Precision targeting of the malignant clone in B-cell malignancies using chimeric antigen receptor T cells against the clonotypic IGHV3-23 B cell receptor

Abstract Introduction: CD19-directed chimeric antigen receptor T-cell (CART19) immunotherapy has revolutionized the treatment of B-cell lymphomas. However, about a third of patients relapse due to antigen-negative escape with loss of CD19 expression. Moreover, responding patients may experience long-term B-cell aplasia due to the fact that CD19 is expressed by all B cells, which results in susceptibility to severe infections. Indeed, the incidence of infections after CART is ~60% and is the leading cause of non-relapse mortality. A significant fraction of B-cell malignancies express B-cell receptors (BcR) that use the IGHV3-23 gene. In particular, IGHV3-23 is the most frequent VH gene segment used for the BcR on lymphoplasmacytic lymphoma/Waldenström macroglobulinemia (~16%, LPL/WM), and accounts for ~18% of ocular adnexal lymphomas (OAL), ~12% of activated B-cell-like large B-cell lymphomas (ABC-LBCL) and germinal center B-cell-like LBCL (GCB-LBCL), ~8% of chronic lymphocytic leukemia (CLL), and ~8% of mantle cell lymphoma (MCL). Specific disease-associated BcR have been shown to induce chronic downstream signaling, supporting that BcR can act as a driver of the disease. As IGHV3-23 is expressed by less than 15% of normal B cells, a CART targeting this antigen would spare the majority of the B-cell repertoire, thus decreasing immunosuppression. Therefore, we hypothesized that anti-IGHV3-23 CAR T cells would be highly effective and safe against B-cell malignancies, as they would: (i) efficiently recognize IGHV3-23+ malignant cells while sparing most normal (IGHV3-23-) B cells; and (ii) target a tumor driver that is essential for malignant B cell survival. Methods and Results: We developed a CAR construct (CD8-41BB-CD3z) targeting the IGHV3-23+ BcR (CART3-23) using a single-chain variable fragment (scFv) derived from the 3C9 monoclonal antibody. As models, we used SU-DHL-6 and CCRF-SB malignant B cells that naturally express the IGHV3-23 BcR. Using luciferase based killing assay, we showed that CART3-23 effectively killed CD19+ IGHV3-23+ SU-DHL-6 and CCRF-SB cell lines, with mean killing efficiencies of 0.553 (95% CI: 0.448-0.658) and 0.721 (95% CI: 0.477-0.965), respectively, compared to CART19 (SU-DHL-6: 0.815, 95% CI: 0.730-0.900; CCRF-SB: 0.971, 95% CI: 0.954-0.988) after 72h of co-culture at a 1:1 effector:target ratio (p=NS). Importantly, CART3-23 spared the CD19+ IGHV3-23- HBL-1 cell line (mean killing: 0.018, 95% CI: -0.130-0.166), whereas CART19 induced near-complete killing (mean: 0.962, 95% CI: 0.935-0.989), highlighting the antigen-specific activity of CART3-23. In line with these observations, CART3-23 demonstrated strong proliferative capacity (CTV dilution) when co-cultured for 120h with irradiated SU-DHL-6 and CCRF-SB cells, with mean proliferation rates of 87.9% (95% CI: 84.8-91.0%) and 97.0% (95% CI: 95.7-98.3%), respectively, which were comparable to those of CART19 (SU-DHL-6: 94.6%, 95% CI: 93.9-95.4%; CCRF-SB: 96.2%, 95% CI: 94.6-97.7%). In contrast, CART3-23 showed limited proliferation when co-cultured with the IGHV3-23-negative cell line HBL-1 cells, highlighting its antigen-specific activation. Lastly, we assessed the specificity and safety of CART3-23. CART3-23 was able to specifically kill IGHV3-23+ B cells from 5 healthy donors while sparing other B cell clones in vitro. Indeed, CART3-23, when co-cultured for 72h with healthy donor PBMCs, selectively eliminated IGHV3-23+ CD20+ B cells with a killing efficiency of 0.902 (95% CI: 0.783–1.022), while largely sparing the broader CD20⁺ B cell population for which killing efficiency was 0.245 (95% CI: 0.144–0.345). As expected, CART19 was responsible for a near complete depletion of all CD20+ B cells, independently of their IGHV3-23 expression. Conclusions: We developed a novel CART strategy to selectively target the malignant B-cell clone by targeting the IGHV3-23, aiming to preserve the majority of the healthy B-cell repertoire. This approach has the potential for clinical targeting of IGHV3-23+ B-cell neoplasms with, conceivably, an improved safety profile.