Investigations Of Molecular Imaging Probes From Radionuclide-and Fluorescent-labeled HER2 Nanobodies

Cancer with high morbidity and mortality is the main cause of serious threats to the population health,and has led to a heavy economic and social burden.It is very essential to develop new technologies for the precise diagnosis and the effective treatment of cancer.HER2 is an important target for cancer diagnosis and treatment,and it is urgent in clinic to develop molecular imaging methods to detect the HER2 expression level of tumor tissue in vivo.This paper aims to develop molecular imaging probes from radionuclides-and fluorescent dye-labeled HER2 nanobodies,therefore providing an effective new molecular imaging paradigm for the diagnosis and treatment of HER2-positive cancers.In this paper,The molecular imaging probes from radionuclides(99mTc?188Re)-and fluorescent dye(Cy7)-labeled HER2 nanobodies were developed and their in vitro characteristics and in vivo small animal SPECT/CT imaging and fluorescence imaging were explored.This dissertation consists of the following five chapters:The first chapter is the introduction of main research background.It first briefly summarizes the relationship between HER2 and tumor,the role and characteristics of molecular imaging technology,the concept and features of nanobodies,and then focuses on the progress and current situation of molecular imaging probes from radionuclides-and fluorescent-labeled HER2 nanobodies and intact antibodies,and finally briefly describes the research contents of this paper.In the second chapter,the molecular imaging probes of radionuclide 99mTc labeled multiple HER2 nanobodies(Nb010,Nb017,Nb023,Nb026)were developed,their tumor model SPECT/CT imaging were explored for screening the properties-optimized candidate HER2 nanobodies,and explore the properties in vivo and in vitro.The tricarbonyl[99mTc]kit labeling method of nanobodies was first established with high synthesis efficiency.This method uses the tricarbonyl kit to synthesize tricarbonyl[99mTc].Compared with the conventional method,the tedious steps of entering CO gas are removed.The labeling eff-iciency of 99mTc nanobodies with His-tag is greater than 95%and the radiochemical purity is greater than 95%.Then tumor-bearing mice SPECT/CT imaging was carried out to evaluate the ability of HER2-positive tumor targeting imaging,to screen the HER2 nanobodies with optimized imaging properties,among which 99mTc-Nb023 and 99mTc-Nb026 SPECT/CT tumor imaging have higher contrast,larger target-to-background ratio,and possess the properties of ideal molecular imaging probes.Therefore,we next explored the in vivo and in vitro properties of molecular imaging probes 99mTc-Nb023 and 99mTc-Nb026,the results show that 99mTc-Nb023 and 99mTc-Nb026 has good properties and potential as a molecular imaging probe.In the third chapter,the preliminary exploration of preparation and SPECT/CT imaging of 188Re-Nb026 for the integration of HER2 targeted radiotherapy.Similar to the above labeling method of tricarbonyl[99mTc],based on the tricarbonyl[188Re]kit,we developed therapeutic radionuclide 188Re-labeled nanobody i.e.188Re-Nb026,with the labeling efficiency of about 85%,and the radiochemical purity of more than 95%after separation.Further SPECT/CT imaging exhibited that 188Re-Nb026 had high uptake in HER2-positive tumor tissues in vivo with strong HER2 targeting and specificity,and similar normal distribution in vivo to that of 99mTc-Nb026.The above experimental results demonstrated that 188Re-Nb026 have great potential of HER2 targeted radioimmunotherapy.In the fourth chapter,the preparation and fluorescence imaging of HER2 targeted fluorescent molecular imaging probe Cy7-Nb017 were preliminarily investigated.The fluorescent molecular probe Cy7-Nb017 was prepared by the reaction of fluorescent dye Sulfo-Cy7-NHS with nanobody Nb017 in light-free and alkaline mediate in room temperature overnight,and separated by PD MiniTrap G-25 column.Mass spectrometry analysis confirmed that the average molar ratio of nanobody Nb017 to Sulfo-Cy7 in the probe Cy7-Nb017 molecule was 1:2.Tumor optical imaging demonstrated that Cy7-Nb017 uptake was high in HER2-positive tumor lesions,but almost no uptake in HER2-negative MDA-MB-468 tumors.Therefore,Cy7-Nb017 as HER2 targeted optical molecular imaging probe is of great potential.Finally,the summary and outlook briefly summarize the main results and findings in this paper.They include the establishment of a universal method of tricarbonyl[99mTc]and[188Re]labeled nanobodies with terminal His-tag,the development of novel molecular imaging probes 99mTc-Nb023,99mTc-Nb026,188Re-Nb026 and Cy7-Nb017 based on HER2 nanobodies,and the evaluation of high contrast SPECT/CT and optical imaging of their HER2-positive tumors.We look forward to the bright prospect of the developed probe for radioimmunoimaging and radioimmunotherapy of HER2-positive cancers,and the great need to evaluate and monitor the efficacy of trastuzumab and pertuzumab for HER2-positive cancers in clinical translation.