Amino acid sequences directed against envelope proteins of a virus and polypeptides comprising the same for the treatment of viral diseases
11518799 · 2022-12-06
Assignee
Inventors
- Catelijne Stortelers (Ghent, BE)
- Peter Vanlandschoot (Bellem, BE)
- Erik Depla (Destelbergen, BE)
- Jose Melero (Madrid, ES)
Cpc classification
C07K2317/569
CHEMISTRY; METALLURGY
C07K2317/94
CHEMISTRY; METALLURGY
A61K2039/507
HUMAN NECESSITIES
C07K2317/24
CHEMISTRY; METALLURGY
C07K16/1027
CHEMISTRY; METALLURGY
C07K2317/64
CHEMISTRY; METALLURGY
C07K2317/76
CHEMISTRY; METALLURGY
C07K2317/92
CHEMISTRY; METALLURGY
C07K2317/22
CHEMISTRY; METALLURGY
International classification
Abstract
The present invention relates in part to amino acid sequences that are directed against and/or that can specifically bind to an envelope protein of a virus, as well as to compounds or constructs, and in particular proteins and polypeptides, that comprise or essentially consist of one or more such amino acid sequences.
Claims
1. A method for the neutralization of different genotypes, different subtypes, different strains, and/or one or more escape mutants of a respiratory syncytial virus (RSV), comprising administering, to a subject in need thereof, a multivalent polypeptide that specifically binds to an F protein of a RSV, said polypeptide comprising three or more immunoglobulin single variable domains, wherein at least one of the immunoglobulin single variable domains comprises a combination of a CDR1, a CDR2 and a CDR3 selected from the following combinations: TABLE-US-00095 Combi- nation CDR1 CDR2 CDR3 1 SEQ ID NO: 722 SEQ ID NO: 1286 SEQ ID NO: 1850 2 SEQ ID NO: 723 SEQ ID NO: 1287 SEQ ID NO: 1851 3 SEQ ID NO: 724 SEQ ID NO: 1288 SEQ ID NO: 1852 4 SEQ ID NO: 725 SEQ ID NO: 1289 SEQ ID NO: 1853 5 SEQ ID NO: 726 SEQ ID NO: 1290 SEQ ID NO: 1854 6 SEQ ID NO: 727 SEQ ID NO: 1291 SEQ ID NO: 1855 7 SEQ ID NO: 728 SEQ ID NO: 1292 SEQ ID NO: 1856 8 SEQ ID NO: 729 SEQ ID NO: 1293 SEQ ID NO: 1857 9 SEQ ID NO: 730 SEQ ID NO: 1294 SEQ ID NO: 1858 10 SEQ ID NO: 731 SEQ ID NO: 1295 SEQ ID NO: 1859 11 SEQ ID NO: 732 SEQ ID NO: 1296 SEQ ID NO: 1860 12 SEQ ID NO: 733 SEQ ID NO: 1297 SEQ ID NO: 1861 13 SEQ ID NO: 734 SEQ ID NO: 1298 SEQ ID NO: 1862 14 SEQ ID NO: 735 SEQ ID NO: 1299 SEQ ID NO: 1863 15 SEQ ID NO: 736 SEQ ID NO: 1300 SEQ ID NO: 1864 16 SEQ ID NO: 737 SEQ ID NO: 1301 SEQ ID NO: 1865 17 SEQ ID NO: 738 SEQ ID NO: 1302 SEQ ID NO: 1866 18 SEQ ID NO: 739 SEQ ID NO: 1303 SEQ ID NO: 1867 19 SEQ ID NO: 740 SEQ ID NO: 1304 SEQ ID NO: 1868 20 SEQ ID NO: 741 SEQ ID NO: 1305 SEQ ID NO: 1869 21 SEQ ID NO: 742 SEQ ID NO: 1306 SEQ ID NO: 1870 22 SEQ ID NO: 743 SEQ ID NO: 1307 SEQ ID NO: 1871 23 SEQ ID NO: 744 SEQ ID NO: 1308 SEQ ID NO: 1872 24 SEQ ID NO: 745 SEQ ID NO: 1309 SEQ ID NO: 1873 25 SEQ ID NO: 746 SEQ ID NO: 1310 SEQ ID NO: 1874 26 SEQ ID NO: 747 SEQ ID NO: 1311 SEQ ID NO: 1875 27 SEQ ID NO: 748 SEQ ID NO: 1312 SEQ ID NO: 1876 28 SEQ ID NO: 749 SEQ ID NO: 1313 SEQ ID NO: 1877 29 SEQ ID NO: 750 SEQ ID NO: 1314 SEQ ID NO: 1878 30 SEQ ID NO: 751 SEQ ID NO: 1315 SEQ ID NO: 1879 31 SEQ ID NO: 752 SEQ ID NO: 1316 SEQ ID NO: 1880 32 SEQ ID NO: 753 SEQ ID NO: 1317 SEQ ID NO: 1881 33 SEQ ID NO: 754 SEQ ID NO: 1318 SEQ ID NO: 1882 34 SEQ ID NO: 755 SEQ ID NO: 1319 SEQ ID NO: 1883 35 SEQ ID NO: 756 SEQ ID NO: 1320 SEQ ID NO: 1884 36 SEQ ID NO: 757 SEQ ID NO: 1321 SEQ ID NO: 1885 37 SEQ ID NO: 758 SEQ ID NO: 1322 SEQ ID NO: 1886 38 SEQ ID NO: 759 SEQ ID NO: 1323 SEQ ID NO: 1887 39 SEQ ID NO: 760 SEQ ID NO: 1324 SEQ ID NO: 1888 40 SEQ ID NO: 761 SEQ ID NO: 1325 SEQ ID NO: 1889 41 SEQ ID NO: 762 SEQ ID NO: 1326 SEQ ID NO: 1890 42 SEQ ID NO: 763 SEQ ID NO: 1327 SEQ ID NO: 1891 43 SEQ ID NO: 764 SEQ ID NO: 1328 SEQ ID NO: 1892 44 SEQ ID NO: 765 SEQ ID NO: 1329 SEQ ID NO: 1893 45 SEQ ID NO: 766 SEQ ID NO: 1330 SEQ ID NO: 1894 46 SEQ ID NO: 767 SEQ ID NO: 1331 SEQ ID NO: 1895 47 SEQ ID NO: 768 SEQ ID NO: 1332 SEQ ID NO: 1896 48 SEQ ID NO: 769 SEQ ID NO: 1333 SEQ ID NO: 1897 49 SEQ ID NO: 770 SEQ ID NO: 1334 SEQ ID NO: 1898 50 SEQ ID NO: 771 SEQ ID NO: 1335 SEQ ID NO: 1899 51 SEQ ID NO: 772 SEQ ID NO: 1336 SEQ ID NO: 1900 52 SEQ ID NO: 773 SEQ ID NO: 1337 SEQ ID NO: 1901 53 SEQ ID NO: 774 SEQ ID NO: 1338 SEQ ID NO: 1902 54 SEQ ID NO: 775 SEQ ID NO: 1339 SEQ ID NO: 1903 55 SEQ ID NO: 776 SEQ ID NO: 1340 SEQ ID NO: 1904 56 SEQ ID NO: 777 SEQ ID NO: 1341 SEQ ID NO: 1905 57 SEQ ID NO: 778 SEQ ID NO: 1342 SEQ ID NO: 1906 58 SEQ ID NO: 779 SEQ ID NO: 1343 SEQ ID NO: 1907 59 SEQ ID NO: 780 SEQ ID NO: 1344 SEQ ID NO: 1908 60 SEQ ID NO: 781 SEQ ID NO: 1345 SEQ ID NO: 1909 61 SEQ ID NO: 782 SEQ ID NO: 1346 SEQ ID NO: 1910 62 SEQ ID NO: 783 SEQ ID NO: 1347 SEQ ID NO: 1911 63 SEQ ID NO: 784 SEQ ID NO: 1348 SEQ ID NO: 1912 64 SEQ ID NO: 785 SEQ ID NO: 1349 SEQ ID NO: 1913 65 SEQ ID NO: 786 SEQ ID NO: 1350 SEQ ID NO: 1914 66 SEQ ID NO: 787 SEQ ID NO: 1351 SEQ ID NO: 1915 67 SEQ ID NO: 788 SEQ ID NO: 1352 SEQ ID NO: 1916 68 SEQ ID NO: 789 SEQ ID NO: 1353 SEQ ID NO: 1917 69 SEQ ID NO: 790 SEQ ID NO: 1354 SEQ ID NO: 1918 70 SEQ ID NO: 791 SEQ ID NO: 1355 SEQ ID NO: 1919 71 SEQ ID NO: 792 SEQ ID NO: 1356 SEQ ID NO: 1920 72 SEQ ID NO: 793 SEQ ID NO: 1357 SEQ ID NO: 1921 73 SEQ ID NO: 794 SEQ ID NO: 1358 SEQ ID NO: 1922 74 SEQ ID NO: 795 SEQ ID NO: 1359 SEQ ID NO: 1923 75 SEQ ID NO: 796 SEQ ID NO: 1360 SEQ ID NO: 1924 76 SEQ ID NO: 797 SEQ ID NO: 1361 SEQ ID NO: 1925 77 SEQ ID NO: 798 SEQ ID NO: 1362 SEQ ID NO: 1926 78 SEQ ID NO: 799 SEQ ID NO: 1363 SEQ ID NO: 1927 79 SEQ ID NO: 800 SEQ ID NO: 1364 SEQ ID NO: 1928 80 SEQ ID NO: 812 SEQ ID NO: 1376 SEQ ID NO: 1940 81 SEQ ID NO: 813 SEQ ID NO: 1377 SEQ ID NO: 1941 82 SEQ ID NO: 814 SEQ ID NO: 1378 SEQ ID NO: 1942 83 SEQ ID NO: 815 SEQ ID NO: 1379 SEQ ID NO: 1943 84 SEQ ID NO: 816 SEQ ID NO: 1380 SEQ ID NO: 1944 85 SEQ ID NO: 817 SEQ ID NO: 1381 SEQ ID NO: 1945 86 SEQ ID NO: 818 SEQ ID NO: 1382 SEQ ID NO: 1946 87 SEQ ID NO: 819 SEQ ID NO: 1383 SEQ ID NO: 1947 88 SEQ ID NO: 820 SEQ ID NO: 1384 SEQ ID NO: 1948 89 SEQ ID NO: 821 SEQ ID NO: 1385 SEQ ID NO: 1949 90 SEQ ID NO: 822 SEQ ID NO: 1386 SEQ ID NO: 1950 91 SEQ ID NO: 823 SEQ ID NO: 1387 SEQ ID NO: 1951 92 SEQ ID NO: 824 SEQ ID NO: 1388 SEQ ID NO: 1952 93 SEQ ID NO: 825 SEQ ID NO: 1389 SEQ ID NO: 1953 94 SEQ ID NO: 826 SEQ ID NO: 1390 SEQ ID NO: 1954 95 SEQ ID NO: 827 SEQ ID NO: 1391 SEQ ID NO: 1955 96 SEQ ID NO: 828 SEQ ID NO: 1392 SEQ ID NO: 1956 97 SEQ ID NO: 829 SEQ ID NO: 1393 SEQ ID NO: 1957 98 SEQ ID NO: 830 SEQ ID NO: 1394 SEQ ID NO: 1958 99 SEQ ID NO: 831 SEQ ID NO: 1395 SEQ ID NO: 1959 100 SEQ ID NO: 832 SEQ ID NO: 1396 SEQ ID NO: 1960 101 SEQ ID NO: 833 SEQ ID NO: 1397 SEQ ID NO: 1961 102 SEQ ID NO: 834 SEQ ID NO: 1398 SEQ ID NO: 1962 103 SEQ ID NO: 835 SEQ ID NO: 1399 SEQ ID NO: 1963 104 SEQ ID NO: 836 SEQ ID NO: 1400 SEQ ID NO: 1964 105 SEQ ID NO: 837 SEQ ID NO: 1401 SEQ ID NO: 1965 106 SEQ ID NO: 838 SEQ ID NO: 1402 SEQ ID NO: 1966 107 SEQ ID NO: 839 SEQ ID NO: 1403 SEQ ID NO: 1967 108 SEQ ID NO: 840 SEQ ID NO: 1404 SEQ ID NO: 1968 109 SEQ ID NO: 841 SEQ ID NO: 1405 SEQ ID NO: 1969 110 SEQ ID NO: 842 SEQ ID NO: 1406 SEQ ID NO: 1970 111 SEQ ID NO: 843 SEQ ID NO: 1407 SEQ ID NO: 1971 112 SEQ ID NO: 844 SEQ ID NO: 1408 SEQ ID NO: 1972 113 SEQ ID NO: 845 SEQ ID NO: 1409 SEQ ID NO: 1973 114 SEQ ID NO: 846 SEQ ID NO: 1410 SEQ ID NO: 1974 115 SEQ ID NO: 847 SEQ ID NO: 1411 SEQ ID NO: 1975 116 SEQ ID NO: 848 SEQ ID NO: 1412 SEQ ID NO: 1976 117 SEQ ID NO: 849 SEQ ID NO: 1413 SEQ ID NO: 1977 118 SEQ ID NO: 850 SEQ ID NO: 1414 SEQ ID NO: 1978 119 SEQ ID NO: 851 SEQ ID NO: 1415 SEQ ID NO: 1979 120 SEQ ID NO: 852 SEQ ID NO: 1416 SEQ ID NO: 1980 121 SEQ ID NO: 853 SEQ ID NO: 1417 SEQ ID NO: 1981 122 SEQ ID NO: 854 SEQ ID NO: 1418 SEQ ID NO: 1982 123 SEQ ID NO: 855 SEQ ID NO: 1419 SEQ ID NO: 1983 124 SEQ ID NO: 856 SEQ ID NO: 1420 SEQ ID NO: 1984 125 SEQ ID NO: 857 SEQ ID NO: 1421 SEQ ID NO: 1985 126 SEQ ID NO: 858 SEQ ID NO: 1422 SEQ ID NO: 1986 127 SEQ ID NO: 859 SEQ ID NO: 1423 SEQ ID NO: 1987 128 SEQ ID NO: 860 SEQ ID NO: 1424 SEQ ID NO: 1988 129 SEQ ID NO: 861 SEQ ID NO: 1425 SEQ ID NO: 1989 130 SEQ ID NO: 862 SEQ ID NO: 1426 SEQ ID NO: 1990 131 SEQ ID NO: 863 SEQ ID NO: 1427 SEQ ID NO: 1991 132 SEQ ID NO: 864 SEQ ID NO: 1428 SEQ ID NO: 1992 133 SEQ ID NO: 865 SEQ ID NO: 1429 SEQ ID NO: 1993 134 SEQ ID NO: 866 SEQ ID NO: 1430 SEQ ID NO: 1994 135 SEQ ID NO: 867 SEQ ID NO: 1431 SEQ ID NO: 1995 136 SEQ ID NO: 868 SEQ ID NO: 1432 SEQ ID NO: 1996 137 SEQ ID NO: 869 SEQ ID NO: 1433 SEQ ID NO: 1997 138 SEQ ID NO: 870 SEQ ID NO: 1434 SEQ ID NO: 1998 139 SEQ ID NO: 871 SEQ ID NO: 1435 SEQ ID NO: 1999 140 SEQ ID NO: 872 SEQ ID NO: 1436 SEQ ID NO: 2000 141 SEQ ID NO: 873 SEQ ID NO: 1437 SEQ ID NO: 2001 142 SEQ ID NO: 874 SEQ ID NO: 1438 SEQ ID NO: 2002 143 SEQ ID NO: 875 SEQ ID NO: 1439 SEQ ID NO: 2003 144 SEQ ID NO: 876 SEQ ID NO: 1440 SEQ ID NO: 2004 145 SEQ ID NO: 877 SEQ ID NO: 1441 SEQ ID NO: 2005 146 SEQ ID NO: 878 SEQ ID NO: 1442 SEQ ID NO: 2006 147 SEQ ID NO: 879 SEQ ID NO: 1443 SEQ ID NO: 2007 148 SEQ ID NO: 880 SEQ ID NO: 1444 SEQ ID NO: 2008 149 SEQ ID NO: 881 SEQ ID NO: 1445 SEQ ID NO: 2009 150 SEQ ID NO: 882 SEQ ID NO: 1446 SEQ ID NO: 2010 151 SEQ ID NO: 883 SEQ ID NO: 1447 SEQ ID NO: 2011 152 SEQ ID NO: 884 SEQ ID NO: 1448 SEQ ID NO: 2012 153 SEQ ID NO: 885 SEQ ID NO: 1449 SEQ ID NO: 2013 154 SEQ ID NO: 886 SEQ ID NO: 1450 SEQ ID NO: 2014 155 SEQ ID NO: 887 SEQ ID NO: 1451 SEQ ID NO: 2015 156 SEQ ID NO: 888 SEQ ID NO: 1452 SEQ ID NO: 2016 157 SEQ ID NO: 889 SEQ ID NO: 1453 SEQ ID NO: 2017 158 SEQ ID NO: 890 SEQ ID NO: 1454 SEQ ID NO: 2018 159 SEQ ID NO: 891 SEQ ID NO: 1455 SEQ ID NO: 2019 160 SEQ ID NO: 892 SEQ ID NO: 1456 SEQ ID NO: 2020 161 SEQ ID NO: 893 SEQ ID NO: 1457 SEQ ID NO: 2021 162 SEQ ID NO: 894 SEQ ID NO: 1458 SEQ ID NO: 2022 163 SEQ ID NO: 895 SEQ ID NO: 1459 SEQ ID NO: 2023 164 SEQ ID NO: 896 SEQ ID NO: 1460 SEQ ID NO: 2024 165 SEQ ID NO: 897 SEQ ID NO: 1461 SEQ ID NO: 2025 166 SEQ ID NO: 898 SEQ ID NO: 1462 SEQ ID NO: 2026 167 SEQ ID NO: 899 SEQ ID NO: 1463 SEQ ID NO: 2027 168 SEQ ID NO: 900 SEQ ID NO: 1464 SEQ ID NO: 2028 169 SEQ ID NO: 901 SEQ ID NO: 1465 SEQ ID NO: 2029 170 SEQ ID NO: 902 SEQ ID NO: 1466 SEQ ID NO: 2030 171 SEQ ID NO: 903 SEQ ID NO: 1467 SEQ ID NO: 2031 172 SEQ ID NO: 904 SEQ ID NO: 1468 SEQ ID NO: 2032 173 SEQ ID NO: 905 SEQ ID NO: 1469 SEQ ID NO: 2033 174 SEQ ID NO: 906 SEQ ID NO: 1470 SEQ ID NO: 2034 175 SEQ ID NO: 907 SEQ ID NO: 1471 SEQ ID NO: 2035 176 SEQ ID NO: 908 SEQ ID NO: 1472 SEQ ID NO: 2036 177 SEQ ID NO: 909 SEQ ID NO: 1473 SEQ ID NO: 2037 178 SEQ ID NO: 910 SEQ ID NO: 1474 SEQ ID NO: 2038 179 SEQ ID NO: 911 SEQ ID NO: 1475 SEQ ID NO: 2039 180 SEQ ID NO: 912 SEQ ID NO: 1476 SEQ ID NO: 2040 181 SEQ ID NO: 913 SEQ ID NO: 1477 SEQ ID NO: 2041 182 SEQ ID NO: 914 SEQ ID NO: 1478 SEQ ID NO: 2042 183 SEQ ID NO: 915 SEQ ID NO: 1479 SEQ ID NO: 2043 184 SEQ ID NO: 916 SEQ ID NO: 1480 SEQ ID NO: 2044 185 SEQ ID NO: 917 SEQ ID NO: 1481 SEQ ID NO: 2045 186 SEQ ID NO: 918 SEQ ID NO: 1482 SEQ ID NO: 2046 187 SEQ ID NO: 919 SEQ ID NO: 1483 SEQ ID NO: 2047 188 SEQ ID NO: 920 SEQ ID NO: 1484 SEQ ID NO: 2048 189 SEQ ID NO: 921 SEQ ID NO: 1485 SEQ ID NO: 2049 190 SEQ ID NO: 922 SEQ ID NO: 1486 SEQ ID NO: 2050 191 SEQ ID NO: 923 SEQ ID NO: 1487 SEQ ID NO: 2051 192 SEQ ID NO: 924 SEQ ID NO: 1488 SEQ ID NO: 2052 193 SEQ ID NO: 925 SEQ ID NO: 1489 SEQ ID NO: 2053 194 SEQ ID NO: 926 SEQ ID NO: 1490 SEQ ID NO: 2054 195 SEQ ID NO: 927 SEQ ID NO: 1491 SEQ ID NO: 2055 196 SEQ ID NO: 928 SEQ ID NO: 1492 SEQ ID NO: 2056 197 SEQ ID NO: 929 SEQ ID NO: 1493 SEQ ID NO: 2057 198 SEQ ID NO: 930 SEQ ID NO: 1494 SEQ ID NO: 2058 199 SEQ ID NO: 931 SEQ ID NO: 1495 SEQ ID NO: 2059 200 SEQ ID NO: 932 SEQ ID NO: 1496 SEQ ID NO: 2060 201 SEQ ID NO: 933 SEQ ID NO: 1497 SEQ ID NO: 2061 202 SEQ ID NO: 934 SEQ ID NO: 1498 SEQ ID NO: 2062 203 SEQ ID NO: 935 SEQ ID NO: 1499 SEQ ID NO: 2063 204 SEQ ID NO: 936 SEQ ID NO: 1500 SEQ ID NO: 2064 205 SEQ ID NO: 937 SEQ ID NO: 1501 SEQ ID NO: 2065 206 SEQ ID NO: 938 SEQ ID NO: 1502 SEQ ID NO: 2066 207 SEQ ID NO: 939 SEQ ID NO: 1503 SEQ ID NO: 2067 208 SEQ ID NO: 940 SEQ ID NO: 1504 SEQ ID NO: 2068 209 SEQ ID NO: 941 SEQ ID NO: 1505 SEQ ID NO: 2069 210 SEQ ID NO: 942 SEQ ID NO: 1506 SEQ ID NO: 2070 211 SEQ ID NO: 943 SEQ ID NO: 1507 SEQ ID NO: 2071 212 SEQ ID NO: 944 SEQ ID NO: 1508 SEQ ID NO: 2072 213 SEQ ID NO: 945 SEQ ID NO: 1509 SEQ ID NO: 2073 214 SEQ ID NO: 946 SEQ ID NO: 1510 SEQ ID NO: 2074 215 SEQ ID NO: 947 SEQ ID NO: 1511 SEQ ID NO: 2075 216 SEQ ID NO: 948 SEQ ID NO: 1512 SEQ ID NO: 2076 217 SEQ ID NO: 949 SEQ ID NO: 1513 SEQ ID NO: 2077 218 SEQ ID NO: 950 SEQ ID NO: 1514 SEQ ID NO: 2078 219 SEQ ID NO: 951 SEQ ID NO: 1515 SEQ ID NO: 2079 220 SEQ ID NO: 952 SEQ ID NO: 1516 SEQ ID NO: 2080 221 SEQ ID NO: 953 SEQ ID NO: 1517 SEQ ID NO: 2081 222 SEQ ID NO: 954 SEQ ID NO: 1518 SEQ ID NO: 2082 223 SEQ ID NO: 955 SEQ ID NO: 1519 SEQ ID NO: 2083 224 SEQ ID NO: 956 SEQ ID NO: 1520 SEQ ID NO: 2084 225 SEQ ID NO: 957 SEQ ID NO: 1521 SEQ ID NO: 2085 226 SEQ ID NO: 958 SEQ ID NO: 1522 SEQ ID NO: 2086 227 SEQ ID NO: 959 SEQ ID NO: 1523 SEQ ID NO: 2087 228 SEQ ID NO: 960 SEQ ID NO: 1524 SEQ ID NO: 2088 229 SEQ ID NO: 961 SEQ ID NO: 1525 SEQ ID NO: 2089 230 SEQ ID NO: 962 SEQ ID NO: 1526 SEQ ID NO: 2090 231 SEQ ID NO: 963 SEQ ID NO: 1527 SEQ ID NO: 2091 232 SEQ ID NO: 964 SEQ ID NO: 1528 SEQ ID NO: 2092 233 SEQ ID NO: 965 SEQ ID NO: 1529 SEQ ID NO: 2093 234 SEQ ID NO: 966 SEQ ID NO: 1530 SEQ ID NO: 2094 235 SEQ ID NO: 967 SEQ ID NO: 1531 SEQ ID NO: 2095 236 SEQ ID NO: 968 SEQ ID NO: 1532 SEQ ID NO: 2096 237 SEQ ID NO: 969 SEQ ID NO: 1533 SEQ ID NO: 2097 238 SEQ ID NO: 970 SEQ ID NO: 1534 SEQ ID NO: 2098 239 SEQ ID NO: 971 SEQ ID NO: 1535 SEQ ID NO: 2099 240 SEQ ID NO: 2484 SEQ ID NO: 2520 SEQ ID NO: 2556 241 SEQ ID NO: 2590 SEQ ID NO: 2606 SEQ ID NO: 2622 242 SEQ ID NO: 2591 SEQ ID NO: 2607 SEQ ID NO: 2623 243 SEQ ID NO: 2592 SEQ ID NO: 2608 SEQ ID NO: 2624 244 SEQ ID NO: 2593 SEQ ID NO: 2609 SEQ ID NO: 2625 245 SEQ ID NO: 2594 SEQ ID NO: 2610 SEQ ID NO: 2626 246 SEQ ID NO: 2596 SEQ ID NO: 2612 SEQ ID NO: 2628 247 SEQ ID NO: 2597 SEQ ID NO: 2613 SEQ ID NO: 2629.
2. The method of claim 1, wherein the escape mutant has a mutation in antigenic site II.
3. The method of claim 1, wherein at least one of the immunoglobulin single variable domains is a domain antibody, a single domain antibody, a V.sub.HH sequence, a partially or fully humanized V.sub.HH sequence, or a camelized V.sub.H sequence.
4. The method of claim 1, wherein at least one of the immunoglobulin single variable domains comprises one or more amino acid sequences having at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NOs: 1 to 22, 158 to 236, 248 to 407, 2448 and 2574 to 2581 in which for the purposes of determining the degree of amino acid identity, the amino acid residues that form the CDR sequences are disregarded; and in which optionally one or more of the amino acid residues at positions 11, 37, 44, 45, 47, 83, 84, 103, 104 and 108 according to the Kabat numbering are: at position 11: L, M, S, V, or W; at position 37:F, Y, H, I, L, or V; at position 44:G, E, A, D, Q, R, S, or L; at position 45: L, R, C, I, L, P, Q, or V; at position 47: W, L, F, A, G, I, M, R, S, V, or Y; at position 83: R, K, N, E, G, I, M, Q, or T; at position 84: P, A, L, R, S, T, D, or V; at position 103: W, P, R, or S; at position 104: G or D; and at position 108: Q, L, or R.
5. The method of claim 1, wherein the multivalent polypeptide comprises two or more immunoglobulin single variable domains selected from the group consisting of SEQ ID NOs: 158 to 236, 248 to 407, 2448 2574 to 2581 and an amino acid sequence which has at least 80% amino acid identity, at least 90% amino acid identity, 95% amino acid identity, 99% amino acid identity or more, or 100% amino acid identity with at least one of the amino acid sequences of SEQ ID NOs: 158 to 236, 248 to 407, 2448 and 2574 to 2581.
6. The method of claim 1, wherein the immunoglobulin single variable domain is a partially or fully humanized V.sub.HH sequence.
7. The method of claim 1, wherein the multivalent polypeptide further comprises one or more other groups, residues, moieties or binding units selected from the group consisting of a domain antibody, a single domain antibody, a V.sub.HH sequence, a partially or fully humanized V.sub.HH sequence, a camelized V.sub.H sequence or an immunoglobulin single variable domain.
8. The method of claim 1, wherein the multivalent polypeptide comprises three immunoglobulin single variable domains.
9. The method of claim 1, wherein the multivalent polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2408 to 2415, 2989 to 2998, 3042 to 3056, 3584 to 3591, and an amino acid sequence having more than 80%, more than 90%, more than 95%, or 99% or more sequence identity with at least one of the amino acid sequences of SEQ ID NOs: 2408 to 2415, 2989 to 2998, 3042 to 3056, and 3584 to 3591.
10. A method for treating a respiratory syncytial virus (RSV) infection, comprising administering, to a subject in need thereof, a multivalent polypeptide that specifically binds to an F protein of a RSV, said polypeptide comprising three or more immunoglobulin single variable domains, wherein at least one of the immunoglobulin single variable domains has the structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, in which CDR1, CDR2 and CDR3 are complementarity determining regions, and FR1, FR2, FR3, and FR4 are framework regions, and comprises a combination of a CDR1, a CDR2 and a CDR3 selected from the following combinations: TABLE-US-00096 Combi- nation CDR1 CDR2 CDR3 1 SEQ ID NO: 722 SEQ ID NO: 1286 SEQ ID NO: 1850 2 SEQ ID NO: 723 SEQ ID NO: 1287 SEQ ID NO: 1851 3 SEQ ID NO: 724 SEQ ID NO: 1288 SEQ ID NO: 1852 4 SEQ ID NO: 725 SEQ ID NO: 1289 SEQ ID NO: 1853 5 SEQ ID NO: 726 SEQ ID NO: 1290 SEQ ID NO: 1854 6 SEQ ID NO: 727 SEQ ID NO: 1291 SEQ ID NO: 1855 7 SEQ ID NO: 728 SEQ ID NO: 1292 SEQ ID NO: 1856 8 SEQ ID NO: 729 SEQ ID NO: 1293 SEQ ID NO: 1857 9 SEQ ID NO: 730 SEQ ID NO: 1294 SEQ ID NO: 1858 10 SEQ ID NO: 731 SEQ ID NO: 1295 SEQ ID NO: 1859 11 SEQ ID NO: 732 SEQ ID NO: 1296 SEQ ID NO: 1860 12 SEQ ID NO: 733 SEQ ID NO: 1297 SEQ ID NO: 1861 13 SEQ ID NO: 734 SEQ ID NO: 1298 SEQ ID NO: 1862 14 SEQ ID NO: 735 SEQ ID NO: 1299 SEQ ID NO: 1863 15 SEQ ID NO: 736 SEQ ID NO: 1300 SEQ ID NO: 1864 16 SEQ ID NO: 737 SEQ ID NO: 1301 SEQ ID NO: 1865 17 SEQ ID NO: 738 SEQ ID NO: 1302 SEQ ID NO: 1866 18 SEQ ID NO: 739 SEQ ID NO: 1303 SEQ ID NO: 1867 19 SEQ ID NO: 740 SEQ ID NO: 1304 SEQ ID NO: 1868 20 SEQ ID NO: 741 SEQ ID NO: 1305 SEQ ID NO: 1869 21 SEQ ID NO: 742 SEQ ID NO: 1306 SEQ ID NO: 1870 22 SEQ ID NO: 743 SEQ ID NO: 1307 SEQ ID NO: 1871 23 SEQ ID NO: 744 SEQ ID NO: 1308 SEQ ID NO: 1872 24 SEQ ID NO: 745 SEQ ID NO: 1309 SEQ ID NO: 1873 25 SEQ ID NO: 746 SEQ ID NO: 1310 SEQ ID NO: 1874 26 SEQ ID NO: 747 SEQ ID NO: 1311 SEQ ID NO: 1875 27 SEQ ID NO: 748 SEQ ID NO: 1312 SEQ ID NO: 1876 28 SEQ ID NO: 749 SEQ ID NO: 1313 SEQ ID NO: 1877 29 SEQ ID NO: 750 SEQ ID NO: 1314 SEQ ID NO: 1878 30 SEQ ID NO: 751 SEQ ID NO: 1315 SEQ ID NO: 1879 31 SEQ ID NO: 752 SEQ ID NO: 1316 SEQ ID NO: 1880 32 SEQ ID NO: 753 SEQ ID NO: 1317 SEQ ID NO: 1881 33 SEQ ID NO: 754 SEQ ID NO: 1318 SEQ ID NO: 1882 34 SEQ ID NO: 755 SEQ ID NO: 1319 SEQ ID NO: 1883 35 SEQ ID NO: 756 SEQ ID NO: 1320 SEQ ID NO: 1884 36 SEQ ID NO: 757 SEQ ID NO: 1321 SEQ ID NO: 1885 37 SEQ ID NO: 758 SEQ ID NO: 1322 SEQ ID NO: 1886 38 SEQ ID NO: 759 SEQ ID NO: 1323 SEQ ID NO: 1887 39 SEQ ID NO: 760 SEQ ID NO: 1324 SEQ ID NO: 1888 40 SEQ ID NO: 761 SEQ ID NO: 1325 SEQ ID NO: 1889 41 SEQ ID NO: 762 SEQ ID NO: 1326 SEQ ID NO: 1890 42 SEQ ID NO: 763 SEQ ID NO: 1327 SEQ ID NO: 1891 43 SEQ ID NO: 764 SEQ ID NO: 1328 SEQ ID NO: 1892 44 SEQ ID NO: 765 SEQ ID NO: 1329 SEQ ID NO: 1893 45 SEQ ID NO: 766 SEQ ID NO: 1330 SEQ ID NO: 1894 46 SEQ ID NO: 767 SEQ ID NO: 1331 SEQ ID NO: 1895 47 SEQ ID NO: 768 SEQ ID NO: 1332 SEQ ID NO: 1896 48 SEQ ID NO: 769 SEQ ID NO: 1333 SEQ ID NO: 1897 49 SEQ ID NO: 770 SEQ ID NO: 1334 SEQ ID NO: 1898 50 SEQ ID NO: 771 SEQ ID NO: 1335 SEQ ID NO: 1899 51 SEQ ID NO: 772 SEQ ID NO: 1336 SEQ ID NO: 1900 52 SEQ ID NO: 773 SEQ ID NO: 1337 SEQ ID NO: 1901 53 SEQ ID NO: 774 SEQ ID NO: 1338 SEQ ID NO: 1902 54 SEQ ID NO: 775 SEQ ID NO: 1339 SEQ ID NO: 1903 55 SEQ ID NO: 776 SEQ ID NO: 1340 SEQ ID NO: 1904 56 SEQ ID NO: 777 SEQ ID NO: 1341 SEQ ID NO: 1905 57 SEQ ID NO: 778 SEQ ID NO: 1342 SEQ ID NO: 1906 58 SEQ ID NO: 779 SEQ ID NO: 1343 SEQ ID NO: 1907 59 SEQ ID NO: 780 SEQ ID NO: 1344 SEQ ID NO: 1908 60 SEQ ID NO: 781 SEQ ID NO: 1345 SEQ ID NO: 1909 61 SEQ ID NO: 782 SEQ ID NO: 1346 SEQ ID NO: 1910 62 SEQ ID NO: 783 SEQ ID NO: 1347 SEQ ID NO: 1911 63 SEQ ID NO: 784 SEQ ID NO: 1348 SEQ ID NO: 1912 64 SEQ ID NO: 785 SEQ ID NO: 1349 SEQ ID NO: 1913 65 SEQ ID NO: 786 SEQ ID NO: 1350 SEQ ID NO: 1914 66 SEQ ID NO: 787 SEQ ID NO: 1351 SEQ ID NO: 1915 67 SEQ ID NO: 788 SEQ ID NO: 1352 SEQ ID NO: 1916 68 SEQ ID NO: 789 SEQ ID NO: 1353 SEQ ID NO: 1917 69 SEQ ID NO: 790 SEQ ID NO: 1354 SEQ ID NO: 1918 70 SEQ ID NO: 791 SEQ ID NO: 1355 SEQ ID NO: 1919 71 SEQ ID NO: 792 SEQ ID NO: 1356 SEQ ID NO: 1920 72 SEQ ID NO: 793 SEQ ID NO: 1357 SEQ ID NO: 1921 73 SEQ ID NO: 794 SEQ ID NO: 1358 SEQ ID NO: 1922 74 SEQ ID NO: 795 SEQ ID NO: 1359 SEQ ID NO: 1923 75 SEQ ID NO: 796 SEQ ID NO: 1360 SEQ ID NO: 1924 76 SEQ ID NO: 797 SEQ ID NO: 1361 SEQ ID NO: 1925 77 SEQ ID NO: 798 SEQ ID NO: 1362 SEQ ID NO: 1926 78 SEQ ID NO: 799 SEQ ID NO: 1363 SEQ ID NO: 1927 79 SEQ ID NO: 800 SEQ ID NO: 1364 SEQ ID NO: 1928 80 SEQ ID NO: 812 SEQ ID NO: 1376 SEQ ID NO: 1940 81 SEQ ID NO: 813 SEQ ID NO: 1377 SEQ ID NO: 1941 82 SEQ ID NO: 814 SEQ ID NO: 1378 SEQ ID NO: 1942 83 SEQ ID NO: 815 SEQ ID NO: 1379 SEQ ID NO: 1943 84 SEQ ID NO: 816 SEQ ID NO: 1380 SEQ ID NO: 1944 85 SEQ ID NO: 817 SEQ ID NO: 1381 SEQ ID NO: 1945 86 SEQ ID NO: 818 SEQ ID NO: 1382 SEQ ID NO: 1946 87 SEQ ID NO: 819 SEQ ID NO: 1383 SEQ ID NO: 1947 88 SEQ ID NO: 820 SEQ ID NO: 1384 SEQ ID NO: 1948 89 SEQ ID NO: 821 SEQ ID NO: 1385 SEQ ID NO: 1949 90 SEQ ID NO: 822 SEQ ID NO: 1386 SEQ ID NO: 1950 91 SEQ ID NO: 823 SEQ ID NO: 1387 SEQ ID NO: 1951 92 SEQ ID NO: 824 SEQ ID NO: 1388 SEQ ID NO: 1952 93 SEQ ID NO: 825 SEQ ID NO: 1389 SEQ ID NO: 1953 94 SEQ ID NO: 826 SEQ ID NO: 1390 SEQ ID NO: 1954 95 SEQ ID NO: 827 SEQ ID NO: 1391 SEQ ID NO: 1955 96 SEQ ID NO: 828 SEQ ID NO: 1392 SEQ ID NO: 1956 97 SEQ ID NO: 829 SEQ ID NO: 1393 SEQ ID NO: 1957 98 SEQ ID NO: 830 SEQ ID NO: 1394 SEQ ID NO: 1958 99 SEQ ID NO: 831 SEQ ID NO: 1395 SEQ ID NO: 1959 100 SEQ ID NO: 832 SEQ ID NO: 1396 SEQ ID NO: 1960 101 SEQ ID NO: 833 SEQ ID NO: 1397 SEQ ID NO: 1961 102 SEQ ID NO: 834 SEQ ID NO: 1398 SEQ ID NO: 1962 103 SEQ ID NO: 835 SEQ ID NO: 1399 SEQ ID NO: 1963 104 SEQ ID NO: 836 SEQ ID NO: 1400 SEQ ID NO: 1964 105 SEQ ID NO: 837 SEQ ID NO: 1401 SEQ ID NO: 1965 106 SEQ ID NO: 838 SEQ ID NO: 1402 SEQ ID NO: 1966 107 SEQ ID NO: 839 SEQ ID NO: 1403 SEQ ID NO: 1967 108 SEQ ID NO: 840 SEQ ID NO: 1404 SEQ ID NO: 1968 109 SEQ ID NO: 841 SEQ ID NO: 1405 SEQ ID NO: 1969 110 SEQ ID NO: 842 SEQ ID NO: 1406 SEQ ID NO: 1970 111 SEQ ID NO: 843 SEQ ID NO: 1407 SEQ ID NO: 1971 112 SEQ ID NO: 844 SEQ ID NO: 1408 SEQ ID NO: 1972 113 SEQ ID NO: 845 SEQ ID NO: 1409 SEQ ID NO: 1973 114 SEQ ID NO: 846 SEQ ID NO: 1410 SEQ ID NO: 1974 115 SEQ ID NO: 847 SEQ ID NO: 1411 SEQ ID NO: 1975 116 SEQ ID NO: 848 SEQ ID NO: 1412 SEQ ID NO: 1976 117 SEQ ID NO: 849 SEQ ID NO: 1413 SEQ ID NO: 1977 118 SEQ ID NO: 850 SEQ ID NO: 1414 SEQ ID NO: 1978 119 SEQ ID NO: 851 SEQ ID NO: 1415 SEQ ID NO: 1979 120 SEQ ID NO: 852 SEQ ID NO: 1416 SEQ ID NO: 1980 121 SEQ ID NO: 853 SEQ ID NO: 1417 SEQ ID NO: 1981 122 SEQ ID NO: 854 SEQ ID NO: 1418 SEQ ID NO: 1982 123 SEQ ID NO: 855 SEQ ID NO: 1419 SEQ ID NO: 1983 124 SEQ ID NO: 856 SEQ ID NO: 1420 SEQ ID NO: 1984 125 SEQ ID NO: 857 SEQ ID NO: 1421 SEQ ID NO: 1985 126 SEQ ID NO: 858 SEQ ID NO: 1422 SEQ ID NO: 1986 127 SEQ ID NO: 859 SEQ ID NO: 1423 SEQ ID NO: 1987 128 SEQ ID NO: 860 SEQ ID NO: 1424 SEQ ID NO: 1988 129 SEQ ID NO: 861 SEQ ID NO: 1425 SEQ ID NO: 1989 130 SEQ ID NO: 862 SEQ ID NO: 1426 SEQ ID NO: 1990 131 SEQ ID NO: 863 SEQ ID NO: 1427 SEQ ID NO: 1991 132 SEQ ID NO: 864 SEQ ID NO: 1428 SEQ ID NO: 1992 133 SEQ ID NO: 865 SEQ ID NO: 1429 SEQ ID NO: 1993 134 SEQ ID NO: 866 SEQ ID NO: 1430 SEQ ID NO: 1994 135 SEQ ID NO: 867 SEQ ID NO: 1431 SEQ ID NO: 1995 136 SEQ ID NO: 868 SEQ ID NO: 1432 SEQ ID NO: 1996 137 SEQ ID NO: 869 SEQ ID NO: 1433 SEQ ID NO: 1997 138 SEQ ID NO: 870 SEQ ID NO: 1434 SEQ ID NO: 1998 139 SEQ ID NO: 871 SEQ ID NO: 1435 SEQ ID NO: 1999 140 SEQ ID NO: 872 SEQ ID NO: 1436 SEQ ID NO: 2000 141 SEQ ID NO: 873 SEQ ID NO: 1437 SEQ ID NO: 2001 142 SEQ ID NO: 874 SEQ ID NO: 1438 SEQ ID NO: 2002 143 SEQ ID NO: 875 SEQ ID NO: 1439 SEQ ID NO: 2003 144 SEQ ID NO: 876 SEQ ID NO: 1440 SEQ ID NO: 2004 145 SEQ ID NO: 877 SEQ ID NO: 1441 SEQ ID NO: 2005 146 SEQ ID NO: 878 SEQ ID NO: 1442 SEQ ID NO: 2006 147 SEQ ID NO: 879 SEQ ID NO: 1443 SEQ ID NO: 2007 148 SEQ ID NO: 880 SEQ ID NO: 1444 SEQ ID NO: 2008 149 SEQ ID NO: 881 SEQ ID NO: 1445 SEQ ID NO: 2009 150 SEQ ID NO: 882 SEQ ID NO: 1446 SEQ ID NO: 2010 151 SEQ ID NO: 883 SEQ ID NO: 1447 SEQ ID NO: 2011 152 SEQ ID NO: 884 SEQ ID NO: 1448 SEQ ID NO: 2012 153 SEQ ID NO: 885 SEQ ID NO: 1449 SEQ ID NO: 2013 154 SEQ ID NO: 886 SEQ ID NO: 1450 SEQ ID NO: 2014 155 SEQ ID NO: 887 SEQ ID NO: 1451 SEQ ID NO: 2015 156 SEQ ID NO: 888 SEQ ID NO: 1452 SEQ ID NO: 2016 157 SEQ ID NO: 889 SEQ ID NO: 1453 SEQ ID NO: 2017 158 SEQ ID NO: 890 SEQ ID NO: 1454 SEQ ID NO: 2018 159 SEQ ID NO: 891 SEQ ID NO: 1455 SEQ ID NO: 2019 160 SEQ ID NO: 892 SEQ ID NO: 1456 SEQ ID NO: 2020 161 SEQ ID NO: 893 SEQ ID NO: 1457 SEQ ID NO: 2021 162 SEQ ID NO: 894 SEQ ID NO: 1458 SEQ ID NO: 2022 163 SEQ ID NO: 895 SEQ ID NO: 1459 SEQ ID NO: 2023 164 SEQ ID NO: 896 SEQ ID NO: 1460 SEQ ID NO: 2024 165 SEQ ID NO: 897 SEQ ID NO: 1461 SEQ ID NO: 2025 166 SEQ ID NO: 898 SEQ ID NO: 1462 SEQ ID NO: 2026 167 SEQ ID NO: 899 SEQ ID NO: 1463 SEQ ID NO: 2027 168 SEQ ID NO: 900 SEQ ID NO: 1464 SEQ ID NO: 2028 169 SEQ ID NO: 901 SEQ ID NO: 1465 SEQ ID NO: 2029 170 SEQ ID NO: 902 SEQ ID NO: 1466 SEQ ID NO: 2030 171 SEQ ID NO: 903 SEQ ID NO: 1467 SEQ ID NO: 2031 172 SEQ ID NO: 904 SEQ ID NO: 1468 SEQ ID NO: 2032 173 SEQ ID NO: 905 SEQ ID NO: 1469 SEQ ID NO: 2033 174 SEQ ID NO: 906 SEQ ID NO: 1470 SEQ ID NO: 2034 175 SEQ ID NO: 907 SEQ ID NO: 1471 SEQ ID NO: 2035 176 SEQ ID NO: 908 SEQ ID NO: 1472 SEQ ID NO: 2036 177 SEQ ID NO: 909 SEQ ID NO: 1473 SEQ ID NO: 2037 178 SEQ ID NO: 910 SEQ ID NO: 1474 SEQ ID NO: 2038 179 SEQ ID NO: 911 SEQ ID NO: 1475 SEQ ID NO: 2039 180 SEQ ID NO: 912 SEQ ID NO: 1476 SEQ ID NO: 2040 181 SEQ ID NO: 913 SEQ ID NO: 1477 SEQ ID NO: 2041 182 SEQ ID NO: 914 SEQ ID NO: 1478 SEQ ID NO: 2042 183 SEQ ID NO: 915 SEQ ID NO: 1479 SEQ ID NO: 2043 184 SEQ ID NO: 916 SEQ ID NO: 1480 SEQ ID NO: 2044 185 SEQ ID NO: 917 SEQ ID NO: 1481 SEQ ID NO: 2045 186 SEQ ID NO: 918 SEQ ID NO: 1482 SEQ ID NO: 2046 187 SEQ ID NO: 919 SEQ ID NO: 1483 SEQ ID NO: 2047 188 SEQ ID NO: 920 SEQ ID NO: 1484 SEQ ID NO: 2048 189 SEQ ID NO: 921 SEQ ID NO: 1485 SEQ ID NO: 2049 190 SEQ ID NO: 922 SEQ ID NO: 1486 SEQ ID NO: 2050 191 SEQ ID NO: 923 SEQ ID NO: 1487 SEQ ID NO: 2051 192 SEQ ID NO: 924 SEQ ID NO: 1488 SEQ ID NO: 2052 193 SEQ ID NO: 925 SEQ ID NO: 1489 SEQ ID NO: 2053 194 SEQ ID NO: 926 SEQ ID NO: 1490 SEQ ID NO: 2054 195 SEQ ID NO: 927 SEQ ID NO: 1491 SEQ ID NO: 2055 196 SEQ ID NO: 928 SEQ ID NO: 1492 SEQ ID NO: 2056 197 SEQ ID NO: 929 SEQ ID NO: 1493 SEQ ID NO: 2057 198 SEQ ID NO: 930 SEQ ID NO: 1494 SEQ ID NO: 2058 199 SEQ ID NO: 931 SEQ ID NO: 1495 SEQ ID NO: 2059 200 SEQ ID NO: 932 SEQ ID NO: 1496 SEQ ID NO: 2060 201 SEQ ID NO: 933 SEQ ID NO: 1497 SEQ ID NO: 2061 202 SEQ ID NO: 934 SEQ ID NO: 1498 SEQ ID NO: 2062 203 SEQ ID NO: 935 SEQ ID NO: 1499 SEQ ID NO: 2063 204 SEQ ID NO: 936 SEQ ID NO: 1500 SEQ ID NO: 2064 205 SEQ ID NO: 937 SEQ ID NO: 1501 SEQ ID NO: 2065 206 SEQ ID NO: 938 SEQ ID NO: 1502 SEQ ID NO: 2066 207 SEQ ID NO: 939 SEQ ID NO: 1503 SEQ ID NO: 2067 208 SEQ ID NO: 940 SEQ ID NO: 1504 SEQ ID NO: 2068 209 SEQ ID NO: 941 SEQ ID NO: 1505 SEQ ID NO: 2069 210 SEQ ID NO: 942 SEQ ID NO: 1506 SEQ ID NO: 2070 211 SEQ ID NO: 943 SEQ ID NO: 1507 SEQ ID NO: 2071 212 SEQ ID NO: 944 SEQ ID NO: 1508 SEQ ID NO: 2072 213 SEQ ID NO: 945 SEQ ID NO: 1509 SEQ ID NO: 2073 214 SEQ ID NO: 946 SEQ ID NO: 1510 SEQ ID NO: 2074 215 SEQ ID NO: 947 SEQ ID NO: 1511 SEQ ID NO: 2075 216 SEQ ID NO: 948 SEQ ID NO: 1512 SEQ ID NO: 2076 217 SEQ ID NO: 949 SEQ ID NO: 1513 SEQ ID NO: 2077 218 SEQ ID NO: 950 SEQ ID NO: 1514 SEQ ID NO: 2078 219 SEQ ID NO: 951 SEQ ID NO: 1515 SEQ ID NO: 2079 220 SEQ ID NO: 952 SEQ ID NO: 1516 SEQ ID NO: 2080 221 SEQ ID NO: 953 SEQ ID NO: 1517 SEQ ID NO: 2081 222 SEQ ID NO: 954 SEQ ID NO: 1518 SEQ ID NO: 2082 223 SEQ ID NO: 955 SEQ ID NO: 1519 SEQ ID NO: 2083 224 SEQ ID NO: 956 SEQ ID NO: 1520 SEQ ID NO: 2084 225 SEQ ID NO: 957 SEQ ID NO: 1521 SEQ ID NO: 2085 226 SEQ ID NO: 958 SEQ ID NO: 1522 SEQ ID NO: 2086 227 SEQ ID NO: 959 SEQ ID NO: 1523 SEQ ID NO: 2087 228 SEQ ID NO: 960 SEQ ID NO: 1524 SEQ ID NO: 2088 229 SEQ ID NO: 961 SEQ ID NO: 1525 SEQ ID NO: 2089 230 SEQ ID NO: 962 SEQ ID NO: 1526 SEQ ID NO: 2090 231 SEQ ID NO: 963 SEQ ID NO: 1527 SEQ ID NO: 2091 232 SEQ ID NO: 964 SEQ ID NO: 1528 SEQ ID NO: 2092 233 SEQ ID NO: 965 SEQ ID NO: 1529 SEQ ID NO: 2093 234 SEQ ID NO: 966 SEQ ID NO: 1530 SEQ ID NO: 2094 235 SEQ ID NO: 967 SEQ ID NO: 1531 SEQ ID NO: 2095 236 SEQ ID NO: 968 SEQ ID NO: 1532 SEQ ID NO: 2096 237 SEQ ID NO: 969 SEQ ID NO: 1533 SEQ ID NO: 2097 238 SEQ ID NO: 970 SEQ ID NO: 1534 SEQ ID NO: 2098 239 SEQ ID NO: 971 SEQ ID NO: 1535 SEQ ID NO: 2099 240 SEQ ID NO: 2484 SEQ ID NO: 2520 SEQ ID NO: 2556 241 SEQ ID NO: 2590 SEQ ID NO: 2606 SEQ ID NO: 2622 242 SEQ ID NO: 2591 SEQ ID NO: 2607 SEQ ID NO: 2623 243 SEQ ID NO: 2592 SEQ ID NO: 2608 SEQ ID NO: 2624 244 SEQ ID NO: 2593 SEQ ID NO: 2609 SEQ ID NO: 2625 245 SEQ ID NO: 2594 SEQ ID NO: 2610 SEQ ID NO: 2626 246 SEQ ID NO: 2596 SEQ ID NO: 2612 SEQ ID NO: 2628 247 SEQ ID NO: 2597 SEQ ID NO: 2613 SEQ ID NO: 2629.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
(1)
(2)
(3)
(4)
(5)
(6)
(7)
(8)
(9)
(10)
(11)
(12)
(13) Family 1 comprises the following NANOBODIES® (V.sub.HH sequences): 192-C10, 206-11H, 206-12F
(14) Family 2 comprises the following NANOBODIES® (V.sub.HH sequences): 192-A8, 206-10F, 206-11D, 206-7E, 207-9G
(15) Family 3 comprises the following NANOBODIES® (V.sub.HH sequences): 192-C4, 206-6A, 206-5A, 206-3A, 206-3D, 206-4G, 192-F2, 206-4D, 192-C6, 192-H2, 206-5E, 206-2A, 207-5D, 206-3E, 206-2G, 206-2H, 206-3C, 191-D3, 206-2F, 207-6B, 206-3F, 207-1D
(16) Family 4 comprises the following NANOBODIES® (V.sub.HH sequences): 191-B9, 207-9A, 207-9B, 207-9H, 206-10C, 206-10D, 192-D3, 206-10B, 207-9D, 207-11D, 207-11E, 206-10E, 191-E4, 207-1C, 207-1F, 207-5C, 207-1E, 207-4D, 206-2E, 206-7H, 207-11F, 207-9F, 207-11H, 192-B1, 206-3B, 207-11B, 207-4H, 192-H1, 206-6D, 206-7B, 207-11A, 207-1A, 207-5B, 207-4A, 207-4B, 207-6A, 207-6D, 207-1B, 207-5A, 207-6C, 207-5E, 207-6E, 207-6F, 207-11G
Family 5 comprises the following NANOBODIES® (V.sub.HH sequences): 207-9C
Family 6 comprises the following NANOBODIES® (V.sub.HH sequences): 206-7G
Family 7 comprises the following NANOBODIES® (V.sub.HH sequences): 207-9E
Family 8 comprises the following NANOBODIES® (V.sub.HH sequences): 206-2C
Family 9 comprises the following NANOBODIES® (V.sub.HH sequences): 206-7C
(17)
(18)
(19) Family 1 comprises the following NANOBODIES® (V.sub.HH sequences): 202-B8
(20) Family 2 comprises the following NANOBODIES® (V.sub.HH sequences): 202-D5
(21) Family 3 comprises the following NANOBODIES® (V.sub.HH sequences): 202-A10, 202-A12, 202-E6, 202-F8
(22) Family 4 comprises the following NANOBODIES® (V.sub.HH sequences): 202-G3
(23) Family 5 comprises the following NANOBODIES® (V.sub.HH sequences): 202-C8
(24) Family 6 comprises the following NANOBODIES® (V.sub.HH sequences): 202-A5, 202-C2, 202-F3, 202-F4, 202-C1, 202-E5, 202-H2
(25) Family 7 comprises the following NANOBODIES® (V.sub.HH sequences): 202-B10, 202-D8, 202-E11, 202-B7, 202-A9, 202-H8, 202-C11, 202-B9, 202-G8, 202-D7, 202-F10, 202-C9, 202-E7, 202-G11, 202-F12, 202-C7
(26)
(27)
(28) Family 1 comprises the following NANOBODIES® (V.sub.HH sequences): 213-B7, 213-D7
(29) Family 2 comprises the following NANOBODIES® (V.sub.HH sequences): 213-E6
(30) Family 3 comprises the following NANOBODIES® (V.sub.HH sequences): 213-H7
(31) Family 4 comprises the following NANOBODIES® (V.sub.HH sequences): 2113-D6, 214-C10
(32) Family 5 comprises the following NANOBODIES® (V.sub.HH sequences): 214-A8, 214-E8, 214-H10
(33) Family 6 comprises the following NANOBODIES® (V.sub.HH sequences): 214-D10
(34) Family 7 comprises the following NANOBODIES® (V.sub.HH sequences): 214-F8
(35)
(36)
(37)
(38)
(39)
(40)
(41)
(42)
(43)
(44)
(45)
(46)
(47)
(48)
(49)
(50)
(51)
(52)
(53)
(54)
(55)
(56)
(57)
(58)
(59)
(60)
(61)
(62)
(63)
(64)
(65)
(66)
(67)
(68)
(69)
(70)
(71)
(72)
(73)
(74)
(75)
(76)
(77)
(78)
(79)
(80)
(81)
(82)
(83)
(84)
(85)
(86)
EXAMPLES
Example 1: Immunizations
(87) Two llamas (156 and 157) were immunized according to standard protocols with 6 boosts of hRSV F.sub.TM-(membrane anchorless form of the fusion protein, 70 kDa; Corrall T. et al. 2007, BMC Biotechnol. 7: 17). Blood was collected from these animals 7 days after boost 6 and 10 days after boost 6.
(88) Two llamas (140 and 163) were immunized according to standard protocols with 6 boosts of H5 Hemagglutinin (HA, A/Vietnam/1203/2004 (H5), Protein Sciences Cat. No. 3006). Blood was collected from these animals 10 days after boost 6.
(89) Two llamas (183 and 196) were immunized according to standard protocols with 6 boosts of Rabies vaccine (inactivated rabies virus; Sanofi Pasteur MSD). Blood was collected from these animals 7 days after boost 6, 17 days after boost 6 and 21 days after boost 6.
Example 2: Library Construction
(90) Peripheral blood mononuclear cells were prepared from blood samples using Ficoll-Hypaque according to the manufacturer's instructions. Next, total RNA was extracted from these cells as well as from the lymph node bow cells and used as starting material for RT-PCR to amplify NANOBODY® (V.sub.HH sequence) encoding gene fragments. These fragments were cloned into phagemid vector derived from pUC119 which contains the LacZ promoter, a coliphage pIII protein coding sequence, a resistance gene for ampicillin or carbenicillin, a multicloning site and the gen3 leader sequence. In frame with the NANOBODY® (V.sub.HH sequence) coding sequence, the vector codes for a C-terminal c-myc tag and a (His)6 tag. Phage was prepared according to standard methods and stored at 4° C. for further use, making phage libraries 156, 157, 140b, 163b, 183 and 196b.
Example 3: Selections Against hRSV
(91) hRSV is a member of the Paramyxoviridae family and is an enveloped virus with two main surface glycoproteins that make the spikes of the virus particle. One of these glycoproteins (protein G) is the attachment protein that mediates binding of the virus to the cell surface. The other glycoprotein (protein F or fusion) mediates fusion of the viral and cell membranes, allowing the entry of the viral nucleocapsid into the cell cytoplasm. Inhibition of the steps mediated by either G or F glycoproteins blocks the initial stages of the infectious cycle and neutralizes virus infectivity. Therefore, antibodies directed against either G or F, and which inhibit their respective activities, neutralize virus infectivity and may protect against a hRSV infection. The F protein is highly conserved and forms trimeric spikes that undergo conformational changes upon activation.
(92) Human respiratory syncytial virus (hRSV) is the leading cause of severe lower respiratory tract infections (bronchiolitis and pneumonia) in infants and very young children and causes annual epidemics during the winter months. The virus also causes a substantial disease burden among the elderly and adults with underlying cardiopulmonary disorders and/or immunosuppressive conditions are also at risk of severe hRSV disease. The immune response does not prevent reinfections.
(93) There is no vaccine available to prevent hRSV infections. The only drug product available in the market is a humanized monoclonal antibody (Synagis®) directed against one of the viral glycoproteins (protein F) which is used prophylactically in children that are at a very high risk of suffering a severe hRSV infection. The restricted use of Synagis® is due, at least in part, to the high cost of this product.
(94) To identify NANOBODIES® (V.sub.HH sequences) recognizing the F.sub.TM-(membrane anchorless form of the fusion protein, 70 kDa, Corrall T. et al. 2007, BMC Biotechnol. 7: 17), libraries 156 and 157 were used. The same antigen was used for selections as for immunizations. The F.sub.TM-protein (25 ng/well) was immobilized on Nunc Maxisorp ELISA plates. A control was included with 0 μg/ml F.sub.TM-. Bound phages were eluted from the F.sub.TM-using trypsin and Synagis® (Palivizumab, MedImmune, humanized monoclonal antibody, described in Zhao & Sullender 2005, J. Virol. 79: 3962) in the first and second round of selections. Remicade (Infliximab, anti-TNF; Centorcor) was used as a control for Synagis®. A 100 molar excess of Synagis® was used in order to identify NANOBODIES® (V.sub.HH sequences) binding specifically at the binding site on RSV. Outputs from the first round selections, eluted with Synagis® were used for second round selections.
(95) Outputs of both rounds of selections were analyzed for enrichment factor (phage present in eluate relative to controls). Based on these parameters the best selections were chosen for further analysis. Individual colonies were picked and grown in 96 deep well plates (1 ml volume) and induced by adding IPTG for NANOBODY® (V.sub.HH sequence) expression. Periplasmic extracts (volume: ˜80 μl) were prepared according to standard methods.
Example 4: Selections Against H5N1
(96) Influenza is an enveloped virus with two main surface antigens, the hemagglutinin (HA) and the neuraminidase (NA). The influenza HA is responsible for virus attachment to target host cells via recognition and binding to sialic acid receptors on membrane-bound proteins of the host cell.
(97) By analysis using monoclonal antibody-resistant mutants it has been shown that neutralizing antibody binding sites map to regions on the surface of the globular membrane distal domains of the HA. Bi- or multispecific NANOBODIES® (V.sub.HH sequences) can exhibit enhanced neutralizing potency and can reduce the incidence of escape mutants in comparison to monospecific NANOBODIES® (V.sub.HH sequences), or currently used monoclonals.
(98) Human infections with avian influenza H5N1 virus were first observed during large scale poultry outbreaks in Hong Kong in 1997. Since its re-emergence in Asia in 2003, 277 laboratory-confirmed human H5N1 cases have been reported from Asia, Europe and Africa of whom 167 have died (WHO, 1 Mar. 2007). In general, humans who catch a humanized Influenza A virus (a human flu virus of type A) usually have symptoms that include fever, cough, sore throat, muscle aches, conjunctivitis and, in severe cases, breathing problems, pneumonia, fever, chills, vomiting and headache. Tissue damage associated with pathogenic flu virus infection can ultimately result in death. The inflammatory cascade triggered by H5N1 has been called a ‘cytokine storm’ by some, because of what seems to be a positive feedback process of damage to the body resulting from immune system stimulation. H5N1 induces higher levels of cytokines than the more common flu virus types. The mortality rate of highly pathogenic H5N1 avian influenza in a human is high; WHO data indicates that 60% of cases classified as H5N1 resulted in death. Influenza virus entry inhibitors may have potential uses as antivirals, prophylactics and as topical treatments (i.e. nasal sprays). These inhibitors may also serve as useful tools in H5N1 vaccine and antiviral research by elucidating novel epitopes involved in protective immune responses against the virus.
(99) To identify NANOBODIES® (V.sub.HH sequences) recognizing the hemagglutinin (HA) of Influenza H5N1, libraries 140b and 163b were used. The same antigen was used for selections as for immunizations. The H5N1 recombinant HA (A/Vietnam/1203/2004 (H5N1), Protein Sciences Cat. No. 3006) was immobilized on Nunc Maxisorp ELISA plates. A control was included with 0 μg/ml HA. Bound phages were eluted from the HA using trypsin in the first and trypsin and VN04-2 (Mouse Monoclonal Anti-H5 Hemagglutinin of A/Vietnam/1203/04 Influenza Virus, Rockland Inc. Cat. No. 200-301-975) in the second round of selections. Mouse IgG was used as an antibody control. A 100 molar excess of the antibody was used in order to identify NANOBODIES® (V.sub.HH sequences) binding specifically at the binding site on influenza HA. Outputs from the first round selections were used for second round selections.
(100) Outputs of both rounds of selections were analyzed for enrichment factor (phage present in eluate relative to controls). Based on these parameters the best selections were chosen for further analysis. Individual colonies were picked and grown in 96 deep well plates (1 ml volume) and induced by adding IPTG for NANOBODY® (V.sub.HH sequence) expression. Periplasmic extracts (volume: ˜80 μl) were prepared according to standard methods.
Example 5: Selections Against Rabies
(101) Rabies is a neurotropic virus that belongs to one of the largest families (Rhabdoviridae) of viruses. It is surrounded by an envelope in which glycoprotein G is embedded. Glycoprotein G is responsible for the induction of protective immunity and contains different motifs that define virulence and pathogenicity.
(102) Glycoprotein G consists of 505 amino-acids and a typical rabies virion contains about 1800 of these proteins. Glycoprotein G binds to the cellular receptor, leading to endocytosis of the virus-receptor complex. Glycoprotein G is the immunodominant antigen of the virus and antibodies are typically directed against 1 of 8 antigenic sites on glycoprotein G, some of which are highly conserved between different strains and genotypes. Neutralizing antibodies prevent binding and entry into the target host cell by blocking binding of viral proteins to the target host cell.
(103) Rabies continues to be a serious worldwide health problem. Each year, an estimated 55,000 people die from rabies and 10 million people are treated after contact with suspected animals.
(104) Rabies virus causes encephalitis in man and animal. The virus is excreted in saliva and transmitted by close contact with infected animals through bites, scratches or licks. Once introduced in a wound, it replicates locally in the muscle cells. After an incubation period of a few days up to several years, the virus crawls up in the peripheral nerves and reaches the brain via retrograde axonal transport. This is followed by extensive replication in the cytoplasm of neurons, brain dysfunction and death. Once symptoms of the disease develop, rabies is fatal.
(105) There is no cure for rabies and once the virus reaches the central nervous system, the patient will die. The present treatment is post-exposure with vaccinations with inactivated virus. Two sources of antibodies are available for passive immunization: human rabies immunoglobulins (HRIG: Imogam, Aventis Pasteur) and equine rabies immunoglobulins (ERIG). These are purified from pooled sera of vaccinated people or horses and administered directly after the bite. Due to technical and economical limitations, the supply of rabies immunoglobulins is limited and there is a worldwide shortage. Immunoglobulins can trigger allergic reactions ranging form skin erythema, fever to anaphylactic shock (as described in the patient information leaflet). The possibility of contamination with blood-borne infectious agents can not be excluded. The WHO strongly recommends that more cost-efficient and safer alternatives should be developed.
(106) To identify NANOBODIES® (V.sub.HH sequences) recognizing the Rabies G protein, libraries 183 and 196b were used. The Rabies virus (rabies inactivated HDCV vaccine; Sanofi Pasteur MSD) was immobilized on Nunc Maxisorp ELISA plates. A control was included with 0 μg/ml. Precoated 8 well strips (Platelia II Rabies plates, BioRad cat no 355-1180) were also used for selections in both first and second round. Phages were preincubated with 100 mg/ml BSA, because the rabies vaccine contained 50 mg/ml HSA. Bound phages were eluted from the virus using trypsin in the first and second round. Bound phages were eluted from the G protein with trypsin or a mouse monoclonal MAb 8-2m or Ab 8-2, a mouse IgG2a (Montaño-Hirose et al. 1993, Vaccine 11: 1259-1266) in the first and second round of selections. A mouse IgG2a was used as an antibody control. A 100 molar excess of the antibody was used in order to identify NANOBODIES® (V.sub.HH sequences) binding specifically at the binding site on rabies virus. Outputs from the first round selections were used for second round selections.
(107) Outputs of both rounds of selections were analyzed for enrichment factor (phage present in eluate relative to controls). Based on these parameters the best selections were chosen for further analysis. Individual colonies were picked and grown in 96 deep well plates (1 ml volume) and induced by adding IPTG for NANOBODY® (V.sub.HH sequence) expression. Periplasmic extracts (volume: ˜80 μl) were prepared according to standard methods.
Example 6: Screening for Binding
(108) In order to determine binding specificity to the viral envelope proteins, the clones were tested in an ELISA binding assay setup. In short, 2 μg/ml of F.sub.TM- or 5 μg/ml H5N1 HA were immobilized directly on Maxisorp microtiter plates (Nunc). Rabies G protein precoated plates from BioRad were used (Cat. No. 355-1180). Free binding sites were blocked using 4% Marvel in PBS. Next, 10 μl of periplasmic extract containing NANOBODY® (V.sub.HH sequence) or monoclonal phages of the different clones in 100 μl 2% Marvel PBST were allowed to bind to the immobilized antigen. After incubation and a wash step, NANOBODY® (V.sub.HH sequence) binding was revealed using a rabbit-anti-V.sub.HH secondary antibody (for the periplasmic fractions) or an anti-M13 antibody against the phages gene3. After a wash step the NANOBODIES® (V.sub.HH sequences) in the periplasmic fractions were detected with a HRP-conjugated goat-anti-rabbit antibody. Binding specificity was determined based on OD values compared to controls having received no NANOBODY® (V.sub.HH sequence).
(109) (a) hRSV
(110) Phage binding ELISA showed binders for both library 156 (61%) and 157 (59%) after the first round of selections and Synagis® elutions.
(111) Phage binding ELISA showed binders for both library 156 (85%) and 157 (50%) after the first round of selections and trypsin elutions.
(112) Periplasmic fraction binding ELISA showed binders for both library 156 (83%) and 157 (78%) after the second round of selections and trypsin elutions.
(113) Periplasmic fraction binding ELISA showed binders for both library 156 (87%) and 157 (68%) after the second round of selections and Synagis® elutions.
(114) (b) H5N1
(115) Periplasmic fraction binding ELISA showed binders for both library 140b (35%) and 163b (24%) after the second round of selections and monoclonal antibody elutions.
(116) Periplasmic fraction binding ELISA showed binders for both library 140b (37%) and 163b (33%) after the second round of selections and trypsins elutions.
(117) (c) Rabies
(118) Periplasmic fraction binding ELISA showed binders for the rabies virus from both library 183 (67%) and 196 (48%) after the second round of selections on virus and trypsin elutions. No binders for the G protein from the virus selected periplasmic fractions. No binders for HSA control.
(119) Periplasmic fraction binding ELISA showed binders for G protein from both library 183 (50%) and 196 (75%) after the second round of selections and trypsins elutions.
(120) Periplasmic fraction binding ELISA showed binders for G protein from library 196 (37%) after the second round of selections and monoclonal antibody elutions.
(121) Sequences of the obtained NANOBODIES® (V.sub.HH sequences) are given in Table A-1.
(122) Clustering of the obtained NANOBODIES® (V.sub.HH sequences) is shown in
Example 7: Screening for Competition
(123) Competition assays were set up with the NANOBODIES® (V.sub.HH sequences) competing with monoclonal, neutralizing antibodies, Synagis® for hRSV, VN04-2 (as described in Hanson et al. 2006, Respiratory Research 7: 126) for H5N1 and a mouse IgG2a monoclonal (as described in Montaño-Hirose et al. 1993, Vaccine 11: 1259-1266) against Rabies. A chessboard ELISA was run to determine the best coating concentration of antigen and the concentration of antibody that gave IC.sub.50.
(124) In short, the antigen was immobilized on Maxisorp microtiter plates (Nunc) and free binding sites were blocked using 4% Marvel in PBS. Next, 100 ng/ml of Synagis®, VN04-2 or mouse IgG2a monoclonal (mab) (dilution 1/10.sup.6) was preincubated with 20 μl of periplasmic extract containing NANOBODY® (V.sub.HH sequence) of the different clones. Control periplasmic fractions selected against other viral coat proteins were included. The competing antibody was allowed to bind to the immobilized antigen with or without NANOBODY® (V.sub.HH sequence). After incubation and a wash step, antibody binding was revealed using a HRP-conjugated goat anti-human Fc antibody (ahFcHRP; Synagis®) or HRP-conjugated donkey anti-mouse antibody (DAMPO; VN04-2 and IgG2a). Binding specificity was determined based on OD values compared to controls having received no NANOBODY® (V.sub.HH sequence) (
Example 8: Determining Competition Efficiency by Titration of Purified NANOBODY® (V.SUB.HH .Sequence)
(125) In order to determine competition efficiency of hRSV F.sub.TM- and H5N1 HA binding NANOBODIES® (V.sub.HH sequences), the positive clones of the binding assay were tested in an ELISA competition assay setup.
(126) In short, 2 μg/ml F.sub.TM- or 2.5 μg/ml HA was immobilized on Maxisorp microtiter plates (Nunc) and free binding sites were blocked using 4% Marvel in PBS. Next, a dilution series of purified NANOBODIES® (V.sub.HH sequences) were allowed to bind to the antigen for 30 minutes before 100 ng/ml (0.67 nM) Synagis® or VN04-2 was incubated. Irrelevant NANOBODIES® (V.sub.HH sequences) against other viral coat proteins were used as negative controls (202 against H5N1 for hRSV competition, 191, and 192 against hRSV for H5N1 competitions). The results are shown in
Example 9: Cell Based and Animal Experiments
(127) To investigate if selected NANOBODIES® (V.sub.HH sequences) recognize different epitopes, epitope mapping could be performed by using monoclonal antibodies which recognize known epitopes. Examples of antibodies against hRSV that may be used are: Synagis® (Palivizumab, MedImmune, humanized monoclonal antibody, as described in Zhao & Sullender 2005, J. Virol. 79: 3962), directed to an epitope in the A antigenic site of the F protein, non-competing with 9C5. 9C5 (HyTest Ltd) (described in Krivitskaia et al. 1999, Vopr. Virusol 44: 279), neutralizing mouse monoclonal, hampers the virus penetration into the cell, recognizes epitope F1a of RSV F-protein, non-competing with Synagis®. 101F (WO 06/050280), humanized mouse monoclonal, directed to an epitope of the RSV F-protein comprising amino acids 423-436 as minimal peptide, non-competing with Synagis® and 9C5.
(128) In vitro neutralization assays of selected NANOBODIES® (V.sub.HH sequences) against virus are used to investigate the neutralizing capacity of the NANOBODIES® (V.sub.HH sequences). One example is the rabies virus neutralization assay, Rapid Fluorescent Focus Inhibition Test (RFFIT) (Standard procedure from WHO Laboratory Techniques in Rabies, 1996), where a standard quantity of free rabies virus is pre-incubated with different dilutions of NANOBODIES® (V.sub.HH sequences). Then the NANOBODY® (V.sub.HH sequence)-virus mixture is added on a monolayer of susceptible Baby Hamster Kidney (BHK) cells. Twenty-four hours later, cells are fixed and stained with a green-fluorescent anti-rabies conjugate to quantify infected cells. Absence of fluorescent cells indicates prior neutralization of the virus inoculum. The neutralizing capacity of a NANOBODY® (V.sub.HH sequence) preparation is expressed in International Units (IU)/ml in reference to the WHO standard (=anti-rabies IgG purified from sera of vaccinated humans).
(129) To investigate the in vivo neutralizing capacity of rabies infection by the NANOBODIES® (V.sub.HH sequences), intracerebral inoculation in mice is used, where both the virus and the NANOBODIES® (V.sub.HH sequences) are administered directly in the brain.
Example 10: Bi- and Trivalent NANOBODIES® (V.SUB.HH .Sequences)
(130) Increased avidity and function have been observed for NANOBODIES® (V.sub.HH sequences) that are bi- or trivalent with either homo- or heteromers of selected NANOBODIES® (V.sub.HH sequences). This is an option to target viral trimeric spikes, either different epitopes or the same epitopes on the spike.
(131) Protocols are available for construction of a trivalent NANOBODY® (V.sub.HH sequence) connected by Gly-Ser linkers of any desired length and composition. It is based on the separate PCR reactions (1 for the N-terminal, 1 for the middle (if trivalent) and 1 for the C-terminal V.sub.HH subunit) using different sets of primers. Different linker lengths can also be introduced by the primers.
Example 11: Screening for NANOBODIES® (V.SUB.HH .Sequences) Binding Different Epitopes of the Trimeric Spike Proteins
(132) For hRSV different monoclonal antibodies are available recognizing different epitopes of the F.sub.TM-protein. In order to screen for NANOBODIES® (V.sub.HH sequences) recognizing three different epitopes the following antibodies or Fab fragments were used: mouse monoclonal 9C5 (3ReS21, Hytest), 101F Fab (WO 2006/050280) and Synagis® (Medimmune). They all bind to different epitopes on the F.sub.TM-protein and were used for competition with selected NANOBODIES® (V.sub.HH sequences). 9C5 is believed to bind to an epitope around amino acid 389, 101F at amino acids 422-438 and Synagis® at amino acids 255-280 (see
(133) For competition with 9C5, 2 μg/ml F.sub.TM-protein was coated in a 96 well plate, blocked and then 20 μl periplasmic fractions was added for 30 minutes before the competitor, 9C5 (100 ng/ml) was added. They were competing for 1 hour before 1/5000 HRP conjugated rabbit anti-mouse antibody was added and incubated for 1 hour. Binding specificity was determined based on OD values compared to controls having received no NANOBODY® (V.sub.HH sequence). Several periplasmic fractions were found to compete with 9C5 indicating recognition of another epitope than Synagis® and 101F (
(134) For competition with 101F Fab, hRSV F.sub.TM-protein was coated in a concentration of 1 g/ml. The plate was blocked with 1% casein and the purified NANOBODIES® (V.sub.HH sequences) were added starting at 500 nM and then diluted 1/3. Three nM of 101F Fab was used for competition for 1 hour before addition of mouse anti-HA (1/2000) was added. After 1 hour, HRP conjugated rabbit anti-mouse antibody was added (0.65 μg/ml). Binding specificity was determined based on OD values compared to controls having received no NANOBODY® (V.sub.HH sequence). Two NANOBODIES® (V.sub.HH sequences) were found to compete with 101F Fab, NB6 (191-E4) and NB4 (192-H1) (
Example 12: Surface Plasmon Resonance for Affinity Measurements
(135) To measure the affinity of selected NANOBODIES® (V.sub.HH sequences), Surface Plasmon resonance was used. For preincubation of the Sensorchip CM5, 10 μg/ml hRSV F.sub.TM-protein was left on for 120 seconds. For immobilization by amine coupling, EDC/NHS was used for activation and ethanolamine HCl for deactivation (Biacore, amine coupling kit). 100 nM Synagis® was added and then 100 nM of the NANOBODIES® (V.sub.HH sequences). Evaluation of the off-rates was performed by fitting a 1:1 interaction model (Langmuir binding model) by Biacore T100 software v1.1. The off-rates and affinity constants are shown in Table C-2. NB6 (191-E4) shows a high off-rate and the Kd was 700 pM. NB2 (191-D3) had a Kd of 2.05 nM. NB6 (191-E4) has been shown to bind to the 101F epitope and NB2 (191-D3) to the Synagis® epitope. Note that NB4 is also competing with Synagis® and may thus be recognizing yet a different epitope.
Example 13: NANOBODIES® (V.SUB.HH .Sequences) Targeting the Sialic Acid Binding Site of Influenza Hemagglutinin
(136) Hemagglutinin (HA) on Influenza viruses binds sialic acid on cells during infection. The sialic acid binding site of the HA forms a pocket which is conserved between Influenza strains. Most HAs of avian influenza viruses preferentially recognize sialic acid receptors containing the α(2,3) linkage to galactose on carbohydrate side chains (human viruses, the α(2,6) linkage). To increase the chance of isolating neutralizing NANOBODIES® (V.sub.HH sequences), a functional selection approach can be used—identify NANOBODIES® (V.sub.HH sequences) that compete with soluble 2,3 sialic acid (or 2,6 sialic acid for some mutational drift variants). This would select for NANOBODIES® (V.sub.HH sequences) targeting the sialic acid binding site of HA. These NANOBODIES® (V.sub.HH sequences) are likely to be the most potent at neutralizing H5N1.
(137) We have selected NANOBODIES® (V.sub.HH sequences) binding to H5N1 HA. To identify, from these NANOBODIES® (V.sub.HH sequences), the NANOBODIES® (V.sub.HH sequences) binding to the sialic acid binding site on hemagglutinin, the following experiments were performed. Fetuin (from fetal calf serum, F2379, Sigma-Aldrich, St. Louis, Mo.) was coated (10 μg/ml) in a 96 well plate and incubated over night at 4° C. The plate was blocked in 2% BSA and then 0.7 μg/ml biotinylated HA (HA-bio) and 10 μl periplasmic fractions of the NANOBODIES® (V.sub.HH sequences) (202-C2; SEQ ID NO: 136, 202-F3; SEQ ID NO: 150, 202-D5; SEQ ID NO: 140, 202-E5; SEQ ID NO: 145, 202-B7; SEQ ID NO: 131, 202-E7; SEQ ID NO: 147, 202-C8; SEQ ID NO: 138, 202-D8; SEQ ID NO: 142, 202-F8; SEQ ID NO: 152, 202-E11; SEQ ID NO: 143) or purified NANOBODY® (V.sub.HH sequence) (203-B1; SEQ ID NO: 2431, 203-H1; SEQ ID NO: 2434, 203-E12; SEQ ID NO: 2435, 203-H9; SEQ ID NO: 2445, 203-B12; SEQ ID NO: 2439, 203-A9; SEQ ID NO: 2438, 203-D9; SEQ ID NO: 2441, 202-C8; SEQ ID NO: 138, 189-E2; SEQ ID NO: 2448) were added for competition. After incubation for 1 hour, HRP conjugated streptavidin was added and incubated for 1 hour. Binding specificity of HA-bio not recognized by periplasmic fractions was determined based on OD values compared to controls having received no NANOBODY® (V.sub.HH sequence). Results of competition between periplasmic fractions and fetuin for binding to HA-bio is shown in
Example 14. In Vitro Neutralization of Virus Infection
(138) To investigate in vitro neutralization of NANOBODIES® (V.sub.HH sequences) in periplasmic fractions against Rabies virus, the rabies virus neutralization assay, Rapid Fluorescent Focus Inhibition Test (RFFIT) (Standard procedure from WHO Laboratory Techniques in Rabies, 1996) was used. A standard quantity of free rabies virus was pre-incubated with different dilutions of NANOBODIES® (V.sub.HH sequences) in periplasmic fractions and then the periplasmic fraction-virus mixture was added on a monolayer of susceptible Baby Hamster Kidney (BHK) cells. Twenty-four hours later, cells were fixed and stained with a green-fluorescent anti-rabies conjugate to quantify infected cells. Absence of fluorescent cells indicated prior neutralization of the virus inoculum. The neutralizing capacity of the NANOBODY® (V.sub.HH sequence) (peri) preparations was expressed in International Units (IU)/ml in reference to the WHO standard (=anti-rabies IgG purified from sera of vaccinated humans). The neutralization assay showed several periplasmic fractions with NANOBODIES® (V.sub.HH sequences) neutralizing the rabies virus (Table C-1). All neutralizing periplasmic fractions were selected against the Rabies G protein (monoclonal antibody and total elution) and showed competition with the mouse monoclonal IgG2a antibody directed against rabies virus and with neutralizing capacity. Llama sera and polyclonal periplasmic fractions selected against the inactivated virus and the G protein were included as well as controls for both the polyclonal periplasmic fractions and the monoclonal periplasmic fractions. Only polyclonal and monoclonal periplasmic fractions selected against the G protein showed neutralization.
Example 15: In Vitro Neutralization of hRSV Infection
(139) The hRSV micro neutralization assay was used to investigate in vitro neutralization capacity of selected purified hRSV NANOBODIES® (V.sub.HH sequences). In here, Hep2 cells were seeded at a concentration of 1.5×10.sup.4 cells/well into 96-well plates in DMEM medium containing 10% fetal calf serum (FCS) supplemented with Penicillin and Streptomycin (100 U/ml and 100 μg/ml, respectively) and incubated for 24 hours at 37° C. in a 5% CO.sub.2 atmosphere. The virus stock used is referred to as hRSV strain long, Long LM-2 and Long M2 (used interchangeably) all referring to a virus stock derived from ATCC VR-26 of which the sequence of the F protein corresponds to P12568 or M22643. The virus stock has been passaged several times from the ATCC stock. The sequence of the F-protein was confirmed to be identical to P12568 (see example 23). A standard quantity of hRSV strain Long was pre-incubated with serial dilutions of purified NANOBODIES® (V.sub.HH sequences) in a total volume of 50 μl for 30 minutes at 37° C. The medium of the Hep2 cells was replaced with the premix to allow infection for 2 hours, after which 0.1 ml of assay medium was added. The assay was performed in DMEM medium supplemented with 2.5% fetal calf serum and Penicillin and Streptomycin (100U/ml and 100 μg/ml, respectively). Cells were incubated for an additional 72 hours at 37° C. in a 5% CO2 atmosphere, after which cells were washed twice with 0.05% Tween-20 in PBS and once with PBS alone, after which cells were fixed with 80% cold acetone (Sigma-Aldrich, St. Louis, Mo.) in PBS (100 μl/well) for 20 minutes at 4° C. and left to dry completely. Next the presence of the F-protein on the cell surface was detected in an ELISA type assay. Thereto, fixed Hep2 cells were blocked with 2% Bovine Serum Albumin (BSA) solution in PBS for 1 hour at room temperature, than incubated for 1 hour with anti-F-protein polyclonal rabbit serum (Corral et al. 2007, BMC Biotechnol. 7: 17) or Synagis® (2 μg/ml). For detection goat Anti-rabbit-HRP conjugated antibodies or goat Anti-Human IgG, Fcγ fragment specific-HRP (Jackson ImmunoResearch, West Grove, Pa.) was used, after which the ELISA was developed according to standard procedures.
(140) The hRSV in vitro neutralization potency of a panel of 15 NANOBODIES® (V.sub.HH sequences) identified in previous examples were analyzed. The NANOBODIES® (V.sub.HH sequences) consisted of 4 groups: Group 1 consisted of hRSV F protein specific NANOBODIES® (V.sub.HH sequences) (192C4; SEQ ID NO: 163, 191D3; SEQ ID NO: 159, 192F2; SEQ ID NO: 167, 192C6; SEQ ID NO: 164, 192H2; SEQ ID NO: 169, 192A8; SEQ ID NO: 160, 192C10; SEQ ID NO: 162) recognizing antigenic site II of the F protein. Antigenic site II (also referred to as site A) was identified by mutations found in the F protein in viral escape mutants and although antigenic site II is often found to be referred to as the region aa 250-275, antibodies typically fail to recognize linear peptides representing this region (Arbiza et al. 1992, J. Gen. Virol. 73: 2225-2234). Antibodies specific to antigenic site II may be neutralizing or not (Garcia-Barreno et al. 1989, J. Virol. 63: 925-932). Palivizumab (Synagis®) is a typical example of a mAb binding to antigenic site II (Zhao and Sullender 2005, J. Virol. 79: 3962-3968). Competition with palivizumab was used to assign the antigenic site for the Nanobod-NANOBODIES® (V.sub.HH sequences)ies (see example 7). Group 2 consisted of hRSV F-protein specific NANOBODIES® (V.sub.HH sequences) (191E4; SEQ ID NO: 166, 192B1; SEQ ID NO: 161, 192C10; SEQ ID NO: 162) recognizing antigenic site IV-VI of the F protein (Lopez et al. 1998, J. Virol. 72: 6922). Antigenic site IV-VI was identified by mutations found in the F protein in viral escape mutants and this site can be found to be referred to as the region aa 423-436. For this antigenic site it has been shown that antibodies may recognize linear peptides (Arbiza et al. 1992, J. Gen. Vir. 73: 2225-2234). Antibodies specific to antigenic site IV-VI may be neutralizing or not (Garcia-Barreno et al. 1989, J. Virol. 63: 925-932). 101F is a typical example of a mAb binding to antigenic site IV-VI (Wu et al. 2007, J. Gen. Virol. 88: 2719-2723). Competition with a Fab of 101F was used to assign the antigenic site for the NANOBODIES® (V.sub.HH sequences) (see example 11). Group 3 consisted of hRSV F-protein specific NANOBODIES® (V.sub.HH sequences) (192H1; SEQ ID NO: 168, 192D3; SEQ ID NO: 165, 192B1; SEQ ID NO: 161) for which the antigenic site could not be attributed, either because NANOBODIES® (V.sub.HH sequences) were not competing with 101F or palivizumab or they were showing competition to both 101F and palivizumab. As controls, 3 NANOBODIES® (V.sub.HH sequences) specific for H5 hemagglutinin from influenza (202A5; SEQ ID NO: 128, 202G3; SEQ ID NO: 154, 202E5; SEQ ID NO: 145) were used.
(141) The neutralization assay showed that NANOBODIES® (V.sub.HH sequences) 191D3, 192C4 and 192F2 can neutralize RSV Long infection, with 191D3 being more potent than Synagis® Fab and 101F Fab (
Example 16: Immunizations
(142) Two llamas (212 and 213) were immunized intramuscularly in the neck with 1 mg of RNA-inactivated RSV strain long A (Hytest, Turku Finland; #8RSV79), followed by 4 boosts of 0.5 mg RSV in a biweekly regimen. Two llamas (206 and 207) were immunized intramuscularly with 1 mg of RNA-inactivated RSV strain long A, boosted with 0.25 mg of RSV after 2 weeks, followed by 3 boosts with 50 μg of recombinant hRSV F.sub.TM-NN (membrane anchorless form of the fusion protein, 70 kDa: Corral et al. 2007; BMC Biotechnol. 7: 17) in a biweekly regimen. For all immunizations the antigens were prepared as oil-PBS emulsions with Stimune as adjuvant.
(143) For library construction, blood was collected from all animals 4 days and 10 days after the fourth immunization, while also a Lymph node biopsy was taken 4 days after the fourth immunization. For the NANOCLONE® procedure, 100 mL blood was collected 11 days after the final boost from llamas 206 and 207.
Example 17: Library Construction
(144) Phage libraries from immune tissues of llamas 206, 207, 212 and 213 were constructed as described in Example 2. Phage was prepared according to standard methods and stored at 4° C. for further use, making phage libraries 206, 207, 212 and 213.
Example 18: NANOBODY® (V.SUB.HH .Sequence) Selection with the F-Protein of hRSV
(145) To identify NANOBODIES® (V.sub.HH sequences) recognizing the fusion protein of RSV, libraries 156, 157, 206, 207, 212 and 213 were used for selection on F.sub.TM-NN (membrane anchorless form of the Long fusion protein, 70 kDa; Corral T. et al. 2007, BMC Biotechnol. 7: 17) as described in Example 3. In addition, selections were done using inactivated hRSV strain Long (Hytest #8RSV79). The F.sub.TM-NN protein (25 ng/well) or RSV (5 to 50 μg/well) was immobilized on Nunc Maxisorp ELISA plates, next to a control with 0 μg/ml antigen. Bound phages were eluted from the F.sub.TM-NN using trypsin, Synagis® (Palivizumab, humanized monoclonal antibody, described in Zhao and Sullender 2005, J.
(146) Virol. 79: 396), or 101F Fab (WO 06/050280, humanized monoclonal antibody) in the first round of selection. Outputs from the first round selections eluted with Synagis® or 101F Fab were used for second round selections, using either Numax Fab (Motavizumab or MEDI-524, a third-generation humanized monoclonal antibody product evolved from palivizumab; WO 06/050166), Synagis® or 101F Fab for elution. Remicade (Infliximab, anti-TNF) was used as a control for Synagis®, while Omnitarg Fab (anti-Her2; PCT/EP2008/066363) served as control for Numax Fab and 101F Fab. A 100 molar excess of Synagis®, Numax Fab or 101F Fab was used in order to identify NANOBODIES® (V.sub.HH sequences) binding specifically to antigenic sites II or IV-VI epitopes on the RSV F-protein. To obtain NANOBODIES® (V.sub.HH sequences) specific for the antigenic site IV-VI, second round selections were performed using two biotinylated peptides: at first, a peptide comprising residues 422-436 of the F-protein (Long) (Abgent, San Diego, Calif.) encompassing the 101F binding epitope (Wu et al. 2007, J. Gen. Virol. 88: 2719-2723), secondly, a peptide mimic of the epitope of Mab19 (HWSISKPQ-PEG4-K-biotin)(Chargelegue et al. 1998, J. Virol. 72: 2040-2056). Outputs of both rounds of selections were analyzed for enrichment factor (phage present in eluate relative to controls). Based on these parameters the best selections were chosen for further analysis. Individual colonies were picked and grown in 96 deep well plates (1 mL volume) and induced by adding IPTG for NANOBODY® (V.sub.HH sequence) expression. Periplasmic extracts (volume: ˜80 μl) were prepared according to standard methods.
(147) For testing of selected clones in RSV neutralization assays, periplasmatic extracts from 10 ml cultures were partially purified by using IMAC PhyTips (Phynexus Inc, San Jose, Calif.). In here 800 μl of periplasmatic extracts was loaded onto Phytips 200+ columns prepacked with immobilized metal affinity chromatography resin, followed by elution of His-tagged NANOBODIES® (V.sub.HH sequences) in 30 μl of 0.1M glycine-HCl/0.15M NaCl (pH3), after which eluates were neutralized with 5 μl of 0.5 M Tris-HCl pH8.5.
Example 19: NANOBODY® (V.SUB.HH .Sequence) Selection with F.SUB.TM.-NN of RSV Using NANOCLONE® Technology
(148) Peripheral blood mononuclear cells (PBMCs) were prepared from blood samples using Ficoll-Hypaque according to the manufacturer's instructions. Antigen specific B-cells expressing heavy chain antibodies on their surface were isolated from the PBMCs via FACS sorting (for a description of the NANOCLONE® technology reference is made to WO 06/079372). Thereto, F.sub.TM-NN protein was labeled with Alexa Fluor 488 dye (Invitrogen, Carlsbad, Calif.; cat. nr. A20000) and subsequently desalted to remove residual non-conjugated Alexa Fluor 488 dye according to the manufacturer's instructions.
(149) Pre-immune (background control) and immune PBMC of a llama were stained with fluorescent dye conjugated IgG1 (conventional heavy+light chain immunoglobulins), IgG2- and IgG3 (heavy chain immunoglobulin classes) specific mouse monoclonal antibodies, fluorescently labeled DH59B antibody (CD172a) (VMRD, Inc. Pullman, Wash.; Cat No. DH59B; Davis et al. 1987, Vet. Immunol. Immunopathol. 15: 337-376) and Alexa 488 labeled antigen. TOPRO3 was included as a live/dead cell discriminator dye. IgG1+B-lymphocytes, monocytes, neutrophils and dead cells were gated out and therefore rejected from sorting. Antigen-specific (A488+) IgG2- or IgG3 positive B cells were single cell sorted individually into separate PCR plate wells containing RT-PCR buffer.
(150) For llama 206, 1.9% antigen positive cells of the total amount of IgG2/IgG3 positive living cells was obtained (1.0% in pre-immune reference sample), for llama 207 4.2% positive cells were obtained (0.7% in pre-immune reference sample). Heavy chain variable region genes were amplified directly from these B-cells by single-cell RT-PCR and nested PCR. PCR products were subsequently cloned into a TOPO-adapted expression vector derived from pUC 119 which contained the LacZ promoter, a resistance gene for ampicillin or carbenicillin, a multicloning site and the gen3 leader sequence. In frame with the NANOBODY® (V.sub.HH sequence) coding sequence, the vector coded for a C-terminal c-myc tag and a (His)6 tag.
(151) The resulting constructs were transformed in TOP 10 Escherichia coli cells via high throughput electroporation. Single clones were grown in 96 deep well plates (1 ml volume) and induced by adding IPTG for NANOBODY® (V.sub.HH sequence) expression. Periplasmic extracts (volume: ˜80 μl) were prepared via osmotic shock and analyzed for binding to F.sub.TM-in a binding ELISA as described in example 6. In total, 8 positive F.sub.TM-NN binders (4 from llama 206, 4 from llama 207) were obtained out of 52 cloned VHHs.
Example 20: Screening for NANOBODIES® (V.SUB.HH .Sequences) that Bind to Antigenic Site II or IV-VI
(152) Periplasmic extracts containing single NANOBODIES® (V.sub.HH sequences) were analyzed for binding to the antigen site II or IV-VI, using an Alphascreen® assay (Perkin Elmer; Waltham, Mass.)(Garcia-Barreno et al. 1989, J. Virol. 63: 925-932). In this setup F.sub.TM-NN is bound simultaneously by Fabs of Synagis® and 101F, allowing detection of NANOBODIES® (V.sub.HH sequences) that interfere with binding of each of the respective antigenic sites II and IV-VI. In here, periplasmic extracts were added to F.sub.TM-NN protein (0.3 nM) and incubated for 15 minutes. Subsequently biotinylated Fab Synagis® (0.3 nM) and Fab 101F conjugated acceptor beads (10 μg/ml) were added and this mixture was incubated for 1 hour. Finally streptavidin-coated donor beads (10 μg/ml) were added and after 1 hour incubation the plate was read on the Envision microplate reader. Periplasmic extracts were diluted 25-fold which corresponds roughly to a final concentration of 40 nM. The assay was validated by titration of the known competitors of Synagis®, mabs 18B2 (Argene, Varilhes, France; 18042 N1902) and 2F7 (Abcam, Cambridge, UK; ab43812). Also Synagis® Fab, Numax Fab, and 101F Fab were analyzed, with Numax Fab having the lowest IC50 value (8.6 E-11 M) followed by Synagis® Fab (5.97 E-10 M) and 101F Fab (1.12 E-9 M). For the screening of periplasmatic extracts (at 1/25 dilution) both Numax Fab (40 nM) and 101F Fab (40 nM) were used as positive controls, while irrelevant periplasmatic extracts served as negative controls. Clones that interfered with binding to F.sub.TM-NN protein in the Alphascreen® more than 75% relative to the negative controls were identified as hit. In total 341 hits were identified out of 1856 clones, derived from all 6 llamas but the majority coming from llamas 206 and 207. In addition, out of 8 clones obtained from NANOCLONE® selections 3 clones showed competition.
(153) The correct antigen site (II or IV-VI) of the competitors was deconvoluted by means of a competition ELISA with biotinylated Synagis® Fab (2 nM) or biotinylated 101F Fab (3 nM) for binding to F.sub.TM-NN protein (1 μg/ml). The protocol is essentially the same as described in example 7, with the following modifications. Periplasmatic extracts were diluted 1/10 and mixed with the biotinylated Fab prior to binding to the immobilized F.sub.TM-NN protein. Detection occurred via Extravidin-HRP conjugated secondary antibodies (Sigma-Aldrich, St. Louis, Mo.; Cat. No. E2886).
(154) All hits were subjected to sequence analysis and classified into families according to their CDR3 sequences. In total 133 unique sequences were derived from llamas 206, 207, 212 and 213, classified into 34 different families (Table C-4). Only 6 families containing 15 unique sequences were classified as binding antigenic site II. All remaining clones were binding antigenic site IV-VI. Eight sequences were non-competing binders identified in binding ELISA to hRSV. Also five new families were identified from libraries 156 and 157, of which one identified as binding antigenic site II, and the remaining as binding antigenic site IV-VI. Also new family members of previously identified families from llamas 156 and 157 were identified.
Example 21: Screening for RSV Neutralizing NANOBODIES® (V.SUB.HH .Sequences)
(155) From all six hRSV libraries 163 unique sequences (160 identified from phage libraries, 3 derived from NANOCLONE®) were analyzed for RSV Long neutralizing capacity in a micro-neutralization assay as partially purified proteins. The screening was essentially performed as described in example 15, using a fixed volume of Phytips purified NANOBODIES® (V.sub.HH sequences) (20 μl). The detection of F-protein on the Hep2 cell surface was done using Synagis® (2 μg/ml), followed by goat Anti-Human IgG, Fcγ fragment specific-HRP (Jackson ImmunoResearch, West Grove, Pa.).
(156) In addition to the previously identified RSV neutralizing NANOBODIES® (V.sub.HH sequences) LG191D3 and LG192C4, which were included as positive controls in the screening, 5 new antigenic site II clones showed strong RSV Long neutralizing activity: 1E4 (also referred to as 207D1; SEQ ID NO: 211), a newly identified family member of 191D3 (SEQ ID NO: 159), 7B2 (SEQ ID NO: 364), NC23 (SEQ ID NO: 380), and two members of the same family 15H8 (SEQ ID NO: 371) and NC41 (SEQ ID NO: 372) (Tables A-1, C-4). None of the antigenic site IV-VI specific NANOBODIES® (V.sub.HH sequences) showed more than very weak neutralizing activity for hRSV Long LM-2 strain.
Example 22: Construction, Production and Characterization of hRSV NANOBODIES® (V.SUB.HH .Sequences)
(157) Five new neutralizing NANOBODIES® (V.sub.HH sequences) selected from the screening described above (1E4, 7B2, 15H8, NC23 and NC41) as well as 2 antigenic site IV-VI NANOBODIES® (V.sub.HH sequences) (NC39; SEQ ID NO: 359, 15B3; SEQ ID NO: 286) were expressed, purified and further characterised. Thereto the encoding sequences were recloned in an expression vector derived from pUC119 which contained the LacZ promoter, a resistance gene for kanamycin, a multicloning site and the OmpA signal peptide sequence. In frame with the NANOBODY® (V.sub.HH sequence) coding sequence, the vector coded for a C-terminal c-myc tag and a (His)6 tag.
(158) Expression occurred in E. coli TG-1 cells as c-myc, His6-tagged proteins in a culture volume of 1 L. Expression was induced by addition of 1 mM IPTG and allowed to continue for 3 hours at 37° C. After spinning the cell cultures, periplasmic extracts were prepared by freeze-thawing the pellets and resuspension in dPBS. These extracts were used as starting material for immobilized metal affinity chromatography (IMAC) using Histrap FF crude columns (GE healthcare, Uppsala, Sweden). NANOBODIES® (V.sub.HH sequences) were eluted from the column with 250 mM imidazole and subsequently desalted towards dPBS.
(159) All purified NANOBODIES® (V.sub.HH sequences) were shown to bind to the F-protein in a binding ELISA to F.sub.TM-NN protein and to hRSV. Results for hRSV binding are shown in Table C-5. In short, 1 μg/ml of F.sub.TM-NN or 5 μg/ml hRSV (Hytest Turku, Finland) were immobilized directly on Maxisorp microtiter plates. Free binding sites were blocked with 1% casein. Serial dilutions of purified NANOBODIES® (V.sub.HH sequences) were allowed to bind the antigen for 1 hour. NANOBODY® (V.sub.HH sequence) binding was revealed using a rabbit-anti-V.sub.HH secondary antibody, and final detection with a HRP-conjugated goat-anti-rabbit antibody. Binding specificity was determined based on OD values compared to irrelevant NANOBODY® (V.sub.HH sequence) controls.
(160) To determine the precise binding affinities of the purified NANOBODIES® (V.sub.HH sequences), a kinetic analysis was performed using Surface Plasmon resonance analysis on the F.sub.TM-NN protein, following the procedure described in example 12. Results are shown in Table C-5.
(161) The ability of purified NANOBODIES® (V.sub.HH sequences) to compete with Synagis® Mab or biotinylated Synagis® Fab for binding to F.sub.TM-NN was determined in ELISA following the procedure described in examples 8 and 20.
(162) NANOBODIES® (V.sub.HH sequences) 15H8 and NC41 also had relatively low affinities (K.sub.D values of 16 and 8.1 nM, respectively). NANOBODIES® (V.sub.HH sequences) 7B2 and NC23 showed off-rates in the 10.sup.−4 (1/s) range, resulting in K.sub.D values around 1 nM, confirming the strong binding to hRSV observed in ELISA. NANOBODIES® (V.sub.HH sequences) 191D3 and 1E4 showed low nM affinities due to very high on-rates. The antigenic site IV-VI binders 15B3 and 191E4 show the highest affinities for F.sub.TM-NN of the panel with sub-nanomolar affinities.
Example 23: In Vitro Micro Neutralization of Distinct hRSV Strains
(163) The potency of purified NANOBODIES® (V.sub.HH sequences) in neutralization of different type A and B RSV strains was tested by the in vitro micro neutralization assay (see example 15). Viral stocks of RSV Long (Accession No. P12568; ATCC VR-26; see example 15), RSV A-2 (ATCC VR-1540; lot nr. 3199840) and RSV B-1 (ATCC VR-1580; lot nr. 5271356) were prepared into Hep2 cells and subsequently titrated to determine the optimal infectious dose for use in the micro neutralization assay. Results of neutralization potencies of the different purified NANOBODIES® (V.sub.HH sequences) are shown in Table C-5. While all six NANOBODIES® (V.sub.HH sequences) that recognize the Synagis® epitope could efficiently neutralize type A strains Long and A-2, they failed to neutralize infection with the B-1 strain or did so at concentrations >1 μM. The 101F competitors 15B3 and 191E4 showed very weak neutralization potency on the B-1 strain only when administrated at μM concentrations.
(164) The sequences of the respective F-proteins of the different RSV strains were verified by means of reverse-transcriptase PCR and subsequent sequence analysis. Briefly, total RNA was isolated from RSV-infected Hep2 cells using RNeasy mini kit (Qiagen, Venlo, Netherlands), after which complementary DNA was prepared using Superscript III reverse transcriptase kit (Invitrogen, Carlsbad, Calif.). The F-protein of RSV A strains was amplified and sequenced using the primers described in Kimura et al. 2004 (Antiviral Research 61: 165-171). For amplification of the RSV B-1 strain F-protein the following primers were used: FB1_outer_for: cttagcagaaaaccgtga (SEQ ID NO: 2419); FB1_outer_rev: tgggttgatttgggattg (SEQ ID NO: 2420); FB1_seq_1123-for: ggactgatagaggatggta (SEQ ID NO: 2421); FB1_seq_1526-rev: gctgacttcacttggtaa (SEQ ID NO: 2422). The sequence of RSV B-1 strain corresponded to Accession nr P13843, with an additional point mutation Ser540Leu. The sequence for the RSV Long M2 strain corresponded completely to the reported sequence (Accession nr M22643). The sequence for the strain RSV A-2 corresponded to Accession M11486. See also Table A-3.
Example 24: Construction, Production and Characterization of Multivalent hRSV NANOBODIES® (V.SUB.HH .Sequences)
(165) Multivalent NANOBODY® (V.sub.HH sequence) constructs connected by Gly-Ser linkers of different lengths and composition were generated by means of separate PCR reactions (1 for the N-terminal, 1 for the middle (in case of trivalent) and 1 for the C-terminal NANOBODY® (V.sub.HH sequence) subunit) using different sets of primers encompassing specific restriction sites. Similarly, multivalent NANOBODY® (V.sub.HH sequence) constructs connected by Ala-Ala-Ala linker were generated. All constructs were cloned into an expression vector derived from pUC119 which contained the LacZ promoter, a resistance gene for kanamycin, a multicloning site and the OmpA signal peptide sequence. In frame with the NANOBODY® (V.sub.HH sequence) coding sequence, the vector coded for a C-terminal c-myc tag and a (His)6 tag. In case a 35 Gly-Ser-linker was present in the multivalent construct, an expression vector was used derived from pUC119 which contained the LacZ promoter, a resistance gene for kanamycin and the OmpA signal peptide sequence. Directly downstream of the signal peptide a multiple cloning site was present for NANOBODY® (V.sub.HH sequence) insertion, followed by a 35Gly-Ser linker encoding DNA sequence and a second multiple cloning site for cloning of a second NANOBODY® (V.sub.HH sequence) sequence. In frame with the resulting NANOBODY® (V.sub.HH sequence)-35Gly-Ser-NANOBODY® (V.sub.HH sequence) coding sequence, the vector coded for a C-terminal c-myc tag and a (His)6 tag. Table C-6 lists the multivalent constructs generated with RSV-specific NANOBODIES® (V.sub.HH sequences). The sequences of the multivalent constructs are shown in Table A-2.
(166) Multivalent RSV NANOBODY® (V.sub.HH sequence) constructs were expressed, purified and further characterized. Production was done in E. coli TG1 cells, followed by purification from the periplasmic fraction via the His-tag by IMAC and desalting, essentially as described in example 22. For certain trivalent constructs (e.g. RSV401, RSV404, RSV406) production was done in P. pastoris followed by purification from the medium fraction. All trivalent NANOBODIES® (V.sub.HH sequences) were subjected to gel filtration as a final step to remove possible bivalent and monovalent degradation products.
(167) Binding of purified multivalent NANOBODIES® (V.sub.HH sequences) to the hRSV F-protein was confirmed in ELISA on both F.sub.TM-protein and on hRSV (see example 22). For the majority of NANOBODIES® (V.sub.HH sequences) the formatting into bivalent and trivalent constructs resulted in a clear but limited (up to 10-fold increase) avidity effect, with the exception of multivalents of 7B2 and NC23 which showed similar EC50 values as their monovalent counterparts (
Example 25: Potency of Bi- and Trivalent Constructs to Neutralize hRSV
(168) The potency of the NANOBODY® (V.sub.HH sequence) constructs was evaluated in the RSV neutralization assay on different RSV strains (see examples 15 and 23). Bivalent NANOBODIES® (V.sub.HH sequences) binding antigenic site II showed marked increases in potencies of 100- to 1000-fold (i.e. much higher than the increase in affinity) in neutralization of Long relative to their monovalent counterparts, with IC50 values ranging from 50-380 pM, being better or similar to Numax Fab. On the RSV B-1 strains however, the potency increase was much less strong, and none of the dimeric constructs was more potent than Synagis®. Surprisingly, this could be overcome by the generation of trivalent constructs, as shown in
(169)
Example 26: Potency of Bi- and Trivalent Biparatopic Constructs to Neutralize hRSV
(170) Biparatopic constructs consisting of one NANOBODY® (V.sub.HH sequence) binding antigenic site II and one NANOBODY® (V.sub.HH sequence) binding antigenic site IV-VI were analysed for neutralization. Biparatopic-bivalent constructs generally showed a flatted curve in the neutralization assay, hampering accurate determination of IC50 values (
(171) Also biparatopic constructs with two different NANOBODIES® (V.sub.HH sequences) binding to antigenic site II, 7B2 and 15H8, were analysed for neutralization (RSV204 and 206). Also in this case significant improvement in potency was noted especially for the B-1 strain were potency increased at least 1000-fold versus the monomeric NANOBODIES® (V.sub.HH sequences).
(172) Trivalent biparatopic constructs of 7B2 and 15B3 were even more potent neutralizers of both Long and B-1 strains and did not show the flattened curves as observed with bivalent biparatopic constructs (
Example 27: Reactivity of Monovalent NANOBODIES® (V.SUB.HH .Sequences) with Escape Mutants of the Long Strain
(173) A number of escape mutants, described in Lopez et al. 1998 (J. Virol. 72: 6922-6928), and specific for antigenic site II (R47F/4, R47F/7, RAK13/4, R7C2/11, R7C2/1) or IV-VI (R7.936/1, R7.936/4, R7.936/6, R7.432/1) or the combination of both (RRA3), were selected for testing their reactivity with 10 monovalent NANOBODIES® (V.sub.HH sequences), including NANOBODY® (V.sub.HH sequence) 191C7 (EVQLVESGGGLVQAGGSLRLSCAASGSSGVINAMAWHRQAPGKERELVAHISSGGS TYYGDFVKGRFTISRDNAKDTVYLQMNSLKPEDTAVYYCHVPWMDYNRRDYWGQ GTQVTVSS; SEQ ID NO: 2423) previously identified as not binding to antigenic sites II or IV-VI.
(174) This assay was performed according to Lopez et al. 1998 (J. Virol. 72: 6922-6928). In brief, each NANOBODY® (V.sub.HH sequence) was tested at 0.2 μg/ml in ELISA using antigen extracts of HEp-2 cells infected with the different escape mutants. Absorbance results were normalized for reactivity on the reference virus strain (Long wild type) strain as well as on the control NANOBODY® (V.sub.HH sequence) 191C7. Results are shown in Table C-7.
(175) A reactivity of >75% is indicated by Δ symbols • symbols correspond to a reactivity between 75 and 50%, .diamond-solid. symbols correspond to a reactivity of 25-50% and less than 25% reactivity is indicated by a blank square. In general NANOBODIES® (V.sub.HH sequences) already identified as antigenic site II binders in previous examples (192C4, 191D3, 191F2, NC23, 15H8, 7B2 and NC41) were found to be sensitive to typical mutations in antigenic site II, while the other NANOBODIES® (V.sub.HH sequences) already identified as antigenic site IV-VI binders were indeed sensitive for mutations in these sites.
Example 28: Reactivity of Multivalent NANOBODIES® (V.SUB.HH .Sequences) with Escape Mutants of the Long Strain
(176) Subsequently a number of multivalent constructs was analyzed on a limited panel of escape viruses to assess binding. This assay was performed according to Lopez et al. 1998 (J. Virol. 72: 6922-6928). In brief, each NANOBODY® (V.sub.HH sequence) was tested at 0.1 μg/ml for monovalent NANOBODIES® (V.sub.HH sequences) and at 0.05 μg/ml for bi- and trivalent NANOBODIES® (V.sub.HH sequences) in ELISA using antigen extracts of HEp-2 cells infected with the different escape mutants. Absorbance results were normalized for reactivity on the reference virus strain (Long wild type) strain as well as on the control NANOBODY® (V.sub.HH sequence) (191E4; SEQ ID NO: 166, in this particular assay). Results are shown in Table C-8.
(177) A reactivity of >75% is indicated by Δ symbols • symbols correspond to a reactivity between 75 and 50%, .diamond-solid. symbols correspond to a reactivity of 25-50% and less than 25% reactivity is indicated by a blank square. Remarkably, multivalent constructs showed improved binding compared to their monovalent counterpart, to the mutant virus R7C2/11. In addition the biparatopic construct RSV403 was not sensitive to any of the mutants.
Example 29: Intranasal Delivery of Bivalent NANOBODY® (V.SUB.HH .Sequence) RSV101
(178) To test the capacity of NANOBODY® (V.sub.HH sequence) RSV101 (SEQ ID NO: 2382) to neutralize virus in vivo, a mouse model was used. In this model, female Balb/c mice (9-10 weeks old) were inoculated intranasally with 100 μg of purified RSV101 dissolved in 50 μl PBS. As an irrelevant NANOBODY® (V.sub.HH sequence) control, the bivalent NANOBODY® (V.sub.HH sequence) 12D2biv was used. In addition, one group of mice received 100 μg Palivizumab (Synagis®) and a fourth group received PBS only. Five hours later, 10.sup.6 infectious units of the RSV A2 strain were administered intranasally. Four days and 1 day before virus infection and 1 and 4 days after infection mice were treated with cyclophosphamide (first dosing at 3 mg/kg; subsequent dosing at 2 mg/kg all administered s.c.) to suppress the immune system and as such to increase virus replication.
(179) Three and 5 days after viral challenge, mice were killed; lungs were removed, homogenized and cleared from tissue by centrifugation. Sub-confluent Hep-2 cells, incubated in serum-free medium, were infected with serial dilutions of cleared lung homogenates. Four hours after infection the medium was removed and replaced by fresh medium containing 1% FCS and 0.5% agarose. Two to three days after infection the agarose overlay was removed to allow staining of RSV-plaques by an anti-RSV antibody.
(180) Infectious virus (pfu/lung) was recovered from all animals in the negative control groups (PBS and 12D2biv) in lung homogenates on day 3 (
Example 30: Functionality of NANOBODY® (V.SUB.HH .Sequence) RSV101 after Intranasal Administration
(181) In order to test whether NANOBODIES® (V.sub.HH sequences) or palivizumab antibodies might still be present in lungs 3 and 5 days after inoculation, lung homogenates of PBS treated mice were pre-incubated for 1 h with the same volume of lung homogenates from the different experimental groups described in Example 29, prepared either three or five days post-infection.
(182) As shown in
(183) Taken together, these data show that the functional bivalent NANOBODY® (V.sub.HH sequence) RSV101 remains present and functionally active in the lungs for at least 72 hours after administration.
(184) To further demonstrate the presence of functional virus-neutralizing NANOBODIES® (V.sub.HH sequences) in the lung homogenates, 500 plaque forming units (pfu) of RSV were incubated with different amounts of lung homogenates. These mixtures were incubated for 90 minutes at room temperature. Next, mixtures were put on HepG2 cells grown in 96 well plates. After 2 hours cells were washed and an overlay of growth medium with 0.5% agarose was added. After three days RSV plaques were visualized (
(185) From the data (
Example 31: Viral RNA is not Detected in the Lungs of Mice Pre-Treated Intranasally with RSV101
(186) The results described in Example 29 demonstrated that no infectious virus was present in the lungs of mice treated with RSV101. However, there was still the possibility that virus had infected cells and that viral genomic RNA was replicated with release of non-infectious viral particles or without release of viral particles. To investigate this possibility, the presence of viral RNA was determined by qPCR. RNA was isolated from 100 μl of each long homogenate (1000 μl) prepared 5 days post-infection. By the use of an M-gene specific primer RSV genomic RNA specific cDNA was synthesized and quantified by qPCR (in duplicate). The level of viral genomic RNA in each lung homogenate was calculated relative to a lung sample which showed the lowest qRT-PCR signal (normalized to value of 1). As shown in Table C-9, the presence of relative viral genomic RNA in lungs of mice treated with RSV 101 and Synagis® was reduced strongly compared to PBS or 12D2biv treated mice.
Example 32: The HA-Pseudotyped Neutralization Assay
(187) A HA pseudotyped neutralization assay was developed as described in Temperton et al. 2007 (Temperton N J, Hoschler K, Major D et al. A sensitive retroviral pseudotype assay for influenza H5N1-neutralizing antibodies. Influenza and Other Respiratory Viruses 2007 1: 105-112). The construction of HA pseudotyped viruses and assays was also done according to Temperton et al. 2007.
(188) Plasmids and Cell Lines
(189) Plasmid pI.18/VN1194 HA was constructed at NIBSC (Hertfordshire, UK). The full-length HA ORF from A/Vietnam/1194/04 was amplified by PCR and cloned into the expression vector p. 18. This backbone plasmid is a pUC-based plasmid incorporating promoter and Intron A elements from human cytomegalovirus.
(190) The MLV and HIV gag/pol constructs have been described previously (Besnier C, Takeuchi Y, Towers G. 2002, Restriction of lentivirus in monkeys. Proc. Natl. Acad. Sci. USA 9: 11920-11925) The luciferase (Luc) reporter construct MLV-Luc has been described in Op De Beeck A, Voisset C, Bartosch B et al. 2004 (Characterization of functional hepatitis C virus envelope glycoproteins. J. Virol. 78: 2994-3002). Vesicular stomatitis virus envelope protein (VSV-G) expression vector pMDG has been described previously (Naldini L, Blomer U, Gallay P et al. 1996, In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector. Science 272: 263-267). All cell lines were cultured in Dulbecco's modified eagle medium (DMEM) with Glutamax and high glucose (Gibco, Paisley, Scotland, UK), supplemented with 10% fetal calf serum and penicillin/streptomycin, except for HEK 293T cells (15% fetal calf serum).
(191) Viral Vector Production and Infection of Target Cells
(192) Confluent plates of 293T cells were split 1:4 the day before transfection. Each plate of 293T cells was transfected with 1 μg gag/pol construct, 1.5 □g Luc reporter construct, and 1.5 □□g HA- or VSV-G-expressing construct by using the Fugene-6 transfection reagent. At 24 h post-transfection, 1 U of exogenous neuraminidase (Sigma, St. Louis, Mo., USA) was added to induce the release of HA-pseudotyped particles from the surface of the producer cells. Supernatant was harvested 48 and 72 h post-transfection, filtered through 0.45-1 m filters, and stored at −80° C. MLV vector titers were measured on human 293T, quail QT6, canine MDCK, porcine PK15 and ST-IOWA cells and are presented as infectious units (IU) per milliliter. Briefly, cells were infected with vector, and Luc titers were determined 72 h later by Luc assay. Titers were expressed as RLU for Luc.
(193) MLV(HA) Pseudotype Neutralization Assay
(194) Serum samples (5 μl) were heat inactivated at 56° C. for 30 min, twofold serially diluted in culture medium, and mixed with MLV(HA) virions (10 000 RLU for Luc) at a 1:1 v/v ratio. Purified NANOBODIES® (V.sub.HH sequences) (10 or 20 μl) were diluted to 100 μl and twofold serially diluted in culture medium, and mixed with MLV(HA) virions (10 000 RLU for Luc) at a 1:1 v/v ratio. After incubation at 37° C. for 1 h, 1×10.sup.4 293T cells were added to each well of a 96-well flat-bottomed plate. Relative light units (RLU) for Luc were evaluated 48 h later by luminometry using the Promega Bright-Glo system (Promega, Madison, Wis., USA) according to the manufacturer's instructions. IC90/IC50-neutralizing antibody titers were determined as the highest serum dilution resulting in a 90/50% reduction of infection (as measured by marker gene transfer) compared with a pseudotype virus only control. For Luc, titers <100 are designated negative.
Example 33: Llamas Develop High Virus-Neutralizing Antibody Titers after Immunizations with Purified H5 HA
(195) Sera taken from immunized llamas before (pre-immune) and 21 and 48 days after the first immunization was tested in the pseudotyped neutralization assay as described in Example 32 (
Example 34: Identification of NANOBODIES® (V.SUB.HH .Sequences) that Neutralize HA Pseudotyped Virus
(196) Several purified NANOBODIES® (V.sub.HH sequences) were tested in the pseudo typed virus neutralization assay described in Example 32. In
Example 35: Combinations of NANOBODIES® (V.SUB.HH .Sequences)
(197) Combined treatment with different virus neutralizing antibodies might results in additive or even synergistic neutralizing effect (Zwick M B, Wang M, Poignard P, Stiegler G. Katinger H, et al. 2001, Neutralization synergy of human immunodeficiency virus type 1 primary isolates by cocktails of broadly neutralizing antibodies. J Virol. 75: 12198-12208; Laal S, Burda S, Gorny M K, Karwowska S. Buchbinder A et al. 1994, Synergistic neutralization of human immunodeficiency virus type 1 by combinations of human monoclonal antibodies. J. Virol. 68: 4001-4008; Li A, Baba T W, Sodroski J, Zolla-Pazner S, Gorny M K et al. 1998, Synergistic neutralization of simian-human immunodeficiency virus SHIV by triple and quadruple combinations of human monoclonal antibodies and high-titer anti-human immunodeficiency. J. Virol. 72: 3235-40). However, this was not observed when combinations of 202-C8 with 203-B12, 202-C8 with 203-H9 or 203-B12 with 203-H9 were tested in the pseudotyped neutralization assay (
Example 36: Bi- and Trivalent NANOBODIES® (V.SUB.HH .Sequences)
(198) Protocols are available for construction of a bivalent or trivalent NANOBODY® (V.sub.HH sequence) connected by Gly-Ser linker(s) of any desired length and composition. It is based on the separate PCR reactions (1 for the N-terminal, 1 for the middle (if trivalent) and 1 for the C-terminal V.sub.HH subunit) using different sets of primers. Different linker lengths can also be introduced by the primers.
(199) Bivalent and trivalent NANOBODIES® (V.sub.HH sequences) with different linker lengths from 202-C8 and 203-B12 and 203-H9 were constructed (SEQ ID NO's: 2423 to 2430; Table A-4). When tested in the pseudotyped neutralization assays all bivalent and trivalent NANOBODIES® (V.sub.HH sequences) showed superior neutralization potencies compared to the monovalent building blocs. (
(200) To test the potency of different NANOBODY® (V.sub.HH sequence) formats against different H5 strain viruses, lentiviral pseudotyped viruses were used. For transfection, 5×10.sup.6 HEK-293T cells were plated 24 h prior to addition of a complex comprising plasmid DNA and Fugene 6™ that facilitated DNA transport into the cells (as described by the manufacturer; Roche, UK). The human immunodeficiency virus type 1 (HIV-1) gag-pol construct pCMV-A8.91 and firefly luciferase reporter construct (pCSLW, where the luciferase gene has been cloned into pCSGW in place of GFP) were transfected concurrently with the required H5 HA envelope construct (p1.18-H5HA from different H5 clades) at a g ratio of 1:1.5:1 respectively. 24 hours post-transfection, 1U exogenous bacterial NA was added to each plate to effect particle release into the supernatant. At 48 and 72 hrs post-transfection, virus was harvested by filtration through a 0.45 uM filter and stored at −80C until needed. Neutralization assays were performed very similar to the previously described MLV(HA) assays (Example 32).
(201) When bivalent and trivalent NANOBODIES® (V.sub.HH sequences) with different linker lengths from 202-C8 and 203-H9 were tested against these different H5 variants using the lentiviral pseudotyped neutralization assays all bivalent and trivalent NANOBODIES® (V.sub.HH sequences) showed superior neutralization potencies compared to the monovalent building blocs (
Example 37: In Vivo Neutralization of Influenza Virus by NANOBODY® (V.SUB.HH .Sequence) 202-C8
(202) To test the capacity of NANOBODY® (V.sub.HH sequence) 202-C8 to neutralize virus in vivo, a mouse model was used. In this model, female Balb/c mice (6-7 weeks old) were inoculated intranasally with 100 μg of purified 202-C8 dissolved in 50 μl PBS. As an irrelevant NANOBODY® (V.sub.HH sequence) control, the RSV NANOBODY® (V.sub.HH sequence) 191-D3 (SEQ ID NO: 159) was used. In addition, one group of mice received PBS only. Four hours later, 1 LD50 of the mouse adapted NIBRG-14 virus (Temperton et al. 2007) was administered intranasally. The NIBRG-14 virus contains the HA (with the polybasic cleavage site removed) and the NA of the A/Vietnam/1194//2004 (H5N1) virus. The internal viral genes are of the A/Puerto Rico/8/1934(H1N1).
(203) Four and six days after viral challenge, mice were killed, lungs were removed and homogenized. Viral titers (TCID50) were determined by infection of MDCK cells with serial dilutions of lung homogenates. The presence of virus in cell supernatant was determined by hemagglutination assays (Table C-10). Titers were calculated according the method of Reed, L. J. and Muench, H. 1938 (A simple method of estimating fifty percent endpoints. The American Journal of Hygiene 27: 493-497). A value of “0” was entered if no virus was detected. The geometric mean and standard deviation are reported for each group at each time point.
(204) Mice treated with 202-C8 never showed any sign of disease during the whole experiment. The PBS and 191-D3-treated mice showed clinical signs, including ruffled fur, inactivity, hunched posture, and depression.
(205) Virus was recovered from all animals in the negative control groups (PBS and 191-D3) in lung homogenates on day 4 and 6 after challenge. None of the animals in the 202-C8-treated group had detectable virus titers on day 4 and 6 post challenge (Table C-10).
Example 38: Functionality of NANOBODY® (V.SUB.HH .Sequence) 202-C8 in the Lungs after Inoculation
(206) To test how long NANOBODY® (V.sub.HH sequence) 202-C8 remains active in the lungs after intranasal inoculation, female Balb/c mice (6-7 weeks old) were inoculated intranasally with 100□g of purified 202-C8 dissolved in 50□l PBS. As an irrelevant NANOBODY® (V.sub.HH sequence) control the RSV NANOBODY® (V.sub.HH sequence) 191-D3 was used. In addition, one group of mice received PBS only. All mice received 1 LD50 of the mouse adapted NIBRG-14 intranasally, but virus was given 4, 24 or 48 hours after inoculation of the NANOBODIES® (V.sub.HH sequences). Four days after viral challenge, mice were killed, lungs were removed and homogenized. Viral titers (TCID50) were determined by infection of MDCK cells with serial dilutions of lung homogenates. The presence of virus in cell supernatant was determined by hemagglutination assays. Titers were calculated according the method of Muench and Reed. A value of “0” was entered if no virus was detected. The geometric mean and standard deviation are reported for each group at each time point (Table C-11).
(207) Mice pretreated with 202-C8 never showed any signs of disease during the whole experiment. The PBS and 19-D3-treated mice showed clinical signs, including ruffled fur, inactivity, hunched posture, and depression and a reduction in body weight (
(208) Virus was recovered from all animals pretreated with the control NANOBODY® (V.sub.HH sequence) 191-D3 or PBS. Virus could not be detected in the lungs of mice that were treated with 202-C8, 4 and 24 hours before virus inoculation. No virus could be detected in lungs of three mice of seven treated with 202-C8 48 hours before virus inoculation (
(209) Taken together, these data show that the monovalent NANOBODY® (V.sub.HH sequence) 202-C8 remains actively present in the lungs for at least 48 hours after intranasal administration.
Example 39: Surface Plasmon Resonance for Affinity Measurements
(210) To measure the affinity of selected NANOBODIES® (V.sub.HH sequences), Surface Plasmon resonance was used. Two thousand Reference units (RU), H5 was coupled on a Sensorchip CM5 in 10 mM sodium acetate pH 5.5 and immobilized by aminecoupling (Biacore, aminecoupling kit). Dilutions of the NANOBODIES® (V.sub.HH sequences) were added at concentrations 250-62.5 nM and run over a reference flow channel with no HA and then over the HA coupled flow channel at a flow rate of 5 μl/min. Evaluation of the KA and KD was performed by fitting a 1:1 interaction model (Langmuir binding model), removing the background from the reference flow channel. The kinetic curves of the NANOBODIES® (V.sub.HH sequences) (62.5 nM) are shown in
Example 40. Determination of Binding Efficacy of Purified Multivalent NANOBODIES® (V.SUB.HH .Sequences) to H5
(211) In order to determine binding specificity to H5, the different multivalent NANOBODIES® (V.sub.HH sequences) were tested in an ELISA binding assay in different concentrations. In short, 2 μg/ml of H5 were immobilized directly on Maxisorp microtiter plates (Nunc). Free binding sites were blocked using 4% Marvel in PBS. Next, Dilutions (1/10) of the NANOBODIES® (V.sub.HH sequences) starting with 10 pM in 100 μl 2% Marvel PBST were allowed to bind to the immobilized antigen. After incubation and a wash step, NANOBODY® (V.sub.HH sequence) binding was revealed using a rabbit-anti-V.sub.HH secondary antibody (a V.sub.HH). After a wash step the NANOBODIES® (V.sub.HH sequences) were detected with a HRP-conjugated goat-anti-rabbit antibody (GARPO). Binding specificity was determined based on OD values compared to controls (192-C4; SEQ ID NO: 163) against HRSV and 213-H7-15GS-213-H7 (SEQ ID NO: 2427) against Rabies). The multivalent NANOBODIES® (V.sub.HH sequences) show higher binding capacity than the monovalent (
Example 41. Multivalent NANOBODIES® (V.SUB.HH .Sequences) Blocking the Interaction of H5 with Sialic Acid on Fetuin
(212) To investigate if the multivalent NANOBODIES® (V.sub.HH sequences) were able to block the interaction of H5 with sialic acid on fetuin, the same experimental set up was used as described in Example 13. Fetuin (from fetal calf serum, F2379, Sigma-Aldrich) was coated (10 μg/ml) in a maxisorb 96 well plate and incubated over night at 4° C. The plate was blocked in 2% BSA and then 0.7 μg/ml biotinylated HA (HA-bio) and different dilutions of purified multivalent NANOBODIES® (V.sub.HH sequences) were added for competition, diluted 1/10, starting with 500 nM. After incubation for 1 hour, HRP conjugated streptavidin was added and incubated for 1 hour. Binding specificity of HA-bio not recognized by purified multivalent NANOBODIES® (V.sub.HH sequences) was determined based on OD values compared to controls having received control NANOBODIES® (V.sub.HH sequences) (192-C4 against HRSV and 213H7-15GS-213H7 against Rabies). Results of competition between the purified multivalent NANOBODIES® (V.sub.HH sequences) and fetuin for binding to HA-bio is shown in
Example 42: Pharmacokinetics of 191D3, ALX-0081 and RANKL008A in the Male Wistar Rat after Single Intratracheal or Intravenous Administration
(213) 42.1: Test Items:
(214) Test items are described in Table C-12.
(215) 42.2 Methods
(216) Animal Model
(217) 101 male Wistar rats (approximately 300 gram and 11 weeks old) were used for this study, a strain bred by Charles River Laboratories, Germany. The animals were held for at least 6 days for adaptation. Following the initial health check, the animals were weighed and allocated by means of a computerised randomisation program to the test groups; only healthy animals were used.
(218) The sterile test substances were thawed in a water bath at 25° C. while swirling gently for 10 minutes. For intratracheal dosing, no further dilutions were required. For intravenous administration, the required amount of test substance was diluted in sterile DPBS ((Dulbecco's modified) Phosphate Buffered Saline) down to the desired concentrations. The test item formulations were freshly prepared within 4 hours prior to dosing.
(219) Dose and Route of Administration
(220) The different test groups and the dose levels are given in Table C-13. The i.v. bolus dose was given into a tail vein. The amount of test item for i.v. administration was adjusted to each animal's current body weight. The i.t. dose was administered intratracheally with a syringe with a blunt stainless steel dosing needle, after deep anaesthetization with isoflurane. The amount of test item for i.t. administration was set to 100 μL/animal, irrespective of body weight. Based on the actual body weights of the animals, an approximate dose in mg/kg could be calculated from the averaged body weights for comparison with the i.v. route: 4 mg/kg for RSV NB2, 5 mg/kg for ALX-0081 and 5 mg/kg for RANKL008a.
(221) The average body weight of intratracheally dosed animals was on average 0.315 kg (RSV NB2 group), 0.317 kg (ALX-0081 group), 0.323 kg (RANKL008a group). As these animals received a fixed dosing of 100 μL/animal, the corresponding mean dose per b.w. were calculated at 3.6 mg/kg (RSV NB2 group), 3.1 mg/kg (ALX-0081 group), 3.2 mg/kg (RANKL008a group).
(222) Blood and BALF Sampling and Processing.
(223) After i.v. dosing, blood was sampled (approximately 300 μL) at 0.05, 0.25, 0.5, 1, 2, 4, 6, and 24 hours from the tail vein of RSV NB2- and ALX-0081-dosed animals and at 0.05, 0.25, 0.5, 1, 2, 4, 8, 24, and 48 hours from RANKL008a-dosed animals. All blood samples were placed on melting ice. Within approximately 30 minutes after sampling, the blood samples were centrifuged at 50C for 10 minutes (1500 g). Citrated plasma was stored in polypropylene tubes at approximately ≤−75° C. until dispatch on dry ice to the Sponsor.
(224) After intratracheal dosing, blood, lungs, and BALF were collected (at necropsy following deep anaesthesia with isoflurane) at 0.05, 0.333, 1, 2, 4, 6, and 24 hours from RSV NB2-dosed rats and ALX-0081-dosed rats and at 0.05, 0.333, 1, 2, 4, 8 and 24 hours from animals dosed with RANKL008a. By means of an aorta punction 4 mL of blood was withdrawn. Within 42 minutes after sampling, the blood samples were centrifuged at 5° C. for 10 minutes (1500 g). Citrated plasma was stored in polypropylene tubes at approximately ≤−75° C. until dispatch on dry ice to the Sponsor. Following the removal of blood, lungs were harvested. First, the lungs including trachea were rinsed with iced DPBS and weighed. Then, BALF was collected. Five mL lavage fluid (DPBS) was carefully put into the lungs. After approximately 10 seconds, as much fluid as possible was returned to the syringe. BALF was transferred to an empty tube and directly stored on melting ice. This procedure was repeated. The second collection of BALF was added to the first collection. The volume of BALF that was collected was documented and reported. Subsequently, BALF was stored at approximately ≤−75° C. until dispatch on dry ice to the Sponsor.
(225) Determination of RSV NB2 in Rat Plasma or BALF
(226) 96-well microtiter plates (Maxisorp, Nunc, Wiesbaden, Germany) were coated overnight at 4° C. with 100 μL hRSV (12.5 μg/mL, Hytest. Turku, Finland). Thereafter wells were aspirated, blocked (RT, 1 h, PBS-0.1% casein) and washed. The standards, QC, and predilutions of the test samples were prepared in a non-coated (polypropylene) plate in 100% rat plasma or BALF and incubated for 30 min at RT while shaking at 600 rpm. A 1/10 dilution of the samples in PBS-0.1% casein (final concentration of rat plasma or BALF is 10%) was transferred to the coated plate and incubated for 1 hr at RT while shaking at 600 rpm. After three washing steps with PBS-0.05% Tween20, the plates were incubated with polyclonal rabbit anti-NANOBODY® (V.sub.HH sequence) monoclonal K1 (1/2000 in PBS-0.1% casein, in-house) for 1 hr at RT while shaking at 600 rpm. After 3 washing steps with PBS-0.05% Tween20, 100 μl horseradish peroxidase (HRP) labeled polyclonal goat anti-rabbit (1/2000 in PBS-0.1% casein, DakoCytomation, Glostrup, Denmark) was incubated for 1 hr at RT while shaking at 600 rpm. Visualization was performed covered from light for 20 min with 100 μL 3,3′,5,5′-tetramethylbenzidine (esTMB, SDT, diluted 1/3). After 20 min, the colouring reaction was stopped with 100 μL 1N HCl. The absorbance was determined at 450 nm, and corrected for background absorbance at 620 nm. Concentration in each sample was determined based on a sigmoidal standard curve. The lower limit of quantification (LLOQ) and upper limit of quantification (ULOQ) of the different assays are listed in Table C-14.
(227) Determination of ALX-0081 in Rat Plasma or BALF
(228) 96-well microtiter plates (Maxisorp, Nunc) were coated overnight at 4° C. with 100 μL vWF in PBS (2.5 μg/mL, Haemate P1200/500—ZLB Behring). Thereafter wells were aspirated, blocked (RT, 1 h, PBS-0.1% casein) and washed. The standards, QC, and predilutions of the test samples were prepared in a non-coated (polypropylene) plate in 100% rat plasma or BALF and incubated for 30 min at RT while shaking at 600 rpm. A 1/5 dilution of the samples in PBS-0.1% casein (final concentration of rat plasma or BALF is 20%) was transferred to the coated plate and incubated for 1 hr at RT while shaking at 600 rpm. After three washing steps with PBS-0.05% Tween20, the plates were incubated with the anti-ALX0081 NB vWF12B2-GS9-12B2-BIO (1 μg/ml in PBS-0.1% casein, in-house) for 30 min at RT while shaking at 600 rpm. After 3 washing steps with PBS-0.05% Tween20, 100 μl streptavidin-HRP (1/2000 in PBS-0.1% casein, DakoCytomation) was incubated for 30 min at RT while shaking at 600 rpm. Visualization was performed covered from light for 15 min with 100 μL 3,3′,5,5′-tetramethylbenzidine (esTMB, SDT, diluted ⅓). After 15 min, the coloring reaction was stopped with 100 μL 1N HCl. The absorbance was determined at 450 nm, and corrected for background absorbance at 620 nm. Concentration in each sample was determined based on a sigmoidal standard curve. The LLOQ and ULOQ of the different assays are listed in Table C-15.
(229) Determination of RANKL008a in Rat Plasma or BALF
(230) 96-well microtiter plates (Maxisorp, Nunc) were coated overnight at 4° C. with 100 μL neutravidin in PBS (2 μg/mL, Pierce, Rockford, Ill.). Wells were aspirated and blocked. After 3 washing steps with PBS-0.05% Tween20, biotinylated RANKL (0.5 μg/mL in PBS-0.1% casein) was captured by incubating 100 μL for 1 hr at RT while shaking at 600 rpm. After this incubation step, wells were washed. The standards, QC, and predilutions of the test samples were prepared in a non-coated (polypropylene) plate in 100% rat plasma or BALF and incubated for 30 min at RT while shaking at 600 rpm. A 1/10 dilution of the samples in PBS-0.1% casein (final concentration of rat plasma or BALF is 10%) was transferred to the coated plate and incubated for 1 hr at RT while shaking at 600 rpm. After three washing steps with PBS-0.05% Tween20, the plates were incubated with polyclonal rabbit anti-NANOBODY® (V.sub.HH sequence) monoclonal R23 (1/2000 in PBS-0.1% casein, in-house) for 1 hr at RT while shaking at 600 rpm. After 3 washing steps with PBS-0.05% Tween20, 100 μl horseradish peroxidase (HRP) labelled polyclonal goat anti-rabbit (1/5000 in PBS-0.1% casein, DakoCytomation, Glostrup, Denmark) was incubated for 1 hr at RT while shaking at 600 rpm. Visualization was performed covered from light for 10 min with 100 μL 3,3′,5,5′-tetramethylbenzidine (esTMB, SDT, diluted 1/3). After 10 min, the coloring reaction was stopped with 100 μL 1N HCl. The absorbance was determined at 450 nm, and corrected for background absorbance at 620 nm. Concentration in each sample was determined based on a sigmoidal standard curve. The LLOQ and ULOQ of the different assays are listed in Table C-16.
(231) Non-Compartmental Pharmacokinetic Data Analysis
(232) Individual plasma and mean BALF concentration-time profiles of all rats were subjected to a non-compartmental pharmacokinetic analysis (NCA) using WinNonlin Professional Software Version 5.1 (Pharsight Corporation, Mountain View Calif., USA). The pre-programmed Models 200 and 201 were used to analyse the intratracheal and intravenous data, respectively. The linear-up/log down trapezoidal rule was used to calculate the area under the concentration-time data.
(233) 1.3 Results
(234) Plasma Concentrations of RSVNB2, ALX-0081 and RANKL008a
(235) The observed plasma concentration-time data of the individual animals after a single i.v. administration and of the mean (n=4 animals/time-point; destructive sampling) plasma concentration-time data after a single i.t. administration of RSV NB2, ALX-0081, and RANKL008a are shown in
(236) Plasma Pharmacokinetic Analysis of RSVNB2, ALX-0081 and RANKL008a
(237) An overview of the basic pharmacokinetic parameters obtained by non-compartmental PK analysis of RSV NB2 (4 mg/kg i.v. & 3.6 mg/kg i.t.), ALX-0081 (5 mg/kg i.v. & 3.1 mg/kg i.t.) and RANKL008a (5 mg/kg i.v. & 3.2 mg/kg i.t.) is given in Tables C-20, C-21 and C-22.
(238) The PK parameters discussed herein were obtained using non-compartmental analysis (NCA). For rat 1 and 2 (RSV NB2 i.v.), rat 6 (ALX-0081 i.v.) and rat 9 (RANKL008a i.v.) difficulties in blood sampling occurred, and due to the limited data, these animals were excluded from subsequent pharmacokinetic calculations. The terminal parameters for some of the animals were calculated based on only two data-points in the terminal phase.
(239) After i.v. administration of RSV NB2 4 mg/kg and ALX-0081 5 mg/kg comparable plasma PK profiles were observed (
(240) After i.t. administration of RSV NB2 (3.6 mg/kg), ALX-0081 (3.1 mg/kg) and RANKL008a (3.2 mg/kg), comparable terminal half-lives in the plasma were observed for the three NANOBODIES® (V.sub.HH sequences) (RSV NB2: 9.48 hr, ALX-0081: 10.5 hr and RANKL008a: 13.0 hr). For RSV NB2 and ALX-0081 the half-lives are longer after i.t. administration than after i.v. administration. It is conceivable that for these rapidly cleared compounds, the absorption is the rate limiting step resulting in flip-flop kinetics (i.e. kinetics are absorption rate controlled and the terminal phase is driven by the slow absorption from the site of administration (the lung) to the systemic circulation.
(241) The exposure after i.t. administration was lower for all NANOBODIES® (V.sub.HH sequences) as compared to that after i.v. administration. This resulting bioavailabilities were 22.1%, 13.9%, and 6.9% for RSV NB2 (16.6 kD), ALX-0081 (27.9 kD), and RANKL008a (40.9 kD), respectively.
(242) For lung topical applications (RSV NB2), a high pulmonary exposure is desired. It could be expected that a faster and more complete absorption (resulting in a higher bioavailability) would not benefit pulmonary exposure. Therefore, RSV NANOBODIES® (V.sub.HH sequences) with a higher molecular weight (f.e. a trivalent RSV NANOBODY® (V.sub.HH sequence)) could possibly lead to enhanced local (pulmonary) exposures.
(243) The current data indicate that systemic exposure to NANOBODIES® (V.sub.HH sequences) can be achieved after intratracheal administration, suggesting that the pulmonary route may be viable as non-invasive method of delivery of NANOBODIES® (V.sub.HH sequences). Notably, the use of specific delivery formulations and/or devices could significantly improve bioavailability after pulmonary application. It is suggested that the bioavailability may be improved around 5 times (i.t. vs aerosol—see e.g. table 2 in Patton J., Fishbum S., Weers J. 2004, The Lung as a Portal of Entry for Systemic Drug Delivery. Proc. Am. Thorac. Soc. 1: 338-344).
(244) BALF Concentrations of RSVNB2, ALX-0081 and RANKL008a
(245) The mean observed BALF concentration-time profiles after a single intratracheal administration of RSV NB2, ALX-0081 and RANKL008a to male rats is shown in
(246) The terminal half-lives of the three NANOBODIES® (V.sub.HH sequences) in BALF were based on the two last data-points only. Of note is also that there was quite some inter-individual variability as indicated by the large standard deviations (see Table C-24). After i.t. administration, comparable terminal half-lives were observed in plasma (RSV NB2 9.48 hr, ALX-0081 10.5 hr and RANKL008a 13.0 hr) and in BALF (RSV NB2 16.0 hr, ALX-0081 9.21 hr and RANKL008a 11.6 hr), supporting the notion that the plasma kinetics are likely absorption rate controlled.
(247) Following intratracheal administration, the RSV NB2, ALX-0081, RANKL008a NANOBODY® (V.sub.HH sequence) exposure in BALF was observed for at least 24 hours (i.e. the last sampling time for BALF).
(248) Amounts of RSVNB2, ALX-0081 and RANKL008a in BALF
(249) After intratracheal dosing broncho-alveolar lavage fluid (BALF) was collected at necropsy as described in detail earlier.
(250) Theoretically, the amount of NANOBODY® (V.sub.HH sequence) in the lung at a given time-point can be obtained by multiplying the measured concentration of each BALF sample by the volume of DPBS added (10 mL), provided that the NANOBODY® (V.sub.HH sequence) is efficiently washed out. These individual calculated amounts and their corresponding mean (+SD) values are listed in Table C-25 and C-26, respectively.
(251) Note however that large variations occurred in the recovery of the BALF. For some animals it was possible to recover 9.5 mL fluid after injecting 10 mL DPBS, while for other animals only 3 mL was recovered. Furthermore, since the lavage is performed twice and combined, in a single vial, it is impossible to determine how much volume was recovered from the first or second lavage separately. Moreover, it is also unknown whether there are differences in the concentration of the first and second lavage.
(252) The result is that overestimations of the true amount of NANOBODY® (V.sub.HH sequence) may occur when multiplying the measured BALF concentrations are simply multiplied with the theoretical volume of 10 mL DPBS.
(253) Alternatively, if the amount of NANOBODY® (V.sub.HH sequence) is estimated by multiplying the measured concentration of each BALF sample by the actual recovered volume of BALF, this may result in underestimations of the actual amount of NANOBODY® (V.sub.HH sequence) in case significant amounts of NANOBODY® (V.sub.HH sequence) are present in unrecovered BALF.
(254) Therefore, the true amount of NANOBODY® (V.sub.HH sequence) in BALF should theoretically be comprised between the amount calculated via the theoretical BALF volume or the actual BALF volume. It is important to note that the larger the recovered volume, the more accurate the calculations are expected to be. Since the average recovered volume is on average ca. 7 mL (Table C-27), both calculation methods should not provide very different results. The individual calculated amounts and mean (+SD) values based on actual recovered volumes are listed in Table C-28 and C-29, respectively.
(255) By dividing the calculated amount of NANOBODY® (V.sub.HH sequence) by the actual amount dosed (RSV NB2: 1.14 mg, ALX-0081: 0.985 mg, RANKL008a: 1.03 mg), the recovered fraction of the dose was calculated. Expressed as a percentage, the dose normalized individual calculated amounts and their corresponding mean (+SD) values based on the theoretical BALF volume (10 mL) and actual recovered volumes are listed in Tables C-30 to C-33.
(256) By dividing the calculated amount of NANOBODY® (V.sub.HH sequence) by the actual amount dosed, the recovered fraction of the dose could be compared across time: The highest mean amount to dose percentages via actual and theoretical volume are 35.7% and 49.5% for RSV NB2 (After 20 minutes), 74.0% and 98.3% for ALX-0081 (After 4 minutes) and 47.1% and 67.4% for RANKL008A (After 1 hour), respectively. Thus for ALX-0081 almost the total fraction of the dose could be recovered in the BALF, while for RSV NB2 and RANKL008a, the fraction was lower: approximately 50% of the dose. The highest individual amount to dose percentages via actual and theoretical volume are 76.6% and 117.3% for RSV NB2, 145% and 182% for ALX-0081 and 84.1% and 120% for RANKL008a at time-point 1 hour post-dose. As expected, the variability was appreciable.
(257) After 24 hours, the fraction of the dose recovered in BALF was lower for all NANOBODIES® (V.sub.HH sequences) than at earlier time-points. The mean fraction recovered ranged from 12.4% to 16.5% via the theoretical volume and ranged from 8.46% to 12.5% via the actual volumes for the three tested NANOBODIES® (V.sub.HH sequences).
(258) 42.3 Conclusions
(259) After i.v. administration to rats, similar PK characteristics were observed for RSV NB2 and ALX-0081. For RANKL008a, substantially lower clearance values and longer terminal half-lives were observed. This may be explained by binding of the anti-HSA NANOBODY® (V.sub.HH sequence) of RANKL008a to rat albumin.
(260) The current data indicate that systemic exposure to NANOBODIES® (V.sub.HH sequences) can be achieved after intra-tracheal administration, suggesting that the pulmonary route may be viable as non-invasive method for the delivery of NANOBODIES® (V.sub.HH sequences). The limited data also suggested that the systemic bioavailability seems to decrease with increasing molecular weight.
(261) After i.t. administration comparable terminal half-lives were observed for the three NANOBODIES® (V.sub.HH sequences). For RSV NB2 and ALX-0081 the half-lives are longer after i.t. administration than after i.v. administration, suggesting that that absorption is the rate limiting step because the drug is slowly absorbed from its site of dosing (i.e. the lung) to the circulation. Comparable terminal half-lives are observed both in plasma and in BALF. This observation further enhances the possibility that the kinetics could be absorption rate controlled.
(262) Following intra-tracheal administration, the RSV NB2, ALX-0081, RANKL008a NANOBODY® (V.sub.HH sequence) exposure in BALF was observed for at least 24 hours (i.e. the last sampling time for BALF).
(263) Following intra-tracheal administration, systemic exposure to the RSV NB2, ALX-0081 NANOBODY® (V.sub.HH sequence) in plasma was observed for at least 24 hours (i.e. the last sampling time of plasma after intra-tracheal administration. Following i.v. administration both of these NANOBODIES® (V.sub.HH sequences) without anti-HSA were no longer detectable at 24 hours.
(264)
Example 43: Further Studies with an Anti-RSV NANOBODY® (V.SUB.HH .Sequence) Construct
(265) Example 43.1:—Prophylactic Study with RSV407 in Cotton Rat
(266) In this study cotton rats are treated either i.m. or intranasally with RSV neutralizing NANOBODY® (V.sub.HH sequence) constructs (RSV 407; SEQ ID NO: 2415) or control (PBS). Viral RSV challenge is administered intranasally 1 hour later. At day 4, animals are sacrificed and RSV titers determined by Q-PCR in nasal and lung washes as well as in nasal and lung tissue.
Example 43.2—Therapeutic Study with RSV407 in Cotton Rat
(267) RSV therapeutic studies have been described in the past; e.g. by Crowe and colleagues (1994, Proc. Nat. Ac. Sci.; 91: 1386-1390) and Prince and colleagues (1987, Journal of Virology 61:1851-1854).
(268) In this study cotton rats are intranasally infected with RSV. Twenty-four hours after infection a first group of animals are treated with RSV neutralizing NANOBODY® (V.sub.HH sequence) constructs (RSV 407) or control (PBS). Treatment is administered to pulmonary tissue by intranasal or aerosol administration. Treatment is repeated at 48 and 72 hours. At day 4 animals are sacrificed and RSV titers determined by Q-PCR in nasal and lung washed as well as in nasal and lung tissue.
(269) In the second group, treatment is only initiated 3 days after infection and repeated at day 4 and 5. Finally, at day 6 animals are sacrificed and RSV titers determined by Q-PCR in nasal and lung washed as well as in nasal and lung tissue.
Example 43.3—Lung to Systemic
(270) In this study the lung tissue of rats is exposed to an RSV neutralizing NANOBODY® (V.sub.HH sequence) (RSV407) by intratracheal or aerosol administration. Serum and BAL samples are taken at regular time points up to 3 days after administration. The NANOBODY® (V.sub.HH sequence) concentration is measured by means of ELISA and samples are subjected to RSV microneutralization as described in Example 15. By combining the information from the ELISA and the neutralization assay the RSV IC50 of each sample can be determined to assess systemic bioavailabilty of functional RSV NANOBODY® (V.sub.HH sequence).
Example 44: Screening Procedures, for Hep2 Cells Infected with RSV B-1
(271) In addition to the identification of NANOBODIES® (V.sub.HH sequences) that are potent neutralizers of RSV Long strain in a microneutralization assay, NANOBODIES® (V.sub.HH sequences) can also be screened for their ability to neutralize RSV B-1. Clones obtained from selections against the F-protein and RSV, specifically from trypsin elutions, competitive elution with 101F Fab or with linear peptides (see Example 18), were subjected to an alternative screening procedure that included binding to the F-protein of RSV B-1.
(272) As a first step, approximately 1000 periplasmatic extracts were analyzed for binding to F.sub.TM-NN protein (1 μg/ml) in ELISA (see Example 20). On average, 44% of all clones were identified as binders (>2-fold over background), with 27% identified as strong binders (>3-fold). Only 10% of all binders originated from llamas 212 and 213.
(273) Binders were subjected to a competition ELISA with Synagis® (67 pM) for binding to RSV Long (10 μg/ml; Hytest #8RSV79) to identify clones of epitope Class II. Detection of Synagis® was done using goat anti-human-HRP conjugated IgG (Jackson ImmunoResearch Laboratories, Inc., Cat. No. 109-035-098). This assay resulted in 9 hits (Table C-34).
(274) In a similar manner, periplasmatic extracts were analyzed in a competition ELISA with 101F Fab to identify clones of epitope Class IV-VI (see Example 20). Detection was done using anti-HA monoclonal antibody (Zymed, 32-6700, 1389267), followed by anti-mouse-HRP conjugated antibody (Dako, Cat. No. P0260). Of the 90 competitors identified, the best 101F Fab competitors were further tested at dilutions ranging from 1/100- 1/1000 to allow differentiation between clones (Table C-34).
(275) As third step, the Class II and IV-VI epitope clones were analyzed for binding to Hep2 cells infected with RSV B-1 strains. In this assay, Hep2 cells were seeded into 96-wells plates and infected with an RSV B-1 strain, essentially following the procedure described for the neutralization assay (see Example 15). After three days the cells were fixed with ice-cold acetone and plates were used in an ELISA assay using periplasmic extracts at different dilutions. NANOBODY® (V.sub.HH sequence) binding to Hep2-B1 infected cells was detected using anti-V.sub.HH rabbit polyclonal antibody, followed by goat Anti-rabbit-HRP conjugated antibodies, after which the ELISA was developed according to standard procedures. In general, the Class II epitope clones proved weaker binders to Hep2-B1 cells than clones of the epitope Class IV-VI (Table C-34).
(276) Sequence analysis reduced the total number of competing NANOBODIES® (V.sub.HH sequences). Clones 8A1 (SEQ ID NO: 249), 8B10 (SEQ ID NO: 342) and 1B2 (SEQ ID NO: 166) were found as multiple copies which were all ranked amongst the strongest binders to Hep2 B-1-infected cells. Clone 1B2 was identical to the sequence of the previous identified 191E4. The unique sequence 19E2 (SEQ ID NO: 301) belongs to the large family 4. From the group of Synagis® competitors, clones 19C4 (also referred to as 15H8; SEQ ID NO: 371) and 1G8 (SEQ ID NO: 2578) were the best RSV B-1 binders. Based on the binding to both RSV long and B-1, on sequence, and on 101F competition, a selection was made from 101F competitors for further analysis as purified proteins (Table C-34).
Example 45: Immunization of Llamas with Rabies Virus
(277) Two llamas were immunised with rabies virus antigen and lymphocytes were collected as a source of virus-specific single-chain antibody mRNA. Immunised llamas had identification numbers 183 and 196, source: N.V. Neerhofdieren Bocholt, location: animal facilities of the Belgian Scientific Institute of Public Health (IPH, authorisation nr. LA1230177). All experimental procedures were approved by the Ethical Committee of the IPH and the Veterinary and Agrochemical Research Centre (VAR) (advice nr. 070515-04).
(278) Inactivated Rabies Vaccine Merieux HDCV, marketed by Sanofi Pasteur MSD for use in humans, was the antigen. This vaccine contains the Wistar strain of the Pitman Moore virus grown on human diploid WI38 lung cells (PM/WI38 1503 3M). It contains human albumin, but no adjuvant. The vaccine was injected in the neck and the suspension divided over two spots (0.5 ml/spot) at day 0, 7, 28, 35, 57. Blood lymphocytes were collected on EDTA on day 42, 49 and 62 (Table C-35).
(279) Both llamas developed protective titers of neutralizing antibodies in the range of 15-35 IU/ml. Lymphocytes were successfully collected from the blood. Lymph nodes were not distinguishably enlarged, which made them difficult to find. For this reason, lymph nodes were not used as a source of lymphocytes.
Example 46: In Vitro Neutralisation Potency of Monovalent NANOBODY® (V.SUB.HH .Sequence) Clones with the RFFIT Assay
(280) The neutralizing potency of NANOBODY® (V.sub.HH sequence) clones was determined and the most potent clones were selected to make bivalent and biparatopic combinations for further in vivo experiments. The clones were pre-selected by their capacity to bind to a substrate of purified glycoprotein G (Platelia II ELISA plates). Some of the selected clones competed with monoclonal antibody 8-2, which recognizes an epitope on the antigenic site IIa of the rabies surface glycoprotein G (Montaño-Hirose J A, Lafage M, Weber P, Badrane H, Tordo N, Lafon M. 1993, Protective activity of a murine monoclonal antibody against European bat lyssavirus 1 (EBL1) infection in mice. Vaccine 11: 1259-66).
(281) The neutralizing potency of NANOBODY® (V.sub.HH sequence) or antibody preparations was determined with the Rapid Fluorescent Focus Inhibition Test (RFFIT). This test is a virus-neutralisation assay which uses Baby Hamster Kidney (BHK)-21 cells as susceptible targets. Infection of cells is visualized by staining with a fluorescein isothiocyanate (FITC)-coupled anti-nucleocapsid conjugate (Bio-Rad Laboratories, France). The virus strain used is the highly virulent and neurotropic Challenge Virus Standard (CVS)-11 (genotype 1 genus Lyssavirus, Family Rhabdoviridae). CVS-11 was obtained from the American Type Culture Collection (ATCC reference VR959). The in vitro neutralizing potency is expressed in International Units (IU)/ml in reference to “The Second International standard for Anti-rabies Immunoglobulin” purchased from the United Kingdom National Institute for Biological Standards and Control. A serum titer of 0.5 IU/ml is considered protective in vivo. RFFIT was performed according to the Manual of Diagnostic Tests and Vaccines for Terrestrial Animals (Office International des Epizooties, 2008) and ISO17052 norms (BELAC Accreditation 081-TEST). The results are shown in Table C-36.
(282) The majority of NANOBODY® (V.sub.HH sequence) clones (15/16), which were isolated from the immunised llamas and selected based on their binding capacities to glycoprotein G, were neutralizing (≥0.50 IU/ml) in the RFFIT. In general, their potency was significantly lower compared to the reference monoclonal antibody (Mab) RV1C5 (0.17 nM IC.sub.50). The clones with the strongest potency were 212-C12 (8 nM IC.sub.50), 213-E6 (14 nM IC.sub.50) and 212-F6 (18 nM IC.sub.50). Control NANOBODIES® (V.sub.HH sequences), which were raised against another virus (human respiratory syncytial virus) or Toll-like receptor 3, were not neutralizing.
Example 47: Potency of Combinations of Monovalent Antibodies
(283) The potency of a combination of two different monovalent NANOBODIES® (V.sub.HH sequences) (no linkage) and the synergistic effect on the neutralizing potency compared to the monovalent clones was investigated.
(284) The neutralizing potency of combinations and single clones was determined by RFFIT. Competition binding experiments showed that clones 213-E6, 214-E8 and 213-H7 bind to the same major epitope on the glycoprotein G, whereas 212-C12 binds to a different major epitope. The results are shown in Table C-37.
(285) All tested combinations of monovalent clones yielded no additive effect on the neutralizing potency. Synergistic effects were not observed even with clones which bind to different major epitopes.
Example 48: Cross-Neutralization of Selected Clones Against Divergent Genotype 1 and 5 Lyssa Virus
(286) Clones that were selected against the genotype 1 CVS-11 strain were examined for their ability to cross-neutralize other genotype 1 lyssaviruses (laboratory strains and street isolates; obtained from Prof S. Van Gucht, Scientific Institute of Public Health, Rabies Laboratory, Brussels, Belgium).
(287) Cross-neutralisation against a genotype 5 lyssavirus (European bat lyssavirus-1, EBLV-1; obtained from Prof S. Van Gucht, Scientific Institute of Public Health, Rabies Laboratory, Brussels, Belgium) was also examined. Most human cases of rabies (>99%) are caused by genotype 1 lyssaviruses. EBLV-1 circulates in certain species of bats (mainly Eptesicus serotinus) in Europe.
(288) Evelyn-Rotnycki-Abelseth (ERA) is an attenuated genotype 1 strain which is used as an oral vaccine for immunisation of wild life (ATCC reference VR322). Chien Beersel (CB)-1 is a virulent genotype 1 virus isolated from the brain of a rabid dog which was imported from Morocco to Belgium (Le Roux I. and Van Gucht S. 2008. Two cases of imported canine rabies in the Brussels area within six months time. WHO Rabies Bulletin 32(1), Quarter 1). The EBLV-1 strain 8919FRA belongs to genotype 5 and was isolated from an Eptesicus serotinus bat in France (Bourhy et al. 1992. Antigenic and molecular characterization of bat rabies virus in Europe. J Clin Microbiol. 30(9):2419-26). The strain was provided by Dr. L. Dacheux form the Pasteur Institute of Paris (MTA DB/EB-08/420). The viral stocks were grown in BHK-21 cells, except for CB-1 which was grown in neuroblastoma N2a cells. The lysates of infected cell cultures were centrifuged at 20000×g for 20 minutes at 4° C. and supernatants were stored at −80° C.
(289) In addition, 7 genotype 1 strains were provided by Dr. L. Dacheux from the Pasteur Institute of Paris in the form of infected mouse brains. Six strains were wild isolates, among which an isolate from a dog from Cambodia (9912CBG, accession nr. EU086169/EU086132), a fox from France (9147FRA, accession nr. EU293115), a raccoon dog from Poland 9722POL), a human patient from Thailand (8740THA), a dog from the Ivory Coast (07059IC, accession nr. EU853615/FJ545659) and a dog from Niger (9009NIG, accession nr. EU853646). One brain was infected with the laboratory CVS IP13 strain.
(290) The neutralizing potency against ERA, CB-1 and EBVL-1 was determined in an RFFIT adapted with the virus of interest. Neutralisation was defined as a minimal neutralizing potency of 0.50 Equivalent Units (EU)/ml.
(291) For the infected brains, an alternative neutralisation assay was developed. Briefly, ten-fold dilutions of the infected brain suspensions were pre-incubated with a 1/50 dilution of the stock solution of NANOBODY® (V.sub.HH sequence) for 90 minutes at 37° C. and 5% CO.sub.2. Then, susceptible neuroblastoma N2a cells were added to the mix. Two days later, infection of the cells was measured by staining with a FITC-coupled anti-nucleocapsid conjugate (Bio-Rad Laboratories, France). Neutralisation was defined as a minimum hundred-fold reduction of the infectious titer in comparison to an irrelevant NANOBODY® (V.sub.HH sequence) control (172-B3 anti-TLR3).
(292) Results are shown in Table C-38 (ERA), Table C-39 (CB-1), Table C-40 (EBLV-1) and Table C-41 (infected brain). Table C-42 gives an overview of the neutralisation profile of all tested clones.
(293) In general, most clones which neutralized the prototype CVS-11 strain also neutralized most other genotype 1 viruses. An exception is clone 212-C12, which proved to be a relative potent neutralizer of CVS-11, but did not neutralize 3 out of 9 other genotype 1 strains. 214-F8 neutralized all 10 genotype 1 strains. 213-E6 neutralized 9 out of 10 genotype 1 strains and 213-H7 neutralized 8 out of 10 genotype 1 strains. Attention should be drawn to the fact that for 213-E6 and 213-H7 a relative low amount of NANOBODY® (V.sub.HH sequence) was used in the assay (respectively 0.1 and 1.7×10.sup.−3 IU). Neutralisation might have been complete if higher amounts had been used. Seven of the sixteen anti-rabies clones, including clones 213-H7 and 214-E8, were also able to neutralize the divergent EBLV-1 strain. This indicates that the epitope recognized by these clones is highly conserved among lyssaviruses.
Example 49: Potency of Bivalent and Biparatopic NANOBODY® (V.SUB.HH .Sequence) Combinations Measured with the RFFIT Assay
(294) The potential synergistic effect on the neutralizing potency of the linkage of two similar (bivalent) or different (biparatopic) NANOBODIES® (V.sub.HH sequences) compared with the monovalent clones was investigated.
(295) The neutralizing potency of bivalent and biparatopic clones was determined using RFFIT as described above. Different fusion proteins were developed with 3 Gly-Ser linkers: 5GS, 15GS or 25GS. Sequences of multivalent NANOBODY® (V.sub.HH sequence) constructs against rabies are given in Table A-6. NB6-18GS-NB6 (RSV115; SEQ ID NO: 2394) is a control bivalent NANOBODY® (V.sub.HH sequence) which was raised against another virus (human respiratory syncytial virus). Data on neutralization of EBLV-1 strain is shown in Table C-40. Data on neutralization of wild type genotype 1 strains and a laboratory CVS strain in suspensions of infected mouse brain is shown in Table C-41. Table C-42 gives an overview of the neutralisation profile of all tested clones. The results of neutralization of CVS-11 are shown in Table C-43.
(296) The majority of the tested bivalent and biparatopic NANOBODIES® (V.sub.HH sequences) had a significantly higher potency than the corresponding monovalent clones. For example, the biparatopic combination 214E8-15GS-213H7 was 600-fold more potent that the monovalent NANOBODIES® (V.sub.HH sequences). In general, the bivalent combinations seemed less potent than the biparatopic combinations. The most potent bivalent combinations had a neutralizing potency between 15 and 36 IU/nM (213H7-15GS-213H7, 213E6-5GS-213E6, 214F8-15GS-214F8). For the most potent biparatopic combinations, this ranges between 80 and 230 IU/nM (213E6-15GS-213H7, 213H7-15GS-214F8, 214E8-15GS-213H7). This is comparable to the neutralizing potency of the anti-rabies monoclonal antibody RV1C5 (Santa Cruz) (194 IU/nM). Most of the potent combinations had a 15GS linker.
Example 50: In Vivo Neutralisation of Virulent CVS-11 with Monovalent/Bivalent NANOBODIES® (V.SUB.HH .Sequences) Using the Brain as the Susceptible Target System: Intracerebral Inoculation in Mice
(297) 50.1 In Vivo Neutralization by Monovalent NANOBODIES® (V.sub.HH Sequences)
(298) Whether NANOBODIES® (V.sub.HH sequences) (monovalent, bivalent or biparatopic), which proved to be potent neutralizers in vitro, can also neutralize the virus in vivo and prevent lethal infection of the brain was investigated. Outbred Swiss mice (5-6 weeks old) were inoculated intracerebrally with rabies virus CVS-11 pre-incubated with 1 IU of NANOBODY® (V.sub.HH sequence), 1 IU of monoclonal antibody (mab 8-2) or PBS (negative control) (6 to 9 mice/group). Prior to inoculation, the mix of virus and NANOBODY® (V.sub.HHsequence) or antibody was incubated at 37° C., 5% CO.sub.2 for 30 min. A volume of 20 μl (10 μl virus+10 μl NANOBODY® (V.sub.HH sequence)) was inoculated into the brain by transcranial introduction of a 26G needle. Neutralizing units (IU) were determined using the in vitro RFFIT assay. A viral dose of 10.sup.1.5 TCID.sub.50/mouse was used based on preliminary experiments with different doses of virus preincubated with 1 IU of mab 8-2. This preliminary work indicated that a dose of 1 IU of mab 8-2 was able to protect all mice from lethal infection (0% mortality) upon intracerebral inoculation with 10.sup.1.5 TCID.sub.50, which was not the case at higher virus doses (10.sup.2 TCID.sub.50 CVS+1 IU mab 8-2=43% mortality). Mice were examined for (rabies) disease signs each work day and a clinical score was given per day per mice. Clinical scores ranged from 0 (no disease signs) to 6 (weight loss, depression, hunched back, wasp waist, incoordination and hind limb paralysis). At score 6, mice were sacrificed by cervical dislocation. The experiment was ended at 28 days post inoculation (DPI).
(299) The results for monovalent antibodies are shown in
(300) The monoclonal antibody (mab 8-2) provided full protection against an intracerebral challenge with 10.sup.1.5 TCID.sub.50 CVS-11. Pre-incubation with an irrelevant NANOBODY® (V.sub.HH sequence) (191-G2) did not protect the mice from lethal infection (100% mortality). Mice which were inoculated with the virus alone developed 71% mortality. The fact that mortality was higher with the irrelevant NANOBODY® (V.sub.HH sequence) was probably a coincidence and not due to a potentially harmful effect of the NANOBODY® (V.sub.HH sequence). In preliminary experiments, mice which received NANOBODY® (V.sub.HH sequence) alone did not develop signs of disease. Also, the clinical course of the mice which received virus+irrelevant NANOBODY® (V.sub.HH sequence) resembled the typical rabies pattern. The anti-rabies NANOBODY® (V.sub.HH sequence) 213-E6 provided a partial protection against the rabies virus with a mortality of 57%. The Kaplan Meier survival curve of 213-E6 resembles a typical “staircase” profile similar to that of the survival curve with monoclonal antibody at higher virus concentrations. Remarkably, anti-rabies NANOBODY® (V.sub.HH sequence) 212-C12 did not protect (100% mortality) in vivo, although this was one of the most potent clones in vitro with BHK cells as the susceptible targets.
(301) This experiment demonstrates that partial protection can be achieved with monovalent NANOBODY® (V.sub.HH sequence) in the intracerebral challenge model. The in vitro and in vivo potencies are poorly correlated. Although the NANOBODIES® (V.sub.HH sequences) and antibody were used at the same in vitro dose of 1 IU, their in vivo potency was clearly different (mab 8-2>213-E6>212-C12).
(302) 50.2 In Vivo Neutralization by Bivalent NANOBODIES® (V.sub.HH Sequences)
(303) Bihead NANOBODIES® (V.sub.HH sequences) were tested using the same intracerebral challenge model. The results for bivalent and biparatopic antibodies are shown in
(304) As in the previous experiment, the monoclonal antibody 8.2 provided full protection against an intracerebral challenge with 10.sup.1.5 TCID.sub.50 CVS-11, whereas high mortality (87.5%) was observed after pre-incubation with an irrelevant NANOBODY® (V.sub.HH sequence) (191-G2). The bivalent combinations 214E8-15GS-214E8 and 213H7-15GS-213H7 and all biparatopic combinations yielded complete protection against the intracerebral rabies virus challenge (0% mortality). The bivalent combination 212C12-15GS-212C12 yielded now clear partial protection (22.2% mortality). Based on the mortality data with both monovalent and bivalent 212-C12, it is likely that the epitope which is recognized by this clone is less suited for neutralisation in brain than in vitro.
(305) Results of a further experiment with bivalent and biapratopic NANOBODIES® (V.sub.HH sequences) are shown in
(306) The combination of NANOBODIES® (V.sub.HH sequences) in a bivalent or biparatopic conformation induces a synergistic increase of both the in vitro and in vivo potencies. A same in vitro dose of 1 IU is much more effective in the bivalent/biparatopic conformation than in the monovalent conformation.
(307) This experiment presents data from day 0-21. We expect that there will be no further changes in clinical signs or mortality in day 21-28.
(308) 50.3 Detection of Virus in Mouse Brains
(309) The brains of the mice inoculated with 10.sup.1.5 TCID.sub.50 CVS-11 mixed with an anti-rabies NANOBODY® (V.sub.HH sequence) (1 IU 213-E6) were stained for the presence of viral antigens. Acetone-fixed brain smears were subjected to immunofluorescence staining with an FITC-conjugated anti-nucleoprotein antibody (FAT).
(310)
(311) 50.4 Intracerebral Inoculation of Mice with Dose of 10.sup.2 TCID.sub.50
(312) Most bivalent and biparatopic NANOBODIES® (V.sub.HH sequences) provide good protection against a viral dose of 10.sup.1.5 TCID.sub.50. In this experiment, we examined whether the bivalent 213E6-15GS-213H7 also offers protection against a dose of 10.sup.2 TCID.sub.50 CVS-11. Mab RV1C5 (anti-G IgG.sub.2a, Santa Cruz sc-57995) was used as a control antibody.
(313) Results are shown in Table C-49 and
Example 51: In Vivo Protection of Mice by Intranasal Application of NANOBODY® (V.SUB.HH .Sequence)
(314) Monovalent NANOBODIES® (V.sub.HH sequences) against rabies were tested in intranasal mice model. The NANOBODIES® (V.sub.HH sequences) were injected intranasally after preincubation with two different virus doses.
(315) Outbred Swiss mice (5-6 weeks old) were inoculated intranasally with rabies virus CVS-11 pre-incubated with 1 IU of NANOBODY® (V.sub.HH sequence) or monoclonal antibody (mab 8-2). Prior to inoculation, the mix of virus and NANOBODY® (V.sub.HH sequence) or antibody was incubated at 37° C., 5% CO.sub.2 for 30 min. Mice were first anesthetized with isoflurane and fixed with the head held up. A volume of 25 μl (12.5 μl virus+12.5 μl NANOBODY® (V.sub.HH sequence)) was inoculated on top of the nostrils with a micropipette. Immediately after application, the inoculum is inhaled in the nose through the rapid and superficial breathing of the anesthetized animal. A viral dose of 10.sup.3 (IN20090310) or 10.sup.2 TCID.sub.50 (IN20090210, IN20090414) was used. Mice were examined for (rabies) disease signs each work day and a clinical score was given per day per mice. Clinical scores ranged from 0 (no disease signs) to 6 (weight loss, depression, hunched back, wasp waist, incoordination and hind limb paralysis). At score 6, mice were sacrificed by cervical dislocation. The experiment ends at 35 DPI.
(316) The results are shown in
(317) Both the monovalent 213-E6 and bivalent 214E8-15GS-213H7 provided full protection against disease in the intranasal inoculation model when introduced together with the virus at a viral dose of 10.sup.2 TCID.sub.50. At a higher dose of 10.sup.3 TCID.sub.50 protection was partial.
(318) Remarkably, the monovalent clone 212-C12 provided relative good protection in this model, whereas in the intracerebral inoculation model we observed no protection with this clone. To confirm this observation, we performed an additional experiment in which we inoculated part of the mice intranasally and part intracerebrally with CVS-11+212-C12 (
(319) The mortality and survival curve of the group inoculated with the mix of virus and irrelevant NANOBODY® (V.sub.HH sequence) 191-D3 is comparable to that of mice inoculated with virus only in previous experiments.
(320) Surprisingly, we observed no protection with the mab 8-2, despite the fact that this mab proved to be a very potent neutralizer in the in vitro models and in the intracerebral inoculation model. In this experiment, the mortality was even higher (89%) and the median survival time was shorter (9 days) than in group with the irrelevant NANOBODY® (V.sub.HHsequence) (respectively 66% and 13 days). This experiment will be repeated with another mab (RV1C5).
Example 52: In Vivo Protection of Mice by Intranasal Application of NANOBODY® (V.SUB.HH .Sequence) Followed One Day Later by Intranasal Challenge with the Virulent Neurotropic CVS-11 Strain
(321) Intranasal challenge with a virulent neurotropic rabies virus quickly leads to invasion of the brain, most probably upon entry and infection of the sensory neurons of the olfactory epithelium.
(322) To examine whether prior intranasal administration of anti-rabies NANOBODIES® (V.sub.HH sequences) can protect mice from an intranasal challenge with rabies virus one day later, outbred Swiss mice (5-6 weeks old) were treated with an intranasal dose of NANOBODY® (V.sub.HH sequence) (1 IU) or mab (1 IU). One day later, the mice received an intranasal challenge of 10.sup.2 TCID.sub.50 CVS-11 per mouse. For intranasal inoculation, a volume of 25 μl/mouse was applied in both nostrils under isoflurane anesthesia. Mice were examined for (rabies) disease signs each work day and a clinical score was given per day per mouse. Clinical scores ranged from 0 (no disease signs) to 7 (conjunctivitis, weight loss, depression, hunched back, wasp waist, incoordination and hind limb paralysis). At score 6, mice were sacrificed by cervical dislocation. The experiment ended at 35 DPI with virus. The results are shown in
(323) Similar to the intracerebral inoculation model, we observed full protection with mab 8-2 (0% mortality), no protection with NANOBODY® (V.sub.HH sequence) 212-C12 (87.5% mortality) and minor protection with NANOBODY® (V.sub.HH sequence) 213-E6 (75% mortality).
Example 53: Generation of NANOBODY® (V.SUB.HH .Sequence) Constructs
(324) For the expression of the NANOBODY® (V.sub.HH sequence) constructs the GS Gene Expression System™ by Lonza (Basel, Switzerland) is used, which comprises the serum-free and suspension-adapted CHOK1SV cell line and the expression plasmid pEE12.4. The starting point of the construction of the NANOBODY® (V.sub.HH sequence) constructs is the reverse translation of the amino acid sequence into the corresponding nucleotide sequence, optimized for expression in a CHO cell line. This optimization for expression can for instance be done by GeneArt (Regensburg, Germany) or by other companies specialized in gene synthesis. On the N-terminal end of the NANOBODY® (V.sub.HH sequence) construct a generic secretion signal is added, which allows for the endogenous protein to be exported into the growth medium and which is cleaved off upon secretion out of the cell. Such a generic signal sequence can, for instance, be the murine heavy chain leader sequence, the murine light chain leader sequence, any other antibody heavy or light chain leader sequence, the IL-2 secretion signal, etc., as are known in the art. Optionally, 5′ to the end of the secretion signal an optimized Kozak sequence is added, which initiates effective translation from the mRNA transcript. The consensus sequence recommended by Lonza consists of a 9-mer (5′-GCCGCCACC-3′; SEQ ID NO: 2638), and directly precedes the ATG start codon. The NANOBODY® (V.sub.HH sequence) construct is terminated by a double stop codon to increase translation efficiency of the construct.
(325) The NANOBODY® (V.sub.HH sequence) construct including all aforementioned features is typically cloned into the HindIII/EcoRI cloning sites; which requires absence of these sites within the NANOBODY® (V.sub.HH sequence) construct. Cloning into the HindIII/EcoRI sites on the pEE12.4 plasmid results in the removal of most of the multiple cloning site. The recombinant plasmid is transformed into an appropriate E. coli strain (e.g., TOP10), and positive clones are selected for by ampicillin or carbenicillin in the growth medium. The plasmid is amplified and isolated using a plasmid isolation kit.
(326) To transfect the cells, the recombinant plasmid DNA is linearized for instance by digestion with a restriction endonuclease (e.g., PvuI) that cuts the DNA only once; this facilitates the recombination of the plasmid DNA into the cells genome. Freshly thawed CHOK1SV cells are kept in culture (e.g., in CD CHO medium, Invitrogen) and are expanded. An aliquot of about 2×10.sup.7 cells is electroporated with 40 □g of linearized plasmid, using e.g., the BioRad electroporation device (Bio-Rad Gene Pulser. Hercules, Calif.). The transfected cells are resuspended in CD CHO medium and after 1 day put under selective pressure, e.g., in glutamine-deficient medium. To increase selective pressure the medium is supplemented with 66.6 μM methionine sulfoximine after 1 culturing day. The cells are kept under selective pressure, and allowed to expand, either as single cell clones (after limiting dilution), or as a batch culture. Expression levels of the recombinant protein are then determined by e.g. a binding ELISA.
(327) The IgG1-hinge region between the NANOBODY® (V.sub.HH sequence) and the immunoglobulin IgG1 constant domain CH2-CH3 can optionally be extended by a 9GS linker (GGGGSGGGS; SEQ ID NO: 2639) or exchanged by another hinge region, e.g., as derived from IgG3 (ELKTPLGDTTHTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCP RCP; SEQ ID NO: 2640). In a format where one NANOBODY® (V.sub.HH sequence) is preceding and another NANOBODY® (V.sub.HH sequence) following the IgG-Fc domain, the second C-terminal NANOBODY® (V.sub.HH sequence) can be fused to the Fc domain either directly (no linker), or e.g., by a 9GS linker.
(328) Non-limiting embodiments of the NANOBODY® (V.sub.HH sequence) Fc fusion construct include:
(329) (1) NC41::15GS::NC41::G1-hinge::IgG1-Fc
(330) (2) NC41::15GS::NC41::9GS-G1-hinge::IgG1-Fc
(331) (3) NC41::15GS::NC41::G3-hinge::IgG1-Fc
(332) (4) NC41::G1-hinge::IgG1-Fc::NC41
(333) (5) NC41::9GS-G1-hinge::IgG1-Fc::NC41
(334) (6) NC41::G3-hinge::IgG1-Fc::NC41
(335) (7) NC41::G1-hinge::IgG1-Fc::9GS::NC41
(336) (8) NC41::9GS-G1-hinge::IgG1-Fc::9GS::NC41
(337) (9) NC41::G3-hinge::IgG1-Fc::9GS::NC41
(338) (10) NC41::G1-hinge::IgG1-Fc::15B3
(339) (11) NC41::9GS-G1-hinge::IgG1-Fc::15B3
(340) (12) NC41::G3-hinge::IgG1-Fc::15B3
(341) (13) NC41::G1-hinge::IgG1-Fc::9GS::15B3
(342) (14) NC41::9GS-G1-hinge::IgG1-Fc::9GS::15B3
(343) (15) NC41::G3-hinge::IgG1-Fc::9GS::15B3
(344) (16) NC41::NC41::IgG1-Fc
(345) (17) NC41::IgG1-Fc::NC41
(346) (18) 191D3::15GS::191E4::G1-hinge::IgG1-Fc
(347) (19) 191D3::15GS::191E4::9GS-G1-hinge::IgG1-Fc
(348) (20) 191D3::15GS::191E4::G3-hinge::IgG1-Fc
(349) (21) 191D3::G1-hinge::IgG1-Fc::NC41
(350) (22) 191D3::9GS-G1-hinge::IgG1-Fc::191E4
(351) (23) 191D3::G3-hinge::IgG1-Fc::191E4
(352) (24) 191D3::G1-hinge::IgG1-Fc::9GS::191E4
(353) (25) 191D3::9GS-G1-hinge::IgG1-Fc::9GS::191E4
(354) (26) 191D3::G3-hinge::IgG1-Fc::9GS::191E4
(355) (27) 191D3::191E4::IgG1-Fc
(356) (28) 191D3::IgG1-Fc::191E4
(357) Non-limiting examples of NANOBODY® (V.sub.HH sequence) constructs of the invention are also provided in
Example 54: Cross-Reactivity of NANOBODY® (V.SUB.HH .Sequence) 202-C8
(358) Cross-Reactivity of Mono-, Bi- and or Trivalent NANOBODY® (V.sub.HH Sequence) 202-C8
(359) Potential heterosubtypic cross-reactivity of monovalent 202-C8 (SEQ ID NO: 138), bivalent 202-C8 (SEQ ID NO's: 2423 to 2424) and trivalent 202-C8 (SEQ ID NO's: 2425 to 2426) is assessed in an in vitro neutralization assay using PR8 (H1N1), X47 (H3N2) and NIBRG-14 (H5N1) viruses. Neutralization is tested in a hemagglutination inhibition assay using chicken red blood cells and in a virus microneutralization assay using MDCK cells as targets.
(360) In Vivo Neutralization of Mono-, Bi- and/or Trivalent NANOBODY® (V.sub.HH Sequence) 202-C8
(361) An in vivo experiment with the 202-C8 variants (mono-, bi- and/or trivalent) that display good cross-reactive potential is performed. Mice are treated with the mono-, bi- and/or trivalent 202-C8 NANOBODIES® (V.sub.HH sequences) and subsequently challenged with 1LD.sub.50 of mouse-adapted PR8, X47 or NIBRG-14 virus.
(362) Groups of 3 mice are used. At t=0 mice receive 100 microgram of 202-C8 (mono-, bi- or trivalent), 100 microgram of 191-D3 (control NANOBODY® (V.sub.HH sequence)) or 50 μl of PBS intranasally. Four hours later mice are challenged with 1 LD.sub.50 of mouse adapted NIBRG-14, PR8 or X47 virus. As an indicator of morbidity, body weight of mice is determined on a daily basis. On day 4 after challenge all mice are sacrificed and lung homogenates prepared in 1 ml PBS. The amount of infectious virus in the lung homogenates is determined by titration on MDCK cells and by a genome specific qRT-PCR. The experiment is repeated at least one time.
Example 55: Evaluation of Proteolytic Resistance of Bivalent RSV NANOBODY® (V.SUB.HH .Sequence) in Mouse Lungs
(363) The proteolytic resistance of the bivalent RSV101 (191D3-15GS-191D3; SEQ ID NO: 2382) in mouse lungs was evaluated by analysis of mouse lung homogenates and compared with control NANOBODY® (V.sub.HH sequence) 12B2biv.
(364) NANOBODY® (V.sub.HH sequence) was administered to mice 5 hours prior to infections with RSV. Lungs were removed and homogenized 3 or 5 days after infection with RSV. In short, lungs from 5 mice were homogenized and 40 μl SDS-sample buffer (6× Laemli/20% β-mercapto) was added to 200 μl homogenate. As a positive control, 100 ng of RSV101 (0.1 mg/ml) in PBS was used to obtain a 10 μg/ml solution (5 μl NB2biv+45 μl PBS+25 μl1 SB (Invitrogen NP0008; Lot 401488)+DTT (10 mg/ml)).
(365) 24 μl1 (=20 μl lung homogenate) of samples and 15 μl of positive control were loaded on a 12% gel (NuPAGE Bis-Tris Invitrogen NP0341BOX; Lot 8031371) and run for 45 min at 200V. As marker Precision Plus Dual Color Protein Standard (Biorad; 161-0374) was used. After the run, the gel was transferred to a nitrocellulose membrane (Invitrogen i-blot dry blotting system; program 2: 6 min at 23V) and blocked with Odyssey blocking buffer (Li-cor 927-40000; Lot 2782) for 1 h at RT. All incubation and wash steps were done on a rolling plate (100 rpm). The membrane was incubated with polyclonal rabbit antiserum K1 (as primary antibody diluted 1/1000 in Odyssey blocking buffer) for 1 h at RT. Washing was carried out 3×5 min with PBS/0.1% Tween20. Detection was done with goat anti-rabbit IgG (H+L)-DyLight800 (Pierce 35571; Lot IH112638; diluted 1/10000 in Odyssey blocking buffer) for 1 h at RT. Subsequent washing was carried out 3×5 min with PBS/0.1% Tween20. The membrane was scanned with the Odyssey Infrared Imager system (in the 800 channel) (Sensitivity on Odyssey: Linear manual 4; Licor Biosciences).
(366) Results of the Western blot are shown in
(367) Determination of the concentration was done with the Odyssey v3.0 software (
Example 56: Neutralization of Escape Mutants of the Long Strain by Formatted NANOBODIES® (V.SUB.HH .Sequences)
(368) In examples 27 and 28, the binding of monovalent NANOBODIES® (V.sub.HH sequences) to typical antigenic site II and/or IV-VI RSV escape mutants has been described. Binding of NANOBODIES® (V.sub.HH sequences) specifically recognizing these antigenic sites was almost lost or significantly reduced. Formatting of these NANOBODIES® (V.sub.HH sequences) into bi- or trivalent constructs partially restored binding activity but not for all three escape mutant viruses. Binding to the escape mutant R7C2/1 (mutation K272E in antigenic site II) remained below the level of 25% for any bi- or trivalent construct consisting solely of antigenic site II binding NANOBODIES® (V.sub.HH sequences). The NANOBODIES® (V.sub.HH sequences) 15B3 and 191E4, which are binding to antigenic site IV-VI, were the only NANOBODIES® (V.sub.HH sequences) (as such or in biparatopic constructs) able to bind this mutant at a level of 75% or more.
(369) More detailed analysis of the data indicated that binding towards R7C2/1 slightly increased when the valency of the NANOBODY® (V.sub.HH sequence) was increased. The binding of 7B2 constructs was 0, 4.4 and 13% respectively for the monovalent, bivalent (RSV 106) and trivalent (RSV400) formats. Such a low level of residual binding is expected to result in very high loss of potency to neutralize RSV.
(370) The neutralizing potency of NANOBODIES® (V.sub.HH sequences) was assessed on the same selected set of escape mutants as described in example 28. For this purpose the monovalent NANOBODIES® (V.sub.HH sequences) 7B2, 15H8 and NC41 were compared to their respective trivalent counterparts, RSV400, RSV 404 and RSV 407. Of note, in example 28 only RSV400 was assessed for binding these escape mutants. In addition also the biparatopic trivalent molecule RSV403 (7B2-15B3-7B2) was analyzed for its neutralizing capacity.
(371) The hRSV micro neutralization assay was essentially performed as described in example 15. In brief, Hep2 cells were seeded at a concentration of 1.5×10.sup.4 cells/well into 96-well plates in DMEM medium containing 10% fetal calf serum (FCS) supplemented with Penicillin and Streptomycin (100 U/ml and 100 μg/ml, respectively) and incubated for 24 hours at 37° C. in a 5% CO.sub.2 atmosphere. Viral stocks of different viruses were prepared into Hep2 cells and subsequently titrated to determine the optimal infectious dose for use in the micro neutralization assay. A standard quantity of the specific hRSV strain was pre-incubated with serial dilutions of purified NANOBODIES® (V.sub.HH sequences) in a total volume of 50 μl for 30 minutes at 37° C. The medium of the Hep2 cells was replaced with the premix to allow infection for 2 hours, after which 0.1 ml of assay medium was added. The assay was performed in DMEM medium supplemented with 2.5% fetal calf serum and Penicillin and Streptomycin (100U/ml and 100 μg/ml, respectively). Cells were incubated for an additional 72 hours at 37° C. in a 5% CO2 atmosphere, after which cells were washed twice with 0.05% Tween-20 in PBS and once with PBS alone, after which the cells were fixed with 80% cold acetone (Sigma-Aldrich, St. Louis, Mo.) in PBS (100 μl/well) for 20 minutes at 4° C. and left to dry completely. Next the presence of the F-protein on the cell surface was detected in an ELISA type assay. Thereto, fixed Hep2 cells were blocked with 5% Porcine Serum Albumin solution in PBS for 1 hour at room temperature, than incubated for 1 hour with anti-F-protein polyclonal rabbit serum (Corral et al. 2007, BMC Biotechnol. 7: 17) or Synagis® (2 μg/ml). For detection goat Anti-rabbit-HRP conjugated antibodies or goat Anti-Human IgG, Fcγ fragment specific-HRP (Jackson ImmunoResearch, West Grove, Pa.) was used, after which the ELISA was developed according to standard procedures.
(372) As shown in
(373) The trivalent molecules however, were potently neutralizing all 3 escape mutants (
(374) The bivalent IgG Palivizumab (Synagis®), also recognizing antigenic site II was not able to block replication of R7C2/1 or R7C2/11 significantly at concentrations of about 0.2 μM. At this concentration an IC50 was not reached while R7.936/4 and wild type Long virus were neutralized with an IC50 of a few nM (data not shown).
Example 57: Screening for NANOBODIES® (V.SUB.HH .Sequences) that Compete with C179 for Binding Hemagglutinin H5 of Influenza
(375) C179 is a mouse monoclonal antibody which neutralizes H1, H2 and H5 subtypes influenza viruses. It does not prevent attachment of viruses to sialic acid, but instead binds to a rather conserved region on the stem of HA. Monoclonal antibody C179 neutralizes virus by stabilizing the metastable HA and prevents as such the low pH-induced conformational change and fusion of viral and cellular membranes. To isolate NANOBODIES® (V.sub.HH sequences) with a similar binding and neutralizing characteristic, competition assays were set up between NANOBODIES® (V.sub.HH sequences) that bind H5 hemagglutinin and the monoclonal, neutralizing antibodies C179 (Okuno et al. 1993, J. Virol. 67: 2552-2558). In short, the H5 antigen was immobilized on Maxisorp microtiter plates (Nunc) and free binding sites were blocked using 4% Marvel in PBS. Next, 125 ng/ml of C179 was preincubated with 10 and 20 μl of periplasmic extract containing NANOBODY® (V.sub.HH sequence) of the different clones. The competing antibody was allowed to bind to the immobilized antigen with or without NANOBODY® (V.sub.HH sequence). After incubation and a wash step, antibody binding was revealed using a HRP-conjugated donkey anti-mouse antibody. Binding specificity was determined based on OD values compared to controls having received no NANOBODY® (V.sub.HH sequence).
(376) This way, 4 NANOBODIES® (V.sub.HH sequences) were identified which competes with C179 (LG203G8; SEQ ID NO: 2683, LG203E7; SEQ ID NO: 2682, LG203H10; SEQ ID NO: 2446 and LG203G3; SEQ ID NO: 2442) (
Example 58: Optimization of Linker Length of NC41 Trivalents
(377) To determine the impact of the linker length of trivalents of NC41, different constructs with linkers ranging from 3Ala, 9GS, 15GS, to 20GS linkers (RSV408, RSV409, RSV407 and RSV410 resp.) were generated. All four NC41 trivalents were able to completely neutralize both RSV B-1 and Long strains (
Example 59: Humanization of NANOBODY® (V.SUB.HH .Sequence) NC41
(378) The sequence of NANOBODY® (V.sub.HH sequence) NC41 was aligned to the human germline V.sub.H3-23. to allow selection of residues suitable for further humanization of the NANOBODY® (V.sub.HH sequence) sequence. In addition, in silico analysis was done to identify residues that are potentially prone to post-translational modifications, such as Asp isomerisation, and to identify mutations that might improve the chemical stability. The CDR regions and the so-called Hallmark residues, which are known to be essential for the stability and potency of NANOBODIES® (V.sub.HH sequences) were excluded for modification.
(379) For NC41 in total 11 positions were selected for mutation to the corresponding human residue: Four mutations were simultaneous introduced (Val5Leu, Ala14Pro, Glu44Gly, Gln108Leu), as these residues were not expected to dramatically affect the NANOBODY® (V.sub.HH sequence) function (based on data from other NANOBODIES® (V.sub.HH sequences)). In this basic variant, seven residues of which it was unknown whether mutation to the human counterpart was allowed (Ser19Arg, Ile201leu, Ala74Ser, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln) were mutated using a library approach, allowing either the wildtype or the corresponding human amino acid at each position. The resulting library, with a theoretical diversity of 128, was generated by gene assembly using overlapping oligonucleotide sequences containing degenerated codon use, and subsequently cloned into an expression vector derived from pUC 119 which contained the LacZ promoter, a resistance gene for kanamycin, a multicloning site and the OmpA leader sequence. In frame with the NANOBODY® (V.sub.HH sequence) coding sequence, the vector coded for a C-terminal c-myc tag and a (His)6 tag. NANOBODIES® (V.sub.HH sequences) were produced in the periplasm of E. Coli (see Example 22). Library diversity was confirmed by sequence analysis.
(380) Periplasmic extracts from 368 individual NC41 variants and wildtype NC41 were generated and subjected to a functional screening cascade to identify the best humanized NC41 variant, in terms of both potency and stability.
(381) In a first step, RSV binding of humanized NC41 variants to RSV Long was determined in ELISA (Hytest, Turku Finland; #8RSV79)(see Example 22).
(382) Moreover, the positive binders were analyzed for binding to Hep2 cells infected with RSV B-1 strain. In here, Hep2 cells were seeded into 96-wells plates and infected with RSV B-1 strain, essentially following the procedure described for the neutralization assay (see Examples 15 and 21). Three days later cells were fixed with ice-cold acetone and plates were used in an ELISA assay using periplasmic extracts at different dilutions. NANOBODY® (V.sub.HH sequence) binding to Hep2-B1 infected cells was detected using anti-V.sub.HH rabbit polyclonal antibody, followed by goat Anti-rabbit-HRP conjugated antibodies, after which the ELISA was developed according to standard procedures.
(383) Additionally, in order to verify if the introduced mutations affected the temperature stability, periplasmatic extracts of all binders were heated to 74° C. for 2 hours, which is 5° C. above the melting temperature of wildtype NC41. The binding to RSV long before and after heating was analyzed in ELISA, and the ratio of binding signal after vs before heating was taken as measure for temperature stability.
(384) Finally, the kinetic off-rates of the variants were determined in Biacore assay on the F.sub.tm-NN protein, as described in Examples 12 and 22.
(385) All binders were sequenced and ranked according to their capacity to bind the F-protein of RSV. When analyzing the sequences of the strongest binders, a clear preference for Gln105 (human residue) was observed in all cases. Whereas the Ile20Leu mutation appeared underrepresented, for all other positions there was no clear preference for either the wild type or the human sequence, with variants containing up to 10 mutations compared to wildtype NC41. Notably, in one variant an additional point mutation (Gly54Asp) within the CDR2 region was observed. This variant, NC41 variant 6, showed the lowest off-rate of all variants and wildtype NC41, resulting in affinity increase.
(386) Based on the sequence and functional data, 18 variants (Table A-8) were selected for further characterization as purified proteins (
(387) For a select panel of NC41 variants the kinetic binding parameters were determined in Biacore on F.sub.tm-NN protein (Table C-52) as described in Example 12 and 22. No significant differences in the calculated data were observed for NC41 and the humanized NC41 variants 6, 8 and 17. It should be noted that the on-rates of all NC41 variants were at the detection limit of the instrument, but the off-rates could be ranked as v06<v17<NC41<v08. The impact of the Gly to Asp mutation in CDR2 (position 54) could be clearly demonstrated when comparing v17 and v18 as this is the only difference in these maximally humanized variants. Neutralization was tested for both the Long strain and the B-1 strain in two independent assays in comparison to the NC41 wild type as shown in table B-5. In both assays NC41v18 was more potent than NC41 on both viruses and in both assays NC41v18 was more potent than NC41v17 on the Long strain. The improved neutralization of NC41v18 was also observed for the B-1 strain in the second assay.
(388) All NC41 variants were subjected to heat-induced unfolding to assess the effect of the introduced mutations on the stability of the protein. Thereto the melting temperature (Tm) was determined by stepwise increase in temperature in presence of Sypro Orange, a dye that binds to Trp residues that become exposed upon unfolding of the protein. All variants showed to have increased Tm relative to wildtype NC41 (69° C.), up to 9° C. for variant 18.
(389) Three NC41 variants were formatted as trivalent constructs using 15GS linkers, NC41 variant 3 (RSV414), variant 6 (RSV426), and variant 18 (RSV427). Sequences are shown in Table A-9. All trivalents were produced and purified as described in Example 22.
Example 60: Immunisation Llamas with Foot-and-Mouth Disease Virus and Avian Influenza Virus
(390) Two llamas were immunized with mixtures of Foot-and-mouth disease virus (FMDV) and avian influenza virus (AIV) strains (Table C-53) within the high containment unit of the Central Veterinary Institute of Wageningen University and Research Centre in Lelystad, the Netherlands. The AIV strains were all low pathogenic avian influenza strains that were propagated on embryonated eggs and were not inactivated. The FMDV strains were propagated on BHK-21 cells, inactivated by treatment with 10 mM binary ethyleneimine, and concentrated by two consecutive PEG6000 precipitations. Both AIV (for protocol see Arora et. al. 1985, Analytical Biochemistry 144: 189-192) and FMDV antigens were finally purified using sucrose density gradients.
(391) A total of three immunizations were given. The second immunization was given 28 days after the first immunization. The third immunization was given 21 days after the second immunization. All immunizations were given intramuscularly using Specol (Stimune) as an adjuvant (Bokhout et al. 1981, Vet. Immunol. Immunopath. 2: 491-500). Six days after the second and third immunization (34 and 55 days post primary immunization [DPI], respectively) 150 ml heparinized blood samples were taken for isolation of peripheral blood lymphocytes (PBLs) using Ficoll Paque Plus (GE Healthcare). Furthermore, serum was collected from both llamas at 0, 34 and 55 DPI.
(392) The antibody response against H5 and H7 type haemagglutinin was determined using a haemagglutination inhibition test (HI) that was performed according to EU council directive 2005/94/EU. In this assay 25 μl HA antigen containing 8 haemagglutinating units was preincubated with 25 μl of a two-fold dilution series of sera for 1 hour at room temperature in a V-bottom shaped 96-well microtiter plate. After addition of 25 μl 1% chicken erythrocyte suspension and incubation at 4° C. for 45 min the HI titer was determined visually. Llama 3049, that was immunized with both H5 and H7 strains, developed HI titers against both H5 and H7 type antigen during the immunization procedure (Table C-54). Llama 3050, that was immunized with H5 but not with H7 type strains only developed a HI response against H5 antigen (Table C-54).
Example 61: Construction of Phage Display Libraries
(393) Total RNA was isolated from about 10.sup.8 PBLs obtained in example 60 using the RNeasy maxi kit (Qiagen). cDNA synthesis was performed using primer NotI-d(T)18 (Table C-55) and Superscript III reverse transcriptase (Invitrogen). The NANOBODY® (V.sub.HH sequence) encoding fragments were amplified by PCR using primer VH2B in combination with either primer lam07, lam08 or BOLI-192 (Table C-55) and Amplitaq Gold DNA polymerase (Applied Biosystems). The PCR fragments were cut with PstI and NotI and ligated to similarly cut phage display vector pRL144 (Harmsen et al. 2005, Vaccine 23: 4926-4934).
(394) By electroporation of Escherichia coli TG1 cells twelve libraries were obtained (Table C-56).
Example 62: Phage Display Selections
(395) Phage libraries obtained in Example 61 were rescued by infection with VCS-M13 helperphage and phage particles were purification by two PEG precipitations (McCafferty and Johnson 1996, Construction and screening of antibody display libraries. In: Kay, B K, Winter, J, and McCafferty, J [eds], Phage display of peptides and proteins. Academic Press, San Diego, pp. 79-111). For phage display selections libraries pAL442, 443, 444, 448, 449 and 450 were pooled. Phage pannings were performed in 96-well polystyrene microtiter plates (Greiner) by direct coating of AI antigen. AI antigen had been obtained from propagation of AI strains on Madin Darby canine kidney (MDCK) cells grown in suspension on serum free medium (SFM4BHK21 medium, a prototype medium developed for BHK21 cells obtained from Hyclone) and that was 20-fold concentrated using a 100-kDa molecular weight cutoff centrifugation-concentration device. Alternatively, phage pannings were performed using recombinant his-tagged HAO trimer from H5N1 strain A/Anhui/1/2005 (Abcam, Cambridge, UK; Cat. No. ab53938) or recombinant his-tagged HA1 from H7N7 strain A/Chicken/Netherlands/01/03 (Abcam, Cambridge, UK; Cat. No. ab61286), both produced by HEK293 cells. For this purpose these recombinant antigens were captured in polystyrene microtiter plates coated with 2 μg/ml affinity purified polyclonal rabbit anti-his6 peptide antibody (Rockland, Cat. No. 600-401-382). Alternatively, phage display selections were performed using Drosophila S2 cell produced strep-tagged recombinant haemagglutinin derived from an H7N2 influenza strain (HAstr H7N2). Antigen concentrations used during panning were either 0.1 or 0.01 μg/ml. Phage libraries were added at 10.sup.10 TU per well. Bound phage were eluted by incubation in 1 mg/ml trypsin in PBS buffer for 30 min.
Example 63: Binding to Influenza Antigens in ELISA
(396) Individual clones binding to influenza antigens in ELISA were screened using soluble NANOBODIES® (V.sub.HH sequences) prepared according to a previously described protocol (McCafferty, J, and Johnson, K S, 1996, Construction and screening of antibody display libraries. In: Kay, B K, Winter, J, and McCafferty, J [eds], Phage display of peptides and proteins. Academic Press, San Diego, pp. 79-111). The influenza antigens were obtained from virus propagated on MDCK cells using serum free medium and further purified by sucrose density gradients. The authentic AIV antigens used in ELISA originated from the strains indicated in Table C-57.
(397) Briefly, 96-well ELISA plates were coated with 1 μg/ml AIV antigen in 50 mM carbonate/bicarbonate buffer pH 9.6. These plates were then incubated with tenfold diluted E. coli culture supernatants in ELISA-buffer (1% skimmed milk; 0.05% Tween-20; 0.5 M NaCl; 2.7 mM KCl; 2.8 mM KH.sub.2PO.sub.4; 8.1 mM Na.sub.2HPO.sub.4; pH 7.4). Bound NANOBODIES® (V.sub.HH sequences) were subsequently detected using a peroxidase-conjugated monoclonal antibody against the c-myc tag (Roche Applied Science, Mannheim, Germany) and stained with 3,3′,5,5′-tetramethylbenzidine.
(398) After screening individual clones for binding to authentic AIV antigens 39 clones binding to AIV antigens from H5 strains and 50 clones binding to AIV antigen from H7 strains were sequenced. Sequence analysis was performed using the ABI3130 capillary sequencer (Applied Biosystems) and primer MPE26 (Table C-55). The 39 H5 binding clones encoded 25 different NANOBODIES® (V.sub.HH sequences) that form six CDR3 groups (Table A-1 and Table C-58). The 50 H7 binding clones encoded 40 different NANOBODIES® (V.sub.HH sequences) that form seven CDR3 groups (Table A-1 and Table C-59). With the exception of clone IV28, all H5 and H7 binding clones encoded NANOBODIES® (V.sub.HH sequences) containing the hallmark amino acid residues typical of single-domain antibodies (Harmsen et al. 2000, Mol. Immunol. 37: 579-590).
(399) Most H7 binding NANOBODIES® (V.sub.HH sequences) of CDR3 group A contain a potential N-glycosylation site at position 84. Most H5 binding clones bind specifically to AIV antigens of three different H5 strains. However, clones of CDR3 group B also bind to antigen of an H1 strain (Table C-58). Furthermore, two clones (IV154 and IV155) that fall into two CDR3 groups bind to AIV antigen of H1, H7 and H5 strains (Table C-58). These clones probably bind to nucleoprotein, which is highly immunogenic and highly conserved between influenza strains of different serotypes. Consistent with this conclusion, these clones were selected in both panning rounds on authentic AIV antigens whereas most other clones were selected using recombinant haemagglutinin in the second round of phage display selection. Almost all 40 H7 binding NANOBODIES® (V.sub.HH sequences) bind to AIV antigen of two H7 strains, but not to AIV antigen of H1 or H5 strains (Table C-59). Only clone IV18 appeared to bind to H5 antigen. However, the two clones that encoded NANOBODIES® (V.sub.HH sequences) that are identical to IV18 did not show such cross reaction to H5 strains, suggesting that this cross reaction is an artifact.
Example 64: Yeast Expression of Selected NANOBODIES® (V.SUB.HH .Sequences)
(400) We selected eight H5 binding NANOBODIES® (V.sub.HH sequences) and eight H7 binding NANOBODIES® (V.sub.HH sequences) for small scale yeast (Saccharomyces cerevisiae) expression using plasmid pRL188 (Harmsen et al. 2007, Vet. Microbiol. 120: 193-206). This plasmid results in NANOBODY® (V.sub.HH sequence) production with a C-terminal extension with amino acid sequence (SEQ ID NO: 3063; EPKTPKPQPQPQPQPQPNPTTESKCPHHHHHH). We preferably selected clones representing all CDR3 groups for such yeast expression. Insertion of the NANOBODY® (V.sub.HH sequence) coding sequence into pRL188 required the presence of a BstEII restriction endonuclease cleavage site in the FR4 coding region. This site was present in most NANOBODY® (V.sub.HH sequence) clones, but not in all (Tables C-58 and C-59). As a result we could not yeast-produce IV151 and IV153, which are unique representatives of two CDR3 groups, in a facile manner. A person skilled in the art could produce such clones suitable for yeast expression by introduction of this BstEII site by site-directed mutagenesis. Furthermore, the subcloning of IV28 into pRL188 was not successful. NANOBODIES® (V.sub.HH sequences) were expressed in S. cerevisiae under control of the GAL7 promoter and directed into the growth medium by fusion to the invertase signal peptide as described previously (Harmsen et al. 2007, Vet. Microbiol. 120: 193-206 and references therein). The NANOBODIES® (V.sub.HH sequences) were purified from culture supernatant using immobilized-metal affinity chromatography. Purified NANOBODIES® (V.sub.HH sequences) were concentrated and the buffer exchanged to phosphate-buffered saline by use of 5-kDa molecular weight cut-off centrifugal concentration devices (Biomax-5 membrane, Millipore, Bedford, Mass.). The protein concentration was determined using the Bio-Rad (Hercules, Calif.) protein assay.
Example 65: Characteristics of Yeast-Produced NANOBODIES® (V.SUB.HH .Sequences)
(401) 65.1 Binding in ELISA
(402) We next analysed the binding of the selected NANOBODIES® (V.sub.HH sequences) to influenza antigens of strains of different serotypes. This ELISA was essentially performed as described in the previous section (Example 63) for screening of E. coli produced NANOBODIES® (V.sub.HH sequences) but using a higher concentration of influenza antigen (see Table C-57) for coating (5 μg/ml) and using a peroxidase-conjugated anti-his6 monoclonal antibody (Roche Applied Science) for NANOBODY® (V.sub.HH sequence) detection. The NANOBODIES® (V.sub.HH sequences) that were selected for binding to H5 strains all react with all three H5 strains used (
(403) We next analysed the binding of NANOBODIES® (V.sub.HH sequences) to selected authentic and recombinant antigens in ELISA by incubation of twofold dilution series of NANOBODIES® (V.sub.HH sequences) with a starting concentration of 10 μg/ml. NANOBODIES® (V.sub.HH sequences) bound to recombinant antigens were detected using a polyclonal rabbit anti-NANOBODY® (V.sub.HH sequence) serum (R907) and peroxidase-conjugated swine anti-rabbit serum (Dako, P217) since the recombinant antigen also contains a his6 tag. After nonlinear regression analysis the NANOBODY® (V.sub.HH sequence) concentration required to obtain an extinction at 450 nm of 0.2 (authentic antigens) or 1.0 (recombinant antigens) was interpolated. All NANOBODIES® (V.sub.HH sequences) selected for binding to H5 strains could bind to H5N9 antigen with titers differing at most 5-fold (Table C-60). Six clones also could bind to two recombinant H5 antigens (Table C-60), demonstrating that they recognized haemagglutinin. Two further clones (IV 154 and IV 155) did not bind to both recombinant haemagglutinins at all. This further suggests that these clones bind to nucleoprotein, as suggested above based on their binding to authentic influenza antigens of many different H and N types. The NANOBODIES® (V.sub.HH sequences) selected for binding to H7 strains all could bind to authentic antigen of two H7 type influenza strains and recombinant HA1 fragment (Table C-61), showing that they bind to haemagglutinin.
(404) 65.2 Virus Neutralization
(405) We next determined the in vitro virus neutralizing capacity of the selected NANOBODIES® (V.sub.HH sequences). For this purpose 100 tissue culture infective doses required to infect 50% of the wells (TCID.sub.50) were preincubated with twofold dilution series of yeast-produced NANOBODIES® (V.sub.HH sequences) for 1 hour at room temperature. These were subsequently added to MDCK cell monolayers in a serum free medium containing 3 μg/ml trypsin to enable virus replication. After two days of growth at 37° C. and 5% CO2 influenza virus antigen in the wells was detected using an immunoperoxidase monolayer assay employing a nucleoprotein specific monoclonal antibody (HB65, also known as H16-L10-4; Yewdell et al. 1981, J. Immunol. 126: 1814-1819). Neutralization titers were calculated according to Reed and Muench (1938, Am. J. Hyg. 27: 493). Only clone IV146 could neutralize both H5 type viruses at the lowest concentration analysed (0.75 μg/ml), whereas all other NANOBODIES® (V.sub.HH sequences) did not neutralize the two virus strains used at the highest concentration analysed of 50 μg/ml (Tables C-60 and C-61).
(406) 65.3 Inhibition of Hemagglutination
(407) We similarly determined the ability of the yeast-produced NANOBODIES® (V.sub.HH sequences) to inhibit haemagglutination using the protocol described (Example 60) above for analysis of llama sera. We could not detect any inhibition of haemagglutination at the highest NANOBODY® (V.sub.HH sequence) concentration analysed (Tables C-60 and C-61).
(408) Thus, clone IV146 neutralizes influenza virus without inhibiting haemagglutination. This is an unexpected finding since most previously isolated conventional monoclonal antibodies that neutralize influenza virus also inhibit haemagglutination. Clone IV146 also cross reacts in ELISA with H1 and H2 strains. This is again unexpected, since most conventional monoclonal antibodies binding haemagglutinin bind specifically to one haemagglutinin type. However, recently, H5 type haemagglutinin binding human monoclonal antibodies that cross react to H1 and H2 type strains, and neutralize virus without inhibiting haemagglutination were found by several groups (Throsby et al. 2008, Plos ONE 3; Sui et al. Nature Struct. Biol. 16: 265-273; Kashyap et al. 2008, Proc. Nat. Acad. Sci. 22: 5986-5991). These human mAbs bind to a relatively conserved epitope mainly present on the HA2 domain that is involved in initiating the fusion of the viral and host cell membranes, which is essential for infection. This epitope can be present in two conformations: a prefusion state which does not enable membrane fusion and another conformation that is competent for membrane fusion. The prefusion state is recognized by such broadly cross reactive neutralizing antibodies (Sui et al. Nature Struct. Biol. 16: 265-273; Ekiert et al., 2009, Science 324: 246-251), suggesting that the mechanism of virus neutralization by such antibodies relies on inhibition of a conformational change of HA into a conformation competent for fusion. The similarity in virus neutralization and strain specificity of IV146 with these human monoclonal antibodies suggests that IV146 also recognizes this conserved epitope on the HA2 domain that is involved in initiating membrane fusion.
(409) Tables
(410) TABLE-US-00025 TABLE A-2 Amino acid sequence of multivalent constructs that bind hRSV (including Myc-His tag SEQ ID Construct NO Sequence RSV101 2382 EVQLVESGGGLVQAGGSLRLSCEASGRTYSRYGMGWFRQAPGKEREFVAAVSRLSGPRT VYADSVKGRFTISRDNAENTVYLQMNSLKPEDTAVYTCAAELTNRNSGAYYYAWAYDYW GQGTQVTVSSGGGGSGGGGSGGGGSEVQLVESGGGLVQAGGSLRLSCEASGRTYSRYGM GWFRQAPGKEREFVAAVSRLSGPRTVYADSVKGRFTISRDNAENTVYLQMNSLKPEDTA VYTCAAELTNRNSGAYYYAWAYDYWGQGTQVTVSSAAAEQKLISEEDLNGAAHHHHHH RSV102 2383 VQLVESGGGLVQAGGSLRLSCEASGRTYSRYGMGWFRQAPGKEREFVAAVSRLSGPRTV YADSVKGRFTISRDNAENTVYLQMNSLKPEDTAVYTCAAELTNRNSGAYYYAWAYDYWG QGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQAGGSLRLSCEAS GRTYSRYGMGWFRQAPGKEREFVAAVSRLSGPRTVYADSVKGRFTISRDNAENTVYLQM NSLKPEDTAVYTCAAELTNRNSGAYYYAWAYDYWGQGTQVTVSSAAAEQKLISEEDLNG AAHHHHHH RSV103 2384 EVQLVESGGGLVQAGGSLRLSCEASGRTYSRYGMGWFRQAPGKEREFVAAVSRLSGPRT VYADSVKGRFTISRDNAENTVYLQMNSLKPEDTAVYTCAAELTNRNSGAYYYAWAYDYW GQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQA GGSLRLSCEASGRTYSRYGMGWFRQAPGKEREFVAAVSRLSGPRTVYADSVKGRFTISR DNAENTVYLQMNSLKPEDTAVYTCAAELTNRNSGAYYYAWAYDYWGQGTQVTVSSAAAE QKLISEEDLNGAAHHHHHH RSV104 2385 EVQLVESGGGLVQAGGSLRLSCEASGRTYSRYGMGWFRQAPGKEREFVAAVSRLSGPRT VYADSVKGRFTISRDNAENTVYLQMNSLKPEDTAVYTCAAELTNRNSGAYYYAWAYDYW GQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQAGGSLRLSCEASGRTYSRYGMGWFRQA PGKEREFVAAVSRLSGPRTVYADSVKGRFTISRDNAENTVYLQMNSLKPEDTAVYTCAA ELTNRNSGAYYYAWAYDYWGQGTQVTVSSAAAEQKLISEEDLNGAAHHHHHH RSV105 2386 EVQLVESGGGLVQAGDSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVAAISWSDGSTY YADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAADLTSTNPGSYIYIWAYDYWG QGTQVTVSSGGGGSGGGSEVQLVESGGGLVQAGDSLRLSCAASGRTFSSYAMGWFRQAP GKEREFVAAISWSDGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAADL TSTNPGSYIYIWAYDYWGQGTQVTVSSAAAEQKLISEEDLNGAAHHHHHH RSV106 2387 EVQLVESGGGLVQAGDSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVAAISWSDGSTY YADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAADLTSTNPGSYIYIWAYDYWG QGTQVTVSSGGGGSGGGGSGGGGSEVQLVESGGGLVQAGDSLRLSCAASGRTFSSYANG WFRQAPGKEREFVAAISWSDGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVY YCAADLTSTNPGSYIYIWAYDYWGQGTQVTVSSAAAEQKLISEEDLNGAAHHHHHH RSV107 2388 EVQLVESGGGLVQAGGSLRLSCAASGRSFSNYVLGWFRQAPGKEREFVAAISFRGDSAI GAPSVEGRFTISRDNAKNTGYLQMNSLVPDDTAVYYCGAGTPLNPGAYIYDWSYDYWGR GTQVTVSSGGGGSGGGSEVQLVESGGGLVQAGGSLRLSCAASGRSFSNYVLGWFRQAPG KEREFVAAISFRGDSAIGAPSVEGRFTISRDNAKNTGYLQMNSLVPDDTAVYYCGAGTP LNPGAYIYDWSYDYWGRGTQVTVSSAAAEQKLISEEDLNGAAHHHHHH RSV108 2389 EVQLVESGGGLVQAGGSLRLSCAASGRSFSNYVLGWFRQAPGKEREFVAAISFRGDSAI GAPSVEGRFTISRDNAKNTGYLQMNSLVPDDTAVYYCGAGTPLNPGAYIYDWSYDYWGR GTQVTVSSGGGGSGGGGSGGGGSEVQLVESGGGLVQAGGSLRLSCAASGRSFSNYVLGW FRQAPGKEREFVAAISFRGDSAIGAPSVEGRFTISRDNAKNTGYLQMNSLVPDDTAVYY CGAGTPLNPGAYIYDWSYDYWGRGTQVTVSSAAAEQKLISEEDLNGAAHHHHHH RSV109 2390 EVQLVESGGGLVQPGGSLRLSCAASGRTFSSIAMGWFRQAPGKEREFVAAISWSRGRTF YADSVKGRFIISRDDAANTAYLQMNSLKPEDTAVYYCAVDTASWNSGSFIYDWAYDHWG QGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGRTFSSIAMGWFRQAP GKEREFVAAISWSRGRTFYADSVKGRFIISRDDAANTAYLQMNSLKPEDTAVYYCAVDT ASWNSGSFIYDWAYDHWGQGTQVTVSSAAAEQKLISEEDLNGAAHHHHH RSV110 2391 EVQLVESGGGLVQPGGSLRLSCAASGRTFSSIAMGWFRQAPGKEREFVAAISWSRGRTF YADSVKGRFIISRDDAANTAYLQMNSLKPEDTAVYYCAVDTASWNSGSFIYDWAYDHWG QGTQVTVSSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGRTFSSIAMG WFRQAPGKEREFVAAISWSRGRTFYADSVKGRFIISRDDAANTAYLQMNSLKPEDTAVY YCAVDTASWNSGSFIYDWAYDHWGQGTQVTVSSAAAEQKLISEEDLNGAAHHHHHH RSV113 2392 EVQLVESGGGLVQPGGSLRLSCAASGLTLDYYALGWFRQAPGKEREGVSCISSSDHST TYTDSVKGRFTISWDNAKNTLYLQMNSLKPGDTAVYYCAADPALGCYSGSYYPRYDYW GQGTQVTVSSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGLTLDYYA LGWFRQAPGKEREGVSCISSSDHSTTYTDSVKGRFTISWDNAKNTLYLQMNSLKPGDT AVYYCAADPALGCYSGSYYPRYDYWGQGTQVTVSSAAAEQKLISEEDLNGAAHHHHHH RSV114 2393 EVQLVESGGGWVQAGGSLRLSCAASGRAFSSYAMGWIRQAPGKEREFVAGIDQSGEST AYGASASGRFIISRDNAKNTVHLLMNSLQSDDTAVYYCVADGVLATTLNWDYWGQGTQ VTVSSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGWVQAGGSLRLSCAASGRAFSSYA MGWIRQAPGKEREFVAGIDQSGESTAYGASASGRFIISRDNAKNTVHLLMNSLQSDDT AVYYCVADGVLATTLNWDYWGQGTQVTVSSAAAEQKLISEEDLNGAAHHHHHH RSV115 2394 EVQLVESGGGLVQAGGSLRLSCAASGPTFSADTMGWFRQAPGKEREFVATIPWSGGIA YYSDSVKGRFTMSRDNAKNTVDLQMNSLKPEDTALYYCAGSSRIYIYSDSLSERSYDY WGQGTQVTVSSGGGGSGGGGSGGGGGGGSEVQLVESGGGLVQAGGSLRLSCAASGPTF SADTMGWFRQAPGKEREFVATIPWSGGIAYYSDSVKGRFTMSRDNAKNTVDLQMNSLK PEDTALYYCAGSSRIYIYSDSLSERSYDYWGQGTQVTVSSAAAEQKLISEEDLNGAAH HHHHH RSV116 2395 EVQLVESGGGLVQAGGSLSISCAASGGSLSNYVLGWFRQAPGKEREFVAAINWRGDIT IGPPNVEGRFTISRDNAKNTGYLQMNSLAPDDTAVYYCGAGTPLNPGAYIYDWSYDYW GRGTQVTVSSGGGGSGGGGSGGGGSEVQLVESGGGLVQAGGSLSISCAASGGSLSNYV LGWFRQAPGKEREFVAAINWRGDITIGPPNVEGRFTISRDNAKNTGYLQMNSLAPDDT AVYYCGAGTPLNPGAYIYDWSYDYWGRGTQVTVSSAAAEQKLISEEDLNGAAHHHHHH RSV201 2396 EVQLVESGGGLVQAGGSLRLSCEASGRTYSRYGMGWFRQAPGKEREFVAAVSRLSGPR TVYADSVKGRFTISRDNAENTVYLQMNSLKPEDTAVYTCAAELTNRNSGAYYYAWAYD YWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQAGGSLRLSCAASGPTFSADTMGWF RQAPGKEREFVATIPWSGGIAYYSDSVKGRFTMSRDNAKNTVDLQMNSLKPEDTALYY CAGSSRIYIYSDSLSERSYDYWGQGTQVTVSSAAAEQKLISEEDLNGAAHHHHHH RSV202 2397 EVQLVESGGGLVQAGGSLRLSCEASGRTYSRYGMGWFRQAPGKEREFVAAVSRLSGPR TVYADSVKGRFTISRDNAENTVYLQMNSLKPEDTAVYTCAAELTNRNSGAYYYAWAYD YWGQGTQVTVSSGGGGSGGGGSGGGGSEVQLVESGGGLVQAGGSLRLSCAASGPTFSA DTMGWFRQAPGKEREFVATIPWSGGIAYYSDSVKGRFTMSRDNAKNTVDLQMNSLKPE DTALYYCAGSSRIYIYSDSLSERSYDYWGQGTQVTVSSAAAEQKLISEEDLNGAAHHH HHH RSV203 2398 EVQLVESGGGLVQAGGSLRLSCEASGRTYSRYGMGWFRQAPGKEREFVAAVSRLSGPR TVYADSVKGRFTISRDNAENTVYLQMNSLKPEDTAVYTCAAELTNRNSGAYYYAWAYD YWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQAGGSLRLS CAASGPTFSADTMGWFRQAPGKEREFVATIPWSGGIAYYSDSVKGRFTMSRDNAKNTV DLQMNSLKPEDTALYYCAGSSRIYIYSDSLSERSYDYWGQGTQVTVSSAAAEQKLISE EDLNGAAHHHHHH RSV204 2399 EVQLVESGGGLVQAGDSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVAAISWSDGST YYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAADLTSTNPGSYIYIWAYDY WGQGTQVTVSSGGGGSGGGGSGGGGSEVQLVESGGGLVQAGGSLRLSCAASGRSFSNY VLGWFRQAPGKEREFVAAISFRGDSAIGAPSVEGRFTISRDNAKNTGYLQMNSLVPDD TAVYYCGAGTPLNPGAYIYDWSYDYWGRGTQVTVSSAAAEQKLISEEDLNGAAHHHHHH RSV205 2400 EVQLVESGGGLVQAGDSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVAAISWSDGST YYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAADLTSTNPGSYIYIWAYDY WGQGTQVTVSSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGLTLDYY ALGWFRQAPGKEREGVSCISSSDHSTTYTDSVKGRFTISWDNAKNTLYLQMNSLKPGD TAVYYCAADPALGCYSGSYYPRYDYWGQGTQVTVSSAAAEQKLISEEDLNGAAHHHHHH RSV206 2401 EVQLVESGGGLVQAGGSLRLSCAASGRSFSNYVLGWFRQAPGKEREFVAAISFRGDSA IGAPSVEGRFTISRDNAKNTGYLQMNSLVPDDTAVYYCGAGTPLNPGAYIYDWSYDYW GRGTQVTVSSGGGGSGGGGSGGGGSEVQLVESGGGLVQAGDSLRLSCAASGRTFSSYA MGWFRQAPGKEREFVAAISWSDGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDT AVYYCAADLTSTNPGSYIYIWAYDYWGQGTQVTVSSAAAEQKLISEEDLNGAAHHHHHH RSV207 2402 EVQLVESGGGLVQAGGSLRLSCAASGRSFSNYVLGWFRQAPGKEREFVAAISFRGDSA IGAPSVEGRFTISRDNAKNTGYLQMNSLVPDDTAVYYCGAGTPLNPGAYIYDWSYDYW GRGTQVTVSSGGGGSGGGGSGGGGSEVQLVESGGGLVQAGDSLRLSCAASGRTFSSYA MGWFRQAPGKEREFVAAISWSDGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDT AVYYCAADLTSTNPGSYIYIWAYDYWGQGTQVTVSSAAAEQKLISEEDLNGAAHHHHHH RSV301 2403 EVQLVESGGGLVQAGGSLRLSCAASGPTFSADTMGWFRQAPGKEREFVATIPWSGGIA YYSDSVKGRFTMSRDNAKNTVDLQMNSLKPEDTALYYCAGSSRIYIYSDSLSERSYDY WGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQAGGSLRLSCEASGRTYSRYGMGWFR QAPGKEREFVAAVSRLSGPRTVYADSVKGRFTISRDNAENTVYLQMNSLKPEDTAVYT CAAELTNRNSGAYYYAWAYDYWGQGTQVTVSSAAAEQKLISEEDLNGAAHHHHHH RSV302 2404 EVQLVESGGGLVQAGGSLRLSCAASGPTFSADTMGWFRQAPGKEREFVATIPWSGGIA YYSDSVKGRFTMSRDNAKNTVDLQMNSLKPEDTALYYCAGSSRIYIYSDSLSERSYDY WGQGTQVTVSSGGGGSGGGGSGGGGSEVQLVESGGGLVQAGGSLRLSCEASGRTYSRY GMGWFRQAPGKEREFVAAVSRLSGPRTVYADSVKGRFTISRDNAENTVYLQMNSLKPE DTAVYTCAAELTNRNSGAYYYAWAYDYWGQGTQVTVSSAAAEQKLISEEDLNGAAHHH HHH RSV303 2405 EVQLVESGGGLVQAGGSLRLSCAASGPTFSADTMGWFRQAPGKEREFVATIPWSGGIA YYSDSVKGRFTMSRDNAKNTVDLQMNSLKPEDTALYYCAGSSRIYIYSDSLSERSYDY WGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQAGGSLRLSC EASGRTYSRYGMGWFRQAPGKEREFVAAVSRLSGPRTVYADSVKGRFTISRDNAENTV YLQMNSLKPEDTAVYTCAAELTNRNSGAYYYAWAYDYWGQGTQVTVSSAAAEQKLISE EDLNGAAHHHHHH RSV305 2406 EVQLVESGGGLVQPGGSLRLSCAASGLTLDYYALGWFRQAPGKEREGVSCISSSDHST TYTDSVKGRFTISWDNAKNTLYLQMNSLKPGDTAVYYCAADPALGCYSGSYYPRYDYW GQGTQVTVSSGGGGSGGGGSGGGGSEVQLVESGGGLVQAGDSLRLSCAASGRTFSSYA MGWFRQAPGKEREFVAAISWSDGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDT AVYYCAADLTSTNPGSYIYIWAYDYWGQGTQVTVSSAAAEQKLISEEDLNGAAHHHHHH RSV306 2407 EVQLVESGGGLVQPGGSLRLSCAASGLTLDYYALGWFRQAPGKEREGVSCISSSDHST TYTDSVKGRFTISWDNAKNTLYLQMNSLKPGDTAVYYCAADPALGCYSGSYYPRYDYW GQGTQVTVSSGGGGSGGGGSGGGGSEVQLVESGGGLVQAGGSLRLSCAASGRSFSNYV LGWFRQAPGKEREFVAAISFRGDSAIGAPSVEGRFTISRDNAKNTGYLQMNSLVPDDT AVYYCGAGTPLNPGAYIYDWSYDYWGRGTQVTVSSAAAEQKLISEEDLNGAAHHHHHH RSV400 2408 EVQLVESGGGLVQAGDSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVAAISWSDGST YYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAADLTSTNPGSYIYIWAYDY WGQGTQVTVSSGGGGSGGGGSGGGGSEVQLVESGGGLVQAGDSLRLSCAASGRTFSSY AMGWFRQAPGKEREFVAAISWSDGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPED TAVYYCAADLTSTNPGSYIYIWAYDYWGQGTQVTVSSGGGGSGGGGSGGGGSEVQLVE SGGGLVQAGDSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVAAISWSDGSTYYADSV KGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAADLTSTNPGSYIYIWAYDYWGQGTQ VTVSSAAAEQKLISEEDLNGAAHHHHHH RSV401 2409 EVQLVESGGGLVQAGDSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVAAISWSDGST YYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAADLTSTNPGSYIYIWAYDY WGQGTQVTVSSGGGGSGGGGSGGGGSEVQLVESGGGLVQAGDSLRLSCAASGRTFSSY AMGWFRQAPGKEREFVAAISWSDGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPED TAVYYCAADLTSTNPGSYIYIWAYDYWGQGTQVTVSSGGGGSGGGGSGGGGSEVQLVE SGGGLVQPGGSLRLSCAASGLTLDYYALGWFRQAPGKEREGVSCISSSDHSTTYTDSV KGRFTISWDNAKNTLYLQMNSLKPGDTAVYYCAADPALGCYSGSYYPRYDYWGQGTQV TVSSAAAEQKLISEEDLNGAAHHHHHH RSV402 2410 EVQLVESGGGLVQPGGSLRLSCAASGLTLDYYALGWFRQAPGKEREGVSCISSSDHST TYTDSVKGRFTISWDNAKNTLYLQMNSLKPGDTAVYYCAADPALGCYSGSYYPRYDYW GQGTQVTVSSGGGGSGGGGSGGGGSEVQLVESGGGLVQAGDSLRLSCAASGRTFSSYA MGWFRQAPGKEREFVAAISWSDGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDT AVYYCAADLTSTNPGSYIYIWAYDYWGQGTQVTVSSGGGGSGGGGSGGGGSEVQLVES GGGLVQAGDSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVAAISWSDGSTYYADSVK GRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAADLTSTNPGSYIYIWAYDYWGQGTQV TVSSAAAEQKLISEEDLNGAAHHHHHH RSV403 2411 EVQLVESGGGLVQAGDSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVAAISWSDGST YYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAADLTSTNPGSYIYIWAYDY WGQGTQVTVSSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGLTLDYY ALGWFRQAPGKEREGVSCISSSDHSTTYTDSVKGRFTISWDNAKNTLYLQMNSLKPGD TAVYYCAADPALGCYSGSYYPRYDYWGQGTQVTVSSGGGGSGGGGSGGGGSEVQLVES GGGLVQAGDSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVAAISWSDGSTYYADSVK GRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAADLTSTNPGSYIYIWAYDYWGQGTQV TVSSAAAEQKLISEEDLNGAAHHHHHH RSV404 2412 EVQLVESGGGLVQAGGSLRLSCAASGRSFSNYVLGWFRQAPGKEREFVAAISFRGDSA IGAPSVEGRFTISRDNAKNTGYLQMNSLVPDDTAVYYCGAGTPLNPGAYIYDWSYDYW GRGTQVTVSSGGGGSGGGGSGGGGSEVQLVESGGGLVQAGGSLRLSCAASGRSFSNYV LGWFRQAPGKEREFVAAISFRGDSAIGAPSVEGRFTISRDNAKNTGYLQMNSLVPDDT AVYYCGAGTPLNPGAYIYDWSYDYWGRGTQVTVSSGGGGSGGGGSGGGGSEVQLVESG GGLVQAGGSLRLSCAASGRSFSNYVLGWFRQAPGKEREFVAAISFRGDSAIGAPSVEG RFTISRDNAKNTGYLQMNSLVPDDTAVYYCGAGTPLNPGAYIYDWSYDYWGRGTQVTV SSAAAEQKLISEEDLNGAAHHHHHH RSV405 2413 EVQLVESGGGLVQAGGSLRLSCEASGRTYSRYGMGWFRQAPGKEREFVAAVSRLSGPR TVYADSVKGRFTISRDNAENTVYLQMNSLKPEDTAVYTCAAELTNRNSGAYYYAWAYD YWGQGTQVTVSSGGGGSGGGGSGGGGSEVQLVESGGGLVQAGGSLRLSCEASGRTYSR YGMGWFRQAPGKEREFVAAVSRLSGPRTVYADSVKGRFTISRDNAENTVYLQMNSLKP EDTAVYTCAAELTNRNSGAYYYAWAYDYWGQGTQVTVSSGGGGSGGGGSGGGGSEVQL VESGGGLVQAGGSLRLSCEASGRTYSRYGMGWFRQAPGKEREFVAAVSRLSGPRTVYA DSVKGRFTISRDNAENTVYLQMNSLKPEDTAVYTCAAELTNRNSGAYYYAWAYDYWGQ GTQVTVSSAAAEQKLISEEDLNGAAHHHHHH RSV406 2414 EVQLVESGGGLVQPGGSLRLSCAASGRTFSSIAMGWFRQAPGKEREFVAAISWSRGRT FYADSVKGRFIISRDDAANTAYLQMNSLKPEDTAVYYCAVDTASWNSGSFIYDWAYDH WGQGTQVTVSSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGRTFSSI AMGWFRQAPGKEREFVAAISWSRGRTFYADSVKGRFIISRDDAANTAYLQMNSLKPED TAVYYCAVDTASWNSGSFIYDWAYDHWGQGTQVTVSSGGGGSGGGGSGGGGSEVQLVE SGGGLVQPGGSLRLSCAASGRTFSSIAMGWFRQAPGKEREFVAAISWSRGRTFYADSV KGRFIISRDDAANTAYLQMNSLKPEDTAVYYCAVDTASWNSGSFIYDWAYDHWGQGTQ VTVSSAAAEQKLISEEDLNGAAHHHHHH RSV407 2415 EVQLVESGGGLVQAGGSLSISCAASGGSLSNYVLGWFRQAPGKEREFVAAINWRGDIT IGPPNVEGRFTISRDNAKNTGYLQMNSLAPDDTAVYYCGAGTPLNPGAYIYDWSYDYW GRGTQVTVSSGGGGSGGGGSGGGGSEVQLVESGGGLVQAGGSLSISCAASGGSLSNYV LGWFRQAPGKEREFVAAINWRGDITIGPPNVEGRFTISRDNAKNTGYLQMNSLAPDDT AVYYCGAGTPLNPGAYIYDWSYDYWGRGTQVTVSSGGGGSGGGGSGGGGSEVQLVESG GGLVQAGGSLSISCAASGGSLSNYVLGWFRQAPGKEREFVAAINWRGDITIGPPNVEG RFTISRDNAKNTGYLQMNSLAPDDTAVYYCGAGTPLNPGAYIYDWSYDYWGRGTQVTV SSAAAEQKLISEEDLNGAAHHHHHH RSV408 2989 EVQLVESGGGLVQAGGSLSISCAASGGSLSNYVLGWFRQAPGKEREFVAAINWRGDIT IGPPNVEGRFTISRDNAKNTGYLQMNSLAPDDTAVYYCGAGTPLNPGAYIYDWSYDYW GRGTQVTVSSAAAEVQLVESGGGLVQAGGSLSISCAASGGSLSNYVLGWFRQAPGKER EFVAAINWRGDITIGPPNVEGRFTISRDNAKNTGYLQMNSLAPDDTAVYYCGAGTPLN PGAYIYDWSYDYWGRGTQVTVSSAAAEVQLVESGGGLVQAGGSLSISCAASGGSLSNY VLGWFRQAPGKEREFVAAINWRGDITIGPPNVEGRFTISRDNAKNTGYLQMNSLAPDD TAVYYCGAGTPLNPGAYIYDWSYDYWGRGTQVTVSSAAAEQKLISEEDLNGAAHHHHHH RSV409 2990 EVQLVESGGGLVQAGGSLSISCAASGGSLSNYVLGWFRQAPGKEREFVAAINWRGDIT IGPPNVEGRFTISRDNAKNTGYLQMNSLAPDDTAVYYCGAGTPLNPGAYIYDWSYDYW GRGTQVTVSSGGGGSGGGSEVQLVESGGGLVQAGGSLSISCAASGGSLSNYVLGWFRQ APGKEREFVAAINWRGDITIGPPNVEGRFTISRDNAKNTGYLQMNSLAPDDTAVYYCG AGTPLNPGAYIYDWSYDYWGRGTQVTVSSGGGGSGGGSEVQLVESGGGLVQAGGSLSI SCAASGGSLSNYVLGWFRQAPGKEREFVAAINWRGDITIGPPNVEGRFTISRDNAKNT GYLQMNSLAPDDTAVYYCGAGTPLNPGAYIYDWSYDYWGRGTQVTVSSAAAEQKLISE EDLNGAAHHHHHH RSV410 2991 EVQLVESGGGLVQAGGSLSISCAASGGSLSNYVLGWFRQAPGKEREFVAAINWRGDIT IGPPNVEGRFTISRDNAKNTGYLQMNSLAPDDTAVYYCGAGTPLNPGAYIYDWSYDYW GRGTQVTVSSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQAGGSLSISCAASGGS LSNYVLGWFRQAPGKEREFVAAINWRGDITIGPPNVEGRFTISRDNAKNTGYLQMNSL APDDTAVYYCGAGTPLNPGAYIYDWSYDYWGRGTQVTVSSGGGGSGGGGSGGGGSGGG GSEVQLVESGGGLVQAGGSLSISCAASGGSLSNYVLGWFRQAPGKEREFVAAINWRGD ITIGPPNVEGRFTISRDNAKNTGYLQMNSLAPDDTAVYYCGAGTPLNPGAYIYDWSYD YWGRGTQVTVSSAAAEQKLISEEDLNGAAHHHHHH RSV411 2992 EVQLVESGGGLVQAGGSLSISCAASGGSLSNYVLGWFRQAPGKEREFVAAINWRGDIT IGPPNVEGRFTISRDNAKNTGYLQMNSLAPDDTAVYYCGAGTPLNPGAYIYDWSYDYW GRGTQVTVSSGGGGSGGGGSGGGGSEVQLVESGGGLVQAGGSLSISCAASGGSLSNYV LGWFRQAPGKEREFVAAINWRGDITIGPPNVEGRFTISRDNAKNTGYLQMNSLAPDDT AVYYCGAGTPLNPGAYIYDWSYDYWGRGTQVTVSSGGGGSGGGGSGGGGSEVQLVESG GGLVQPGGSLRLSCAASGLTLDYYALGWFRQAPGKEREGVSCISSSDHSTTYTDSVKG RFTISWDNAKNTLYLQMNSLKPGDTAVYYCAADPALGCYSGSYYPRYDYWGQGTQVTV SSAAAEQKLISEEDLNGAAHHHHHH RSV412 2993 EVQLVESGGGLVQPGGSLRLSCAASGLTLDYYALGWFRQAPGKEREGVSCISSSDHST TYTDSVKGRFTISWDNAKNTLYLQMNSLKPGDTAVYYCAADPALGCYSGSYYPRYDYW GQGTQVTVSSGGGGSGGGGSGGGGSEVQLVESGGGLVQAGGSLSISCAASGGSLSNYV LGWFRQAPGKEREFVAAINWRGDITIGPPNVEGRFTISRDNAKNTGYLQMNSLAPDDT AVYYCGAGTPLNPGAYIYDWSYDYWGRGTQVTVSSGGGGSGGGGSGGGGSEVQLVESG GGLVQAGGSLSISCAASGGSLSNYVLGWFRQAPGKEREFVAAINWRGDITIGPPNVEG RFTISRDNAKNTGYLQMNSLAPDDTAVYYCGAGTPLNPGAYIYDWSYDYWGRGTQVTV SSAAAEQKLISEEDLNGAAHHHHHH RSV413 2994 EVQLVESGGGLVQAGGSLSISCAASGGSLSNYVLGWFRQAPGKEREFVAAINWRGDIT IGPPNVEGRFTISRDNAKNTGYLQMNSLAPDDTAVYYCGAGTPLNPGAYIYDWSYDYW GRGTQVTVSSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGLTLDYYA LGWFRQAPGKEREGVSCISSSDHSTTYTDSVKGRFTISWDNAKNTLYLQMNSLKPGDT AVYYCAADPALGCYSGSYYPRYDYWGQGTQVTVSSGGGGSGGGGSGGGGSEVQLVESG GGLVQAGGSLSISCAASGGSLSNYVLGWFRQAPGKEREFVAAINWRGDITIGPPNVEG RFTISRDNAKNTGYLQMNSLAPDDTAVYYCGAGTPLNPGAYIYDWSYDYWGRGTQVTV SSAAAEQKLISEEDLNGAAHHHHHH RSV502 2995 EVQLVESGGGLVQAGGSLRLSCEASGRTFSSYGMGWFRQAPGKEREFVAAVSRLSGPR TVYADSVKGRFTISRDNAENTVYLQMNSLKPEDTAVYTCAAELTNRNPGAYYYTWAYD YWGQGTQVTVSSGGGGSGGGGSGGGGSEVQLVESGGGLVQAGGSLRLSCEASGRTFSS YGMGWFRQAPGKEREFVAAVSRLSGPRTVYADSVKGRFTISRDNAENTVYLQMNSLKP EDTAVYTCAAELTNRNPGAYYYTWAYDYWGQGTQVTVSSGGGGSGGGGSGGGGSEVQL VESGGGLVQAGGSLRLSCEASGRTFSSYGMGWFRQAPGKEREFVAAVSRLSGPRTVYA DSVKGRFTISRDNAENTVYLQMNSLKPEDTAVYTCAAELTNRNPGAYYYTWAYDYWGQ GTQVTVSSAAAEQKLISEEDLNGAAHHHHHH RSV513 3584 EVQLVESGGGLVQAGDSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVAAISWSDGST YYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAADLTSTNPGSYIYIWAYDY WGQGTQVTVSSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGLTLDYY ALGWFRQAPGKEREGVSCISSSDHTTTYTDSVKGRFTISWDNAKNTLYLQMNSLKPED TAVYYCAADPALGCYSGSYYPRYDFWGQGTQVTVSSGGGGSGGGGSGGGGSEVQLVES GGGLVQAGDSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVAAISWSDGSTYYADSVK GRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAADLTSTNPGSYIYIWAYDYWGQGTQV TVSSAAAEQKLISEEDLNGAAHHHHHH RSV514 3585 EVQLVESGGGLVQAGDSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVAAISWSDGST YYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAADLTSTNPGSYIYIWAYDY WGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGLTLDYYALGWFR QAPGKEREGVSCISSSDHTTTYTDSVKGRFTISWDNAKNTLYLQMNSLKPEDTAVYYC AADPALGCYSGSYYPRYDFWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQAGDSLR LSCAASGRTFSSYAMGWFRQAPGKEREFVAAISWSDGSTYYADSVKGRFTISRDNAKN TVYLQMNSLKPEDTAVYYCAADLTSTNPGSYIYIWAYDYWGQGTQVTVSSAAAEQKLI SEEDLNGAAHHHHHH RSV515 3586 EVQLVESGGGLVQAGDSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVAAISWSDGST YYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAADLTSTNPGSYIYIWAYDY WGQGTQVTVSSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRVSCAASGFTFNDY IMGWFRQAPGKERMFIAAISGTGTIKYYGDLVRGRFTISRDNAKNTVYLRIDSLNPED TAVYYCAARQDYGLGYRESHEYDYWGQGTQVTVSSGGGGSGGGGSGGGGSEVQLVESG GGLVQAGDSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVAAISWSDGSTYYADSVKG RFTISRDNAKNTVYLQMNSLKPEDTAVYYCAADLTSTNPGSYIYIWAYDYWGQGTQVT VSSAAAEQKLISEEDLNGAAHHHHHH RSV516 3587 EVQLVESGGGLVQAGDSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVAAISWSDGST YYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAADLTSTNPGSYIYIWAYDY WGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRVSCAASGFTFNDYIMGWFR QAPGKERMFIAAISGTGTIKYYGDLVRGRFTISRDNAKNTVYLRIDSLNPEDTAVYYC AARQDYGLGYRESHEYDYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQAGDSLRL SCAASGRTFSSYAMGWFRQAPGKEREFVAAISWSDGSTYYADSVKGRFTISRDNAKNT VYLQMNSLKPEDTAVYYCAADLTSTNPGSYIYIWAYDYWGQGTQVTVSSAAAEQKLIS EEDLNGAAHHHHHH
(411) TABLE-US-00026 TABLE A-3 F-protein sequences F-protein SEQ ID NO Sequence RSV LONG M-2 2416 MELPILKANAITTILAAVTFCFASSQNITEEFYQSTCSAVSKG YLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKY KNAVTELQLLMQSTPAANNRARRELPRFMNYTLNNTKKTNVTL SKKRKRRFLGFLLGVGSAIASGTAVSKVLHLEGEVNKIKSALL STNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCRIS NIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELL SLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVV QLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCD NAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIF NPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRG IIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEP IINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLHHV NAGKSTTNIMITTIIIVIIVILLSLIAVGLLLYCKARSTPVTL SKDQLSGINNIAFSN RSV A-2 2417 MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKG YLSALRTGWYTSVITIELSNIKKNKCNGTDAKVKLIKQELDKY KNAVTELQLLMQSTQATNNRARRELPRFMNYTLNNAKKTNVTL SKKRKRRFLGFLLGVGSAIASGVAVSKVLHLEGEVNKIKSALL STNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSIS NIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELL SLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVV QLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCD NAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIF NPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRG IIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEP IINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLHNV NAGKSTTNIMITTIIIVIIVILLSLIAVGLLLYCKARSTPVTL SKDQLSGINNIAFSN RSV B-1 2418 MELLIHRSSAIFLTLAVNALYLTSSQNITEEFYQSTCSAVSRG YFSALRTGWYTSVITIELSNIKETKCNGTDTKVKLIKQELDKY KNAVTELQLLMQNTPAANNRARREAPQYMNYTINTTKNLNVSI SKKRKRRFLGFLLGVGSAIASGIAVSKVLHLEGEVNKIKNALL STNKAVVSLSNGVSVLTSKVLDLKNYINNRLLPIVNQQSCRIS NIETVIEFQQMNSRLLEITREFSVNAGVTTPLSTYMLTNSELL SLINDMPITNDQKKLMSSNVQIVRQQSYSIMSIIKEEVLAYVV QLPIYGVIDTPCWKLHTSPLCTTNIKEGSNICLTRTDRGWYCD NAGSVSFFPQADTCKVQSNRVFCDTMNSLTLPSEVSLCNTDIF NSKYDCKIMTSKTDISSSVITSLGAIVSCYGKTKCTASNKNRG IIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKLEGKNLYVKGEP IINYYDPLVFPSDEFDASISQVNEKINQSLAFIRRSDELLHNV NTGKSTTNIMITTIIIVIIVVLLLLIAIGLLLYCKAKNTPVTL SKDQLSGINNIAFSK
(412) TABLE-US-00027 TABLE A-4 Amino acid sequence of multivalent constructs that bind hemagglutinin H5 of influenza Construct SEQ ID NO Sequence 202-C8-9GS- 2423 EVQLVESGGGLVQPGGSLRLSCTGSGFTFSSYWMDWVRQTPGKDLEYVSG 202-C8 ISPSGSNTDYADSVKGRFTISRDNAKNTLYLQMNSLKPEDTALYYCRRSL TLTDSPDLRSQGTQVTVSSGGGSGGGGSEVQLVESGGGLVQPGGSLRLSC TGSGFTFSSYWMDWVRQTPGKDLEYVSGISPSGSNTDYADSVKGRFTISR DNAKNTLYLQMNSLKPEDTALYYCRRSLTLTDSPDLRSQGTQVTVSS 202-C8-15GS- 2424 EVQLVESGGGLVQPGGSLRLSCTGSGFTFSSYWMDWVRQTPGKDLEYVSG 202-C8 ISPSGSNTDYADSVKGRFTISRDNAKNTLYLQMNSLKPEDTALYYCRRSL TLTDSPDLRSQGTQVTVSSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGG SLRLSCTGSGFTFSSYWMDWVRQTPGKDLEYVSGISPSGSNTDYADSVKG RFTISRDNAKNTLYLQMNSLKPEDTALYYCRRSLTLTDSPDLRSQGTQVT VSS 202-C8-10GS- 2425 EVQLVESGGGLVQPGGSLRLSCTGSGFTFSSYWMDWVRQTPGKDLEYVSG 202-C8-10GS- ISPSGSNTDYADSVKGRFTISRDNAKNTLYLQMNSLKPEDTALYYCRRSL 202-C8 TLTDSPDLRSQGTQVTVSSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLS CTGSGFTFSSYWMDWVRQTPGKDLEYVSGISPSGSNTDYADSVKGRFTIS RDNAKNTLYLQMNSLKPEDTALYYCRRSLTLTDSPDLRSQGTQVTVSSGG GGSGGGGSEVQLVESGGGLVQPGGSLRLSCTGSGFTFSSYWMDWVRQTPG KDLEYVSGISPSGSNTDYADSVKGRFTISRDNAKNTLYLQMNSLKPEDTA LYYCRRSLTLTDSPDLRSQGTQVTVSS 202-C8-20GS- 2426 EVQLVESGGGLVQPGGSLRLSCTGSGFTFSSYWMDWVRQTPGKDLEYVSG 202-C8-20GS- ISPSGSNTDYADSVKGRFTISRDNAKNTLYLQMNSLKPEDTALYYCRRSL 202-C8 TLTDSPDLRSQGTQVTVSSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGL VQPGGSLRLSCTGSGFTFSSYWMDWVRQTPGKDLEYVSGISPSGSNTDYA DSVKGRFTISRDNAKNTLYLQMNSLKPEDTALYYCRRSLTLTDSPDLRSQ GTQVTVSSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSC TGSGFTFSSYWMDWVRQTPGKDLEYVSGISPSGSNTDYADSVKGRFTISR DNAKNTLYLQMNSLKPEDTALYYCRRSLTLTDSPDLRSQGTQVTVSS 203-B12- 2428 EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMGWVRRAPGEGLEWVSS 15GS-203-B12 ISSGGALPTYADSVKGRFTISRDNVKNTLYLQMNSLKPEDTAVYSCEKYA GSMWTSERDAWGQGTQVTVSSGGGGSGGGGSGGGGSEVQLVESGGGLVQP GGSLRLSCAASGFTFSSYAMGWVRRAPGEGLEWVSSISSGGALPTYADSV KGRFTISRDNVKNTLYLQMNSLKPEDTAVYSCEKYAGSMWTSERDAWGQG TQVTVSS 203-H9-5GS- 2429 EVQLVESGGGLVQPGGSLRLSCTGSGFTFSSYWMDWVRQTPGKDLEYVSG 203-H9 ISPSGGNTDYADSVKGRFTISRDNAKNTLYLQMNSLQPEDTALYYCRRSL TLTDSPDLRSQGTQVTVSSGGGGSEVQLVESGGGLVQPGGSLRLSCTGSG FTFSSYWMDWVRQTPGKDLEYVSGISPSGGNTDYADSVKGRFTISRDNAK NTLYLQMNSLQPEDTALYYCRRSLTLTDSPDLRSQGTQVTVSS 203-H9-25GS- 2430 EVQLVESGGGLVQPGGSLRLSCTGSGFTFSSYWMDWVRQTPGKDLEYVSG 203-H9 ISPSGGNTDYADSVKGRFTISRDNAKNTLYLQMNSLQPEDTALYYCRRSL TLTDSPDLRSQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVE SGGGLVQPGGSLRLSCTGSGFTFSSYWMDWVRQTPGKDLEYVSGISPSGG NTDYADSVKGRFTISRDNAKNTLYLQMNSLQPEDTALYYCRRSLTLTDSP DLRSQGTQVTVSS
(413) TABLE-US-00028 TABLE A-5 Sequences of multivalent Fc constructs SEQ Construct ID NO Sequence NC41::15GS::NC41::G1- 2641 EVQLVESGGGLVQAGGSLSISCAASGGSLSNYVLGWFRQAPGKERE hinge::IgG1-Fc FVAAINWRGDITIGPPNVEGRFTISRDNAKNTGYLQMNSLAPDDTA VYYCGAGTPLNPGAYIYDWSYDYWGRGTQVTVSSGGGGSGGGGSGG GGSEVQLVESGGGLVQAGGSLSISCAASGGSLSNYVLGWFRQAPGK EREFVAAINWRGDITIGPPNVEGRFTISRDNAKNTGYLQMNSLAPD DTAVYYCGAGTPLNPGAYIYDWSYDYWGRGTQVTVSSEPKSCDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHED PEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNG KEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQ VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK NC41::15GS::NC41::9G 2642 EVQLVESGGGLVQAGGSLSISCAASGGSLSNYVLGWFRQAPGKERE S-G1-hinge::IgG1-Fc FVAAINWRGDITIGPPNVEGRFTISRDNAKNTGYLQMNSLAPDDTA VYYCGAGTPLNPGAYIYDWSYDYWGRGTQVTVSSGGGGSGGGGSGG GGSEVQLVESGGGLVQAGGSLSISCAASGGSLSNYVLGWFRQAPGK EREFVAAINWRGDITIGPPNVEGRFTISRDNAKNTGYLQMNSLAPD DTAVYYCGAGTPLNPGAYIYDWSYDYWGRGTQVTVSSGGGGSGGGS EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTC VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLT VLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPP SRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP GK NC41::15GS::NC41::G3- 2643 EVQLVESGGGLVQAGGSLSISCAASGGSLSNYVLGWFRQAPGKERE hinge::IgG1-Fc FVAAINWRGDITIGPPNVEGRFTISRDNAKNTGYLQMNSLAPDDTA VYYCGAGTPLNPGAYIYDWSYDYWGRGTQVTVSSGGGGSGGGGSGG GGSEVQLVESGGGLVQAGGSLSISCAASGGSLSNYVLGWFRQAPGK EREFVAAINWRGDITIGPPNVEGRFTISRDNAKNTGYLQMNSLAPD DTAVYYCGAGTPLNPGAYIYDWSYDYWGRGTQVTVSSELKTPLGDT THTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPEPKSCDTP PPCPRCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHE DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLN GKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKN QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK NC41::G1-hinge:: 2644 EVQLVESGGGLVQAGGSLSISCAASGGSLSNYVLGWFRQAPGKERE IgG1-Fc::NC41 FVAAINWRGDITIGPPNVEGRFTISRDNAKNTGYLQMNSLAPDDTA VYYCGAGTPLNPGAYIYDWSYDYWGRGTQVTVSSEPKSCDKTHTCP PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV KFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSL TCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLT VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKEVQLVESGGG LVQAGGSLSISCAASGGSLSNYVLGWFRQAPGKEREFVAAINWRGD ITIGPPNVEGRFTISRDNAKNTGYLQMNSLAPDDTAVYYCGAGTPL NPGAYIYDWSYDYWGRGTQVTVSS NC41::9GS-G1-hinge 2645 EVQLVESGGGLVQAGGSLSISCAASGGSLSNYVLGWFRQAPGKERE IgG1-Fc::NC41 FVAAINWRGDITIGPPNVEGRFTISRDNAKNTGYLQMNSLAPDDTA VYYCGAGTPLNPGAYIYDWSYDYWGRGTQVTVSSGGGGSGGGSEPK SCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVV DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLH QDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRD ELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG SFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKE VQLVESGGGLVQAGGSLSISCAASGGSLSNYVLGWFRQAPGKEREF VAAINWRGDITIGPPNVEGRFTISRDNAKNTGYLQMNSLAPDDTAV YYCGAGTPLNPGAYIYDWSYDYWGRGTQVTVSS NC41::G3-hinge:: 2646 EVQLVESGGGLVQAGGSLSISCAASGGSLSNYVLGWFRQAPGKERE IgG1-Fc::NC41 FVAAINWRGDITIGPPNVEGRFTISRDNAKNTGYLQMNSLAPDDTA VYYCGAGTPLNPGAYIYDWSYDYWGRGTQVTVSSELKTPLGDTTHT CPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPC PRCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKE YKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKL TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKEVQLVESGG GLVQAGGSLSISCAASGGSLSNYVLGWFRQAPGKEREFVAAINWRG DITIGPPNVEGRFTISRDNAKNTGYLQMNSLAPDDTAVYYCGAGTP LNPGAYIYDWSYDYWGRGTQVTVSS NC41::G1-hinge:: 2647 EVQLVESGGGLVQAGGSLSISCAASGGSLSNYVLGWFRQAPGKERE IgG1-Fc::9GS::NC41 FVAAINWRGDITIGPPNVEGRFTISRDNAKNTGYLQMNSLAPDDTA VYYCGAGTPLNPGAYIYDWSYDYWGRGTQVTVSSEPKSCDKTHTCP PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV KFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSL TCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLT VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGSE VQLVESGGGLVQAGGSLSISCAASGGSLSNYVLGWFRQAPGKEREF VAAINWRGDITIGPPNVEGRFTISRDNAKNTGYLQMNSLAPDDTAV YYCGAGTPLNPGAYIYDWSYDYWGRGTQVTVSS NC41::9Gs-G1-hinge 2648 EVQLVESGGGLVQAGGSLSISCAASGGSLSNYVLGWFRQAPGKERE IgG1-Fc::9GS::NC41 FVAAINWRGDITIGPPNVEGRFTISRDNAKNTGYLQMNSLAPDDTA VYYCGAGTPLNPGAYIYDWSYDYWGRGTQVTVSSGGGGSGGGSEPK SCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVV DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLH QDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRD ELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG SFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKG GGGSGGGSEVQLVESGGGLVQAGGSLSISCAASGGSLSNYVLGWFR QAPGKEREFVAAINWRGDITIGPPNVEGRFTISRDNAKNTGYLQMN SLAPDDTAVYYCGAGTPLNPGAYIYDWSYDYWGRGTQVTVSS NC41::G3-hinge:: 2649 EVQLVESGGGLVQAGGSLSISCAASGGSLSNYVLGWFRQAPGKERE IgG1-Fc::9GS::NC41 FVAAINWRGDITIGPPNVEGRFTISRDNAKNTGYLQMNSLAPDDTA VYYCGAGTPLNPGAYIYDWSYDYWGRGTQVTVSSELKTPLGDTTHT CPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPC PRCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKE YKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKL TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGS EVQLVESGGGLVQAGGSLSISCAASGGSLSNYVLGWFRQAPGKERE FVAAINWRGDITIGPPNVEGRFTISRDNAKNTGYLQMNSLAPDDTA VYYCGAGTPLNPGAYIYDWSYDYWGRGTQVTVSS NC41::G1-hinge:: 2650 EVQLVESGGGLVQAGGSLSISCAASGGSLSNYVLGWFRQAPGKERE IgG1-Fc::15B3 FVAAINWRGDITIGPPNVEGRFTISRDNAKNTGYLQMNSLAPDDTA VYYCGAGTPLNPGAYIYDWSYDYWGRGTQVTVSSEPKSCDKTHTCP PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV KFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSL TCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLT VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKEVQLVESGGG LVQPGGSLRLSCAASGLTLDYYALGWFRQAPGKEREGVSCISSSDH STTYTDSVKGRFTISWDNAKNTLYLQMNSLKPGDTAVYYCAADPAL GCYSGSYYPRYDYWGQGTQVTVSS NC41::9GS-G1-hinge 2651 EVQLVESGGGLVQAGGSLSISCAASGGSLSNYVLGWFRQAPGKERE ::IgG1-Fc::15B3 FVAAINWRGDITIGPPNVEGRFTISRDNAKNTGYLQMNSLAPDDT AVYYCGAGTPLNPGAYIYDWSYDYWGRGTQVTVSSGGGGSGGGSE PKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTC VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVL TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPGKEVQLVESGGGLVQPGGSLRLSCAASGLTLDYYALGWFRQ APGKEREGVSCISSSDHSTTYTDSVKGRFTISWDNAKNTLYLQMN SLKPGDTAVYYCAADPALGCYSGSYYPRYDYWGQGTQVTVSS NC41::G3-hinge:: 2652 EVQLVESGGGLVQAGGSLSISCAASGGSLSNYVLGWFRQAPGKER IgG1-Fc::15B3 EFVAAINWRGDITIGPPNVEGRFTISRDNAKNTGYLQMNSLAPDD TAVYYCGAGTPLNPGAYIYDWSYDYWGRGTQVTVSSELKTPLGDT THTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPEPKSCDT PPPCPRCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDE LTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG SFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK EVQLVESGGGLVQPGGSLRLSCAASGLTLDYYALGWFRQAPGKER EGVSCISSSDHSTTYTDSVKGRFTISWDNAKNTLYLQMNSLKPGD TAVYYCAADPALGCYSGSYYPRYDYWGQGTQVTVSS NC41::G1-hinge:: 2653 EVQLVESGGGLVQAGGSLSISCAASGGSLSNYVLGWFRQAPGKER IgG1-Fc::9GS::15B3 EFVAAINWRGDITIGPPNVEGRFTISRDNAKNTGYLQMNSLAPDD TAVYYCGAGTPLNPGAYIYDWSYDYWGRGTQVTVSSEPKSCDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHE DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWL NGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELT KNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGG GGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGLTLDYYALGWFR QAPGKEREGVSCISSSDHSTTYTDSVKGRFTISWDNAKNTLYLQM NSLKPGDTAVYYCAADPALGCYSGSYYPRYDYWGQGTQVTVSS NC41::9GS-G1-hinge 2654 EVQLVESGGGLVQAGGSLSISCAASGGSLSNYVLGWFRQAPGKER ::IgG1-Fc::9GS::15B3 EFVAAINWRGDITIGPPNVEGRFTISRDNAKNTGYLQMNSLAPDD TAVYYCGAGTPLNPGAYIYDWSYDYWGRGTQVTVSSGGGGSGGGS EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVT CVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYT LPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGKGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGLTL DYYALGWFRQAPGKEREGVSCISSSDHSTTYTDSVKGRFTISWDN AKNTLYLQMNSLKPGDTAVYYCAADPALGCYSGSYYPRYDYWGQG TQVTVSS NC41::G3-hinge:: 2655 EVQLVESGGGLVQAGGSLSISCAASGGSLSNYVLGWFRQAPGKER IgG1-Fc::9GS::15B3 EFVAAINWRGDITIGPPNVEGRFTISRDNAKNTGYLQMNSLAPDD TAVYYCGAGTPLNPGAYIYDWSYDYWGRGTQVTVSSELKTPLGDT THTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPEPKSCDT PPPCPRCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDE LTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG SFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK GGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGLTLDYYALGW FRQAPGKEREGVSCISSSDHSTTYTDSVKGRFTISWDNAKNTLYL QMNSLKPGDTAVYYCAADPALGCYSGSYYPRYDYWGQGTQVTVSS NC41::NC41::IgG1-Fc 2656 EVQLVESGGGLVQAGGSLSISCAASGGSLSNYVLGWFRQAPGKERE FVAAINWRGDITIGPPNVEGRFTISRDNAKNTGYLQMNSLAPDDTA VYYCGAGTPLNPGAYIYDWSYDYWGRGTQVTVSSGGGGSGGGGSGG GGSEVQLVESGGGLVQAGGSLSISCAASGGSLSNYVLGWFRQAPGK EREFVAAINWRGDITIGPPNVEGRFTISRDNAKNTGYLQMNSLAPD DTAVYYCGAGTPLNPGAYIYDWSYDYWGRGTQVTVSSEPKSCDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHED PEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNG KEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQ VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK NC41::IgG1-Fc::NC41 2657 EVQLVESGGGLVQAGGSLSISCAASGGSLSNYVLGWFRQAPGKERE FVAAINWRGDITIGPPNVEGRFTISRDNAKNTGYLQMNSLAPDDTA VYYCGAGTPLNPGAYIYDWSYDYWGRGTQVTVSSEPKSCDKTHTCP PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV KFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSL TCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLT VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKEVQLVESGGG LVQAGGSLSISCAASGGSLSNYVLGWFRQAPGKEREFVAAINWRGD ITIGPPNVEGRFTISRDNAKNTGYLQMNSLAPDDTAVYYCGAGTPL NPGAYIYDWSYDYWGRGTQVTVSS NC41::NC41::IgG1-Fc 2658 GAAGTACAACTAGTTGAGTCTGGGGGTGGTCTTGTGCAGGCCGGGG GTAGCTTGTCCATTTCATGTGCAGCGAGTGGAGGGAGCCTGTCGAA CTACGTTCTGGGTTGGTTCAGACAAGCTCCTGGGAAGGAAAGAGAA TTTGTCGCTGCAATTAACTGGAGAGGTGATATAACTATTGGCCCTC CAAATGTGGAAGGCCGGTTTACTATTTCCAGGGACAATGCTAAAAA CACGGGTTATCTCCAGATGAACTCCTTGGCTCCGGACGACACTGCC GTGTACTATTGTGGAGCCGGTACCCCCCTCAACCCCGGCGCGTACA TATACGACTGGTCTTACGACTATTGGGGACGGGGCACGCAGGTAAC CGTTAGCAGCGGAGGCGGGGGATCGGGAGGCGGTGGGAGCGGTGGT GGCGGGTCAGAGGTACAACTAGTGGAGAGTGGTGGAGGTCTCGTCC AAGCTGGGGGTTCATTGTCTATTTCGTGTGCTGCCAGCGGAGGATC GCTCAGTAATTACGTGTTAGGCTGGTTTCGCCAAGCACCTGGGAAA GAACGAGAGTTCGTCGCTGCAATCAACTGGCGAGGGGACATAACCA TAGGTCCACCTAATGTTGAGGGTAGGTTCACAATCTCTCGGGACAA TGCGAAGAACACAGGATATCTTCAGATGAATAGTCTTGCCCCAGAC GATACGGCTGTTTATTATTGCGGTGCAGGGACCCCCCTGAATCCGG GGGCCTACATTTATGATTGGTCATACGATTATTGGGGACGTGGGAC CCAAGTTACTGTGTCTTCGGAACCAAAGTCGTGCGATAAGACCCAT ACCTGTCCGCCCTGTCCTGCTCCGGAACTTCTAGGCGGCCCCTCTG TGTTTCTTTTCCCACCCAAGCCGAAGGATACGCTTATGATTTCTCG CACCCCAGAAGTGACGTGTGTTGTCGTCGACGTTAGTCATGAAGAC CCAGAGGTCAAATTTAATTGGTACGTCGACGGGGTCGAAGTCCACA ATGCGAAAACTAAACCTAGGGAGGAGCAATACAACTCGACATATCG TGTAGTCAGCGTCCTGACTGTCTTACATCAGGACTGGCTCAACGGT AAAGAATATAAATGTAAGGTCTCTAACAAAGCTTTGCCTGCGCCGA TTGAAAAGACCATATCTAAAGCGAAGGGACAACCAAGAGAACCACA AGTGTATACGTTACCGCCGTCACGAGACGAACTGACAAAGAACCAG GTCTCTCTCACCTGCCTGGTCAAGGGGTTTTACCCTAGCGACATTG CCGTCGAGTGGGAATCCAACGGACAGCCCGAAAATAACTACAAGAC AACTCCCCCGGTTTTAGATTCGGACGGGAGTTTTTTTCTGTATAGT AAACTTACGGTTGATAAGTCGCGCTGGCAGCAAGGCAACGTCTTCT CTTGTTCTGTGATGCATGAGGCGCTCCACAATCACTATACCCAAAA ATCGCTCTCCTTGTCGCCAGGCAAATGA NC41::IgG1-Fc::NC41 2659 GAGGTGCAATTGGTAGAGAGTGGCGGAGGTCTAGTGCAAGCGGGAG GCTCGCTGAGCATTAGCTGCGCAGCATCGGGCGGATCGTTGTCTAA CTACGTTCTGGGCTGGTTTAGGCAAGCGCCAGGGAAAGAGAGAGAG TTCGTCGCTGCGATAAACTGGCGCGGTGACATAACGATCGGACCTC CAAATGTAGAAGGAAGATTCACCATTAGCAGAGACAATGCAAAGAA CACGGGTTACCTACAGATGAACTCACTGGCTCCGGACGACACTGCA GTGTACTACTGTGGTGCAGGGACTCCCCTAAACCCAGGGGCATATA TTTATGACTGGTCATACGATTATTGGGGCAGAGGAACGCAAGTGAC CGTCAGCAGTGAACCCAAAAGCTGTGACAAGACCCATACATGCCCT CCCTGTCCAGCGCCCGAACTGCTTGGAGGACCAAGTGTTTTCTTAT TCCCGCCAAAGCCCAAGGACACGTTGATGATTAGCAGGACCCCGGA AGTGACATGCGTAGTTGTAGATGTAAGCCACGAAGATCCGGAGGTC AAGTTCAATTGGTATGTTGATGGGGTGGAAGTGCATAACGCTAAAA CTAAACCACGTGAGGAACAGTACAACTCTACTTACAGGGTAGTGTC GGTATTGACAGTTCTGCATCAAGATTGGCTAAACGGCAAAGAATAT AAGTGTAAAGTAAGTAATAAAGCGCTCCCCGCACCCATTGAAAAGA CCATTTCGAAGGCAAAGGGTCAGCCACGCGAGCCGCAGGTGTATAC ACTGCCCCCTTCCAGGGACGAGCTTACGAAGAACCAGGTTAGCTTG ACTTGCCTTGTAAAGGGATTCTACCCCAGTGACATAGCAGTAGAAT GGGAATCGAACGGGCAACCCGAAAACAATTACAAGACAACCCCACC GGTCTTGGACTCTGATGGCTCTTTCTTCTTGTACTCCAAGTTAACC GTAGACAAATCGAGGTGGCAGCAAGGAAACGTTTTCTCGTGCTCTG TAATGCATGAGGCGTTGCATAACCATTATACTCAGAAGAGCCTGTC ACTGTCGCCGGGTAAAGAAGTGCAGCTTGTGGAATCAGGAGGGGGG CTCGTTCAAGCTGGAGGGAGCCTGTCGATCAGCTGCGCAGCGTCCG GAGGCTCGCTAAGTAACTACGTCCTCGGTTGGTTTAGACAGGCCCC AGGCAAGGAAAGGGAATTTGTTGCGGCAATAAATTGGCGAGGAGAT ATAACCATCGGGCCACCCAATGTAGAAGGAAGGTTCACTATTTCGC GGGATAACGCGAAGAATACGGGCTATCTTCAGATGAATTCATTGGC TCCGGACGACACTGCCGTTTACTATTGCGGTGCAGGGACACCGTTG AACCCAGGCGCGTACATTTACGACTGGTCCTACGATTACTGGGGGC GCGGCACGCAAGTTACCGTGTCCAGCTGA 191D3::15GS::191E4:: 2978 EVQLVESGGGLVQAGGSLRLSCEASGRTYSRYGMGWFRQAPGKERE G1-hinge::IgG1-Fc FVAAVSRLSGPRTVYADSVKGRFTISRDNAENTVYLQMNSLKPEDT AVYTCAAELTNRNSGAYYYAWAYDYWGQGTQVTVSSGGGGSGGGGS GGGGSEVQLVESGGGLVQAGGSLRLSCAASGPTFSADTMGWFRQAP GKEREFVATIPWSGGIAYYSDSVKGRFTMSRDNAKNTVDLQMNSLK PEDTALYYCAGSSRIYIYSDSLSERSYDYWGQGTQVTVSSEPKSCD KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDW LNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELT KNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF LYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 191D3::15GS::191E4:: 2979 EVQLVESGGGLVQAGGSLRLSCEASGRTYSRYGMGWFRQAPGKERE 9GS-G1-hinge::IgG1- FVAAVSRLSGPRTVYADSVKGRFTISRDNAENTVYLQMNSLKPEDT Fc AVYTCAAELTNRNSGAYYYAWAYDYWGQGTQVTVSSGGGGSGGGGS GGGGSEVQLVESGGGLVQAGGSLRLSCAASGPTFSADTMGWFRQAP GKEREFVATIPWSGGIAYYSDSVKGRFTMSRDNAKNTVDLQMNSLK PEDTALYYCAGSSRIYIYSDSLSERSYDYWGQGTQVTVSSGGGGSG GGSEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPE VTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYT LPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGK 191D3::15GS::191E4:: 2980 EVQLVESGGGLVQAGGSLRLSCEASGRTYSRYGMGWFRQAPGKERE G3-hinge::IgG1-Fc FVAAVSRLSGPRTVYADSVKGRFTISRDNAENTVYLQMNSLKPEDT AVYTCAAELTNRNSGAYYYAWAYDYWGQGTQVTVSSGGGGSGGGGS GGGGSEVQLVESGGGLVQAGGSLRLSCAASGPTFSADTMGWFRQAP GKEREFVATIPWSGGIAYYSDSVKGRFTMSRDNAKNTVDLQMNSLK PEDTALYYCAGSSRIYIYSDSLSERSYDYWGQGTQVTVSSELKTPL GDTTHTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPEPKSC DTPPPCPRCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDV SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDEL TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 191D3::G1-hinge:: 2981 EVQLVESGGGLVQAGGSLRLSCEASGRTYSRYGMGWFRQAPGKERE IgG1-Fc::191E4 FVAAVSRLSGPRTVYADSVKGRFTISRDNAENTVYLQMNSLKPEDT AVYTCAAELTNRNSGAYYYAWAYDYWGQGTQVTVSSEPKSCDKTHT CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDP EVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGK EYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQV SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSK LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKEVQLVESG GGLVQAGGSLRLSCAASGPTFSADTMGWFRQAPGKEREFVATIPWS GGIAYYSDSVKGRFTMSRDNAKNTVDLQMNSLKPEDTALYYCAGSS RIYIYSDSLSERSYDYWGQGTQVTVSS 191D3::9GS-G1-hinge 2982 EVQLVESGGGLVQAGGSLRLSCEASGRTYSRYGMGWFRQAPGKERE IgG1-Fc::191E4 FVAAVSRLSGPRTVYADSVKGRFTISRDNAENTVYLQMNSLKPEDT AVYTCAAELTNRNSGAYYYAWAYDYWGQGTQVTVSSGGGGSGGGSE PKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCV VVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTV LHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS RDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG KEVQLVESGGGLVQAGGSLRLSCAASGPTFSADTMGWFRQAPGKER EFVATIPWSGGIAYYSDSVKGRFTMSRDNAKNTVDLQMNSLKPEDT ALYYCAGSSRIYIYSDSLSERSYDYWGQGTQVTVSS 191D3::G3-hinge:: 2983 EVQLVESGGGLVQAGGSLRLSCEASGRTYSRYGMGWFRQAPGKERE IgG1-Fc::191E4 FVAAVSRLSGPRTVYADSVKGRFTISRDNAENTVYLQMNSLKPEDT AVYTCAAELTNRNSGAYYYAWAYDYWGQGTQVTVSSELKTPLGDTT HTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPEPKSCDTPP PCPRCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHED PEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNG KEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQ VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKEVQLVES GGGLVQAGGSLRLSCAASGPTFSADTMGWFRQAPGKEREFVATIPW SGGIAYYSDSVKGRFTMSRDNAKNTVDLQMNSLKPEDTALYYCAGS SRIYIYSDSLSERSYDYWGQGTQVTVSS 191D3::G1-hinge:: 2984 EVQLVESGGGLVQAGGSLRLSCEASGRTYSRYGMGWFRQAPGKERE IgG1-Fc::9GS::191E4 FVAAVSRLSGPRTVYADSVKGRFTISRDNAENTVYLQMNSLKPEDT AVYTCAAELTNRNSGAYYYAWAYDYWGQGTQVTVSSEPKSCDKTHT CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDP EVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGK EYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQV SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSK LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGG SEVQLVESGGGLVQAGGSLRLSCAASGPTFSADTMGWFRQAPGKER EFVATIPWSGGIAYYSDSVKGRFTMSRDNAKNTVDLQMNSLKPEDT ALYYCAGSSRIYIYSDSLSERSYDYWGQGTQVTVSS 191D3::9GS-G1-hinge 2985 EVQLVESGGGLVQAGGSLRLSCEASGRTYSRYGMGWFRQAPGKERE IgG1- FVAAVSRLSGPRTVYADSVKGRFTISRDNAENTVYLQMNSLKPEDT Fc::9GS::191E4 AVYTCAAELTNRNSGAYYYAWAYDYWGQGTQVTVSSGGGGSGGGSE PKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCV VVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTV LHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS RDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG KGGGGSGGGSEVQLVESGGGLVQAGGSLRLSCAASGPTFSADTMGW FRQAPGKEREFVATIPWSGGIAYYSDSVKGRFTMSRDNAKNTVDLQ MNSLKPEDTALYYCAGSSRIYIYSDSLSERSYDYWGQGTQVTVSS 191D3::G3-hinge:: 2986 EVQLVESGGGLVQAGGSLRLSCEASGRTYSRYGMGWFRQAPGKERE IgG1-Fc::9GS::191E4 FVAAVSRLSGPRTVYADSVKGRFTISRDNAENTVYLQMNSLKPEDT AVYTCAAELTNRNSGAYYYAWAYDYWGQGTQVTVSSELKTPLGDTT HTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPEPKSCDTPP PCPRCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHED PEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNG KEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQ VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGG GSEVQLVESGGGLVQAGGSLRLSCAASGPTFSADTMGWFRQAPGKE REFVATIPWSGGIAYYSDSVKGRFTMSRDNAKNTVDLQMNSLKPED TALYYCAGSSRIYIYSDSLSERSYDYWGQGTQVTVSS 191D3::191E4::IgG1- 2987 EVQLVESGGGLVQAGGSLRLSCEASGRTYSRYGMGWFRQAPGKERE Fc FVAAVSRLSGPRTVYADSVKGRFTISRDNAENTVYLQMNSLKPEDT AVYTCAAELTNRNSGAYYYAWAYDYWGQGTQVTVSSGGGGSGGGGS GGGGSEVQLVESGGGLVQAGGSLRLSCAASGPTFSADTMGWFRQAP GKEREFVATIPWSGGIAYYSDSVKGRFTMSRDNAKNTVDLQMNSLK PEDTALYYCAGSSRIYIYSDSLSERSYDYWGQGTQVTVSSEPKSCD KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDW LNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELT KNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF LYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 191D3::IgG1- 2988 EVQLVESGGGLVQAGGSLRLSCEASGRTYSRYGMGWFRQAPGKERE Fc::191E4 FVAAVSRLSGPRTVYADSVKGRFTISRDNAENTVYLQMNSLKPEDT AVYTCAAELTNRNSGAYYYAWAYDYWGQGTQVTVSSEPKSCDKTHT CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDP EVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGK EYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQV SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSK LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKEVQLVESG GGLVQAGGSLRLSCAASGPTFSADTMGWFRQAPGKEREFVATIPWS GGIAYYSDSVKGRFTMSRDNAKNTVDLQMNSLKPEDTALYYCAGSS RIYIYSDSLSERSYDYWGQGTQVTVSS
(414) TABLE-US-00029 TABLE A-6 Amino acid sequence of multivalent NANOBODY® (V.sub.HH sequence) constructs that bind rabies virus SEQ ID Construct NO: Sequence 213H7-15GS- 2427 EVQLVESGGGLVQAGGSLRLSCAASGRTLSSYRMGWFRQAPGKEREFISTIS 213H7 WNGRSTYYADSVKGRFIFSEDNAKNTVYLQMNSLKPEDTAVYYCAAALIGGY YSDVDAWSYWGPGTQVTVSSGGGGSGGGGSGGGGSEVQLVESGGGLVQAGGS LRLSCAASGRTLSSYRMGWFRQAPGKEREFISTISWNGRSTYYADSVKGRFI FSEDNAKNTVYLQMNSLKPEDTAVYYCAAALIGGYYSDVDAWSYWGPGTQVT VSS 214E8-15GS- 2663 EVQLVESGGGSVQAGGSLRLSCAASGGTFNPYVMAWFRQAPGNEREFVARIR 214-E8 WSGGDAYYDDSVKGRFAITRDAAKNTVHLQMNSLKPEDTAVYYCAAATYGYG SYTYGGSYDLWGQGTQVTVSSGGGGSGGGGSGGGGSEVQLVESGGGSVQAGG SLRLSCAASGGTFNPYVMAWFRQAPGNEREFVARIRWSGGDAYYDDSVKGRF AITRDAAKNTVHLQMNSLKPEDTAVYYCAAATYGYGSYTYGGSYDLWGQGTQ VTVSS 212C12-15GS- 2664 EVQLVESGGGLVQPGGSLRLSCAASGFTFGSSDMSWVRQAPGKGPEWVSGIN 212C12 SGGGRTLYADSVKGRFTISRDNAKNTLYLQMNSLKSEDTAVYYCATDLYGSS WYTDYWSQGTQVTVSSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLS CAASGFTFGSSDMSWVRQAPGKGPEWVSGINSGGGRTLYADSVKGRFTISRD NAKNTLYLQMNSLKSEDTAVYYCATDLYGSSWYTDYWSQGTQVTVSS 213E6-5GS- 2665 EVQLVESGGGLVQAGASLRLSCAASGSTLSRYGVGWFRQAPGKERELVASVD 213E6 WSGSRTYYADSVKGRFTISRDNAKNTGYLQMNSLKPDDTAVYYCAADSSVVP GIEKYDDWGLGTQVTVSSGGGGSEVQLVESGGGLVQAGASLRLSCAASGSTL SRYGVGWFRQAPGKERELVASVDWSGSRTYYADSVKGRFTISRDNAKNTGYL QMNSLKPDDTAVYYCAADSSVVPGIEKYDDWGLGTQVTVSS 213E6-25GS- 2666 EVQLVESGGGLVQAGASLRLSCAASGSTLSRYGVGWFRQAPGKERELVASVD 213E6 WSGSRTYYADSVKGRFTISRDNAKNTGYLQMNSLKPDDTAVYYCAADSSVVP GIEKYDDWGLGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGG GLVQAGASLRLSCAASGSTLSRYGVGWFRQAPGKERELVASVDWSGSRTYYA DSVKGRFTISRDNAKNTGYLQMNSLKPDDTAVYYCAADSSVVPGIEKYDDWG LGTQVTVSS 214F8-15GS- 2667 EVQLVESGGDLVQAGGSLRLSCVASGSTYSINAMGWYRQAPGKLRELVAAFR 214F8 TGGSTDYADSVKGRFTISRDTAKNTVYLQMNSLKPEDTAVYYCNAEVIYYPY DYWGQGTQVTVSSGGGGSGGGGSGGGGSEVQLVESGGDLVQAGGSLRLSCVA SGSTYSINAMGWYRQAPGKLRELVAAFRTGGSTDYADSVKGRFTISRDTAKN TVYLQMNSLKPEDTAVYYCNAEVIYYPYDYWGQGTQVTVSS 213E6-5GS- 2668 EVQLVESGGGLVQAGASLRLSCAASGSTLSRYGVGWFRQAPGKERELVASVD 212C12 WSGSRTYYADSVKGRFTISRDNAKNTGYLQMNSLKPDDTAVYYCAADSSVVP GIEKYDDWGLGTQVTVSSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTF GSSDMSWVRQAPGKGPEWVSGINSGGGRTLYADSVKGRFTISRDNAKNTLYL QMNSLKSEDTAVYYCATDLYGSSWYTDYWSQGTQVTVSS 213E6-25GS- 2669 EVQLVESGGGLVQAGASLRLSCAASGSTLSRYGVGWFRQAPGKERELVASVD 212C12 WSGSRTYYADSVKGRFTISRDNAKNTGYLQMNSLKPDDTAVYYCAADSSVVP GIEKYDDWGLGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGG GLVQPGGSLRLSCAASGFTFGSSDMSWVRQAPGKGPEWVSGINSGGGRTLYA DSVKGRFTISRDNAKNTLYLQMNSLKSEDTAVYYCATDLYGSSWYTDYWSQG TQVTVSS 213E6-25GS- 2670 EVQLVESGGGLVQAGASLRLSCAASGSTLSRYGVGWFRQAPGKERELVASVD 214E8 WSGSRTYYADSVKGRFTISRDNAKNTGYLQMNSLKPDDTAVYYCAADSSVVP GIEKYDDWGLGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGG GSVQAGGSLRLSCAASGGTFNPYVMAWFRQAPGNEREFVARIRWSGGDAYYD DSVKGRFAITRDAAKNTVHLQMNSLKPEDTAVYYCAAATYGYGSYTYGGSYD LWGQGTQVTVSS 213E6-15GS- 2671 EVQLVESGGGLVQAGASLRLSCAASGSTLSRYGVGWFRQAPGKERELVASVD 213H7 WSGSRTYYADSVKGRFTISRDNAKNTGYLQMNSLKPDDTAVYYCAADSSVVP GIEKYDDWGLGTQVTVSSGGGGSGGGGSGGGGSEVQLVESGGGLVQAGGSLR LSCAASGRTLSSYRMGWFRQAPGKEREFISTISWNGRSTYYADSVKGRFIFS EDNAKNTVYLQMNSLKPEDTAVYYCAAALIGGYYSDVDAWSYWGPGTQVTVS S 214E8-5GS- 2672 EVQLVESGGGSVQAGGSLRLSCAASGGTFNPYVMAWFRQAPGNEREFVARIR 212C12 WSGGDAYYDDSVKGRFAITRDAAKNTVHLQMNSLKPEDTAVYYCAAATYGYG SYTYGGSYDLWGQGTQVTVSSGGGGSEVQLVESGGGLVQPGGSLRLSCAASG FTFGSSDMSWVRQAPGKGPEWVSGINSGGGRTLYADSVKGRFTISRDNAKNT LYLQMNSLKSEDTAVYYCATDLYGSSWYTDYWSQGTQVTVSS 214E8-15GS- 2673 EVQLVESGGGSVQAGGSLRLSCAASGGTFNPYVMAWFRQAPGNEREFVARIR 212C12 WSGGDAYYDDSVKGRFAITRDAAKNTVHLQMNSLKPEDTAVYYCAAATYGYG SYTYGGSYDLWGQGTQVTVSSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGG SLRLSCAASGFTFGSSDMSWVRQAPGKGPEWVSGINSGGGRTLYADSVKGRF TISRDNAKNTLYLQMNSLKSEDTAVYYCATDLYGSSWYTDYWSQGTQVTVSS 214E8-25GS- 2674 EVQLVESGGGSVQAGGSLRLSCAASGGTFNPYVMAWFRQAPGNEREFVARIR 212C12 WSGGDAYYDDSVKGRFAITRDAAKNTVHLQMNSLKPEDTAVYYCAAATYGYG SYTYGGSYDLWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVE SGGGLVQPGGSLRLSCAASGFTFGSSDMSWVRQAPGKGPEWVSGINSGGGRT LYADSVKGRFTISRDNAKNTLYLQMNSLKSEDTAVYYCATDLYGSSWYTDYW SQGTQVTVSS 214E8-15GS- 2675 EVQLVESGGGSVQAGGSLRLSCAASGGTFNPYVMAWFRQAPGNEREFVARIR 213H7 WSGGDAYYDDSVKGRFAITRDAAKNTVHLQMNSLKPEDTAVYYCAAATYGYG SYTYGGSYDLWGQGTQVTVSSGGGGSGGGGSGGGGSEVQLVESGGGLVQAGG SLRLSCAASGRTLSSYRMGWFRQAPGKEREFISTISWNGRSTYYADSVKGRF IFSEDNAKNTVYLQMNSLKPEDTAVYYCAAALIGGYYSDVDAWSYWGPGTQV TVSS 213H7-15GS- 2676 EVQLVESGGGLVQAGGSLRLSCAASGRTLSSYRMGWFRQAPGKEREFISTIS 214 F8 WNGRSTYYADSVKGRFIFSEDNAKNTVYLQMNSLKPEDTAVYYCAAALIGGY YSDVDAWSYWGPGTQVTVSSGGGGSGGGGSGGGGSEVQLVESGGDLVQAGGS LRLSCVASGSTYSINAMGWYRQAPGKLRELVAAFRTGGSTDYADSVKGRFTI SRDTAKNTVYLQMNSLKPEDTAVYYCNAEVIYYPYDYWGQGTQVTVSS 213E6-15GS- 2677 EVQLVESGGGLVQAGASLRLSCAASGSTLSRYGVGWFRQAPGKERELVASVD 214E8 WSGSRTYYADSVKGRFTISRDNAKNTGYLQMNSLKPDDTAVYYCAADSSVVP GIEKYDDWGLGTQVTVSSGGGGSGGGGSGGGGSEVQLVESGGGSVQAGGSLR LSCAASGGTFNPYVMAWFRQAPGNEREFVARIRWSGGDAYYDDSVKGRFAIT RDAAKNTVHLQMNSLKPEDTAVYYCAAATYGYGSYTYGGSYDLWGQGTQVTV SS 214E8-15GS- 2678 EVQLVESGGGSVQAGGSLRLSCAASGGTFNPYVMAWFRQAPGNEREFVARIR 213E6 WSGGDAYYDDSVKGRFAITRDAAKNTVHLQMNSLKPEDTAVYYCAAATYGYG SYTYGGSYDLWGQGTQVTVSSGGGGSGGGGSGGGGSEVQLVESGGGLVQAGA SLRLSCAASGSTLSRYGVGWFRQAPGKERELVASVDWSGSRTYYADSVKGRF TISRDNAKNTGYLQMNSLKPDDTAVYYCAADSSVVPGIEKYDDWGLGTQVTV SS 214F8-15GS- 2679 EVQLVESGGDLVQAGGSLRLSCVASGSTYSINAMGWYRQAPGKLRELVAAFR 213H7 TGGSTDYADSVKGRFTISRDTAKNTVYLQMNSLKPEDTAVYYCNAEVIYYPY DYWGQGTQVTVSSGGGGSGGGGSGGGGSEVQLVESGGGLVQAGGSLRLSCAA SGRTLSSYRMGWFRQAPGKEREFISTISWNGRSTYYADSVKGRFIFSEDNAK NTVYLQMNSLKPEDTAVYYCAAALIGGYYSDVDAWSYWGPGTQVTVSS 214E8-5GS- 2680 EVQLVESGGGSVQAGGSLRLSCAASGGTFNPYVMAWFRQAPGNEREFVARIR 214E8 WSGGDAYYDDSVKGRFAITRDAAKNTVHLQMNSLKPEDTAVYYCAAATYGYG SYTYGGSYDLWGQGTQVTVSSGGGGSEVQLVESGGGSVQAGGSLRLSCAASG GTFNPYVMAWFRQAPGNEREFVARIRWSGGDAYYDDSVKGRFAITRDAAKNT VHLQMNSLKPEDTAVYYCAAATYGYGSYTYGGSYDLWGQGTQVTVSS 212C12-5GS- 2681 EVQLVESGGGLVQPGGSLRLSCAASGFTFGSSDMSWVRQAPGKGPEWVSGIN 212C12 SGGGRTLYADSVKGRFTISRDNAKNTLYLQMNSLKSEDTAVYYCATDLYGSS WYTDYWSQGTQVTVSSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFGS SDMSWVRQAPGKGPEWVSGINSGGGRTLYADSVKGRFTISRDNAKNTLYLQM NSLKSEDTAVYYCATDLYGSSWYTDYWSQGTQVTVSS
(415) TABLE-US-00030 TABLE A-7 Linker sequences SEQ ID Linker NO: Sequences 5GS 2970 GGGGS 7GS 2971 SGGSGGS 9GS 2639 GGGGSGGGS 10GS 2972 GGGGSGGGGS 15GS 2662 GGGGSGGGGSGGGGS 18GS 2973 GGGGSGGGGSGGGGGGGS 20GS 2974 GGGGSGGGGSGGGGSGGGGS 25GS 2975 GGGGSGGGGSGGGGSGGGGSGGGGS 30GS 2976 GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS 35GS 2977 GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS GGGGS G1 hinge 2660 EPKSCDKTHTCPPCP 9GS-G1 hinge 2661 GGGGSGGGSEPKSCDKTHTCPPCP G3 hinge 2640 ELKTPLGDTTHTCPRCPEPKSCDTPPPCPR CPEPKSCDTPPPCPRCPEPKSCDTPPPCPR CP
(416) TABLE-US-00031 TABLE A-8 Sequences of humanized NC41 variants NANOBODY® (V.sub.HH sequence) SEQ ID NO: Sequence NC41 372 EVQLVESGGGLVQAGGSLSISCAASGGSLSNYVLGWFRQAPGKEREFVA AINWRGDITIGPPNVEGRFTISRDNAKNTGYLQMNSLAPDDTAVYYCGA GTPLNPGAYIYDWSYDYWGRGTQVTVSS NC41v01 2999 EVQLLESGGGLVQPGGSLRLSCAASGGSLSNYVLGWFRQAPGKGREFVA AINWRGDITIGPPNVEGRFTISRDNAKNTLYLQMNSLAPEDTAVYYCGA GTPLNPGAYIYDWSYDYWGQGTLVTVSS NC41v02 3000 EVQLLESGGGLVQPGGSLRLSCAASGGSLSNYVLGWFRQAPGKGREFVA AINWRGDITIGPPNVEGRFTISRDNSKNTLYLQMNSLAPEDTAVYYCGA GTPLNPGAYIYDWSYDYWGQGTLVTVSS NC41v03 3001 EVQLLESGGGLVQPGGSLRISCAASGGSLSNYVLGWFRQAPGKGREFVA AINWRGDITIGPPNVEGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCGA GTPLNPGAYIYDWSYDYWGQGTLVTVSS NC41v04 3002 EVQLLESGGGLVQPGGSLSISCAASGGSLSNYVLGWFRQAPGKGREFVA AINWRGDITIGPPNVEGRFTISRDNSKNTLYLQMNSLRPDDTAVYYCGA GTPLNPGAYIYDWSYDYWGQGTLVTVSS NC41v05 3003 EVQLLESGGGLVQPGGSLSISCAASGGSLSNYVLGWFRQAPGKGREFVA AINWRGDITIGPPNVEGRFTISRDNSKNTLYLQMNSLAPEDTAVYYCGA GTPLNPGAYIYDWSYDYWGQGTLVTVSS NC41v06 3004 EVQLLESGGGLVQPGGSLPLSCAASGGSLSNYVLGWFRQAPGKGREFVA AINWRDDITIGPPNVEGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCGA GTPLNPGAYIYDWSYDYWGQGTLVTVSS NC41v07 3005 EVQLLESGGGLVQPGGSLSISCAASGGSLSNYVLGWFRQAPGKGREFVA AINWRGDITIGPPNVEGRFTISRDNAKNTLYLQMNSLAPDDTAVYYCGA GTPLNPGAYIYDWSYDYWGQGTLVTVSS NC41v08 3006 EVQLLESGGGLVQPGGSLSISCAASGGSLSNYVLGWFRQAPGKGREFVA AINWRGDITIGPPNVEGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCGA GTPLNPGAYIYDWSYDYWGQGTLVTVSS NC41v09 3007 EVQLLESGGGLVQPGGSLSISCAASGGSLSNYVLGWFRQAPGKGREFVA AINWRGDITIGPPNVEGRFTISRDNSKNTLYLQMNSLRPDDTAVYYCGA GTPLNPGAYIYDWSYDYWGQGTLVTVSS NC41v10 3008 EVQLLESGGGLVQPGGSLSISCAASGGSLSNYVLGWFRQAPGKGREFVA AINWRGDITIGPPNVEGRFTISRDNAKNTGYLQMNSLAPDDTAVYYCGA GTPLNPGAYIYDWSYDYWGQGTLVTVSS NC41v11 3009 EVQLLESGGGLVQAGGSLSISCAASGGSLSNYVLGWFRQAPGKGREFVA AINWRGDITIGPPNVEGRFTISRDNAKNTGYLQMNSLAPDDTAVYYCGA GTPLNPGAYIYDWSYDYWGQGTLVTVSS NC41v12 3010 EVQLLESGGGLVQPGGSLSISCAASGGSLSNYVLGWFRQAPGKGREFVA AINWRGDITIGPPNVEGRFTISRDNAKNTGYLQMNSLAPDDTAVYYCGA GTPLNPGAYIYDWSYDYWGQGTLVTVSS NC41v13 3011 EVQLLESGGGLVQPGGSLPLSCAASGGSLSNYVLGWFRQAPGKGREFVA AINWRGDITIGPPNVEGRFTISRDNAKNTGYLQMNSLAPEDTAVYYCGA GTPLNPGAYIYDWSYDYWGQGTLVTVSS NC41v14 3012 EVQLLESGGGLVQPGGSLPLSCAASGGSLSNYVLGWFRQAPGKGREFVA AINWRGDITIGPPNVEGRFTISRDNSKNTLYLQMNSLAPEDTAVYYCGA GTPLNPGAYIYDWSYDYWGQGTLVTVSS NC41v15 3013 EVQLLESGGGLVQAGGSLRLSCAASGGSLSNYVLGWFRQAPGKGREFVA AINWRGDITIGPPNVEGRFTISRDNAKNTLYLQMNSLAPEDTAVYYCGA GTPLNPGAYIYDWSYDYWGQGTLVTVSS NC41v17 3014 EVQLLESGGGLVQPGGSLRLSCAASGGSLSNYVLGWFRQAPGKGREFVA AINWRGDITIGPPNVEGRFTISRDNSKNTLYLQMNSLAPEDTAVYYCGA GTPLNPGAYIYDWSYDYWGQGTLVTVSS NC41v18 3015 EVQLLESGGGLVQPGGSLRLSCAASGGSLSNYVLGWFRQAPGKGREFVA AINWRDDITIGPPNVEGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCGA GTPLNPGAYIYDWSYDYWGQGTLVTVSS
(417) TABLE-US-00032 TABLE A-9 Amino acid sequence of multivalent humanized constructs that bind hRSV NANOBODY® SEQ (V.sub.HH ID sequence) NO: Sequence RSV414 2996 EVQLLESGGGLVQPGGSLRISCAASGGSLSNYVLGWFRQAPGKGREFVAAINWR GDITIGPPNVEGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCGAGTPLNPGAYI YDWSYDYWGQGTLVTVSSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRIS CAASGGSLSNYVLGWFRQAPGKGREFVAAINWRGDITIGPPNVEGRFTISRDNS KNTLYLQMNSLRPEDTAVYYCGAGTPLNPGAYIYDWSYDYWGQGTLVTVSSGGG GSGGGGSGGGGSEVQLLESGGGLVQPGGSLRISCAASGGSLSNYVLGWFRQAPG KGREFVAAINWRGDITIGPPNVEGRFTISRDNSKNTLYLQMNSLRPEDTAVYYC GAGTPLNPGAYIYDWSYDYWGQGTLVTVSS RSV426 2997 EVQLLESGGGLVQPGGSLRLSCAASGGSLSNYVLGWFRQAPGKGREFVAAINWR DDITIGPPNVEGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCGAGTPLNPGAYI YDWSYDYWGQGTLVTVSSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLS CAASGGSLSNYVLGWFRQAPGKGREFVAAINWRDDITIGPPNVEGRFTISRDNA KNTLYLQMNSLRPEDTAVYYCGAGTPLNPGAYIYDWSYDYWGQGTLVTVSSGGG GSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGGSLSNYVLGWFRQAPG KGREFVAAINWRDDITIGPPNVEGRFTISRDNAKNTLYLQMNSLRPEDTAVYYC GAGTPLNPGAYIYDWSYDYWGQGTLVTVSS RSV427 2998 EVQLLESGGGLVQPGGSLRLSCAASGGSLSNYVLGWFRQAPGKGREFVAAINWR DDITIGPPNVEGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCGAGTPLNPGAYI YDWSYDYWGQGTLVTVSSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLS CAASGGSLSNYVLGWFRQAPGKGREFVAAINWRDDITIGPPNVEGRFTISRDNS KNTLYLQMNSLRPEDTAVYYCGAGTPLNPGAYIYDWSYDYWGQGTLVTVSSGGG GSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGGSLSNYVLGWFRQAPG KGREFVAAINWRDDITIGPPNVEGRFTISRDNSKNTLYLQMNSLRPEDTAVYYC GAGTPLNPGAYIYDWSYDYWGQGTLVTVSS
(418) TABLE-US-00033 TABLE A-10 Amino acid sequence of multivalent constructs that bind hRSV SEQ ID Construct NO Sequence RSV101 3016 EVQLVESGGGLVQAGGSLRLSCEASGRTYSRYGMGWFRQAPGKEREFVAAVSRLSGPRT VYADSVKGRFTISRDNAENTVYLQMNSLKPEDTAVYTCAAELTNRNSGAYYYAWAYDYW GQGTQVTVSSGGGGSGGGGSGGGGSEVQLVESGGGLVQAGGSLRLSCEASGRTYSRYGM GWFRQAPGKEREFVAAVSRLSGPRTVYADSVKGRFTISRDNAENTVYLQMNSLKPEDTA VYTCAAELTNRNSGAYYYAWAYDYWGQGTQVTVSS RSV102 3017 EVQLVESGGGLVQAGGSLRLSCEASGRTYSRYGMGWFRQAPGKEREFVAAVSRLSGPRTV YADSVKGRFTISRDNAENTVYLQMNSLKPEDTAVYTCAAELTNRNSGAYYYAWAYDYWG QGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQAGGSLRLSCEAS GRTYSRYGMGWFRQAPGKEREFVAAVSRLSGPRTVYADSVKGRFTISRDNAENTVYLQM NSLKPEDTAVYTCAAELTNRNSGAYYYAWAYDYWGQGTQVTVSS RSV103 3018 EVQLVESGGGLVQAGGSLRLSCEASGRTYSRYGMGWFRQAPGKEREFVAAVSRLSGPRT VYADSVKGRFTISRDNAENTVYLQMNSLKPEDTAVYTCAAELTNRNSGAYYYAWAYDYW GQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQA GGSLRLSCEASGRTYSRYGMGWFRQAPGKEREFVAAVSRLSGPRTVYADSVKGRFTISR DNAENTVYLQMNSLKPEDTAVYTCAAELTNRNSGAYYYAWAYDYWGQGTQVTVSS RSV104 3019 EVQLVESGGGLVQAGGSLRLSCEASGRTYSRYGMGWFRQAPGKEREFVAAVSRLSGPRT VYADSVKGRFTISRDNAENTVYLQMNSLKPEDTAVYTCAAELTNRNSGAYYYAWAYDYW GQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQAGGSLRLSCEASGRTYSRYGMGWFRQA PGKEREFVAAVSRLSGPRTVYADSVKGRFTISRDNAENTVYLQMNSLKPEDTAVYTCAA ELTNRNSGAYYYAWAYDYWGQGTQVTVSS RSV105 3020 EVQLVESGGGLVQAGDSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVAAISWSDGSTY YADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAADLTSTNPGSYIYIWAYDYWG QGTQVTVSSGGGGSGGGSEVQLVESGGGLVQAGDSLRLSCAASGRTFSSYAMGWFRQAP GKEREFVAAISWSDGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAADL TSTNPGSYIYIWAYDYWGQGTQVTVSS RSV106 3021 EVQLVESGGGLVQAGDSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVAAISWSDGSTY YADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAADLTSTNPGSYIYIWAYDYWG QGTQVTVSSGGGGSGGGGSGGGGSEVQLVESGGGLVQAGDSLRLSCAASGRTFSSYANG WFRQAPGKEREFVAAISWSDGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVY YCAADLTSTNPGSYIYIWAYDYWGQGTQVTVSS RSV107 3022 EVQLVESGGGLVQAGGSLRLSCAASGRSFSNYVLGWFRQAPGKEREFVAAISFRGDSAI GAPSVEGRFTISRDNAKNTGYLQMNSLVPDDTAVYYCGAGTPLNPGAYIYDWSYDYWGR GTQVTVSSGGGGSGGGSEVQLVESGGGLVQAGGSLRLSCAASGRSFSNYVLGWFRQAPG KEREFVAAISFRGDSAIGAPSVEGRFTISRDNAKNTGYLQMNSLVPDDTAVYYCGAGTP LNPGAYIYDWSYDYWGRGTQVTVSS RSV108 3023 EVQLVESGGGLVQAGGSLRLSCAASGRSFSNYVLGWFRQAPGKEREFVAAISFRGDSAI GAPSVEGRFTISRDNAKNTGYLQMNSLVPDDTAVYYCGAGTPLNPGAYIYDWSYDYWGR GTQVTVSSGGGGSGGGGSGGGGSEVQLVESGGGLVQAGGSLRLSCAASGRSFSNYVLGW FRQAPGKEREFVAAISFRGDSAIGAPSVEGRFTISRDNAKNTGYLQMNSLVPDDTAVYY CGAGTPLNPGAYIYDWSYDYWGRGTQVTVSS RSV109 3024 EVQLVESGGGLVQPGGSLRLSCAASGRTFSSIAMGWFRQAPGKEREFVAAISWSRGRTF YADSVKGRFIISRDDAANTAYLQMNSLKPEDTAVYYCAVDTASWNSGSFIYDWAYDHWG QGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGRTFSSIAMGWFRQAP GKEREFVAAISWSRGRTFYADSVKGRFIISRDDAANTAYLQMNSLKPEDTAVYYCAVDT ASWNSGSFIYDWAYDHWGQGTQVTVSS RSV110 3025 EVQLVESGGGLVQPGGSLRLSCAASGRTFSSIAMGWFRQAPGKEREFVAAISWSRGRTF YADSVKGRFIISRDDAANTAYLQMNSLKPEDTAVYYCAVDTASWNSGSFIYDWAYDHWG QGTQVTVSSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGRTFSSIAMG WFRQAPGKEREFVAAISWSRGRTFYADSVKGRFIISRDDAANTAYLQMNSLKPEDTAVY YCAVDTASWNSGSFIYDWAYDHWGQGTQVTVSS RSV113 3026 EVQLVESGGGLVQPGGSLRLSCAASGLTLDYYALGWFRQAPGKEREGVSCISSSDHST TYTDSVKGRFTISWDNAKNTLYLQMNSLKPGDTAVYYCAADPALGCYSGSYYPRYDYW GQGTQVTVSSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGLTLDYYA LGWFRQAPGKEREGVSCISSSDHSTTYTDSVKGRFTISWDNAKNTLYLQMNSLKPGDT AVYYCAADPALGCYSGSYYPRYDYWGQGTQVTVSS RSV114 3027 EVQLVESGGGWVQAGGSLRLSCAASGRAFSSYAMGWIRQAPGKEREFVAGIDQSGEST AYGASASGRFIISRDNAKNTVHLLMNSLQSDDTAVYYCVADGVLATTLNWDYWGQGTQ VTVSSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGWVQAGGSLRLSCAASGRAFSSYA MGWIRQAPGKEREFVAGIDQSGESTAYGASASGRFIISRDNAKNTVHLLMNSLQSDDT AVYYCVADGVLATTLNWDYWGQGTQVTVSS RSV115 3028 EVQLVESGGGLVQAGGSLRLSCAASGPTFSADTMGWFRQAPGKEREFVATIPWSGGIA YYSDSVKGRFTMSRDNAKNTVDLQMNSLKPEDTALYYCAGSSRIYIYSDSLSERSYDY WGQGTQVTVSSGGGGSGGGGSGGGGGGGSEVQLVESGGGLVQAGGSLRLSCAASGPTF SADTMGWFRQAPGKEREFVATIPWSGGIAYYSDSVKGRFTMSRDNAKNTVDLQMNSLK PEDTALYYCAGSSRIYIYSDSLSERSYDYWGQGTQVTVSS RSV116 3029 EVQLVESGGGLVQAGGSLSISCAASGGSLSNYVLGWFRQAPGKEREFVAAINWRGDIT IGPPNVEGRFTISRDNAKNTGYLQMNSLAPDDTAVYYCGAGTPLNPGAYIYDWSYDYW GRGTQVTVSSGGGGSGGGGSGGGGSEVQLVESGGGLVQAGGSLSISCAASGGSLSNYV LGWFRQAPGKEREFVAAINWRGDITIGPPNVEGRFTISRDNAKNTGYLQMNSLAPDDT AVYYCGAGTPLNPGAYIYDWSYDYWGRGTQVTVSS RSV201 3030 EVQLVESGGGLVQAGGSLRLSCEASGRTYSRYGMGWFRQAPGKEREFVAAVSRLSGPR TVYADSVKGRFTISRDNAENTVYLQMNSLKPEDTAVYTCAAELTNRNSGAYYYAWAYD YWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQAGGSLRLSCAASGPTFSADTMGWF RQAPGKEREFVATIPWSGGIAYYSDSVKGRFTMSRDNAKNTVDLQMNSLKPEDTALYY CAGSSRIYIYSDSLSERSYDYWGQGTQVTVSS RSV202 3031 EVQLVESGGGLVQAGGSLRLSCEASGRTYSRYGMGWFRQAPGKEREFVAAVSRLSGPR TVYADSVKGRFTISRDNAENTVYLQMNSLKPEDTAVYTCAAELTNRNSGAYYYAWAYD YWGQGTQVTVSSGGGGSGGGGSGGGGSEVQLVESGGGLVQAGGSLRLSCAASGPTFSA DTMGWFRQAPGKEREFVATIPWSGGIAYYSDSVKGRFTMSRDNAKNTVDLQMNSLKPE DTALYYCAGSSRIYIYSDSLSERSYDYWGQGTQVTVSS RSV203 3032 EVQLVESGGGLVQAGGSLRLSCEASGRTYSRYGMGWFRQAPGKEREFVAAVSRLSGPR TVYADSVKGRFTISRDNAENTVYLQMNSLKPEDTAVYTCAAELTNRNSGAYYYAWAYD YWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQAGGSLRLS CAASGPTFSADTMGWFRQAPGKEREFVATIPWSGGIAYYSDSVKGRFTMSRDNAKNTV DLQMNSLKPEDTALYYCAGSSRIYIYSDSLSERSYDYWGQGTQVTVSS RSV204 3033 EVQLVESGGGLVQAGDSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVAAISWSDGST YYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAADLTSTNPGSYIYIWAYDY WGQGTQVTVSSGGGGSGGGGSGGGGSEVQLVESGGGLVQAGGSLRLSCAASGRSFSNY VLGWFRQAPGKEREFVAAISFRGDSAIGAPSVEGRFTISRDNAKNTGYLQMNSLVPDD TAVYYCGAGTPLNPGAYIYDWSYDYWGRGTQVTVSS RSV205 3034 EVQLVESGGGLVQAGDSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVAAISWSDGST YYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAADLTSTNPGSYIYIWAYDY WGQGTQVTVSSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGLTLDYY ALGWFRQAPGKEREGVSCISSSDHSTTYTDSVKGRFTISWDNAKNTLYLQMNSLKPGD TAVYYCAADPALGCYSGSYYPRYDYWGQGTQVTVSS RSV206 3035 EVQLVESGGGLVQAGGSLRLSCAASGRSFSNYVLGWFRQAPGKEREFVAAISFRGDSA IGAPSVEGRFTISRDNAKNTGYLQMNSLVPDDTAVYYCGAGTPLNPGAYIYDWSYDYW GRGTQVTVSSGGGGSGGGGSGGGGSEVQLVESGGGLVQAGDSLRLSCAASGRTFSSYA MGWFRQAPGKEREFVAAISWSDGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDT AVYYCAADLTSTNPGSYIYIWAYDYWGQGTQVTVSS RSV207 3036 EVQLVESGGGLVQAGGSLRLSCAASGRSFSNYVLGWFRQAPGKEREFVAAISFRGDSA IGAPSVEGRFTISRDNAKNTGYLQMNSLVPDDTAVYYCGAGTPLNPGAYIYDWSYDYW GRGTQVTVSSGGGGSGGGGSGGGGSEVQLVESGGGLVQAGDSLRLSCAASGRTFSSYA MGWFRQAPGKEREFVAAISWSDGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDT AVYYCAADLTSTNPGSYIYIWAYDYWGQGTQVTVSS RSV301 3037 EVQLVESGGGLVQAGGSLRLSCAASGPTFSADTMGWFRQAPGKEREFVATIPWSGGIA YYSDSVKGRFTMSRDNAKNTVDLQMNSLKPEDTALYYCAGSSRIYIYSDSLSERSYDY WGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQAGGSLRLSCEASGRTYSRYGMGWFR QAPGKEREFVAAVSRLSGPRTVYADSVKGRFTISRDNAENTVYLQMNSLKPEDTAVYT CAAELTNRNSGAYYYAWAYDYWGQGTQVTVSS RSV302 3038 EVQLVESGGGLVQAGGSLRLSCAASGPTFSADTMGWFRQAPGKEREFVATIPWSGGIA YYSDSVKGRFTMSRDNAKNTVDLQMNSLKPEDTALYYCAGSSRIYIYSDSLSERSYDY WGQGTQVTVSSGGGGSGGGGSGGGGSEVQLVESGGGLVQAGGSLRLSCEASGRTYSRY GMGWFRQAPGKEREFVAAVSRLSGPRTVYADSVKGRFTISRDNAENTVYLQMNSLKPE DTAVYTCAAELTNRNSGAYYYAWAYDYWGQGTQVTVSS HHH RSV303 3039 EVQLVESGGGLVQAGGSLRLSCAASGPTFSADTMGWFRQAPGKEREFVATIPWSGGIA YYSDSVKGRFTMSRDNAKNTVDLQMNSLKPEDTALYYCAGSSRIYIYSDSLSERSYDY WGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQAGGSLRLSC EASGRTYSRYGMGWFRQAPGKEREFVAAVSRLSGPRTVYADSVKGRFTISRDNAENTV YLQMNSLKPEDTAVYTCAAELTNRNSGAYYYAWAYDYWGQGTQVTVSS RSV305 3040 EVQLVESGGGLVQPGGSLRLSCAASGLTLDYYALGWFRQAPGKEREGVSCISSSDHST TYTDSVKGRFTISWDNAKNTLYLQMNSLKPGDTAVYYCAADPALGCYSGSYYPRYDYW GQGTQVTVSSGGGGSGGGGSGGGGSEVQLVESGGGLVQAGDSLRLSCAASGRTFSSYA MGWFRQAPGKEREFVAAISWSDGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDT AVYYCAADLTSTNPGSYIYIWAYDYWGQGTQVTVSS RSV306 3041 EVQLVESGGGLVQPGGSLRLSCAASGLTLDYYALGWFRQAPGKEREGVSCISSSDHST TYTDSVKGRFTISWDNAKNTLYLQMNSLKPGDTAVYYCAADPALGCYSGSYYPRYDYW GQGTQVTVSSGGGGSGGGGSGGGGSEVQLVESGGGLVQAGGSLRLSCAASGRSFSNYV LGWFRQAPGKEREFVAAISFRGDSAIGAPSVEGRFTISRDNAKNTGYLQMNSLVPDDT AVYYCGAGTPLNPGAYIYDWSYDYWGRGTQVTVSS RSV400 3042 EVQLVESGGGLVQAGDSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVAAISWSDGST YYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAADLTSTNPGSYIYIWAYDY WGQGTQVTVSSGGGGSGGGGSGGGGSEVQLVESGGGLVQAGDSLRLSCAASGRTFSSY AMGWFRQAPGKEREFVAAISWSDGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPED TAVYYCAADLTSTNPGSYIYIWAYDYWGQGTQVTVSSGGGGSGGGGSGGGGSEVQLVE SGGGLVQAGDSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVAAISWSDGSTYYADSV KGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAADLTSTNPGSYIYIWAYDYWGQGTQ VTVSS RSV401 3043 EVQLVESGGGLVQAGDSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVAAISWSDGST YYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAADLTSTNPGSYIYIWAYDY WGQGTQVTVSSGGGGSGGGGSGGGGSEVQLVESGGGLVQAGDSLRLSCAASGRTFSSY AMGWFRQAPGKEREFVAAISWSDGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPED TAVYYCAADLTSTNPGSYIYIWAYDYWGQGTQVTVSSGGGGSGGGGSGGGGSEVQLVE SGGGLVQPGGSLRLSCAASGLTLDYYALGWFRQAPGKEREGVSCISSSDHSTTYTDSV KGRFTISWDNAKNTLYLQMNSLKPGDTAVYYCAADPALGCYSGSYYPRYDYWGQGTQV TVSS RSV402 3044 EVQLVESGGGLVQPGGSLRLSCAASGLTLDYYALGWFRQAPGKEREGVSCISSSDHST TYTDSVKGRFTISWDNAKNTLYLQMNSLKPGDTAVYYCAADPALGCYSGSYYPRYDYW GQGTQVTVSSGGGGSGGGGSGGGGSEVQLVESGGGLVQAGDSLRLSCAASGRTFSSYA MGWFRQAPGKEREFVAAISWSDGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDT AVYYCAADLTSTNPGSYIYIWAYDYWGQGTQVTVSSGGGGSGGGGSGGGGSEVQLVES GGGLVQAGDSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVAAISWSDGSTYYADSVK GRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAADLTSTNPGSYIYIWAYDYWGQGTQV TVSS RSV403 3045 EVQLVESGGGLVQAGDSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVAAISWSDGST YYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAADLTSTNPGSYIYIWAYDY WGQGTQVTVSSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGLTLDYY ALGWFRQAPGKEREGVSCISSSDHSTTYTDSVKGRFTISWDNAKNTLYLQMNSLKPGD TAVYYCAADPALGCYSGSYYPRYDYWGQGTQVTVSSGGGGSGGGGSGGGGSEVQLVES GGGLVQAGDSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVAAISWSDGSTYYADSVK GRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAADLTSTNPGSYIYIWAYDYWGQGTQV TVSS RSV404 3046 EVQLVESGGGLVQAGGSLRLSCAASGRSFSNYVLGWFRQAPGKEREFVAAISFRGDSA IGAPSVEGRFTISRDNAKNTGYLQMNSLVPDDTAVYYCGAGTPLNPGAYIYDWSYDYW GRGTQVTVSSGGGGSGGGGSGGGGSEVQLVESGGGLVQAGGSLRLSCAASGRSFSNYV LGWFRQAPGKEREFVAAISFRGDSAIGAPSVEGRFTISRDNAKNTGYLQMNSLVPDDT AVYYCGAGTPLNPGAYIYDWSYDYWGRGTQVTVSSGGGGSGGGGSGGGGSEVQLVESG GGLVQAGGSLRLSCAASGRSFSNYVLGWFRQAPGKEREFVAAISFRGDSAIGAPSVEG RFTISRDNAKNTGYLQMNSLVPDDTAVYYCGAGTPLNPGAYIYDWSYDYWGRGTQVTV SS RSV405 3047 EVQLVESGGGLVQAGGSLRLSCEASGRTYSRYGMGWFRQAPGKEREFVAAVSRLSGPR TVYADSVKGRFTISRDNAENTVYLQMNSLKPEDTAVYTCAAELTNRNSGAYYYAWAYD YWGQGTQVTVSSGGGGSGGGGSGGGGSEVQLVESGGGLVQAGGSLRLSCEASGRTYSR YGMGWFRQAPGKEREFVAAVSRLSGPRTVYADSVKGRFTISRDNAENTVYLQMNSLKP EDTAVYTCAAELTNRNSGAYYYAWAYDYWGQGTQVTVSSGGGGSGGGGSGGGGSEVQL VESGGGLVQAGGSLRLSCEASGRTYSRYGMGWFRQAPGKEREFVAAVSRLSGPRTVYA DSVKGRFTISRDNAENTVYLQMNSLKPEDTAVYTCAAELTNRNSGAYYYAWAYDYWGQ GTQVTVSS RSV406 3048 EVQLVESGGGLVQPGGSLRLSCAASGRTFSSIAMGWFRQAPGKEREFVAAISWSRGRT FYADSVKGRFIISRDDAANTAYLQMNSLKPEDTAVYYCAVDTASWNSGSFIYDWAYDH WGQGTQVTVSSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGRTFSSI AMGWFRQAPGKEREFVAAISWSRGRTFYADSVKGRFIISRDDAANTAYLQMNSLKPED TAVYYCAVDTASWNSGSFIYDWAYDHWGQGTQVTVSSGGGGSGGGGSGGGGSEVQLVE SGGGLVQPGGSLRLSCAASGRTFSSIAMGWFRQAPGKEREFVAAISWSRGRTFYADSV KGRFIISRDDAANTAYLQMNSLKPEDTAVYYCAVDTASWNSGSFIYDWAYDHWGQGTQ VTVSS RSV407 3049 EVQLVESGGGLVQAGGSLSISCAASGGSLSNYVLGWFRQAPGKEREFVAAINWRGDIT IGPPNVEGRFTISRDNAKNTGYLQMNSLAPDDTAVYYCGAGTPLNPGAYIYDWSYDYW GRGTQVTVSSGGGGSGGGGSGGGGSEVQLVESGGGLVQAGGSLSISCAASGGSLSNYV LGWFRQAPGKEREFVAAINWRGDITIGPPNVEGRFTISRDNAKNTGYLQMNSLAPDDT AVYYCGAGTPLNPGAYIYDWSYDYWGRGTQVTVSSGGGGSGGGGSGGGGSEVQLVESG GGLVQAGGSLSISCAASGGSLSNYVLGWFRQAPGKEREFVAAINWRGDITIGPPNVEG RFTISRDNAKNTGYLQMNSLAPDDTAVYYCGAGTPLNPGAYIYDWSYDYWGRGTQVTV SS RSV408 3050 EVQLVESGGGLVQAGGSLSISCAASGGSLSNYVLGWFRQAPGKEREFVAAINWRGDIT IGPPNVEGRFTISRDNAKNTGYLQMNSLAPDDTAVYYCGAGTPLNPGAYIYDWSYDYW GRGTQVTVSSAAAEVQLVESGGGLVQAGGSLSISCAASGGSLSNYVLGWFRQAPGKER EFVAAINWRGDITIGPPNVEGRFTISRDNAKNTGYLQMNSLAPDDTAVYYCGAGTPLN PGAYIYDWSYDYWGRGTQVTVSSAAAEVQLVESGGGLVQAGGSLSISCAASGGSLSNY VLGWFRQAPGKEREFVAAINWRGDITIGPPNVEGRFTISRDNAKNTGYLQMNSLAPDD TAVYYCGAGTPLNPGAYIYDWSYDYWGRGTQVTVSS RSV409 3051 EVQLVESGGGLVQAGGSLSISCAASGGSLSNYVLGWFRQAPGKEREFVAAINWRGDIT IGPPNVEGRFTISRDNAKNTGYLQMNSLAPDDTAVYYCGAGTPLNPGAYIYDWSYDYW GRGTQVTVSSGGGGSGGGSEVQLVESGGGLVQAGGSLSISCAASGGSLSNYVLGWFRQ APGKEREFVAAINWRGDITIGPPNVEGRFTISRDNAKNTGYLQMNSLAPDDTAVYYCG AGTPLNPGAYIYDWSYDYWGRGTQVTVSSGGGGSGGGSEVQLVESGGGLVQAGGSLSI SCAASGGSLSNYVLGWFRQAPGKEREFVAAINWRGDITIGPPNVEGRFTISRDNAKNT GYLQMNSLAPDDTAVYYCGAGTPLNPGAYIYDWSYDYWGRGTQVTVSS RSV410 3052 EVQLVESGGGLVQAGGSLSISCAASGGSLSNYVLGWFRQAPGKEREFVAAINWRGDIT IGPPNVEGRFTISRDNAKNTGYLQMNSLAPDDTAVYYCGAGTPLNPGAYIYDWSYDYW GRGTQVTVSSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQAGGSLSISCAASGGS LSNYVLGWFRQAPGKEREFVAAINWRGDITIGPPNVEGRFTISRDNAKNTGYLQMNSL APDDTAVYYCGAGTPLNPGAYIYDWSYDYWGRGTQVTVSSGGGGSGGGGSGGGGSGGG GSEVQLVESGGGLVQAGGSLSISCAASGGSLSNYVLGWFRQAPGKEREFVAAINWRGD ITIGPPNVEGRFTISRDNAKNTGYLQMNSLAPDDTAVYYCGAGTPLNPGAYIYDWSYD YWGRGTQVTVSS RSV411 3053 EVQLVESGGGLVQAGGSLSISCAASGGSLSNYVLGWFRQAPGKEREFVAAINWRGDIT IGPPNVEGRFTISRDNAKNTGYLQMNSLAPDDTAVYYCGAGTPLNPGAYIYDWSYDYW GRGTQVTVSSGGGGSGGGGSGGGGSEVQLVESGGGLVQAGGSLSISCAASGGSLSNYV LGWFRQAPGKEREFVAAINWRGDITIGPPNVEGRFTISRDNAKNTGYLQMNSLAPDDT AVYYCGAGTPLNPGAYIYDWSYDYWGRGTQVTVSSGGGGSGGGGSGGGGSEVQLVESG GGLVQPGGSLRLSCAASGLTLDYYALGWFRQAPGKEREGVSCISSSDHSTTYTDSVKG RFTISWDNAKNTLYLQMNSLKPGDTAVYYCAADPALGCYSGSYYPRYDYWGQGTQVTV SS RSV412 3054 EVQLVESGGGLVQPGGSLRLSCAASGLTLDYYALGWFRQAPGKEREGVSCISSSDHST TYTDSVKGRFTISWDNAKNTLYLQMNSLKPGDTAVYYCAADPALGCYSGSYYPRYDYW GQGTQVTVSSGGGGSGGGGSGGGGSEVQLVESGGGLVQAGGSLSISCAASGGSLSNYV LGWFRQAPGKEREFVAAINWRGDITIGPPNVEGRFTISRDNAKNTGYLQMNSLAPDDT AVYYCGAGTPLNPGAYIYDWSYDYWGRGTQVTVSSGGGGSGGGGSGGGGSEVQLVESG GGLVQAGGSLSISCAASGGSLSNYVLGWFRQAPGKEREFVAAINWRGDITIGPPNVEG RFTISRDNAKNTGYLQMNSLAPDDTAVYYCGAGTPLNPGAYIYDWSYDYWGRGTQVTV SS RSV413 3055 EVQLVESGGGLVQAGGSLSISCAASGGSLSNYVLGWFRQAPGKEREFVAAINWRGDIT IGPPNVEGRFTISRDNAKNTGYLQMNSLAPDDTAVYYCGAGTPLNPGAYIYDWSYDYW GRGTQVTVSSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGLTLDYYA LGWFRQAPGKEREGVSCISSSDHSTTYTDSVKGRFTISWDNAKNTLYLQMNSLKPGDT AVYYCAADPALGCYSGSYYPRYDYWGQGTQVTVSSGGGGSGGGGSGGGGSEVQLVESG GGLVQAGGSLSISCAASGGSLSNYVLGWFRQAPGKEREFVAAINWRGDITIGPPNVEG RFTISRDNAKNTGYLQMNSLAPDDTAVYYCGAGTPLNPGAYIYDWSYDYWGRGTQVTV SS RSV502 3056 EVQLVESGGGLVQAGGSLRLSCEASGRTFSSYGMGWFRQAPGKEREFVAAVSRLSGPR TVYADSVKGRFTISRDNAENTVYLQMNSLKPEDTAVYTCAAELTNRNPGAYYYTWAYD YWGQGTQVTVSSGGGGSGGGGSGGGGSEVQLVESGGGLVQAGGSLRLSCEASGRTFSS YGMGWFRQAPGKEREFVAAVSRLSGPRTVYADSVKGRFTISRDNAENTVYLQMNSLKP EDTAVYTCAAELTNRNPGAYYYTWAYDYWGQGTQVTVSSGGGGSGGGGSGGGGSEVQL VESGGGLVQAGGSLRLSCEASGRTFSSYGMGWFRQAPGKEREFVAAVSRLSGPRTVYA DSVKGRFTISRDNAENTVYLQMNSLKPEDTAVYTCAAELTNRNPGAYYYTWAYDYWGQ GTQVTVSS RSV513 3588 EVQLVESGGGLVQAGDSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVAAISWSDGST YYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAADLTSTNPGSYIYIWAYDY WGQGTQVTVSSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGLTLDYY ALGWFRQAPGKEREGVSCISSSDHTTTYTDSVKGRFTISWDNAKNTLYLQMNSLKPED TAVYYCAADPALGCYSGSYYPRYDFWGQGTQVTVSSGGGGSGGGGSGGGGSEVQLVES GGGLVQAGDSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVAAISWSDGSTYYADSVK GRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAADLTSTNPGSYIYIWAYDYWGQGTQV TVSS RSV514 3589 EVQLVESGGGLVQAGDSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVAAISWSDGST YYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAADLTSTNPGSYIYIWAYDY WGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGLTLDYYALGWFR QAPGKEREGVSCISSSDHTTTYTDSVKGRFTISWDNAKNTLYLQMNSLKPEDTAVYYC AADPALGCYSGSYYPRYDFWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQAGDSLR LSCAASGRTFSSYAMGWFRQAPGKEREFVAAISWSDGSTYYADSVKGRFTISRDNAKN TVYLQMNSLKPEDTAVYYCAADLTSTNPGSYIYIWAYDYWGQGTQVTVSS RSV515 3590 EVQLVESGGGLVQAGDSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVAAISWSDGST YYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAADLTSTNPGSYIYIWAYDY WGQGTQVTVSSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRVSCAASGFTFNDY IMGWFRQAPGKERMFIAAISGTGTIKYYGDLVRGRFTISRDNAKNTVYLRIDSLNPED TAVYYCAARQDYGLGYRESHEYDYWGQGTQVTVSSGGGGSGGGGSGGGGSEVQLVESG GGLVQAGDSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVAAISWSDGSTYYADSVKG RFTISRDNAKNTVYLQMNSLKPEDTAVYYCAADLTSTNPGSYIYIWAYDYWGQGTQVT VSS RSV516 3591 EVQLVESGGGLVQAGDSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVAAISWSDGST YYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAADLTSTNPGSYIYIWAYDY WGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRVSCAASGFTFNDYIMGWFR QAPGKERMFIAAISGTGTIKYYGDLVRGRFTISRDNAKNTVYLRIDSLNPEDTAVYYC AARQDYGLGYRESHEYDYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQAGDSLRL SCAASGRTFSSYAMGWFRQAPGKEREFVAAISWSDGSTYYADSVKGRFTISRDNAKNT VYLQMNSLKPEDTAVYYCAADLTSTNPGSYIYIWAYDYWGQGTQVTVSS
(419) TABLE-US-00034 TABLE C-1 Overview of the RFFIT tests on periplasmic fractions of the NANOBODIES ® (V.sub.HH sequences) of the invention as described in Example 14. Rabies neutralizing antibody titer Sample (50% dilution) Remark Polyclonal anti- Lama C <0.5 IU/ml (<1/9) no neutralisation hRSV periplasmic fractions Polyclonal anti- Lama 1 <0.5 IU/ml (<1/9) no neutralisation rabies vaccine 210 virus periplasmic fractions Polyclonal Lama 2 3.18 IU/ml (1/88) strong anti-rabies 211 neutralisation glycoprotein G periplasmic fractions Monoclonal 192-D3 <0.5 IU/ml (<1/9) no neutralisation anti-hRSV 192-B6 <0.5 IU/ml (<1/9) no neutralisation periplasmic 192-C4 <0.5 IU/ml (<1/9) no neutralisation fractions Monoclonal 202-C1 <0.5 IU/ml (<1/9) no neutralisation anti-H5N1 202-F4 <0.5 IU/ml (<1/9) no neutralisation periplasmic 202-B7 <0.5 IU/ml (<1/9) no neutralisation fractions Anti-rabies 213-D6 <0.5 IU/ml (<1/9) no neutralisation glycoprotein G 213-E6 5.31 (1/140) strong periplasmic neutralisation fractions, 213-B7 0.62 (1/16) neutralisation total elution 213-D7 0.62 (1/16) neutralisation 213-H7 0.83 (1/22) neutralisation Anti-rabies 214-A8 1.42 (1/38) neutralisation glycoprotein G 214-E8 <0.5 IU/ml (1/11) 0.42 = minor periplasmic neutralisation, fractions, but below cut-off monoclonal 214-F8 0.65 (1/17) neutralisation antibody 214-C10 <0.5 IU/ml (<1/9) 0.25 = minor eluted neutralisation, but below cut-off 214-D10 <0.5 IU/ml (<1/9) 0.25 = minor neutralisation, but below cut-off 214-H10 0.67 (1/18) neutralisation Anti-“other 202-D4 <0.5 IU/ml (<1/9) no neutralisation viral coat 202-F7 <0.5 IU/ml (<1/9) no neutralisation protein” 192-D2 <0.5 IU/ml (<1/9) no neutralisation control 192-F4 <0.5 IU/ml (<1/9) no neutralisation periplasmic fractions
(420) TABLE-US-00035 TABLE C-2 Binding of selected NANOBODIES ® (V.sub.HH sequences) to immobilized F.sub.TM-protein in Surface Plasmon Resonance. name clone ka (1/Ms) kd (1/s) KD (M) NB1 192-C4 1.13E+06 8.46E−03 7.47E−09 NB2 191-D3 1.59E+06 3.24E−03 2.05E−09 NB4 192-H1 1.65E+06 6.11E−03 3.72E−09 NB5 192-A8 3.22E+05 9.37E−04 2.91E−09 NB6 191-E4 2.98E+05 2.08E−04 7.00E−10 NB9 192-C6 1.15E+06 8.08E−03 7.00E−09 NB10 192-F2 8.07E+05 5.77E−03 7.14E−09 NB11 191-B9 1.94E+05 4.92E−03 2.54E−08 NB13 192-H2 8.29E+05 1.28E−02 1.54E−08 NB14 192-B1 2.29E+05 1.27E−02 5.55E−08 NB15 192-C10 1.75E+05 6.13E−04 3.49E−09
(421) TABLE-US-00036 TABLE C-3 Classification of viral fusion proteins based on the structural motifs of their post-fusion conformations Protein Virus family Virus species database code Class I Orthomyxoviridae Influenza A virus HA 1HA0, 3HMG, Influenza C virus HEF 1HTM, 1QU1, 1FLC Paramyxoviridae Simian parainfluenza virus 5 F 2B9B, 1SVF Human parainfluenza virus F 1ZTM Newcastle disease virus F 1G5G Respiratory syncytial F 1G2C Measles F2 Sendai F2 Filoviridae Ebola virus gp2 1EBO, 2EBO Retroviridae Moloney murine leukemia virus TM 1AOL Human immunodeficiency 1ENV, 1AIK virus 1 gp41 Simian immunodeficiency virus gp41 2SIV, 2EZO Human T cell leukemia virus 1 gp21 1MG1 Human syncytin-2 TM 1Y4M Visna virus TM 1JEK Coronaviridae Mouse hepatitis virus S2 1WDG SARS corona virus E2 2BEQ, 1WYY Class II Flaviviridae Tick-borne encephalitis virus E 1URZ, 1SVB Dengue 2 and 3 virus E2 1OK8 IUZG, Yellow Fever E 10AN, 1TG8 West Nile E Togaviridae Semliki forest virus E1 1E9W, 1RER Sindbis E1 Class III Rhabdoviridae Rabies virus G 2GUM Vesicular stomatitis virus G Herpesviridae Herpes simplex virus gB 2CMZ
(422) TABLE-US-00037 TABLE C-4 Sequence analysis of hRSV NANOBODIES ® (Vim sequences) from new libraries 206 207 212 213 clone family epitope clone family epitope clone family epitope clone family epitope clone family epitope 5C1 1 IV-VI 5A1 4sub3 IV-VI 8C8 2 IV-VI 5A8 7 binder 7B9 18 IV-VI 8A1 n = 4 5G2 n = 13 5A6 3 IV-VI 5A10 8 binder 7E7 20 binder 8G1 5H1 8E11 n = 9 14A6 n = 4 25B3 6B1 8F11 16A6 5A2 4sub1 IV-VI 8H2 13F11 22D56 5B2 n = 36 8H3 15B8 7G1 15 binder 5V3 13A3 15G11 5A8 16 II 5D2 13C5 17C10 7B2 n = 5 5E2 13H1 21E7 22A4 5F3 13H2 21F8 22E10 5G3 15E6 5G4 6 IV-VI 22H4 5H2 17A3 6G5 n = 5 14H3 21 IV-VI 5H3 25G8 8E6 24D6 22 IV-VI 5C1 6D1 5 IV-VI 13A10 23E5 23 IV-VI 8F2 8D5 n = 12 21H10 14E2 25 IV-VI 8G4 13B4 5C8 11 IV-VI 23G1 28 binder 13A1 13B6 6D4 n = 6 13A4 13E6 8B10 13B1 13F4 8E10 13B2 15H3 15A7 13C1 17E5 15E10 13C3 19D3 13C7 12 IV-VI 13D6 19F3 15A9 13E2 25C4 15F11 13E3 25E3 17A9 14 IV-VI 15A5 8E2 9 IV-VI 15E11 19 IV-VI 15A6 8C6 10 IV-VI 19A5 27 IV-VI 15B2 15A1 13 II 15H8 29 II 15B3 6H2 NC41 15E5 15C5 17 IV-VI 6A8 30 IV-VI 17C2 NC39 8B11 32 IV-VI 17D4 8A6 24 IV-VI 17G4 25F3 26 II 19B2 25H9 31 IV-VI 25A4 17E1 33 n = 4 II 25A9 21A4 25B5 25A11 25G2 25C8 25H5 NC23 34 II 25E11 8G3 4sub2 13B5 n = 5 15F2 19E2 25D1
(423) TABLE-US-00038 TABLE C-5 Characteristics of NANOBODIES ® (Vim sequences) that bind hRSV F- protein Competition Binding Synagis ® RSV neutralization hRSV Fab kinetic analysis IC50 (nM) (n = 2) Clone Family Epitope EC50 EC50 ka (1/Ms) kd (1/s) KD Long A-2 B1 191D3 LG 3sub2 II 1.5E−10 5.9E−09 1.5E+06 2.8E−03 1.9E−09 253 227 — 1E4 LG 3sub2 II 6.6E−11 4.5E−09 8.0E+05 1.3E−03 1.6E−09 380 298 ND 7B2 16 II 9.0E−11 1.9E−09 5.7E+05 6.5E−04 1.1E−09 91 177 2690 NC23 34 II 1.0E−10 2.3E−09 8.0E+05 7.4E−04 9.2E−10 144 109 — 15H8 29 II 8.3E−10 3.9E−08 1.2E+06 2.1E−02 1.6E−08 200 218 2340 NC41 29 II 4.1E−10 3.2E−08 8.2E+05 6.7E−03 8.1E−09 58 26 4000 15133 4sub1 IV-VI 5.8E−11 — 4.1E+05 2.7E−04 6.7E−10 — — 1274 191E4 LG 21 IV-VI 8.3E−11 — 5.7E+05 1.5E−04 2.7E−10 — — 4327 Synagis ® II 2.8E+05 1.8E−04 6.4E−10 4 2.5 1.7
(424) TABLE-US-00039 TABLE C-6 Nomenclature for multivalent NANOBODIES ® (V.sub.HH sequences) directed against hRSV F-protein SEQ ID Type Name Construct NO: Bivalent RSV101 191D3-15GS-191D3 2382 RSV102 191D3-25GS-191D3 2383 RSV103 191D3-35GS-191D3 2384 RSV104 191D3-9GS-191D3 2385 RSV105 7B2-9GS-7B2 2386 RSV106 7B2-15GS-7B2 2387 RSV107 15H8-9GS-15H8 2388 RSV108 15H8-15GS-15H8 2389 RSV109 NC23-9GS-NC23 2390 RSV110 NC23-15GS-NC23 2391 RSV113 15B3-15GS-15B3 2392 RSV114 NC39-20GS-NC39 2393 RSV115 191E4-18GS-191E4 2394 RSV116 NC41-15GS-NC41 2395 Biparatope RSV201 191D3-9GS-191E4 2396 RSV202 191D3-15GS-191E4 2397 RSV203 191D3-25GS-191E4 2398 RSV204 7B2-15GS-15H8 2399 RSV205 7B2-15GS-15B3 2400 RSV206 15H8-15GS-15B3 2401 RSV207 15H8-15GS-7B2 2402 RSV301 191E4-9GS-191D3 2403 RSV302 191E4-15GS-191D3 2404 RSV303 191E4-25GS-191D3 2405 RSV305 15B3-15GS-7B2 2406 RSV306 15B3-15GS-15H8 2407 RSV513 7B2-15GS-19E2-15GS-7B2 3584 RSV514 7B2-9GS-19E2-9GS-7B2 3585 RSV515 7B2-15GS-8A1 -15GS-7B2 3586 RSV516 7B2-9GS-8M-9GS-7B2 3587 Trivalent RSV400 7B2-15GS-7B2-15GS-7B2 2408 RSV401 7B2-15GS-7B2-15GS-15B3 2409 RSV402 15B3-15GS-7B2-15GS-7B2 2410 RSV403 7B2-15GS-15B3-15GS-7B2 2411 RSV404 15H8-15GS-15H8-15GS-15H8 2412 RSV405 191D3-15GS-191D3-15GS-191D3 2413 RSV406 NC23-15GS-NC23-15GS-NC23 2414 RSV407 NC41-15GS-NC41-15GS-NC41 2415 RSV408 NC41-AAA-NC41-AAA-NC41 2989 RSV409 NC41-9GS-NC41-9GS-NC41 2990 RSV410 NC41-20GS-NC41-20GS-NC41 2991 RSV411 NC41-15GS-NC41-15GS-15B3 2992 RSV412 15B3-15GS-NC41-15GS-NC41 2993 RSV413 NC41-15GS-15B3-15GS-NC41 2994 RSV414 NC41v03-15GS-NC41v03-15GS-NC41v03 2996 RSV426 NC41v06-15GS-NC41v06-15GS-NC41v06 2997 RSV427 NC41v18-15GS-NC41v18-15GS-NC41v18 2998 RSV502 1E4-15GS-1E4-15GS-1E4 2995
(425) TABLE-US-00040 TABLE C-7 Reactivity of monovalent NANOBODIES ® (Vim sequences) with antigen extracts of HEp-2 cells infected with different escape mutants of the Long strain NANOBODY ® Virus (V.sub.HH sequence) R47F/4 R47F/7 RAK13/4 R7C2/11 R7C2/1 R7.936/1 R7.936/4 R7.936/6 R9.432/1 RRA3 192C4 Δ Δ Δ Δ 191D3 • Δ Δ Δ Δ 191E4 Δ Δ Δ Δ Δ .diamond-solid. • • Δ Δ 192F2 Δ Δ Δ Δ 191C7 Δ Δ Δ Δ Δ Δ Δ Δ Δ Δ 15B3 Δ Δ Δ Δ Δ Δ .diamond-solid. Δ Δ NC23 Δ • Δ Δ Δ Δ 15H8 Δ Δ Δ Δ 7B2 .diamond-solid. Δ Δ Δ Δ NC41 .diamond-solid. Δ Δ Δ Δ aa substituion N262Y N2681 N216D/ K272T K272E V447A K433T K432T S436F N262Y/ N262Y R429S
(426) TABLE-US-00041 TABLE C-8 Reactivity of monovalent and bivalent NANOBODIES ® (V.sub.HH sequences) with antigen extracts of HEp-2 cells infected with different escape mutants of the Long strain Virus NANOBODY ® (V.sub.HH sequence) R7C2/11 R7C2/1 R7.936/4 7B2 Δ RSV106: 7B2-7B2 .diamond-solid. Δ RSV400: 7B2-7B2-7B2 Δ Δ RSV403: 7B2-15B3-7B2 Δ Δ Δ 15B3 Δ Δ Δ RSV113: 15B3-15B3 Δ Δ .diamond-solid. 191D3 Δ RSV101: 191D3-191D3 Δ 15H8 Δ RSV108: 15H8-15H8 .diamond-solid. Δ NC23 • Δ RSV110: NC23-NC23 • Δ 191E4 Δ Δ Δ aa substitution K272T K272E K433T
(427) TABLE-US-00042 TABLE C-9 Relative viral genomic RNA in lungs of treated mice 3 and 5 days post viral inoculation 3 days post viral inoculation relative gRNA level PBS LGB1 LGB2 Synagis Mouse 1 8.64 6.31 45.80 2.13 Mouse 2 13.09 3.23 45.90 1.97 Mouse 3 43.23 2.94 8.50 4.01 Mouse 4 12.10 1.01 32.99 1.63 Mouse 5 31.79 2.42 60.99 0.00 Average 21.77 3.18 38.84 1.95 SD 13.43 1.74 17.57 1.28 5 days post viral inoculation relative gRNA level PBS RSV101 12D2biv Synagis Mouse 1 170.69 16.96 214.74 4.82 Mouse 2 53.45 10.96 466.40 4.81 Mouse 3 471.42 3.84 350.39 7.20 Mouse 4 404.66 5.60 418.76 6.32 Mouse 5 342.39 2.19 193.26 4.15 Average 288.52 7.91 328.71 5.46 SD 172.47 6.04 121.32 1.25
(428) TABLE-US-00043 TABLE C-10 Viral titers in mouse treated with 202-C8, 191-D3 or only PBS, 4 and 6 days post virus inoculation as described in Example 37 Day 4 lung titers (TCID50/ml lung homogenate) Group Mouse 1 Mouse 2 Mouse 3 Geo. Mean StDev PBS (n = 3) 355656 63246 63246 160716 137843 191D3 (n = 3) 112468 112468 632456 285797 245124 202-C8 (n = 3) 0 0 0 0 0 Day 6 lung titers (TCID50/ml lung homogenate) Group Mouse 1 Mouse 2 Mouse 3 Geo. Mean StDev PBS (n = 3) 63426 112468 112468 96121 23119 191-D3 (n = 3) 63246 112468 112468 96061 23203 202-C8 (n = 3) 0 0 0 0 0
(429) TABLE-US-00044 TABLE C-11 Animal weight and viral titers after intranasal administration of NANOBODY ® (Vim sequence) into mice challenged with virus at different time points after inoculation of the NANOBODY ® (Vim sequence) (see Example 38) Weight Weight Weight Weight Weight Day 0 Day 1 Day 2 Day 3 Day 4 Lung titer Day 4 202-C8 4 h mouse 1 18.15 18.32 17.67 18.5 18.23 0 202-C8 4 h mouse 2 20.67 20.42 20.43 20.94 20.93 0 202-C8 4 h mouse 3 19.72 19.67 18.97 19.68 19.77 0 Average 19.51 19.47 19.02 19.71 19.64 0 St. Dev. 1.27 1.06 1.38 1.22 1.35 0 202-C8 24 h mouse 1 18.76 18.81 18.52 18.83 18.85 0 202-C8 24 h mouse 2 19.48 19.62 18.99 18.96 19.13 0 202-C8 24 h mouse 3 18.73 18.55 18.18 18.34 18.32 0 202-C8 24 h mouse 4 19.19 19.27 18.9 19.48 19.32 0 202-C8 24 h mouse 5 18.95 19.24 18.36 18.96 19.06 0 202-C8 24 h mouse 6 18.99 18.81 18.21 18.66 18.91 0 average 19.02 19.05 18.53 18.87 18.93 0 St. Dev. 0.28 0.39 0.35 0.38 0.34 0 202-C8 48 h mouse 1 17.88 17.5 17.44 17.43 17.81 9355 202-C8 48 h mouse 2 17.29 17.01 16.94 17.11 17.37 355656 202-C8 48 h mouse 3 19.42 19.08 19.2 19.33 19.44 93550 202-C8 48 h mouse 4 19.47 19.53 18.89 19.31 19.51 0 202-C8 48 h mouse 5 19.73 19.55 19.34 19.54 20.02 0 202-C8 48 h mouse 6 18.92 18.84 18.72 18.47 18.91 63250 202-C8 48 h mouse 7 17.94 17.65 17.82 17.74 19.49 0 average 18.66 18.45 18.34 18.42 18.94 74544 St. Dev. 0.95 1.04 0.93 1.00 0.98 129378 PBS 4 h mouse 1 18.97 18.89 18.69 18.05 16.95 3556500 PBS 4 h mouse 2 18.15 18.36 18.13 17.32 15.95 6325000 PBS 4 h mouse 3 19.54 19.9 19.68 18.11 16.87 6325000 Average 18.89 19.05 18.83 17.83 16.59 5402167 St. Dev. 0.70 0.78 0.78 0.44 0.56 1598394 PBS 48 h mouse 1 20.01 19.73 19.59 18.76 17.66 3556500 PBS 48 h mouse 2 21.43 21.68 20.9 20.06 19.39 632500 PBS 48 h mouse 3 18.78 19.02 18.74 17.67 16.8 632500 average 20.07 20.14 19.74 18.83 17.95 1607167 St. Dev. 1.33 1.38 1.09 1.20 1.32 1688172 191-D3 4 h mouse 1 20.3 20.42 20.11 19.72 19.28 6324600 191-D3 4 h mouse 2 18.39 18.54 18.66 18.38 18.33 9355000 191-D3 4 h mouse 3 18.39 18.82 18.44 17.77 16.3 3556500 Average 19.03 19.26 19.07 18.62 17.97 6412033 St. Dev. 1.10 1.01 0.91 1.00 1.52 2900239 191-D3 24 h mouse 1 18.94 18.63 18.62 18.21 18.29 6324600 191-D3 24 h mouse 2 19.46 19.62 19.4 18.48 18.09 63250000 191-D3 24 h mouse 3 19.63 19.58 19.83 19.18 18.51 2000000 191-D3 24 h mouse 4 19.03 18.94 19.07 18.45 17.49 6325000 191-D3 24 h mouse 5 18.91 18.72 19 17.84 17.32 935500 average 19.19 19.10 19.18 18.43 17.94 15767020 St. Dev. 0.33 0.47 0.46 0.49 0.51 26657313 191-D3 48 h mouse 1 19.5 19.39 18.93 19.04 18 3556500 191-D3 48 h mouse 2 19.53 19.3 19.2 18.76 17.94 3556500 191-D3 48 h mouse 3 20.02 20.23 20.46 19.81 19.26 9355000 191-D3 48 h mouse 4 18.21 18.09 18.12 17.75 17.29 935500 191-D3 48 h mouse 5 18.38 18.17 18.32 17.92 16.53 6325000 191-D3 48 h mouse 6 21.19 20.83 20.55 20.34 18.98 632460 average 19.47 19.34 19.26 18.94 18.00 4060160 St. Dev. 1.10 1.09 1.04 1.02 1.02 3322192
(430) TABLE-US-00045 TABLE C-12 Test items for use in the study described in Example 42 Alternative Name names Reference RSV NB2 191D3 SEQ ID NO: 159 in present application ALX-0081 12A2H1-3a- SEQ ID NO: 98 in WO 06/122825 12A2H1 RANKL008a SEQ ID NO: 759 in WO 08/142164
(431) TABLE-US-00046 TABLE C-13 Study design for study described in Example 42 Single Dose Number of Group Substance Route (mg/kg) animals 1 RSV NB2 i.v. 4 3 2 ALX-0081 i.v. 5 3 3 RANKL008A i.v. 5 3 4 RSV NB2 i.t. 3.6 28 5 ALX-0081 i.t. 3.1 28 6 RANKL008A i.t. 3.2 28 7 — — — 8
(432) TABLE-US-00047 TABLE C-14 LLOQ and ULOQ for determination of RSV NB2 in rat plasma and BALF samples as described in Example 42 LLOQ (ng/ml) ULOQ (ng/ml) Plasma/BALF Plasma/BALF PK ELISA Plate level level Plate level level RSV NB2 0.4 4.0 20.0 200.0
(433) TABLE-US-00048 TABLE C-15 LLOQ and ULOQ for determination of ALX-0081 in rat plasma and BALF sample as described in Example 42 LLOQ (ng/ml) ULOQ (ng/ml) PK ELISA Plate level Plasma/BALF Plate level Plasma/BALF ALX-0081 0.75 3.75 40.0 200.0
(434) TABLE-US-00049 TABLE C-16 LLOQ and ULOQ for determination of RANKL008A in rat plasma and BALF samples as described in Example 42 LLOQ (ng/ml) ULOQ (ng/ml) Plasma/BALF Plasma/BALF PK ELISA Plate level level Plate level level RANKL008A 0.1 1.0 7.5 75.0
(435) TABLE-US-00050 TABLE C-17 Individual plasma concentration-time data of RSV NB2, ALX-0081, and RANKL008A after a single i.v. bolus dose of RSV NB2 (4 mg/kg), ALX-0081 (5 mg/kg) and RANKL008A (5 mg/kg), respectively to male Wistar rats Plasma concentration after i.v. administration (μg/mL) Nominal RSV NB2 ALX-0081 RANKL008A Time ID 1 ID 2 ID 3 ID 4 ID 5 ID 6 ID 7 ID 8 ID 9 3 min 23.6 34.5 32.1 60.4 63.2 NS 94.3 107 100 (5 min) 15 min 5.16 10.7 10.6 9.18 14.1 NS 95.7 94.8 92.8 30 min 3.61 5.91 3 3.15 3.37 4.55 88.4 85.9 74.1 1 hr NS 5.12 2.36 1.09 1.31 1.84 81.5 73.8 NS 2 hr NS NS 0.763 0.498 0.594 NS 58.7 55.9 NS 4 hr NS NS 0.161 0.219 0.315 0.328 35.8 35.1 NS 6 hr NS NS 0.056 0.125 0.161 0.116 / / / 8 hr / / / / / / 17.1 18.8 NS 24 hr BQL NS BQL BQL BQL BQL 3.17 3.94 NS 48 hr / / / / / / 0.902 0.988 NS NS: No sample could be obtained (refer to in vivo report) BQL: Below Quantification Limit
(436) TABLE-US-00051 TABLE C-18 Individual plasma concentration-time data of RSV NB2, ALX-0081, and RANKL008A after a single i.t. dose of RSV NB2 (3.6 mg/kg), ALX-0081 (3.1 mg/kg) and RANKL008A (3.2 mg/kg), respectively to male Wistar rats. 6/8 hr time-point: 6 hr for RSV NB2 and ALX-0081, 8 hr for RANKL008A Plasma concentration after i.t. administration (μg/mL) RSV NB2 ALX-0081 RANKL008A Time ID Concentration ID Concentration ID Concentration 3 min 10 0.158 38 0.056 66 0.004 11 0.085 39 0.013 67 0.030 12 0.081 40 0.029 68 0.006 13 0.127 41 0.077 69 0.005 20 min 14 0.204 42 0.102 70 0.072 15 0.167 43 0.102 71 0.081 16 0.131 44 0.097 72 0.151 17 0.267 45 0.070 73 0.083 1 hr 18 0.202 46 0.122 74 0.401 19 0.167 47 0.112 75 0.541 20 0.120 48 0.049 76 0.305 21 0.120 49 0.109 77 1.077 2 hr 22 BQL 50 0.041 78 0.279 23 0.230 51 0.100 79 0.389 24 0.091 52 0.084 80 0.705 25 0.202 53 0.091 81 0.489 4 hr 26 0.113 54 0.069 82 0.965 27 0.150 55 0.077 83 0.601 28 0.080 56 0.053 84 0.934 29 0.129 57 0.085 85 0.672 6/8 hr 30 0.125 58 0.034 86 0.869 31 0.071 59 0.048 87 1.42 32 0.108 60 0.070 88 1.16 33 0.091 61 0.059 89 0.606 24 hr 34 0.024 62 0.014 90 0.493 35 0.024 63 0.022 91 0.450 36 0.025 64 0.014 92 0.434 37 0.036 65 0.020 93 0.342
(437) TABLE-US-00052 TABLE C-19 Mean plasma concentration-time data of RSV NB2, ALX-0081, and RANKL008A after a single i.t. dose of RSV NB2 (3.6 mg/kg), ALX-0081 (3.1 mg/kg) and RANKL008A (3.2 mg/kg), respectively to male Wistar rats Plasma concentration after i.t. administration (μg/mL) RSV NB2 ALX-0081 RANKL008A (ID 10-37) (ID 38-65) (ID 66-93) Time Average SD Average SD Average SD 3 min 0.113 0.037 0.044 0.028 0.012 0.013 20 min 0.192 0.058 0.093 0.015 0.097 0.037 1 hr 0.152 0.040 0.098 0.033 0.581 0.345 2 hr 0.175 0.074 0.079 0.026 0.465 0.181 4 hr 0.118 0.030 0.071 0.014 0.793 0.184 6 hr 0.099 0.023 0.052 0.015 / / 8 hr / / / / 1.01 0.35 24 hr 0.027 0.006 0.018 0.004 0.430 0.063
(438) TABLE-US-00053 TABLE C-20 Individual Basic Pharmacokinetic parameters of RSV NB2, ALX-0081, and RANKL008A after a single i.v. dose of RSV NB2 (4 mg/kg), ALX-0081 (5 mg/kg) and RANKL008A (5 mg/kg) to Wistar Rats. i.v.: RSV NB2 4 mg/kg; ALX-0081/RANKL008A 5 mg/kg ALX-0081 ALX-0081 RANKL008A RANKL008A RSV NB2 Parameter Unit ID 4 ID 5 ID 7 ID 8 ID 3 C(0) ug/mL 96.7 92.0 94.3 110 42.3 Vss mL/kg 255 250 91.5 92.8 250 CL mL/hr/kg 363 311 9.17 8.82 363 MRT hr 0.702 0.804 9.98 10.5 0.690 t½ λz hr 2.01 2.12 13.2 12.0 0.926 λz Lower hr 2 2 24 24 0.5 λz Upper hr 6 6 48 48 6 AUClast hr * ug/mL 13.4 15.6 528 550 11.0 AUCextrap % 2.51 3.09 3.16 3.03 0.560 AUCinf hr * ug/mL 13.8 16.1 545 567 11.0 AUCinfiD hr * kg/mL 0.0028 0.0032 0.1091 0.1134 0.0028
(439) TABLE-US-00054 TABLE C-21 Mean Basic Pharmacokinetic parameters of RSV NB2, ALX-0081, and RANKL008A after a single i.v. dose of RSV NB2 (4 mg/kg), ALX-0081 (5 mg/kg) and RANKL008A (5 mg/kg) to Wistar Rats i.v.: RSV NB2 4 mg/kg; ALX-0081/RANKL008A 5 mg/kg ALX-0081 RANKL008A CV CV Parameter Unit Average % Average % RSV NB2 C(0) ug/mL 94.3 4 102 11 42.3 Vss mL/kg 252 1 92.1 1 250 CL mL/hr/kg 337 11 9.00 3 363 MRT hr 0.753 10 10.2 4 0.690 t½ λz hr 2.06 4 12.6 7 0.926 λz Lower hr 2 0 24 0 0.5 λz Upper hr 6 0 48 0 6 AUClast hr * ug/mL 14.5 10 539 3 11.0 AUCextrap % 2.80 15 3.09 3 0.560 AUCinf hr * ug/mL 14.9 11 556 3 11.0 AUCinf/D hr * kg/mL 0.003 9 0.111 3 0.003
(440) TABLE-US-00055 TABLE C-22 Basic Pharmacokinetic parameters of RSV NB2, ALX-0081, and RANKL008A after a single i.v. dose of RSV NB2 (3.6 mg/kg), ALX-0081 (3.1 mg/kg) and RANKL008A (3.2 mg/kg) to Wistar Rats i.t. administration ALX-0081 RANKL008A RSV NB2 Parameter Unit 3.1 mg/kg 3.2 mg/kg 3.6 mg/kg Vss/F mL/kg 36339 2833 21853 CL/F mL/hr/kg 2407 130 1641 MRT hr 15.1 21.7 13.3 t½ λz hr 10.5 13.0 9.48 λz Lower hr 2 8 4 λz Upper hr 24 24 24 t½ λz 0.979 1.000 0.999 AUClast hr*ug/mL 1.02 16.5 1.83 AUCextrap % 20.8 32.8 16.8 AUCinf hr*ug/mL 1.29 24.6 2.19 tmax hr 1 8 0.330 Cmax ug/ml 0.098 1.01 0.192 AUCinf/D hr*kg/mL 0.0004 0.0077 0.0006 F % 13.9 6.90 22.1 Vss/F = MRT*CL (MRT not corrected for MAT) Estimation F incorrect if CL i.v. and CL i.t. are different; Note dose i.v. ≠ i.t.
(441) TABLE-US-00056 TABLE C-23 Individual observed BALF concentrations of RSV NB2, ALX-0081, and RANKL008A after a single intratracheal administration of RSV NB2 (3.6 mg/kg), ALX-0081 (3.1 mg/kg) and RANKL008A (3.2 mg/kg) to male rats Nomi- BALF concentrations after i.t. administration (μg/mL) nal RSV NB2 ALX-0081 RANKL008A Time ID Concentration ID Concentration ID Concentration 3 min 10 46.2 38 145 66 32.3 11 65.0 39 57.9 67 56.1 12 23.0 40 69.2 68 27.0 13 36.7 41 115 69 80.2 20 min 14 32.8 42 40.4 70 14.4 15 54.8 43 148 71 87.9 16 70.2 44 93.4 72 43.3 17 68.1 45 55.7 73 22.4 1 hr 18 134 46 179 74 124 19 50.7 47 80.6 75 70.3 20 35.8 48 62.4 76 33.8 21 18.4 49 35.8 77 49.8 2 hr 22 BQL 50 33.7 78 16.1 23 22.1 51 36.9 79 58.3 24 26.1 52 111 80 49.0 25 32.6 53 37.1 81 22.3 4 hr 26 14.9 54 32.7 82 24.8 27 60.9 55 2.44 83 11.4 28 45.0 56 85.1 84 95.0 29 4.81 57 50.5 85 24.9 6/8 hr 30 24.4 58 36.2 86 15.6 31 43.6 59 90.1 87 42.1 32 21.6 60 51.9 88 72.4 33 33.1 61 74.6 89 30.2 24 hr 34 9.53 62 20.9 90 32.7 35 19.1 63 13.2 91 14.6 36 10.7 64 16.5 92 7.48 37 17.0 65 14.6 93 6.91 BQL: below the quantification limit
(442) TABLE-US-00057 TABLE C-24 Mean observed BALF concentrations of RSV NB2, ALX-0081, and RANKL008A after a single intratracheal administration of RSV NB2 (3.6 mg/kg), ALX-0081 (3.1 mg/kg) and RANKL008A (3.2 mg/kg) to male rats BALF concentration after i.t. administration (μg/mL) RSV NB2 ALX-0081 RANKL008A (ID 10-37) (ID 38-65) (ID 66-93) Nominal Time Average SD Average SD Average SD 3 min 96.8 40.4 48.9 24.4 42.7 17.6 20 min 84.3 47.9 35.7 32.9 56.5 17.2 1 hr 89.4 62.4 69.4 39.2 59.7 51.1 2 hr 54.6 37.5 36.4 20.4 26.9 5.3 4 hr 42.7 34.6 39 37.9 31.4 26.1 6 hr 63.2 23.9 40.1 24.1 / / 8 hr / / / / 30.7 9.9 24 hr 16.3 3.4 15.4 12.1 14.1 4.7
(443) TABLE-US-00058 TABLE C-25 Individual theoretical amount (BALF Concentration × 10 mL) of RSV NB2, ALX-0081, and RANKL008A in BALF after single intratracheal administration of RSV NB2 (3.6 mg/kg), ALX-0081 (3.1 mg/kg) and RANKL008A (3.2 mg/kg) to male rats BALF Theoretical Amount after i.t. Administration (μg) RSV NB2 ALX-0081 RANKL008A Nominal Time ID Amount ID Amount ID Amount 4 min 10 462 38 1446 66 323 11 650 39 579 67 561 12 230 40 692 68 270 13 367 41 1155 69 802 20 min 14 328 42 404 70 144 15 548 43 1479 71 879 16 702 44 934 72 433 17 681 45 557 73 224 1 hr 18 1338 46 1788 74 1238 19 507 47 806 75 703 20 358 48 624 76 338 21 184 49 358 77 498 2 hr 22 BQL 50 337 78 161 23 221 51 369 79 583 24 261 52 1109 80 490 25 326 53 371 81 223 4 hr 26 149 54 327 82 248 27 609 55 24.4 83 114 28 450 56 851 84 950 29 48.1 57 505 85 249 6/8 hr 30 244 58 362 86 156 31 436 59 901 87 421 32 216 60 519 88 724 33 331 61 746 89 302 24 hr 34 95.3 62 209 90 327 35 191 63 132 91 146 36 107 64 165 92 74.8 37 170 65 146 93 69.1 BQL: below the quantification limit
(444) TABLE-US-00059 TABLE C-26 Mean (+SD) theoretical amount (BALF Concentration × 10 mL) of RSV NB2 (3.6 mg/kg), ALX-0081 (3.1 mg/kg) and RANKL008A (3.2 mg/kg) in BALF after intratracheal administration BALF theoretical amount after i.t. administration (μg) RSV NB2 ALX-0081 RANKL008A (ID 10-37) (ID 38-65) (ID 66-93) Nominal Time Average SD Average SD Average SD 4 min 427 176 968 404 489 244 20 min 565 172 843 479 420 329 1 hr 597 511 894 624 694 392 2 hr 269 53 546 375 364 204 4 hr 314 261 427 346 390 379 6 hr 307 99 632 239 / / 8 hr / / / / 401 241 24 hr 141.0 47.2 163 34 154 121
(445) TABLE-US-00060 TABLE C-27 Individual recovered volume of BALF after two lavages with DPBS (2 × 5mL) after a single intratracheal administration of RSV NB2 (3.6 mg/kg), ALX-0081 (3.1 mg/kg) and RANKL008A (3.2 mg/kg) to male rats Recovered Volume of BALF after lavages Nominal RSV NB2 ALX-0081 RANKL008A Time ID BALF (mL) ID BALF (mL) ID BALF (mL) 4 min 10 5.5 38 7.5 66 8.0 11 6.5 39 6.5 67 8.0 12 8.5 40 8.5 68 4.0 13 7.5 41 7.5 69 8.5 20 min 14 8.0 42 7.0 70 7.5 15 6.0 43 8.0 71 3.0 16 6.5 44 8.0 72 6.0 17 8.5 45 7.5 73 8.0 1 hr 18 6.5 46 8.0 74 7.0 19 6.5 47 7.5 75 6.0 20 7.5 48 8.0 76 7.5 21 7.5 49 7.0 77 8.0 2 hr 22 5.5 50 8.0 78 6.0 23 6.0 51 8.0 79 7.5 24 6.5 52 6.5 80 8.0 25 7.0 53 7.5 81 8.0 4 hr 26 5.5 54 8.0 82 7.0 27 5.0 55 8.0 83 6.5 28 9.5 56 9.0 84 7.0 29 8.0 57 7.5 85 7.5 6/8 hr 30 7.0 58 8.0 86 7.0 31 7.0 59 9.0 87 6.5 32 7.0 60 6.0 88 7.5 33 8.5 61 8.5 89 9.0 24 hr 34 6.5 62 7.5 90 8.0 35 6.5 63 7.5 91 7.5 36 7.5 64 8.5 92 8.0 37 7.0 65 6.5 93 5.5
(446) TABLE-US-00061 TABLE C-28 Individual actual amount (BALF Concentration x recovered volume) of RSV NB2, ALX-0081, and RANKL008A in BALF after a single intratracheal administration of RSV NB2 (3.6 mg/kg), ALX-0081 (3.1 mg/kg) and RANKL008A (3.2 mg/kg) to male rats BALF Actual Amount after i.t. Administration (μg) RSV NB2 ALX-0081 RANKL008A Nominal Time ID Amount ID Amount ID Amount 4 min 10 254 38 1084 66 258 11 422 39 377 67 449 12 195 40 588 68 108 13 275 41 866 69 682 20 min 14 262 42 283 70 108 15 329 43 1183 71 264 16 456 44 747 72 260 17 579 45 418 73 179 1 hr 18 869 46 1430 74 867 19 330 47 605 75 422 20 269 48 499 76 254 21 138 49 250 77 399 2 hr 22 BQL 50 270 78 96.4 23 132 51 295 79 438 24 170 52 721 80 392 25 228 53 278 81 179 4 hr 26 81.9 54 262 82 174 27 305 55 19.5 83 74.3 28 428 56 766 84 665 29 38.5 57 379 85 187 6/8 hr 30 171 58 289 86 109 31 305 59 811 87 274 32 151 60 311 88 543 33 281 61 634 89 272 24 hr 34 62.0 62 157 90 262 35 124 63 98.7 91 110 36 80.0 64 140 92 59.9 37 119 65 95.2 93 38.0 BQL: below the quantification limit
(447) TABLE-US-00062 TABLE C-29 Mean actual amount (BALF Concentration × recovered volume) of RSV NB2, ALX-0081, and RANKL008A in BALF after a single intratracheal administration RSV NB2 (3.6 mg/kg), ALX-0081 (3.1 mg/kg) and RANKL008A (3.2 mg/kg) to male rats BALF actual amount after i.t. Administration (μg) RSV NB2 ALX-0081 RANKL008A (ID 10-37) (ID 38-65) (ID 66-93) Nominal Time Average SD Average SD Average SD 4 min 287 97 729 310 374 248 20 min 406 140 658 401 203 74 1 hr 401 322 696 512 485 265 2 hr 177 48 391 220 276 165 4 hr 213 185 357 311 275 265 6 hr 227 77 512 254 / / 8 hr / / / / 299 180 24 hr 96.5 30.4 123 30 117 101
(448) TABLE-US-00063 TABLE C-30 Individual theoretical amount (BALF Concentration × 10 mL) normalized by dose (%) of RSV NB2, ALX-0081, and RANKL008A in BALF after a single intratracheal administration of RSV NB2 (3.6 mg/kg), ALX-0081 (3.1 mg/kg) and RANKL008A (3.2 mg/kg) to male rats BALF Theoretical Amount normalized by dose (%) RANKL008A Nominal RSV NB2 ALX-0081 Amount/D Time ID Amount/D (%) ID Amount/D (%) ID (%) 4 min 10 40.5 38 147 66 31.3 11 57.0 39 58.8 67 54.4 12 20.2 40 70.2 68 26.2 13 32.2 41 117 69 77.8 20 min 14 28.7 42 41.0 70 14.0 15 48.1 43 150 71 85.4 16 61.6 44 94.8 72 42.0 17 59.7 45 56.5 73 21.8 1 hr 18 117.3 46 182 74 120 19 44.5 47 81.8 75 68.3 20 31.4 48 63.3 76 32.8 21 16.2 49 36.3 77 48.4 2 hr 22 BQL 50 34.3 78 15.6 23 19.3 51 37.5 79 56.6 24 22.9 52 113 80 47.6 25 28.6 53 37.6 81 21.7 4 hr 26 13.1 54 33.2 82 24.1 27 53.4 55 2.48 83 11.1 28 39.5 56 86.4 84 92.3 29 4.22 57 51.3 85 24.2 6/8 hr 30 21.4 58 36.7 86 15.1 31 38.3 59 91.5 87 40.9 32 18.9 60 52.7 88 70.3 33 29.0 61 75.8 89 29.3 24 hr 34 8.36 62 21.2 90 31.8 35 16.8 63 13.4 91 14.2 36 9.36 64 16.7 92 7.26 37 15.0 65 14.9 93 6.71 BQL: below the quantification limit
(449) TABLE-US-00064 TABLE C-31 Individual actual amount (BALF Concentration × recovered volume) normalized by dose (%) of RSV NB2 (3.6 mg/kg), ALX-0081 (3.1 mg/kg) and RANKL008A (3.2 mg/kg) in BALF after intratracheal administration BALF Actual Amount normalized by dose (%) RSV NB2 ALX-0081 RANKL008A Time ID Amount/D (%) ID Amount/D (%) ID Amount/D (%) 4 min 10 22.3 38 110 66 25.1 11 37.0 39 38.2 67 43.6 12 17.1 40 59.7 68 10.5 13 24.1 41 87.9 69 66.2 20 min 14 23.0 42 28.7 70 10.5 15 28.8 43 120 71 25.6 16 40.0 44 75.8 72 25.2 17 50.8 45 42.4 73 17.4 1 hr 18 76.3 46 145 74 84.1 19 28.9 47 61.4 75 41.0 20 23.6 48 50.6 76 24.6 21 12.1 49 25.4 77 38.7 2 hr 22 BQL 50 27.4 78 9.4 23 11.6 51 30.0 79 42.5 24 14.9 52 73.2 80 38.1 25 20.0 53 28.2 81 17.3 4 hr 26 7.19 54 26.6 82 16.9 27 26.7 55 1.98 83 7.21 28 37.5 56 77.8 84 64.6 29 3.37 57 38.5 85 18.1 6/8 hr 30 15.0 58 29.4 86 10.6 31 26.8 59 82.3 87 26.6 32 13.2 60 31.6 88 52.7 33 24.6 61 64.4 89 26.4 24 hr 34 5.44 62 15.9 90 25.4 35 10.9 63 10.0 91 10.6 36 7.02 64 14.2 92 5.81 37 10.5 65 9.66 93 3.69 BQL: below the quantification limit
(450) TABLE-US-00065 TABLE C-32 Mean (+SD) theoretical amount (BALF Concentration × 10 mL) normalized by dose (%) of RSV NB2 (3.6 mg/kg), ALX-0081 (3.1 mg/kg) and RANKL008A (3.2 mg/kg) in BALF after intratracheal administration BALF theoretical amount/Dose (%) RSV NB2 ALX-0081 RANKL008A (ID 10-37) (ID 38-65) (ID 66-93) Time Average SD Average SD Average SD 4 min 37.5 15.5 98.3 41.0 47.5 23.7 20 min 49.5 15.1 85.6 48.6 40.8 32.0 1 hr 52.3 44.8 90.7 63.3 67.4 38.0 2 hr 23.6 4.7 55.5 38.1 35.4 19.8 4 hr 27.6 22.9 43.4 35.1 37.9 36.8 6 hr 26.9 8.7 64.2 24.3 / / 8 hr / / / / 38.9 23.4 24 hr 12.4 4.1 16.5 3.4 15.0 11.7
(451) TABLE-US-00066 TABLE C-33 Mean actual amount (BALF Concentration × recovered volume) normalized by dose (%) of RSV NB2 (3.6 mg/kg), ALX-0081 (3.1 mg/kg) and RANKL008A (3.2 mg/kg) in BALF after intratracheal administration BALF actual amount/Dose (%) RSV NB2 ALX-0081 RANKL008A (ID 10-37) (ID 38-65) (ID 66-93) Time Average SD Average SD Average SD 4 min 25.1 8.5 74.0 31.5 36.3 24.1 20 min 35.7 12.3 66.8 40.7 19.7 7.2 1 hr 35.2 28.2 70.7 51.9 47.1 25.7 2 hr 15.5 4.2 39.7 22.3 26.8 16.0 4 hr 18.7 16.2 36.2 31.6 26.7 25.7 6 hr 19.9 6.8 51.9 25.8 / / 8 hr / / / / 29.1 17.5 24 hr 8.46 2.66 12.5 3.1 11.4 9.8
(452) TABLE-US-00067 TABLE C-34 Alternative screening of NANOBODIES ® (V.sub.HH sequences) described in Example 44 % Binding % Binding % Inhibition 101F % SEQ RSV-A Hep2-B1 Fab Inhibition ID Previous Fold 1:200 1:100 1:300 1:1000 Synagis Clone NO: Llama Selection Family screen Epitope blanc 1:50 PE PE PE PE PE 1:10 PE PMP8A1 249 206 R1 trypsin 1 101F 3.7 98% 92% nd 55% 20% PMP8B10 342 207 R1 tryspin 11 101F 3.5 94% 84% 56% 31% 6% PMP13A1 274 206 R1 + 2 101F 4sub1 101F 2.9 84% 65% 74% 46% 13% PMP13B4 318 206 R1 + 2 101F 5 101F 2.7 75% 56% 82% 47% 20% PMP13C1 278 206 R1 + 2 101F 4sub1 101F 3.0 104% 86% 57% 37% 9% PMP19E2 301 206 R1 101F; R2 peptide 4sub2 101F 3.5 87% 58% 74% 27% 5% PMP13D1 308 206 R1 + 2 101F 4sub3 101F 3.1 93% 75% 78% 52% 16% PMP13E12 2580 207 R1 + 2 101F 14 101F 3.7 97% 75% 74% 28% 8% PMP23E5 365 212 R1 + 2 RSV 101F 23 101F 3.4 103% 82% 37% 16% nd PMP1B2 166 156 R1 RSV trypsin LG21 LG191E4 101F 3.8 88% 85% 82% 58% 25% PMP1A2 389 156 RQ RSV trypsin LG34 101F 4.0 86% 66% 82% 27% 5% PMP7B2 354 212 R1 trypsin 16 Synagis 4.7 61% 41% 70% PMP19C4 371 207 R1 101F; R2 peptide 29 15H8 Synagis 2.5 72% 50% 39% PMP1A6 404 156 R1 RSV trypsin LG Synagis 4.2 57% 39% 67% PMP1G8 2578 156 R1 RSV trypsin LG Synagis 3.7 73% 43% 57% PMP1E4 211 156 R1 RSV trypsin LG3-2 Synagis 3.6 55% 55% 55% PMP1G3 159 156 R1 RSV trypsin LG3-2 LG191D3 Synagis 3.4 52% 45% 52% PMP1E5 167 156 R1 RSV trypsin LG3-1 Synagis 3.4 54% 37% 41% PMP20B2 2576 156 R1 101F LG3-1 Synagis 3.0 32% 32% 33% PMP20C1 2577 156 R1 101F LG40 Synagis 2.7 37% 35% 33%
(453) TABLE-US-00068 TABLE C-35 Overview of immunizations, sampling and neutralizing antibody titers of the llamas. Immunisation experiment 75a 75b Cocktail nr C127 C127 RFFIT titer (50% dilution) Date Day Llama 183 Llama 196 Tissue collection Llama 183 Llama 196 Start immunisation Day 25/07/07 0 2.5 IU 2.5 IU 10 ml pre-immune blood <0.50 IU/ml <0.50 IU/ml (<1/9) (<1/9) Day 01/08/07 7 2.5 IU 2.5 IU — Day 21/08/07 27 10 ml immune blood 2 IU/ml 6 IU/ml (1/66) (1/179) Day 22/08/07 28 2.5 IU 2.5 IU — Day 29/08/07 35 2.5 IU 2.5 IU Day 31/08/07 37 10 ml immune blood 22 IU/ml 27 IU/ml (1/674) (1/789) Day 05/09/07 42 150 ml immune blood (PBL1) 37 IU/ml 33 IU/ml lymph node biopsy: unsuccessful (1/989) (1/896) Day 12/09/07 49 150 ml immune blood (PBL2) 22.72 IU/ml 14.86 IU/ml (1/674) (1/441) Day 20/09/07 57 2.5 IU 2.5 IU Day 25/09/07 62 150 ml immune blood (PBL3) 22.25 IU/ml 35.35 IU/ml (1/673) (1/1071)
(454) TABLE-US-00069 TABLE C-36 In vitro neutralizing potency of monovalent NANOBODY ® (V.sub.HH sequence) clones with the RFFIT assay CVS-11 neutralizing antibody titer NANOBODY ® (V.sub.HH sequence) ATCC VR 959, sequence G protein: NCBI EU126641 Clone Elusion 50% dilution IU.sup.a/ml IU/mg IU/μM.sup.b nM IC.sub.50.sup.c Mab 8-2 Ascites mouse 1/303250 10108.33 nd.sup.d nd nd Mab RV1C5 100 μg IgG.sub.2a/ml PBS (Santa 1/4985 165.15 1651.5 193500 0.17 Cruz sc-57995) 214-C10 trypsin 1.sup.st + mab 2.sup.d round 1/122 4.24 10.60 0.16 219.67 214-F8 trypsin 1.sup.st + mab 2.sup.d round 1/33 1.15 7.19 0.11 324.85 214-A8 trypsin 1.sup.st + mab 2.sup.d round 1/263 9.12 7.93 0.12 292.97 214-E8 trypsin.sup.st + mab 2.sup.d round 1/140 4.87 9.37 0.14 248.86 213-E6 Mab 1.sup.st + trypsin 2.sup.d round 1/3238 112.33 170.20 2.54 13.66 213-B7 Mab 1.sup.st + trypsin 2.sup.d round 1/140 4.87 7.38 0.11 315.86 213-D7 Mab 1.sup.st + trypsin 2.sup.d round 1/147 5.10 7.61 0.11 305.37 213-D6 Mab 1.sup.st + trypsin 2.sup.d round <1/9 <0.50 <0.48 <0.01 >7816.67 213-H7 Mab 1.sup.st + trypsin 2.sup.d round 1/49 1.71 12.21 0.18 191.43 192-C4 Anti HRSV.sup.e <1/9 <0.50 <0.63 <0.01 >5881.11 192-A8 Anti HRSV <1/9 <0.50 <0.77 <0.02 >4838.89 191-E4 Anti HRSV <1/9 <0.50 <0.63 <0.01 >5955.56 212-A2 Trypsin 1.sup.st and 2.sup.d round 1/47 1.62 1.72 0.03 1340.00 212-B2 Trypsin 1.sup.st and 2.sup.d round 1/75 2.60 3.66 0.05 634.27 212-G2 Trypsin 1.sup.st and 2.sup.d round 1/263 9.12 9.31 0.14 249.66 212-F6 Trypsin 1.sup.st and 2.sup.d round 1/4057 122.43 114.42 1.71 17.67 212-612 Trypsin 1.sup.st and 2.sup.d round 1/1028 31.00 20.00 0.30 101.02 212-C12 Trypsin 1.sup.st and 2.sup.d round 1/11363 394.26 308.02 4.60 7.55 214-H10 trypsin 1.sup.st + mab 2.sup.d round 1/330 11.44 8.17 0.12 284.24 .sup.aInternational Unit (IU) .sup.b1 mg NANOBODY ® (V.sub.HH sequence)/ml = 67 μM .sup.c= mg/ml × 50% dilution × 67000 .sup.dnot determined .sup.ehuman respiratory syncytial virus
(455) TABLE-US-00070 TABLE C-37 Effect of combinations of NANOBODIES ® (V.sub.HH sequences) on the neutralizing potency compared to single NANOBODIES ® (V.sub.HH sequences). CVS neutralizing antibody titer strain CVS-11, ATCC VR 959, sequence Combinations of NANOBODIES ® G protein: NCBI EU126641 (V.sub.HH sequences) 50% dilution IU.sup.a/ml IU/mg IU/μM.sup.b nM IC.sub.50.sup.c 10 μl 212-C12 + 10 μl medium 1/19426 643.58 205.62 3.07 10.80 10 μl medium + 10 μl 213-E6 1/2987 98.64 65.76 0.98 33.65 10 μl 212-C12 + 10 μl 213-E6 1/10757 356.35 153.93 2.30 14.42 10 μl 212-C12 + 10 μl medium 1/8302 232.64 85.85 1.28 21.87 10 μl medium + 10 μl 213-H7 1/150 4.22 30.14 0.45 62.53 10 μl 212-C12 + 10 μl 213-H7 1/4346 122.3 85.52 1.28 22.05 10 μl 212-C12 + 10 μl medium 1/21220 597.18 220.36 3.29 8.56 10 μl medium + 10 μl 214-E8 1/280 7.38 14.19 0.21 124.43 10 μl 212-C12 + 10 μl 214-E8 1/8635 243.01 150.01 2.24 12.57 10 μl 212-C12 + 10 μl medium 1/14380 404.70 149.34 2.23 12.63 10 μl medium + 10 μl 172-B3.sup.d <1/9 <0.50 <0.14 <0.01 >26948.89 10 μl 212-C12 + 10 μl 172-B3 1/8902 250.54 79.03 1.18 23.86 10 μl 214-E8 + 10 μl medium 1/178 5.26 10.12 0.15 195.73 10 μl medium + 10 μl 213-H7 1/60 1.76 12.57 0.19 156.33 10 μl 214-E8 + 10 μl 213-H7 1/131 3.88 11.76 0.18 168.78 10 μl 214-E8 + 10 μl medium 1/108 3.18 6.12 0.09 322.59 10 μl medium + 10 μl 213-E6 1/5252 155.78 83.75 1.25 23.73 10 μl 214-E8 + 10 μl 213-E6 1/2022 59.96 50.39 0.75 39.43 10 μl 1214-H10 + 10 μl medium 1/842 24.96 17.83 0.27 111.40 10 μl medium + 10 μl 213-E6 1/6166 182.84 98.30 1.47 20.21 10 μl 214-H10 + 10 μl 213-E6 1/1611 47.8 29.33 0.44 67.79 .sup.aInternational Unit (IU) .sup.b1 mg NANOBODY ® (V.sub.HH sequence)/ml = 67 μM .sup.c= mg/ml × 50% dilution × 67000 .sup.d172-B3 = control NANOBODY ®(V.sub.HH sequence) directed against TLR-3
(456) TABLE-US-00071 TABLE C-38 Cross-neutralisation potency of monovalent NANOBODY ® (V.sub.HH sequence) clones: neutralization of the genotype 1 ERA strain ERA neutralizing antibody titer Attenuated vaccine strain, ATCC VR332, complete genome: NCBI EF206707 Sample 88% nucleotide identity with G of CVS-11 Interpretation cross- Clone Elusion 50% dilution EU.sup.a/ml EU/mg EU/μM.sup.b nM IC.sub.50.sup.c neutralisation Mab 8-2 Ascites mouse 1/506795 16895.00 nd.sup.d nd nd Yes OIE 0.5 IU/ml Canine reference serum 1/47 1.56 nd nd nd 3 × stronger compared to CVS WHO 0.5 IU/ml Human reference serum 1/20 0.66 nd nd nd Similar to CVS WHO 6 IU/ml Human reference serum 1/192 6.40 nd nd nd Similar to CVS 192-C4 Anti-HRSV.sup.e <1/9 <0.50 <0.63 <0.01 >5881.11 No 192-A8 Anti-HRSV <1/9 <0.50 <0.77 <0.02 >4838.89 No 191-E4 Anti-HRSV <1/9 <0.50 <0.63 <0.01 >5955.56 No 214-C10 Anti-rabies.sup.f 1/421 14.03 35.08 0.52 63.66 Yes 214-F8 Anti-rabies 1/114 3.81 23.81 0.36 94.04 Yes 214-A8 Anti-rabies <1/9 <0.50 <0.43 <0.01 >8561.11 No 214-E8 Anti-rabies <1/9 <0.50 <0.96 <0.02 >3871.11 No 213-E6 Anti-rabies 1/8635 287.83 154.75 2.31 14.43 Yes 213-B7 Anti-rabies 1/165 5.51 8.35 0.12 268.00 Yes 213-D7 Anti-rabies 1/179 5.97 8.91 0.13 250.78 Yes 213-D6 Anti-rabies <1/9 <0.50 <0.48 <0.01 >7816.67 No 213-H7 Anti-rabies 1/367 12.23 87.36 1.30 25.56 Yes 212-A2 Anti-rabies 1/16 0.52 0.55 0.01 3936.25 Yes 212-B2 Anti-rabies 1/55 1.84 2.59 0.04 864.91 Yes 212-G2 Anti-rabies <1/9 <0.50 <0.51 <0.01 >7295.56 No 212-F6 Anti-rabies 1/30 0.99 0.93 0.01 2389.67 Yes 212-B12 Anti-rabies 1/14 0.45 0.29 <0.01 7417.86 No 212-C12 Anti-rabies 1/27367 912.23 336.62 5.02 6.63 Yes 214-H10 Anti-rabies <1/9 <0.50 <0.36 <0.01 >10422.22 No .sup.a1 Equivalent Unit (EU) is comparable to the neutralizing potency of 1 International Unit (IU) .sup.b1 mg NANOBODY ® (V.sub.HH sequence)/ml = 67 μM .sup.c= mg/ml × 50% dilution × 67000 .sup.dnot determined .sup.econtrol NANOBODY ® (V.sub.HH sequence) raised against human respiratory syncytial virus .sup.fNANOBODY ® (V.sub.HH sequence) raised against rabies virus
(457) TABLE-US-00072 TABLE C-39 Cross-neutralisation potency of monovalent NANOBODY ® (V.sub.HH sequence) clones: neutralization of wild type genotype 1 strain CB-1 Chien Beersel-1 (CB-1) neutralizing antibody titer Belgian isolate of a genotype 1 canine rabies virus (Le Roux I. & Van Gucht S, WHO Rabies Bulletin 2008, 32(1), Quarter 1) Interpretation cross- NANOBODY ® (V.sub.HH sequence) 50% dilution EU.sup.a/ml EU/mg EU/μM.sup.b nM IC.sub.50.sup.c neutralisation Mab 8-2 Ascites mouse 1/881758 29391.92 nd.sup.d nd nd Very strong OIE 0.5 IU/ml Canine reference serum 1/36 1.18 nd nd nd 2 × stronger compared to CVS WHO 0.5 IU/ml Human reference serum 1/47 1.56 nd nd nd 3 × stronger compared to CVS WHO 6 IU/ml Human reference serum 1/402 13.40 nd nd nd 2 × stronger compared to CVS 192-C4 Anti-HRSV.sup.e <1/9 <0.50 <0.63 <0.01 >5881.11 Absent 192-A8 Anti-HRSV <1/9 <0.50 <0.77 <0.011 >4838.89 Absent 191-E4 Anti-HRSV <1/9 <0.50 <0.63 <0.01 >5955.56 Absent 214-C10 Anti-rabies.sup.f 1/653 21.77 54.43 0.81 41.04 Strong 214-F8 Anti-rabies 1/593 19.78 123.63 1.85 18.08 Very strong 214-A8 Anti-rabies 1/2768 92.25 80.22 1.20 27.84 Strong 214-E8 Anti-rabies 1/1906 63.55 122.21 1.82 18.28 Very strong 213-E6 Anti-rabies 1/10610 353.66 535.85 8.00 4.17 Very strong 213-B7 Anti-rabies 1/1263 42.09 63.77 0.95 35.01 Strong 213-D7 Anti-rabies 1/1996 66.52 99.28 1.48 22.49 Strong 213-D6 Anti-rabies 1/73 2.42 2.30 0.034 963.70 Weak 213-H7 Anti-rabies 1/8902 296.74 2119.57 31.64 1.05 Very strong 212-A2 Anti-rabies 1/524 17.48 18.60 0.28 120.19 Strong 212-B2 Anti-rabies 1/1384 46.12 64.96 0.97 34.37 Strong 212-G2 Anti-rabies 1/483 16.09 16.42 0.25 135.94 Strong 212-F6 Anti-rabies 1/1959 65.32 61.05 0.91 36.60 Strong 212-B12 Anti-rabies 1/11364 378.80 244.39 3.65 9.14 Very strong 212-C12 Anti-rabies 1/17635 587.84 459.25 6.85 4.86 Very strong 214-H10 Anti-rabies 1/4985 166.18 118.70 1.77 18.82 Very strong .sup.a1 Equivalent Unit (EU) is comparable to the neutralizing potency of 1 International Unit (IU) .sup.b1 mg NANOBODY ® (V.sub.HH sequence)/ml = 67 μM .sup.c= mg/ml × 50% dilution × 67000 .sup.dnot applicable .sup.econtrol NANOBODY ® (V.sub.HH sequence) raised against human respiratory syncytial virus .sup.fNANOBODY ® (V.sub.HH sequence) raised against rabies virus
(458) TABLE-US-00073 TABLE C-40 Cross-neutralisation potency of monovalent and bivalent NANOBODY ® (V.sub.HH sequence) clones: neutralization of EBLV-1 strain EBLV-1 neutralizing antibody titer Genotype 5, strain 8918FRA, complete genome: NCBI EU293112 Interpretation Sample 71% nucleotide identity with G of CVS-11 cross- Clone Elusion 50% dilution EU.sup.b/ml EU/mg EU/μM.sup.c nM IC.sub.50.sup.d neutralisation Mab 8-2 Ascites mouse 1/627878 20929.27 na na na Yes OIE 0.5 IU/ml Canine reference serum <1/9 <0.50 na na na No WHO 0.5 IU/ml Human reference serum <1/9 <0.50 na na na No WHO 6 IU/ml Human reference serum 1/37 1.22 na Na na 5 × weaker compared to CVS- 11 214-C10 trypsin 1.sup.st + mab 2.sup.d round <1/9 <0.50 <1.25 <0.02 >2977.78 No 214-F8 trypsin 1.sup.st + mab 2.sup.d round <1/9 <0.50 <3.13 <0.05 >1191.11 No 214-A8 trypsin 1.sup.st + mab 2.sup.d round 1/25 0.83 0.72 0.02 3082.00 Yes 214-E8 trypsin.sup.st + mab 2.sup.d round 1/67 2.25 4.33 0.06 520.00 Yes 213-E6 Mab 1.sup.st + tiypsin 2.sup.d round <1/9 <0.50 <0.76 <0.02 >4913.33 No 213-B7 Mab 1.sup.st + tiypsin 2.sup.d round 1/38 1.27 1.92 0.03 1163.68 Yes 213-D7 Mab 1.sup.st + tiypsin 2.sup.d round 1/41 1.38 2.06 0.03 1094.88 Yes 213-D6 Mab 1.sup.st + tiypsin 2.sup.d round <1/9 <0.50 <0.48 <0.01 >7816.67 No 213-H7 Mab 1.sup.st + tiypsin 2.sup.d round 1/16 0.52 3.71 0.06 586.25 Yes 192-C4 Anti HRSV <1/9 <0.50 <0.63 <0.01 >5881.11 No 192-A8 Anti HRSV <1/9 <0.50 <0.77 <0.02 >4838.89 No 191-E4 Anti HRSV <1/9 <0.50 <0.63 <0.01 >5955.56 No 212-A2 Trypsin 1.sup.st and 2.sup.d round 1/25 0.83 0.88 0.01 2519.20 Yes 212-B2 Trypsin 1.sup.st and 2.sup.d round <1/9 <0.50 <0.70 <0.02 >5285.56 No 212-G2 Trypsin 1.sup.st and 2.sup.d round <1/9 <0.50 <0.51 <0.01 >7295.56 No 212-F6 Trypsin 1.sup.st and 2.sup.d round <1/9 <0.50 <0.47 <0.01 >7965.56 No 212-B12 Trypsin 1.sup.st and 2.sup.d round <1/9 <0.50 <0.32 <0.01 >11538.89 No 212-C12 Trypsin 1.sup.st and 2.sup.d round <1/9 <0.50 <0.39 <0.01 >9528.89 No 214-H10 trypsin 1.sup.st + mab 2.sup.d round 1/41 1.36 0.97 0.01 2287.80 Yes 212-C12 15GS 212-C12 <1/9 <0.50 <0.50 <0.013 >4166.61 No 213-E6 5GS 213-E6 <1/9 <0.50 <0.53 <0.028 >4001.35 No 213-E6 15GS 213-H7 1/63 2.04 4.86 0.14 236.70 Yes 214-E8 15GS 213-H7 1/2187 70.15 305 8.52 3.76 Yes (potent) 213-H7 15GS 214-F8 1/41 1.32 11 0.30 107.74 Yes .sup.aserial dilution with different tips .sup.b1 Equivalent Unit (EU) can inhibit 50% of 10.sup.4.54 TCID.sub.50 of EBLV-1 on BHK cells; this is comparable to the neutralizing potency of 1 International Unit (IU) against CVS-11 .sup.c1 mg NANOBODY ® (V.sub.HH sequence)/ml = 67 μM .sup.d=mg/ml × 50% dilution × 67000 .sup.enot applicable
(459) TABLE-US-00074 TABLE C-41 Cross-neutralisation potency of monovalent and bivalent NANOBODY ® (V.sub.HH sequence) clones: neutralization of wild type genotype 1 strains and a laboratory CVS strain in suspensions of infected mouse brain using neuroblastoma cells as the susceptible target system Virus titer (TCID.sub.50.sup.a/ml) in brain suspension infected with strain . . . after pre-incubation with NANOBODY ® (V.sub.HH sequence) 9912CBG 9147FRA 9722P0L 8740THA NANOBODY ® (V.sub.HH Dog Fox CVS Raccoon dog Human 07059IC 9009NIG sequence) Cambodia France Strain IP13 Poland Thailand Dog Ivory Coast Dog Niger 172-B3 Anti-TLR3 4582 1125 ≤632456 2730 805 6325 780 Mab 8-2 Anti-rabies ≤80.sup.b* ≤67* 169643 ≤70* ≤63* ≤63* ≤63* 214-F8 Anti-rabies ≤78* ≤63* 1465* ≤63* ≤63* ≤75* ≤70* 213-E6 Anti-rabies ≤63* ≤63* ≤63* ≤63* ≤63* ≤63* 719 213-H7 Anti-rabies 379 ≤63* 170* ≤63* ≤63* 155 ≤78* 212-C12 Anti-rabies 733 5750 2000* 1411 ≤78* 452* ≤69* 212-C12-15GS-212-C12 ≤63* ≤63* ≤63* 213-E6-5GS-213-E6 ≤63* ≤63* ≤63* 213-E6-15GS-213-H7 ≤63* ≤63* ≤63* 214-E8-15GS-213-H7 ≤63* ≤63* ≤63* 213-H7-15GS-214-F8 ≤63* ≤63* ≤63* .sup.aTissue Culture Infectious Dose 50%: this corresponds with the dilution of the infected brain suspension - NANOBODY ® (V.sub.HH sequence) mixture which yields 50% infection in neuroblastoma cells .sup.bTiters with asterisks following the number correspond with a minimum hundredfold reduction of virus infectivity compared to control clone 172-B3 (anti-TLR3)
(460) TABLE-US-00075 TABLE C-42 Overview of the neutralisation potency of monovalent and bivalent NANOBODY ® (V.sub.HH sequence) clones: neutralization profile against different rabies virus strains and isolates. Neutralisation.sup.a of Genotype 1 9912CBG 9147FRA CVS 9722P0L 8740THA 07059IC NANOBODY ® (V.sub.HH Dog Fox Strain Raccoon Human Dog Ivory 9009NIG Genotype 5 sequence) CVS ERA CB-1 Cambodia France IP13 dog Poland Thailand Coast Dog Niger EBLV-1 Mab 8-2 Ascites mouse Yesb Yes Yes Yes Yes No Yes Yes Yes Yes Yes OIE 0.5 Canine ref. Yes Yes Yes nt.sup.c nt nt nt nt nt nt No IU/ml serum WHO 0.5 Human ref. Yes Yes Yes nt nt nt nt nt nt nt No IU/ml serum WHO 6 Human ref. Yes Yes Yes nt nt nt nt nt nt nt Yes IU/ml serum 192-C4 Anti-HRSV.sup.d No No No nt nt nt nt nt nt nt No 192-A8 Anti-HRSV No No No nt nt nt nt nt nt nt No 191-E4 Anti-HRSV No No No nt nt nt nt nt nt nt No 172-B3 Anti-TLR3.sup.e No nt nt No No No No No No No nt 214-F8 Anti-rabies Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes No 213-E6 Anti-rabies Yes Yes Yes Yes Yes Yes Yes Yes Yes No No 213-H7 Anti-rabies Yes Yes Yes No Yes Yes Yes Yes No Yes Yes 212-C12 Anti-rabies Yes Yes Yes No No Yes No Yes Yes Yes No 214-E8 Anti-rabies Yes No Yes nt nt nt nt nt nt nt Yes 214-C10 Anti-rabies Yes Yes Yes nt nt nt nt nt nt nt No 214-A8 Anti-rabies Yes No Yes nt nt nt nt nt nt nt Yes 213-B7 Anti-rabies Yes Yes Yes nt nt nt nt nt nt nt Yes 213-D7 Anti-rabies Yes Yes Yes nt nt nt nt nt nt nt Yes 213-D6 Anti-rabies No No Yes nt nt nt nt nt nt nt No 212-A2 Anti-rabies Yes Yes Yes nt nt nt nt nt nt nt Yes 212-B2 Anti-rabies Yes Yes Yes nt nt nt nt nt nt nt No 212-G2 Anti-rabies Yes No Yes nt nt nt nt nt nt nt No 212-F6 Anti-rabies Yes Yes Yes nt nt nt nt nt nt nt No 212-B12 Anti-rabies Yes No Yes nt nt nt nt nt nt nt No 214-H10 Anti-rabies Yes No Yes nt nt nt nt nt nt nt Yes 212-C12 15GS 212-C12 Yes nt nt Yes nt nt nt nt Yes Yes EBLV-1 213-E6 5GS 213-E6 Yes nt nt Yes nt nt nt nt Yes Yes No 213-E6 15GS 213-H7 Yes nt nt Yes nt nt nt nt Yes Yes Yes 214-E8 15GS 213-H7 Yes nt nt Yes nt nt nt nt Yes Yes Yes 213-H7 15GS 214-F8 Yes nt nt Yes nt nt nt nt Yes Yes Yes .sup.aNeutralisation is defined as an RFFIT titer of ≥50 IU or EU/ml (CVS, ERA, CB-1, EBLV-1), or a minimum hundredfold reduction of virus infectivity of a mixture of infected brain and NANOBODY ® (V.sub.HH sequence) in the neuroblastoma assay .sup.bYes in bold means a relative strong neutralizing potency: ≥100 IU or EU/mg in the RFFIT assay or ≤100 TCID.sub.50/ml in the neuroblastoma assay .sup.cNot tested .sup.dControl NANOBODY ® (V.sub.HH sequence) raised against human respiratory syncytial virus .sup.eControl NANOBODY ® (V.sub.HH sequence) raised against Toll-like receptor 3
(461) TABLE-US-00076 TABLE C-43 Effect of linking NANOBODIES® (V.sub.HH sequences) in bivalent or biparatopic combinations on the neutralizing potency. CVS neutralizing antibody titre Potency strain CVS-11, ATCC VR 959, sequence (IU/nM) G protein: NCBI EU126641 increase 50% versus Stock Nanobodies dilution IU.sup.a/ml IU/mg IU/nM.sup.b nM IC50.sup.c monovalent Bivalent 17/09/08 NB6 18GS NB6 10 <0.50 <2.38 <0.07 >725 — 17/09/08 213-H7 15GS 213-H7 12839 412 549 15.38 2.09 34.2 17/09/08 214-E8 15GS 214-E8 14156 454 349 9.78 3.28 31.5 17/09/08 212-C12 15GS 212-C12 10284 330 330 8.57 3.74 4.6 25/02/09 213-E6 5GS 213-E6 41075 1292 1297 36 0.88 27.7 30/10/08 213-E6 25GS 213-E6 674 21 300 8.29 3.76 6.4 30/10/08 214-F8 15GS 214-F8 421 13 650 17.2 1.79 63.7 Biparatopic 17/09/08 213-E6 SGS 212-C12 12006 385 385 10 3.21 6.3 17/09/08 213-E6 25GS 212-C12 40199 1289 248 6.70 4.79 4.2 30/10/08 213-E6 25GS 214-E8 1489 46 657 1.84 1.68 2.3 03/02/09 213-E6 15GS 213-H7 125670 3763 4252 93.7 0.26 107.1 17/09/08 214-E8 SGS 212-C12 5340 171 214 5.68 5.65 5.2 17/09/08 214-E8 15GS 212-C12 31109 998 322 8.70 3.69 8 30/10/08 214-E8 25GS 212-C12 2767 70.5 573 1.60 1.94 1.5 25/02/09 214-E8 15GS 213-H7 59651 1890 8215 230 0.14 605.3 25/02/09 213-H7 15GS 214-F8 13532 429 3575 97.5 0.33 270.8 .sup.aInternational Unit (IU) .sup.b1 mg bihead Nanobody/ml = 35.7 to 38.5 μM .sup.c= mg/ml × 1/50% dilution × (35700 to 38500)
(462) TABLE-US-00077 TABLE C-44 Synthesis of the peak clinical score, mortality and survival time in different groups of mice as described in Example 50 Peak clinical Mean time for Median Nr. of Inoculum score.sup.a Mortality mice death survival time.sup.b mice Virus Pre-incubated with (mean/mouse) (%) (days) (days) 7 10.sup.1.5 TCID.sub.50.sup.c — PBS 4.3 71 7.4 ± 0.89 7 7 10.sup.1.5 TCID.sub.50 1 IU mab 8-2 0 0 Na.sup.d na 6 10.sup.1.5 TCID.sub.50 6.4 μg 191-G2 5.3 100 7.3 ± 0.52 7 7 10.sup.1.5 TCID.sub.50 1 IU 212-C12 6 100 7.4 ± 0.53 7 7 10.sup.1.5 TCID.sub.50 1 IU 213-E6 3.4 57 6.75 ± 0.96 9 .sup.aclinical scores range from 0 (no disease) to 6 (weight loss, depression, hunched back, wasp waist, incoordination and hind limb paralysis) .sup.bthe median survival time is the time at which half of the mice have died on the Kaplan Meier curve (survival curve) .sup.aTCID.sub.50: tissue culture infectious dose 50%, .sup.dnot applicable
(463) TABLE-US-00078 TABLE C-45 Synthesis of peak clinical score, mortality and survival time in different groups of mice as described in Example 50 Mean time Peak clinical scored.sup.a Mortality for mice death Median survival time.sup.b Nr. of mice Inoculum Pre-incubated (mean/mouse) (%) (days) (days) 8 10.sup.1.5 TCID.sub.50.sup.c 191-G2 1 IU 5.25 ± 2.12 87.5 7.29 ± 1.25 8 9 10.sup.1.5 TCID.sub.50 Mab 8-2 1 IU 0 0 0 na.sup.d 9 10.sup.1.5 TCID.sub.50 212-C12 15GS 212-C12 1 IU 1.33 ± 2.65 22.2 9 ± 1.4 na 9 10.sup.1.5 TCID.sub.50 214-E8 15GS 214-E8 1 IU 0 0 0 na 9 10.sup.1.5 TCID.sub.50 213-H7 15GS 213-H7 1 IU 0 0 0 na 9 10.sup.1.5 TCID.sub.50 214-E8 15GS 212-C12 1 IU 0 0 0 na 9 10.sup.1.5 TCID.sub.50 213-E6 25GS 212-C12 1 IU 0 0 0 na 8 10.sup.1.5 TCID.sub.50 213-E6 SGS 212-C12 1 IU 0 0 0 na 9 10.sup.1.5 TCID.sub.50 213-E6 15GS 213-H7 1 IU 0 0 0 na .sup.aclinical scores range from 0 (no disease) to 6 (weight loss, depression, hunched back, wasp waist, incoordination and hind limb paralysis) .sup.bthe median survival time is the time at which half of the mice have died on the Kaplan Meier curve (survival curve) .sup.cTCID .sub.50: tissue culture infectious dose 50%, .sup.dnot applicable
(464) TABLE-US-00079 TABLE C-46 Synthesis of peak clinical score, mortality and survival time in different groups of mice as described in Example 52 Antibody/Nanobody Peak clinical Mean time for mice Median survival Nr. of IN injection on Virus IN injection on score Mortality death time mice day −1 day 0 (mean/mouse).sup.a (%) (days) (days).sup.b 8 191-D3 1 IU 10.sup.2 TCID.sub.50.sup.c 6.1 ± 2.5 87.5 9.9 ± 1.4 9 8 Mab 8-2 1 IU 10.sup.2 TCID.sub.50 0 0 0 Na.sup.d 8 212-C12 1 IU 10.sup.2 TCID.sub.50 6.1 ± 2.5 87.5 10.2 ± 1.6 12 8 213-E6 1 IU 10.sup.2 TCID.sub.50 5.25 ± 3.2 75 11.8 ± 1.6 12 .sup.aclinical scores range from 0 (no disease) to 7 (conjunctivitis, weight loss, depression, hunched back, wasp waist, incoordination and hind limb paralysis) .sup.bthe median survival time is the time at which half of the mice have died on the Kaplan Meier curve (survival curve) .sup.cTCID.sub.50: tissue culture infectious dose 50% .sup.dnot applicable
(465) TABLE-US-00080 TABLE C-47 Synthesis of peak clinical score, mortality and survival time upon intranasal inoculation of a mix of virus and NANOBODY® (V.sub.HH sequence) or antibody as described in Example 51 Peak clinical Mean time for Median Nr. of score Mortality mice death survival Exp mice Inoculum (mean/mouse).sup.a (%) (days) time (days).sup.b I 8 CVS 10.sup.3 TCID.sub.50.sup.c + 191-D3 6.5 ± 0.53 100 8.75 ± 0.46 9 9 CVS 10.sup.3 TCID.sup.50 + 212-C12 3.78 ± 3.6 55.6 11.6 ± 1.52 13 9 CVS 10.sup.3 TCID.sup.50 + 213-E6 3 ± 3.57 44.4 12.5 ± 1 na.sup.d II 8 CVS 10.sup.2 TCID.sup.50 + PBS 6.12 ± 2.5 87.5 12 ± 0 12 8 CVS 10.sup.2 TCID.sup.50 + Mab 8-2 6 ± 2.5 87.5 10.3 ± 1.6 10.5 8 CVS 10.sup.2 TCID.sup.50 + 212-C12 0 0 0 na 8 CVS 10.sup.2 TCID.sup.50 + 213-E6 0 0 0 na III 8 CVS 10.sup.2 TCID.sup.50 + 191-D3 4.22 ± 3.23 66 11.3 ± 3.14 13 8 CVS 10.sup.2 TCID.sup.50 + Mab8-2 6.11 ± 2.3 89 9.25 ± 0.46 9 8 CVS 10.sup.2 TCID.sup.50 + 212-C12 2.33 ± 3.5 33 11.7 ± 2.3 na 8 CVS 10.sup.2 TCID.sup.50 + 213-E6 0 0 0 na 8 CVS 10.sup.2 TCID.sup.50 + 0 0 0 na 214E8-15G5-213-H7 .sup.aclinical scores range from 0 (no disease) to 6 (weight loss, depression, hunched back, wasp waist, incoordination and hind limb paralysis), .sup.bthe median survival time is the time at which half of the mice have died on the Kaplan Meier curve (survival curve), .sup.cTCID.sub.50: tissue culture infectious dose 50%, .sup.dnot applicable
(466) TABLE-US-00081 TABLE C-48 Synthesis of peak clinical score, mortality and survival time in different groups of mice as described in Example 50.2 Mean time Peak clinical score.sup.b Mortality for mice death Median survival time.sup.c Nr. of mice Inoculum Pre-incubated (mean/mouse) (%) (days) (days) 9 10.sup.1.5 TCID.sub.50.sup.d NB6-18GS-NB6 1 IU 5.33 ± 2 88.9 7.12 ± 2.42 6 9 10.sup.1.5 TCID.sub.50 Mab 8-2 1 IU 0 0 0 na.sup.e 10 10.sup.1.5 TCID.sub.50 214-E8 15GS 212-C12 0 0 0 na 9 10.sup.1.5 TCID.sub.50 213-E6 25GS 212-C12 1 IU 0 0 0 na 7 10.sup.1.5 TCID.sub.50 213-E6 5GS 212-C12 1 IU 0.86 ± 2.27 14.3 21 na 9 10.sup.1.5 TCID.sub.50 213-E6 15GS 213-H7 1 IU 0 0 0 na 10 10.sup.1.5 TCID.sub.50 213-E6 5GS 213-E6 1 IU 0 0 0 na 9 10.sup.1.5 TCID.sub.50 213-E6 15GS 214-E8 1 IU 4 ± 3 66.7 12.5 ± 1.22 13 10 1015 TC1D5o 214-E8 15GS 213-E6 11U 0 0 0 na .sup.bclinical scores range from 0 (no disease) to 6 (weight loss, depression, hunched back, wasp waist, incoordination and hind limb paralysis) .sup.cthe median survival time is the time at which half of the mice have died on the Kaplan Meier curve (survival curve) .sup.dTCID.sub.50: tissue culture infectious dose 50%, .sup.enot applicable
(467) TABLE-US-00082 TABLE C-49 Synthesis of peak clinical score, mortality and survival time in different groups of mice as described in Example 50.4 Median Peak clinical score.sup.a Mortality Mean time for mice death survival time Nr. of mice Inoculum Pre-incubated (mean/mouse) (%) (days) (days) 9 10.sup.2 TCID.sub.50.sup.c PBS 6 ± 0 100 6.11 ± 0.33 6 8 10.sup.2 TCID.sub.50 RV1C5 1 IU 0 0 0 na.sup.d 9 10.sup.2 TCID.sub.50 213E6-15G5-213H7 1 IU 0 0 0 na .sup.aclinical scores range from 0 (no disease) to 6 (weight loss, depression, hunched back, wasp waist, incoordination and hind limb paralysis) .sup.bthe median survival time is the time at which half of the mice have died on the Kaplan Meier curve (survival curve) .sup.cTCID.sub.50: Tissue Culture Infectious Dose 50%, .sup.dnot applicable
(468) TABLE-US-00083 TABLE C-50 Synthesis of peak clinical score, mortality and survival time upon intranasal or intracerebral inoculation of 10.sup.2 TCID.sub.50 CVS-11 mixed with 1 IU 212-C12. Mean time Median Peak clinical for mice survival Nr. of Route of score.sup.a Mortality death time.sup.b mice Inoculum Pre-incubated inoculation (mean/mouse) (%) (days) (days) 9 10.sup.2 TCID.sub.50.sup.c 212-C12 1 IU IC 6 ± 0 100 7.22 ± 0.44 7 9 10.sup.2 TCID.sub.50 212-C12 1 IU IN 0 0 0 na.sup.d .sup.aclinical scores range from 0 (no disease) to 6 (weight loss, depression, hunched back, wasp waist, incoordination and hind limb paralysis) .sup.bthe median survival time is the time at which half of the mice have died on the Kaplan Meier curve (survival curve) .sup.cTCID.sub.50: tissue culture infectious dose 50% .sup.dnot applicable
(469) TABLE-US-00084 TABLE C-51 Concentration (ng) of NANOBODY® (V.sub.HH sequence) RSV101 or 12B2biv in lung homogenates of mice inoculated with NANOBODY® (V.sub.HH sequence) 3 and 5 days after administering of the NANOBODY® (V.sub.HH sequence) and infection with RSV as described in Example 55. Day 3 Day 5 Mouse RSV101 12B2biv PBS RSV101 12B2biv PBS 1 17.47 36.42 <5 5.8 19.15 6.68 2 14.21 27.07 8.46 <5 10.21 3 29.69 15.92 <5 16.56 4 31.69 45.74 <5 14.86 5 19.55 27.59 <5 21.51
(470) TABLE-US-00085 TABLE C-52 Neutralization and kinetic binding parameters for selected NC41 variants Neutralization IC50 (nM) Biacore (F.sub.tm-NN) Name Long B-1 Long B-1 ka (1/Ms) kd (1/s) KD (M) NC41 202 4707 122 3291 1.7E+06 6.70E−03 4.00E−09 NC41v03 255 1507 nd nd nd nd nd NC41v06 111 806 nd nd 2.0E+06 4.80E−03 2.50E−09 NC41v17 249 677 149 346 1.9E+06 5.90E−03 3.20E−09 NC41v18 116 728 98 194 nd nd nd Synagis 7.3 2.1 6.0 2.9
(471) TABLE-US-00086 TABLE C-53 Antigens used for llama immunization Virus strain Serotype Amount.sup.a (μg) Llama 3049 A/Chicken/Italy/1067/1999 H7N1 100 A/Mallard/Netherlands/2/2005 H5N2 100 A/Swan/Netherlands/06003448/2006 H7N7 100 FMDV Asia 1 Shamir Asia 1 50 FMDV A24 Cruzeiro A 15 Llama 3050 A/Ostrich/Netherlands/03006814/2003 H2N3 100 A/Mallard/Netherlands/06026212/2006 H8N4 100 A/Ty/Netherlands/06001571-041Tr/2006 H6N5 100 A/Chearwater/Australia/2576/02 H15N6 100 A/Mallard/Netherlands/06014516/2006 H10N8 100 A/Chicken/Ita1y/22A/98 H5N9 100 FMDV SAT2 SAT2 50 .sup.aAmount of antigen for each individual immunization.
(472) TABLE-US-00087 TABLE C-54 Analysis of llama antibody response by haemagglutination inhibition test H7N1 HI titer 2log H5N7 HI titer 2log Immunised with 55 34 55 llama H5 and H7 strains 0 DPI 34 DPI DPI 0 DPI DPI DPI 3049 H7N1/H5N2/H7N7 — 7 9 — 3 5 3050 H5N9 — — — — 7 11
(473) TABLE-US-00088 TABLE C-55 Oligonucleotides used for the construction of phage display libraries and sequencing as described in example 61 SEQ ID Primer NO: Sequence (5′- 3′) NotI- 3057 AACTGGAAGAATTCGCGGCCGCAGGAATTTTTTTTTT d(T)18 TTTTTTTT VH2B 3058 AGGTSMARCTGCAGSAGTCWGG lam07 3059 AACAGTTAAGCTTCCGCTTGCGGCCGCGGAGCTGGGG TCTTCGCTGTGGTGCG lam08 3060 AACAGTTAAGCTTCCGCTTGCGGCCGCTGGTTGTGGT TTTGGTGTCTTGGGTT BOLI-192 3061 AACAGTTAAGCTTCCGCTTGCGGCCGCTACTTCATTC GTTCCTGAGGAGACGGT MPE26 3062 GGATAACAATTTCACACAGGA
(474) TABLE-US-00089 TABLE C-56 Phage display libraries obtained as described in Example 61 Days post Hinge Library Library Llama immunisation primer Size.sup.a pAL439 3049 34 lam07 4.7 × 10.sup.6 pAL440 3049 34 lam08 8.0 × 10.sup.6 pAL441 3049 34 BOLI-192 6.1 × 10.sup.6 pAL442 3049 55 lam07 6.7 × 10.sup.6 pAL443 3049 55 lam08 7.6 × 10.sup.6 pAL444 3049 55 BOLI-192 1.1 × 10.sup.7 pAL445 3050 34 lam07 1.0 × 10.sup.7 pAL446 3050 34 lam08 9.8 × 10.sup.6 pAL447 3050 34 BOLI-192 8.0 × 10.sup.6 pAL448 3050 55 lam07 5.4 × 10.sup.6 pAL449 3050 55 lam08 9.5 × 10.sup.6 pAL450 3050 55 BOLI-192 5.3 × 10.sup.6 .sup.aThe number of colonies obtained after transformation of E. coli TG1.
(475) TABLE-US-00090 TABLE C-57 Influenza strains used for antigen preparation as described in Example 63 Influenza strain Serotype A/PR/8/34 (ATCC VR-1469) H1N1 A/Mallard/Netherlands/2/05 H5N2 A/Mallard/Denmark/75-64650/03 H5N7 A/Turkey/Wisconsin/68 H5N9 A/Chicken/Italy/1067/V99 H7N1 A/Swan/Netherlands/06003448/06 H7N7 A/Ostrich/Netherlands/03006814/03 H2N3 A/Ty/Netherlands/06001571-041Tr/06 H6N5 A/Mallard/Netherlands/06026212-002/06 H8N4 A/Duck/Germany/R113/95 H9N2 A/Mallard/Netherlands/06014516/06 H10N8 A/Chearwater/Australia/2576/02 H15N6
(476) TABLE-US-00091 TABLE C-58 Sequence characteristics, panning history and binding to influenza antigens of selected putative H5 binding NANOBODIES® (V.sub.HH sequences) Number Potential Panning of N- BstEII round Extinction at 450 nm in identical CDR3 glycosy- site 1 on Panning round ELISA on AIV antigens.sup.e Expressed Clone clones.sup.a Group.sup.b lation site.sup.c in FR4.sup.d antigen.sup.e 2 on antigen.sup.e H1N1 H7N7 H5N2 H5N9 H5N7 in yeast IV121 3 A None present H5N2 HAhis6 H5N1 0.085 0.053 0.101 0.099 0.449 not done IV122 2 A None present H5N2 HAhis6 H5N1 0.06 0.055 0.168 0.129 0.937 not done IV123 1 A None present H5N2 HAhis6 H5N1 0.065 0.06 0.12 0.188 0.487 not done IV126 1 A None present H5N2 HAhis6 H5N1 0.142 0.06 0.202 0.33 0.883 not done IV127 2 A None present H5N2 HAhis6 H5N1 0.113 0.106 0.216 0.443 1.15 not done IV131 1 A None present H5N2 HAhis6 H5N1 0.047 0.046 0.216 0.398 0.936 done IV132 1 A None present H5N2 HAhis6 H5N1 0.048 0.048 0.072 0.113 0.33 not done IV133 1 A None present H5N2 HAhis6 H5N1 0.048 0.051 0.243 0.377 1.206 done IV134 2 A None present H5N2 HAhis6 H5N1 0.049 0.049 0.106 0.194 0.95 not done IV135 1 A None present H5N2 HAhis6 H5N1 0.053 0.049 0.195 0.169 0.832 not done IV136 1 A None present H5N2 HAhis6 H5N1 0.088 0.123 0.182 0.372 0.953 not done IV140 3 A None present H5N2 HAhis6 H5N1 0.047 0.048 0.117 0.099 0.834 not done IV144 3 A None present H5N2 HAhis6 H5N1 0.12 0.089 0.407 0.656 1.282 done IV156 1 A None present H5N9 H5N7 0.048 0.054 0.401 0.649 1.418 done IV157 1 A None present H5N9 H5N7 0.046 0.049 0.352 0.336 1.375 done IV160 1 A None present H5N9 HAhis6 H5N1 0.052 0.053 0.283 0.312 1.243 not done IV124 2 B None present H5N2 HAhis6 H5N1 0.413 0.063 0.274 0.429 0.868 not done IV125 1 B None present H5N2 HAhis6 H5N1 0.461 0.076 0.272 0.413 0.801 not done IV145 1 B None present H5N2 HAhis6 H5N1 0.204 0.056 0.162 0.183 0.746 not done IV146 1 B None present H5N2 HAhis6 H5N1 0.299 0.051 0.223 0.285 0.744 done IV147 5 B None present H5N2 HAhis6 H5N1 0.216 0.047 0.182 0.197 0.599 not done IV151 1 C None absent H5N2 HAhis6 H5N1 0.172 0.106 0.164 0.181 0.709 not done IV153 1 D None absent H5N7 H5N2 0.045 0.048 0.436 0.05 0.056 not done IV154 1 E None present H5N9 H5N2 0.843 0.961 1.594 0.566 1.35 done IV155 1 F None present H5N9 H5N2 0.759 1.059 1.641 0.449 1.243 done .sup.aNumber of times a clone was isolated that encodes an identical NANOBODY® (V.sub.HH sequence). .sup.bClones belonging to the same CDR3 group have highly similar CDR3 sequences and identical CDR3 length. .sup.cPotential N-glycosylation sites (Asn—X—Ser/Thr, where X is not Pro) are either absent or present at the indicated position (MGT numbering). .sup.dThe presence of a unique BstEII restriction endonuclease cleavage site present in the FR4 encoding region and suitable for subcloning into yeast expression vector pRL188 is indicated. .sup.eH1N1, H7N7, H5N2, H5N7 and H5N9 refer to authentic influenza antigen produced by MDCK cells; HAhis6 H5N1 was from Abcam (cat. No. ab53938).
(477) TABLE-US-00092 TABLE C-59 Sequence characteristics, panning history and binding to influenza antigens of selected putative 117 binding NANOBODIES® (V.sub.HH sequences) Number of Potential iden- N- BstEII Panning tical CDR3 glycosy- site in Panning round round 2 on Extinction at 450 nm in ELISA on AIV antigens.sup.e Expressed Clone clones.sup.a Group.sup.b lation site.sup.c FR4.sup.d 1 on antigen.sup.e antigen.sup.e H1N1 H5N2 H5N7 H5N9 H7N1 H7N7 in yeast IV1 1 A None present H7N1 or H7N7 HAstr H7N2 0.056 0.051 0.057 0.052 1.277 1.096 done IV2 1 A 84 present H7N1 or H7N7 HAstr H7N2 0.048 0.05 0.048 0.045 1.366 0.814 not done IV3 1 A 84 present H7N1 or H7N7 HAstr H7N2 0.048 0.049 0.048 0.047 1.161 0.832 not done IV4 1 A 84 present H7N1 or H7N7 HAstr H7N2 0.047 0.05 0.048 0.047 1.158 0.945 not done IV6 2 A 84 present H7N1 or H7N7 HAstr H7N2 0.048 0.051 0.05 0.054 0.92 0.724 not done IV7 1 A 84 present H7N1 or H7N7 HAstr H7N2 0.048 0.054 0.05 0.047 1.2 0.806 not done IV9 1 A 84 present H7N1 or H7N7 HAstr H7N2 0.046 0.051 0.047 0.047 1.008 0.939 not done IV10 1 A 84 present H7N1 or H7N7 HAstr H7N2 0.047 0.052 0.047 0.048 1.133 1.078 not done IV11 1 A 84 present H7N1 or H7N7 HAstr H7N2 0.047 0.05 0.053 0.051 0.912 0.762 not done IV12 1 A 84 present H7N1 or H7N7 HAstr H7N2 0.065 0.123 0.195 0.078 0.956 0.984 not done IV16 1 A 84 present H7N1 HAlhis H7N7 0.048 0.05 0.05 0.045 1.071 0.789 not done IV24 1 A 84 present H7N7 HAlhis H7N7 0.05 0.049 0.051 0.047 1.166 1.032 not done IV26 1 A 84 present H7N7 HAlhis H7N7 0.061 0.109 0.114 0.097 1.127 1.003 done IV30 1 A 84 present H7N1 HAlhis H7N7 0.054 0.054 0.072 0.053 0.844 0.32 not done IV34 1 A 84 present H7N1 HAlhis H7N7 0.05 0.108 0.076 0.079 1.097 0.95 not done IV14 1 B None present H7N1 HAlhis H7N7 0.054 0.05 0.052 0.048 1.191 0.969 not done IV15 1 B None present H7N1 HAlhis H7N7 0.046 0.05 0.053 0.05 0.551 0.502 not done IV17 7 B None present H7N1 HAlhis H7N7 0.046 0.05 0.048 0.046 0.67 0.593 not done IV18 3 B None present H7N1 HAlhis H7N7 0.051 1.503 0.516 0.098 0.927 0.608 not done IV29 1 B None present H7N1 HAlhis H7N7 0.053 0.049 0.054 0.048 0.946 1.002 done IV31 1 B None present H7N1 HAlhis H7N7 0.045 0.051 0.05 0.049 1.013 1.043 not done IV33 1 B None present H7N1 HAlhis H7N7 0.045 0.049 0.047 0.047 0.885 0.762 not done IV35 1 B None present H7N7 HAlhis H7N7 0.065 0.054 0.054 0.047 1.121 0.907 not done IV36 1 B None present H7N7 HAlhis H7N7 0.048 0.048 0.048 0.047 1.029 0.999 not done IV40 1 B None absent H7N7 HAlhis H7N7 0.048 0.05 0.05 0.047 1.021 0.667 not done IV42 1 B None present H7N1 HAlhis H7N7 0.06 0.049 0.052 0.048 0.741 0.797 not done IV8 1 C None present H7N1 or H7N7 HAstr H7N2 0.047 0.05 0.049 0.045 1.077 0.456 not done IV21 1 C None present H7N7 HAlhis H7N7 0.047 0.047 0.047 0.05 0.945 0.565 done IV23 1 C None present H7N7 HAlhis H7N7 0.047 0.048 0.049 0.046 1.052 0.616 not done IV45 1 C None present H7N1 HAlhis H7N7 0.05 0.052 0.05 0.047 0.59 0.217 not done IV47 1 C None present H7N7 HAlhis H7N7 0.07 0.055 0.054 0.05 1.077 0.668 not done IV48 1 C None present H7N7 HAlhis H7N7 0.061 0.051 0.052 0.048 0.939 0.442 not done IV50 1 C None present H7N7 HAlhis H7N7 0.056 0.055 0.052 0.049 0.814 0.32 not done IV22 2 D None present H7N7 HAlhis H7N7 0.051 0.05 0.051 0.053 1.001 0.976 not done IV37 1 D None present H7N7 HAlhis H7N7 0.048 0.049 0.05 0.048 1.001 0.978 done IV38 1 D None present H7N7 HAlhis H7N7 0.047 0.051 0.05 0.047 0.915 0.99 not done IV5 1 E None present H7N1 or H7N7 HAstr H7N2 0.054 0.049 0.05 0.049 1.171 1.092 done IV27 1 E None present H7N1 HAlhis H7N7 0.054 0.047 0.051 0.048 1.321 1.165 not done IV25 1 F None present H7N7 HAlhis H7N7 0.046 0.05 0.048 0.047 0.706 0.797 done IV28 1 G None present H7N1 HAlhis H7N7 0.049 0.049 0.049 0.047 0.704 0.714 failed .sup.aNumber of times a clone was isolated that encodes an identical NANOBODY® (V.sub.HH sequence). .sup.bClones belonging to the same CDR3 group have highly similar CDR3 sequences and identical CDR3 length. .sup.cPotential N-glycosylation sites (Asn—X—Ser/Thr, where X is not Pro) are either absent or present at the indicated position (MGT numbering). .sup.dThe presence of a unique BstEII restriction endonuclease cleavage site present in the FR4 encoding region and suitable for subcloning into yeast expression vector pRL188 is indicated. .sup.eH1N1, H7N1, H7N7, H5N2, H5N7 and H5N9 refer to authentic influenza antigen produced by MDCK cells; HAlhis H7N7 was from Abcam (Abcam, Cat. No. ab61286).
(478) TABLE-US-00093 TABLE C-60 Antigen binding characteristics of yeast-produced NANOBODIES® (V.sub.HH sequences) binding to H5 strains ELISA titers.sup.c (ng/ml) VNT titer.sup.d CDR3 HA0his6 H5, HA1his6 H7, (ug/ml) HI titer.sup.e (ug/ml) Clone group H5N9.sup.a ab53938b ab53875b H5N7 H5N9 H5N7 H5N9 IV131 A 19.5 36.8 32 >50 >50 >1000 >1000 IV133 A 29.4 39.1 32.5 >50 >50 >1000 >1000 IV144 A 31.6 33.1 34.8 >50 >50 >1000 >1000 IV156 A 14.4 51.9 33 >50 >50 >1000 >1000 IV157 A 14.5 30.6 9.9 >50 >50 >1000 >1000 IV146 B 43.0 161.5 62.9 <0.75 <0.75 >1000 >1000 IV154 E 8.3 >10000 >10000 >50 >50 >1000 >1000 IV155 F 34.3 >10000 >10000 >50 >50 >1000 >1000 .sup.aELISA titers were determined on authentic MV antigens of strains shown in Table C-57 using a peroxidase-conjugated anti-h1s6 monoclonal antibody. NANOBODY® (V.sub.HH sequence) concentrations resulting in an extinction of 0.2 were interpolated. .sup.bELISA titers were determined on recombinant haemagglutinins derived from two different H5 influenza strains derived from Abcam. NANOBODY® (V.sub.HH sequence) concentrations resulting in an extinction of 1 were interpolated. .sup.c>10000 indicates extinctions below the value used for interpolation of titer at the highest NANOBODY® (V.sub.HH sequence) concentration analysed. .sup.d>50, no virus neutralization at the highest NANOBODY® (V.sub.HH sequence) concentration analysed; <0.75, neutralization at the lowest NANOBODY® (V.sub.HH sequence) concentration analysed. .sup.e>1000, no inhibition of haemagglutination at the highest NANOBODY® (V.sub.HH sequence) concentration analysed.
(479) TABLE-US-00094 TABLE C-61 Antigen binding characteristics of yeast-produced NANOBODIES® (V.sub.HH sequences) binding to H7 strains ELISA titers (ng/ml) VNT titer.sup.c CDR3 HA1his6 H7, (pg/ml) HI titer.sup.d (μg/ml) Clone group H7N1.sup.a H7N7.sup.a ab61286b H7N1 H7N7 H7N1 H7N7 IV1 A 11.2 66.4 62.2 >50 >50 >600 >600 IV26 A 14.1 147 80.6 >50 >50 >1000 >1000 IV29 B 6.8 9.3 7.2 >50 >50 >1000 >1000 IV21 C 85.8 1969 69 >50 >50 >1000 >1000 IV37 D 46 141 31.4 >50 >50 >1000 >1000 IV5 E 5.0 12.9 30.7 >50 >50 >1000 >1000 IV25 F 18.8 22.8 27.8 >50 >50 >400 >400 .sup.aELISA titers were determined on authentic MV antigens of strains shown in Table C-57 using a peroxidase-conjugated anti-his6 monoclonal antibody. NANOBODY® (V.sub.HH sequence) concentrations resulting in an extinction of 0.2 were interpolated. .sup.bELISA titers were determined on recombinant haemagglutinin derived from Abcam (Cat. No. ab61286). NANOBODY® (V.sub.HH sequence) concentrations resulting in an extinction of 1 were interpolated. .sup.c>50, no virus neutralization at the highest NANOBODY® (V.sub.HH sequence) concentration analysed. .sup.d>1000, >600 or >400, no inhibition of haemagglutination at the highest NANOBODY® (V.sub.HH sequence) concentration analysed.
The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, it being recognized that various modifications are possible within the scope of the invention.
(480) All references disclosed herein are incorporated by reference, in particular for the teaching that is referenced hereinabove.