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4802 Enhancing CAR T Cell Efficacy By Modulating PI3K Signaling Via a Synthetic CD28 Rheostat

Program: Oral and Poster Abstracts
Session: 702. CAR-T Cell Therapies: Basic and Translational: Poster III
Hematology Disease Topics & Pathways:
Research, Translational Research
Monday, December 9, 2024, 6:00 PM-8:00 PM

Sanam Shahid, MD1, Winson Cai2*, Erin R. Burns2*, Jasmine S. Um2*, Serena Mathew2*, Sydney Souness2*, Sarah Yoo2*, Bing-Yi Chen2*, Takeshi Fujino2*, Renier Brentjens, MD PhD3 and Anthony F. Daniyan, MD4,5*

1Pediatrics, Memorial Sloan Kettering Cancer Center, New York, NY
2Memorial Sloan Kettering Cancer Center, New York, NY
3Department of Medicine, Roswell Park Comprehensive Cancer Center, Buffalo, NY
4Department of Medicine, Cell Therapy Service, Memorial Sloan Kettering Cancer Center, New York, NY
5Department of Medicine, Leukemia Service, Memorial Sloan Kettering Cancer Center, New York, NY

Introduction: CD28 is a costimulatory protein incorporated into two FDA-approved second-generation CD19-directed CAR T cell products and utilized extensively preclinically. Within CAR T cells, CD28 induces complex signaling including activation of the PI3K/AKT/mTOR pathway, with ingrained signaling redundancies. Although PI3K signaling enhances proliferation, excessive growth signals may lead to T cell exhaustion and terminal differentiation, correlating with suboptimal clinical responses. Importantly, PI3K and AKT inhibitors have been used to modulate CAR T cell signaling, enriching memory-like and reducing highly differentiated T cells, leading to a more potent cellular product. We aimed to attenuate CD28 signaling redundancies through the development of a “synthetic rheostat” that tempers PI3K/AKT/mTOR pathway signaling.

Methods: We mutated CD28 to reduce PI3K signaling and assessed the function of this mutant (referred to as SAVVY) compared to a native CD28 in second-generation CD19-targeting CAR T cells against NALM6, a B cell acute lymphoblastic leukemia (B-ALL) cell line. CD19-targeting CAR T cells were produced by gammaretroviral transduction of peripheral blood-derived primary T cells. In vitro, we evaluated CAR T cell proliferation, target lysis, CAR recycling, T cell mitochondrial mass/function, T cell immunophenotype (memory/exhaustion), and T cell cytokine secretion. In vivo, we evaluated tumor control and overall survival in cell-line derived xenograft murine models. In vitro and in vivo, models of normal antigen density, low antigen expression, and PD-L1 overexpression were evaluated given the clinical relevance and prevalence of antigen-positive relapses, as well as CD19 antigen-low/negative relapses post-treatment with FDA-approved CAR T cell therapies.

Results: Although CD19-targeting SAVVY-CAR T cells demonstrated no significant difference in long-term anti-tumor efficacy compared to native CD28-CAR T cells against NALM6 (effector:target (E:T) ranging from 1:5 to 1:40), they exhibited superior proliferation in vitro. When cocultured long-term in vitro with low antigen density tumor (NALM6-CD19low) and PD-L1-overexpressing tumor (NALM6-PD-L1+), SAVVY-CAR T cells demonstrated comparable cytotoxicity and proliferation at an E:T of 1:5 but superior tumor control at a lower E:T of 1:40. The SAVVY modification did not perturb blastogenesis or expression of early activation markers (CD69), but it protracted late activation marker expression (CD25, CD71, HLA-DR). SAVVY-CAR T cell activation protraction was not dependent on CAR recycling, mitochondrial mass, or mitochondrial transmembrane potential. SAVVY-CAR T cells demonstrated lower expression of co-inhibitory markers on excessive antigen encounter, and there was no significant difference in cytokine secretion (interferon-gamma, IL-2, TNF-a, granzyme B) when cocultured with NALM6 or NALM6-CD19low. CD8+ SAVVY-CAR T cells retained a memory immunophenotype (CD62L+) after antigen encounter relative to native CD8+ CD28-CAR T cells. SAVVY-CAR T cells showed superiority in vivo compared to native CD28-CD19 CAR T cells in xenograft models utilizing disseminated NALM6, NALM6-CD19low, and NALM6-PD-L1+, with mice exhibiting significantly prolonged survival in all three models.

Conclusions: The SAVVY modification imparts enhanced functionality to second-generation CAR T cells evident in vitro and in vivo, leading to significantly improved tumor control in an aggressive disseminated B-ALL model under normal, low antigen-density, and PD-L1-high conditions. Recent findings have shown that CD28 is more active in low antigen density disease compared to 4-1BB, raising concerns about attenuating the former’s signaling. However, SAVVY-CAR T cells demonstrate enhanced anti-tumor effects compared to native CD28-CAR T cells in a low antigen density model, which could potentially lead to longer-lasting remissions and reduced antigen-low relapses. Given these robust findings, SAVVY has been incorporated into a CD371 (CLL-1, CLEC12A)-directed CAR T cell product with constitutive secretion of interleukin-18, which is currently being investigated in a phase 1 trial for relapsed/refractory AML patients (CLEAR-AML; NCT06017258). Furthermore, the impact of SAVVY is currently being evaluated in other CAR constructs targeting various hematologic and solid tumor malignancies.

Disclosures: Brentjens: Triumvira, Cargo Tx, CoImmune: Membership on an entity's Board of Directors or advisory committees; BMS: Research Funding; BMS, Caribou, Sanofi: Other: licensed intellectual property to and collects royalties from; BMS, Atara Biotherapeutics Inc. and was a consultant for Triumvira, Cargo Tx, CoImmune: Consultancy. Daniyan: Tigen Pharma SA: Patents & Royalties: Intellectual Property Rights; Promicell Therapeutics, Inc.: Consultancy, Current holder of stock options in a privately-held company; Shoreline Biosciences, Inc.: Consultancy; Caribou Biosciences, Inc: Patents & Royalties: Intellectual Property Rights.

*signifies non-member of ASH