The Construction Of A Novel HER2 Targeting Affinity Probe And The Study Of Tumor PET Imaging

Background:Overexpression of human epidermal growth factor receptor-2(HER2)in tumor is associated with poor prognosis and malignant behavior.HER2 becomes an important biomarker for tumor diagnosis and therapy.Evaluation of the expression of HER2 in clinic was lack of specificity,timeliness and comprehensiveness.Non-invasive PET imaging modality targeting HER2 provides a new method for real-time and accurate determining HER2 levels.Compared with antibodies,affibody is an ideal vector for HER2 probe with the advantages of easily synthesis,high specific binding to receptor and strong tissue penetration etc.However,tedious synthesis producers and unfavorable hepatic uptake might hinder the widely spread of the agent in clinic.A HER2 affibody,ZHER2:342,was modified with a hydrophilic linker,GGGRDN,to improve the pharmacokinetic property of the probe.Meanwhile,a cysteine was also introduced to facilitate the in situ labeling.The resulting analog was Cys-MZHER2:342,denoted as Cys-MZHER2.Cys-MZHER2 was coupled to the bifunctional chelators and easily labeled with PET nuclides including 18F、68Ga,89Zr etc and a series of novel HER2 PET probes were obtained.In vitro cells experiments and in vivo imaging in tumor models was performed to evaluate the targeting and imaging characters of the probes.Also,the clinic trail was performed to further investigate the application value.Methods:NOTA/DFO-MAL-Cys-MZHER2 was synthesized by coupling Cys-MZHER2 to NOTA/DFO-MAL,and the specific probes,18FA1/68Ga NOTA-MAL-Cys-MZHER2 and 89Zr-DFO-MAL-Cys-MZHER2 were obtained by one step labeling strategy.Quality control and in vitro stability were performed.In vitro cell uptake assay and in vivo PET imaging were used to investigate the imaging properties of the probes.Estimation of the dosimetry and toxicity experiments were also tested.A clinic trail was firstly practiced in domestic hospitals.Results:The precursors,NOTA/DFO-MAL-Cys-MZHER2,were obtained with the yields of nearly 50%.Under optimal conditions,the yields of 18FAl/68Ga NOTA-MAL-Cys-MZHER2 and 89Zr-DFO-MAL-Cys-MZHER2 were18±2,1%、81±5%and 90.2±1.90%%respectively.The radiopurities were more than 95%.The total synthesis time was only 30 minutes.The probes was stable in PBS and serum.The tracers accumulated in HER2 overexpression human ovarian cancer SKOV-3 cells and the uptakes were significantly higher than those in block group.In vivo studies in mice bearing tumors showed that these probes highly retained in SKOV-3 xenografts and the longest time was 48 hours.The tumors were clearly visualized with good contrast to normal tissue.ROI analysis revealed that the average uptakes in the tumor was greater than 5%ID/g.On the contrary,the counterparts of MCF-7 tumors kept low levels(～1%ID/g).The outcome was consistent with the immunohistochemical analysis.It was confirmed that 18FA1/68Ga-NOTA-MAL-Cys-MZHER2 and 89Zr-DFO-MAL-Cys-MZHER2 specifically bound to HER2 with excellent sensitivity.The uptakes of the probes in the tumors were significantly positive correlated to the HER2 levels.The probes quickly cleared from normal organs resulting in low abdomen backgrounds.The kidney was the main metabolic organ since more radioactivity was found.Estimation of the dosimetry and toxicity experiments showed that the probes were safe.Clinic PET/CT imaging in the volunteers preliminarily revealed that 68Ga-NOTA-MAL-Cys-MZHER2 can be used to significantly discrimin ate HER2 levels in the primary focus.The metastasis was also detected.The tracer was tolerable in the human body and no adverse events were observed.Conclusion:The novel HER2 affibodis for PET imaging were prepared in a simple straightforward labeling procedures and suit for clinical routine application with the satisfactory yields and radiochemical purity.The imaging properties was good and the target organ was clearly visualized.It may be benefit for tumor diagnosis,choosing the therapy schedule and monitoring effects of the therapy etc.