Low Target Antigen Expression Mediates Resistance to BCMA CAR T Cell Therapy

Introduction: BCMA-targeted CAR T cell therapies have shown promising responses, but many patients have residual disease post therapy, and relapses are common. Prior studies with CD19-targeted CAR T cells have shown that antigen density is an important determinant of response, with CARs requiring several hundred (Mansilla-Soto et al. Nat Med 2021) to a few thousand (Spiegel et al. Nat Med 2021) target molecules per cell for effective tumor recognition and control. Methods: To determine how antigen levels affect response to BCMA-directed CAR T-cell therapy, we performed quantitative flow cytometric measurements of BCMA antibody binding capacity (ABC) on fresh tumor samples from patients with rel/refractory multiple myeloma (RRMM), including 27 patients treated with idecabtagene vicleucel or ciltacabtagene autoleucel. ABC of GPRC5d was measured as control. To model the impact of antigen levels on CAR T cell response, we generated CRISPR-Cas9 edited leukemia (NALM6) and myeloma (MM1s) clones with varying levels of CD19 and BCMA antigen expression, respectively, and quantitatively assessed CAR T cell function against these target antigen-titrated lines in vitro and in vivo. Results: Antigen levels were measured in a cohort of 32 patients with RRMM with a median of 6 (range 3-12) prior lines of therapy, including seven patients previously treated with BCMA targeted antibody-drug conjugates. Median BCMA ABC was 670 mol/cell (range undetectable - 2460 mol/cell), consistent with prior estimates (Salem et al. Leuk Res 2018). Among patients treated with commercial BCMA-targeted CAR T-cell therapy (n=27), median BCMA expression was 670 (IQR 380-850) mol/cell prior to treatment. At the time of measurable residual disease or progression, median antigen abundance had decreased to 390 (IQR 290-490) mol/cell (15 patients, p = 0.011). The majority of relapsed patients displayed low-level detectable BCMA. Expression of control antigen GPRC5d was unchanged pre- and post-treatment. In vitro modeling showed that CAR T cell tumor lysis and cytokine production (IFNγ, IL-2) were gradually impaired as antigen levels decreased from 2000 to 200 mol/cell, and were absent at < 50 mol/cell. In an in vivo xenograft model, BCMA-directed CAR T cells cured an unmodified MM1s line expressing > 5,000 BCMA mol/cell, whereas animals bearing an MM1s.13 antigen low cell line expressing 600 (+/- 150) mol/cell initially controlled tumor but relapsed by day 40. Conclusion: BCMA expression in patients with RRMM is lower than CD19 expression typically seen in patients with B cell malignancies. Furthermore, while complete loss of BCMA after CAR T cell therapy is uncommon, resistant or relapsed disease frequently expresses low levels of antigen. In vitro and in vivo modeling show that these levels are below the threshold required for optimal CAR T cell function. These data suggest that low antigen expression may be an important mechanism of relapse in BCMA CAR T cell therapy for multiple myeloma.