Universal Anti-CD7 CAR-T Cells Targeting T-ALL and Functional Analysis of CD7 Antigen on T/CAR-T Cells

Background Patients with relapsed/refractory acute T lymphoblastic leukemia (T-ALL) suffer from unsatisfactory prognosis and limited treatment options. Recently, the application of chimeric antigen receptor T cells (CAR-T) turns the scale. Pan-T cell markers like CD2, CD5 and CD7 have been harnessed for CAR-T cell therapy with the protein expression block or gene knockout of targeted antigens on T cells. However, the potential effects of CD7 antigen loss on T and CAR-T cells still need further elucidation. Herein, we constructed universal CAR-T cells targeting the pan-T cell marker-CD7, and explored the biological function of CD7 antigen on both T and CAR-T cells.

Methods Genetic editing approach (CRISPR/Cas9) and lentiviral transduction method were applied to produce universal anti-CD7 CAR-T cells (TRAC^-/-CD7^-/-,CD7UCAR). In vitro CAR-T cell phenotype and cytotoxicity were detected via flow cytometry, co-culture killing assay, degranulation assay and cytometric bead array. NSG mice were inoculated with Jurkat cells to establish xenograft mouse model, and the tumor load, body weight and survival time were dynamically monitored after treatment with T/CAR-T cells. To determine the impacts of CD7 antigen loss on T/CAR-T cells, CRISPR-based CD7 knockout was applied on primary T cells and anti-CD19 CAR-T cells. CD7⁺ and CD7^- T or CAR-T cells were sorted via flow cytometry, and their phenotypes were measured in terms of cell differentiation, inhibitory and active marker expression. Cell function was detected based on short- and long-term killing assay, degranulation assay and ELISA. RNA-seq was further applied to clarify the role of CD7 antigen on T cells.

Results The CD7 UCAR-T was successfully produced with our institute previously established CD7 monoclonal antibody HIT7 clone derived single-chain variable fragment (scFv) and second-generation CAR backbone. The CD7 UCAR-T cells can efficiently proliferate and specifically induce primary T-ALL tumor cell lysis in vitro, with elevated degranulation level and proinflammatory cytokines secretion. The UCAR-T cells are also able to significantly reduce tumor load and extend mice survival time. As for the function of CD7 antigen on T cells and anti-CD19 CAR-T cells, T/CAR-T cells tended to be CD8 negative with the loss of CD7 antigen expression, while fewer differences on other phenotypes like active or inhibitory marker expression were detected. CD7^-/- CAR-T cells seem to perform slightly better on target cell cytotoxicity at long-term killing assay with slightly higher cytokine secretion, especially on TNF-a. RNA-seq analysis showed an elevated proportion of activated CD4 memory cell population among CD7^-/- CAR-T cells, with gene enrichment on cytokine signaling pathway and T cells co-stimulatory pathways on CD7^-/- T and CAR-T cells, respectively. Besides, transcriptional detection showed limited distinction on crucial regulatory genes of T/CAR-T cells, indicating a potential translational regulation characteristic of CD7 antigen and the safety of CD7 knockout on primary T cells.

Conclusions CD7 UCAR-T, constructed with CD7 monoclonal antibody HIT7 hybridoma clone, showed strong specific cytotoxicity against CD7⁺ leukemia cells in comprehensive function studies, may become a promising strategy for T-ALL treatment. In addition, CD7 knockout leads to elevation of CD4 memory cell population without impairing CAR-T cells function. Whether CD7 knockout will benefit the establishment of long-term memory is worth further evaluation.

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