Poster S4 - Improving the anti-angiogenic and anti-tumorigenic properties of collagen-based peptides through structure activity relationship studies

Introduction: Peptides have the potential to be effective anti-cancer therapeutics. They are significantly smaller than antibodies but can have similar binding affinities, thus they can have higher bioavailability in tumor tissues. Additionally, they can be synthesized relatively inexpensively through solid-phase synthesis and being composed of natural amino acids, can have low immunogenicity and cytotoxicity. Our lab has identified and developed several integrin-binding peptides based on type IV collagen that serve as inhibitors of tumor growth in triple-negative breast cancers (TNBC). By binding to integrins at the surface, this family of peptides disrupts their association with growth factor receptors, leading to disruption of their downstream signaling and inhibiting angiogenesis, lymphangiogenesis, inflammation, and metastasis. However, it remains unclear what the precise molecular mechanism of these interactions are and whether they can be improved. In order to understand this, we designed a library of similar peptides using a hypothesis-driven approach and evaluated their activities in a variety of functional assays.

Materials and

Methods: We designed a library of 40 candidate peptides by making hypothesis-driven 1-2 amino acid substitutions and deletions from the original 20-mer peptide SP2036. We assessed anti-angiogenic and anti-lymphangiogenic activity by analyzing inhibition of adhesion and migration on human umbilical vein endothelial cells (HUVECs) and lymphatic endothelial cells (LECs) in vitro using the Agilent xCelligence Real Time Cell Analyzer (RTCA). We further tested inhibition of endothelial cell function using the in vitro tube formation assay and quantified the results using the ImageJ angiogenesis analyzer toolbox. To investigate effects on inhibition of growth factor signaling and inflammation, we performed Western blots against phospho-Met, the receptor for hepatocyte growth factor (HGF), and IκBα, the inhibitor of tumor necrosis factor alpha (TNFα) signaling. To further examine the anti-inflammatory effects of peptide treatment, we observed changes in NFκB nuclear translocation using immunofluorescence microscopy. Finally, we tested the overall anti-tumor activity of the most promising candidate peptide using the 4T1 syngeneic TNBC mouse model.

Results, Conclusions, and Discussions: After performing the preliminary in vitro assays on all candidate peptides, we identified 12 peptides that, on average, outperformed the original peptide in each assay. Of these, there were two sets of changes that improved activity. One was the substitution or deletion of the terminal phenylalanine residue. We believe that this residue is responsible for aggregation of peptide molecules due to π–π interactions between adjacent peptide molecules, so its elimination resulted in increased solubility of the peptide and thus increased bioavailability to cell surface integrins. The other set of beneficial changes were modifications to an asparagine-rich region of the peptide, specifically replacement of asparagine residues with aspartic acid. We believe that this region is directly involved in integrin-binding, so small changes to this region have proportionally large effects to the overall activity. The best performing peptide, 2-028, exhibited significantly higher activity in every assay compared to the original peptide. Interestingly, it differs by only one amino acid: an arginine substitution on the C-terminus. Preliminary in vivo data show that 2-028 has potent anti-tumor activity in vivo, achieving a 25% tumor volume reduction in a 4T1 mouse model. Overall, this study significantly improved our understanding of this family of peptides by highlighting which regions and specific residues are responsible for their activity. Moreover, it produced a promising new therapeutic candidate for further study.

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