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2834 CD19 CAR T Cells Drive a Remodeling of the Immune Microenvironment Associated with T-Cell Dysfunction in Acute Lymphoblastic Leukemia

Program: Oral and Poster Abstracts
Session: 614. Acute Lymphoblastic Leukemias: Biomarkers, Molecular Markers, and Minimal Residual Disease in Diagnosis and Prognosis: Poster II
Hematology Disease Topics & Pathways:
Research, Lymphoid Leukemias, ALL, Translational Research, Chimeric Antigen Receptor (CAR)-T Cell Therapies, Diseases, Treatment Considerations, Biological therapies, Immunology, Lymphoid Malignancies, Biological Processes, Technology and Procedures, Omics technologies
Sunday, December 8, 2024, 6:00 PM-8:00 PM

Marianna Ponzo, PhD1,2*, Lorenzo Drufuca, PhD3*, Chiara Buracchi, PhD1,4*, Marco M Sindoni1,4*, Silvia Nucera, MD1,4*, Cristina Bugarin, MSc1*, Ramona Bason, PhD3*, Grazisa Rossetti, PhD3*, Raul Bonnal3*, Cristian Meli5*, Benedetta Rambaldi5*, Federico Lussana6*, Silvia Ferrari, MD5*, Alex Moretti, MD1*, Giulia Risca7*, Christian Pellegrino, PhD2*, Markus G. Manz, MD2, Stefania Galimberti7*, Alessandro Rambaldi5, Andrea Biondi1,4,8, Giuseppe Gaipa1, Massimiliano Pagani, PhD3,9* and Chiara F. Magnani, PhD1,2

1Tettamanti Center, Fondazione IRCCS San Gerardo dei Tintori, Monza, Italy
2Department of Medical Oncology and Hematology, University Hospital Zurich and University of Zurich, Zurich, Switzerland
3The AIRC Institute of Molecular Oncology (IFOM), Milan, Italy
4School of Medicine and Surgery, University of Milano-Bicocca, Monza, Italy
5Department of Oncology-Hematology, Azienda Socio Sanitaria Territoriale Papa Giovanni XXIII, Bergamo, Italy
6Department of Oncology and Hematology, University of Milan and ASST Papa Giovanni XXIII, Bergamo, Italy
7Bicocca Bioinformatics, Biostatistics and Bioimaging Centre, Department of Medicine and Surgery, University of Milano - Bicocca, Milan, Italy
8Pediatrics, Fondazione IRCCS San Gerardo dei Tintori, Monza, Italy
9Department of Medical Biotechnology and Translational Medicine, Università degli Studi, Milan, Italy

Introduction

Chimeric Antigen Receptor (CAR) T cells induce durable responses in B-cell acute lymphoblastic leukemia (B-ALL). However, a substantial proportion of patients remain refractory or relapse with suboptimal CAR T cell activity. The contribution of the tumor microenvironment to the therapeutic response after CAR T cells remains incompletely understood. We hypothesized that the bone marrow (BM) immunological niche reacts to CAR T cell-mediated inflammation by activating inhibitory pathways and molecules. To verify this hypothesis, we performed a transcriptional analysis of CAR T cells and the BM microenvironment from treated patients and validated findings by flow cytometry.

Methods

Our group developed anti-CD19 CAR T cells generated with the non-viral Sleeping Beauty (SB) transposon vector from donor T cells in patients relapsed after allogeneic hematopoietic stem cell transplantation (Magnani CF et al, J Clin Invest. 2020 Nov 2; 130(11): 6021–6033). We performed single-cell RNA sequencing and spectral flow cytometry-based analysis of BM-resident immune cells from patients with B-ALL. Data in this study were generated from patients enrolled in the FT01CARCIK Phase I/IIb clinical trial (NCT03389035), in the FT03CARCIK Phase II study (NCT05252403) or who received autologous CAR T cells as part of a Phase I/II trial or commercial cell therapy. Mechanistic insights into the pathways and molecules involved were validated in tumor-bearing humanized mice.

Results

We analyzed hematological laboratory parameters from patients after treatment and we found that CAR T cells elicit a bystander activation of endogenous immune cells, involving both innate and adaptive immunity, which is actively regulated by a process of immune regulation. To elucidate the cellular and molecular mediators involved in dampening CAR T-cell-mediated immune activation, we performed scRNA-seq of BM samples collected at early time points after treatment (1-2 months). Eighteen sequencing libraries were analyzed, for a total of 71407 high-quality cells. Unbiased clustering of BM cells and infusion products was performed by UMAP embedding. More representative clusters were classified into infusion product, endogenous CD4 and CD8 population, B cells, myeloid cells, pDC, NK, and NK-T cells. We observed profound changes in the microenvironment in response to CAR T-cell-mediated inflammation, and an increase in myeloid cells. Significant enrichment in IFN response, hypoxia, and TGF-β-signaling was associated with the expansion of myeloid-derived suppressor cells (MDSCs) and endogenous exhausted CD8+ T cells, findings validated with a cohort of additional 20 matched BM samples pre- and post-CAR T-cell treatment acquired using spectral flow cytometry. By modeling intercellular communications, we revealed that HIF1α, VEGF, and TGFBR2 are key players in the intercellular communication between CAR T cells and the immune niche, driving widespread T-cell exhaustion, and affecting both CAR T cells and endogenous T cells. Moreover, infusion of anti-CD19 CAR T cells leads to increased accumulation of human MDSCs and exacerbation of a hypoxic environment in HSPC-humanized mice bearing a human tumor. Finally, we also assessed the role of tumor microenvironmental factors on the duration of response (DOR) and found that PD1 expression in endogenous T cells post-treatment was associated with a lack of durable response.

Conclusions

This study highlights the critical role of the tumor microenvironment on CAR T-cell fate and endogenous immunity in B-ALL. We demonstrate that IFN response, hypoxia, and TGF-β signaling lead to general immune suppression after treatment, resulting in endogenous T-cell exhaustion and compromising CAR T-cell efficacy.

Disclosures: Lussana: Amgen: Speakers Bureau; Abbvie: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Incyte: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Pfizer: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Clinigen: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Biondi: CoImmune, Galapagos, Amgen, Novartis, BMS: Consultancy, Research Funding, Speakers Bureau. Pagani: CheckmAb: Membership on an entity's Board of Directors or advisory committees.

*signifies non-member of ASH