(F-229) Optimizing Folate-Peptide Intermediate for Universal Chimeric Antigen Receptor T-Cell Therapy

Chimeric antigen receptor (CAR) T-cell therapy is a revolutionary cancer treatment with promising clinical efficacy in treating liquid cancers such as relapsed/refractory CD19+ B-cell leukemia and lymphoma. Yet, current single-antigen targeted CAR T cells have had little success treating solid-tumors due to tumor heterogeneity and plasticity. To address these limitations, we aim to develop a universal CAR T-cell system that utilizes cancer-targeting intermediates conjugated onto CARs to mediate tumor cell binding. We are engineering a SpyCatcher003 CAR T-cell system (DB5 CARs) and employ a synthetic, high-affinity biomaterial to bind folate receptor alpha (FOLR1) for targeted killing of FOLR1+ tumor cells (e.g., ovarian and breast tumors). Our approach for universal CAR T-cell therapy could provide safe, cost-effective, and broad-targeting treatments for patients with heterogeneous solid tumors.

Our DB5 CAR T-cell platform utilizes a protein-peptide pair described in the literature (SpyCatcher003 and SpyTag003, respectively) that spontaneously form an isopeptide bond, which we exploit to conjugate synthetic biomaterial and direct T cell binding to cancer cells. Donor T cells are isolated and genetically engineered using a lentiviral vector (Figure 1.A) encoding the DB5 CARs . The folate-SpyTag003 intermediate is synthesized by standard solid-phase peptide synthesis. After HPLC purification, the heterobifunctional folate-SpyTag003 is tested for loading onto DB5 CAR T cells and binding to FOLR1+ cancer cells by flow cytometry based assays (Figure 1.B). Separate CD4+ and CD8+ DB5 CAR T cells functional assays are performed to measure the activation of DB5 CAR T cells. For the CD4+ DB5 CAR T cells, an intracellular cytokine stain (ICCS) is used to measure cytokines produced by CD4+ T cells when co-cultured with FOLR1+ target cells for 5 h. To assess CD8+ DB5 CAR T cell activation, a flow cytometry-based cytotoxicity assay is used to measure specific lysis of dye-labeled FOLR1+ cancer cells when co-cultured with the CD8+ T cells for 18 h. Data from binding and functional studies are analyzed using FlowJo and GraphPad Prism software.

The efficacy of our universal CAR T cell system requires an intermediate which covalently associates with the CAR and binds cancer antigens for targeted T-cell killing of tumor cells. Triplicate studies quantified the strong affinity of folate-SpyTag003 for loading DB5 CARs (Figure 2) and binding high FOLR1-expressing KB cells in vitro (Figure 3). After being co-cultured with tumor cells, CD8+ and CD4+ DB5 CAR T cells armed with folate-SpyTag003 showed increased cytotoxic activity and cytokine responses (Figure 4). Thus, we demonstrate that folate-SpyTag003 guides DB5 CAR T cells and selectively activates CD4+ T-cell cytokine production in the presence of FOLR1+ tumor cells. While the low-FOLR1 expressing HCC1143 cells induced less significant cytokine responses, KB cells elicited robust CD4+ T cell response.

In this study, we have developed a heterobifunctional folate-peptide chimera to target and activate DB5 CAR T cells. This project aims to expand the synthetic toolkit for intermediates such as peptides, small molecules, and aptamers which could be used in parallel with universal CARs to achieve multiplexed antigen targeting. As opposed to commonly used biological molecules (e.g., antibodies), these synthetic molecules can be manufactured at lower costs. Further, the dosage of small fast-circulating intermediates can be controlled to readily switch CAR T-cell activity on/off and thus mitigate T-cell exhaustion and treatment-associated toxicities. The versatility of synthetic intermediates can allow researchers and healthcare providers to create a treatment regimen specific to a patient’s tumor heterogeneity and could be altered in response to tumor plasticity, thus providing a more personalized and effective cancer treatment.

I would like to acknowledge the Mary Gates Endowment for supporting my research with the MGE Research Scholarship.