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 patien