Targeted Radiotherapy With Tumor Vascular Homing Peptide GEBP11Evaluated By Multimodality Imaging For Gastric Cancer

【Background】Angiogenesis, the growth of new blood vessels, plays a critical role in theprogression of tumor growth and metastasis, making it an attractive target forboth cancer imaging and therapy. Thus, for over last couple of decades, there hasbeen a robust activity aimed towards the discovery of angiogenesis inhibitors.Most of the endogenous anti-angiogenic proteins are too large and complex sothey have difficulty penetrating tissues and are also too costly to be produced inlarge quantities necessary for mainstream therapies. As a result, significant effortis being placed into developing small peptide. Peptide has attached attentionbecause of its good penetration of tissues, metabolic stability and especially thetolerance towards bulky modifcations. Pegylation, encapsulation in liposomesand conjugation with radio-labeled molecules are common modifications to beconsidered. Targeted delivery of radionuclides by peptide can specifically deliverradiation to tumors, while sparing the normal organs and tissues. Compared with conventional therapy, peptide-based radioimmunotherapy is a safer and less toxictherapy for diseases associated with abnormal angiogenesis. To date, numerousanti-angiogenic peptides have been investigated and some of them are tested inclinical trials.GEBP11is a nine amino acid vascular homing peptide, which was screenedand identified by our research group using phage display technology.¹³¹I-GEBP11demonstrated signifcant anti-tumor toxicity and low side effects.However, peptide GEBP11suffered from short serum half-life, low bindingaffinity and rapid tumor wash out, which makes it not to be an ideal therapeuticagent. In this investigation, we applied the polyvalency principle to develop anovel PEGylated trimeric GEBP11peptide to improve the peptide-receptorbinding affinity and pharmacokinetics. By labeled with radionuclides,2PEG-（GEBP11）₃acquired both imaging and targeting therapeutic abilities.¹³¹I-2PEG-（GEBP11）₃could be a promising candidate for peptide-based targetingtherapy for gastric cancer.【Objectives】The aim of this study is to investigate the targeting characteristics of¹³¹I-2PEG-（GEBP11）₃as a potential agent for diagnosis and receptor-mediatedtargeted radiotherapy of gastric cancer.【Methods】1.2PEG-（GEBP11）₃was synthesized by amide linkage between carboxy ofbifid PEG and GEBP11, which was identified using mass spectrum.Immunofuorescence staining was conducted to confirm the binding specificityofpeptide GEBP11with Co-HUVECs. GEBP11and2PEG-（GEBP11）₃werelabeled with¹³¹I using Iodogen tube method, then the labeled GEBP11peptideswere validate for radiochemical yield, specific activity and metabolic stability. The binding affnity and specifcity of GEBP11peptides were assessed via cellcompetitive binding assay and autoradiography.2. SPECT, Cerenkov imaging and biodistribution studies were performed onSGC7901-Luc gastric carcinoma-bearing mice to compare the targeting ability ofradiolabeled GEBP11peptide to tumor tissues.3. MTT and cell apoptosis on Co-HUVECs and anti-tumor activity in nudemice bearing SGC7901-Luc tumor xenografts of human gastric cancer wereconducted to evaluated the effect of¹³¹I-GEBP11and¹³¹I-2PEG-（GEBP11）₃onCo-HUVECs and xenografts. H&E and Immunohistochemistry staining andTUNEL assay were conducted to assess the cell apoptosis and vascular volumechange in tumors after treatment.【Results】1. Mass spectrum confirmed the identity of2PEG-（GEBP11）₃.Immunofuorescence staining showed Co-HUVECs had stronger FITC-GEBP11staining than HUVECs, whereas HUVECs and SGC7901had no or only weakbackground staining. By directly labeling GEBP11peptides with Na¹³¹I, weacquired high radiolabeling effciency93%-98%and specifc activity beyond10Ci/mmol. For in vitro stability test, the radiolabeling efficiency of radiolabeledGEBP11peptides was above83%when they were incubated with differentsolutes including fresh human serum,0.9%saline, EDTA and cysteine at24h. Allof these indicted the satisfactory stability of radiolabeling GEBP11peptides,which could be used as radiotracers for cancer imaging and therapy. The cellcompetitive binding assay and autoradiography demonstrated that2PEG-（GEBP11）₃had higher binding affinity to Co-HUVECs than GEBP11, andthe labeling process had not obvious effect on the receptor-binding affinity.2. The localization of radiolabeled GEBP11in human SGC7901-Luc tumor-bearing nude mice was performed by Cerenkov imaging and multipletime-point static SPECT scans. The tumor could be clearly visualized at4h andthe mouse injected with¹³¹I-2PEG-（GEBP11）₃had the highesttumor-to-contralateral background contrast. Both SPECT and Cerenkov imagingrevealed that high activity was mainly observed from the tumor sample of bothtracers, and the higher signal was also observed from the tumor of¹³¹I-2PEG-（GEBP11）₃. The CLI intensity was well correlated with radioactivitycalculated by nuclear imaging studies. Biodistribution studies showed higheractivity accumulation of¹³¹I-2PEG-（GEBP11）₃in the tumor than other organssuch as muscle, gastric and intestine. The tumor uptake was2.22±0.13%ID/g at4h after injection and slowly decreased to1.61±0.05%ID/g at24h. Theradiotracer was eliminated through kidney and liver pathway with the tumorslower than non-target organs leading to high tumor-to-nontumor ratios for thetrimeric GEBP11peptide.3. MTT and cell apoptosis showed¹³¹I-2PEG-（GEBP11）₃exhibited highercell apoptosis and cytotoxicity against Co-HUVECs than¹³¹I-GEBP11, whileNa¹³¹I had minimal effect on the cell viability. Tumors in mice treated with¹³¹I-2PEG-（GEBP11）₃showed a slower growth, and tumors were signifcantlysmaller than those in¹³¹I-GEBP11treating group.¹³¹I-URP grew somewhatslower than those in PBS control group. Kaplan-Meier curves showed that micebearing SGC7901-Luc tumor treated with radiolabeled GEBP11peptides hadsignificant effect on overall survival rate. Mice treated with¹³¹I-GEBP11prolonged survival from35to51days, whereas¹³¹I-2PEG-（GEBP11）₃extendedsurvival to75days. Immunohistochemical analysis with TUNEL assay and H&Estaining revealed that¹³¹I-2PEG-（GEBP11）₃induced higher levels of cellapoptosis and inhibited tumor angiogenesis than¹³¹I-GEBP11, while¹³¹I-URP did not induce change of tumor cell and tumor blood density. Moreover, there is noobvious liver and kidney toxicity in every treated group.【Conclusion】1. By directly labeling GEBP11peptides with Na¹³¹I, we acquired highradiolabeling effciency and specifc activity and satisfactory metabolic stability.¹³¹I-2PEG-（GEBP11）₃had higher binding affinity and specificity to Co-HUVECsthan¹³¹I-GEBP11.2. Compared with¹³¹I-GEBP11,¹³¹I-2PEG-（GEBP11）₃had higher tumoruptake, significant tumor growth delay and lower side effects, which indicated¹³¹I-2PEG-（GEBP11）₃could be a promising candidate for peptide-based targetingtherapy for gastric cancer.