Single-Cell Analysis of CAR-T Cell Products Idecabtagene Vicleucel and Ciltacabtagene Autoleucel Reveals Factors That Influence Clinical Outcomes

Background: The introduction of chimeric antigen receptor autologous T-cells (CAR-T) targeting B-cell maturation antigen (BCMA) has changed the paradigm for patients with relapsed and refractory multiple myeloma. Both idecabtagene vicleucel (ide-cel) and ciltacabtagene autoleucel (cilta-cel) have outperformed prior standards of care in randomized trials and indirect comparisons suggest differential behavior, particularly depth and durability of response, favoring cilta-cel. We sought to understand these differences by sequencing infusion product (IP) of ide-cel and CAR-enriched peripheral blood mononuclear cells (PBMCs) during expansion in both ide-cel and cilta-cel.

Methods: Single cell RNAseq (scRNAseq) and VDJ libraries were generated from 52 samples in total; composed of 40 unique patient ide-cel IP and CAR-enriched PBMCs from 6 matched ide-cel and 6 unique cilta-cel patients. Patients were stratified as having durable response (DR) if they were alive and without evidence of progression at 9 months following infusion, and non-durable response (NDR) if they died from myeloma or had evidence of disease progression before this cutoff. Single-cell raw sequencing reads were aligned to a modified GRCh38 reference containing sequences of ide-cel and cilta-cel constructs. Following quality control procedures, CD4 and CD8 cells were identified using an in silico gating method and analyzed separately. Differential gene expression and pathway analysis was done using FindMarkers in Seurat v5 followed by gene set enrichment analysis. Sample-level pathway signatures were generated by aggregating read counts of CAR positive (CAR+) cells in each sample followed by single sample gene set enrichment analysis (ssgsea).

Results: We found that ide-cel IP was composed of primarily CD4 cells whereas post-infusion PBMCs were primarily CD8 cells regardless of product, confirmed by high correlation with flow cytometry phenotyping. Ribosomal and MHC class II genes were significantly upregulated in CAR+ CD8 IP cells from DRs, whereas metabolic pathways (Glycolysis, TCA cycle) and pathways associated with cell cycle progression were upregulated in CAR+ CD4 cells of NDRs. In addition, we noted higher expression of the ide-cel construct in CAR+ CD4 IP cells of DRs. This expression correlated with NFKB signaling and anti-apoptotic gene signatures, both of which were significantly associated with greater progression free and overall survival. In CAR+ ide-cel PBMCs, ribosomal genes were again upregulated in DRs in addition to higher expression of CD27, KLF2, TCF7, and the ide-cel construct.

Directly comparing the two products in PBMCs, we noted that compared to ide-cel, CAR+ CD4 cells from cilta-cel had a higher proportion of CD4 effector memory cells (p=0.01) and lower proportion of CD4 stem central memory cells (p=0.04). In contrast CD8 stem central memory cells were higher in cilta-cel (p=0.01). Compared to CAR+ cells of ide-cel, cilta-cel CAR+ cells had higher expression of CD27, GZMK, and TCF7. Additionally, expression of the respective CAR construct was approximately 4-fold higher in cilta-cel via scRNAseq and flow cytometry using florescent tagged recombinant BCMA, suggesting more CAR per cell even when accounting for the additional binding domains of the cilta-cel construct. Ssgsea confirmed NFKB signaling and ribosomal pathways to be significantly higher in cilta-cel when compared with ide-cel. Finally, we noted that the TCR repertoire of cilta-cel was far less clonal than ide-cel and was significantly more diverse in CAR+ CD8 cells of cilta-cel.

Conclusion: To our knowledge this study presents the largest single-cell analysis of approved anti-BCMA CAR-T products to date and first single-cell analysis of ide-cel infusion product. The results provide a biological narrative highlighting the differences in make-up of ide-cel and cilta-cel, which helps inform the understanding of differences in clinical efficacy. Ide-cel infusion product characteristics associated with durable responses included higher CAR expression levels, enhanced NFKB signaling and anti-apoptosis signatures. CAR+ cells from PBMCs after ide-cel infusion associated with durable response were enriched for ribosomal and stemness genes. Relative to ide-cel, CAR+ cilta-cel PBMCs exhibit more NFKB signaling, CAR construct expression, and higher expression of ribosomal, stemness, and persistence genes.

Puglianini:Legend Biotech USA Inc: Consultancy, Honoraria. Liu:BioLineRx: Consultancy, Honoraria. Alsina:Sanofi: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees. Nishihori:ImmunoGen: Consultancy; Medexus: Membership on an entity's Board of Directors or advisory committees; Karyopharm: Other: drug only supply to the institution; Novartis: Research Funding. Shain:Abbvie: Research Funding; BMS: Consultancy, Research Funding; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Research Funding; Adaptive Biotech: Consultancy; Karyopharm, Janssen, Adaptive Biotechnologies, GlaxoSmithKline, BMS, Sanofi, and Regeneron: Honoraria; Karyopharm: Research Funding; Takeda: Consultancy; Sanofi: Consultancy; Glaxo Smith Kline: Consultancy, Membership on an entity's Board of Directors or advisory committees. Baz:Karyopharm Therapeutics: Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Research Funding; Bristol-Myers Squibb: Research Funding; Cellectar: Membership on an entity's Board of Directors or advisory committees; AbbVie: Research Funding; Regeneron: Research Funding; Pfizer: Membership on an entity's Board of Directors or advisory committees. Blue:Pfizer Pharmaceuticals, Oncopeptides, Takeda, Abbvie, Janssen, and Kite Pharmaceuticals: Consultancy; Sanofi: Speakers Bureau. Grajales-Cruz:Amgen, Sanovi: Speakers Bureau; Cellectar, Janssen, Sanofi: Membership on an entity's Board of Directors or advisory committees. Hansen:BMS: Consultancy, Research Funding; Janssen: Consultancy; Pfizer: Consultancy; Karyopharm: Consultancy, Research Funding. Locke:Emerging Therapy Solutions Gerson Lehman Group: Consultancy; BMS: Consultancy, Research Funding; ASH: Honoraria, Other: Travel support; Janssen: Consultancy; Celgene: Consultancy; Bluebird Bio: Consultancy, Research Funding; Legend Biotech: Consultancy; Gilead Company: Consultancy; Calibr: Consultancy; Allogene: Consultancy, Research Funding; Cowen: Consultancy; Cellular Biomedicine Group: Consultancy; Amgen: Consultancy; Umoja: Consultancy; Iovance: Consultancy; National Cancer Institute: Research Funding; 2SeventyBio: Research Funding; CERo Therapeutics: Research Funding; Pfizer: Consultancy; Sana: Consultancy; Gerson Lehrman Group (GLG): Consultancy; Caribou: Consultancy; A2: Consultancy; Society for Immunotherapy of Cancer: Honoraria; iMedX: Honoraria; Clinical Care Options Oncology: Honoraria; Communications CARE Education: Honoraria; BioPharma: Honoraria; Aptitude Health: Honoraria; Moffit Cancer Center: Patents & Royalties: cellular immunotherapy; Wugen: Consultancy; Novartis: Consultancy, Research Funding; Kite, a Gilead Company: Consultancy, Other: Travel support, Research Funding; GammaDelta Therapeutics: Consultancy; ecoR1: Consultancy; Leukemia and Lymphoma Society Scholar in Clinical Research: Research Funding; Aptitude Health: Honoraria. Freeman:BMS: Consultancy, Honoraria, Research Funding; Seattle Genetics: Consultancy; Celgene: Consultancy; Abbvie: Consultancy; ONK therapeutics: Consultancy; Incyte: Consultancy; Amgen: Consultancy; Janssen: Consultancy, Research Funding; Sanofi: Consultancy; Roche/Genentech: Research Funding.