A novel targeted protein degradation platform based on internalization-enhancing peptide for lysosomal degradation of cell-surface proteins

Targeted protein degradation (TPD) is an emerging therapeutic approach that enables the selective elimination of undesirable proteins with high specificity. Although TPD holds great promise for targeting traditionally undruggable proteins and overcoming resistance often seen with inhibition-based therapies, several challenges remain, including suboptimal pharmacokinetics, limited internalization, and the dependency on E3 ligases or lysosome-targeting receptors. Here, we present a novel TPD platform based on internalization-enhancing peptide (IEP). IEP binds tightly yet non-covalently to antibodies (Abs) without altering their structure and enhances Ab internalization. We hypothesized that IEP could promote lysosomal degradation of cell-surface proteins by facilitating internalization. To demonstrate its potential, Atezolizumab (ATZ), an FDA-approved monoclonal antibody targeting PD-L1, was used as a model. Since ATZ alone does not strongly induce PD-L1 internalization, it provides an ideal system to test IEP-mediated degradation. Indeed, PD-L1 protein levels were markedly reduced in MDA-MB-231 (TNBC) cells treated with ATZ-IEP (a non-covalent ATZ/IEP complex), whereas no reduction was observed with ATZ or IEP alone. The decrease in PD-L1 was blocked by BafA1, a lysosomal proteolysis inhibitor, but not by MG132, a proteasome inhibitor-indicating that ATZ-IEP mediates lysosomal, rather than proteasomal, degradation. To assess the generality of this effect, Durvalumab (DUR), another FDA-approved anti-PD-L1 antibody, and HCC827 (NSCLC) cells were tested. PD-L1 levels were reduced with DUR-IEP (a non-covalent DUR/IEP complex) to a similar extent as with ATZ-IEP, and ATZ-IEP also decreased PD-L1 expression in HCC827 cells, comparable to the effect observed in MDA-MB-231 cells. These results indicate that IEP functions broadly across different antibodies and tumor types. Intriguingly, using transgenic CHO cell lines with varying PD-L1 expression, we found that the extent of PD-L1 reduction increased as baseline PD-L1 expression decreased (~70 % in PD-L1 Low vs. ~20 % in PD-L1 High ). This finding suggests that IEP may help overcome anti-PD-L1 resistance in tumors with low PD-L1 expression. In summary, ATZ-IEP mediates lysosomal degradation of PD-L1 and demonstrates broad applicability across antibodies and tumor types. Ongoing in vivo studies aim to confirm the efficacy of ATZ-IEP, further supporting the versatility of IEP as a novel TPD platform.