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3439 Phenotypic, Transcriptomic and Proteomic Characteristics of CAR T-Cell Dysfunction Are Associated with Inferior CAR T-Cell Expansion and Treatment Failure in r/r B-NHL

Program: Oral and Poster Abstracts
Session: 703. Cellular Immunotherapies: Basic and Translational: Poster II
Hematology Disease Topics & Pathways:
Research, Biological therapies, Translational Research, Lymphomas, B Cell lymphoma, Chimeric Antigen Receptor (CAR)-T Cell Therapies, Diseases, Therapies, immunology, Lymphoid Malignancies, Biological Processes
Sunday, December 10, 2023, 6:00 PM-8:00 PM

Viktoria Blumenberg, MD1,2,3,4, Maryam Kazerani, Ph.D.2*, Stephan Baumann5*, Tobias Straub, Ph.D.6*, Francesco Corrado, MD2*, Jan Wulf2*, Galina Busch5*, Kai Rejeski, MD3,4,5,7, Lisa Frölich8,9*, Christian Schmidt, MD9*, Michael von Bergwelt-Baildon1,8,10*, Veit L. Buecklein, MD1,2,3* and Marion Subklewe, MD4,5,11,12

1Department of Medicine III, LMU University Hospital, LMU Munich, Munich, Germany
2Laboratory for Translational Cancer Immunology, Gene Center, LMU Munich, Munich, Germany
3Bavarian Cancer Research Center (BZKF), Munich, Germany
4German Cancer Consortium (DKTK), Partner Site Munich, Munich, Germany
5Laboratory for Translational Cancer Immunology, LMU Gene Center, LMU Munich, Munich, Germany
6Core Facility Bioinformatics, LMU Biomedical Center, LMU Munich, Munich, Germany
7LMU University Hospital, Munich, Germany
8German Cancer Consortium (DKTK), partner site Munich, Munich, Germany
9Department of Medicine III, University Hospital, LMU Munich, Munich, Germany
10Bavarian Cancer Research Center (BZKF), partner site Munich, Munich, Germany
11Bavarian Cancer Research Center (BZKF), R/R ALL Study Group, Bavaria, Germany
12Department of Medicine III, LMU University Hospital, LMU Munich, Muenchen, Bavaria, Germany

Introduction: Despite the curative potential of CAR T-cells, a significant number of patients fail to respond or relapse early. Recently, we and others showed that early expansion failure, and particularly CAR T-cell levels at day 7, are strong predictors for treatment resistance (Blumenberg et al, Blood Supplement 2022; Locke et al, Blood Adv 2020). Here we dissected early characteristics of CAR T-cell dysfunction based on immune checkpoint expression, transcriptomic signatures and cytokine profiles and investigated their link to CAR T-cell expansion failure and patient outcome of CD19-targeted CAR T-cell therapy.

Methods: Patients with r/r B-NHL who underwent treatment with axi-cel or tisa-cel between January 2019 and November 2021 in the third- or later-line setting at the LMU in Munich were included (n=55). EDTA-anticoagulated peripheral blood and serum was collected. CD19 CAR and immune checkpoint expression (IC; PD-1, TIM-3, LAG-3 and CD224) was assessed by multiparameter flow cytometry on day 14 after CAR T-cell infusion. A next-generation targeted single-cell proteogenomics approach (BD RhapsodyTM) to analyze the transcriptome of distinct CAR T-cell subpopulations on day 7 and day 28 after CAR T-cell infusion. A total of 92 human immuno-oncology related proteins were simultaneously measured using the Olink® Immuno-Oncology panel on day 14 after infusion. Signatures of CAR T-cell dysfunction have been compared between patients with low (loEx) or high (hiEx; < vs ≥ 19 CAR T-cells/µl at day 7 post infusion)CAR T-cell expansion and were put into context of clinical outcome measures.

Results: CAR T-cell levels at day 7 were inversely correlated to co-expression of PD-1 (r = -0.4923, p = 0.0011, n = 41), TIM-3 (r = -0.3634, p = 0.0195, n = 41) and LAG-3 (r = -0.5412, p = 0.0003, n = 41) on CAR T-cells at day 14, suggestive of an association of early expansion failure and dysfunction of CAR T-cells. Indeed, loEx (n=26) showed higher frequencies of CAR T-cells coexpressing several IC, including LAG-3+ (p=0.0036), LAG-3+PD-1+ (p = 0.0048), or LAG-3+TIM-3+ (p = 0.0065) compared to hiEx (n=15) on day 14 after transfusion. Conversely, we detected higher frequencies of non-dysfunctional CAR T-cell phenotypes negative for several IC, such as CD244+PD-1TIM-3(p = 0.0006), CD244+LAG-3PD-1TIM-3(p = 0.001) and CD244+LAG-3+PD-1TIM-3(p = 0.011) in hiEx. Next, we applied uniform manifold approximation and projection (UMAP) data to examine the surface expression of IC on CAR T-cells at day 14 post-infusion according to treatment response. Unsupervised clustering revealed a higher abundance of CAR T-cell clusters positive for several IC (CD244+LAG-3PD-1+TIM-3+) in NR (n = 16) compared to R (n = 23). By contrast, CAR T-cell clusters containing CAR T-cells expressing no IC were more abundant in R compared to NR. In addition, we found significantly increased levels of a highly dysfunctional CAR T-cell phenotype positive for all four IC in NR compared to R at day 14 post infusion (p = 0.0178, n = 46). When specifically comparing the transcriptome of CAR T-cells between NR (n = 6) and R (n = 6), immune inhibitory and exhaustion-related genes were upregulated in NR, such as GZMK, KLRC1 and NR4A2 (figure 1, cluster 1). Conversely, CAR T-cells in R overexpressed genes related to T-cell effector function and cell differentiation, such as TIMP-1, TNFDF10 and MYC (figure 1, cluster 0). Consistent with these findings, a multiplexed proteomics assay for immunomodulatory serum factors in day 14 samples revealed an upregulation of proteins linked to an immune inhibitory and inflamed milieu in patients with highly dysfunctional CAR T-cells (e.g., sPD-L1 and sLAG-3, p = 0.0235 and p = 0.0264, respectively, n = 46) as well as in non-responding patients (e.g., sPD-L1 and IL-6, p = 0.0036 and p = 0.0053, respectively, n = 55).

Conclusion: We were able to characterize a phenotypic, transcriptomic and proteomic signature of CAR T-cell dysfunction in post-infusion samples of both patients experiencing CAR T-cell expansion failure as well as lack of radiographic response. Our data show that treatment failure might be predicted not only by CAR T-cell kinetics but also by early assessment of the CAR T-cell phenotype, transcriptome and secretome, and thus enables identification of patients in need of salvage treatment.

Disclosures: Blumenberg: Gilead: Consultancy, Research Funding; Novartis: Honoraria, Research Funding; BMS: Research Funding; Janssen: Honoraria, Research Funding; Roche: Consultancy, Research Funding. Rejeski: Kite/Gilead: Other: Travel Support, Research Funding; Pierre-Fabre: Other: Travel Support; Novartis: Honoraria; BMS/CELGENE: Consultancy, Honoraria. von Bergwelt-Baildon: Novartis: Research Funding, Speakers Bureau; Astellas: Research Funding, Speakers Bureau; BMS: Research Funding, Speakers Bureau; Mologen: Research Funding, Speakers Bureau; Roche: Research Funding, Speakers Bureau; MSD Sharpe & Dohme: Research Funding, Speakers Bureau; Gilead: Research Funding, Speakers Bureau; Miltenyi Biotec: Research Funding, Speakers Bureau. Buecklein: Gilead/Kite: Other: Travel Funding, Research Funding; Roche: Honoraria, Research Funding; Miltenyi Biotech: Research Funding; BMS: Research Funding; Pfizer: Consultancy, Honoraria, Speakers Bureau; Amgen: Consultancy; Pierre Fabre: Other: Travel Funding; Priothera: Consultancy. Subklewe: Amgen: Consultancy, Honoraria, Research Funding; Ichnos Sciences: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding, Speakers Bureau; Seagen: Research Funding; Molecular Partners: Consultancy, Honoraria, Research Funding; Gilead/Kite: Consultancy, Honoraria, Other: Travel Support, Research Funding, Speakers Bureau; AstraZeneca: Speakers Bureau; Takeda: Consultancy, Honoraria, Research Funding; BMS/Celgene: Consultancy, Honoraria, Research Funding, Speakers Bureau; AvenCell: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Research Funding, Speakers Bureau; Miltenyi Biotec: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria, Other: Travel Support, Speakers Bureau; Roche: Consultancy, Honoraria, Other: Travel Support, Research Funding, Speakers Bureau; Incyte Biosciences: Consultancy, Honoraria; GSK: Speakers Bureau; LAWG: Speakers Bureau; Springer Healthcare: Speakers Bureau; AbbVie: Consultancy, Honoraria; Autolus: Consultancy, Honoraria; advesya (CanCell Therapeutics): Consultancy, Honoraria; Genmab US: Consultancy, Honoraria; Interius BioTherapeutics: Consultancy, Honoraria; Nektar Therapeutics: Consultancy, Honoraria; Orbital Therapeutics: Consultancy, Honoraria; Sanofi: Consultancy, Honoraria; Scare: Consultancy, Honoraria.

*signifies non-member of ASH