(PA-085) TRAIL-CD28 Armoring Enhances anti-BCMA CAR-T Cell Function and Overcomes Resistance in Multiple Myeloma

While BCMA-targeted CAR-T cell therapies have demonstrated encouraging response rates in multiple myeloma (MM), most patients eventually relapse. Our transcriptomic single cell analysis of patient samples treated with anti-BCMA CAR-T cells (Ide-cel) revealed that responders exhibited higher expression of genes encoding survival and activation-related proteins such BclxL, TRAIL, and CD28. Cognizant of the role of death receptor ligands in mediating activated T cells cytolytic activity, we here investigated whether TRAIL or TRAIL-CD28 chimeric construct armoring of anti-BCMA CAR-T cells enhanced their fitness and anti-tumoral activity.

Methods: Single-cell RNA sequencing (CITE-seq, 10x Genomics) was used to analyze CAR-T cells from responders and non-responders to Ide-cel. CAR constructs with anti-BCMA scFv, 4-1BB and CD3z were cloned into the pLX307 lentiviral backbone, with variants co-expressing either TRAIL or a chimeric TRAIL-CD28 molecule. Functional assessment was performed using MM cell lines under standard and exhaustion-inducing conditions. Resistant model included in-house derived teclistamab-resistant MM cell lines and NFkB activated cells (Cas9 TRAF3 Knock-out) with acquired cross-resistance to variable anti-BCMA and anti-GPRC5D T cell engagers and CAR-T cells. Flow cytometry was performed using CytoFLEX and spectral Sony ID7000 systems to assess tumor clearance, T cells phenotype, and cells viability.

Results: TRAIL armoring significantly enhanced BCMA CAR-T cell functionality across MM cell lines, under conditions of chronic antigenic stimulation and high disease burden (low E:T ratio). TRAIL-armored CAR-T cells effectively eradicated teclistamab-resistant and NFκB-activated MM cells, a capacity that was notably absent in unarmored CAR-T cells. However, under very high-density culture conditions, TRAIL expression induced mild fratricide (~10%) among CAR-T cells. To mitigate this effect, we engineered a TRAIL-CD28 chimera, fusing the extracellular and transmembrane TRAIL domains with CD28 intracellular signaling domain. This chimeric construct not only significantly mitigated fratricide, but also potently enhanced engineered T cells survival through CD28-mediated pro-survival signaling. TRAIL-CD28–armored CAR-T cells exhibited enhanced fitness profiles with preserved naïve- and central memory-like phenotypes and sustained BclxL expression—hallmarks of durable T cell memory function. Importantly, TRAIL-CD28 armoring of anti-BCMA CAR-T cells did not result in cytolysis of healthy donors PBMCs, consistent with their undetectable or low death receptors 4/5 expression. Ongoing animal studies with luciferase stably expressing MM cells are ongoing to evaluate the in vivo activity of these armored CAR-T cells.

Conclusions: TRAIL-CD28 armoring of CAR T cells represents a promising strategy to enhance the efficacy of anti-BCMA CAR-T cells. These findings warrant further preclinical and clinical validation as a next-generation CAR-T for relapsed/refractory MM.