Development Of Series Molecular Imaging Probes For Gastric Cancer Diagnosis And Imaging Study

?Background? Gastric cancer is one of the malignancies that severely threatens human health.Delayed diagnosis attributed to its high mortality rate,thus early detection is the key to improve survival.However,current clinical methods are ineffective in early diagnosis because of lacking sensitivity and specificity.MG7 Ab is a gastric cancer specific monoclonal antibody.It has great potential to be a gastric cancer diagnostic marker with high binding specificity and affinity to gastric cancer cells.Molecular imaging,being a new tool in basic research,could detect molecule changes in a continuous and kinetic fashion and has promising further in clinical use.Multi-modality imaging could complement each modality and acquire more precise and detailed information,providing more solid evidence in disease diagnosis.?Aim? To develop series of gastric cancer specific,multi-modality probes MG7Ab-Cy5.5,64Cu-NODAGA-MG7 Ab and MG7Ab-ICG based on monoclonal antibody MG7 Ab as guiding molecule;to characterize the probes and verify their gastric cancer imaging ability in in vitro and in vivo settings.?Method? 1.Development and characterization of gastric cancer specific MG7Ab: The MG7 Ab monoclonal antibody was developed through hybridoma and purified by octoic acid and ammonium sulphate.Western Blot assays and immunofluorescence staining experiments were used to verify the antibody's binding specificity and affinity.2.Development and characterization of PET/CT imaging tracers: Bifunctional chelators NODAGA-NHS ester and p-NCS-benzyl-NODAGA were successfully conjugated onto MG7 Ab and further used for 64 Cu radiolabeling.PD-10 column was used to purify the radiotracers and determine the labeling yield.Tracers stability were tested with both PBS and FBS incubation.3.Development and characterization of MG7Ab-ICG tracers: ICGs in different forms were conjugated onto MG7 Ab through different reaction strategies.UV-Vis absorption was detected to characterize the tracers.Tracers stability were tested with PBS incubation and photoacoustic imaging ability was determined by imaging.4.In vitro cell uptake and imaging experiments: Cell uptake experiments were implemented using all the tracers developed above.Tracers binding pattern were acquired and analyzed using laser confocal microscope.Competing experiments were carried out to verify the tracers specificity.5.In vivo imaging and biodistribution studies: Nude mice bearing gastric cancer model were established.Near infrared imaging,PET/CT imaging,photoacoustic imaging and near infrared endoscopy were conducted.Images at different time points post tracers injection were acquired and used for data analysis.Biodistribution studies were carried out for each of the tracers developed..?Results? 1.Characterization of the probes: Gastric cancer specific monoclonal antibody MG7 Ab was developed from hybridoma and ascites system and further purified using octoic acid and ammonium sulphate.8.67 mg of purified MG7 Ab was obtained.Western Blot assays were taken out using multiple gastric cancer cell lines and epithelium cell line.Results showed that using MG7 Ab as primary antibody,cancer cell lines SGC-7901,KATO III and MKN-28 exhibited predominant 130 k Da band,where in AGS,weak band was shown and in MKN-45 and GES cell lines,no obvious band at 130 k Da was observed.Immunofluorescence imaging revealed the MG7 Ab mainly binding to cell membrane in SGC-7901 and MKN-28 cells.Whereas in GES cell,no fluorescence was detected.2.Characterization of MG7Ab-Cy5.5 tracer,in vitro and in vivo imaging studies: Cy5.5-NHS ester dye was successfully conjugated onto MG7 Ab with approximately 2.5 molar dye per molar antibody.Cell uptake experiments showed that MG7Ab-Cy5.5 tracer could specifically bind to SGC-7901 and MKN-28 cell lines instead of GES cell line.Laser confocal microscopy was implemented and revealed the tracers was mainly bound to cell membrane.In vivo near infrared imaging exhibited specific tumor uptake of the tracer.Biodistribution assays revealed the tracers binding was tumor specific and the tracers were metabolized mainly through liver.3.Characterization of 64Cu-NODAGA-MG7 Ab tracers,in vitro and in vivo imaging studies: Bifunctional chelators NODAGA-NHS ester and p-NCS-benzyl-NODAGA were conjugated onto MG7 Ab under various reaction conditions.64 Cu was further radiolabeled on both precursors.The radiolabeling yield for the two precursors were 85.7 % and 80.9 %.Stability test in both PBS and FBS solutions indicated both tracers were stable with integrity of 96.37 % vs.94.60 %(in PBS)and 95.39 % vs.94.75 %(in 40 %FBS)at 240 min after incubation.Cell uptake assays revealed both tracers could specifically bind to gastric cancer cell line SGC-7901.For example,at 120 min post incubation,cell uptake of tracer 1 was 13.93 ± 0.45 %ID and of tracer 2 was 10.62 ± 0.07 %ID.Cell uptakes were significantly decreased when competing MG7 Ab was added.At 60 min post incubation,the cell uptake before and after competing MG7 Ab addition was 12.29 ± 0.27 %ID vs.3.85 ± 0.21 %ID for tracer 1 and 10.39 ± 0.04 %ID vs.3.26 ± 0.04 %ID for tracer 2.Binding affinity tests showed the antibody's dissociation constant KD = 1.15 ± 0.17 ?M.In vivo imaging studies indicated the tracer 1 were better than tracer 2 in metabolic behavior and imaging contrast.At 24 h post injection,tumor uptake of the two tracers was 2.27 ± 0.54 %ID/g and 0.25 ± 0.13 %ID/g,respectively.Biodistribution analysis showed the tumor uptake could be effectively inhibited by competing MG7 Ab.At 24 h post injection,tumor uptake without and with competing MG7 Ab was 3.44 ± 0.29 %ID/g and 1.01 ± 0.08 %ID/g.4.Characterization of MG7Ab-ICG tracers,in vitro and in vivo imaging studies: ICGs in various forms were covalently and non-covalently conjugated onto MG7 Ab.UV-Vis absorption curves showed a 5.4:1 ICG to MG7 Ab ration in covalently conjugated tracer and 12.3:1 in non-covalently conjugated tracer.Photoacoustic imaging revealed a linear relationship between the photoacoustic signal and the tracer concentration with R2 = 0.9984.In vitro stability experiments indicated the covalently conjugated tracer had better stability than non-covalently conjugated tracer.The former tracer had a integrity of 79.6 %,75.9 % and 72.2 % at 12 h,24 h and 48 h post PBS incubation,whereas the later only had 31.7 %,26.0 % and 25.2 %.However,the tracer components at 48 h post incubation were similar for both tracers with a ICG to MG7 Ab ratio ranging from 3 to 4.Cell uptake experiments showed the signals were 1.37*106 ± 1.93*105 p/s/cm2/sr for tracer group,1.04*106 ± 1.05*105 p/s/cm2/sr for competing group and 1.07*106 ± 1.28*105 p/s/cm2/sr for negative control group.In vivo photoacoustic imaging exhibited a significant signal increase at tumor region in tracer group than in competing group,with 667.3 % ± 102.2 % vs.339.8 ± 41.3 % in signal.Infrared endoscopy showed that 15 min after tracer injection,the specific signals and unspecific signals were distinguishable at the tumor sites.?Conclusion? In the present study,we have produced and purified a batch of gastric cancer specific monoclonal antibody MG7 Ab.Based on MG7 Ab,series of gastric cancer targeting molecular imaging tracers(MG7Ab-Cy5.5,64Cu-NODAGA-MG7 Abs and MG7Ab-ICGs)were developed.Both in vitro and in vivo experiments testified and verified the tracers imaging capability.Imaging conditions were also explored and optimized during the experiments.The newly developed series of molecular imaging tracers have great potential in clinical translation and could help improve the diagnosis of gastric cancer.