Table 1.
Binding and functional properties of hits derived from the full naïve campaign.
| Hit Identification Screen (FMAT) CHO-hCCR1 vs CHO-hCCR5 |
Potency GTPγS assay hCCR1/hMIP-1α |
||||||
|---|---|---|---|---|---|---|---|
| Clone Name | Specificity Rank | Binding Ratio | Apparent Affinity EC50 (nM) CHO-hCCR1 |
Mean IC50 (nM) | IC50 range Confidence intervals (nM) | Selectivity Binding to hCCR2, 3, 5 |
Cross-reactivity Rodent, minipig, and cyno CCR1 |
| 14Y029-130A06 | High | 103.9 | 153.1 | Inactive | >3548 | NB | NB |
| 14Y029-133D02 | High | 103.3 | 7.09 | 23.0 | 17–32 | NB | Mini-pig |
| 14Y029-141A10 | High | 79.8 | 9.2 | 64.5 | 35–118 | NB | NB |
| 14Y029-142B03 | High | 75.7 | 57.4 | 156.7 | 102–241 | NB | NB |
| 14Y029-142A07 | High | 70.5 | 180.4 | 827.9 | 314–2183 | NB | Mini-pig |
| 14Y029-131H05 | Medium | 62.2 | 97.9 | 1240.3 | 209–7374 | NB | Mini-pig and Rat |
| 14Y029-131C10 | Medium | 54.9 | 6.2 | 247.7 | 75–821 | NB | NB |
| 14Y029-125A08 | Low | 33.0 | 16.2 | 1753.0 | 473–6503 | NB | NB |
| 14Y029-131E11 | Low | 24.6 | 286.6 | 2952.8 | 2199–3966 | NB | Minipig |
| 14Y029-137B01 | Negative | 0.88 | NB | 324.4 | 170–619 | NB | NB |
| Isotype control | - | - | NB | Inactive | 1265–2412 | NB | NB |
A small subset of hits derived from the full naïve campaign was assessed for binding to CHO-hCCR1, selectivity over related chemokine receptors, cross-reactivity to relevant preclinical orthologues, and potency in a GTPγS assay format. Specificity rank was set arbitrarily based on the CHO-hCCR1:CHO-hCCR5 binding ratio as determined using the FMAT screen: ‘High’ >70, ‘Medium’ 40–70, ‘Low’ <40, or ‘Negative’ ≤1. A total of 9 hCCR1 binders and 1 internal negative control mAb were characterized. Apparent affinity (EC50) was assessed by flow cytometry using CHO-hCCR1 cells. Selectivity and cross-reactivity were assessed by flow cytometry at a single concentration (10 µg/ml) of each mAb on suitable cell lines overexpressing paralogues or orthologues; appropriate control mAbs demonstrated expression of each chemokine receptor and binding to CHO-hCCR1 was confirmed in parallel. Potency was measured using a GTPγS assay format with signaling via CCR1 driven by MIP-1α/CCL3. EC50 and IC50 values were calculated using GraphPad Prism. Where Mean IC50 values are quoted, the standard lower and upper confidence intervals were also calculated from the standard error. NB – no binding observed above background.