Cetuximab-based PROteolysis targeting chimera for effectual downregulation of NSCLC with varied EGFR mutations

Area of Science:

• Oncology
• Molecular Biology
• Drug Development

Background:

• PROteolysis Targeting Chimeras (PROTACs) are bifunctional molecules that harness the body's natural protein degradation pathways.
• PROTACs have shown promise in degrading oncoproteins, including those previously considered undruggable.
• Epidermal growth factor receptor (EGFR) is a key oncoprotein driving cancer proliferation and progression, particularly in non-small cell lung cancer (NSCLC).

Purpose of the Study:

• To synthesize and characterize an antibody-based PROTAC (ABTAC) designed to degrade EGFR.
• To evaluate the efficacy of the EGFR-degrading ABTAC in non-small cell lung cancer (NSCLC) models.

Main Methods:

• Conjugation of Cetuximab (antibody targeting EGFR) with Pomalidomide (E3 ligase recruiting ligand) using lysine conjugation and copper-free click chemistry (CuAAC).
• Analytical characterization via reverse-phase liquid chromatography and mass spectrometry to confirm conjugation and stoichiometry.
• In vitro efficacy assessment using IC50 determination in EGFR-sensitive and resistant NSCLC cell lines, 3D spheroid assays, circular dichroism, and surface plasmon resonance (SPR).

Main Results:

• Successful synthesis and characterization of an EGFR-degrading ABTAC, with approximately five E3-ligase inhibitor molecules conjugated per antibody.
• Significant reduction in IC50 values (10-30 fold) for the ABTAC compared to Cetuximab alone in both EGFR-sensitive (HCC827) and resistant (H1650) NSCLC cells.
• 3D spheroid assays demonstrated that the ABTAC induced significant apoptosis and inhibited cell proliferation, with minimal structural or binding alterations to the antibody.

Conclusions:

• The developed EGFR-degrading ABTAC represents a promising therapeutic strategy for non-small cell lung cancer.
• ABTACs offer a potent approach to target and degrade key oncoproteins like EGFR, overcoming resistance mechanisms.
• The conjugation strategy and characterization methods provide a robust framework for developing future antibody-based targeted protein degraders.