Development of a Novel Humanized BCMA-Directed CAR for Multiple Myeloma: Robust Activity and Low Toxicity in Preclinical Studies

Background: Chimeric antigen receptor T (CAR-T) cell therapies targeting B cell maturation antigen (BCMA) have emerged as a promising intervention for relapsed/refractory multiple myeloma (r/r MM). However, challenges such as treatment-related inflammatory toxicities [cytokine release syndrome (CRS) and neurotoxicity], relapse, limited availability, and the exorbitant costs need to be addressed to expand their benefits to more patients. Here, we designed and tested several humanized BCMA-targeted CARs through extensive in vitro and in vivo studies and identified a novel BCMA CAR candidate with the best safety and efficacy profile. Methods: We developed four novel BCMA-specific CAR constructs (h1 to h4 CAR) using a humanization platform previously established in our laboratory (Dwivedi et al, Mol Cancer Ther.2021, Jain et al, ASH 2022). BCMA CAR-T cells were generated using lentiviral-mediated transduction and screened for in vitro functional attributes (binding affinity, cytotoxicity, and cytokine production) including repeat antigen stimulation assay. T cell subset differentiation and checkpoint marker expression were analyzed using multiparametric flow cytometry. For in vivo efficacy studies, luciferase-labeled BCMA+ tumor cells (5e5) were intravenously injected into immunodeficient mice, followed by CAR-T cell infusion (5e6). Hematological analysis and histopathological examinations were also performed to verify the safety and biodistribution of CAR-T cells. Results: All four novel CAR constructs (h1 to h4 CAR) were initially screened for their lentiviral vector production feasibility, CAR surface expression, and ex vivo CAR-T cell expansion. Among these, the h1CAR and h2CAR showed significantly higher viral titers (>4e6 TU/ml) and excellent BCMA CAR-T proliferative capabilities compared to the others. Subsequently, we assessed their binding strength to the BCMA antigen, of which h1CAR (KD: 0.76nM) and h2CAR (KD: 0.55nM) demonstrated affinities comparable to the high-binding murine CAR (KD: 0.57nM), which was used as a positive control. Based on these preliminary results, h1CAR and h2CAR were selected for further functional analysis. To evaluate their antigen-dependent activity in an ex vivo setting, we co-cultured CAR-T cells with tumor cells exhibiting varying surface BCMA antigen densities. Specifically, h2CAR-T cells demonstrated robust in vitro cytotoxicity across all BCMA-expressing cell lines (Mean ± SEM; RPMI8226Low: 88.33%±2.14, Nalm6-BCMAMed: 88.55%±3.01, K562-BCMAHigh: 76.43%±5.08), comparable to murine anti-BCMA CAR-T cells (mCAR-T cells, positive control) (RPMI8226Low: 86.30%±2.50, Nalm6-BCMAMed: 86.08%±4.36, K562-BCMAHigh: 76.78%±5.40), with negligible killing of BCMA-negative cell lines (Nalm6: 14.68%±11.62, K562: 0.62%±10.07). Moreover, the cytokine analysis revealed significantly lower secretion of IFN-γ (p=0.0195), TNF-α (p=0.0011), IL-2 (p<0.001), IL-6 (p=0.0352) and IL-1β (p=0.019) with h2CAR-T cells compared to mCAR-T cells. This suggests that h2CAR could be a potential low-toxicity CAR, as cytokines associated with CRS were present at lower levels than with mCAR. To further evaluate the robustness of CAR-T cells, we conducted an in vitro antigen rechallenge assay, during which h2CAR-T cells showed superior expansion capacity over mCAR-T cells with complete target elimination at each interval. The h2CAR-T also exhibited less exhaustion, as indicated by lower TIM3 expression (p=0.0034) and maintained a more stable memory phenotype compared to other CARs. In a pre-established myeloma xenograft model, h2CAR-T cells effectively suppressed tumor growth for over 90 days, while all mice treated with mCAR-T relapsed by day 35. More importantly, h2CAR-T treatment prolonged animal survival relative to mock controls and mCAR-T treated groups (Median survival time: 123 days, p<0.001). No systemic or organ-related toxicities were observed post CAR-T infusion. Conclusion: The novel h2CAR demonstrated robust preclinical affinity, efficacy, and a favorable safety profile. These results provide a strong foundation for a first-in-human clinical trial with h2CAR as a promising candidate for multiple myeloma.