Fig 1: Optimization of humanized anti-CD169 mAbs and functional validation. A. Potential sequence liabilities found in CDR-engrafted humanized antibodies define variants indicated by suggested amino acid exchanges. B. Competition assay with Raji-Siglec-1 cell binding and HIV-1Gag-eGFP VLPs to test anti-CD169 mAb variants to eliminate selected potential liabilities. C. Competition for Raji-Siglec-1 cell binding between HIV-1Gag-eGFP VLPs and anti-CD169 mAbs. We tested the three different forms of #1F5 and #3F1 antibodies: murine (m-, triangle and lighter color), humanized (h0-, square), and stabilized (h1-, diamond). The comparison between these antibodies is shown with a non-linear fit to a variable response curve from one representative experiment out of two. D. IC50 values of individual mAbs were calculated with a non-linear fit to a variable response curve. Data from two independent experiments.
Fig 2: Humanization of anti-CD169-mAbs and functional validation. A. Diagram illustrating the design of humanized anti-CD169 mAbs. Created with Biorender. B. C. D. Competition for Raji-Siglec-1 cells binding between HIV-1Gag-eGFP VLPs and 3 different anti-CD169 mAbs for 1 h at 37°C. The antibody isotype control was used as a reference (IsoC, with grey star symbols). A comparison between original murine antibody (m-, triangle and lighter color), chimeric antibody (c-, circle), and humanized CDR-engrafted antibody (h0, square and darker color) is shown for each antibody clone (#1F5, B; #3F1, C; #5B10, D [corresponding to a non-linear fit to a variable response curve from one representative experiment out of two]). E. IC50 values of individual mAbs that achieved a total HIV-1Gag-eGFP VLP blocking effect. Data from two independent experiments.
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