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. Pre-clinical 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 – 2.2×105 antigens/cell) tissues both in vitro and in vivo.

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.

Multicellular biology, as well as new cell based therapies, are dependent on the control of cell-cell interactions. Herein, we detail the use of a multivalent lipidated scaffold for the rapid...

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.

Yeast surface display selections against mammalian cell monolayers have proven effective in isolating proteins with novel binding activity. Recent advances in this technique allow for recovery of clones with even micromolar binding affinity. However, no efficient method has been shown for affinity based selection in this context. This study demonstrates the effectiveness of titratable avidity reduction using dithiothreitol (DTT) to achieve this goal. A series of epidermal growth factor receptor binding fibronectin domains with a range of affinities are used to quantitatively identify the number of ligands per yeast cell that yield the strongest selectivity between strong, moderate, and weak affinities. Notably, reduction of ligand display to 3,000 – 6,000 ligands per yeast cell yields 16-fold selectivity of a 2 nM binder relative to a 17 nM binder. These lessons are applied to affinity maturation of an EpCAM-binding fibronectin population, yielding an enriched pool of ligands with significantly stronger affinity than an analogous pool sorted by standard cellular selection methods. Collectively, this study offers a facile approach for affinity selection of yeast displayed ligands against full length cellular targets and demonstrates the effectiveness of this method by generating EpCAM-binding ligands that are promising for further applications.