Exploratory proteomic and bioinformatics analysis unveils epitope pairing between IGHV3-64 and K-Ras for polyclonal drug conjugation in colorectal cancer

Aim: Colorectal cancer (CRC) remains the second leading cause of cancer-related mortality worldwide. While antibody-drug conjugates (ADCs) offer targeted therapeutic options, they are often limited by toxicity, immunogenicity, complex pharmacokinetics, and high production costs. Polyclonal antibodies—capable of recognizing multiple epitopes—present a promising, yet underexplored, alternative for targeted drug delivery. The stage-specific presence of secreted, stable immunoglobulins (IGs) in CRC and their potential utility in drug conjugation strategies remain largely uncharacterized. Methods: This study utilized electrospray ionization nano-liquid chromatography tandem mass spectrometry (ESI-nanoLC-MS/MS) proteomic analysis on pre-treatment plasma samples across CRC stages to identify stage-specific IGs. Venn diagram comparisons refined IG candidates, while protein stability was assessed using ProtParam. Molecular docking simulations (via CB-Dock2), epitope mapping (via CABS-dock), and cell-penetrating peptide (CPP) prediction were integrated to explore epitope pairing between IGs and Kirsten rat sarcoma viral oncogene homolog (K-Ras) neoantigen, evaluating their potential for polyclonal drug conjugates (pPDCs). Results: A total of 325 secreted IGs were initially identified, with 46 found to be stage-specific. Protein stability analysis shortlisted 5 IGs—3 for early-stage and 2 for advanced-stage CRC. Molecular docking revealed that IG heavy variable 3-64 (IGHV3-64) exhibited high-affinity binding with Irinotecan [binding free energy (ΔG) = −10.0 kcal/mol] and showed epitope-level pairing with K-Ras at residues 2–17 and 106–114. Additional CPP motif analysis supported the potential of IGHV3-64-derived peptides for intracellular delivery, reinforcing their promise in pPDC development. Conclusions: IGHV3-64 emerges as a strong candidate biomarker for advanced-stage CRC, demonstrating consistent binding affinity to Irinotecan and epitope pairing with K-Ras. Its inherent CPP features further support its potential for targeted, intracellular delivery in pPDCs design. These findings highlight a novel direction in personalized cancer immunotherapy, warranting further in vitro and in vivo validation to confirm clinical utility.