^99mTc-Z_HER2:V2 Molecular Imaging And Its Value In Monitoring The Early Efficacy In HER2 Positive Tumors' Treatment

Part one:Preparation and in vitro study of ^99mTc labeled Z_HER2:V2Objective The research aimed to replace-VEN with-AEN at the N-terminal for affibody Z_HER2:342ER2:342 of HER2 and to use four amino acids-G?Gly?GGC?Cys?as chelating agent for decoration at the C-terminal.Direct labeling method was used to label radionuclide ^99mTc,to prepare affibody molecule imaging probe ^99mTc-Z_HER2:V2,and to analyze its HER2 binding characteristics in vitro.Method Fmoc/tBu solid-phase synthesis is used to synthesize Z_HER2:V2?sequence:AENKFNKEMRNAYWEIALLPNLNNQQKRAFIRSLYDDPSQSANLLAE AKKLNDAQ?and four amino acids?-GGGC?are connected at C-terminal so as to form an oligopeptide which can be strongly chelate with the structure being similar to N₃S.In addition,ligand-exchange chromatography is used to label ^99mTc.Reverse-phase high performance liquid chromatography?RP-HPLC?is used to determine labeling rate and radiochemical purity of the probe.Stability in vitro and its binding capacity and retention rate in vitro with HER2 over-expression ovarian cancer SKOV3 cells are analyzed.Meanwhile,comparison with non-interruption?unsaturation?group which wasused excessively unlabelled Z_HER2:V2ER2:V2 to pre-interrupt HER2 receptors of SKOV3cells in vitro,so as to research binding characteristics of the molecular probe.Result Radioactive peak of molecular probe ^99mTc-Z_HER2:V2 is a single peak,retention time of radioactive peak is 12min,andlabeling rate is98.99%±0.99%?n=6?.Radiochemical rate is more than 96%,not only^99mTc-Z_HER2:V2 mixed with normal saline but also mixed with fresh serum within 8h;moreover,position of radioactive peak is stable.^99mTc-Z_HER2:V2inHER2 over-expression ovarian cancer SKOV3 cells has higher binding rate which is the highest at the 24h with binding rate of 6.15%±0.18%in vitro.^99mTc-Z_HER2:V2 has relatively higher retention rate in the cells,which can be75.26%±3.25%at the highest.In case there is about 35.16%±11.23%of^99mTc-Z_HER2:V2left in the cells after 24h,it indicates that the molecular probe can be highly taken in by HER2 over-expression SKOV3 cells and can be left in the cells for a long time.Binding rate of cells membrane is in curve decline trend with time extension.However,there is about 70%of ^99mTc-Z_HER2:V2 are bound on cells membrane after 24h,which indicates that ^99mTc-Z_HER2:V2 can be bound with HER2 receptors on the cells membrane and can be transferred into the cells,but it is a slow process.In addition,after 500 times and 1000 times of excessive unlabelled Z_HER2:V2 are respectively added to SKOV3 cells for interruption,binding rate between molecular probe and SKOV3 cells will be decreased with increase of interruption times,which is respectively1.33%±0.21%and 0.94%±0.13%,and indicates that specificity binding characterize of molecular probe ^99mTc-Z_HER2:V2.Part two:The distribution and imaging studies of ^99mTc-Z_HER2:V2 in vivoObjective Body distribution of ^99mTc-Z_HER2:V2 in nude mice bearing the HER2 over-expression SKOV3 xenografts was researched;meanwhile,comparative research was made for the molecular probe in SKOV3 nude mice?HER2 over-expression?and that in MCF-7 nude mice of?HER2 low expression?.Method Adherence method is used to culture HER2 over-expression ovarian cancer SKOV3 cells and HER2 low expression breast cancer MCF-7cells.5×10⁶ ovarian cancer SKOV3 cells or MCF-7 cells are subject to external subcutaneous inoculation of right fore limb for nude mice so as to prepare HER2 over-expression and low expression xenografts animal model.The nude mice with full,no necrosis,and no significant differences in size should be selected to be incorporated in the experiment when tumor diameter is grown to 1.5².0cm 3 or 4 weeks later.Sixteen SKOV3 nude mice with the no obvious differences tumor size should be selected,which should be randomly divided into 4 groups.^99mTc-Z_HER2:V2ER2:V2 should be injected into each nude mice through caudal vein.A group of nude mice should be randomly taken at the 1^st hour,the 2^nd hour,the 4^th hour,and the 6^th hour after injection.Then,they should be subject to vena ophthalmica so as to collect blood and should be killed with the way of cervical dislocation.Heart,liver,spleen,lung,kidney,stomach,small intestine,cerebrum,muscle,skeleton,and tumor tissue should be taken so as to respective weigh quality.Moreover,its radioactivity counts should be determined;percentage injection rate which is%ID/g of tissue per gram and ratio between tumor and muscular tissue?tumor to muscle,T/M?should be calculated.Moreover,metabolism of^99mTc-Z_HER2:V2 in nude mice with ovarian cancer SKOV3 cells and radioactive distribution in different organs or tissues should be analyzed.Five nude mice with HER2over-expression of SKOV3 ovarian cancer cells and five nude mice with HER2 low expression of MCF-7 breast cancer should be randomly selected;^99mTc-Z_HER2:V2 should be injected into the nude mice through caudal vein.SPECT imaging should be taken 1^st hour,the 2^nd hour,the 4^th hour,the 6^th hour and the 8^th hour after injection.Later after injection so as to observe concentration of the probe in the tumors and to respectively calculate theT/NT ratio between tumor and contralateral area with the same sizeat different time point.Comparative analysis of SPECT imaging for ^99mTc-Z_HER2:V2 in nude mice with HER2 over-expression of SKOV3 ovarian cancer cells and nude mice with HER2 low expression of MCF-7 breast cancer cells.In addition,5models ofnude mice with SKOV3 ovarian cancer are taken;each nude mice should be subject to injection of excessively unlabelled HER2 affibody Z_HER2:V2ER2:V2 so as to close the HER2 receptors of tumor tissue through caudal vein.^99mTc-Z_HER2:V2 should be injected 5min later.SPECT imaging should be conducted 1^st hour,the 2^nd hour,the 4^th hour,the 6^th hour and the 8^th hour later after injection according to above-mentioned SPECT imaging method so as to make comparative observation of radioactivity concentration for tumor site of nude mice in the interruption group and the non-interruption group,and analyzed the binding characteristics of ^99mTc-Z_HER2:V2in vivo.The value of HER2 expression detection in vivo for using molecular probe ^99mTc-Z_HER2:V2should be discussed.Result1. In vivo,the uptake of HER2 over-expression SKOV3 tumor to^99mTc-Z_HER2:V2ER2:V2 will be risen with injection time extension and will reach the peak which is 9.38±1.22%ID/g at the time of 6h.It indicates that^99mTc-Z_HER2:V2ER2:V2 distributed in tumor of SKOV3 nude mice will be gradually increased with time extension.Such as 6h,T/Mratio between tumor and contralateral muscle is 14.31±3.16,which indicates that tumor has high uptakeof molecular probe.In addition,^99mTc-Z_HER2:V2 has the highest radioactive distribution in the kidneys.Meanwhile,radioactive distribution in the liver is obviously lower than kidney,which indicates that most of the molecular probe is subject to metabolism through kidney;liver has a low uptake rate;radioactive distribution in heart,skeleton,lung,and muscular tissue is low.Radioactivity in the blood is low,which indicates that the molecular probe can be quickly eliminate from blood.2.Tumor can be seen in SPECT imaging of 1h later after SKOV3nude mice are subject to intravenous injection of probe^99mTc-Z_HER2:V2.Moreover,radioactivity concentration will be gradually increased with imaging time extension.In addition,there is obvious radionuclide concentration in both kidneys and bladder,while there is no obvious radionuclide concentration in liver,thyroid,and other positions.The highest T/NT ratio,which is 14.13±1.22 at 8h measured by ROI technology.2.SKOV3 nude mice should be injected with excessively unlabelled Z_HER2:V2 in advance so as to conduct SPECT imaging after HER2 receptorsare closed.Obvious uptake of tumor tissue is not seen,which indicates that unlabelled Z_HER2:V2 can close HER2 receptors of SKOV3 tumor;^99mTc-Z_HER2:V2 have specificity binding characteristics in vivo.3.SPECT imaging for MCF-7nude mice at different time points after injection of ^99mTc-Z_HER2:V2 should be conducted.Radionuclide concentration can be obviously seen in kidney and bladder;there is no obvious imaging of tumor at different time points.T/NT ratio is obviously lower than SKOV3nude mice,and there is significant difference at different time points?t value is respectively 23.595,38.678,14.44,12.00,and 12.80,while P value is respectively 0.000,0.000,0.000,0.000,and 0.000?.This result can be included ^99mTc-Z_HER2:V2could be uptook by HER2 over-expression's tumors in vivo.Part three:The study of ^99mTc-Z_HER2:V2 SPECT imaging to monitoring the early efficacy in HER2 positive tumors'treatmentObjective This study will investigate the value of molecular probe^99mTc-Z_HER2:V2 SPECT imaging in the evaluation of the efficacy of chemotherapy combined with herceptin in the treatment of HER2over-expression tumors.Method The nude miceare divided into two groups.Each treatment group has 3 nude mice.Treatment group 1?chemotherapy group?:they should be respectively subject to intraperitoneal injection of 3mg/kg cis-platinum and20mg/kg paclitaxel at the 1d,the 4d,the 8d,the 11d of treatment;Treatment group 2?chemotherapy allies anti-HER2 group?:they should be respectively subject to intraperitoneal injection of 3mg/kg cis-platinum and 20mg/kg paclitaxel at the 1d and the 4d of treatment and should be subject to intraperitoneal injection of 40mg/kg trastuzumab at the 8d of treatment.Weight and tumor volume of nude mice should be determined per 3d during the treatment period.Vernier caliper should be used to measure the longest diameter?L?and the shortest diameter?D?of tumor.Tumor volume should be calculated according to the following formula:tumor volume=L×D²×1/2.Each group of nude mice should be respectively subject to ^99mTc-Z_HER2:V2SPECT imaging before treatment and the 8d and the 15d later after treatment.The supine position imaging should be taken after molecular probe^99mTc-Z_HER2:V2was subject to tail intravenous injection 4h later.Radioactivity concentration of tumor for each group of SKOV3 nude mice should be observed.Meanwhile,region of interest?ROI?should be used to respectively calculate T/NT?target to nontarget?ratio and to make comparative analysis of the treatment group.All groups of nude mice should be killed after termination of SPECT imaging at the 15d of treatment;tumor tissue should be taken out;general form of tumor tissue should be observed so as to make immunohistochemical analysis.HER2 expression after treatment of tumor tissue should be observed.Finally,value of ^99mTc-Z_HER2:V2SPECT imaging in therapeutic evaluation of HER2 over-expression tumor for chemotherapy combined with herceptin treatment should be observed.Result Weight of nude mice in two treatment groups will be subject to statistical analysis after termination of treatment with no significant differences?F=0.579,P=0.689;F=0.462,P=0.762?,which indicates that the weight of nude mice was not influence by chemotherapy or chemotherapy allies anti-HER2.Tumor volume of nude mice in the two treatment groups is gradually increased in the first 7d of treatment,and has not significant differences?t=0.165,P=0.877>0.05?after termination of 7d treatment.Tumor volume of nude mice in the treatment group 2 is gradually shrunk in the last8d of treatment,while in the treatment group 1 is continued to be increased.Tumor volume of nude mice in the treatment group 2 is obviously smaller than treatment group 1?t=18.318,P=0.000<0.05?.The result indicates that chemotherapy can be used to shrink volume of tumor after being combined with the antiHER2 drug so as to reach the purpose of treatment.Treatment group 1 and treatment group 2 can be subject to ^99mTc-Z_HER2:V2ER2:V2 SPECT imaging before treatment and on the 8d,15d after treatment.Radionuclide concentration can be seen in the tumor of SKOV3nude mice in different groups before treatment.Radionuclide concentration of tumor position in treatment group 1 can be firstly reduced?the first 7d of treatment?and can be enhanced?the last 8d of treatment?after treatment.Radionuclide concentration of tumor in treatment group 2 can be gradually reduced.T/NT ratio of tumor for nude mice in the two groups of treatment has no obvious statistical differences?t=1.518,P=0.204>0.05?.T/NT ratio of tumor for nude mice in the treatment group 1 and the treatment group 2 is obviously decreased.T/NT ratio of tumor for nude mice in the treatment group 2 is continued to be decreased in the last 8d treatment;T/NT ratio of tumor for nude mice in the treatment group 1 is increased than that in the first 7d of treatment.T/NT ratio of tumor for nude mice in the two treatment groups are compared after termination of treatment,which has statistic differences?t=7.962,P=0.001<0.05?.In addition,T/NT ratio of tumor position for nude mice in the treatment group 2 is lower than that in the treatment group1.Iimmunohistochemical analysis of tumor tissue after treatment of SKOV3nude mice with HER2 over-expression in treatment group 1,and treatment group 2:brown dye to a middle extent can be seen in the treatment group 1;brown dye to the rare extent can be seen in the treatment group 2.The result indicates that combined anti HER2 treatment with chemotherapy can be used to effectively reduce HER2 expression's level.Meanwhile,molecular probe ^99mTc-Z_HER2:V2 is proved to be as an excellentmolecular probe to monitor the early efficacy of tumor treatment.Conclusion:1.The research successfully to prepare HER2 small molecular targeted binding protein Z_HER2:V2labelled by Radionuclide^99mTc.The labeling method is simple and feasible with a high labeling rate and a good stability in vitro,which is not required to be purified.It can be specificity bound with HER2over-expression ovarian cancer SKOV3cellsin vitro,and can be retention in the cells for a long time,thus it is a potential molecular imaging probe.2.The molecular probe ^99mTc-Z_HER2:V2 has some advance in vivo,such as,good bio-distribution;tumor uptake of SKOV3nude mice is obvious,most of which are excreted through kidneysand with less uptake in the liver,quick blood elimination,and good image quality.^99mTc-Z_HER2:V2 has good specificity binding characteristics ofHER2receptors in vivo.Detection HER2 expression of molecular probe ^99mTc-Z_HER2:V2 in vivo nidus is feasible.3.Treatment of chemotherapy combined with trastuzumab has more obviousefficacytoHER2over-expressionovariancancer SKOV3xenograftstumor than that of single chemotherapy.In addition,it can significantly shrink tumor volume and can obviously reduce HER2 expression level of tumor tissue.So that thetherapeutic efficacy could be monitored by molecular probe ^99mTc-Z_HER2:V2.