Nfkb signaling in idecabtagene vicleucel infusion product drives response in relapsed/refractory multiple myeloma

Abstract

Abstract Background: B-cell maturation antigen (BCMA) directed chimeric antigen receptor T-cell (CAR-T) therapy has revolutionized the treatment of relapsed/refractory multiple myeloma (RRMM). However, responses to CAR-T in RRMM are heterogenous and CAR-T infusion product (IP) features determinant of efficacy remain poorly defined. We performed single-cell RNA sequencing (scRNA-seq) on idecabtagene vicleucel (ide-cel) infusion product of RRMM patients to elucidate transcription profiles associated with CAR-T efficacy. Methods: ScRNA-seq libraries were generated from 40 patient IPs using Cell Ranger (v7.1.0, 10X Genomics), and aligned against a modified GRCh38 reference containing the ide-cel construct. Poor quality cells and doublets were then removed. CD4 and CD8 cells were identified and analyzed separately using an established in-silico gating method (Li, Cancer cell 2023). Harmony within Seurat v5 allowed integration, and cell subtype clusters were annotated by highly expressed genes. Samples collected shortly before CAR-T were analyzed by flow cytometry, including material used to make ide-cel (pre-treatment apheresis) and the tumor microenvironment (TME; bone marrow depleted of CD138+ cells), with the latter also being subject to Nanostring PanCancer Immune Profiling gene expression. Patients were stratified as durable response (DR) if still alive and without progression at 9 months or a non-durable response (NDR) if they died or had evidence of disease progression prior to this cutoff. For NF-κB inhibition assays, BCMA CAR-T with CD3z/4-1BB co-stimulatory domain resembling ide-cel, were manufactured from healthy donor T cells. After manufacture, cells were treated for two hours with BMS-345541, a selective IKKβ inhibitor, or left untreated. NF-κB activation was assessed by quantifying phosphorylation of the p65 subunit at Ser536 using the PathScan® Phospho–NF-κB p65 (Ser536) ELISA in parallel with Total NF-κB p65 ELISA. Results: Following quality control and batch correction, 184,398 cells, including 105,951 CD4 and 38,721 CD8 T cells from ide-cel IP were analyzed. Ide-cel was composed of primarily CD4 cells and cell cluster composition differed minimally between DR and NDR patients. One cluster characterized by expression of glycolytic genes was upregulated in NDR patients (p-value 0.0006). Pseudobulk aggregation followed by single-sample gene set enrichment (ssGSEA) of CAR positive (CAR+) CD4 cells from DR patients exhibited increased NF-κB signaling (p-value 0.02) – a pleiotropic activator of cellular responses including survival, proliferation, and inflammation. Pro-survival genes and tonic signaling pathways, and expression of the ide-cel construct, were also upregulated in DR and highly correlated with NF-κB signaling, progression-free survival, and overall survival. Analysis of apheresis samples matched to IP, revealed NF-κB signaling in CAR+CD4 IP cells positively correlated with flow cytometry defined apheresis CD4 T cell central memory cells (CD45RO+ CCR7+), TCF1, and absence of checkpoint ligands (PD1- TIGIT- LAG3-). Conversely lower NF-κB signaling in CAR+CD4 IP negatively correlated with flow defined apheresis CD4 expression of PD1 and CD39. Apheresis BM derived TME phenotypes were similar by flow cytometry. Nanostring of TME demonstrated similar NF-κB pathway correlation with TME CD4 T cell phenotype by flow cytometry, suggesting that NF-κB findings in ide-cel IP are informed by the TME prior to CAR-T manufacture. Pharmacological inhibition of NF-κB reduced CAR T cell function, resulting in impaired cytotoxicity against the H929-GFP-FFL multiple myeloma cell line, as measured by a luciferase-based bioluminescence assay, and decreased secretion of IFN-γ, TNF-α, IL-2, and IL-6, as determined by the ELLA platform. Conclusion: This study presents the first scRNAseq analyses of ide-cel infusion product and elucidates product specific factors that associate with efficacy in RRMM. NF-κB signaling, pro-survival genes, tonic signaling signatures, and ide-cel construct expression in the ide-cel infusion product CD4 CAR-T cells were significantly associated with improved depth and durability of responses. NF-κB signatures were particularly impactful and correlated with apheresis and TME T cells being more central memory-like and less exhausted. This suggests CAR-T product fitness is impacted by the pre-existing T cell fitness and informed by the TME. FLL and CLF contributed equally.