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Abstract The rational design and implementation of synthetic mammalian communication systems can unravel fundamental design principles of cell communication circuits and offer a framework for engineering of designer cell consortia with potential applications in cell therapeutics. Here, we develop the foundations of an orthogonal, and scalable mammalian synthetic communication platform that exploits the programmability of synthetic receptors and selective affinity and tunability of diffusing coiled-coil peptides. Leveraging the ability of coiled-coils to exclusively bind to a cognate receptor, we demonstrate orthogonal receptor activation and Boolean logic operations at the receptor level. We show intercellular communication based on synthetic receptors and secreted multidomain coiled-coils and demonstrate a three-cell population system that can perform AND gate logic. Finally, we show CC-GEMS receptor-dependent therapeutic protein expression. Our work provides a modular and scalable framework for the engineering of complex cell consortia, with the potential to expand the aptitude of cell therapeutics and diagnostics.

Targeted molecular radiation therapy is a promising emerging treatment modality in oncology, and peptide synthesis may shorten the time to reach the clinical stage. In this study, we have explored Chemo-Enzymatic Peptide Synthesis, or CEPS, as a new means of producing a therapeutic HER2 targeted Affibody® molecule, comprising a C-terminal albumin binding domain (ABD) for half-life extension and a total length of 108 amino acids. In addition, a DOTA moiety could be incorporated at N-terminus directly during the synthesis step and subsequently utilized for site-specific radiolabeling with the therapeutic radionuclide 177Lu. Retained thermodynamic stability as well as retained binding to both HER2 and albumin was verified. Furthermore, HER2 binding specificity of the radiolabeled Affibody molecule was confirmed by an in vitro saturation assay showing a significantly higher cell-bound activity of SKOV-3 (high HER2 expression) compared with BxPC3 (low HER2 expression), both in the presence and absence of HSA. In vivo evaluation in mice bearing HER2 expressing xenografts also showed specific tumor targeting as well as extended time in circulation and reduced kidney uptake compared with a HER2 targeted Affibody molecule without the ABD moiety. To conclude, we have demonstrated that CEPS can be used for production of Affibody-fusion molecules with retained in vitro and in vivo functionality.

Hepatitis C virus remains a global threat, despite the availability of highly effective direct-acting antiviral (DAA) drugs. With thousands of new infections annually, the need for a prophylactic vaccine is evident. However, traditional vaccine design has been unable to provide effective vaccines so far. Therefore, alternative strategies need to be investigated. In this work, a chemistry-based approach is explored towards fully synthetic peptide-based vaccines using epitope mimicry, by focusing on highly effective and conserved amino acid sequences in HCV, which, upon antibody binding, inhibit its bio-activity. Continuous and discontinuous epitope mimics were both chemically synthesized based on the HCV-E2 glycoprotein while using designed fully synthetic cyclic peptides. These cyclic epitope mimics were assembled on an orthogonally protected scaffold. The scaffolded epitope mimics have been assessed in immunization experiments to investigate the elicitation of anti-HCV-E2 glycoprotein antibodies. The neutralizing potential of the elicited antibodies was investigated, representing a first step in employing chemically synthesized epitope mimics as a novel strategy towards vaccine design.