Experimental Study Of Bispecific Peptide Heterodimer Molecular Imaging Tracers For Cancer Imaging

Objective: Integrin α_vβ₃ and aminopeptidase N（APN,also known as CD13）are two important targets involved in the regulation of angiogenesis,tumor proliferation,invasion and metastasis.In this study,we developed a heterodimeric tracer ⁶⁸Ga-NGR-RGD,consisting of NGR（Asp-Gly-Arg）and RGD（Arg-Gly-Asp）peptides targeting CD13 and integrin α_vβ₃,respectively.This heterodimeric tracer was compared with its monospecific peers,⁶⁸Ga-NGR and ⁶⁸Ga-RGD,exploring the feasibility and advantages of dual-receptor-targeted heterodimer in breast cancer imaging.Methods: NGR peptide was first modified with bifunctional chelator NO₂A_tBu-N₃ and then conjugated to BCN-PEG4-c（RGDyK）via copper free click chemistry.The resulting precursor,c（RGDyK）-PEG4-NOTA-click-PEG4-c（CNGRC）（referred to as NGR-RGD）was purified and radiolabeled with ⁶⁸Ga.The in vitro and in vivo stability were determined by analytical radio-high performance liquid chromatography（radio-HPLC）.Human breast cancer cell line MCF-7 was used for cell uptake and block assays of ⁶⁸Ga-NGR-RGD,⁶⁸Ga-NGR and ⁶⁸Ga-RGD.Meanwhile,small animal PET/CT scan and post-imaging biodistribution studies of the probes were performed in MCF-7 subcutaneous xenograft models.Four human breast cancer cell lines,MCF-7,MDA-MB-231,MDA-MB-468 and MX-1 were applied for ⁶⁸Ga-NGR-RGD cell uptake assay,PET/CT scan of tumor models and post-imaging biodistribution studies.⁶⁸Ga-NGR-RGD imaging of mice with MCF-7 pulmonary metastases were performed.The expression levels of integrin α_vβ₃ and CD13 in cells and tumors were checked via western blot and immunochemical staining,respectively.Results: The heterodimeric tracer was successfully synthesized and radiolabeled with ⁶⁸Ga at a molar activity of 45¹00 MBq/nmol（n = 6）.The radiochemical yield and purity of ⁶⁸Ga-NGR-RGD were over 99%（n = 6）.It demonstrated high in vitro and in vivo stability.CD13 was highly expressed in MCF-7 cell relatively,weakly in MDA-MB-468,none in MDA-MB-231 and MX-1.MDA-MB-231 showed highest expression level of integrin α_ⅴ and β₃,followed by MCF-7 and MDA-MB-468,while MX-1 showed no expression.Cell assays showed that the uptake of ⁶⁸Ga-NGR-RGD by MCF-7 cells was significantly higher than that of single-targeted probes ⁶⁸Ga-NGR（P < 0.001）and ⁶⁸Ga-RGD（P < 0.001）.The uptake of ⁶⁸Ga-NGR-RGD could be blocked by excess amounts of unlabeled NGR-RGD,NGR+RGD,NGR and RGD.At 2h,the uptake of ⁶⁸Ga-NGR-RGD by MCF-7 cells was higher than MDA-MB-231（P < 0.01）,MDA-MB-468（P < 0.001）and MX-1（P < 0.001）.In static PET/CT scan,MCF-7 tumor could be clearly visualized and exhibited higher uptake at 30 min post injection（p.i.）of ⁶⁸Ga-NGR-RGD than that of either ⁶⁸Ga-RGD（P < 0.05）or ⁶⁸Ga-NGR（P < 0.001）.High specificity was shown in blocking studies.MCF-7 tumor exhibited the highest uptake of ⁶⁸Ga-NGR-RGD,followed by MDA-MB-231,MDA-MB-468,and MX-1 tumors.This was consistent with their expression levels of CD13 and α_vβ₃ as confirmed by western blot and immunohistochemical staining.Metastatic lesions in lung were clearly detectable on ⁶⁸Ga-NGR-RGD PET/CT imaging in tumor models of pulmonary metastases.Conclusions: In this study,a CD13 and α_vβ₃ dual-receptor targeting tracer,⁶⁸Ga-NGR-RGD was successfully developed and showed higher tumor uptake,targeting efficacy and longer tumor retention compared with monomeric ⁶⁸Ga-NGR and ⁶⁸Ga-RGD,clearly detecting metastatic lesions in lung.Its promising in vivo performance makes it an ideal candidate for future clinical translation.Objective: The CXC chemokine receptor 4（CXCR4）and integrin α_vβ₃ play an important role in tumor development,progression,invasion and metastasis,which are overexpressed in various types of human cancers.Studies have indicated that probes which can recognize multiple targets could achieve higher targeting efficacy than their monospecific peers.Therefore,in this study,we synthesized a peptide-based antagonist for CXCR4,cyclic peptide cyclo（D-Tyr-N-Me-D-Lys-Arg-2-Nal-Gly）（referred to as y G5）,an analogy of peptide FC131.y G5 was linked to c（RGDyK）to construct a heterodimer y G5-RGD.The heterodimer was compared with the single-targeted tracers to explore the feasibility and advantages of the prepared dual-targeted probe ⁶⁸Ga-y G5-RGD in pancreatic cancer imaging.Methods: y G5 peptide was first modified with bifunctional chelator NO₂A_tBu-N₃ and then conjugated to BCN-PEG4-c（RGDyK）via copper free click chemistry.The resulting precursor was identified and radiolabeled with ⁶⁸Ga.The in vitro and in vivo stability was confirmed by radio-high performance liquid chromatography（radio-HPLC）.The relative expression levels of both CXCR4 and integrin α_vβ₃ of BXPC3 and MX-1 cells and corresponding tumor tissues were characterized by western blot and immunohistochemical staining.The in vitro cell studies were performed.PET/CT imaging and biodistrbution of ⁶⁸Ga-y G5-RGD,⁶⁸Ga-y G5 and ⁶⁸Ga-RGD were performed using human pancreatic cancer cell BXPC3 xenograft tumor models.As control,the in vivo performance of ⁶⁸Ga-y G5-RGD was also evaluated in human breast cancer cell MX-1 tumor-bearing mice.Results: y G5 and y G5-RGD were successfully synthesized and labeled with ⁶⁸Gawith high yield（> 99%）and purity（> 99%）at molar activity of 74⁹2 MBq/nmol（n = 6）.Less than 1% disassociation was observed after incubation in PBS and fresh human serum at 37 ℃ for 2h in vitro.⁶⁸Ga-y G5-RGD also showed high in vivo metabolic stability.BXPC3 showed moderate expression level of CXCR4 as well as integrin α_vβ₃,while MX-1 low or not.The uptake of ⁶⁸Ga-y G5-RGD was higher than ⁶⁸Ga-y G5（P < 0.001）and ⁶⁸Ga-RGD（P < 0.001）and could be blocked by excess amounts of AMD3100（FDA-approved CXCR4 antagonist）or unlabeled RGD（P < 0.001）.On small animal PET/CT images,⁶⁸Ga-y G5-RGD was mainly excreted via liver and kidney.BXPC3 tumor was visualized clearlier post injection of ⁶⁸Ga-y G5-RGD than that of ⁶⁸Ga-y G5 and ⁶⁸Ga-RGD during observation period.What’s more,BXPC3 tumors were still clearly visible at 2h post injection of ⁶⁸Ga-y G5-RGD.In addition,the blocking study in BXPC3 tumor showed that the tumor visualization could be blocked at 30 min post co-injection of ⁶⁸Ga-y G5-RGD and block agents.MX-1 tumor was not visible by ⁶⁸Ga-y G5-RGD.The biodistribution results were consistent with imaging.Conclusions: In our study,a peptide-based heterodimeric tracer ⁶⁸Ga-y G5-RGD targeting CXCR4 and integrin α_vβ₃ was successfully developed with excellent in vitro and in vivo performance.The dual-receptor targeting tracer showed higher targeting efficacy and longer tumor retention,showing promising application for cancer imaging.