Research On PET Probe In Colorectal Cancer For Amplification Pretargeting

Colorectal cancer is a malignant disease with both high incidence and mortality rates that keep rising over time because of hard diagnosis at an early stage and easy postoperative metastasis and recurrence.TAG-72,a tumor-associated glycoprotein,is overexpressed in colorectal cancer and underexpressed in normal tissues.CC49,a second-generation anti TAG-72 monoclonal antibody,has been used as a radiolabeled targeting agent in nuclear medicine imaging and could be considered as a suitable agent for future clinical application.There are some limitations of conventional nuclear medicine imaging of tumors with radiolabeled macromolecular compounds,such as prolonged targeting time and slow blood clearance.Pretargeting is a useful approach to improve tumor/nontumor ratios rapidly.It requires two steps,where the modified targeting macromolecular was administrated first followed by the radioactive effector designed for rapid clearance from all tissues except the tumor.The recognition system of PNA/c PNA was considered suitable for pretargeting,because of the high affinity,great enzyme resistance,no immunogenicity and no biological toxicity.In order to improve the radioactivity uptake in tumor,the three-step amplification pretargeting approach was applied in this work.The dendrimer PAMAM G4 was conjugated with multiple copies of PNAs as a signal amplification platform,which could combine with the antibody CC49-c PNA and the tracer of ¹⁸F-c PNA.¹⁸F-cPNA,as a nucleic acid PET probe,was synthesized through Cu AAC click reaction between c PNA-hexynoic and ¹⁸F-fluoroethyl azide at 80°C for 20 min via the catalyst of copper sulfate and sodium L-ascorbate solution.The product of ¹⁸F-c PNA was prepared with>30 GBq/μmol of the molar activity and>99%of the radiochemica1 purity in 80～90 minutes of total synthesis time（including purification time）.The measured Log D_7.4 value of ¹⁸F-c PNA was-1.70±0.25（n=6）,which reflected that the hydrophilicity of the probe.The probe of ¹⁸F-c PNA displayed the great stability in NBCS and PBS at 37°C for 2 hours.The pharmacokinetics experiment showed that the ¹⁸F-c PNA demonstrated an expected rapid blood clearance(t_1/2,α=1.9min,t_1/2,β=16.0 min).The biodistribution study in normal BALB/c mice exhibited the rapid distribution and clearance characteristics ¹⁸F-c PNA.There was little radioactivity accumulation in most organs and tissues（except bone and marrow）,which proved that¹⁸F-c PNA was suitable for further pretargeting strategy.The amplification platform of PAMAM G4-PNA was synthesis by coupling PAMAM G4 and PNA at the different molar ratio by the catalyst of EDC-methiodide,following purification by the ultrafiltration centrifugation.The SE-HPLC and MALDI-TOF MS spectra exhibited the optimum molar ratio of PNA/PAMAM G4 was 20:1.The average GPM of PNA in polymer was calculated to be 11.7 based on MALDI-TOF MS spectrum.The retention time of PAMAM G4-PNA/¹⁸F-c PNA（36.5 min）was earlier than ¹⁸F-c PNA（38.1 min）,which indicated the successful hybridization to duplex.The accessibility rate of ¹⁸F-c PNA to PAMAM G4-PNA was more than 99%because there was no radioactivity peak of free ¹⁸F-c PNA.The targeting agent of CC49-（c）PNA was prepared by conjugating CC49 and（c）PNA via the commercial Hydralink kit.The intensity of the fluorescent signal of CC49-c PNA and CC49-PNA appeared to be identical to that of native CC49 by flow cytometry analysis.The results indicated that the immunoreactive fraction of CC49-c PNA and CC49-PNA preserved more than 95%compared with the native CC49.The probe of ¹²⁵I-CC49-c PNA was prepared by the iodogen method with high radiochemical purity（>95%）.The measured Log D_7.4 value of the probe was-1.70±0.25（n=6）,which indicated that ¹²⁵I-CC49-c PNA was hydrophilic.The probe of ¹²⁵I-CC49-c PNA showed the satisfying in vitro stability and slow blood clearance(t_1/2,α=1.1 h,t_1/2,β=24.5 h).The biodistribution of ¹²⁵I-CC49-c PNA was assessed in LS174T tumor-bearing nude BALB/c mice to evaluate the optimum pretargeting interval.The results showed the high radioactivity uptake in tumor and little accumulation in non-target organs.The 24-hour point was chosen as the optimum pretargeting interval because the T/NT ratio reached the highest at this time point.The amplification pretargeting experiments was carried out based on the successful synthesis of ¹⁸F-c PNA,PAMAM G4-PNA and CC49-c PNA.All of them were mixed in the solution and the mixture was identified by SE-HPLC in radioactivity signal.The retention time of the CC49-c PNA/G4-PNA/¹⁸F-c PNA complex was 23.9min,which was earlier than PAMAM G4-PNA/¹⁸F-c PNA（36.5 min）and ¹⁸F-c PNA（38.1 min）.The single peak in radioactivity signal indicated the complete hybridization between ¹⁸F-c PNA and CC49-c PNA/G4-PNA in solution.The signal amplification study in cells demonstrated there was significant difference between amplification pretargeting group（10.09%）and conventional pretargeting group（6.09%）.The value of amplification factor（AF）was calculated as approximately 3.5 in LS174T cells.The PET/CT study of LS174T tumor-bearing nude BALB/c mice exhibited that there was higher tumor uptake of ¹⁸F-c PNA in amplification group(SUV_max=2.6%ID/g and SUV_ave=2.5%ID/g)than that in pretargeting group(SUV_max=2.1%ID/g and SUV_ave=1.6%ID/g)and nonspecific group(SUV_max=0.64%ID/g and SUV_ave=0.47%ID/g).The value of AF was calculated to be 1.8 according to PET/CT imaging.The results of biodistribution in LS174T xenograft mice was similar to the above results.The tumor uptake in amplification group was 3.21±0.77%ID/g,which was amplified approximately 2.7 times.This work demonstrated the three-step amplification pretargeting strategy was successfully applied in nuclear medicine imaging,which has great potential for diagnosis of colorectal cancer.