Improving BCMA-targeted CAR-T cell therapy of myeloma using TGF-beta resistance and inducible production of IL-18

Background: Although immunotherapy with B cell maturation antigen (BCMA)-targeted chimeric antigen receptor (CAR) T cells, or BCMA CARTs for short, has yielded unprecedented treatment responses in patients with relapsed and/or refractory multiple myeloma (RRMM), efficacy is limited by lack of durability in most cases. This has been attributed, in large part, to the immunosuppressive bone marrow environment (BME) of RRMM, leading to inactivation, exhaustion and attrition of BCMA CARTs. Next-generation CARTs, capable of overcoming BME-dependent inhibitory constraints, may lead to more durable treatment options for RRMM. Methods: To inform the design of BCMA CARTs that can maintain long-term activity in the myeloma-laden bone marrow, we leveraged a recently published single-cell RNA sequencing dataset, GSE210079, of the BME from 9 patients with RRMM. Based on clinical outcomes, patients were stratified into early relapse (<3 months) and late relapse (>12 months) groups. Biocomputational analyses revealed that activation of TGF-beta signaling and T cell exhaustion pathways, along with suppression of IL-18-driven immune responses, were implicated in the mechanisms driving early-relapse post-BCMA CAR-T treatment. Inspired by these findings, we employed standard molecular biology tools to manufacture BCMA CARTs that feature both TGF-beta resistance and simultaneously disrupt PD‑1 expression and enabling inducible IL‑18 secretion. Result: Compared to T cells from late-relapse myeloma, T cells from early-relapse myeloma demonstrated upregulated oncogenic signaling circuits (p53, FoxO, AMPK), elevated expression of exhaustion-related genes ( PDCD1 , HAVCR2 , LAG3 , CTLA4, TIGIT; p < 0.01), and increased mRNA levels of key TGF-beta pathway genes ( TGFB1 , SMAD2/3 / 4 , TGFBR2 ). Early-relapse myeloma exhibits reduced TCR signaling and Th1/Th2 differentiation, which are regulated by IL-18. BCMA CARTs were rendered resistant to TGF-beta by enforced expression of a dominant-negative ligand-binding but non-signaling transforming growth factor beta receptor 2 (dnTFGBR2). Monoallelic “knock-in” of an IL-18 encoding gene in the PDCD1 locus relied on CRISPR/Cas9-mediated editing. The genetically modified BCMA CAR-T cells demonstrated potent cytotoxic activity against myeloma cells in vitro, increased resistance to TGF-beta-mediated suppression, and inducible secretion of IL-18 driven by the endogenous PDCD1 promoter. IL‑18 levels increased in correlation with T cell activation, accompanied by a concomitant silencing of PD‑1 expression. Conclusion: TGF-beta-resistant BCMA CARTs that produce IL-18 when the cells are challenged by exhaustion in the BME, and thus disrupt PD-1, may be viable way forward to a more potent and durable therapy for patients with RRMM than available today. This hypothesis will be evaluated in greater depth in preclinical in vivo studies ongoing in our laboratory.