A Novel ⁶⁸Ga-labeled Affibody For Breast Cancer HER2 Pet Imaging

Background:Overexpression of HER2 in breast cancer is associated with poor prognosis.There may be differences between primary tumors and metastases and changes during treatment.Targeted HER2 Affibody PET imaging may evaluate HER2 expression in vivo in real-time.Our group previously used the ¹⁸FA1 one-step strategy to label the affibody MZHER_2:342 with easy synthesis and good pharmacokinetics,but the labeling yield was low,limiting the clinical spread.In addition,more and more PET/CT centers are not equipped with cyclotrons,based on the production of ⁶⁸Ga tracer will enhance the function of these PET/CT.Therefore,in this study,we use the ⁶⁸Ga labeling NOTA-MAL-MZHER_2:342,examining the labeling yield,monitoring the targeting and HER2-positive breast cancer xenografts in vivo.Method:The Affibody MZHER_2:342 was coupled to the chelator MAL-NOTA under standard reaction conditions.The affibody molecule was then labeled with the ⁶⁸Ga eluted by a generator.The product ⁶⁸Ga-NOTA-MAL-MZHER_2:342 was subjected to quality control and in vitro stability analysis.In vitro cell uptake blockade verified the binding specificity of the probe to HER2.The expression of HER2 in tumor-bearing mice was detected by immunohistochemistry.After injection of the probe,MicroPET imaging was performed in tumor-bearing mice（BT474 and MCF-7）,the region of interest was delineated,and tumor uptake values were calculated.The biodistribution confirmed the distribution of the probe in various organs in the body and verified the imaging results of MicroPET.Results:The precursor NOTA-MAL-MZHER_2:342 was labeled with ⁶⁸Ga by single-step method with a labeling yield of 81±5%and a radiochemical purity of>95%.The stability was good in plasma and PBS.Immunohistochemistry confirmed that human breast cancer cells BT474 and MCF-7 were HER2 positive and negative,respectively.In vitro uptake inhibition experiments showed that the uptake values of BT474 cells at 15 min and 60 min were 2.32±0.20 and 3.50±0.20%AD/10⁶cells,respectively,and after the blocker was added in advance,the uptake decreased to 1.36±0.18%AD/106 cells at 15 min and maintained at a low level.The BT474tumor-bearing mice underwent microPET imaging.The tumor uptake value was as high as 15.26±2.73%ID/g at 30 minutes and maintained at a high level,which was significantly different from the surrounding normal tissues.The in vivo biodistribution was consistent with the results of microPET imaging,indicating that the probe is highly radioactive uptake in HER2-positive tumors.The tracer quickly removes from the blood and normal tissues,resulting in a high tumor to blood ratio.The highest normal tissue radioactivity concentration was observed in the kidneys.Conclusions:⁶⁸Ga-NOTA-MAL-MZHER_2:342 has the advantages of simple synthesis,markedly increased labeling yield,high radiochemical purity,and high specificity for tumors and good target-to-locality in HER2-positive tumor-bearing mice,and is suitable for clinical promotion.