Marked Impact of Different Cytokines on Phenotype and Cytotoxic Activity of CD19-Specific CAR T Cells

Introduction

The advent of T cells expressing chimeric antigen receptors (CART) for the treatment of cancer patients has been a milestone in the field of immunotherapy. For clinical application, reproducibility and safe generation of CART must be guaranteed. We investigated how different culture conditions of human peripheral blood mononuclear cells (PBMCs) influence the phenotype, i.e. naïve (N), central / effector memory (CM/EM), effector (E) cells, and the cellular composition of the resulting CART preparation, among others: the percentage of CD56+, γ/δ TCR+ and CD4+CD8+ double positive cells. Eventually, freezing/thawing of CART might also have an impact on their number, phenotype and function.

Methods

CART were generated by transducing human PBMCs from healthy donors with a CD19.CAR-CD28-CD137zeta vector (kindly provided by the Center for Cell and Gene Therapy, Houston) under two different stimulating culture conditions - anti-CD3/anti-CD28 antibodies with the addition of either IL-7/IL-15 or IL-2. CART generation lasted for 15 days. Cytotoxic ability of the generated CART was assessed by a standard chromium (Cr-51) release assay. CD19+ (Daudi) and CD19- (K562) cells were labeled for 2h with Cr-51 and coincubated with CART for 4h. Cr-51 release was measured in a liquid scintillation counter. In addition, multi-parametric flow cytometry was performed using a FACS LSR device.

Results

Overall, the transduction rate for both cytokine cocktails was around 50% of all CD3+ T cells. Freezing/thawing of CART reduced their cytotoxic activity: Lysis of the CD19+ target cell line (Daudi) reached a maximum of 80% for fresh cells and 50% for freshly thawed cells, both stimulated with IL-7/IL-15. FACS analysis revealed that freshly prepared cells showed an increased percentage of activated lymphocytes compared to cryopreserved cells. The majority of these activated lymphocytes were CD4+CD8+ double positive. Using freshly thawed cells, CART stimulated with IL-7/IL-15 showed higher cytotoxic activity than CART stimulated with IL-2: 50% vs. 38% (Daudi) and 7% vs. 5% (K562). Stimulation with IL-7/IL-15 led to an increase in the CD56+ CAR NK T cell population (20%) compared to IL-2 (8%). γ/δ TCR+ CART differed as well (3% for IL-7/IL-15, 8% for IL-2). The percentage of CD8+ and CD4+ CART was similar for both cytokine cocktails with around 60% and 30%, respectively. However, stimulation with IL-7/IL-15 showed a trend towards generation of a lower percentage of CD4+CD8+ double positive CART than stimulation with IL-2 (2% vs. 6%). The analysis of the different CART phenotypes showed significant differences: N 24% vs. 3%, CM 21% vs. 13%, EM 24% vs. 77%, E 31% vs. 7%, for IL-7/IL-15 vs. IL-2, respectively. A general phenomenon observed for both cytokine cocktails, was the difference in phenotype for CD4+ vs. CD8+ CART: CD4+ CART showed a more distinct memory phenotype, i.e. central and effector memory cells. Correspondingly, the CD8+ CART shifted more towards the naïve and effector phenotype. The percentage of highly effective stem cell memory CART (T_SCM: naïve CD27+CD95+) was assessed. Using IL-7/IL-15, CAR T_SCM cells accounted for 4.8%, whereas for IL-2, 2.7% had a T_SCM phenotype. 75% of the CART were HLA-DR positive (activation marker) and 100% were positive for CD95 (FAS receptor, inhibition marker). Using PBMCs from healthy donor samples, the generated CART products did not contain any contamination of either B (CD19+) or stem cells (CD34+).

Conclusions

We have established a combination of immunophenotyping and cytotoxicity assays for CART as a preclinical potency assay. Stimulation with IL-7/IL-15 led to a balanced phenotype expression, with a similar number of N, CM, EM and E CART, whereas the majority of CART under IL-2 stimulation adopted an EM phenotype. Correlation of these data with the clinical outcome of patients receiving the corresponding cell products will allow optimization and standardization of CART therapy.