The Preparation And Preclinical Evaluation Of Bispecific PSMA/FAP Heterodimers

Objective:Malignant tumors often have tumor heterogeneity and complex tumorinterstitial interaction systems,which limit the efficiency of single-specific tracers in tumor diagnosis and treatment.Since prostate-specific membrane antigen(PSMA)is overexpressed in a variety of solid tumor cells and fibroblast activated protein(FAP)is upregulated in the tumor matrix,we suggest that heterodimer probes targeting PSMA and FAP receptors can improve the diagnostic performance of tumors.In this study,we conjugated lysine-urea-glutamic acid(PSMA)pharmacophore and quinoline FAP pharmacophore through different linkers and NOTA chelating agents,and labeled them by AL18F-NOTA chelating chemistry.Two PSMA/FAP dual target probes[18F]AIFPSMA-FAPI-01 and[18F]AlF-PSMA-FAPI-02 were prepared.In this study,the two bispecific probes will be preevaluated from the aspects of radiosynthesis,in vitro characterization,cell characteristics,in vivo biological distribution and micro-PET/CT imaging,and compared with the corresponding monomer probes[18F]AlF-PSMABCH and[18F]FAPI-42.Methods:Two novel bispecific PSMA/FAP heterodimers were prepared by artificial solid-phase polypeptide synthesis and characterized by high resolution mass spectrometry.Al18F-NOTA chelation method was used for radioactive labeling,and radioanalytical high performance liquid chromatography(HPLC)was used for identification.Lipid water partition coefficient and in vitro and in vivo stability of the two isodimers were determined.The binding,competition,internalization and effection properties of 18F-labeled heterodimers[18F]AIF-PSMA-FAPI-01 and[18F]AIF-PSMAFAPI-02 were determined by 22Rv1 cells with high PSMA expression and A549-FAP transfection cells with overexpression of FAP.Biological distribution studies and PET imaging studies in vivo of small animals in tumor-bearing nude mice carrying 22Rv1 or A549-FAP were performed and compared with corresponding single-specific tracers[18F]AlF-PSMA-BCH and[18F]FAPI-42.Results:Two kinds of 18F-labeled heterodimer probes[18F]AIF-PSMA-FAPI-01 and[18F]AIF-PSMA-FAPI-02 were successfully prepared,and the whole labeling process was about 35min.The unattenuated corrected radiochemical yields were 25.7±4.1%(n=5)and 17.5±1.4%(n=7),respectively.The radiochemical purity was>99%,the specific activity was about 10-30 GBq/μmol,and the stability in vivo and in vitro was good.All of them had good hydrophilic properties(logD7.4 values were2.74±0.02 and-2.04±0.04,respectively).The IC50 values of the two heterodimer fusion peptides and the corresponding monomer peptide NOTA-FAPI-42 in A549-FAP cells were 1.7±0.2 nM,5.8±0.6 nM and 0.75±0.3 nM,respectively.In 22Rv1 cells,the IC50 values of the two heterodimer fusion peptides and the corresponding monomer peptide NOTA-PSMA-BCH were 33.7±0.9 nM,9.8±0.7 nM and 2.5±0.3 nM,respectively.The IC50 values of the two groups of cells were all in the nanomoll level,indicating that the heterodimer peptides NOTA-PSMA-FAPI-01 and NOTA-PSMA-FAPI-02 had similar binding affinity for FAP receptor and PSMA target compared with the corresponding unmodified monomers.Several block experiments demonstrated that the two heterodimer probes had specific dual targets of PSMA and FAP in vitro and in vivo.Biodistribution studies in tumor bearing mice and PET imaging studies in small animals showed that these two 18F-labeled heterodimers have a high uptake rate in tumors,are metabolized mainly through the kidney and can be rapidly cleared of activity from the blood and normal organs.Compared with single specific tracer[18F]AlF-PSMA-BCH or[18F]FAPI-42,both 18F-labeled heterodimer probes showed higher tumor uptake and longer tumor retention time,thus achieving relatively high imaging quality.Conclusion:Based on the above results,these two new heterodimeric probes[18F]AlF-PSMA-FAPI-01 and[18F]AlF-PSMA-FAPI-02 showed high specificity and affinity targeting to PSMA and FAP in vitro and in vivo.Moreover,the two probes have excellent properties,such as easy synthesis,high tumor absorption and good pharmacokinetic characteristics,so that they can be considered as promising candidates for PET tracers.Double targeted tumor imaging can improve diagnostic sensitivity and specificity.