RESUMO
Recently, phage display technology has been announced as the recipient of Nobel Prize in Chemistry 2018. Phage display technique allows high affinity target-binding peptides to be selected from a complex mixture pool of billions of displayed peptides on phage in a combinatorial library and could be further enriched through the biopanning process; proving to be a powerful technique in the screening of peptide with high affinity and selectivity. In this review, we will first discuss the modifications in phage display techniques used to isolate various cancer-specific ligands by in situ, in vitro, in vivo, and ex vivo screening methods. We will then discuss prominent examples of solid tumor targeting-peptides; namely peptide targeting tumor vasculature, tumor microenvironment (TME) and over-expressed receptors on cancer cells identified through phage display screening. We will also discuss the current challenges and future outlook for targeting peptide-based therapeutics in the clinics.
RESUMO
Background and purpose: Tumor vasculature is increasingly recognized as a target for cancer therapy. In recent years, a fusion protein consisting of the extra cellular domain of tissue factor (truncated tissue factor, tTF) was fused to the antibody selectively binding to tumor vasculature. Antibody-truncated tissue factor(Ab-tTF) fusion protein specifically induced thrombotic occlusion of tumor vessels resulting in tumor growth retardation or regression in some types of solid tumors. However, there were still some disadvantages in the above approach. We constructed and expressed that the (RGD)_3-tTF fusion protein with peptides arginine-glycine-aspartic acid (GRGDSP, abbr. RGD)as the carrier of tTF to explore whether it bad the capability of targeting to tumor vasculature in the colonic carcinoma model. Methods: The (RGD)_3-tTF fusion gene consisting of the tTF was fused to three series-wound peptides RGD. The (RGD)_3-tTF construct was expressed in Escherichia coil BL21(DE_3). The fusion protein was purified through Nickel affinity chromatography column. The activity of inducing blood coagulation was detected by clotting assay and coagulation factor X (FX) activation assay. The specific binding to integrins α_vβ_3 was analyzed by indirect enzyme linked immunosorbent assay (ELISA). All these were compared with the fusion protein RGD-tTE Colonic nude mice models were randomly divided into 3 groups (1 nude mice per group).Tumors were stained by the (RGD)_3-tTE RGD-tTF fusion protein and tTF which were labeled with Fluorescein Isothiocyanate(FITC). The location of the (RGD)_3-tTF fusion protein in the colonic carcinoma bearing nude mice tissue was analyzed by immunofluorescence assay. Results: The (RGD)_3-tTF fusion protein retained tissue factor thrombogenic activities. With increasing concentration, the clotting time was shortened correspondingly. Under the conditions of Ca~(2+), the clotting time was 9.96±0.56 min when the concentration was 6 μmol/L(P<0.01). The (RGD)_3-tTF fusion protein could activise F X above 6 μmol/L concentration, which was similar to RGD-tTF fusion (F=0.147, P>0.05). The ability of the (RGD)_3-tTF fusion protein binding specifically to integrins α_vβ_3 was stronger than that of the RGD-tTF fusion protein in the same concentration (F=164.81, P<0.01), which was apparently indicated by the A_(405nm) 1.25 and 0.95 when the concentration was 0.24 μmol/L. Immunofluorescence assay showed that the (RGD)_3-tTF fusion protein was assembling in the tumor vasculature of the colonic carcinoma bearing nude mice. Conclusion: The (RGD)_3-tTF fusion protein which retained tissue factor thrombogenic activities could bind specifically and efficiently to tumor vasculature in the colonic carcinoma bearing mice through binding to the tumor marker integrins α_vβ_3. It might be a promising foundation for further studies on the colon cancer molecular targeted therapy with tTF as an effective factor.