Fig. 6: Development of AT118i4h32 variants with reduced polyspecificity.
From: An in silico method to assess antibody fragment polyreactivity
a Electrostatic surface of AT118i4h32. CDR1, CDR2, and CDR3 are colored blue, green, and orange. All positions substituted to produce variants of AT118i4h32 with reduced polyreactivity are shown in sticks with atomic coloring b AT118i4h32 structure as colored in a. G26D27 and T57I65 substitutions are boxed. c PSR staining of yeast displaying AT118i4h32 variants. All amino acid substitutions decrease polyreactivity. Data in c comprise the mean +/− SEM of four independent experiments, each performed in technical triplicate. CDRs are colored as in a. d Binding of AT118i4h32 variants to HEK293 suspension cells expressing FLAG-AT1R. Cells were stained with AT118i4h32-V5-His variants, AlexaFlour-488 conjugated anti-FLAG, and AlexaFlour-647 conjugated anti-V5 antibodies, then analyzed by flow cytometry. Data in d is the average of three independent experiments performed in technical triplicate, error bars are shown as SEM. e Radioligand competition binding of AT118i4h32 variants or the small molecule antagonist losartan and [3H]-olmesartan to AT1R in cell membranes. Like WT AT118i4h32, the G26D27, T57I65, and G26D27 T57I65 variants compete with olmesartan for binding to the AT1R. Data in e is the average of three independent experiments performed in technical triplicate, error bars are shown as SEM. f Suppression of Gq-mediated inositol monophosphate production by AT118i4h32 in response to AngII stimulation. HEK293 suspension cells expressing FLAG-AT1R were treated with 5 μM AT118i4h32 or no nanobody prior to AngII stimulation. Data in d is the average of three independent experiments performed in technical triplicate, error bars are shown as SEM. Ki values are reported in Supplementary Table 6.
