Rational Design of a Bifunctional and-Gate Ligand to Modulate Cell-Cell Interactions

Much of synthetic biology focuses on the engineering of gene expression systems, using linear relationships between nucleic acid sequences and output proteins, plus simple pathway relationships that can be modeled by one-dimensional interactions. In contrast, Nature is three-dimensional, and evolution uses spatial geometry to construct optimized systems. The molecular geometry of domains in engineered fusion proteins is crucial for designing artificial synthetic-biological systems, such as chimeric antigen receptor T cells and bispecific T cell engagers. Of particular interest are fusion proteins composed of two or more domains that deliver a therapeutic activity to target cells while minimizing undesired action on other cells that may lead to side effects. Implicit in this design is the potential to crosslink cells, whether desired or not. Here, we constructed a bifunctional AND-gate protein, which activates a subset of cells that bear two different receptors and avoids action on other cells. These experiments address how protein structure and geometry can inform the rational design of a synthetic system. Specifically, erythropoietin (EPO) was directed to red blood cell (RBC) precursors by fusion to an anti-glycophorin A (GPA) antibody V region to stimulate RBC production without causing thrombosis. Several different forms of anti-GPA-EPO fusion proteins stimulated RBC and not platelet production in mice. However, they also caused adhesion between RBCs and EPO receptor-bearing cells in vitro and enhanced thrombosis in mice. Based on the protein structural model of the RBC surface, we rationally designed a form of an anti-GPA-EPO fusion protein that does not mediate cell-cell adhesion or enhance thrombosis in mice. We found that the linker element of the fusion protein, the position of the antibody binding epitope, and the target cell surface proteins synergistically contribute to cell-cell interaction mediated by the fusion protein. This work shows how synthetic biology can use nano-scale three-dimensional engineering.