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The therapeutic promise of chimeric antigen receptor (CAR) T cells was realized when complete remission rates of 90% were reported after treating B cell acute lymphoblastic leukemia (B-ALL) with CD19-targeted CAR T cells. However, with increasing numbers of patients treated, challenges have become evident, especially regarding poor CAR T cell responses, which might be caused by exhaustion. Detailed understanding of the mechanism of CD28-dependent exhaustion in CAR T cells will allow the design of a CAR less prone to exhaustion and reduce relapse rates. We hypothesized that by mutating the CD28 subdomains YMNM and PRRP and leaving only the PYAP CD28 subdomain active we would optimize CAR T cell signaling and reduce exhaustion. We found mutated CAR T cells with only a functional PYAP subdomain secrete 50% less IFNγ, IL6, and TNFα after 24hr stimulation as well as having significantly less TCR signaling as measured by Nur77 compared to non-mutated CD28 CAR T cells. Additionally, mutated CAR T cells injected into mice had decreased expression of PD1 (82% to 62%) in the BM after 2 months. Studies in Rag−/− mice also showed mutated CAR T cells were increased 35% in the BM and 92% in the spleen compared to non-mutated CD28 CAR T cells. After challenge with tumor, exhausted mutant CAR T cells had increased IFNγ (42%), TNFα (62%) and IL2 (73%) secretion compared to exhausted non-mutated CD28 CAR T cells. This suggests that CAR T cells with only the PYAP subdomain are able to persist and be effective longer because they have optimized CD28 signaling. This work allows for development of an enhanced 2nd generation CAR T cell therapy for B cell malignancies by optimizing CAR T cell activation and persistence which may reduce relapse rates and severe toxicities.

Chimeric antigen receptor (CAR) T cells have mediated exciting results for patients. The CAR is a novel antigen-receptor that includes an antigen-binding domain, derived from an antibody, linked to an intracellular T cell activation domain. This 1st generation CAR design worked well in vitro but mediated poor results in vivo. Modification of the CAR to include a co-stimulatory domain, classified as a 2nd generation CAR design, achieved optimal T cell persistence and tumor killing in mice, which served as the rationale for evaluation of CAR T cells in patients. The most common clinically evaluated CARs target CD19 and include CD28 or 4-1BB co-stimulatory domains. However, there has been no extensive comparison of 2nd generation CAR T cells in mice, which could serve as a model for how these domains impact CAR T cell function in patients. Therefore, we evaluated and compared the function of mouse CD19 targeted CARs modified with a CD28 or 4-1BB domain. In contrast to clinical results, mouse CD28 CAR T cells imparted better leukemia-killing and persistence than mouse 4-1BB CAR T cells. We identified a defect in CAR T cell signaling mediated by mouse 4-1BB and mutation of this domain restored CAR signaling and in vivo leukemia killing. Also, we determined that CAR T cells with the mutation-optimized 4-1BB co-stimulatory domain proliferated in response to antigen in the bone marrow (BM) after a > 1-month period of rest, while CAR T cells with the CD28 co-stimulatory domain did not. We determined that this is likely due to the acquisition of an exhausted phenotype in the CD28 CAR T cells. Our results illustrate how co-stimulation modulates CAR T cell function in mice and suggest mechanisms for its impact on persistence and cancer-killing in patients.