Both APRIL and antibody-fragment-based CAR T cells for myeloma induce BCMA downmodulation by trogocytosis and internalization

Abstract

BackgroundChimeric antigen receptor (CAR) T cell therapy targeting B cell maturation antigen (BCMA) on multiple myeloma (MM) produces fast but not long-lasting responses. Reasons for treatment failure are poorly understood. CARs simultaneously targeting two antigens may represent an alternative. Here, we (1) designed and characterized novel A proliferation inducing ligand (APRIL) based dual-antigen targeting CARs, and (2) investigated mechanisms of resistance to CAR T cells with three different BCMA-binding moieties (APRIL, single-chain-variable-fragment, heavy-chain-only).MethodsThree new APRIL-CARs were designed and characterized. Human APRIL-CAR T cells were evaluated for their cytotoxic function in vitro and in vivo, for their polyfunctionality, immune synapse formation, memory, exhaustion phenotype and tonic signaling activity. To investigate resistance mechanisms, we analyzed BCMA levels and cellular localization and quantified CAR T cell–target cell interactions by live microscopy. Impact on pathway activation and tumor cell proliferation was assessed in vitro and in vivo.ResultsAPRIL-CAR T cells in a trimeric ligand binding conformation conferred fast but not sustained antitumor responses in vivo in mouse xenograft models. In vitro trimer-BBζ CAR T cells were more polyfunctional and formed stronger immune synapses than monomer-BBζ CAR T cells. After CAR T cell–myeloma cell contact, BCMA was rapidly downmodulated on target cells with all evaluated binding moieties. CAR T cells acquired BCMA by trogocytosis, and BCMA on MM cells was rapidly internalized. Since BCMA can be re-expressed during progression and persisting CAR T cells may not protect patients from relapse, we investigated whether non-functional CAR T cells play a role in tumor progression. While CAR T cell–MM cell interactions activated BCMA pathway, we did not find enhanced tumor growth in vitro or in vivo.ConclusionAntitumor responses with APRIL-CAR T cells were fast but not sustained. Rapid BCMA downmodulation occurred independently of whether an APRIL or antibody-based binding moiety was used. BCMA internalization mostly contributed to this effect, but trogocytosis by CAR T cells was also observed. Our study sheds light on the mechanisms underlying CAR T cell failure in MM when targeting BCMA and can inform the development of improved treatment strategies.

Structure-guided engineering of immunotherapies targeting TRBC1 and TRBC2 in T cell malignancies

“…Fratricide could also arise from trogocytosis, a process involving stripping of membranes from target cells by CAR T cells. As previously reported for several CAR T cell products 32 – 35 , trogocytosis can contribute to diminished in vivo persistence by stimulating fratricide against antigen-positive CAR T cells. Additionally, it may also facilitate tumor escape due to transient downregulation of target antigen.…”

Essential procedures of single-cell RNA sequencing in multiple myeloma and its translational value

“…[181][182][183] A recent study found that MM relapse following CAR-T therapy may relate to the trogocytosis and internalization or biallelic loss of BCMA. 130,137,184 Most of the current therapies target either tumor cells or immune cells, but inflammatory MSCs in the BM microenvironment remain unaffected by successful antitumor therapy. This may contribute to disease persistence or relapse and suggests a potential avenue for treating MM.…”

Innovation in BCMA CAR-T therapy: Building beyond the Model T

“…Avoiding overly strong CAR affinity may lower T-cell exhaustion as well ( 20 ). Interestingly, lowering CAR affinity for target antigens may also lower the risk of trogocytosis, a complex process by which the CAR-T cell strips BCMA from the target cell before itself expressing the same molecule (and thus becoming a potential victim to fratricide by other CAR-T cells) ( 21 , 22 ).…”