Fig. 6: Emibetuzumab mutants co-optimized for affinity and specificity also display high bioactivity and stability. | Nature Communications

Fig. 6: Emibetuzumab mutants co-optimized for affinity and specificity also display high bioactivity and stability.

From: Co-optimization of therapeutic antibody affinity and specificity using machine learning models that generalize to novel mutational space

Fig. 6

A Antigen (HGFR, 1 nM) and non-specific (ovalbumin, 0.1 mg/mL) binding measurements for soluble IgGs. B Concentration-dependent antigen binding of antibody mutants (EM1 and EM2) relative to wild-type. C Non-specific binding analysis of wild-type and mutant IgGs to a second polyspecificity reagent (soluble membrane proteins, 0.1 mg/mL). D Antibody-mediated inhibition of human cancer cell growth in response to hepatocyte growth factor (HGF). E Antibody self-association measurements (CS-SINS scores) in a standard formulation condition (pH 6 and 10 mM histidine). F Apparent melting temperature (Tm) of the antibody mutants. In (A), the data are averages of three independent experiments and the error bars are standard deviations. In (B), the binding experiments were performed two or three times, representative binding curves are shown, the EC50 values are averages of the independent experiments, and the EC50 errors are standard deviations. The p value comparing WT to EM1 is 0.026 and the p value comparing WT to EM2 is 0.022. In (C), the binding experiments were performed three times and the average curve is shown. In (D), five independent experiments were performed (three technical replicates per experiment), the average value of each independent experiment is shown as a different symbol, the overall average values are shown as bars, the errors are standard deviations, and CTL is a control IgG (elotuzumab) that does not bind HGFR. The p values comparing WT, EM1, and EM2 to HGF are 4 × 10−4, 2 × 10−6, and 2 × 10−7, respectively. The p values comparing WT, EM1, and EM2 to CTL are 3 × 10−3, 2 × 10−4, and 2 × 10−4, respectively. In (E), the values are averages of three independent experiments, the error bars are standard deviations, and CS-SINS scores less than 0.35 correspond to IgG1s predicted to display low viscosity (<30 cP) and opalescence (<12 NTU) when concentrated to 150 mg/mL13,44. The p value comparing WT to EM2 is 0.0009 and the p value comparing EM1 to EM2 is 0.0002. In (F), the data are averages of three independent experiments and the error bars are standard deviations. The p value comparing WT to EM1 is 0.015. In (B) and (D–F), independent two-sided t-tests were performed to determine significance and the p values are <0.05 (*), <0.01 (**) and <0.001 (***).

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