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As assessed by BLI analysis using the Forté Bio™ Octet system, ALPN-101 bound human recombinant CD28, ICOS, and CTLA-4 with KD values of 192, 707, and 241 pM, respectively (Table 7, Figure 4, Figure 5, and Figure 6).
Of note, ALPN-101 demonstrates a rapid on-rate and gradual off-rate for human CD28, ICOS, and CTLA-4 (Figure 4, Figure 5, and Figure 6). The residual view in Figure 6 shows the difference between the fit line and the actual data, with each line ideally being 0 or random oscillation around 0. Any patterning can indicate...
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10. CONCLUSIONS
Under the conditions tested, flow cytometric analyses confirmed that ALPN-101 binds to its human cell-expressed counter-structures CD28, ICOS, and CTLA-4 with approximately equivalent binding affinities, with EC50s ranging from 633-680 pM. The affinity (KD) measurements of ALPN-101 binding to its three-human recombinan...
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11. REFERENCES
1. Yao S, Zhu Y, Zhu G, Augustine M, Zheng L, Goode DJ, Broadwater M, Ruff W, Flies S, Xu H, Flies D, Luo L, Wang S, Chen L (2011) B7-H2 is a costimulatory ligand for CD28 in human. Immunity 34(5):729-40.
2. Rixon M. (2018). ALPN-101 Structure and Amino Acid Sequence. AIS Report ALPN100-CMC-002.
3. Rixon M. (2018). Fc1.1 Structure and Amino Acid Sequence. AIS Report ALPN100-CMC-001.
4. Rixon M. (2018). ALPN-101 sequence alignments: ICOSL, CD28, ICOS, and CTLA-4 - homologies. AIS Report ALPN100-CMC-003.
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12. APPENDICES
Table 8: ALPN-101 Flow Cytometric Raw Binding Values (MFI): Human Counter Structures
Counter Structure Protein Conc. (pM) ALPN-101 ALPN-101 WT ICOSL-Fc1.1 WT ICOSL-Fc1.1 Fc 1.1 Fc 1.1
CD28 100000 32075 32885 6134 6018 542 648
20000 33589 34080 1282 1494 242 226
4000 35645 35040 1224 1283 197 156
800 18228 18597 1144 1094 152 174
160.0 4260 4762 585 615 160 153
32.0 1289 1311 466 387 295 155
6.4 445 429 176 198 189 198
1.3 222 233 312 179 139 146
ICOS 100000 73257 69358 71858 55642 234 649
20000 61738 65646 55462 65453 158 238
4000 72227 65955 57521 53948 122 134
800 38680 40497 37836 36318 125 177
160.0 11220 10709 11112 11132 204 169
32.0 2927 2957 3120 2937 126 476
6.4 890 846 1016 895 233 712
1.3 387 523 615 391 142 583
CTLA-4 100000 25302 22494 7628 8822 381 295
20000 25580 24675 1299 1404 166 170
4000 28635 26502 374 612 142 156
800 14977 14272 661 531 241 287
160.0 3795 3694 358 319 122 328
32.0 1067 1122 187 361 179 296
6.4 386 388 147 139 154 174
1.3 197 189 323 192 148 348
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Table 9: ALPN-101 Flow Cytometric Raw Binding Values (MFI): Cynomolgus Counter Structures
Counter Structure Protein Conc. (pM) ALPN-101 ALPN-101 WT ICOSL-Fc1.1 WT ICOSL-Fc1.1 Fc 1.1 Fc 1.1
CD28 100000 24410 24406 4935 4821 194 262
20000 24120 24200 1899 1834 126 124
4000 24518 24455 1033 1916 114 113
800 17694 16153 1213 1515 139 112
160.0 4961 5412 917 1060 116 137
32.0 1546 1401 292 437 106 109
6.4 546 413 195 199 125 116
1.3 216 228 166 189 109 129
ICOS 100000 73101 69247 54471 62260 217 317
20000 64179 67335 56975 58953 124 134
4000 58616 57905 38429 40834 112 121
800 44955 40784 27770 26762 120 144
160.0 11053 11622 9096 9035 112 117
32.0 3274 2870 2860 2598 168 208
6.4 1038 941 831 799 111 408
1.3 391 442 427 278 181 454
CTLA-4 100000 18016 17676 4866 5242 171 192
20000 17974 18321 832 814 127 169
4000 17796 18756 567 827 137 110
800 10062 9853 674 532 112 132
160.0 2860 2718 432 524 115 114
32.0 806 810 227 260 115 123
6.4 378 320 180 151 108 115
1.3 297 222 166 130 115 134
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Table 10: ALPN-101 Flow Cytometric Raw Binding Values (MFI): Rat Counter Structures
Counter Structure Protein Conc. (pM) ALPN-101 ALPN-101 WT ICOSL-Fc1.1 WT ICOSL-Fc1.1 Fc 1.1 Fc 1.1
CD28 100000 80195 78791 7454 7415 702 816
20000 85084 84062 5402 5371 340 656
4000 95160 91524 4817 5891 349 426
800 24943 25244 5995 4876 500 343
160.0 6125 6122 2961 2690 407 431
32.0 1741 1820 1009 1035 299 363
6.4 880 849 553 604 360 458
1.3 641 504 472 444 286 466
ICOS 100000 98614 74753 88764 91881 346 338
20000 105843 108277 101601 99196 189 273
4000 116478 112805 112726 108867 173 167
800 27240 29128 27322 27776 237 352
160.0 6202 6390 6312 6659 167 158
32.0 2019 2336 2295 2162 291 1862
6.4 824 976 984 897 195 464
1.3 449 690 849 547 151 27980
CTLA-4 100000 39728 47060 1820 1775 321 273
20000 45720 40828 1102 1255 343 476
4000 53506 54048 1450 1180 189 300
800 19778 18124 1605 1201 211 230
160.0 4692 4829 1362 1266 271 438
32.0 1942 1678 709 849 293 200
6.4 1074 759 919 909 221 248
1.3 985 1065 695 862 284 609
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