Figure 2.

CD5 antigen binding assay and competitive binding analysis of V_H and validation of anti-CD5 biepitopic antibodies

(A) Schematic diagram of the anti-CD5 V_H binding using a competitive fluorescence-activated cell sorting assay. (B) Representative flow cytometry analysis shows that H65, fully human (FH)V_H1, and FHV_H3 CAR-T cells all bind to CD5 antigen. FHV_H3 recognizes the overlapped epitope of CD5 as H65, whereas FHV_H1 binds different CD5 antigen epitopes with FHV_H3 and H65. (C) Binding specificities of anti-CD5 biepitopic antibodies are shown. CD5⁺ cell lines (Jurkat and CCRF-CEM) and CD5⁻ cell lines (CCRF-CD5KO and Raji) were stained with isotype control, FHV_H1-hFc, FHV_H3-hFc, FHV_H1/V_H3-hFc, and FHV_H3/V_H1-hFc antibody, respectively, followed by a secondary APC-labeled anti-human IgG, and analyzed using FACS. (D) Binding of FHV_H3/V_H1 to QT6 cells individually expressing 5,900 full-length human membrane proteins as determined by flow cytometry is shown.