Armored Multivalent CAR-T Cells Targeting BCMA, GPRC5D, and CD38 Eradicate Antigen-Heterogeneous Multiple Myeloma, Address Antigen Escape and Enhance Expansion

Introduction: The expression of BCMA, GPRC5D, and CD38 is highly prevalent and independent across multiple myeloma plasma cells (MMPC), with rare occurrence of double- or triple-negative MMPCs in patients (Dekhtiarenko et al. ASH 2023). While targeted antibodies and CAR-T therapies have transformed the treatment landscape for MM, many patients still developed resistance to treatment and relapsed. In relapsed MM, CAR-T therapy demonstrated superior ORR in the entire cohort, as well as in cohorts with extramedullary disease (EMD) and high-risk cytogenetics, compared to bispecific antibodies (Vegivinti et al. ASH 2023). The heterogeneity of antigens and the suppressive tumor microenvironment in EMD pose significant clinical challenges. Moreover, clinical studies have demonstrated that antigen loss due to internalization, shedding or clonal mutation reveals non-overlapping mechanism of drug resistance (Samur et al. ASH 2022, Lee et al. ASH 2023). The response to CAR-T cells is influenced by both tumor antigen expression and baseline immune fitness (Freeman et al. ASH 2023). To address these challenges, we have developed multi-specific CAR-T targeting antigens – BCMA, GPRC5D and/or CD38, and incorporating novel armor, in order to enhance anti-tumor efficacy in the presence of soluble BCMA (sBCMA), reduce risk of Ag escape, preserve T cell fitness and promote expansion. We aim to achieve broad and deep tumor depletion, improve clinical outcome and durability for R/R MM.

Methods: We designed mono-, bi- and tri-specific CAR-T constructs targeting BCMA, GPRC5D, and/or CD38 with a membrane-bound armor in a lentiviral vector. The multi-valent targeting strategy incorporates novel bi-paratopic VHH domains specific to BCMA and GPRC5D, as well as a novel mono-VHH domain for CD38 recognition. We hypothesized that our leading multi-specific VHH CAR-T(s) would augment T-cell engagement and sensitivity with MM cells, counteract antigen escape, alleviate the inhibitory effect of "antigen sink" caused by the elevated sBCMA in MM, exhibit no toxicity towards only CD38-positive normal cells, and additionally, the use of a novel armor would preserve T fitness and facilitate expansion. We conducted preclinical tests to confirm the functionality of these features, by comparing the in vitro cytotoxicity, cytokine release, and T cell phenotype of multi-specific CAR-Ts to benchmarks against antigen-heterogeneous MM cells expressing varying levels of antigens. Additionally, we investigated in vivo treatment efficacy and persistence of CAR-T cells in a NCG murine model xenograft with heterogeneous MM cell lines.

Results: Engagement with BCMA, GPRC5D and/or CD38 resulted in multi-specific CAR-T cells exhibiting improved immune cell activation, increased degranulation upon activation, comparable or increased cytotoxicity and cytokine production, as well as enhanced target-dependent expansion under repetitive tumor challenges. Importantly, the improved designs and novel VHH binders of multi-specific CAR-Ts mitigated the inhibitory effects of sBCMA. To address a potential safety concern, the toxicity of multi-specific CAR-Ts was specifically assessed on only CD38-positive cells. Furthermore, in a model predicting CRS risk, upon antigen-specific stimulation, the multi-specific CAR-Ts induced low level of IL-6 release via monocytes. In addition, an in vivo comparison of the multi-specific CAR-Ts with mono- or dual-specific CAR-Ts showed that the multi-specific CAR-Ts displayed enhanced T cell expansion and superior efficacy in tumor elimination.

Conclusions: The preclinical data of the multi-specific CAR-Ts exhibit their potent anti-tumor efficacy and persistence, addressing antigen escape and preserving potency in the presence of sBCMA. Furthermore, the incorporation of innovative armor enhances T cell fitness and reduces exhaustion. Our findings advocate for further exploration of multi-specific targeting as a promising strategy to facilitate rapid and profound tumor clearance and achieve durable responses in R/R MM, potentially circumventing clonal selection. Further investigation into baseline and longitudinal correlations between immune and tumor profiles with clinical response to the multi-specific CAR-T may provide insights into mechanisms of response and resistance, and inform a development of novel therapy.