ATRA Augments BCMA Expression on Myeloma Cells and Enhances Recognition By BCMA-CAR T-Cells
Authors: Garcia-Guerrero Estefania , Rodríguez-Lobato Luis Gerardo , Danhof Sophia , Sierro-Martínez Belén , Goetz Ralph , Sauer Markus , Perez-Simon Jose A. , Einsele Hermann , Hudecek Michael , Prommersbe Sabrina 
Published: November 2020

Background:
B cell maturation antigen (BCMA) is a B-lineage antigen that is retained on malignant plasma cells in multiple myeloma (MM), and is under investigation as a target antigen for humoral and cellular immunotherapy. Targeting BCMA with chimeric antigen receptor (CAR) T-cells, T-cell engaging antibodies and antibody-drug conjugates has resulted in high rates of clinical responses however, the depth and durability of these responses is still not satisfactory and most patients ultimately relapse. This has been attributed at least in part to low or non-uniform BCMA expression on MM cells, as well as MM cell escape after BCMA down-regulation or even loss. Here, we show that epigenetic modulation with all-trans retinoic acid (ATRA) augments BCMA expression at the gene (and protein) level and leads to enhanced BCMA molecule density on the surface of MM cells that translates into increased anti-MM potency of BCMA CAR T-cells.
Methods:
Primary MM cells and myeloma cell lines were treated with titrated doses of ATRA (25, 50, 100 nM), alone and in combination with the g-secretase inhibitor crenigacestat (10 nM). BCMA expression was analyzed by flow cytometry, RT-qPCR and direct stochastic optical reconstruction microscopy (dSTORM). BCMA CAR T-cells were derived from healthy donors and MM patients (n&gt;6) and their anti-MM function analyzed in vitro and in the NSG/MM.1S murine xenograft model in vivo.
Results:
By RT-qPCR, we observed a 1.8-fold (MM.1S) and 2.1-fold (OPM-2) increase in BCMA gene expression after treatment with 50 nM ATRA for 72 hours. By flow-cytometry, we confirmed increased BCMA protein expression, with 1.9-fold (MM.1S and OPM-2) increase in mean fluorescence intensity relative to isotype control staining. Super-resolution dSTORM microscopy on MM.1S cells confirmed the increase in BCMA protein expression and showed a homogenous distribution pattern of BCMA molecules across the cell surface without an increase in cluster formation. These data were confirmed with primary MM cells from patients with newly diagnosed (n=7) and relapsed/refractory (n=11) MM. The increase in MFI for BCMA expression on primary MM cells after ATRA treatment was 1.2-fold - 2.2-fold (mean: 1.6-fold; p=.01 at 50 nM ATRA). By ELISA, we did not detect increased levels of soluble BCMA protein in supernatant of MM.1S cells after ATRA treatment. Accordingly, we found superior cytolytic activity, cytokine secretion and proliferation of CD8+ and CD4+BCMA CAR T-cells in response to ATRA-treated vs. non-treated primary MM cells and MM cell lines. In the NSG/MM.1S xenograft model, we confirmed increased BCMA expression on MM.1S after systemic treatment with ATRA, and superior anti-MM activity after adoptive transfer of BCMA CAR T-cells. Further, we confirmed that epigenetic modulation of BCMA-expression with ATRA works synergistically with g-secretase inhibitor treatment that has recently been shown to prevent cleavage of BCMA molecules from the surface of MM cells (Pont Blood 2019). Combination treatment with ATRA and the g-secretase inhibitor crenigacestat led to higher BCMA density on primary MM cells (and cell lines) than each single-agent treatment alone, resulting in maximum reactivity of by BCMA CAR T-cells in vitro and in vivo.
Conclusions:
Taken together, the data show that BCMA expression on MM cells can be increased by epigenetic modulation with ATRA. After ATRA treatment, MM cells have increased susceptibility to BCMA CAR T-cell treatment in pre-clinical models vitro and in vivo, that can be increased even further by combination treatment of ATRA and g-secretase inhibitors. These data suggest the potential to improve responses (depth and durability) of immunotherapies directed against BCMA.

Disclosures
Einsele: Takeda: Consultancy, Honoraria, Speakers Bureau; Bristol-Myers Squibb: Consultancy, Honoraria, Research Funding, Speakers Bureau; Amgen: Consultancy, Honoraria, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Research Funding, Speakers Bureau; Janssen: Consultancy, Honoraria, Research Funding, Speakers Bureau; Novartis: Honoraria, Speakers Bureau; Sanofi: Consultancy, Honoraria, Research Funding, Speakers Bureau; GlaxoSmithKline: Honoraria, Research Funding, Speakers Bureau.