NGR peptide ligands for targeting CD13/APN identified through peptide array screening resemble fibronectin sequences

Area of Science:

• Biochemistry
• Molecular Biology
• Oncology

Background:

• Peptides with the Asn-Gly-Arg (NGR) motif target CD13 isoforms on tumor blood vessels, aiding tumor-specific drug delivery.
• The flanking residues of the NGR sequence significantly influence its binding affinity and specificity to the CD13 receptor.

Purpose of the Study:

• To screen a library of NGR peptides with varied flanking residues using a peptide array-whole cell binding assay.
• To identify novel NGR peptides with improved binding affinity and specificity for CD13 on tumor vasculature.

Main Methods:

• Synthesis of a 45-peptide NGR library on a cellulose membrane.
• Screening the peptide library against CD13-positive (HUVEC, HT-1080) and CD13-negative (MDA-MB-435, MDA-MB-231) cell lines.
• Validation of high-affinity binders using flow cytometry and fluorescence microscopy for cellular uptake and specificity.

Main Results:

• Identification of five cyclic and acyclic NGR peptides with up to 5-fold higher binding to CD13-positive cells compared to the lead peptide.
• Negligible binding of the identified peptides to CD13-negative cell lines, indicating high specificity.
• Demonstrated improved in vitro cellular uptake and specificity of the selected NGR peptides.
• Discovered that the most effective NGR peptides share sequences with human fibronectin.

Conclusions:

• Novel NGR peptides with enhanced CD13 binding affinity and specificity have been identified.
• These peptides exhibit promising characteristics for targeted tumor vasculature drug delivery, imaging, and therapeutic applications.
• The identified peptides, resembling human fibronectin sequences, offer a potential for developing cancer therapeutics with reduced systemic toxicity.