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

Nature uses multivalency to govern many biological processes. The development of macromolecular and cellular therapies has largely been dependent on engineering similar polyvalent interactions to enable effective targeting. Such therapeutics typically utilize high-affinity binding domains that have the propensity to recognize both antigen-overexpressing tumors and normal-expressing tissues, leading to "on-target, off-tumor" toxicities. One strategy to improve these agents' selectivity is to reduce the binding affinity, such that biologically relevant interactions between the therapeutic and target cell will only exist under conditions of high avidity. Preclinical studies have validated this principle of avidity optimization in the context of chimeric antigen receptor (CAR) T cells; however, a rigorous analysis of this approach in the context of soluble multivalent targeting scaffolds has yet to be undertaken. Using a modular protein nanoring capable of displaying ≤8 fibronectin domains with engineered specificity for a model antigen, epithelial cell adhesion molecule (EpCAM), this study demonstrates that binding affinity and ligand valency can be optimized to afford discrimination between EpCAMHigh (2.8-3.8 × 106 antigens/cell) and EpCAMLow (5.2 × 104 to 2.2 × 105 antigens/cell) tissues both in vitro and in vivo.

Programming Cell-Cell Interactions through Non-genetic Membrane Engineering.

The ability to direct targeted intercellular interactions has the potential to enable and expand the use of cell-based therapies for regenerative medicine, tissue engineering, and immunotherapy. While genetic engineering approaches have proven effective, these techniques are not amenable to all cell types and often yield permanent modifications with potentially long-lasting adverse effects, restricting their application. To circumvent these limitations, there is intense interest in developing non-genetic methods to modify cell membranes with functional groups that will enable the recognition of target cells. While many such techniques have been developed, relatively few have been applied to directing specific cell-cell interactions. This review details these non-genetic membrane engineering approaches-namely, hydrophobic membrane insertion, chemical modification, liposome fusion, metabolic engineering, and enzymatic remodeling-and summarizes their major applications. Based on this analysis, perspective is provided on the ideal features of these systems with an emphasis on the potential for clinical translation.

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

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.

Engineering Reversible Cell-Cell Interactions with Lipid Anchored Prosthetic Receptors.

Membrane-engineered cells displaying antigen-targeting ligands are useful as both scientific tools and clinical therapeutics. While genetically encoded artificial receptors have proven efficacious, their scope remains limited, as this approach is not amenable to all cell types and the modification is often permanent. Our group has developed a nongenetic method to rapidly, stably, and reversibly modify any cell membrane with a chemically self-assembled nanoring (CSAN) that can function as a prosthetic receptor. Bifunctional CSANs displaying epithelial cell adhesion molecule (EpCAM)-targeted fibronectin domains were installed on the cell membrane through hydrophobic insertion and remained stably bound for ≥72 h in vitro. These CSAN-labeled cells were capable of recognizing EpCAM-expressing target cells, forming intercellular interactions that were subsequently reversed by disassembling the nanoring with the FDA-approved antibiotic, trimethoprim. This study demonstrates the use of this system to engineer cell surfaces with prosthetic receptors capable of directing specific and reversible cell-cell interactions.