Eradication of Established Tumors by Chemically Self-Assembled Nanoring Labeled T Cells

Abstract

Our laboratory has developed chemically self-assembled nanorings (CSANs) as prosthetic antigen receptors (PARs) for the nongenetic modification of T cell surfaces. PARs have been successfully employed in vitro to activate T cells for the selective killing of leukemia cells. However, PAR efficacy has yet to be evaluated in vivo or against solid tumors. Therefore, we developed bispecific PARs that selectively target the human CD3 receptor and human epithelial cell adhesion molecule (EpCAM), which is overexpressed on multiple carcinomas and cancer stem cells. The αEpCAM/αCD3 PARs were found to stably bind T cells for >4 days, and treating EpCAM MCF-7 breast cancer cells with αEpCAM/αCD3 PAR-functionalized T cells resulted in the induction of IL-2, IFN-γ, and MCF-7 cytotoxicity. Furthermore, an orthotopic breast cancer model validated the ability of αEpCAM/αCD3 PAR therapy to direct T cell lytic activity toward EpCAM breast cancer cells in vivo, leading to tumor eradication. In vivo biodistribution studies demonstrated that PAR-T cells were formed in vivo and persist for over 48 h with rapid accumulation in tumor tissue. Following PAR treatment, the production of IL-2, IFN-γ, IL-6, and TNF-α could be significantly reduced by an infusion of clinically relevant concentrations of the FDA-approved antibiotic, trimethoprim, signaling pharmacologic PAR deactivation. Importantly, CSANs did not induce naïve T cell activation and thus exhibit a limited potential to induce naïve T cell anergy. In addition, murine immunogenicity studies demonstrated that CSANs do not induce a significant antibody response nor do they activate splenic cells. Collectively, our results demonstrate that bispecific CSANs are able to nongenetically generate reversibly modified T cells that are capable of eradicating targeted solid tumors.

Multivalent Ligand Binding to Cell Membrane Antigens: Defining the Interplay of Affinity, Valency, and Expression Density

“…SEC and DLS results were consistent with our previous analyses of Fn3-targeted CSANs, including cryo-electron microscopy (cryo-EM) evidence of nanoring formation. 43, 44…”

Anti-EGFR Fibronectin Bispecific Chemically Self-Assembling Nanorings (CSANs) Induce Potent T Cell-Mediated Antitumor Responses and Downregulation of EGFR Signaling and PD-1/PD-L1 Expression

“…The enhanced avidity of the rings provides enhanced binding and selectivity to T cells and the targeted antigen of interest (28). We have successfully demonstrated the in vitro and in vivo efficacy of scFv-based PARs (26,29). However, generating scFv-based monomers to form the rings is dependent on refolding steps that increases the time required for purification with significantly reduced protein yields (29,30).…”

Advanced biomaterials for cancer immunotherapy

“…In recent years, biologically derived nanobiomaterials, such as cancer cell membranes, viral proteins, and DNAs, have started to be used for new cancer nanovaccines [ 260 – 263 ]. Researchers found that tumor antigens and subcellular particles in the cancer membrane, such as melanoma cells, could be loaded into various NPs, which induce specific cellular and humoral responses, thus preventing tumor growth [ 157 , 264 , 265 ].…”