Enhanced Antitumoral Activity of the Academic CAR-T ARI0002h Against Normal and Low BCMA Expressing Myeloma Cells After Incorporating a Transmembrane CD28 Domain

BCMA is the main target for CAR-T cells in multiple myeloma (MM), demonstrating promising outcomes. However, unlike what happens with CART19 in lymphoblastic leukemia and non-hodgkin lymphoma, a high proportion of patients will relapse after CART BCMA therapy due to insufficient antigen expression, low CAR-T cell persistence and/or T-cell exhaustion. CD28 transmembrane domains (CD28-TMD) in CAR receptors promote dimerization with CD28, forming CAR-CD28 heterodimers that enhance sensitivity to low-abundance antigens. In other B cell malignancies, second-generation anti-CD19 4-1BB CARs with CD28-TMD have shown high efficacy and a favorable toxicity profile. We have developed a second-generation CD8α-TMD BCMA-4-1BBζ CAR-T product, ARI0002h (ARI2h; cesni-cel) for patients with relapsed/refractory MM. We hypothesized that replacing the TMD of ARI0002h with a CD28-TMD could increase efficacy and reduce tumor escape while maintaining a tolerable toxicity profile. We generated CAR-T cells using T-cells isolated from buffy coats and evaluated the efficacy and fitness of CAR-Ts at day 8-10 of expansion against several MM cell lines. In vitro analyses included cytotoxicity, proliferation, cytokine secretion, T-cell subset markers, activation and exhaustion profiling, metabolomic assays, and RNA-seq after multiple tumor challenges. In in vivo xenograft studies using NSG mice, with tumor cells expressing GFP-ffLuc, disease progression was monitored weekly via bioluminescence imaging.