通过肽诱导清除实现细胞表面蛋白的靶向蛋白降解

In recent years, several modalities have emerged that enable the clearance of cell surface proteins. Contributing to the current toolbox, we previously reported the development of an antibody-based degrader platform that enables the repurposing of the Wnt-responsive cell surface E3 ubiquitin ligases, RNF43 and ZNRF3, for the degradation of plasma membrane proteins. In particular, through the design of recombinant bispecific antibodies that we termed Proteolysis Targeting Antibodies (PROTABs), we facilitate the tethering of RNF43 or ZNRF3 to the extracellular domains of different receptors, including IGF1R, HER2 and PD-L1. Consequently, this drives efficient “on demand” target degradation both in vitro and in vivo . We also demonstrate the broad applicability of this platform through the identification of additional E3 ubiquitin ligases that can be repurposed as cell surface protein degraders. Stemming from this work, we profiled the cell surface proteome in a panel of isogenic ‘Wnt-low' and ‘Wnt-high' Colorectal Cancer (CRC) cell lines. Focusing on differentially regulated proteins, we demonstrate that the Transferrin Receptor (TfR1) has increased cell surface expression under ‘Wnt-high' conditions, a known hallmark of CRC. Consistently, several studies show that TfR1 is commonly upregulated in cancer, which is often attributed to its role in mediating iron uptake. Indeed, the Cancer Dependency Map (DepMap) Project reveals that the viability of a large number of cancer cell lines is dependent on TfR1, highlighting its therapeutic potential. To explore this, we generated anti-TfR1 PROTABs. In an exogenous ligase expression setting, anti-TfR1 PROTABs, induce target degradation, dependent on RNF43/ZNRF3 levels, concomitant with cell viability defects. Intriguingly, while non-targeting bivalent TfR1 bispecific antibodies drive minimal target clearance, treatment induces viability aberrations comparable to that seen with anti-TfR1 PROTABs. We, therefore postulated that TfR1 cell surface clustering might be sufficient to affect cell fitness. To investigate this, we explored alternative target clustering approaches. Excitingly, this led to the development of a multimeric peptide-based modality that mediates efficient target cell surface clearance and protein degradation. Importantly, through testing a large panel of cancer cell lines, we show a dose-dependent effect on cell viability that spans multiple indications beyond CRC. Together, this highlights the therapeutic potential of leveraging anti-TfR1 multivalent peptides as an alternative targeted protein degradation approach.