+1+2Lo design exhibited 10-100-fold lower cytotoxic potency in cells expressing each individual antigen compared to target cells expressing both antigens simultaneously. A homodimeric design for either GD2 or HER2 would not be expected to exhibit such selectivity.

These results demonstrate the selective cytotoxicity could be attained with the 1+1+2Lo design by targeting cells expressing each individual antigen with 10-100-fold lower cytotoxic potency than targets expressing both antigens simultaneously.

Accordingly, the 1+1+2Lo format antibodies of the present technology are useful in methods for treating a disease or condition, such as cancer.

Example 13: Binding Affinity and Cytotoxic Dual Specificity of the 1+1+2Hi Format Antibodies of the Present Technology

To assess the binding affinity of the heterodimeric 1+1+2Hi format antibodies of the present technology, the combined binding effect of HER2/EGFR 1+1+2Hi, a heterodimeric 1+1+2Hi format antibody, which can bind both HER2 and EGFR, either simultaneously or separately, was analyzed. Homodimeric formats against HER2 and EGFR were included for reference. Desmoplastic Small Cell Round 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. As shown in FIG. 14, the binding of the high affinity 1+1+2 heterodimer antibody was stronger than that of either anti-HER2 or anti-EGFR homodimeric antibodies, while maintaining specificity for both antigens, thus demonstrating cooperative binding.

HER2/GPA33 1+1+2 Hi, a heterodimeric 1+1+2Hi format antibody, which can bind both GPA33 and HER2 either simultaneously or separately, was compared with the homodimeric format antibodies against GPA33 and HER2, and monovalent control antibodies against GPA33 or HER2. To compare the combined binding effect, 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 the final wash, the cells were analyzed using flow cytometry. HER2/GPA33 1+1+2 Hi antibody bound both HER2 and GPA33 on Colo205 cells, either simultaneously or separately (FIG. 17b). As shown in FIG. 17b, the binding affinity of the 1+1+2Hi heterodimer antibody was stronger than either anti-HER2 or anti-GPA33 homodimeric and monovalent control antibodies, while maintaining specificity for both antigens, thus demonstrating cooperative binding.

To evaluate the cytotoxic specificity of the HER2/GPA33 1+1+2Hi format antibody, colon cancer cells (Colo205) were first incubated with ⁵¹Cr for one hour. After the incubation, the ⁵¹Cr labeled target cells were mixed with serial dilutions of the indicated antibody and activated human T-cells for four hours at 37° C. After four hours, the supernatant was harvested and read on a gamma counter to quantify the released ⁵¹Cr. Cytotoxicity was measured as the % of released ⁵¹Cr from maximum release. As shown in FIG. 17a, the high affinity 1+1+2 heterodimer antibody killed Colo205 cells as effectively as the anti-GPA33 homodimeric antibody, but with greater potency than the anti-HER2 homodimeric antibody and monovalent control antibodies. These results demonstrate functional cooperativity between the HER2 and GPA33 antigen binding domains, and illustrate that the dual specificity of a 1+1+2Hi format does not significantly compromise its cytotoxicity against either antigen individually.

Accordingly, the 1+1+2Hi format antibodies of the present technology are useful in methods for treating a disease or condition, such as cancer.

Example 14: Combined Binding Effects and Cytokine Release Induced by the 2+1+1 Format Antibodies of the Present Technology

To evaluate the combined binding effects of the heterodimeric 2+1+1 format antibodies of the present technology, several heterodimeric 2+1+1 format antibodies were compared with their corresponding homodimeric format antibodies and monovalent control antibodies. For example, CD3/CD4 2+1+1, a heterodimeric 2+1+1 format antibody that can bind both CD3 and CD4 simultaneously was compared with its corresponding bivalent format antibodies against CD3 and CD4, and a monomeric CD3 binder (2+1). For this binding assay, active 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 the final wash, the cells were analyzed using flow cytometry. As shown in FIG. 19, the binding of CD3/CD4 2+1+1 antibodies showed enhanced binding compared to the bivalent CD4 antibody and monomeric CD3 binder (2+1), thus demonstrating cooperative binding.

Similarly, binding of CD3/PD-1 2+1+1, a heterodimeric 2+1+1 format antibody that can bind both CD3 and PD-1 simultaneously, was compared with homodimeric anti-PD-1 and anti-CD3 antibodies, and with an anti-CD3 monomeric (2+1) binder. For this binding assay active 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 the final wash, the cells were analyzed using flow cytometry. As shown in FIG. 20, the 2+1+1 heterodimer antibody bound cells better than either anti-PD-1 homodimeric antibody or anti-CD3 monomeric (2+1) binder, thus demonstrating cooperative binding. Collectively, these data demonstrate that a heterodimeric 2+1+1 format antibody of the present technology binds its target better than the corresponding weaker-binding homodimeric antibody and its corresponding monomeric (2+1) binder, thus demonstrating cooperative binding.

Next, cytokine release induced by CD3/CD28 2+1+1, a heterodimeric 2+1+1 format antibody, was analyzed. The homodimeric format antibodies against CD3 and CD28 were included for reference. Naïve human T-cells and melanoma tumor cells (M14) were co-cultured along with the indicated BsAb for 20 hours. Culture supernatants were harvested following the incubation and analyzed for secreted cytokine IL-2 by FACS. Data were normalized to T-cell cytokine release after 20 hours without target cells or antibody. As shown in FIG. 18, the CD3/CD28 2+1+1 antibody showed more potent cytokine release activity compared to either CD3 or CD28 engagement alone, illustrating cooperative activity from dual CD3/CD28 engagement. These results demonstrate the utility of a heterodimeric 2+1+1 design that can bind both CD3 and CD28 on T-cells.

Accordingly, the 2+1+1 format antibodies of the present technology are useful in methods for treating a disease or condition, such as cancer.

Example 15: Comparison of the IgG-L-scFv Format of the Present Technology with BiTE-Fc and IgG-H-scFv Formats

The IgG-L-scFv design was next compared with two other common dual bivalent design strategies: the BiTE-Fc and the IgG-H-scFv formats. First, to compare cytokine release induced by IgG-L-scFv design compared to BiTE-Fc and the IgG-H-scFv, naïve T-cells and melanoma tumor cells (M14) were co-cultured along with each BsAb for 20 hours. Culture supernatants were harvested and analyzed for secreted cytokine IL-2. Data were normalized to T-cell cytokine release after 20 hours without target cells or antibody. As shown in FIG. 21a, the IgG-L-scFv design (2+2) exhibited unusually potent T-cell functional activity compared to other dual bivalent T-cell bispecific antibody formats.

To compare binding intensity, T-cells and melanoma tumor cells (M14) were separately 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. As shown in FIG. 21b (upper panel), IgG-L-scFv design showed unusually weak T-cell binding activity compared to other dual bivalent T-cell bispecific antibody formats. In contrast to their GD2 binding activity (FIG. 21b (middle panel)), each BsAb demonstrated quite different T-cell binding activities. These data demonstrated how the IgG-L-scFv design is uniquely different than other dual-bivalent designs, with each scFv showing incomplete bivalent binding. Although the inclusion of two scFv domains in the IgG-L-scFv did result in an improvement over monovalent designs, it still did not compare to the binding activity of the 2+2 IgG-H-scFv or 2+2 BiTE-Fc designs, illustrating the sterically hindered binding of this format.

The effect of the observed binding and cytokine release profiles on the in vivo antitumor activity was explored next. Immunodeficient mice (Balb/c IL-2Rgc−/−, Rag2−/−) were implanted with neuroblastoma cells (IMR32) subcutaneously and treated with intravenous activated T-cells and antibody (2-times per week). Tumors sizes were measured by caliper. As shown in FIG. 21c, the IgG-L-scFv design antibodies inhibited tumor growth. In comparison, the IgG-H-scFv and BiTE-Fc design antibodies showed a borderline in vivo effect. Therefore, in contrast to the IgG-H-scFv (2+2HC) and the BiTE-Fc (2+2B) designs, the IgG-L-scFv format (2+2) demonstrated significant cytokine IL-2 responses in vitro (FIG. 21a), which correlated with stronger in vivo activity (FIG. 21c).

Collectively, these data demonstrate the in vivo superiority of the IgG-L-scFv format antibodies in that only the IgG-L-scFv format antibodies were capable of inhibiting tumor growth in animals in contrast to other dual bivalent designs.

These results demonstrate that the HDTVS antibodies disclosed herein are useful in methods for treating a disease or condition, such as cancer.

Example 16: Importance of Cis-Oriented Binding Domains with Respect to In Vitro Properties of an Anti-IgG-[L]-scFv Antibody

To further understand the in vitro properties of antibodies of various designs, a anti-CD33 IgG-[L]-scFv panel was created, and the in vitro cytotoxicity EC₅₀, fold-difference in EC₅₀, antigen valency, heterodimer design and protein purity were examined. FIG. 22 summarizes the data. Fold change was based on the EC₅₀of 2+2. Purity was calculated as the fraction of protein at correct elution time out of the total protein by area under the curve of the SEC-HPLC chromatogram. For the cytotoxicity assays, CD33-transfected cells (Nalm6) were first incubated with ⁵¹Cr for one hour. Afterwards, ⁵¹Cr labeled target cells were mixed with serial titrations of the indicated antibody and activated human T-cells for four hours at 37° C. The supernatant was harvested and analyzed on a gamma counter to quantify the released ⁵¹Cr. Cytotoxicity was measured as the % of released ⁵¹Cr from maximum release. These results shown in FIG. 22 confirm the relative importance of cis-oriented binding domains in an additional antigen system (CD33) which is much more membrane distal than GD2 (see FIG. 5).

These results demonstrate that the HDTVS antibodies disclosed herein are useful in methods for treating a disease or condition, such as cancer.

EQUIVALENTS

The present technology is not to be limited in terms of the particular embodiments described in this application, which are intended as single illustrations of individual aspects of the present technology. Many modifications and variations of this present technology can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. Functionally equivalent methods and apparatuses within the scope of the present technology, in addition to those enumerated herein, will be apparent to those skilled in the art from the foregoing descriptions. Such modifications and variations are intended to fall within the scope of the present technology. It is to be understood that this present technology is not limited to particular methods, reagents, compounds compositions or biological systems, which can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

In addition, where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group.

As will be understood by one skilled in the art, for any and all purposes, particularly in terms of providing a written description, all ranges disclosed herein also encompass any and all possible subranges and combinations of subranges thereof. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art all language such as “up to,” “at least,” “greater than,” “less than,” and the like, include the number recited and refer to ranges which can be subsequently broken down into subranges as discussed above. Finally, as will be understood by one skilled in the art, a range includes each individual member. Thus, for example, a group having 1-3 cells refers to groups having 1, 2, or 3 cells. Similarly, a group having 1-5 cells refers to groups having 1, 2, 3, 4, or 5 cells, and so forth.

All patents, patent applications, provisional applications, and publications referred to or cited herein are incorporated by reference in their entirety, including all figures and tables, to the extent they are not inconsistent with the explicit teachings of this specification.

	Number	Date	Country
	62774111	Nov 2018	US
	62794523	Jan 2019	US

HETERODIMERIC TETRAVALENCY AND SPECIFICITY ANTIBODY COMPOSITIONS AND USES THEREOF

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