Chimeric antigen receptor (CAR) modified T Cells in acute myeloid leukemia: limitations and expectations.

Acute myeloid leukemia (AML) is an aggressive hematologic malignancy with a poor prognosis despite the advent of novel therapies. Consequently, a major need exists for new therapeutic options, particularly for patients with relapsed/refractory (R/R) AML. In recent years, it has been possible to individualize the treatment of a subgroup of patients, particularly with the emergence of multiple targeted therapies. Nonetheless, a considerable number of patients remain without therapeutic options, and overall prognosis remains poor because of a high rate of disease relapse. In this sense, cellular therapies, especially chimeric antigen receptor (CAR)-T cell therapy, have dramatically shifted the therapeutic options for other hematologic malignancies, such as diffuse large B cell lymphoma and acute lymphoblastic leukemia. In contrast, effectively treating AML with CAR-based immunotherapy poses major biological and clinical challenges, most of them derived from the unmet need to identify target antigens with expression restricted to the AML blast without compromising the viability of the normal hematopoietic stem cell counterpart. Although those limitations have hampered CAR-T cell therapy translation to the clinic, there are several clinical trials where target antigens, such as CD123, CLL-1 or CD33 are being used to treat AML patients showing promising results. Moreover, there are continuing efforts to enhance the specificity and efficacy of CAR-T cell therapy in AML. These endeavors encompass the exploration of novel avenues, including the development of dual CAR-T cells and next-generation CAR-T cells, as well as the utilization of gene editing tools to mitigate off-tumor toxicities. In this review, we will summarize the ongoing clinical studies and the early clinical results reported with CAR-T cells in AML, as well as highlight CAR-T cell limitations and the most recent approaches to overcome these barriers. We will also discuss how and when CAR-T cells should be used in the context of AML.

Monitoring of kinetics and exhaustion markers of circulating CAR-T cells as early predictive factors in patients with B-cell malignancies.

Purpose: CAR-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 design: In 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. Results: Validation 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. Conclusions: These 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.

All-trans retinoic acid works synergistically with the γ-secretase inhibitor crenigacestat to augment BCMA on multiple myeloma and the efficacy of BCMA-CAR T cells.

B-cell maturation antigen (BCMA) is the lead antigen for chimeric antigen receptor (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 reverse transcription polymerase chain reaction 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/MM.1S). 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.

Overcoming Chimeric Antigen Receptor (CAR) Modified T-Cell Therapy Limitations in Multiple Myeloma.

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.