Poster S3 - Unlock the potential of leucine rich repeats as peptide drugs to address the resistance to EGFR targeted-therapy

Introduction: Targeted therapy employing EGFR-targeted tyrosine kinase (TK) inhibitor chemotherapy has demonstrated significant efficacy in treating non-small cell lung cancer (NSCLC) patients harboring mutated EGFR within cancer cells. However, despite initial efficacy, nearly all patients eventually develop resistance to these small molecular drugs within 9-14 months. Mechanisms of resistance encompass new EGFR mutations and alternative signaling pathways through other TKs such as MET, IGFR1, AXL, and HER2. To overcome this resistance, second- and third-generation small molecular TK inhibitors have been developed, yet these new drugs encounter issues such as high toxicity and emergence of new therapy resistance via similar mechanisms. Thus, there is an urgent need for developing novel TK inhibitor drugs to confront these challenges.

Small decorin proteins serve as endogenous pan-TK inhibitors, impeding the activity of EGFR and most TKs implicated in resistance to small molecular TK inhibitors. The leucine-rich repeats (LRR) peptide motifs within the core of decorin bind to and counteract various TKs, thereby hindering cancer cell proliferation. These LRRs exhibit considerable promise as peptide drugs for addressing resistance to EGFR-targeted TK inhibitors, offering superiority over small molecular drugs due to their reduced likelihood of therapy resistance stemming from a more intricate binding pattern to TKs. Despite their potential, the development of LRR-based TK inhibitors remains largely unexplored. Challenges include elucidating which LRR binds to which TK and devising an optimal delivery system for these hydrophobic peptides to overcome hurdles in their in vivo application, such as challenging delivery, limited tissue penetration, and short half-life.

Materials and

Methods: We utilized a novel polypeptide nanoparticle platform for the precise delivery of decorin-derived LRRs, facilitating their molecular analysis to discern individual LRRs' binding partners and enabling their effective in vivo application to overcome hurdles associated with short hydrophobic peptide drugs. Our nanoparticle is a 40 nm diameter micelle self-assembled with elastin-like polypeptides (ELP) at physiological temperature. ELP, a thermo-responsive polypeptide, undergoes a transition from soluble to insoluble state above a specific transition temperature dictated by its sequence. The recombinant fusion protein of ELP with decorin-derived LRR peptides was synthetically produced in E. coli, with purification achieved chromatography-free by inducing temperature-induced phase separation of ELP-LRR, separating it from impurities. Following purification, these ELP-LRR recombinant proteins self-assemble into 40 nm diameter spherical micelles at temperatures above 20 degrees Celsius, each nanoparticle comprising 40 ELP units, irrespective of the attached peptide sequence.

To ensure the optimal presentation of hydrophobic LRRs on the nanoparticle surface, we optimized our ELP nanoparticle with a hydrophilic peptide linker. We generated a comprehensive library of ELP-LRR micelles encompassing all decorin-derived LRRs. Subsequently, employing cancer cells overexpressing specific TK receptors, we identified LRRs exhibiting binding and antagonistic activity towards these TKs. Utilizing surface plasmon resonance, we quantified the binding kinetics and affinity of LRR-TK interactions, including all the LRRs against TKs implicated in resistance to EGFR-targeted TK inhibitors. Further, we prepared mixed micelles incorporating these bioactive ELP-LRRs and evaluated their antitumor efficacy in cancer cells resistant to small molecule TK inhibitors.

Results, Conclusions, and Discussions: We effectively pinpointed decorin-derived leucine-rich repeats (LRRs) responsible for targeting and counteracting EGFR, IGFR1, AXL, and HER2, respectively. These LRRs individually impede the proliferation of cancer cells resistant to TK inhibitors, while their synergistic combination manifests a heightened antitumor effect. Notably, ELP-LRR nanoparticles outperform free LRR peptides, demonstrating enhanced TK antagonism and prolonged circulation in vivo, leveraging the multivalency effects and elevated molecular weight of nanoparticles, respectively. Combining experimental and computational methodologies, we further delineated the specific fragments and key amino acids within LRRs crucial for TK antagonist activity. In sum, our efforts culminate in the successful development of innovative peptide therapeutics to combat resistance to EGFR-targeted small molecular TK inhibitors in cancer cells. Their delivery via ELP nanoparticles streamlines their discovery process and augments their in vivo efficacy.

In contrast to multi-target and multi-functional decorin proteins, LRR peptide drugs exhibit precise targeting and tailored bioactivity, mitigating unforeseen side effects associated with interactions with other receptors. Additionally, compared to small molecular TK inhibitors, peptide drugs engage in a more intricate interaction pattern with TKs, thus reducing the likelihood of therapy resistance emergence.

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