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4 Ketogenic Diet Enhances CAR T Cell Antitumor Function Via β-Hydroxybutyrate

Program: Marquee Sessions
Session: Plenary Scientific Session
Hematology Disease Topics & Pathways:
Research, Fundamental Science, ALL, Translational Research, Non-Hodgkin lymphoma, B Cell lymphoma, Chimeric Antigen Receptor (CAR)-T Cell Therapies, Diseases, Immunology, Treatment Considerations, Biological therapies, Lymphoid Malignancies, Metabolism, Biological Processes
Sunday, December 8, 2024, 2:00 PM-4:00 PM

Shan Liu1*, Puneeth Guruprasad2, Kecheng Han1*, Luca Paruzzo, MD3*, Alexander Shestov, PhD3*, Andre Kelly4*, Kevin R. Amses, PhD1,3*, Amichay Afriat, PhD5*, Bhoomi Madhu, PhD1*, Lev Litichevskiy, PhD1*, Ezra Dubowitz1*, Neil Tangal1*, Alana McSween1*, Melody Tan3*, Alberto Carturan3*, Andrew Lee3*, Yunlin Zhang, MS3*, Giulia Gabrielli, MD3*, Raymone Pajarillo, MS3*, Ruchi P. Patel, PhD3, Guido Ghilardi, MD3*, Patrizia Porazzi, PhD3*, Stephen J. Schuster, MD3,6,7,8, Roddy S. O'Connor, PhD4*, Michael C. Milone, MD, PhD3*, Joshua Rabinowitz, MD, PhD5*, Maayan Levy, PhD1* and Marco Ruella, MD3,6,7,8

1Department of Microbiology, University of Pennsylvania, Philadelphia, PA
2Center for Cellular Immunotherapies, Perelman School of Medicine, University of Pennsylvania, North Wales, PA
3Center for Cellular Immunotherapies, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA
4Center for Cellular Immunotherapies, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA
5Lewis-Sigler Institute for Integrative Genomics, Princeton University, Princeton, NJ
6Lymphoma Program, Abramson Cancer Center, University of Pennsylvania, Philadelphia, PA
7Division of Hematology/Oncology, Hospital of the University of Pennsylvania, Philadelphia, PA
8Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA

Introduction. Several mechanisms of resistance have been identified for chimeric antigen receptor (CAR) T cell therapy; however, there are limited data on the impact of lifestyle factors, such as diet, on CAR T cell efficacy. Indeed, diet-derived metabolites can modulate T cell functions through the regulation of metabolic, epigenetic, and transcriptional processes. Here, we investigated whether diet can affect responses to CAR T cell treatment.

Methods and Results. To this end, we implanted diffuse large B-cell lymphoma (DLBCL) tumors (A20) into immunocompetent mice (Balb/c), and fed them one of five representative diets, including ketogenic, high-fiber, high-fat, high-protein, Western (i.e., high cholesterol), and a macro- and micronutrient content-matched control diet. Upon tumor engraftment, we infused murine anti-CD19 CAR T cells (CART19). Mice fed a ketogenic diet showed improved tumor control and overall survival relative to all other screened diets. As expected, the level of β-hydroxybutyrate (BHB), the principal metabolite produced during ketosis, was significantly higher in ketogenic diet-fed mice compared to other groups. Since effector T cells can utilize BHB to fuel the citric acid cycle, we hypothesized that BHB can serve as a more efficient energy source than glucose for active CAR T cells, leading to more pronounced effector responses.

Therefore, we tested the effect of BHB alone on several xenograft cancer models in immunodeficient NOD-SCID gamma chain-deficient (NSG) mice. First, DLBCL tumors were implanted into NSG mice (5x106 CD19+ OCI-Ly18 cells, subcutaneous), and BHB was oral gavaged daily. On day 11 post-implantation, a suboptimal dose of CART19 (3x106 CAR+ cells) was infused. Mice receiving both oral BHB and CART19 demonstrated potent tumor control (complete response, CR 6/7) compared to mice receiving a vehicle gavage (CR 1/7). BHB+CART19-treated mice also showed higher peripheral CAR T cell expansion and elevated serum effector cytokines. Furthermore, BHB given ad libitum in the drinking water led to improved tumor control of anti-mesothelin CAR-T in AsPC-1 (pancreatic cancer, subcutaneous) and anti-CD19 CAR-T in Nalm6 (B cell acute lymphoblastic leukemia, intravenous) xenografts.

Mechanistically, using carbon-13 (13C) isotope labeling, we found that antigen-activated CAR T cells preferentially integrate carbon from BHB into the citric acid cycle components over that of standard glucose. This process fuels CAR-T oxidative phosphorylation and increases their oxygen consumption, as confirmed by the Seahorse Assay (Agilent Seahorse XF). To determine whether BHB modulates epigenetic accessibility in CAR T cells, we performed an assay for transposase-accessible chromatin with sequencing (ATAC-seq) on CAR T cells treated with or without BHB. We found that BHB-treatment exposed crucial effector and memory genes (e.g., FOXO1, TCF7, GZMB) in CAR T cells. This likely reflects increased Acetyl-CoA from BHB entering the CAR-T nucleus and acetylating histones. Finally, we found that deleting BDH1, the key enzyme involved in BHB metabolism to Acetyl-CoA, in CAR-T reduces the functional benefit of BHB. This further confirms that BHB enhances CAR-T functions through metabolic modulation.

To test the translational relevance of these findings, we retrospectively analyzed CART19 patient serum (n=17, all LBCL) taken 7 days post-infusion from the NCT02030834 trial using mass spectrometry. In line with our preclinical results that BHB can support CAR-T proliferation, BHB serum concentration positively correlated with CART19 expansion (P = 0.0164). Lastly, we produced CAR T cells from the leukapheresis products of two CART19 patients. We found that providing BHB in the cell culture media during ex vivo manufacturing boosted patient T cell proliferation, resulting in a ~4X increase in absolute cell count.

Conclusions. Our results demonstrate that ketogenic diet-derived BHB can be provided as a dietary intervention to augment CAR-T function in multiple cancer models. The key mechanism involves the enhancement of the citric acid cycle and histone modifications during CAR T cell activation. The results of this study will be translated into a first-in-human clinical trial of BHB-supplementation during CART19 treatment for relapsed or refractory B cell lymphoma.

*S. Liu and P. Guruprasad contributed equally.

**M. Ruella and M. Levy jointly supervised this work.

Disclosures: Patel: Vittoria Biotherapeutics: Current Employment. Ghilardi: Vittoria Biotherapeutics: Honoraria. Schuster: viTToria biotherapeutics: Consultancy; Nordic Nanovector: Honoraria, Membership on an entity's Board of Directors or advisory committees; Merck: Research Funding; Legend Biotech: Consultancy, Honoraria; Kite Pharmaceuticals: Consultancy; Janssen: Consultancy, Honoraria, Research Funding; Genmab: Consultancy; AstraZeneca: Consultancy, Honoraria; AbbVie: Consultancy; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Genentech/Roche: Consultancy, Honoraria, Research Funding; Celgene/Juno Therapeutics: Consultancy, Honoraria, Research Funding; Caribou Biosciences: Consultancy, Membership on an entity's Board of Directors or advisory committees; BioNTech: Consultancy; BeiGene: Consultancy, Honoraria; Pharmacyclics: Consultancy, Research Funding; Gilead: Research Funding; Acerta: Consultancy. Ruella: AbClon Inc.: Other: Consultancy, Research Funding; Vittoria Biotherapeutics: Current equity holder in private company, Patents & Royalties.

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