Fig. 1

scFV sequence optimization yields an anti-CD74 CAR (74bbz)-T cell with superior functions. A Design of CD74 CAR lentiviral vector. CD74 scFV: anti-CD74 scFV ended with CD8α hinge region; TM: CD8α transmembrane domain; 4-1BB: signaling domain of 4-1BB molecule; CD3ζ: CD3 zeta chain; T2A: self-cleaving peptide from thosea asigna virus 2A; reporter: GFP or tEGFR reporter gene. B Distribution of mutations on the V_H/V_L chain (right) and framework (FR)/complementarity-determining region (CDR) of the scFV on the 74bbz CAR. C Binding affinity of 74bbz CAR mutants displayed on Jurkat cell to CD74 ECD-Fc. The Jurkat CAR mutant cells were stained with CD74-ECD-Fc followed by anti-Fc-APC for flow cytometric analysis. The red bar is the parent 74bbz. All the numbers on the x-axis are the CAR clone numbers. D Early T cell activation marker CD69 expression on Jurkat CAR mutant cells. The Jurkat CAR mutant cells were incubated with Mino cells at an effector-to-target (ET) ratio of 1:2 for 6 h before the Jurkat CAR mutant cells were stained for CD69. E Repeated antigen stimulation assay for Jurkat CAR mutant cells. The Jurkat CAR mutant cells were stimulated weekly with irradiated Mino cells for three weeks and cultured under suboptimal culture condition with reduced FBS at 1%. The cell proliferation on Jurkat CAR mutant cells were assayed and the percentage of increased cell numbers were normalized as fold increase compared to Jurkat cells with parent 74bbz cells. F Specific lysis of the Mino cells by Jurkat CAR mutant cells. Jurkat CAR mutant cells were cocultured with Mino cells at E:T ratio of 5:1 for 24 h before the specific lysis were analyzed. G A four-way Venn diagram identified 2 clones of 74bbz CAR with the superiority over four aspects of CAR-T cell: CD69 activation (yellow), cytotoxicity (green), proliferation (red), and binding to CD74 (blue)