Fig. 1. Identification of antitumor scFvs optimal for CAR-T cells using a T-cell-based scFv generation system.

Each of the steps and times required for preparation of the CAR library, generation of CAR-library T cells, antigen stimulation of CAR-library T cells, and isolation of new scFvs are summarized. a Variable region libraries derived from the human immunoglobulin light chain (hL, hK) or heavy chain (hH) were prepared. These libraries were fused with the variable region of an existing antitumor antibody (VH, e.g. 3M4E5-H, 35-G01-H; VL, e.g. 3M4E5-L, 35-G01-L, indicated in blue and red, respectively) to generate antitumor scFv libraries. Then, a CAR library encoding the scFv library followed by the transmembrane/intracellular domains of a CAR (CD28ζ) construct was prepared. b A CAR library was retrovirally transduced into human peripheral blood T cells to generate antitumor CAR-library T cells. ScFv-optimized (dark red), scFv-suboptimal (light red), and non-reactive (gray) CAR-T cells can be included among CAR-library T cells. c Antitumor CAR-library T cells were stimulated with tumor cells expressing a target antigen, such as A2/NY-ESO-1₁₅₇ and CD19, to concentrate the scFvs for delivery of activation signals to peripheral blood T cells via a CAR construct. d After isolation of antigen-specific CAR-T cells by flow cytometry using A2/NY-ESO-1₁₅₇ tetramer and soluble CD19 dimer, cDNA was synthesized and the primary structures of new scFvs that would be optimal for CAR-T cells were determined.