Pre-targeted immuno-PET based on bioorthogonal chemistry for imaging PD-1 positive non-small cell lung cancer

1518 Objectives: The long-lived radionuclides labeled pembrolizumab are used to visualize PD-1 expression through nuclear imaging. The sluggish pharmacokinetic of long-lived radionuclides labeled pembrolizumab demonstrated sustained radiation to the patient and dampen its translation to the clinic for imaging of cancer. Compared with the traditional immuno-PET strategy, a pre-targeting bioorthogonal chemistry approach of utilizing short-lived radionuclide (F-18, Ga-68, Cu-64, etc.) labeled small molecules which can rapidly and site-specifically conjugate with pembrolizumab pre-injected in vivosignificantly could decrease radiation exposure of healthy tissues. Moreover, due to the rapid elimination of radiolabeled small molecules, this approach could result in a high tumor-to-background contrast. Our study aimed to develop a pre-targeted immuno-PET PD-1 positive non-small cell lung cancer imaging using Reppe anhydride/ tetrazine bioorthogonal chemistry. Methods: Reppe anhydride was labeled with 64Cu by NOTA. Tetrazines were conjugated with pembrolizumab using EDC/NHS chemistry. PD-1 positive non-small cell lung cancer cell lines A549 were used in this study. The pre-targeted immuno-PET study was performed in mice bearing tumors, and the tumor/muscle (T/M) and tumor/blood (T/B) ratio were calculated. The tetrazine modified pembrolizumab was injected intravenously into A549 tumor xenograft models. 64Cu labeled Reppe anhydride was injected 24 h after pembrolizumab-tetrazine pre-treatment.Results: 64Cu labeled Reppe anhydride was obtained with a radiochemical yield of 65 ± 3.3% with a synthesis duration of 1 h. The radiochemical purity was 97.1 ± 0.7%, and the specific activity was up to 3.7 ± 1.5 GBq/μmol. The PET probe was stable in fetal bovine serum (FBS) and phosphate buffered saline (PBS, pH = 7.4). The pre-targeted immuno-PET PET/CT imaging revealed that the accumulation of PET probe in A549 tumors (5.6 ± 1.2 %ID/g, 4 h p.i.) pre-treated with pembrolizumab-tetrazine was significantly higher compared to the control group without antibody pre-treatment (0.5 ± 0.3 %ID/g, 4 h p.i.). Meanwhile, the T/NT ratios (tumor-to-muscle, 8.1 ± 1.5, 4 h p.i.) of PET probe were much higher than control group (0.8 ± 0.3, 4 h p.i.). The accumulation of probe in gallbladder (6.3 ± 1.8 and 4.7 ± 1.2 %ID/g at 4 h p.i., respectively) and kidney (7.3 ± 1.2 and 6.8 ± 1.1 %ID/g at 4 h p.i., respectively) were observed. Conclusion: The visualization of PD-1 positive non-small cell lung cancerusing pre-targeted immuno-PET imaging through bioorthogonal chemistry is technically feasible. The radioisotope labeled Reppe anhydride proved was as efficient as radiolabeled tetrazine modified PD-1 specific monoclonal antibodies pembrolizumabin vivo. Furthermore, this pre-targeting strategy resulted in low radiation exposure to healthy tissues in PET imaging due to the specificity of the in vivoPET probe. The tumor-specific accumulation and high tumor-to-background contrast indicated that pre-targeted immuno-PET imaging are powerful tools to visualize PD-1 expression in tumor models, and potentially humans.Acknowledgements: This work is supported by Natural Science Foundation of Beijing Municipality (Nos 7194295).