Balancing efficacy and safety: IL-15 BCMA CAR-T cells vs. 2nd generation CAR-T in preclinical myeloma model

Abstract

Abstract Multiple myeloma (MM) is the second most frequent hematologic malignancy worldwide and remains incurable. Current treatments are costly, have limited availability, and many patients either fail to respond or relapse. Chimeric antigen receptor (CAR) T-cell therapy has shown remarkable clinical responses, spurring the development of improved products to enhance cell potency, safety, and accessibility, especially in low-income countries. BCMA has emerged as an ideal target, being highly expressed on myeloma cells and largely restricted to plasma cells and B cells. CAR-T cells engineered to express cytokines like interleukin-15 (IL-15) show enhanced expansion, tumor infiltration, and antitumor activity. We hypothesized that the incorporation of soluble Interleukin-15 (sIL-15) production would enhance the functional capacity of BCMA-targeting CAR-T cells, resulting superior antitumor efficacy in preclinical models. Thus, we generated primary human T cells to target BCMA-expressing tumor cells through lentivirus transduction with synthetic constructs encoding either a 2nd-generation CAR (BCMA-4-1BB-CD3ζ) or a 4th-generation CAR (BCMA-4-1BB-CD3ζ-IL-15). Peripheral blood T cells were isolated from healthy donors, activated, and transduced. Both 2nd- and 4th-generation BCMA CAR-T cells achieved high transduction efficiencies (mean CAR⁺ frequency: 49.9% 2nd- BCMA CAR-T; 80.7% 4th- BCMA CAR-T) and up to 400-fold expansion, maintaining upper to 90% viability. Co-culture assays with BCMA-expressing myeloma cell lines (MM1.S, U266) demonstrated robust antigen-specific cytotoxicity at effector:target ratios of 1, 2, 5, and 10, tested compared to non-transduced (NT) T cells and CD19-CAR-T cells controls (p-value<0.01), whereas no cytotoxic activity was detected against BCMA-negative K562 cells. Additionally, the tested CAR-T cells exhibited robust IFN-γ and TNF-α production against BCMA⁺ cell lines (p-value<0.001) and no significant expression of T-cell exhaustion markers with PD-1, TIM-3, and LAG-3 levels comparable to NT group, indicating preserved functionality and sustained T-cell activation. We also characterized the memory phenotype of these CAR-T cells. Importantly, IL-15-producing BCMA CAR-T cells displayed increased expansion of central memory (p-value<0.05), suggesting that sIL-15 supports the maintenance of memory phenotypes during expansion. To evaluate the impact of IL-15 on the antitumor efficacy of different BCMA-CAR-T cells in vivo, we performed survival studies in NSG mice engrafted with MM1.S tumor cells and treated with 5 × 10⁶ 2nd or 4th BCMA-CAR-T cells. Groups of mice were treated with saline, non-transduced T cells, and CD19 CAR-T cells as control groups (n = 5 per group). In vivo, both BCMA-targeting CAR-T cells therapy demonstrated potent antitumor activity compared to the NT and CD19 control groups. Flow cytometry analysis confirmed the persistence of CD45+CD3+CAR+ cells in bone marrow and spleen of mice treated with 4th BCMA-CAR-T cells. In contrast, these cells were markedly reduced in the 2nd-generation BCMA CAR-T group and undetectable in the NT, CD19. Although the 4th-generation CAR-T cells achieved full tumor protection, mice experienced significant toxicity, leading to mortality within 35 days. In contrast, 2nd generation CAR-T cells therapy induced potent tumor control and survival for up to 140 days (median survival= 125 days), maintaining weight with no severe toxicity. The long-term protection was associated with CD3⁺ T-cell infiltration into the bone marrow and spleen of mice euthanized 140 days post-infusion, at the end of the observation period. As expected, mice treated with CD19-CART cell, NT cells or from saline group showed progressive tumor growth and none of the mice survival. Our findings demonstrate that IL15 production enhances the potency and expansion of BCMA-CAR-T cells but may also drive toxicity. While 4th-generation CAR-T cells rapidly eliminated tumor burden, 2nd-generation CAR-T cells provided durable, safe, and effective tumor control in vivo. These results support the continued development of optimized cytokine-secreting CAR-T platforms, with a critical need to fine-tune IL-15 expression to balance efficacy and safety, particularly in settings where long-term remission with minimal toxicity is desired. Importantly, our findings highlight the promise of second-generation BCMA CAR-T cells as both an academic innovation and a scalable therapeutic option for global use.