(PA-005) The Role of Trogocytosis in BCMA CAR T Cell Therapy with Gamma-Secretase Inhbition

BCMA CAR T cells have substantial efficacy in relapsed/refractory multiple myeloma (MM).γ-Secretase inhibitors (GSIs) increase surface BCMA, reduce antigen shedding and improve cytotoxicity in preclinical studies.Increased BCMA density after GSI has correlated with improved outcomes (ORR, PFS and OS) in a phase I clinical trial.Nonetheless, most patients relapse. Trogocytosis—the transfer of membrane-bound antigens from tumor to CAR T cells—has emerged as a potential mechanism of antigen downmodulation, T cell dysfunction, and fratricide. We investigated the effects of GSI on trogocytosis kinetics and its consequences in vitro, and in concert with clinical data from two Phase I trials of BCMA-targeted CAR T therapy.

Methods:

Human T cells transduced with BCMA-directed 4-1BB/CD3ζ CAR (EGFRt⁺), were expanded and sorted. MM cell lines (H929, MM.1R, MOLP8) and BCMA-mCherry⁺ K562 cells were co-cultured with CAR T cells (E:T 1:1) for 10 min–24h ± GSI [crenigacestat] (0.1 μM, 24h).BCMA density and trogocytosis were evaluated by flow cytometric (FC) analysis.Latrunculin A (1 μM) was used to inhibit trogocytosis.BCMA-acquiring CAR T cells (Trogo⁺) were sorted after 6h co-culture and re-plated with naïve CAR T cells for 24h to assess fratricide.Patient samples from two Phase I trials—FCARH143 (NCT03338972) and FCARH143+GSI (NCT03502577) were analyzed for trogocytosis, CAR T persistence (qPCR) and clinical response.

Results:

GSI significantly increased BCMA density, enhanced CAR T cell cytotoxicity, but also increased trogocytosis, particularly in high-antigen-density cell lines (H929+GSI vs H929; 30min (P < 0.0001),1h (P < 0.0001),2h (P < 0.0001),6h (P < 0.0001),and 24h (P < 0.0001) and in CD4⁺ CAR T cells (CD4 vs CD8 CAR T cells; 10min (P=0.01), 2h (P=0.01), and 6h (P=0.004).CAR T Trogo⁺ cells exhibited reduced proliferative capacity, diminished cytotoxic function (CAR T Trogo+ vs CAR T;(P=0.0002), and higher exhaustion/activation markers (CD4+ CAR T Trogo+ vs CD4 CAR T; PD-1+LAG-3+, PD-1+TIM-3+, and TIM-3+LAG-3+;(P=0.04, P=0.03, and P=0.002). In fratricide assays, CAR T Trogo⁺ cells were susceptible to killing by naïve CAR T cells (CAR T Trogo+ alone vs CAR T Trogo+ and CAR T, P=0.0005).Confocal microscopy and live imaging confirmed trogocytosis and fratricide.BCMA trogocytosis was found in patient samples, although an association with reduced CAR T cell persistence and treatment response did not reach statistical significance.

Conclusions:

We report disparate effects of GSI on BCMA CAR T cell therapy including enhancement of tumor targeting and promotion of trogocytosis-associated dysfunction.Trogocytosis may contribute to antigen modulation, CAR T cell exhaustion, and fratricide, potentially limiting full realization of the benefits from increased antigen density.Future clinical trials incorporating early time-point sampling and mechanistic assessments may facilitate further optimization of GSI administration and inform strategies to mitigate trogocytosis-associated resistance mechanisms.