Enhanced Antitumoral Efficacy of ARI0002h CAR-T with CD28-TMD in Normal and Low BCMA Expressing Myeloma Cells
Authors: Rodríguez-Lobato Luis Gerardo , Cardus Oriol , Mañé Pujol Joan , Battram Anthony , Moreno David F. , Perez-Amill Lorena , Martín-Antonio Beatriz , Oliver-Caldés Aina , De Daniel Anna , Tovar Natalia , Cibeira López M Teresa , Rosinol Dachs Laura , Juan Manel , Urbano-Ispizua Alvaro , Fernández de Larrea Carlos 
Published: December 2024

INTRODUCION
Multiple myeloma (MM) remains incurable despite significant therapeutic advancements. Consequently, there is considerable interest in developing new immunotherapies, such as CAR-T cells. BCMA is the primary target for CAR-T cells in MM, showing promising outcomes. However, despite high initial response rates, many patients experience relapse (Munshi, NEJM 2021; Berdeja, Lancet 2021) after CAR-T cells targeting BCMA; this may be attributed to low CAR-T cell persistence, low antigen expression and/or T-cell exhaustion.
Recent research has revealed that CD28 transmembrane domains (CD28-TMD) in CAR receptors induce dimerization with CD28, forming CAR-CD28 heterodimers that enhance sensitivity to low-abundance antigens. Clinically, approved anti-CD19 CAR-T cells for B-cell malignancies have demonstrated that second-generation 4-1BB CARs with CD28-TMD offer high efficacy rates and a favorable toxicity profile.
At our institution, we have developed a 2nd generation CD8α-TMD BCMA-4-1BBζ CAR-T product (ARI0002h; cesni-cel) for patients with relapsed/refractory MM. This product has shown encouraging clinical results (Oliver-Caldés, Lancet Oncol 2023).
Our goal is to create an enhanced anti-BCMA ARI0002h CAR incorporating a CD28-TMD domain to bolster T-cell activation and responsiveness, thereby increasing efficacy against MM cells with low BCMA expression.
METHODS:
T-cells were isolated from PBMCs of healthy donors, activated with CD3/CD28 Dynabeads, and transduced with lentivirus. CAR expression was evaluated on day 8, and subsequent experiments were performed after 8-10 days of T-cell expansion. BCMA CAR-T cells were tested against MM cell lines (U266, MM.1S, and MM.1S BCMAdim). In vitro analyses included cytotoxicity, proliferation, cytokine secretion, and markers of T-cell subsets, activation and exhaustion. In vivo xenograft studies utilized NSG mice. Tumor cells expressing GFP-ffLuc were injected intravenously on day 0. On day 14, mice were randomized to receive effector cells. Disease progression was monitored weekly using bioluminescence imaging.
RESULTS:
The anti-BCMA ARI0002h CAR construct was modified by replacing the CD8α-TMD with a CD28-TMD domain to create ARI2h-TM28. Following lentiviral transduction, ARI2h-TM28 CARs were expressed on the T-cell membrane with a transduction rate comparable to the original ARI0002h CAR's.
Antigen detection, tumor cell lysis, cytokine secretion, and proliferation were confirmed in response to MM cell lines expressing normal levels of BCMA (U266 and MM.1S). In vivo activity was assessed using NSG mice engrafted with MM.1S cells. A high dose of tumor cells and a low dose of CAR-T cells were injected to create a stress MM model. Tumor control was superior in the ARI2h-TM28 group compared to the ARI0002h group, with a significant survival advantage observed in the ARI2h-TM28 group.
The capabilities of ARI2h-TM28 against a MM cell line with reduced BCMA expression (MM.1S-BCMAdim) were also analyzed. In vitro, the killing capacity of ARI2h-TM28 was approximately 45% higher than that of ARI0002h. Furthermore, in the in vivo NSG mice model with reduced BCMA expression, ARI2h-TM28 demonstrated better myeloma control and extended survival compared to ARI0002h.
To elucidate the mechanisms behind the increased activity in both normal and reduced BCMA expression scenarios, preliminary data indicated that after four challenges with tumor cells expressing normal BCMA levels, expansion rates were similar between ARI0002h and ARI2h-TM28. PD-1 expression increased in ARI0002h, while TIGIT increased and LAG3 decreased in ARI2h-TM28. No significant differences in activation markers were noted. However, after four challenges, ARI2h-TM28 CD8 cells exhibited a more balanced effector memory and effector cell percentage compared to ARI0002h. The characterization of ARI2h-TM28 T cells regarding metabolism, transcriptomic, cytokine profiles, activation, exhaustion markers, and T-cell subsets is ongoing.
CONCLUSION:
The incorporation of a CD28-TMD into the ARI0002h CAR enhances its potency against MM tumor cell lines with both normal and reduced BCMA expression, demonstrating superior in vitro and in vivo efficacy. This modification enables better control of higher tumor burdens and offers improved long-term disease management compared to CARs with CD8α-TMD.