Smita S. Chandran

Engineered T and NK cell therapies have widely been used to treat hematologic malignancies and solid tumors, with promising clinical results. Current chimeric antigen receptor (CAR) T cell therapeutics have, however, been associated with treatment-related adverse events such as cytokine release syndrome (CRS) and are prone to immunologic exhaustion. CAR-NK therapeutics, while not associated with CRS, have limited in vivo persistence. We now demonstrate that an NK-like TCRαβ ⁺ CD8 T cell subset, identified and expanded ex vivo through its expression of the activating receptor NKG2C (NKG2C ⁺ NK-like T cells), can be transduced to express a second-generation CD19 CAR (1928z), resulting in superior tumor clearance, longer persistence and decreased exhaustion compared to conventional 1928z CAR ⁺ CD8 T cells and 1928z CAR+ NK cells. Moreover, CAR-modified NKG2C ⁺ NK-like T cells resulted in significantly reduced CRS compared to conventional CAR ⁺ CD8 T cells. Similarly, NKG2C ⁺ NK-like T cells engineered with a TCR targeting the NY-ESO-1 antigen exhibit robust tumor control and minimal exhaustion compared to TCR-engineered conventional CD8 T cells. These data establish NKG2C ⁺ NK-like T cells as a robust platform for cell engineering, and offer a safer, more durable alternative to conventional CAR-T and CAR-NK therapies.

Chimeric antigen receptor (CAR)-engineered T and NK cells can cause durable remission of B-cell malignancies; however, limited persistence restrains the full potential of these therapies in many patients. The FAS ligand (FAS-L)/FAS pathway governs naturally-occurring lymphocyte homeostasis, yet knowledge of which cells express FAS-L in patients and whether these sources compromise CAR persistence remains incomplete. Here, we constructed a single-cell atlas of diverse cancer types to identify cellular subsets expressing FASLG , the gene encoding FAS-L. We discovered that FASLG is limited primarily to endogenous T cells, NK cells, and CAR-T cells while tumor and stromal cells express minimal FASLG . To establish whether CAR-T/NK cell survival is regulated through FAS-L, we performed competitive fitness assays using lymphocytes modified with or without a FAS dominant negative receptor (ΔFAS). Following adoptive transfer, ΔFAS-expressing CAR-T and CAR-NK cells became enriched across multiple tissues, a phenomenon that mechanistically was reverted through FASLG knockout. By contrast, FASLG was dispensable for CAR-mediated tumor killing. In multiple models, ΔFAS co-expression by CAR-T and CAR-NK enhanced antitumor efficacy compared with CAR cells alone. Together, these findings reveal that CAR-engineered lymphocyte persistence is governed by a FAS-L/FAS auto-regulatory circuit.

The optimal T-cell attributes for adoptive cancer immunotherapy are unclear. Recent clinical trials of ex vivo–expanded tumor-infiltrating lymphocytes indicated that differentiated T effector cells can elicit durable antitumor responses in some patients with cancer, with their antitumor activity tightly correlated with their persistence in the host. Thus, there is great interest in the definition of intrinsic biomarkers that can predict the conversion of short-lived tumor antigen–specific T effector cells into long-lived T memory cells. Long-term persistence of ex vivo–expanded tumor-specific CD8+ T effector clones has been reported in refractory metastatic melanoma patients after adoptive T-cell transfer. By using highly homogeneous clone populations from these preparations, we performed a comparative transcriptional profiling to define preinfusion molecular attributes that can be ascribed to an effector-to-memory transition. Through this route, we discovered that preinfusion T-cell clones that expressed the IL7 receptor (IL7R) and c-myc were more likely to persist longer after adoptive transfer to patients. The predictive value of these two biomarkers was strengthened by using IL7R protein, IL7-induced pSTAT5, and c-myc mRNA expression to prospectively identify human tumor-specific T effector clones capable of engraftment into immunodeficient mice. Overall, our findings reveal IL7R and c-myc expression as intrinsic biomarkers that can predict the fate of CD8+ T effector cells after adoptive transfer. Cancer Res; 75(16); 3216–26. ©2015 AACR.