Distinct Signaling By Chimeric Antigen Receptors (CARs) Containing CD28 Signaling Domain Versus 4-1BB In Primary Human T Cells

Chimeric Antigen Receptors (CARs) have shown great promise in the field of targeted adoptive immunotherapy against cancer. These receptors are specific for tumor antigens and have the binding properties of monoclonal antibodies with signaling molecules of T cells. When expressed on T cells, these receptors help the cells home to tumor targets and perform their cytotoxic functions. CARs containing the 4-1BB signaling domain have been used against B-cell chronic lymphocytic leukemia and have shown the most clinical success in terms of tumor targeting and persistence in patients upon engraftment. In contrast, their CD28-containing CAR counterpart failed to show comparable persistence in patients. Despite extensive clinical use, the detailed molecular mechanisms involved in the activation of CAR-grafted T cells remain elusive. To address this, we hypothesize that CARs take advantage of the endogenous T cell receptor (TCR) signaling pathways in a manner unique to their analogous intracellular domains.

By electroporation of CAR encoding in vitro transcribed RNA into primary human T cells, we achieved >90% CAR-positive T cell population. We expressed different CARs constructs, all specific for a widely expressed tumor antigen - mesothelin. Keeping the scFv region constant to SS1 that is specific for mesothelin, we varied the intracellular signaling domains (ICDs) ranging from first generation CARs (containing only the CD3z ICD) to the second generation CARs (CD28-CD3z or 41BB-CD3z ICDs) Upon verifying CAR expression by flow cytometry, these T cells were stimulated with mesothelin antigen to analyze differences in signaling between the different CAR groups.

Here we report that CARs with CD28 show stronger activation of T cells when compared to CARs with 4-1BB or CD3z alone. Stimulation of different CAR constructs revealed that the antigen-specific activation threshold for CAR-T cells is greatly reduced when the CD28 endodomain is included in the CAR architecture. This activation state, measured by the activation of proximal signaling proteins, as well as the MAPK and Akt signaling pathways continues to increase and persist for longer time durations in T cells with the CD28-containing CAR construct. Co-immunoprecipitation studies reveal direct interaction of CARs with pZAP70 and TRAF proteins, but not other known signaling molecules of the TCR complex. T cells with CARs containing CD28 intracellular domain showed a high and sustained level of calcium flux in comparison to T cells with the 4-1BB containing CARs. Experiments to determine the molecular signatures of CAR-grafted T cells stimulated with cognate antigen for longer time durations are currently underway. Taken together, these studies have significant impact on the future design of CARs and adoptive immunotherapy.

Kawalekar:Novartis: Research Funding. Posey:Novartis: Research Funding. Fraietta:Novartis: Research Funding. Lee:Novartis: Research Funding. Zhao:Novartis: Research Funding. June:Novartis: Research Funding.