Improved Expansion and Function of CAR T Cell Products from Cultures Initiated at Defined CD4:CD8 Ratios

BACKGROUND: Current manufacturing paradigms for chimeric antigen receptor (CAR) modified T cells require ex vivo T cell activation, genetic modification, and expansion in cytokine-containing cell cultures. Most CAR T cell studies infuse cell products generated from unselected cells, in which the CD4:CD8 ratio is determined by what is collected during leukapheresis. The proportion of each subset can vary greatly in these products, reflecting the high heterogeneity of the CD4:CD8 ratio present in patients' (pts) blood at the time of treatment. Preclinical data demonstrate superior in vivo anti-tumor efficacy of cell products consisting of equal numbers of CD4⁺ and CD8⁺ T cells, and a recent clinical trial, in which CD19-targeted CAR T cell products were infused at a 1:1 ratio of CD4:CD8 T cells, showed clear dose-response and dose-toxicity relationships. However, CAR T cell manufacturing using parallel CD4⁺ and CD8⁺ CAR T cell cultures adds significant cost and complexity compared with a single-stream culture. Additionally, we have found that CD8⁺ T cell cultures from heavily treated pts often exhibit suboptimal expansion. We therefore evaluated whether CAR T cell products with approximately equal ratios of CD4⁺ to CD8⁺ cells could be generated by mixing CD4⁺ and CD8⁺ cells at a defined ratio at culture initiation, and whether the presence of ex vivo CD4⁺ help can improve CD8⁺ cell expansion.

METHODS: CD4⁺ and CD8⁺ T cells were isolated from apheresis peripheral blood mononuclear cell products of lymphoma pts (n=15) treated in one of three CD20-targeted CAR T cell trials or healthy donors (n=5) using positive (CD4) and negative (CD8) immunomagnetic bead selection. Cell cultures (1x10⁶ cells each) were established by activating CD4⁺ and CD8⁺ cells with anti-CD3/CD28-coated paramagnetic beads, mixing them at various ratios in 24-well plates, and transducing 24 hours later with a lentivirus encoding the 1.5.3-NQ-28-BB-z CD20 CAR. Beads were removed at day 4, and cells were expanded in IL-2 containing medium. At day 7, cells were counted and analyzed by flow cytometry for CD4, CD8, and CAR expression, and then restimulated 1:1 with irradiated CD20⁺ lymphoblastoid cells to boost growth. On day 14 cells were again counted and analyzed by flow cytometry for CD4, CD8, and CAR expression, as well as immunophenotype. In some cases CAR⁺CD4⁺ and CAR⁺CD8⁺ cells were flow-sorted from either CD4-only, CD8-only, and mixed cultures, stained with CFSE, and restimulated with irradiated CD20⁺ Raji tumor cells. Proliferation and cytokine secretion were measured by flow cytometry and Luminex, respectively. In vivo T cell activity was assessed using an NSG mouse model in which T cells were infused 7 days after i.v. injection of Raji-ffLuc cells. Suboptimal doses of T cells were used to distinguish differences between conditions, since higher doses cure most mice. Mice received either: (1) 70:30 mixed CD4:CD8 CAR⁺ cultures (n=8), (2) 1:1 ratio of CD4:CD8 CAR⁺ cells grown separately (n=8), (3) Mixed CD4:CD8 empty vector cells (n=5), or (4) no treatment (n=5).

RESULTS: An initial CD4:CD8 ratio of 70:30 yielded a median CD4/CD8 ratio of 1.1 for pts (Figure 1A) and 1.3 for donors at day 7, and a ratio of 0.6 and 1.0 for pts and donors, respectively, at day 14. Mixed cultures resulted in CD8⁺ cell expansion that was significantly higher than in separate cultures. At day 7, mean CD8⁺ cell expansion was 12.1-fold vs. 4.6-fold for 70:30 mixed vs. separate cultures for donors, and 2.9-fold vs. 0.7-fold for pts (Figure 1B). At day 14 CD8⁺ mean cell expansion was 105-fold (70:30 mixed) vs. 25-fold (separate) for donors and 40-fold vs. 1.9-fold for pts. CD8⁺ cells grown in mixed cultures also exhibited higher expression of memory markers (CD62L and CCR7), lower levels of exhaustion markers (Lag-3 and PD-1), and better proliferation compared with CD8 cells grown in separate cultures. In the mice we found that tumor growth inhibition was superior in the 70:30 mixed culture group than in mice receiving a 1:1 ratio of CD4:CD8 cells grown separately (Figure 1C).

CONCLUSIONS: A single-stream CAR T cell culture initiated at a defined CD4:CD8 ratio of 70:30 yielded on average approximately equal numbers of CD4⁺ and CD8⁺ cells in the final cell product. Mixed CD4⁺ and CD8⁺ cultures generated CD8⁺ T cells with a less differentiated phenotype, and superior expansion, proliferative capacity, and in vivo activity compared with CD4⁺ and CD8⁺ cells manufactured in parallel.

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