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CD28 192 1.92E-10 1.187E-11 135400 938.3 0.000026 1.598E-06 0.8865 |
CTLA-4 240.5 2.405E-10 3.745E-11 412400 21110 9.916E-05 1.459E-05 0.9065 |
Pharmacology Study Report Alpine Immune Sciences, Inc. ALPN100-PHRM-001 |
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Figure 3: General Description of Binding Sensorgrams |
ALPN-101 was loaded onto sensors at 10µg/mL via anti-human Ig capture. Flag-His or His-tagged monomeric CD28, ICOS or CTLA-4 counter structure titrations were then associated for 300 seconds. Samples were then allowed to dissociate equal to the time required to achieve a 10% decrease in signal: i.e. 900 seconds for ICO... |
Figure 4: CD28 Binding Sensorgram |
As described in Figure 3, ALPN-101 was loaded onto sensors via anti-human Ig capture and then CD28-Flag-His protein associated for 300 seconds. Samples were then allowed to dissociate equal to the time required to achieve a 10% decrease in signal: i.e. 3600 seconds for CD28. |
Pharmacology Study Report Alpine Immune Sciences, Inc. ALPN100-PHRM-001 |
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Figure 5: ICOS Binding Sensorgram |
As described in Figure 3, ALPN-101 was loaded onto sensors via anti-human Ig capture and then ICOS-Flag-His protein associated for 300 seconds. Samples were then allowed to dissociate equal to the time required to achieve a 10% decrease in signal: i.e. 900 seconds for ICOS. |
Figure 6: CTLA-4 Binding Sensorgram |
As described in Figure 3, ALPN-101 was loaded onto sensors via anti-human Ig capture and then CTLA-4-His protein associated for 300 seconds. Samples were then allowed to dissociate equal to the time required to achieve a 10% decrease in signal: i.e. 900 seconds for CTLA-4. |
Alpine Immune Sciences, Inc. |
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Pharmacology Study Report ALPN100-PHRM-001 |
8. STORAGE OF DATA |
Confirmation of cell surface expression of human CD28, ICOS, and CTLA-4 on transiently transfected Expi293F cells: Notebook reference # ALPINE.LSE.2 (p.15-18). |
Transfection Notebook reference #: EAR.007 Exp#TF20171204 (p. 88-89). |
Binding Notebook reference #: ALPINE.LSE.004 (p. 82-90). |
BLI KD determination Notebook reference #: JEH04-84 & 85. |
All raw data generated for this study is securely stored on the AIS server. |
Pharmacology Study Report Alpine Immune Sciences, Inc. ALPN100-PHRM-001 |
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9. RESULTS |
ALPN-101 bound human CD28, ICOS, and CTLA-4 in flow cytometric assays with EC50s of 648, 633, and 680 pM, respectively. ALPN-101 bound cynomolgus monkey homologs of its target molecules with affinities (440, 579, 704 pM) similar to those for the human targets, and ALPN-101 bound rat (1064, 1028, 793 pM) and mouse (1153... |
Weak WT ICOSL binding to CD28 and CTLA-4 has been demonstrated previously by flow cytometric binding, surface plasmon resonance (SPR), and mutational analysis (Yao et al., 2011). In the studies described herein, ALPN-101 exhibited improved CD28 binding compared to WT ICOSL-Fc1.1, demonstrated by a >10-fold increase in ... |
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... |
Pharmacology Study Report Alpine Immune Sciences, Inc. ALPN100-PHRM-001 |
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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... |
Pharmacology Study Report Alpine Immune Sciences, Inc. ALPN100-PHRM-001 |
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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. |
Pharmacology Study Report Alpine Immune Sciences, Inc. ALPN100-PHRM-001 |
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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 |
Pharmacology Study Report Alpine Immune Sciences, Inc. ALPN100-PHRM-001 |
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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 |
Pharmacology Study Report Alpine Immune Sciences, Inc. ALPN100-PHRM-001 |
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