Introduction: Robust biomarkers of response and resistance to chimeric antigen receptor (CAR) T cell therapy in patients with relapsed/refractory multiple myeloma (RRMM) are lacking. We conducted a longitudinal single-cell multi-omics study to identify factors predicting response to BCMA-directed CAR T cells.
Methods: Peripheral blood samples from 10 patients (8 Ide-cel; 2 Cilta-cel) were collected on the day of leukapheresis and 30 days after CAR T cell treatment. Bone marrow biopsies were performed on day 30 after CAR T cell therapy. We used 57 oligonucleotide-coupled antibodies for surface proteome analysis. Libraries for single cell BCR, TCR and RNA were generated using the 10x genomics 5’ chemistry. Cell types were annotated with Seurat and WNN. scCODA identified cell type composition changes. InferCNV detected CNVs in malignant plasma cells. Ligand-receptor signaling was inferred with iTalk and CellPhoneDB. CAR T cell in vitro functionality was tested through cytotoxicity assays. For analyses patients were divided by their response to CAR T cell therapy on day 30 after infusion (CR: n=5, no CR: n=5).
Results: We sequenced 178,142 cells (median 7,990 cells/sample, range 1,569-10,972 cells) and already observed differences between patients in CR and no CR at leukapheresis. CR patients harbored more CD8+ TEM and NK cells but fewer monocytes. Non-responders showed recurrently higher PIM kinase expression in monocytes, dendritic and NK cells as well as higher protein expression of immune checkpoints on monocytes (CD39) and NK cells (CD94). Cell-cell interaction analysis identified inhibitory communication of monocytes with NK and CD8+ T cells in non-responders. Since we detected an immunosuppressive environment in non-responders at leukapheresis, we aimed at characterizing the functionality of manufactured CAR T cells. CAR T cells isolated from patients in CR and no CR at day 7 post-infusion, effectively eliminated MM cells (U-266), indicating that also CAR T cells from non-responders remained functional in vitro. Comparing single-cell transcriptomes of CAR T cells isolated from patients in CR who received Cilta-cel or Ide-cel, we noticed an upregulation of genes associated with cell cycle regulation, exhaustion/senescence, and chemotaxis in Cilta-cel CAR T cells. Surface proteomics revealed that hyperexpanded CAR T cells displayed a more exhausted and senescent phenotype, characterized by higher expression levels of immune checkpoints and NK cell receptors (PD1, CD57, CD94), as well as lower expression levels of markers associated with activation. In contrast, non-hyperexpanded CAR T cell clonotypes exhibited a protein expression profile marked by downregulation of exhaustion/senescence markers (e.g., TIM-3, KLRB1, KLRG1, and CD337).
Conclusions: Our study indicates an association between an immunosuppressive microenvironment that is already present at time of leukapheresis and resistance to CAR T cell therapy in RRMM.
