Nuclide Labeling And SPECT/CT Molecular Imaging Of HER2 Targeting Biomolecules

HER2 is a member of the human epidermal growth factor receptor family and is overexpressed in most common cancers,such as breast cancer,ovarian cancer,gastric cancer and lung cancer,yet almost no expression in normal cells.Previous studies have shown that the expression level of HER2 is closely related to proliferation,invasion,survival and prognosis of cancers.Therefore,HER2 has become a very important research targets and areas for molecular imaging,diagnosis,treatment and biomarkers of cancers.Molecular imaging provides a noninvasive,quantitative,molecular/cell-level functional imaging method for the diagnosis and evaluation of cancer.It can achieve early diagnosis of cancer,and timely,dynamic prognosis evaluation,and can significantly improve the quality of treatment and survival rate of cancer patients.Therefore,using HER2 as a target,developing HER2 specific molecular imaging probe can hold great potential of clinical translation for cancer imaging,diagnosis and treatment.This dissertation aims to investigate the radionuclide labeling of HER2-specific targeting biomolecules and the corresponding SPECT/CT imaging,in order to develop molecular probes with clinical application prospect.This dissertation consists of four chapters.The first chapter is the introduction.It mainly introduces the background and current situation of the research.It summarizes the important role of HER2 in cancer diagnosis and treatment and the progress of HER2-targeted molecular probes.The second chapter is the radiolabeling of trastuzumab using radionuclide ¹²⁵I and ^99mTc,and their SPECT/CT imaging.First,we used a simple method for the quick¹²⁵I-radioiodination of trastuzumab,which achieved excellent labeling yield.Subsequently,we studied the uptake of ¹²⁵I-Tz in tumor cells with HER2 high and low expression respectively,demonstrating the binding specificity of trastuzumab to HER2.Finally,¹²⁵I-Tz was intravenously injected into the mice bearing HER2 highly expressed tumors to perform SPECT/CT molecular imaging for real-time monitoring.The imaging results showed that ¹²⁵I-Tz had the ability to specifically target tumor.The biodistribution study showed the uptake of ¹²⁵I-Tz in normal tissues and organs was relatively low,suggesting the clearance rate in vivo was great.Then,we performed ^99mTc labeling of trastuzumab which requires suitable chelating agents.Therefore,we first successfully conjugated DTPA to trastuzumab.After optimizing the labeling conditions,the ^99mTc labeling of DTPA-Tz obtained better labeling yield and radiochemical purity.The SPECT/CT imaging results showed that tumor uptake of ^99mTc-DTPA-Tz was relatively low,probably due to the inconsistency between the short half-life of ^99mTc and the long circulation cycle in vivo of trastuzumab.Thus we focused on the smaller-sized HER2 targeting biomolecules at later stages.The third chapter is the use of radionuclide ¹²⁵I and ^99mTc to label nanobodies and to perform SPECT/CT imaging.First,the screened nanobodies with HER2 targeting capability were screened and analyzed for their purity by HPLC.After that,nanobodies N214 and N216 without His tag were easily labeled using ¹²⁵I,which obtained excellent labeling yields.Accordingly,SPECT/CT imaging of ¹²⁵I labeled nanobodies was conducted.The results of the imaging showed that the tumor uptake of ¹²⁵I-N214 and ¹²⁵I-N216 was not high,and the specific targeting abilities were not shown.Then,we used the His-tagged nanobody His-Nb108 for ^99mTc labeling.The radiolabeling was successful using ^99mTc^??CO?₃?H₂O?₃⁺labeling method.The SPECT/CT imaging results of ^99mTc-His-Nb108 showed that its tumor uptake was low,and its metabolic clearance was fast.The higher uptake in the kidney indicated that it excreted mainly through the urinary system.As the screening of nanobodies is in the initial stage and the screening cycle is rather long,we selected the reported peptides for the following research object.The fourth chapter is the ^99mTc labeling of selected peptides and their SPECT/CT imaging.Here,we chose HYNIC as a chelating agent,EDDA and Tricine as co-ligands,and ^99mTc was labeled with HYNIC conjugated peptide LTVSPWY and YLFFVFER respectively.The ITLC results showed that the labeling yield of HYNIC-LTVSPWY was great,while the labeling yield of HYNIC-YLFFVFER was medium.Therefore,only ^99mTc-HYNIC-LTVSPWY was studied for the changes of the radiochemical purity in saline and serum,suggesting good stability in vitro.Finally,we performed SPECT/CT imaging with ^99mTc-HYNIC-LTVSPWY and^99mTc-HYNIC-YLFFVFER respectively.The imaging results showed that both of them circulated quickly in vivo and reached the tumor sites in a short period of time.The stability of ^99mTc-HYNIC-LTVSPWY in vivo is better than another one,and its tumor targeting ability is better also.The fifth chapter is the summary and prospect.This chapter summarizes the results and the corresponding analysis of the experimental parts,describing the different HER2 targeted biomolecules for ¹²⁵I and ^99mTc radiolabeling and their SPECT/CT imaging outcomes,and further prospects for the development of HER2targeted molecular probes.