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. 2021 Sep 12;9(9):e002737. doi: 10.1136/jitc-2021-002737

Figure 1.

Figure 1

Bispecific antibody binder enhances specificity and transduction efficiency of the mutant lentivirus. (A) Schematic of bispecific antibody in tandem Fab format (tFab) used for redirecting mutant Sindbis lentiviral vector (SINV-LV) to CD3+ T cells for targeted transduction. Orthogonal amino acid mutation sets are shown for constant and variable domains of each Fab to ensure correct pairing of heavy and light chains. (B) Binding affinity of control IgG (blue) and tFab (red) to human CD3ε analyzed by ELISA (n=2). (C) Binding affinity of tFab (red) to mutant Sindbis E2 glycoprotein analyzed by ELISA (n=2). (D) SINV-GFP transduction to CD3+ T cells is enhanced by addition of the tFab molecule in a concentration-dependent manner. At all tested concentrations, excess anti-CD3 IgG of the same clone blocked tFab-mediated SINV-GFP transduction, suggesting that transduction was specifically mediated via tFab binding CD3 in T cells. Data represent results of three independent experiments performed in triplicate (MOI=25) and analyzed using a two-way ANOVA with a post hoc Tukey’s test for multiple comparisons (****p<0.0001). (E) addition of tFab to mutated Sindbis pseudotyped lentiviral vector encoding green fluorescent protein (SINV-GFP) redirects the mutant lentiviral vector to CD3+ T cells in a mixed culture (CD3+ and CD3− cells together) demonstrating the specificity towards CD3+ T cells. (F) in mixed cultures of CD3+ (Sup-T1) and CD3− (BV-173) cells, SINV-GFP plus tFab demonstrated substantial selectivity towards CD3+ T cells as indicated by the increase in percentage of GFP+ cells. data represent results of three independent experiments performed in triplicate (MOI=25; (tFab)=30 nM) and analyzed using a two-way ANOVA with a post hoc Tukey’s test for multiple comparisons (****p<0.0001). ANOVA, analysis of variance; MOI, multiplicity of infection.