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558 Dual Inhibition of PI3KDelta/Gamma during Manufacturing Reprograms Metabolism of CAR T Cells to Enhance Expansion and Cytotoxicity Against CLL

Program: Oral and Poster Abstracts
Type: Oral
Session: 703. Adoptive Immunotherapy: Mechanisms and New Approaches: Optimizing CAR T cells for Improved Outcomes
Hematology Disease Topics & Pathways:
Biological, Therapies, CAR-Ts, Technology and Procedures, cell expansion, Clinically relevant, flow cytometry, mass cytometry
Monday, December 7, 2020: 8:00 AM

Christopher Ronald Funk1, Shuhua Wang, MD1*, Alex Waller, BS2*, Claudia Edgar, BS1*, Aditi Sharma, PhD1*, Kevin Chen, BS1*, Vikas Gupta, MD, PhD3*, Shanmuganathan Chandrakasan, MD4*, Jaquelyn Zoine5,6*, Andrew Fedanov, BS5,6*, Sunil S. Raikar, MD7, Jean L. Koff, MD1, Christopher Flowers, MD, MS8, Silvia Coma, PhD9*, Jonathan A. Pachter, PhD9*, Sruthi Ravindranathan, PhD1*, H. Trent Spencer, Ph.D.5,10*, Mala Shanmugam, PhD1 and Edmund K. Waller, PhD, MD11

1Department of Hematology and Medical Oncology, Emory University School of Medicine, Atlanta, GA
2Department of Lymphoma/Myeloma, Division of Cancer Medicine, The University of Texas MD Anderson Cancer Center, Houston, TX
3Emory University, Atlanta, GA
4Division of Bone Marrow Transplant, Department of Pediatrics, Emory University School of Medicine, Atlanta, GA
5Aflac Cancer and Blood Disorders Center, Children's Healthcare of Atlanta, Atlanta, GA
6Department of Pediatrics, Emory University School of Medicine, Atlanta
7Aflac Cancer and Blood Disorders Center, Emory University/Children's Healthcare of Atlanta, Atlanta, GA
8Department of Lymphoma and Myeloma, The University of Texas MD Anderson Cancer Center, Houston, TX
9Verastem Oncology, Inc., Needham, MA
10Department of Pediatrics, Emory University School of Medicine, Atlanta, GA
11Department of Hematology and Medical Oncology, Winship Cancer Institute, Emory University, Atlanta, GA

Chronic lymphocytic leukemia (CLL) creates an immune-suppressive microenvironment that induces terminal T-cell differentiation and T-cell exhaustion, thereby limiting the efficacy of chimeric antigen receptor (CAR) T cells. Indeed, analysis of CLL patients has shown that a high frequency of senescent T-cells (dashed line, Figure 1A) prior to CAR T-cell manufacturing was associated with disease progression and failure to benefit from CAR T (Fraietta et al. 2018). In a cohort of early stage untreated CLL patients, we show that 55% of patients had a predominant senescent phenotype (Figure 1A). This striking observation highlights the need for strategies to enrich non-senescent T cells for CAR T therapy in CLL patients. To address this need, we studied the effect of adding phosphoinositide 3-kinase inhibitors (PI3Ki) during ex vivo CAR T cell-manufacturing on T cell phenotype, metabolism, in vivo expansion, persistence and anti-CLL cytotoxicity in NOG mice bearing the human OSU-CLL compared with conventional CART.

To investigate the relative influence of PI3Kd-selective versus dual PI3K-d/g inhibition, we first cultured T cells from CLL patients activated with anti-CD3/CD28 beads across logarithmic dose scales of duvelisib or idelalisib. With dual-PI3K-d/g inhibition, CLL donor T cell expansion was 150% of conventionally expanded CART (Figure 1B) with dose-dependent decreases in expression of exhaustion markers TIM-3 and LAG-3 (Figure 1C). PI3K-d/g blockade increased frequencies of CD8+ CAR T cells, thus normalizing the ratio of CD4:CD8 CAR T cells (Figure 1D). To further characterize the effects of dual PI3K-d/g inhibition on T-cell phenotype, mass cytometry time-of-flight (CyTOF) analyzed the phenotype of CAR T cells cultured with or without duvelisib. Unsupervised clustering algorithms showed duvelisib increased frequencies and numbers of T-stem cell memory (Tscm), naïve, and central memory CD8+ CAR T cells (data not shown). Finally, unbiased clustering algorithms identified increases in frequencies of CD27+CD45RO- CD8+ CAR T cells which was confirmed in additional patients using flow cytometry (Figure 1E). In summary, duvelisib enriched populations of less differentiated, more potent CD8+ CAR T cells during manufacturing.

To assess whether these phenotypic changes conferred functional benefit, the cytotoxicity of CAR T cells cultured with or without duvelisib was compared using the OSU-CLL cell line. Duvelisib-cultured CAR T cells (Duv-CAR T cells) had greater cytotoxicity against OSU-CLL cell line (Figure 1F). Subsequently, gene expression profiling of Duv-CAR T cells showed decreased glycolysis pathway scores, down-regulated glucose transporters, and up-regulated glutamine transporters relative to control CAR T cells, leading us to explore T cell metabolism. Elevated protein expression of PGC1-a, a critical regulator of mitochondrial biogenesis and autophagy, was associated with increased staining with NAO, a measure of mitochondrial mass in Duv-CAR T cells (data not shown). To directly measure mitochondrial content of cells, transmission electron microscopy of control CAR T and Duv-CAR T cells generated from CLL patients was performed after 14 days of culture with representative images shown in Figure 1H. A 1.45-fold increase in mitochondrial cross-sectional area was observed (representative images in Figure 1G), which imputes a 175% increase in mitochondrial volume for duvelisib cultured CAR T cells relative to control CAR T cells (p=0.0013; Figure 1G).

Following transfer to NOG mice engrafted with a human CLL cell line, Duv-CAR T cells demonstrated greater in vivo expansion (Figure 1H), faster elimination of CLL (data not shown), and improved mouse survival (Figure 1I) in a model of high disease burden OSU-CLL. In summary, dual PI3K-d/g inhibition during CLL patient-derived CAR T-cell manufacturing increased yields of Tscm, naïve, and central memory CD8+ Duv-CAR T cells with greater mitochondrial mass and enhanced efficacy in eliminating CLL in a mouse model.

Disclosures: Funk: Verastem Oncology: Research Funding. Flowers: BeiGene: Consultancy; Bayer: Consultancy; Denovo Biopharma: Consultancy; V Foundation: Research Funding; National Cancer Institute: Research Funding; Eastern Cooperative Oncology Group: Research Funding; Burroughs Wellcome Fund: Research Funding; AbbVie: Consultancy, Research Funding; Leukemia and Lymphoma Society: Membership on an entity's Board of Directors or advisory committees; Cancer Prevention and Research Institute of Texas: Research Funding; TG Therapeutics: Research Funding; Millennium/Takeda: Consultancy, Research Funding; Spectrum: Consultancy; Acerta: Research Funding; Pharmacyclics/Janssen: Consultancy; Karyopharm: Consultancy; OptumRx: Consultancy; Genentech, Inc./F. Hoffmann-La Roche Ltd: Consultancy, Research Funding; Kite: Research Funding; Celgene: Consultancy, Research Funding; Gilead: Consultancy, Research Funding. Coma: Verastem Oncology, Inc: Current Employment, Current equity holder in publicly-traded company. Pachter: Verastem Oncology, Inc: Current Employment, Current equity holder in publicly-traded company. Waller: Verastem Oncology, Inc: Consultancy, Research Funding.

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