An Integrated Experimental Study On Diagnosis And Treatment Of Ovarian Cancer Based On ^99mTc And ¹³¹I Labeled HER2 Affibody

Objective:To prepare HER2 affibody with high stability and high radiolabeling rate with ^99mTc or ¹³¹I for single-photon radionuclide imaging.The biological distribution,safety,and SPECT static imaging of radiolabelled affibody were studied in nude mouse model of SKOV3 ovarian cancer to explore the potential diagnostic and therapeutic value in HER2-positive ovarian cancer patients.Methods:The amino terminus and carboxyl terminus of the HER2 affibody were reshaped for convenient labelling with ^99mTc and ¹³¹I by gene recombination and expression.The ^99mTc/¹³¹I-labeled HER2 affibody was prepared under the optimal experimental conditions,and the radiochemical purity,stability and equilibrium dissociation constant（Kd）were identified.TwentyμCi（29.6MBq/Kg）of ^99mTc/¹³¹I-labeled molecular probe was injected into the tail vein of the SKOV3 ovarian cancer model,and the mice were sacrificed and dissected at different times after drug injection,and tissues samples（heart,lung,liver,spleen,kidney,stomach,intestine,muscle,bone,thyroid,brain,blood）were weighed and the radioactivity count of each sample was measured with a gamma-counter.After time decay correction,the radioactive uptake value（%ID/g,the percentage of injected dose per gram of tissue）was calculated,and the data were processed and analyzed with SPSS18.0 software.SPECT static imaging was performed at different times after injection of ^99mTc-（HE）₃Z_HER2:2395G₃C or ¹³¹I-Y（HE）₃Z_HER2:2395G₃C.HER2expression was determined by immunohistopathological examination.Results:The HER2 affibody（HE）₃Z_HER2:2395G₃C with reshaped amino terminus and carboxyl terminus was prepared by gene recombinant expression for convenient ^99mTc labeling.The affibody Y（HE）₃Z_HER2:2395G₃C was recombinantly prepared for ¹³¹I labeling.The ^99mTc/¹³¹I labeling of the affibodies can be completed at room temperature,with a labeling rate of over 90%,and the radiochemical purity can be maintained at over 90%after mixing with serum and normal saline after incubating at 37℃for 120 min and 240min.In animal models of SKOV3 ovarian cancer with high HER2 expression,^99mTc and¹³¹I labeled affibodies was injected into the tail vein for imaging,and the targeted uptake of molecular probes was observed.^99mTc and ¹³¹I-labeled affibodies quickly bind to HER2-positive SKOV3 ovarian cancer tissues after entering the blood circulation system,^99mTc-（HE）₃Z_HER2:2395G₃C is mainly cleared from the urinary system,¹³¹I-Y（HE）₃Z_HER2:2395G₃C is mainly excreted through the urinary system and gastrointestinal tract.Conclusion:HEHEHE was added to the amino terminus of HER2 affibody Z_HER2:2395,and the carboxyl terminus was linked to GGGC,and a ^99mTc-labeled affibody（HE）₃Z_HER2:2395G₃C was successfully prepared.Affibody Y（HE）₃Z_HER2:2395G₃C can be prepared by introducing one more tyrosine at the amino terminus of affibody（HE）₃Z_HER2:2395G₃C,which is easily labeled with ¹³¹I.After optimizing the experimental conditions,molecular probes ^99mTc-（HE）₃Z_HER2:2395G₃C and ¹³¹I-Y（HE）₃Z_HER2:2395G₃C with high labeling rate can be prepared,which are stable in serum and normal saline.The^99mTc-labeled molecular probe ^99mTc-（HE）₃Z_HER2:2395G₃C was able to specifically image nude mice with HER2-positive ovarian cancer,and radioactivity uptake was seen in tumors as small as 4 mm in diameter.Tumors can also be targeted with ¹³¹I-Y（HE）₃Z_HER2:2395G₃C,and significant radioactivity uptake was still observed 120 h after injection of the molecular probe,suggesting that it may be one of the potential drugs for the targeted treatment of HER2-positive ovarian cancer.