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. 2021 Mar 2;4:273. doi: 10.1038/s42003-021-01791-1

Fig. 4. Target reactivity of CAR-T cells expressing new scFvs isolated from CD19 CAR-library T cells.

Fig. 4

a CD69 upregulation of Jurkat 76/35-G01-LH CAR cells and control Jurkat 76 cells in response to K562, K562/CD19, and Raji cells was measured by flow cytometry. The experiments were performed in triplicate, and error bars show the SD. b A construct of the soluble CD19 (sCD19) dimer is shown (top). Jurkat 76/CAR transfectants and control Jurkat 76 cells were stained with 40 μg/mL sCD19 dimer or PE-anti-his mAb alone (bottom). c CD19-specific scFv libraries possessing each human kappa chain (hK), lambda chain (hL) library derived from two different donors along with 35-G01-H, indicated as hL/35-G01-H and hK/35-G01-H, were individually generated. A second-generation CAR library encoding each CD19-specific scFv library together with CD28ζ was transduced into peripheral blood T cells. CD19 CAR-library T cells similarly stimulated three times were stained with 40 μg/mL sCD19 dimer or PE-anti-his mAb alone. Representative stainings of CD19 CAR-library CD8+ T cells (hL/35-G01-H and hK/35-G01-H) derived from donors 1 and 2, and CD4+ T cells (hL/35-G01-H) derived from the same donors are depicted. Each percentage of sCD19 dimer-positive cells among CAR-library T cells is shown. d sCD19 dimer positivity in CAR-library (hK/35-G01-H, hL/35-G01-H, and hH/35-G01-L) CD8+ T cells and CD4+ T cells is summarized. Each dot represents each library source derived from the different donors. The original 35-G01 CAR-T cells and control T cells were prepared as a positive and a negative control, respectively. e Newly isolated CD19 CARs were individually reconstituted in Jurkat 76 cells. Jurkat 76/CAR transfectants expressing each different VL (L16, L7, L17, L4, K4, K5, K9, and K6) chain paired with 35-G01-H were stained with 40 μg/mL sCD19 dimer or PE-anti-his mAb alone. L and K indicate lambda and kappa chain, respectively. f Jurkat 76/CAR transfectants were incubated with the indicated target cells, and their CD69 upregulation was measured by flow cytometry. The experiments were performed in triplicate, and error bars show the SD. g Jurkat 76/CAR transfectants were stained with graded concentrations of sCD19 dimer. Percentage maximal staining was calculated relative to the staining pattern of each transfectant with 40 μg/mL sCD19 dimer as 100%.