Extended Data Fig. 1: Generation of Fc-silent mAbs.
From: CD47 masks pro-phagocytic ligands in cis on tumor cells to suppress antitumor immunity
a, The three mutations (‘LALAPG’) introduced in the Fc portion of mAbs to render them Fc-silent are depicted. LALAPG is L234A (‘LA’), L235A (‘LA’), and P329G (‘PG’). b, Binding assay of Fc-intact and Fc-silent variants of mIgG2a (mAb MOPC21) or hIgG1 (mAb Z10) to FcRs on BMDMs assessed by flow cytometry. c, Binding of Fc-silent mSIRPα mAbs #23, #27 and MY-1 to EL-4 cells and EL-4 cells expressing full-length mSIRPα or a variant of mSIRPα containing only the first Ig-like variable (V) domain assessed by flow cytometry. d, Binding of a soluble mCD47-Fc fusion protein to EL-4 cells, expressing or not expressing mSIRPα, studied by flow cytometry. MFI, mean fluorescence intensity. e, Binding of Fc-silent mSIRPα mAbs #23, #27 and MY-1 to mSIRPα-Fc, mSIRPβ1a-Fc, mSIRPβ1b-Fc and mSIRPβ1c-Fc, determined by ELISA. f, Binding of Fc-silent Ctrl IgG MOPC21, hCD47 mAbs B6H12 and AO-176 or hSIRPα-Fc fusion proteins TTI-621 and ALX148 to WT and CD47−/− Daudi cells evaluated by flow cytometry. g, Binding of Fc-silent hSIRPα mAbs to Sirpa-/- BMDMs, transduced with retroviruses encoding or not encoding the V1 or V2 version of hSIRPα, studied by flow cytometry. All data are means ± s.e.m. ****p < 0.0001. Flow cytometry profiles are representative of 3 (c,f, g) or 2 (b) independent experiments. Results are pooled from 5 (d) and 3 (e) independent experiments.
