Fig. 2. Target reactivity of CAR-T cells expressing new scFvs identified from A2/NY-ESO-1₁₅₇ CAR-library T cells.

a A2/NY-ESO-1₁₅₇-specific scFv libraries possessing the variable and J regions of each human kappa chain (hK), lambda chain (hL) or heavy chain (hH) library derived from four different donors along with the A2/NY-ESO-1₁₅₇-specific 3M4E5-H or 3M4E5-L, indicated as hK/3M4E5-H, hL/3M4E5-H, and hH/3M4E5-L, were individually generated. A second-generation CAR library encoding each A2/NY-ESO-1₁₅₇-specific scFv library together with CD28ζ was transduced into peripheral blood T cells. A2/NY-ESO-1₁₅₇ CAR-library T cells similarly stimulated three times were stained with 20 μg/mL A2/NY-ESO-1₁₅₇ or A2/HIV Gag₇₇ tetramer. Representative stainings of A2/NY-ESO-1₁₅₇ CAR-library CD8⁺ T cells (hL/3M4E5-H and hH/3M4E5-L) derived from donors 1 and 2, and CD4⁺ T cells (hL/3M4E5-H) derived from donors 3 and 4 are shown. Each percentage of A2/NY-ESO-1₁₅₇ tetramer-positive cells among CAR-library T cells is indicated. b A2/NY-ESO-1₁₅₇ tetramer positivity in CAR-library CD8⁺ T cells and CD4⁺ T cells is summarized. Each dot represents each library source derived from the different donors. The original 3M4E5 CAR-T cells and control T cells were prepared as a positive and a negative control, respectively. Two-tailed Mann–Whitney test was performed to compare two different groups. *p < 0.05. c Control T cells, or hL/3M4E5-H CAR-library CD8⁺ T cells (donor 1) were incubated with T2 cells pulsed with 10 μg/mL NY-ESO-1₁₅₇ peptide or HIV Gag₇₇ peptide. Multiple cytokine production by these CAR-T cells was measured by intracellular cytokine staining via flow cytometry. TNFα⁺IFNγ⁺IL2⁺ cells in ΔNGFR⁺CD8⁺ T cells are shown. The experiments were performed in triplicate, and error bars show the SD. Welch’s t test (two-sided) was performed for comparison. *p < 0.05. d, e Newly identified A2/NY-ESO-1₁₅₇ CARs were individually reconstituted in Jurkat 76 cells. Jurkat 76/CAR transfectants expressing each different VL (3M4E5-L, L1, L73, L88, L66, or L80) or VH (H73 or H1) chain paired with 3M4E5-H or 3M4E5-L were stained with 5 μg/mL A2/NY-ESO-1₁₅₇ or A2/HIV Gag₇₇ tetramer. Staining of control Jurkat 76 cells is also shown as a negative control (d). Jurkat 76/CAR transfectants were cocultured with T2 cells loaded with 10 μg/mL NY-ESO-1₁₅₇ peptide or HIV Gag₇₇ peptide. CD69 upregulation of Jurkat 76/CAR transfectants was measured by flow cytometry. The experiments were performed in triplicate, and error bars depict the SD (e). f Jurkat 76/CAR transfectants were stained with graded concentrations of A2/NY-ESO-1₁₅₇ tetramer (top). They were also incubated with T2 cells pulsed with graded concentrations of A2/NY-ESO-1₁₅₇ peptide (bottom). Percentage maximal staining of each indicated transfectant was calculated relative to the staining pattern of each transfectant with 10 μg/mL A2/NY-ESO-1₁₅₇ tetramer as 100% (top). Percentage maximal reactivity of each transfectant was measured relative to the CD69 upregulation of each transfectant for 10 μg/mL NY-ESO-1₁₅₇ peptide loaded onto T2 cells as 100% (bottom). g Structural avidity, shown as EC₅₀ values in μg/mL, was calculated as the concentration of A2/NY-ESO-1₁₅₇ tetramer required to achieve 50% of the maximal staining. Functional avidity, expressed as EC₅₀ values in μg/mL, was estimated as the concentration of NY-ESO-1₁₅₇ peptide required to obtain 50% of the maximal reactivity. Each dot represents each Jurkat 76/CAR transfectant. The nonparametric Spearman correlation coefficient was calculated. r and p values are shown.