Extended Data Fig. 3: Characterization of anti-CD40 hIgG2 mAb ChiLob 7/4 affinity mutants.
From: Reducing affinity as a strategy to boost immunomodulatory antibody agonism
a, SPR of various ChiLob 7/4 h2 affinity mutants injected at 250, 50, 10, 2, 0.4, and 0 nM binding to CD40ECD. Data representative of 3 independent experiments. b, ChiLob 7/4 h2 affinity mutants were evaluated for their binding affinity for CD40ECD by SPR as indicated in a, with affinity constants (ka, kd and KD) calculated. Fold change indicates affinity change compared with WT ChiLob 7/4 h2. c, Ramos cells were incubated with 0.5 μg/mL of AF647-labelled ChiLob 7/4 h1 and various concentrations of competing ChiLob 7/4 h2 affinity mutants as indicated and then washed and bound AF647-labelled ChiLob 7/4 h1 detected. Means ± SEM, n = 3, data representative of 3 independent experiments. d, e, Purified hCD40Tg mouse B cells were incubated with ChiLob 7/4 h2 affinity mutants as indicated for 2 days and then stained for surface expression of CD23 (d, left plot, exemplar raw data) and CD86 (d, right plot, exemplar raw data). B cell proliferation was assessed by 3H-thymidine incorporation (e). Plots show affinity (KD) vs maximum CD23 MFI, maximum CD86 MFI or maximum proliferation. Means ± SEM, n = 3, data representative of 3 independent experiments. f, OTI cells were adoptively transferred into hCD40Tg mice 1 day before treatment with ChiLob 7/4 h2 mutants with peripheral SIINFEKL+ CD8 cells identified by flow cytometry on day 4. Mean ± SEM, n = 7, data pooled from two independent experiments. Each dot represents one mouse. Two-tailed, non-paired Student’s t test, the p values for WT h2 vs FW-12 h2, vs FW-22 h2, vs FW-16 h2 are (from left to right) 0.0023, 0.0023 and 0.0023. g, Purified human B cells were incubated with ChiLob 7/4 h2 affinity mutants for 2 days and then stained for surface expression of CD23 (left plot, exemplar raw data) and CD86 (right plot, exemplar raw data). h, Purified human B cells were incubated with ChiLob 7/4 h2 mutants as indicated for 3 days and then 3H-thymidine was added for 18 h to measure proliferation. Inset cell culture images were taken on day 2. For g, h, Means ± SEM, n = 3, data representative of 3 independent experiments. i, Purified human B cells were incubated with ChiLob 7/4 h2 affinity mutants as indicated for 2 days. B cell proliferation was assessed by 3H-thymidine incorporation. Plots show affinity (KD) vs maximum CD23 MFI, maximum CD86 MFI or maximum proliferation. Means ± SEM, n = 3, data representative of 3 independent experiments.
