Multiple myeloma (MM) remains an incurable disease regardless of recent advances in the field. Therefore, a substantial unmet need exists to treat patients with relapsed/refractory myeloma. The use of novel agents such as daratumumab, elotuzumab, carfilzomib, or pomalidomide, among others, usually cannot completely eradicate myeloma cells. Although these new drugs have had a significant impact on the prognosis of MM patients, the vast majority ultimately become refractory or can no longer be treated due to toxicity of prior treatment, and thus succumb to the disease. Cellular therapies represent a novel approach with a unique mechanism of action against myeloma with the potential to defeat drug resistance and achieve long-term remissions. Genetic modification of cells to express a novel receptor with tumor antigen specificity is currently being explored in myeloma. Chimeric antigen receptor gene-modified T-cells (CAR T-cells) have shown to be the most promising approach so far. CAR T-cells have shown to induce durable complete remissions in other advanced hematologic malignancies like acute lymphocytic leukemia (ALL) and diffuse large B-cell lymphoma (DLBCL). With this background, significant efforts are underway to develop CAR-based therapies for MM. Currently, several antigen targets, including CD138, CD19, immunoglobulin kappa (Ig-Kappa) and B-cell maturation antigen (BCMA), are being used in clinical trials to treat myeloma patients. Some of these trials have shown promising results, especially in terms of response rates. However, the absence of a plateau is observed in most studies which correlates with the absence of durable remissions. Therefore, several potential limitations such as lack of effectiveness, off-tumor toxicities, and antigen loss or interference with soluble proteins could hamper the efficacy of CAR T-cells in myeloma. In this review, we will focus on clinical outcomes reported with CAR T-cells in myeloma, as well as on CAR T-cell limitations and how to overcome them with next generation of CAR T-cells.

PurposeCAR-T cell therapy has proven to be a disruptive treatment in the hematology field, however, less than 50% of patients maintain long-term response and early predictors of outcome are still inconsistently defined. Here, we aimed to optimize the detection of CD19 CAR-T cells in blood and to identify phenotypic features as early biomarkers associated with toxicity and outcomes.Experimental designIn this study, monitoring by flow cytometry and digital PCR (dPCR), and immunophenotypic characterization of circulating CAR-T cells from 48 patients treated with Tisa-cel or Axi-cel was performed.ResultsValidation of the flow cytometry reagent for the detection of CAR-T cells in blood revealed CD19 protein conjugated with streptavidin as the optimal detection method. Kinetics of CAR-T cell expansion in blood confirmed median day of peak expansion at seven days post-infusion by both flow cytometry and digital PCR. Circulating CAR-T cells showed an activated, proliferative, and exhausted phenotype at the time of peak expansion. Patients with increased expansion showed more severe CRS and ICANs. Immunophenotypic characterization of CAR-T cells at the peak expansion identified the increased expression of co-inhibitory molecules PD1 and LAG3 and reduced levels of the cytotoxicity marker CD107a as predictors of a better long-term disease control. ConclusionsThese data show the importance of CAR-T cells in vivo monitoring and identify the expression of PD1LAG3 and CD107a as early biomarkers of long-term disease control after CAR-T cell therapy.

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>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.

B-cell maturation antigen (BCMA) is the lead antigen for CAR T-cell therapy in multiple myeloma (MM). A challenge is inter- and intra-patient heterogeneity in BCMA expression on MM cells and BCMA downmodulation under therapeutic pressure. Accordingly, there is a desire to augment and sustain BCMA expression on MM cells in patients that receive BCMA-CAR T-cell therapy. We used all-trans retinoic acid (ATRA) to augment BCMA expression on MM cells and to increase the efficacy of BCMA-CAR T-cells in pre-clinical models. We show that ATRA treatment leads to an increase in BCMA transcripts by quantitative PCR and an increase in BCMA protein expression by flow cytometry in MM cell lines and primary MM cells. Analyses with super-resolution microscopy confirmed increased BCMA protein expression and revealed an even distribution of non-clustered BCMA molecules on the MM cell membrane after ATRA treatment. The enhanced BCMA expression on MM cells after ATRA treatment led to enhanced cytolysis, cytokine secretion and proliferation of BCMA-CAR T-cells in vitro, and increased efficacy of BCMA-CAR T-cell therapy in a murine xenograft model of MM in vivo (NSG/MM1.S). Combination treatment of MM cells with ATRA and the γ-secretase inhibitor crenigacestat further enhanced BCMA expression and the efficacy of BCMA-CAR T-cell therapy in vitro and in vivo. Taken together, the data show that ATRA treatment leads to enhanced BCMA expression on MM cells and consecutively, enhanced reactivity of BCMA-CAR T-cells. The data support the clinical evaluation of ATRA in combination with BCMA-CAR T-cell therapy and potentially, other BCMA-directed immunotherapies.