Abstract 3996: Secreted cytokine-αPDL1 fusion proteins improve solid tumor chimeric antigen receptor (CAR) T-cell therapy
Authors: Christian Lea , Murad John P. , Lopez Lupita , Park Anthony , Yang Jason , Lee Eric , Trac Candi , Forman Stephen , Priceman Saul J. 
Published: March 2024

Abstract
Background: Success of Chimeric Antigen Receptor (CAR) T cells therapy for solid tumors are limited by the suppressive solid tumor microenvironment (TME), T cell exhaustion, lack of persistence, and poor trafficking to tumors. Strategies to improve this include therapeutic combinations, such as checkpoint inhibition (CPI) or cytokine support. However, CPI therapy has largely failed because of refractory and resistant tumors and cytokine administration can lead to lethal toxicities. To this end, we propose that enabling CAR T cells to secrete bi-functional fusion proteins consisting of cytokine modifiers (ie: IL12, IL15, or TGFβ-trap) combined with checkpoint inhibition (αPDL1 scFv) can provide tumor PDL1 sequestered cytokine activity and local tumor-immune modulation to boost solid tumor CAR T cell efficacy, enhance CPI impacts, and safely improve durable outcomes. Methods: Mouse T cells dual transduced to express surface CAR targeting prostate or ovarian solid tumors in addition to secretable cytokine fused to an αPDL1 scFv were generated. In vitro, the PDL1 blockade capacity and tumor-surface cytokine presentation of supernatants from dual transduced CAR T cells was assessed on PDL1 induced tumor cells. Further, secreted fusion protein transduced CAR T cells were assessed for function, phenotype, and cytokine release in a repetitive tumor rechallenge assay. In vivo, mouse CAR T cells dual transduced with fusion proteins and appropriate controls were assessed for anti-tumor efficacy, survival kinetics, and toxicity in immunocompetent solid tumor mouse models of prostate cancer and intraperitoneal disseminated ovarian cancer. Results: CAR T cells were shown to be successfully dual transduced, and secreted αPDL1-cytokine fusion proteins exhibiting functional PDL1 binding characteristics in vitro. CAR T cells engineered with αPDL1-IL12 fusion protein had greater anti-tumor activity and CAR specific expansion compared to other tested fusion proteins in in vitro co-culture assays. In a syngeneic prostate tumor model, mice receiving CAR T cells with αPDL1-IL12 fusion T cells out competed other cytokine fusion combinations. In a syngeneic ovarian tumor model we safely achieved 100% curative response rate with CAR secreting αPDL1-IL12 fusion in contrast to CAR with αPDL1mutIL12 control along with tumor regional PDL1 blockade on myeloid subsets within the TME. Conclusions: Our findings suggest that CAR T cells engineered to secrete αPD-L1-IL12 fusion protein show improved tumor control, less systemic toxicity, and enhanced expansion which promotes eradication of disease in two independent in vivo tumor models with two unique solid tumor CAR targets. We believe this strategy has the potential to improve solid tumor CAR T-cell efficacy and enhance durable innate immune responses.
Citation Format: Lea Christian, John P. Murad, Lupita Lopez, Anthony Park, Jason Yang, Eric Lee, Candi Trac, Stephen Forman, Saul J. Priceman. Secreted cytokine-αPDL1 fusion proteins improve solid tumor chimeric antigen receptor (CAR) T-cell therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3996.