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CAR‐T cells derived from multiple myeloma patients at diagnosis have improved cytotoxic functions compared to those produced at relapse or following daratumumab treatment

Abstract Chimeric antigen receptor T cells (CAR‐T) have provided promising results in multiple myeloma (MM). However, many patients still relapse, pointing toward the need of improving this therapy. Here, we analyzed peripheral blood T cells from MM patients at different stages of the disease and investigated their phenotype and capacity to generate functional CAR‐T directed against CS1 or B Cell Maturation antigen. We found a decrease in naive T cells and elevated frequencies of exhaustion markers in T cells from treated MM patients. Interestingly, individuals treated with daratumumab display elevated ratios of central memory T cells. CAR‐T derived from patients at relapse show reduced in vitro expansion and cytotoxic capacities in response to MM cells compared to those produced at diagnosis. Of note, CAR‐T from daratumumab treated patients display intermediate defects. Reduced anti‐myeloma activity of CAR T cells from treated patients was also observed in a mouse model. Our findings suggest that T cell defects in MM patients, specifically during relapse, have a major impact on their capacity to generate efficient therapeutic CAR‐T. Selecting naive or central memory T cell subsets to generate therapeutic T cells could improve the CAR‐T therapy for MM.

free survival remains relatively short, ranging from 11 to 15 months depending on the study [7,8].
Progression of MM is associated with major changes in the T cell compartment in blood and bone marrow of patients [9]. Decreased CD4/CD8 T cell ratio, elevation of regulatory T cells (Treg) over Th17 T cells, and expression of exhaustion markers have been reported [10].
Furthermore, many MM therapies, such as autologous stem cell transplantation or exposure to Immunomodulatory imide drugs (IMIDs), are thought to impact on the function of T lymphocytes [11]. In line with this, it has recently been suggested that CAR-T cells manufactured from MM patients following induction therapy could be more efficient than those generated from relapsed individuals [12]. Here, we further investigated the impact of treatments and disease progression on blood T cell populations and on the activity of CAR-T generated from MM patients at different stage of the disease.

MATERIALS AND METHODS
Details of patients included in the study are shown in Table S1. Detailed experimental description of sample collection, fluorescence-activated cell sorting (FACS), production of CAR T cells, and functional assays are provided in Supplementary Information.

RESULTS AND DISCUSSION
To directly investigate T cell populations in MM, we prospectively constituted four cohorts of patients at diagnosis (Diag), at relapse (RL), upon daratumumab treatment (Dara) and at relapse after daratumumab treatment (RDara) (patient characteristics are detailed in Table S1A), as well as from non age-matched healthy donors (Don). As previously reported [11], FACS analysis on peripheral blood mononuclear cells from RL patients showed that the ratios of CD4/CD8 T cell were inverted compared to healthy donors and Diag patients. However, we observed that this defect was amplified in the Dara and RDara cohorts ( Figure 1A) suggesting that antibody treatments worsen this feature. Analysis of CD45RO and CD62L expression revealed that the percentages of CD4 + CD62L + CD45ROnaïve T cells (NT) in RL were reduced compared to Diag (25% vs 37%), and this reduction was more pronounced in Dara and RDara patients (16% and 14% respectively) ( Figure 1B). Conversely, the percentages of central memory  Figure 1C). Furthermore, analysis of PD-1 and LAG3, whose expression correlates with loss of T cell cytotoxicity showed that CD4 + T cells from Dara and RDara patients expressed higher percentages of both exhaustion markers compared to Diag (43% and 36% vs 21% for PD1 and 10% and 7% vs 4% for LAG3) ( Figure 1D).
Altogether, our results indicate that reduced naïve populations and elevated frequencies of exhaustion markers are common features of T cells in treated MM patients. However, daratumumab-treated patients display specific alterations in T-cell subsets, such as elevated ratio of CM T cells, while those who relapse after daratumumab therapy have increased frequencies of effector subsets.
Given these phenotypic alterations, we investigated the function of anti-CS1 CAR-T 5 generated by lentiviral transduction of peripheral T cells from MM patients included in each cohort (see Table S1B for patient characteristics). We first established an in vitro assay to measure the expansion of the anti-CS1 or scFv deficient (Mock) CAR-T cells cultured on CS1 expressing NIH3T3 cells. After 7 days in culture, we observed no proliferation of CAR-T derived from RL, while those generated from Diag and Don expanded 12 and 8 fold, respectively ( Figure 2A). Interestingly, CAR-T produced from Dara patients expanded around three-fold, whereas those derived from RDara cohort displayed no sign of proliferation. We next analyzed the potential of anti-CS1 CAR-T, produced from MM patients (see Table   S1C for