Cite-Seq Profiling of T Cells in Multiple Myeloma Patients Undergoing BCMA Targeting CAR-T or Bites Immunotherapy

Abstract

Adaptive T cell therapy using chimeric antigen receptor (CAR) T cells and bispecific T cell engagers (BiTEs) have demonstrated encouraging responses in heavily pre-treated multiple myeloma (MM) patients. However, the cellular and molecular predictors of clinical response are not fully understood as well as the mediators of acquired resistance remain elusive. Local immune suppression and T cell exhaustion are important mediators of responses therefore, it is plausible to speculate that a tolerant tumor microenvironment and the expansion of specific T cell populations may dictate clinical responses. In this study, we performed at the single cell level a broad immunophenotypic and transcriptomic characterization of the blood and bone marrow (BM) T cells of sensitive and resistant MM patients treated with adaptive T cell therapies. Using cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) we measured the expansion of variable T cell subsets, T cell specific activation and inhibitor markers and their functional states in order to identify cellular mediators of resistance to these adoptive immune therapies. Serial blood samples and BM aspirates (n=12) were collected from patients treated with anti-BCMA CAR-T or BCMA-CD3 BiTEs at variable time points, prior and post initiation of therapy and at relapse. Bone marrow mononuclear fractions were isolated through ficoll density gradients coupled with magnetic sorting of CD3pos T cells. Unbiased mRNA profiling coupled with feature barcoding technology for cell surface protein (TotalSeq-B) of BM CD3pos T cells was then performed by using the chromium single cell (10x Genomics). Paired-end sequencing was performed on Illumina platform. Cell Ranger and Seurat pipeline were used for sample de-multiplexing, barcode processing, single-cell 3′ gene counting, cell surface protein expression and data analysis. CAR-T cells were identified by the expression of the chimeric CAR-T cell transcript. The parallel measurement of transcripts and cell surface protein phenotypes of CD3pos T cells using a panel of 19 immune surface markers underlined the T cell repertoire diversity and identified different T cell subsets among the CD8pos and CD4pos T cells. Notably, the cell surface protein information overlaid on the transcript-generated UMA allowed accurate identification of all main immune clusters, in particular for the CD45RA and CD45RO positive cells. Comparison of CITE-Seq features revealed that the T cells composition of the blood and BM niches differed significantly between sensitive and resistant patients. As such an enrichment of CD4pos T cells with a higher CD4:CD8 ratio was noted in responding patients. Phenotypic (CD45RA, CD45RO, CD95, CCR7, CD62L, CD28, CD27) and transcriptional signatures (TCF7, LEF1, GATA3, EOMES, TBX21, PRDM1) also identified a higher proportion of memory like T cells (Tscm, Tcm) in responding patients. In contrast, T cells of resistant patients were enriched with terminally exhausted (Tex) and senescent cells with loss of CD28, high GMZHand GMZB, CD57pos, CD69pos and CD160pos as well as upregulation of TBX21. Expression of T cell checkpoint inhibitors such as LAG3, TIGIT and PD1 was high in these Tex cells as well as in some Tem. Of note, ex vivo T cell activation studies with TIGIT blockade demonstrated T cell activation in an autologous MM and T cell co-culture system with enhanced MM cells death. An expanded cluster of regulatory T cells (Treg) FOXP3pos,CD25pos was also observed in two resistant patients. Of note, no loss of BCMA transcript or surface expression was noted in MM cells at the time of acquired resistance. Single cell transcriptome of primary MM cells and chromatin accessibility (ATAC-seq) analyses of T cells of these patients are ongoing to investigate the transcriptional programs and epigenetic factors underlying the immune escape. Combined single cell features profiling of the transcriptome and surface protein expression of T cells from MM patients receiving BCMA targeted CAR-T or BiTEs therapies revealed potential mediators of resistance. In particular, T cells composition (low CD4:CD8 ratio and reduced population of Tscm, Tcm) along with an enrichment of terminally exhausted T cells are the main features observed in resistant patients. Delineating these mechanisms will guide future T cells engineering studies to enhance the efficacy and response durability of adoptive immunotherapy in MM. Disclosures McCulloch: Amgen: Honoraria; Sanofi: Honoraria; Celgene: Honoraria; Janssen: Honoraria. Duggan:Jannsen: Consultancy; Amgen: Consultancy; Novartis: Honoraria; Celgene: Consultancy; Astra Zeneca: Consultancy. Jimenez-Zepeda:Janssen, Celgene, Amgen, Takeda: Honoraria. Bahlis:AbbVie: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; GSK: Consultancy, Honoraria; Genentech: Consultancy, Honoraria; BMS/Celgene and Janssen: Consultancy, Honoraria, Other: Travel, Accomodations, Research Funding; Karyopharm Therapeutics: Consultancy, Honoraria; Sanofi: Consultancy, Honoraria. Neri:Celgene/BMS: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Amgen: Consultancy, Honoraria.

Determinants of Response and Mechanisms of Resistance of CAR T-cell Therapy in Multiple Myeloma

“…The mechanism whereby BCMA CAR T cells reduce BCMA cell-surface expression levels probably includes selection of cells with lower BCMA levels, whereas tumor cells with higher BCMA expression are eliminated. Furthermore, biallelic BCMA deletions, resulting in lack of BCMA expression, have also recently been found to trigger resistance to BCMA CAR T cells (27)(28)(29). Although BCMA antigen loss seems to be an uncommon mechanism of escape from BCMA-directed CAR T-cell therapy (4% in the KarMMa study; ref.…”

“…There is increasing evidence that the heterogeneity of T-cell subsets in the apheresis product explains part of the variability of the activity of the CAR T cells infused to patients in clinical trials. First, several BCMA CAR T-cell studies show that patients with MM with a high frequency of early memory T cells in the leukapheresis product experience a higher response rate and superior peak CAR T-cell expansion when compared with patients with a low frequency of these cells (9,27,65,66). Similarly, the presence of early memory T cells in the leukapheresis product was correlated with response in patients with chronic lymphocytic leukemia (CLL), ALL, and lymphoma treated with CD19 CAR T cells (65,67,68).…”

“…CD4 + T cells promote the proliferation, survival, and activity of CD8 + T cells by providing a variety of cytokines including IL2, which explains the synergy between CD4 + and CD8 + CAR T cells in mediating antitumor responses (70,71). Furthermore, T cells from BCMA CAR T-cell-resistant patients were enriched with terminally exhausted and senescent cells with high expression of inhibitory immune checkpoint receptors, such as LAG-3, TIGIT, and PD-1 (27). Altogether, this indicates that premanufacturing T-cell characteristics are important determinants of response to CAR T-cell therapy.…”

“…Immune suppressor cells impair CAR T-cell activity in different types of cancers (122)(123)(124)(125). Although regulatory T cell (Treg) expansion has been described in patients with MM without response to BCMA CAR T-cell therapy (27), the precise role of Tregs in mediating CAR T-cell resistance remains unclear. The impact of other immune suppressor cells, such as myeloid-derived suppressor cells (MDSC) and immunosuppressive macrophages, on CAR T-cell activity is currently unknown in MM, and therefore all ongoing CAR T-cell trials should be accompanied by immune monitoring studies REVIEW JULY 2021 blood CANCER dISCoVERY | OF11 to increase our understanding of the potential ability of immune suppressor cells to impair both CAR T-cell function and persistence.…”

Effective anti-BCMA retreatment in multiple myeloma

“…In the context of CAR T cells or BsAbs, responses have been associated with a higher CD4/CD8 ratio and an increased frequency of CD45RO 2 CD27 1 CD8 1 T cells, reflective of stem memory T cells. 24,25 In contrast, T cells with exhausted or senescent phenotypes are enriched in patients who are resistant to anti-BCMA BsAb or CAR T cells. 25 This indicates that the efficacy of BCMA-targeting therapies may be greater during early stages of the disease when patients are less immunosuppressed.…”