Screening and Identification of a Phage Display Derived Peptide That Specifically Binds to the CD44 Protein Region Encoded by Variable Exons

Abstract

CD44, especially the isoforms with variable exons (CD44v), is a promising biomarker for the detection of cancer. To develop a CD44v-specific probe, we screened a 7-mer phage peptide library against the CD44v3-v10 protein using an improved subtractive method. The consensus sequences with the highest frequency (designated CV-1) emerged after four rounds of panning. The binding affinity and specificity of the CV-1 phage and the synthesized peptide for the region of CD44 encoded by the variable exons were confirmed using enzyme-linked immunosorbent assay and competitive inhibition assays. Furthermore, the binding of the CV-1 probe to gastric cancer cells and tissues was validated using immunofluorescence and immunohistochemistry assays. CV-1 sensitively and specifically bound to CD44v on cancer cells and tissues. Thus, CV-1 has the potential to serve as a promising probe for cancer molecular imaging and target therapy.

Screening and identification of a CD44v6 specific peptide using improved phage display for gastric cancer targeting

“…We utilized the recombinant protein with a eukaryotic expression host, human HEK-293 cells for the advanced structure of CD44 on cell surface. Overexpression of CD44v3-v10 was validated using Western blot in our former work (27). For every round of panning, recovery rate (output phages/input phages) increased from 3.05×10 -6 to 6.70×10 -3 (Figure 1A), indicating enrichment of positive clones.…”

“…To the contrary, region of CD44 encoded by variable exons maintains high cancer specificity. To improve diagnosis accuracy, we also screened a peptide targeting v3-v10 exons encoded region with an improved biopanning strategy (27). Considering most reported researches suggested the high sensitivity and specificity of v6 exon for GC, we aimed to identify peptide targeting this region.…”

“…Our team used to screen and characterize several peptides targeting cancer. Peptide ligands were screened using phage display biopanned on cancer tissues, cancer cells and recombinant protein (25)(26)(27)(28). Even though target molecules expressed on cells can maximize their original structure, receptors of screened peptides were still unknown based on the traditional screen protocol.…”

“…Even though target molecules expressed on cells can maximize their original structure, receptors of screened peptides were still unknown based on the traditional screen protocol. Using recombinant protein as target, we obtained two CD44 specific peptides, one probed pan-CD44, one aimed CD44v3-v10 (27,28). Then we focus on CD44v6, the optimal CD44 variant as a GC biomarker.…”

Application of Phage-Displayed Peptides in Tumor Imaging Diagnosis and Targeting Therapy

“…CD44, a cancer-associated membrane glycoprotein involved in cell adhesion, tumor progression and drug-resistance, is a potential diagnostic target for cancer detection. A peptide CV-1 (THENWPA) specifically bound to gastric cancer cell surface marker CD44 was selected, identified and demonstrated as a promising probe for early molecular imaging and monitoring CD44v-positive stomach tumors (Zhang et al 2016 ).…”