Fig. 2

Generation and Characterization of CD70 CAR-T Cells A The amino acid sequences of CDR1-3 region of novel nanobody isolated from the phage library and identified as the specific binder to CD70. B CAR construct with CD8α signal peptide (SP), scFv (41D12) or VHH (Nanobody), CD8α hinge and transmembrane domain, intracellular 4-1BB co-stimulatory domain, and intracellular CD3ζ activation domain. C CD70 CAR expression levels of 5 different donors measured by flow cytometry after transduction of activated T cells. CAR detected by biotin-labeled CD70 protein and BV421-conjugated streptavidin. D The percentage of CD70 CAR expressing T cells from 5 different health donors. All CAR highly expressed on T cell surface. E Antigen specific cytotoxicity of CAR-T cells. As CD70 positive cell lines (786-O, 769-P, ACHN, A498, K562 transduced with CD70) and CD70-negative cell lines (K562-WT, ACHN-CD70 KO) as a control, assessed by luciferase assay. All CAR-T cells were potent to kill CD70 positive target cell without cytotoxicity to antigen negative cells. F Antigen specific cytotoxicity of CD70 CAR-T cells against CD70-positive RCC cell lines (786-O, 769-P, ACHN, A498) during serial killing assays using luciferase assays. *P < 0.05, ****P < 0.0001 by two-way ANOVA with Dunnett's multiple comparisons test adjusted p value. G Fold change of CD3 + T cells in CD70 CAR-T co-culture with RCC cell lines during antigen stimulation in repetitive co-culture assay. H Analysis of cytokine production in the supernatants from repetitive co-cultures of RCC cells and CAR-T cells. The heatmap showed an increase in cytokines, including IL-2, TNF-α, IFN-γ, in the CD70 CAR-T cell groups