Abstract 4099: Dual targeting of tumor cells and cancer associated fibroblasts enhances CAR T efficacy in solid tumors
Authors: Huang Bo , Zheng Suilan , Chu Haiyan , Mukkamala Ramesh , Bowroju Suresh K. , Singh Yashapal , Kasireddy Sudarsan R. , Bari Md Sazzadul , Srinivasarao Madduri , Beitz Laurie , Ryu Byoung , Jensen Michael , Scharenberg Andrew M. , Low Philip S. 
Published: April 2023

Abstract
CAR T cell therapies have demonstrated considerable potency in treating hematologic cancers, but only limited efficacy in eliminating solid tumors. One reason for this discrepancy may derive from the immunologic and physical barriers created by infiltration of cancer associated fibroblasts (CAFs) into solid tumors. These CAFs, which can comprise 15-85% of the stromal cells in a tumor mass, are correlated with poor patient survival and can reduce CAR T cell efficacies by secreting immunosuppressive cytokines, stimulating tumor cell proliferation, and depositing fibrotic barriers to CAR T cell penetration. Here, we are pursuing a method to suppress or eliminate CAF activities in the tumor microenvironment (TME).
A potential CAR T cell target that is uniquely expressed on CAF surfaces is fibroblast activation protein (FAP). In this study, we have employed a novel, highly specific FAP targeting small molecule to direct our universal CAR T cells to CAFs. For this purpose, we have designed a CAR that contains an otherwise classical CAR except the extracellular scFv specifically binds fluorescein. Upon addition of a bispecific adaptor comprised of fluorescein linked via a short spacer to our FAP ligand, a bridge is formed between the anti-fluorescein CAR on the T cell and FAP on the CAF, resulting in formation of an immunologic synapse between the CAR T cell and CAF that triggers CAF destruction and CAR T cell proliferation. Importantly, this CAR T cell design also permits simultaneous administration of a second bispecific adaptor (i.e. fluorescein linked via a short spacer to a cancer-specific ligand) that can enable the same CAR T cell to concurrently kill adjacent cancer cells.
To test whether this universal CAR T cell can eliminate both cancer cells and CAFs, we have implanted KB cells (i.e. a cell line that creates an immunologically “cold” folate receptor (FR) expressing solid tumor) into NSG mice and quantitated the CAR T cell’s toxicity in the presence of one or both bispecific adapters. While administration of the universal CAR T cells followed by intravenous injection of an FR-targeting bispecific adaptor achieved significant anti-tumor efficacy, co-injection of a FAP-targeted bispecific adaptor enhanced this efficacy without apparent toxicity. Analyses of tumor masses from the therapy further revealed that co-administration of the FAP-targeted bispecific adaptor not only promoted CAF elimination but also enhanced CAR T infiltration and activation. Importantly, a similar improvement in anti-tumor efficacy could be readily demonstrated in a second immunologically “cold” PSMA-expressing solid tumor model. Taken together, we conclude that the bispecific adaptor/universal CAR T approach offers a unique opportunity to concurrently eradicate cancer cells and tumor-supporting CAFs in a manner that can improve the overall performance of the CAR T cells in solid tumors.
Citation Format: Bo Huang, Suilan Zheng, Haiyan Chu, Ramesh Mukkamala, Suresh K. Bowroju, Yashapal Singh, Sudarsan R. Kasireddy, Md Sazzadul Bari, Madduri Srinivasarao, Laurie Beitz, Byoung Ryu, Michael Jensen, Andrew M. Scharenberg, Philip S. Low. Dual targeting of tumor cells and cancer associated fibroblasts enhances CAR T efficacy in solid tumors. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4099.