Senescent/Exhausted Phenotype of CAR T Cells and Induction of Immunoregulatory Environment Correlate with Reduced Response to CAR T Cell Therapy in Relapsed/Refractory B Cell Malignancies

Chimeric antigen receptor (CAR) T cells have shown promising results in patients (pts) with B cell malignancies, yet approximately 60% of pts with diffuse large B cell lymphoma (DLBCL) will relapse. Therefore, future efforts are needed to improve the outcomes of these pts. A total of 18 pts with relapsed/refractory B cell malignancies, DLBCL (n=17) and ALL (n=1), were enrolled on a phase 1b/2 study (NCT02772198) of locally produced CD19 CAR T cells. Clinical response was determined at 28 days following cell administration. Blood samples obtained prior to the lymphodepleting conditioning and at days 7, 14, 21, 30 and 60 after CAR T administration were collected. The manufactured CAR T products (n=9) were also subjected to immunophenotypic analysis. Clinically, 11 of 18 pts (61%) responded to CAR T therapy, 6 (33%) with complete response (CR), and 5 (28%) with partial response (PR). Analysis of manufactured CAR T products revealed high CXCR3 expression (77% and 96% positive within CD4+ and CD8+ subsets), indicating high migratory capacity of CAR T cells toward inflamed tissues. Furthermore, co-expression of CXCR4 (56% and 54% positive within CD4+ and CD8+ cells) suggests increased homing ability of the manufactured CAR T toward CXCL12-rich bone marrow and lymph nodes. Interestingly, higher CCR7 expression (32% vs 8.5%) and lower CCR6 levels (15% vs 28%) were detected on CD8+ CAR T cells from responding pts who achieved CR and PR (n=6) in comparison to non-responders (n=3), suggesting that less differentiated phenotype together with increased trafficking of CAR T to lymphoid tissue corresponds with improved clinical outcome. Additionally, we assessed the immunoregulatory and senescent/exhausted phenotype in CAR T products. Low percentage of CD4+CD25+CD127- Treg cells (13.5%) was detected, with no correlation to clinical response. However, significantly higher frequency of exhausted CD57+CD39+CD28- cytotoxic CD8+ cells stand out as signature population in CAR T products of non-responders in comparison to CR pts (37% vs 9.5%, p It is known that immunosuppressive environment affects CAR T cell activation. Notably, responding and non-responding pts presented distinct Treg patterns. Pts achieving CR demonstrated modest and delayed increase in Treg cells. In contrast, non-responders possessed rapidly increasing percentage of Treg cells at day 14 post infusion. In line with this finding, notable increase in proportion of immunosuppressive CD11b+CD14+CD163+CD206+ myeloid cells was detected in blood of non-responders, while pts achieving CR experienced transient increase in myeloid suppressor cells at day 7 that went back to normal levels at day 14. Overall, these results elucidate in part the mechanisms of CAR T traffic, immunosuppressive responses as well as induction of T cell senescence/exhaustion that most probably downregulate CAR T effectiveness as observed in non-responding pts.