Immunophenotypic profiling of K12 CAR T-Cells for CD7+ malignancies: lab- vs. bioreactor-based manufacturing 3506

Abstract Description T-cell malignancies remain challenging to treat with standard therapies. Development of Chimeric Antigen Receptor (CAR) T cell therapies against CD7 holds promise to increase current therapeutic outcome. To address the shared expression of CD7 with normal T cells and resultant fratricide effect, several strategies including the natural selection towards CD7Low/Neg cells have been proposed. Here, three batches of a ligand-based 3rd generation CAR against CD7, termed K12CAR-T, were manufactured in a GMP bioreactor-setting (BR-batches) and compared to matched-donor batches generated in the lab with in-depth immunophenotyping using full spectrum flow cytometry. On day 6, the maturation state and expression of prominent markers of exhaustion/activation were assessed. On average, 80% of the cells retained a TCM phenotype (CD62L+/CD45RA-) in BR-batches, with an ∼48% increase in %TCM cells and ∼9-fold increase in CD62L MFI levels compared to Lab-batches. Contrary to Lab-batches, CD62L expression in BR-batches was not significantly different to CD19CAR-Ts. BR-batches cells also had a more advantageous phenotype, exemplified by 67% lower expression of PD-1 and 39% higher expression of CD28. BR-batches had prominent CD7-restricted cytotoxic activity towards T-ALL cell lines with slightly higher IFN-? secretion. Overall, GMP bioreactor manufacturing yielded improved K12CAR-Ts compared to lab-based production, with a good phenotype suitable for use in a subsequent phase I clinical trial. Funding Sources Just Transition Fund (JTF) by the European Union Topic Categories Tumor Immunology: Checkpoints, Prevention, and Treatment (TIPT)