Engineering next-generation fusion proteins to dress the cell membrane with functionally enhanced receptors

Projektinformationen

DRESSCODE

ID Finanzhilfevereinbarung: 101116941

DOI

10.3030/101116941

EK-Unterschriftsdatum 18 Oktober 2023

Startdatum 1 Januar 2024

Enddatum 31 Dezember 2028

Finanziert unter

• European Research Council (ERC)

Gesamtkosten

€ 1 497 778,00

EU-Beitrag

€ 1 497 778,00

Koordiniert durch

UNIVERSITAETSKLINIKUM FREIBURG
Germany

Periodic Reporting for period 1 - DRESSCODE (Engineering next-generation fusion proteins to dress the cell membrane with functionally enhanced receptors)

Berichtszeitraum: 2024-01-01 bis 2026-06-30

Zusammenfassung vom Kontext und den Gesamtzielen des Projekts

Most protein-based drugs currently in clinical use are based on soluble proteins that are administered to patients as bulk formulations or via smart drug-delivery systems with controlled release kinetics. To date, these delivery systems do not enable the use of transmembrane proteins as drugs, particularly synthetic signalling receptors. Nevertheless, recent advances in gene therapy and cell-based therapy have demonstrated that such synthetic receptors could be highly promising therapeutic agents. For example, chimeric antigen receptor (CAR) T-cell therapy against cancer has revolutionised cancer treatment. However, both gene and cell-based therapies rely on genetic engineering of cells, which remains expensive and is not widely accessible.

The challenge of DRESSCODE is to push the boundaries of protein engineering, by developing innovative protein-based drug-delivery systems capable of delivering functional transmembrane receptors to cells without altering the cell genome. Such delivery systems would offer facilitated manufacturing, improved cost-effectiveness, and off-the-shelf therapeutic products for clinical use. The overall objective of the project is to provide proof-of-concept for the successful delivery of (1) a functional CAR receptor to T cells for cancer immunotherapy and (2) a functional VEGFR-2 receptor to endothelial cells to enhance angiogenesis in wound healing—two high-impact medical applications. Using a step-by-step approach involving multivalency-based proteins, membrane-insertion peptides, cell-penetrating peptides, delivery of signalling domains and protein–protein fusions, we aim to reconstruct the extracellular, transmembrane, and intracellular domains of single-pass transmembrane receptors.

The success of DRESSCODE will establish a novel category of therapeutic proteins—recombinant transmembrane receptors—as drugs for clinical application.

Arbeit, die ab Beginn des Projekts bis zum Ende des durch den Bericht erfassten Berichtszeitraums geleistet wurde, und die wichtigsten bis dahin erzielten Ergebnisse

The project is divided into 3 main goals, respectively engineering the delivery of the 1) extracellular, 2) intracellular/signaling and 3) transmembrane part of the synthetic receptor. In each aim, novel assays and methodologies have to be setup, before validation in vitro and in vivo in mouse model of cancer and wound healing. Currently, all 3 aims of DRESSCODE have been initiated: assays and methodologies for protein delivery have been attempted and are currently under optimization. In vitro proof-of-concept on the CAR-T receptor is expected in the upcoming year. In vivo experimentation will take place in year 4-5 of the project.

Main achievements until now are:
- The development of a highly-sensitive reporter cell lines to assess the efficacy of fusion proteins containing cell-penetrating domain in the cytosol. We showed that we could successfully designed fusion proteins to penetrate the cell-membrane, with at least 2 different cell-penetrating domains. We are currently quantifying the efficacy and designing fusion to active signalling domains, instead of model proteins.
- The validation of membrane retention of membrane insertion-fused proteins. We have shown that membrane insertion fusion proteins insert in the membrane of multiple cancerous and non-cancerous cell lines at pH 5.5 with good viability. We are currently assessing what protein tags could be translocated into the cell cytoplasm.
- The production of an engineered multivalent scaffolds for enhanced receptor avidity. Production yields are relatively low, yet the purification of the scaffold remains the most challenging process. We have now designed multiple purification strategies now and are currently optimising them. We showed that the scaffold, despite low purity, can be successfully used for further functionalisation. In parallel, we have started the exploration of alternative multi-avidity scaffold.

Note: an extension of the project timeline will be requested to fulfil the research program despite some initial delays.

Fortschritte, die über den aktuellen Stand der Technik hinausgehen und voraussichtliche potenzielle Auswirkungen (einschließlich der bis dato erzielten sozioökonomischen Auswirkungen und weiter gefassten gesellschaftlichen Auswirkungen des Projekts)

We have been so far focusing our efforts on the establishment of essential in vitro models to demonstrate the proper reconstruction and functionality of protein-based delivered transmembrane receptors, as well as on the characterization and optimization of the different technologies needed to re-assemble synthetic receptors. Further research is needed to provide results beyond of the current state-of-the-art, yet we expect to achieve them in the upcoming years. IPR will be filled before publication of such results.