-Author name in bold denotes the presenting author
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1963 Targetable Cellular Etiology of Prolonged Cytopenia Following CD19 CAR T-Cell TherapyClinically Relevant Abstract

Program: Oral and Poster Abstracts
Session: 703. Cellular Immunotherapies: Basic and Translational: Poster I
Hematology Disease Topics & Pathways:
Research, Biological therapies, Translational Research, Chimeric Antigen Receptor (CAR)-T Cell Therapies, immune mechanism, Therapies, Adverse Events, Biological Processes
Saturday, December 10, 2022, 5:30 PM-7:30 PM

Xubin Li1*, Qing Deng, PhD2*, Jared Henderson, PhD2*, Grace Watson3*, Laurel Deaton4*, Taylor Cain1*, Luis Fayad, MD5, Swaminathan Padmanabhan Iyer, MD5*, Fredrick B. Hagemeister, MD6, Loretta J. Nastoupil, MD7, Nathan H. Fowler, MD5, Jason Westin, MD8, Raphael Steiner, MD5*, Ranjit Nair, MD9, Sairah Ahmed, MD8, Gheath Alatrash, PhD, DO10, Sattva S. Neelapu, MD11, Paolo Strati, MD5 and Michael R. Green, PhD12

1MD Anderson Cancer Center, Houston
2Department of Lymphoma & Myeloma, The University of Texas MD Anderson Cancer Center, Houston, TX
31400 Holcombe Blvd, MD Anderson Cancer Center, Houston, TX
4Department of Lymphoma & Myeloma, University of Texas MD Anderson Cancer Center, Houston, TX
5Department of Lymphoma and Myeloma, The University of Texas MD Anderson Cancer Center, Houston, TX
6Department of Lymphoma and Myeloma, The University of Texas, MD Anderson Cancer Center, Houston, TX
7Department of Lymphoma and Myeloma, MD Anderson Cancer Center, Houston, TX
8The University of Texas MD Anderson Cancer Center, Houston, TX
9Department of Lymphoma/Myeloma, The University of Texas MD Anderson Cancer Center, Pearland, TX
10Department of Hematopoietic Biology & Malignancy, The University of Texas MD Anderson Cancer Center, Pearland, TX
11Department of Lymphoma and Myeloma, M.D. Anderson Cancer Center, Houston, TX
12Department of Lymphoma and Myeloma, The University of Texas MD Anderson Cancer Center, Friendswood, TX

Introduction: Anti-CD19 chimeric antigen receptor T-cell (CART) therapy is highly effective in relapsed/refractory large B-cell lymphoma (rrLBCL), but is associated with toxicities that are a clinical challenge and delay recovery. While the biological mechanisms of cytokine release syndrome and neurological toxicity have been investigated, the pathophysiology of prolonged cytopenia (grade ³3 cytopenia lasting beyond day 30) observed in about a third of the patients is poorly understood. Prolonged cytopenia associates with increase transfusional needs and infectious complications, in addition to preventing initiation of subsequent treatment in case of relapse. Here, we performed scRNA-seq analysis of bone marrow samples to identify cellular and molecular features associated with CART-associated prolonged cytopenia.

Methods: Bone marrow (BM) aspirates were collected from 16 patients with rrLBCL treated with axi-cel, in which 11 patients had grade 3-4 cytopenia at day 30 and 5 patients did not. 5 healthy donor BM samples and 1 rrLBCL patient with chemotherapy-associated cytopenia were included as controls. All 22 samples were analyzed by 10X with 5’GEX plus BCR&TCR and utilized for clustering, but 2 patients with cytopenia post-CART were excluded from subsequent analysis due to BM involvement with acute myeloid leukemia/LBCL. Stringent quality control and doublet removal was performed followed by unsupervised clustering and dimension reduction, and differential gene expression analysis using Seurat. Harmony was used to integrate data from different patients so that batch effects across patients were mitigated. scCODA was used to perform cell composition analysis. SCPA was used for pathway analysis.

Results: After stringent QC, we obtained scRNA-seq data for 92,676 cells from BM aspirates of 22 patients. The cells were partitioned into 26 clusters, corresponding to distinct cell types including CD8 T cells, CD4 T cells, natural killer (NK) cells, B cells, myeloid cells, hematopoietic progenitor cells (HPCs), and hematopoietic stem cells (HSCs). To find characteristics associated with CART-associated cytopenia, we compared cellular and molecular features between cells from patients with (n=9) or without cytopenia after CART (n=5). scCODA analysis of all cells and cell clusters identified a significant over-representation of GZMH+ FGFBP2+ CD8 T cells within patients with CART-associated cytopenia (FDR<0.02). To further study these cells, we re-clustered CD8 T cells and identified 11 subsets including a refined GZMH+ FGFBP2+ CD8 T cell cluster that was significantly over-represented in BM samples from patients with CART-associated cytopenia compared with patients without cytopenia following CART (FDR<0.03). The GZMH+ FGFBP2+ CD8 T cells highly expressed cytotoxic effector molecules and were strongly enriched for a previously defined cytotoxicity signature. Analysis of scTCR data identified a higher degree of clonal expansion within CD8 T-cells in patients with CART-associated cytopenia compared to patients without. The top expanded TCR clones were enriched within the GZMH+ FGFBP2+ CD8 T cell subset, and did not express the CAR transcript. Pathway enrichment analysis identified a significant enrichment of interferon (IFN) signaling and inflammatory pathways within T cells, NK cells, myeloid cells, HPCs, and HSCs (adjusted P<0.05). The majority of IFNG-expressing cells resided within the GZMH+ FGFBP2+ CD8 T cell cluster that was over-represented in patients with CART-associated cytopenia, and which also expressed high levels of TBX21 – the master regulator of IFNG expression.

Conclusions: These data represent the first single cell analysis of BM samples from CART-treated patients with prolonged severe cytopenia. We show for the first time that a clonally-expanded IFNG-expressing cytotoxic T-cells and an enrichment of IFN signaling within the HSCs of BM aspirates may be responsible for CART-associated cytopenia. Previous studies have reported that IFNG can impair self-renewal and skew the differentiation of HSCs, providing a possible mechanistic link between these cell subsets and the observed cytopenias. Importantly, IFN signaling can be targeted using IFNG-neutralizing antibodies or eltrombopag, suggesting potential targeted interventions that may improve the outcomes of patients with CART-associated prolonged cytopenias.

Disclosures: Nastoupil: ADC Therapeutics, BMS, Caribou Biosciences, Epizyme, Genentech/Roche, Gilead/Kite, Genmab, Janssen, MEI, Morphosys, Novartis, Takeda: Honoraria; Genentech/Roche, MEI, Takeda: Other: DSMC; BMS, Caribou Biosciences, Epizyme, Genentech, Gilead/Kite, Genmab, Janssen, IGM Biosciences, Novartis, Takeda: Research Funding. Fowler: Roche: Consultancy, Research Funding; Gilead: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; BostonGene Corporation: Current Employment, Other: Leadership. Westin: Kite, a Gilead Company: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Iksuda: Consultancy; Merck: Consultancy; Bristol Myers Squibb: Consultancy, Research Funding; Calithera: Consultancy, Research Funding; Genentech/Roche: Consultancy, Research Funding; MorphoSys/Incyte Corporation: Consultancy, Research Funding; AstraZeneca: Consultancy, Research Funding; ADC Therapeutics: Consultancy, Research Funding; MonteRosa: Consultancy; Abbvie/GenMab: Consultancy; SeaGen: Consultancy. Steiner: Seagen: Research Funding; BMS: Research Funding; GSK: Research Funding; Rafael Pharmaceuticals: Research Funding. Nair: Incyte Corporation: Honoraria. Ahmed: Servier: Membership on an entity's Board of Directors or advisory committees; Xencor: Research Funding; Chimagen: Consultancy, Research Funding; Myeloid Therapeutics: Consultancy; Merck: Research Funding; Seagen: Research Funding; Tessa Therapeutics: Consultancy, Research Funding. Neelapu: Adicet Bio: Consultancy, Honoraria, Other: Personal fees, Research Funding; Calibr: Consultancy, Honoraria, Other: Personal fees; Incyte: Consultancy, Honoraria, Other: Personal fees; Cell Medica/Kuur: Consultancy, Honoraria, Other: Personal fees; Bluebird Bio: Consultancy, Honoraria; Medscape: Consultancy, Honoraria; Unum Therapeutics: Consultancy, Honoraria, Other: Personal fees, Research Funding; Takeda Pharmaceuticals: Patents & Royalties: related to cell therapy.; Acerta: Research Funding; Cellectis: Research Funding; Karus Therapeutics: Research Funding; Precision Biosciences: Consultancy, Honoraria, Other: Personal fees, Research Funding; Legend Biotech: Consultancy, Honoraria, Other: Personal fees; Aptitude Health: Consultancy, Research Funding; Bio Ascend: Consultancy, Honoraria; Poseida: Research Funding; Allogene Therapeutics: Consultancy, Honoraria, Other: Personal fees, Research Funding; Pfizer: Consultancy, Honoraria, Other: Personal fees; Celgene: Consultancy, Honoraria, Other: Personal fees, Research Funding; Novartis: Consultancy, Honoraria, Other: Personal fees; Merck: Consultancy, Honoraria, Other: Personal fees, Research Funding; Bristol Myers Squibb: Consultancy, Honoraria, Other: Personal fees, Research Funding; Kite: Consultancy, Honoraria, Other: Personal fees, Research Funding. Strati: Roche Genentech: Consultancy; Hutchinson MediPharma: Consultancy; ADC Therapeutics: Consultancy, Research Funding; Kite Gilead: Consultancy; TG Therapeutics: Consultancy; Astrazeneca Acerta: Research Funding; ALX Oncology: Research Funding; Sobi: Research Funding. Green: Tessa Therapeutics: Honoraria; KDAc Therapeutics: Current holder of stock options in a privately-held company; Monte Rosa Therapeutics: Honoraria; Allogene: Research Funding; Kite/Gilead: Research Funding; Sanofi: Research Funding; Abbvie: Research Funding; Daiichi Sankyo: Consultancy, Honoraria.

*signifies non-member of ASH