CS1 Targeted Chimeric Antigen Receptors (CAR) for Treatment of Multiple Myeloma

Outcomes for myeloma patients have improved, however, most patients suffer fatal relapse. Although CD19 CAR-Ts for B cell malignancies have attained cure, BCMA CAR-T treatment results in 30-70% complete responses but responses are often not durable. Compared to BCMA, CS1 (SLAMF7) is more uniformly expressed on myeloma cells, and is also expressed on light chain amyloidosis cells. CS1 has limited expression on normal cell types, making it a good CAR-T target. Patients with relapsed myeloma, light chain amyloidosis, and those who relapse after BCMA-CAR-T might benefit from CS1-CAR-Ts. CS1-CAR-Ts expressing the scFv derived from Elotuzumab antibody (clone HuLuc63) show efficacy in preclinical myeloma models. Here, we report testing of CS1-CAR-Ts with the scFv's derived from a separate antibody clone (Luc90). The MM.1S human myeloma cell line (CS1+; expressing luciferase and GFP; MM.1S-CG) were used as target cells in four hour Chromium 51 release killing assays. At a 10:1 effector: target ratio, CS1-CAR-Ts killed 65%+/-11 (sd) of MM.1S-CGs while CD19-CAR-T controls showed minimal killing (10%+/-8). MM.1S cells lacking CS1 (MM.1S-CG-deltaCS1) were not efficiently killed by CS1-CAR-Ts (24%+/-12), showing specificity (Fig. 1A). We tested in vivo efficacy of our CS1-CAR-T by injecting 500,000 MM.1S-CG into NSG mice and 28 days later when tumor burden was high (BLI signal 10 10 photon flux), injected 2X10^6 CS1-CAR-T or negative control CD19-CAR-Ts. Bioluminescent imaging (BLI) showed CS1-CAR-Ts treated mice had a three log decrease in photon flux and clearance of marrow tumor (Fig 1B). All mice treated with CS1-CAR-Ts (n = 10) lived >100 days (Fig. 1C) while median survival of CD19-control mice (n = 8) was 43 days. A subset of CS1-CAR-T mice developed extramedullary tumors that retained expression of CS1, suggesting antigen escape did not occur. Mice engrafted with MM.1S-CG-deltaCS1 cells and treated with CS1-CAR-T had similar survival to control mice (49 days), demonstrating in vivo specificity (Fig. 1C). CS1 is expressed on CD8+ T-cells. The percent of CD8+ cells in control CD19-CAR-T cultures was 28%+/-10 and 9%+/-2 in CS1-CAR-T cultures, suggesting fratricide. We hypothesized deleting CS1 from T cells (using CRISPR/CAS9) prior to transduction with CS1-CAR would generate more effective CS1 CAR-Ts (delta-CS1-CAR-T-CS1) since fratricide would not occur during expansion. Generation of delta-CS1-CAR-T-CS1 restored CD8+ cells (30%+/-7). Although the percentage of CD8+ increased in delta-CS1-CAR-T-CS1, we observed similar killing to CS1-CAR-Ts in chromium assays. Experiments comparing efficacy of delta-CS1-CAR-T-CS1 vs CS1-CAR-T in vivo are ongoing. We have generated highly effective CAR-T cells targeting CS1, demonstrated by their ability to specifically kill CS1+ myeloma cells in vitro and in vivo. Our work further validates the utility of CS1 as an immunotherapeutic target for myeloma.Figure 1Figure 1