Fig. 2: Selectivity of designed binders for αvβ6 and αvβ8.

a The A39K mutation confers selectivity towards αvβ6 compared to αvβ8 where there is a charge reversal (Glu316 for β6 shown as a gray stick, Lys304 for β8 shown as a blue stick). b Cell surface titration of B6B8_BP and B6_BP against K562 cells stably transfected with αvβ8. B6B8_BP lacking the A39K mutation binds to αvβ8 with a Kd of ~7.3 nM whereas B6_BP containing the A39K mutation binds to αvβ8 > 500 nM. c Cell surface titration of AlexaFluor-488-labeled B6B8_BP and B6_BP using αvβ6 (+) human epidermoid A431 carcinoma cells. B6_BP binds to A431 cells with higher potency than B6B8_BP (30 (±0.004) pM vs 167 (±0.028) pM). Data are presented as mean values +/− SD (n = 3 independent experiments). d B6_BP_dslf selectively inhibits αvβ6-mediated TGF-β1 activation. αvβ6 and αvβ8 transfectants were co-incubated with CAGA-reporter cells and GARP/TGF-β1 transfectants and inhibitors. B6_BP_dslf inhibits TGF-β activation with an IC50 of 32.8 (±3.4) nM. Data are presented as mean values +/− SD. e Binding affinities (Kd) of B8_BP_dslf (MAVY) point mutants to integrins αvβ6 and αvβ8, determined by BLI. Each experiment was repeated at least twice (n = 2). The LATI motif is in native L-TGF-β1. f, g Competitive inhibition of h-LAP₁ binding to (f) αvβ6 and (g) αvβ8 by designed inhibitors and control small molecule PLN-74809²⁰. Each experiment was performed at least three times (n = 3). h Heatmap of IC₅₀ values for h-LAP₁ binding assays in f and g. i Binding affinities (Kd) and fold-selectivity values of B6_BP_dslf and B8_BP-LATY to all eight RGD integrins compared to small molecules PLN-74809²⁰ and GSK3008348²¹. Binding data for PLN-74809 and GSK3008348 are taken from Decaris et al. 2021²⁰. Rows shaded in gray indicate the RGD integrin(s) for which each molecule is selective (i.e B6_BP_dslf and B8_BP-LATY are both mono-selective whereas PLN-74809 and GSK3008348 are dual- and tri-selective, respectively). n/a not available.