Interrogating and targeting co-inhibitory and co-stimulatory molecules in CAR T-cell Therapies

Adoptive cell therapies (ACT) with chimeric antigen receptors (CAR) hold promise to treat cancer, yet several challenges must be overcome to achieve this goal. Clinical evidence attributes treatment failure and resistance to the lack of functional T-cell persistence, antigen escape, and a suppressive tumor microenvironment replete with inhibitory but not co-stimulatory molecules. Investigating response and relapse mechanisms and effectively targeting tumor resistance mediators are urgently needed to improve current ACT. Our analysis of clinical CAR-T products revealed intrinsic PD-L1 (int-PD-L1) expression on the T cells and trogocytic acquisition of tumor-derived PD-L1 (extrinsic; ext-PD-L1). However, the functional role of PD-L1 on CAR-T remains unknown. We hypothesize that both int-PD-L1 and ext-PD-L1 limit CAR-T activity, and that rewiring PD-L1 and CD80 signaling with synthetic receptors will boost antitumor efficacy across ACT platforms. Aim 1 will determine how int-PD-L1 regulates CAR-T under optimal and suboptimal antigen stimulation; Aim 2 will define the impact of trogocytic ext-PD-L1 transfer on CAR-T function using in vitro assays and in vivo leukemia and melanoma models. To enhance T-cell persistence and function, we developed 80BB, a synthetic dual-costimulatory receptor fusing the CD80 ectodomain to the 4-1BB endodomain. 80BB delivers CD28 and 4- 1BB signals and functions as a CTLA-4 switch receptor, driving tumor control across diverse ACT platforms. Although CD80 interaction with CD28 and CTLA-4 underlies these benefits, CD80 also binds PD-L1, yet 80BB did not protect CAR-T from PD-L1/PD-1-mediated inhibition. We therefore engineered a novel 80BB-based platform that retains CD80 interaction with CD28 and CTLA-4 while redirecting PD-L1 engagement, to overcome PD-L1-mediated suppression. Aim 3 will determine mechanisms of action and therapeutic benefit of this novel synthetic costimulatory receptor in B-cell malignancy and solid-tumor models. Our multidisciplinary approach combines basic immunology with translational immunoengineering to overcome ACT barriers. We aim to advance next-generation engineered T-cells and expand the scope of ACT, improving the survival and quality of life of cancer patients. Project Number: 1R37CA300795-01A1 | Fiscal Year: 2026 | NIH Institute/Center: National Cancer Institute (NCI) | Principal Investigator: Mohamad Hamieh | Institution: WEILL MEDICAL COLL OF CORNELL UNIV, NEW YORK, NY | Award Amount: $716,183 | Activity Code: R37 | Study Section: Cellular Immunotherapy of Cancer Study Section[CIC] View on NIH RePORTER: https://reporter.nih.gov/project-details/11368643