CD8 chimeric antigen receptor T cells manufactured in absence of CD4 cells exhibit hypofunctional phenotype.

Background: Cell culture conditions during manufacturing can impact the clinical efficacy of chimeric antigen receptor (CAR) T cell products. Production methods have not been standardized because the optimal approach remains unknown. Separate CD4 and CD8 cultures offer a potential advantage but complicate manufacturing and may affect cell expansion and function. In a phase 1/2 clinical trial, we observed poor expansion of separate CD8 cell cultures and hypothesized that coculture of CD4 cells and CD8 cells at a defined ratio at culture initiation would enhance CD8 cell expansion and simplify manufacturing.

Methods: We generated CAR T cells either as separate CD4 and CD8 cells, or as combined cultures mixed in defined CD4:CD8 ratios at culture initiation. We assessed CAR T cell expansion, phenotype, function, gene expression, and in vivo activity of CAR T cells and compared these between separately expanded or mixed CAR T cell cultures.

Results: We found that the coculture of CD8 CAR T cells with CD4 cells markedly improves CD8 cell expansion, and further discovered that CD8 cells cultured in isolation exhibit a hypofunctional phenotype and transcriptional signature compared with those in mixed cultures with CD4 cells. Cocultured CAR T cells also confer superior antitumor activity in vivo compared with separately expanded cells. The positive impact of CD4 cells on CD8 cells was mediated through both cytokines and direct cell contact, including CD40L-CD40 and CD70-CD27 interactions.

Conclusions: Our data indicate that CD4 cell help during cell culture maintains robust CD8 CAR T cell function, with implications for clinical cell manufacturing.