What question did this study set out to answer?

To assess the efficacy of the CAR-T cell therapy ARI0002h with a CD28 transmembrane domain against myeloma cells expressing varying BCMA levels.

March 5, 2026Open Access

Enhanced antitumoral activity of the academic CAR-T ARI0002h against normal and low BCMA-expressing myeloma cells after incorporating a transmembrane CD28 domain

Key Points

To assess the efficacy of the CAR-T cell therapy ARI0002h with a CD28 transmembrane domain against myeloma cells expressing varying BCMA levels.
Generated CAR-T cells from isolated T-cells and evaluated their performance against multiple myeloma cell lines.
Conducted in vitro analyses including cytotoxicity, proliferation, and cytokine secretion.
Performed in vivo xenograft studies using NSG mice and monitored disease progression through bioluminescence imaging.
In vitro, ARI2h-TM28 showed similar cytotoxicity and proliferation but better efficacy against low BCMA-expressing cells.
In vivo, ARI2h-TM28 exhibited superior tumor control and improved survival in relapse models.
The modified CAR-T demonstrated enhanced metabolic profiles and reduced T-cell exhaustion, increasing persistence and efficacy.

Abstract

Background B-cell maturation antigen (BCMA) is the main target for chimeric antigen receptor (CAR)-T cells in multiple myeloma (MM), demonstrating promising outcomes. However, unlike what happens with CART19 in lymphoblastic leukemia and non-Hodgkin's lymphoma, a high proportion of patients will relapse after CAR-T BCMA therapy due to insufficient antigen expression, low CAR-T cell persistence and/or T-cell exhaustion. In other B cell malignancies, second-generation anti-CD19 4-1BB CARs with CD28-transmembrane domain (TMD) have shown high efficacy and a favorable toxicity profile. We have developed a second-generation CD8α-TM BCMA-4-1BBζ CAR-T product, ARI0002h (Cesnicabtagene-autoleucel) 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. Methods 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. Results Despite showing similar in vitro performance regarding cytotoxicity, proliferation and cytokine production, ARI2h-TM28 outperforms ARI0002h in a low BCMA expression setting and achieves superior in vivo tumor control and survival in relapse models with antigen downregulation. Furthermore, ARI2h-TM28 showed an optimized metabolic profile, more oxidative and energetic compared with ARI0002h, with downregulation of proinflammatory genes in CD8 T cells, contributing altogether both to reduced exhaustion and increased persistence of the CARs, improving their efficacy in preclinical models. Conclusions Incorporating a CD28-TMD into the ARI0002h CAR enhances tumor control even in relapse models with downregulation of the target antigen, offering improved long-term disease management. This modification increases potency against MM tumor cell lines with both normal and reduced BCMA expression, demonstrating superior metabolic endurance and in vivo activity.

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