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1 upporting Information Pretargeted Immuno-PET based on Bioorthogonal Chemistry for Imaging EGFR Positive Colorectal Cancer Xudong hi, Kai Gao, Hao Huang and Ran Gao* *Institute of Laboratory Animal cience, Chinese Academy of Medical ciences (CAM) & Comparative Medicine Center, Peking Union Medical Collage (PUMC), Beijing, 10021, People s Republic of China gaoran201708@163.com Experiment section Materials Bioorthogonal ligation agent carboxyl-tetrazine (4-(6-methyl-1,2,4,5-tetrazin-3-yl) benzoic acid) and Reppe anhydride derivatives (7,8--[tert-butyl(2-aminoethyl) carbamate]succinimide endo-tricyclo-[ ]deca-3,9-diene) were purchased from Bioorth Biotech Inc. (anjing, China). The p-c-bn-ta was purchased from Macrocyclics, Inc. (Dallas, TX). 1-ethyl-3-[3-(dimethylamino)propyl] carbodiimide (EDC), -Hydroxysuccinimide (H), aluminum chloride hexahydrate (AlCl 3 6H 2 ) and other solvents and reagents were purchased from igma-aldrich. Chemical ynthesis and Radiochemistry -Boc protected Reppe anhydride derivative 1 (10.0 mg, 0.03 mmol) was dissolved in CH 3 H (1.0 ml). CF 3 CH (0.5 ml) was added and stirred 4 hours at room temperature and then evaporated to afford amino functionalized Reppe anhydride TFA salt. The resulting salt was dissolved in CH 3 C, after which Et 3 (10.0 mg, 0.10 mmol) was added, followed by p-c-bn-ta (17.0 mg, 0.03 mmol). This resulting solution was stirred for 1 hour at room temperature and then evaporated. The crude product was purified by flash column chromatography on silica gel (mobile phase: CH 3 H: CH 2 Cl 2 = 4:1) to afford 12.0 mg product as a pink solid in

2 55% yield. [M+a] + m/z calcd. for [C34H4268Ha] , found The 18 F-radiolabeling reaction was achieved in one step via the simple [ 18 F]AlF chelation reaction in aqueous phase. Briefly, 18 F was eluted from a QMA ep-pak light cartridge with 0.4 M KHC 3, and 200 µl fractions were collected. 3 µl AlCl 3 (2mM) mixed with 100 µl Reppe anhydride-ta (1 mg/ml) in 0.1 M sodium acetate (ph 4.0) buffer, 185 MBq of 18 F-fluoride were added. The reaction mixture was incubated at 90 C for 20 min. The radiolabeled Reppe anhydride-ta were purified using a HLB cartridge. The mixture was loaded onto the asis HLB 3-cc cartridge (Waters). The cartridge was washed with 1 ml of 0.9% saline solution to eliminate unreacted [ 18 F]AlF. The final product was eluted with 200 µl 80% EtH into the product vial containing 2 ml of 0.9% saline solution ready for further in vitro and in vivo experiments. The radiochemical purity was confirmed by Radio-TLC (thin layer chromatography). Then octanol-water partition coefficient value was calculated by determining the radioactivity in octanol and PB. Briefly, 3.7 MBq [ 18 F]AlF-TA-Reppe anhydride was added to a vial containing 0.5 ml of octanol and 0.5 ml of PB (ph = 7.4). The mixture was then vortexed vigorously for 10 min. ubsequently, the mixture was centrifuged at 12,500 rpm for 5 min to ensure the complete separation of layers. Aliquots of the aqueous and octanol layers were collected, measured in a gamma counter (n=3). The octanol-water partition coefficient A value was calculated as log P= log octonal (A octanal : radioactivity in octanal, A water : A water radioactivity in water). In vitro stability The stability of PET probe was determined by mixing 3.7 MBq (100 µl) of [ 18 F]AlF-TA-Reppe anhydride with 900 µl of human serum at room temperature with vortexing. The radiochemical purity was measured by radio-tlc at different time points. Antibody bioconjugation Carboxyl-tetrazine (4-(6-methyl-1,2,4,5-tetrazin-3-yl)benzoic acid) was activated by EDC and H in CH 3 C for 2 h with a Tz: EDC: H molar ratio of 1:2:2. The

3 tetrazine succinimidyl ester was obtained without purification. Therapeutic monoclonal antibodies cetuximab or panitumumab (200 µg) in 200 µl DM was incubated for 3 h at 4 C with 20 molar excess of crude tetrazine succinimidyl ester. The antibody-tetrazine conjugates were then purified using a PD-10 column with PB buffer as the mobile phase. on-immunized control antibody IgG from mouse serum (migg) was modified in a similar manner. The molar ratio of conjugated tetrazine to cetuximab and panitumumab was determined according to the UV-visible absorbance of tetrazine at 540 nm and antibody at 280 nm, respectively. The IED-DAR reaction between [ 18 F]AlF-TA-Reppe anhydride and the tetrazine modified mabs was demonstrated by incubating an equimolar amount of [ 18 F]AlF-TA-Reppe anhydride (7.4 MBq, 50 µl PB buffer, ph = 7.4) and cetuximab-tetrazine conjugates (50 µg in 100 µl PB buffer) at room temperature for 10 min. A small aliquot of the reaction mixture was spotted onto a C18 TLC plate. TLC was performed using 90% MeC in H 2 as mobile phase and analyzed by radio-tlc. Cell lines and animal model HCT116 cell lines were cultured in DMEM (dulbecco's modified eagle medium) containing and supplemented with 10% fetal bovine, 100 µg/ml penicillin, and 100 µg/ml streptomycin. All cells were incubated at 37 C in a humidified 5% C 2 incubator. HCT116 cells ( ) were resuspended in 100 µl PB and injected subcutaneously into the left hind leg of nude mice (BALB/c). Tumors were measured every 5 days with precision calipers. The tumors were calculated using the equation V=0.5 D d 2 (V, tumor volume; D, tumor length; d, tumor width). After about 20 days, mice were used for experiments when the tumor size reached 100 mm 3. In vivo PET/CT Imaging and biodistribution experiment 14.9 ± 0.5 MBq [ 18 F]AlF-TA-Reppe anhydride was injected intravenously via tail vein into HCT116 tumor-bearing nude mice. At the time points of 15, 30 and 120 minutes post-injection, the mice (n=5) were anesthetized with isoflurane (2% in 100% medical 2 at 2ml/min) and placed on small animal PET/CT (iemens Inveon MM,

4 Germany). After 15 min scan, images were obtained after reconstruction using filtered back projection (FBP) algorithm. For quantitative analysis, RIs (region of interests) were drawn manually in the tumor, normal tissue, and major organs of each animal, using CT as guide. The percent injected dose per gram (%ID/g) was computed using Inveon Research Workplace 2.0 provided by iemens. Pretargeted immuno-positron emission tomography (PET) was also performed. ude mice with HCT116 xenografts were administered mabs-tetrazine (cetuximab-tetrazine or panitumumab-tetrazine, 200 µg in 200 µl of 0.9% saline) 48 h prior to injection of [ 18 F]AlF-TA-Reppe anhydride (14.9 ± 0.5 MBq) via tail vein. Control group of nude mice treated with non-immunized antibody migg-tetrazine was also injected of [ 18 F]AlF-TA-Reppe anhydride with same dose. Images were acquired at 120 minutes after probe injection. PET/CT scans and image analysis was performed using the same method as above. The mabs-tetrazine (cetuximab-tetrazine or panitumumab-tetrazine, 200 µg in 200 µl of 0.9% saline) pre-treated HCT116 tumor-bearing mice were injected intravenously through tail vein with [ 18 F]AlF-TA-Reppe anhydride (3.7 MBq, 100 µl). At the time points of 15, 30 and 120 minutes post-injection, the mice (n=5) was sacrificed and major organs (tumor, blood, liver, intestine, kidney, muscle, bone etc.) were picked to measure their weight and radioactivity (1480 Wizard γ-counter, Perkin Elmer), respectively. The uptake of [ 18 F]AlF-TA-Reppe anhydride in organs were calculated as %ID/g (the percent injected dose per gram). Using these data, tumor-to-blood ratios and tumor-to-muscle ratios were calculated.

5 a b HBoc 1) 20% CF 3 CH CH3H, rt, 4 h 2) p-c-bn-ta CH 3 C, rt, 1 h H H 1 2 H H 55% H AlCl 3, 18 F-fluoride sodium acetate buffer 90 C,20 min H H TD-TA-Al- 18 F 18 F H Al 30% RCY ε H2 Lys + CH EDC/H CH 3 C/DM 4 C,5 h Lys ε H mabs mabs-tetrazine c ε H Lys mabs-tetrazine H H 18 F H Al PB buffer, ph=7.4 ε H Lys H Al Figure 1. (a) chematic of the synthesis of TA-Reppe anhydrides and 18 F-radiolabeled Reppe anhydride derivatives. (b) ynthesis of mabs-tetrazine conjugates where mabs represents cetuximab or panitumumab. (c) Conjugation of mabs-tetrazine with [ 18 F]AlF-TA-Reppe anhydride gives conjugates rt,10 min 18 F H H Figure 2. The image of MALDI-TF mass spectrum of TA-Reppe anhydride

6 Figure 3. Radio-TLC characterization of before and after purification of [ 18 F]AlF-TA-Reppe anhydride Figure 4. In vitro stability of [ 18 F]AlF-TA-Reppe anhydride in human serum at room temperature.