Abstract 2209: Costimulation of T cells by CD81 enhances CAR transduction of naive T cells
Authors: Schultz Liora M. , Czerwinski Debra , Kuo Chiung-Chi , Levy Shoshana , Levy Ronald 
Published: July 2016

Abstract
T cells genetically modified to express chimeric antigen receptors (CARs) mediate the eradication of antigen expressing tumor cells. The process of ex vivo T cell transduction to express CARs influences the phenotype, function and ultimate fate of the final CAR+ T cell product infused into patients. Pre-clinical data supports that naive-derived effector T cells have superior anti-tumor effect and enhanced persistence upon adoptive transfer as compared to memory-derived effector T cells. CD81 is a member of the tetraspanin family that functions as a T cell costimulator with activity independent of and additive to CD28. The addition of CD81 to CD3 and CD28 costimulation specifically induces increased activation of naive T cells. Retroviral genetic engineering methods require cells to be activated and in cell cycle to permit optimal gene integration. Here we demonstrate that introducing CD81 costimulation to the process of CAR transduction results in enhanced activation of naive T cells and resultant improved CAR transduction of the naive T cell subset.
Human naive T cells were isolated from healthy donors using magnetic bead negative selection. The purity of isolation was confirmed using flow cytometry showing CD62L+, 45RO- surface expression of the selected T cells. Naive T cells were differentially activated using CD3 antibody alone or in combination with CD28 or dual CD28 and CD81 costimulation. Using CD69 as a measure of activation, we demonstrate that stimulation with anti-CD3 alone or CD3 and CD28 antibodies does not activate isolated naive T cells. The addition of a CD81 antibody induces activation of both CD4+ and CD8+ naive T cells at 24 and 48 hours. We then transduced these activated naive T cells with a retrovirus encoding1928z, a CD19 specific second generation CAR. We used flow cytometry to confirm CAR transduction and compared the transduction efficiencies of the differentially costimulated groups.
We demonstrate that adding CD81 costimulation to CD3 and CD28 T cell activation permits retroviral-mediated CAR transduction of naive T cells. In contrast, stimulating CD3 alone or CD3 and CD28 does not induce activation, nor CAR transduction of isolated naive T cells. Transduction efficiency correlated with the degree of activation pre-transduction. Although the starting population was comprised of purified naive T cells, the activation and retroviral transduction process induced a change in surface markers. Surface expression of CD45RO increased and CD62L decreased, generating a final population of both naive T cells and naive-derived memory and effector T cells. This confirms that phenotyping T cells using surface markers following transduction is not reflective of the original T cell parent population.
We conclude that inclusion of CD81 co-stimulation into the process of CAR transduction improves transduction of naive T cells resulting in a final CAR+ T cell pool enriched for more potent naive derived CAR+ T cells.
Citation Format: Liora M. Schultz, Debra Czerwinski, Chiung-Chi Kuo, Shoshana Levy, Ronald Levy. Costimulation of T cells by CD81 enhances CAR transduction of naive T cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2209.