The Identification Of Vasculature Homing Peptide GX1 Specifically To Tumor Endothelial Cells In Vitro And In Vivo

Angiogenesis plays a pivotal role in tumor growth and metastasis. Now angiogenesis-targeted therapy has become a promising new approach for cancer therapy. To deliver drugs specificlly targeting to tumor vasculature endothelial cells remains to be a key issue of antiangiogenesis. It is evident that the vasculature is highly specialized in individual tissues. In particular, tumor vasculature undergoing angiogenesis express specific endothelial surface markers (''vascular zip codes'') that are absent or barely detectable in mature vessels, and those markers might enable direct therapeutic targeting. Therefore, finding these specific"vascular zip codes"to improve the targeting ability of drug plays a critical role in antiangiogenesis. In vivo phage display technology isolates peptides that bind selectively to target receptors expressed only on certain blood vessels in patient or mouse model. So far, many angiogenic cell surface receptors/ homing motifs have been found including avβ3 integrin/ RGD-4C, CD13/NGR, APA/CPRECES, MMP-2/CTTHWGFTLC and HSP90/ CVPELGHEC.Using in vivo phage display technology in immunosuppressed murine tumorigenesis models with human gastric cancer xenograft by subrenal capsule assay (SRCA), we obtained a cyclic 7-mer peptide motif CGNSNPKSC that homing specifically to vasculature endothelial cells of human gastric cancer. Then we performed histological identification in vitro. Histochemistry in mouse and human tissue showed that this phage peptide only bound to the endothelial cells of human gastric cancer and colon cancer. The receptor of GX1 was overexpressed in tumor vascular endothelial cells and the expression level correlated with differentiation of gastric cancer. The expression of GX1 receptor in poor-differentiated gastric cancer tissues was significantly higher than that in well-differentiated tissues and moderate-differentiated tissues. This peptide was also observed only specific binding to HUVEC not to SGC-7901, Eca-109, LoVo and Hep-G2 by ELISA.Histological identification in vitro showed that this phage only bound to the endothelial cells of human gastric cancer and colon cancer. To further valuate the value of GX1 in antiangiogenesis therapy, peptide CGNSNPKSC displayed on the surface of GX1 phage need to be further identified. It depended on three elements for a new phage displayed peptide to be valuated for the pharmaproject of antiangiogenesis therapy, those were the binding specificity of GX1 to endothelial cells, the binding affinity of GX1 to endothelial cells, and the specific targeting ability of GX1 to tumor tissue in vivo. To evaluate the ability of GX1 as a targeting peptide to be used for the discovery of antiangiogenesis drugs, this study was performed.【Objectives】To validate the binding specificity and affinity of GX1 to endothelial cells and the targeting ability of GX1 to vasculature of human gastric cancer in vivo.【Methods】 1. GX1 was labeled with 99TcmO4- using direct labeling method, and the labeled peptide was validated for radiochemical yield, specific actibity, and in vitro stability. The bioactivity of 99Tcm-GX1 was validated by cell receptor autoradiography in cultured HUVEC cells.2. To analyze quantitively the binding specificity and affinity of GX1 to HUVEC, the receptor intensity on HUVEC, and the difference of affinity of GX1 binding to Co-HUVEC and HUVEC, receptor autoradiography, receptor binding assay and competitive inhibition assay, and phage binding assay were performed in vitro.3. The radioactivity of all orgens of nude mice injected with 99Tcm-GX1 were determined and the the radioactivity of all orgens per g (%ID/g)were calculated to analyze the specific distribution of 99Tcm-GX1.4. Gamma camera images of nude mice bearing tumor xenografts of human gastric carcinoma injected with 99Tcm-GX1 was obtained 0.5h~24h after injected to identify the targeting ability of labeled peptide to tumor tissues. 99Tcm-Oxytocin was used as a control.【Results】1. By direct labeling GX1 with 99TcmO4-, we acquired high radiolabeling efficiency at 90-98%, as well as high specific activity beyond 200Ci / mmol. In vitro stability test, the radiolabeling efficiency was above 95% in stock solution, 0.9% saline, cysteine and EDTA, and 89% in fresh human serum at 24h. There was no obvious reduction in radiolabeling efficiency in all solutes, which indicated that 99Tcm -GX1 has fine in vitro stability. Receptor autoradiography showed that there were obvious silver particles on HUVEC, the intensity of which beyond 5 folds of background. Compared with 99TcmO4-, uptake of 99Tcm-GX1 by HUVEC was significantly increased, which indicated that 99Tcm-GX1 has fine biological activity.2. Microautoradiography, receptor binding assay and competitive inhibition assay, phage binding assay in vitro showed that GX1 peptide or phage could bind specifically to HUVEC, not to SGC7901 cells, LoVo cells, and GES cells., which confirmed the specifically binding ability of GX1 to vascular endothelial cells. The binding of GX1 by Co-HUVEC was higher than that of HUVEC. The binding constant of 99Tcm -GX1 was calculated by Scatchard analysis. The Kd value of Co-HUVEC and HUVEC were determined to be 3062pM and 3831pM. The number of binding sites for the labeled peptide (receptor density) was estimated as 1.17×104 per Co-HUVEC and 0.88×104 per HUVEC respectively.3. Biodistribution data of 99Tcm-GX1 in nude mice bearing tumour xenografts of human gastric carcinoma showed that the radiotracer exhibited a quick decrease in radioactivity over time in blood and primary organs. Highest activity concentration is observed in kidneys. Tumor accumulation in the tumour xenografts decreased from 1.19±0.08% ID/g 30 min p. i. to 0.74±0.02% ID/g 24h p. i, 11 times higher than that of muscle. The resulting tumor/non-tumor (muscle, blood, liver, kidney) ratios steadily increased over time, which showed that 99Tcm-GX1 had the ability of specifically targeting to tumor tissues in vivo.4. Gamma camera images of nude mice bearing tumor xenografts of human gastric carcinoma injected with 99Tcm-GX1 showed the tumor on the right flank could be visualized as early as 8h and the activity was higher than that of heart until 24h. The most clearly visualized imaging appeared at 18h. Compared to 99Tcm-GX1, the activity of tumor in nude mice injected 99Tcm-OXT was constantly lower than that of heart since 8h. T/H ratios of tumor-bearing nude mice injected 99Tcm-GX1 were beyond 1.0, which were higher than that of mice injected 99Tcm–OXT (<1.0). It also could be seen that there were obvious 99Tcm-GX1 uptake in the tumor in nude mice bearing tumor xenografts of human colon cancer. 【Conclusion】1. We successfully labeled GX1 with 99TcmO4- at high labeling efficiency(>90%),favorable specific activity, fine in vitro stability and perfect biological activity, which confirmed the qualification of 99Tcm -GX1 for ECT imaging.2. GX1 exhibited excellent receptor-binding affinity and specificity to endothelial cells with a Kd of 3831 pM and binding sites of 0.88×104 / HUVEC.3. GX1 showed better binding affinity on the co-culture model of tumor vasculature endothelial cells (Co-HUVEC) than HUVEC.4. GX1 showed the ability of specific targeting of gastric cancer angiogenesis in tumor-bearing nude mice.