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New fluorine-18 pretargeting PET imaging by bioorthogonal chlorosydnone-cycloalkyne click reaction Mylène Richard,a Charles Truillet,a Vu Long Tran,a Hui Liu,b Karine Porte,b Davide Audisio, b Mélanie Roche,a Benoit Jego,a Sophie Cholet,c François Fenaille,c Bertrand Kuhnast,a Frédéric Taranb* and Simon Specklina*

a.UMR 1023 IMIV, Service Hospitalier Frédéric Joliot (SHFJ), CEA, Inserm, Université Paris Sud, CNRS, Université Paris-Saclay, Orsay, France. Email: simon.specklin@cea.fr b.Service de Chimie Bio-organique et Marquage DRF-JOLIOT-SCBM, CEA, Université Paris-Saclay, 91191 Gif-sur-Yvette (France). Email: frederic.taran@cea.fr c. Service de Pharmacologie et d’Immunoanalyse (SPI), CEA/DRF/JOLIOT, Université Paris Saclay, F-91191 Gif-sur-Yvette, France.

Supplementary Information

Table of content

I. General Information S2

II. Synthesis of chloro-sydnones S4

III. Synthesis and radiolabeling of [18F]-2 S5

IV. Kinetic studies S10

V. Preparation and in vitro labeling of CTX-Syd conjugate S12

VI. Cetuximab radiolabeling with 89Zr S16

VII. In vivo imaging S17

VIII. 1H NMR and 13C NMR spectra S19

Electronic Supplementary Material (ESI) for ChemComm.This journal is © The Royal Society of Chemistry 2019

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I. General Information

1. Synthesis

Organic solvents (Aldrich) were used without further purification. Unless otherwise noted, all other commercially available reagents and solvents were used without further purification. Purifications of reactions products were carried out by flash chromatography using Merck silica gel (40-63 µm). Preparative TLC was done on silica gel GF 1000µm 20X20cm from Analtech 1H NMR (400 MHz), 13C NMR (100 MHz) were measured on a Brucker Avance 400 MHz spectrometer. Chemical shifts are reported in parts per million (ppm, δ) downfield from residual solvents peaks and coupling constants are reported as Hertz (Hz). Splitting patterns are designated as singlet (s), broad singlet (br. s), doublet (d), triplet (t), quartet (q) and quintet (quin). Splitting patterns that could not be interpreted or easily visualized are designated as multiplet (m). Electrospray mass spectra were obtained using an ESI-Quadripole autopurify, Waters (pump: 2545, mass: ZQ2000) mass Spectrometer. Absorbances were measured on a Varian Cary® 50 UV-Vis spectrophotometer. Fluorescence spectra were obtained on a HORIBA FluoroMax®-4 fluorimeter. Fluorescence analyses for kinetic studies were recorded on a molecular Device SpectraMax® M5e. MALDI-TOF spectra were obtained on an UltrafleXtreme MALDI-TOF mass spectrometer from Bruker Daltonics (Bremen, Germany) using 2,4,6-trihydroxyacetophenone as matrix (10 mg/mL in water/acetonitrile, 50:50, v/v containing 0.1% TFA).

2. Radiochemistry

No-carrier-added aqueous [18F]fluoride ion was produced via the [18O(p,n)18F] nuclear reaction by irradiation of a 2 mL [18O]water target (> 97%-enriched, CortecNet) on an Cyclone-18/9 cyclotron (18 MeV proton beam, IBA) and was transferred to the appropriate hot cell. Target hardware: commercial, 2-mL, two-port, stainless steel target holder equipped with a domed-end niobium cylinder insert. Target to hot cell liquid-transfer system: 60 m PTFE line (0.8 mm internal diameter ; 1/16 inch external diameter), 2.0 bar helium drive pressure, transfer time 3-6 min. Typical production of [18F]fluoride ion at the end of bombardment for a 20 µA, 30 minutes irradiation: 27-30 GBq. Radiosyntheses using fluorine-18, including the HPLC purifications, were performed in a 5-cm-lead shielded cell using a TRACERLab FX-FN or FX N Pro (GE Medical Systems). Zirconium-89 was purchased from PerkinElmer.

3. In vivo imaging

Cell line A431: A431 cell line is established from an epidermoid carcinoma (epidermis) of an 85-year-old female patient. It expresses abnormally high levels of Epidermal growth factor receptor (EGFR) and is thus used as a positive control for EGFR expression. It is also a cetuximab-sensitive cancer cell line. A431 was obtained from DSMZ (Braunschweig, Germany), and cultured in DMEM supplemented with 10% fetal bovine serum (FBS, PAA, Coelbe, Germany), L-glutamine, penicillin and streptomycin (Invitrogen, Frankfurt, Germany).

Animal experiments: All experiments involving animals (rodents) were conducted according to the European directive 2010/63/EU and its transposition in the French law (Décret n° 2013-118). Animal experiments were conducted at the imaging facility CEA-SHFJ (authorization D91-471-105/ethic committee n°44). Five to seven week old male nu/nu mice were purchased from Charles River. Mice received a subcutaneous bolus of 5 million of A431 in the flank for tumor imaging studies (n=6 for each radiotracer condition studied).

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Image reconstruction: All the images were reconstructed using a 2D OSEM iterative algorithm. The regions of interest (ROI) corresponding to significant uptake of each tracer were delineated in the images using pMOD software. The activity in the organs was plotted versus time to generate a time activity curve.

Statistics: All statistical analysis was performed used PRISM software. Statistically significant differences in the data were determined using an unpaired Student’s t test. Changes at the 95% confidence level (P < 0.05) were qualified as statistically significant.

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II. Synthesis of chloro-sydnone

Chlorosydnone 3 was synthesized according to our previously described procedure.1

4-chloro-sydnone, 4

To a solution of 3 (712 mg, 3 mmol, 1 eq.) in 10 mL DMF was added NHS (414 mg, 3.6 mmol, 1.2 eq.) and DCC (1236 mg, 6 mmol, 2 eq.). The mixture was stirred overnight, and complete conversion has been shown by LC-MS analysis. After addition of water, the mixture was extracted with EtOAc (3x 50 mL). The crude was purified with EtOAc:hexane=1:2 to 1:1 and the compound SI1 was obtained as a yellow solid (470 mg, 46 %).

SI1 (101.1 mg, 0.3 mmol, 1 eq.) and SI2 (79.5 mg, 0.3 mmol, 1 eq.) were dissolved in 3 mL DCM at room temperature and NMM (60.6 mg, 0.6 mmol, 2 eq.) was added. The mixture was stirred at room temperature overnight. The solvent was removed under vacuum and the crude mixture was purified by preparative TLC (DCM:MeOH 95:5) to give compound 4 as a yellow oil (31 mg, 0.09 mmol, 30 %).

1H NMR (MeOD, 400 MHz): δ 8.17 (d, J = 8.7 Hz, 2H), 7.90 (d, J = 8.7 Hz, 2H), 3.74-3.60 (m, 20H).

LC-MS: C20H26ClN3O9 [M+H]+ 488.1.

HRMS: calcd for C20H27ClN3O9Na (M+H+): 488.1430. Found: 488.1426.

1 L. Plougastel, O. Koniev, S. Specklin, E. Decuypere, C. Créminon, D. A. Buisson, A. Wagner, S. Kolodych, F. Taran, Chem.

Commun. 2014, 50 (66), 9376.

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III. Synthesis and radiolabeling of [18F]-2

1. Synthesis of precursor 1 and non-radioactive reference 2

tert-Butyl 3-(2-(2-hydroxyethoxy)ethoxy)propanoate (SI3).2

To a suspension of NaH (660 mg, 27.5 mmol, 1.1 eq) in anhydrous THF (50 mL) was added diethylene glycol (9.28 g, 87 mmol, 3.5 eq) at 0 °C. After 15 min at 0 °C, tert-butyl acrylate (3.20 g, 25 mmol, 1 eq) was added and the mixture was stirred 16 h at RT. An HCl 1 M solution was added dropwise until pH~1 is reached and a saturated solution of NaHCO3 was then slowly added. The resulting mixture was extracted with DCM and the combined organic extracts were dried over Na2SO4 and concentrated under reduced pressure. The crude residue was purified by column chromatography (heptane/acetone 80/20 to 60/40) to afford SI3 (2.531 g, 43%) as a colorless oil.

1H NMR (CDCl3, 400 MHz): δ 3.76-3.68 (m, 4H), 3.67-3.58 (m, 6H), 2.53-2.47 (m, 3H), 1.44 (s, 9H).

13C NMR (CDCl3, 100 MHz): δ 170.9, 80.6, 72.4, 70.3 (2C), 66.8, 61.7, 36.1, 28.0 (3C).

tert-Butyl 3-(2-(2-(tosyloxy)ethoxy)ethoxy)propanoate (SI4).3

To a solution of SI3 (1.17 g, 5.00 mmol, 1 eq) in DCM (10 mL) were added tosyl chloride (1.33 mg, 7.00 mmol, 1.4 eq), Et3N (0.71 mL, 7.00 mmol, 1.1 eq) and DMAP (61 mg, 0.50 mmol, 0.1 eq). The mixture was stirred 16 h at RT and a saturated solution of NH4Cl was then added. The mixture was extracted with DCM and the combined organic extracts were dried over Na2SO4 and concentrated under reduced pressure. The crude residue was purified by column chromatography (heptane/EtOAc 75/25) to afford SI4 (1.81 g, 93%) as a colorless oil.

1H NMR (CDCl3, 400 MHz): δ 7.78 (d, 3J=8.4 Hz, 2H), 7.33 (d, 3J=8.4 Hz, 2H), 4.14 (t, 3J=4.8 Hz, 2H), 3.69-3.63 (m, 4H), 3.54 (m, 4H), 2.47 (t, 3J=6.4 Hz, 2H), 2.44 (s, 3H), 1.43 (s, 9H).

13C NMR (CDCl3, 100 MHz): δ 170.8, 144.7, 132.9, 129.8 (2C), 127.9 (2C), 80.5, 70.6, 70.2, 69.2, 68.6, 66.8, 36.1, 28.0 (3C), 21.6.

3-(2-(2-Fluoroethoxy)ethoxy)propanoic acid (SI5).

2 Werther, P.; Möhler, J. S.; Wombacher, R. Chem. Eur. J. 2017, 23, 18216–18224. 3 Wosnick, J. H.; Mello, C. M.; Swager, T. M. J. Am. Chem. Soc. 2005, 127, 3400–3405.

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To a solution of SI4 (775 mg, 1.99 mmol, 1 eq) in THF (1 mL) was added a solution of TBAF (1 M in THF, 5.0 mL, 5.0 mmol, 2.5 eq) and the resulting mixture was then stirred at 50 °C for 2 h. After cooling to RT, the mixture was concentrated under reduced pressure and the crude residue was purified by column chromatography (heptane/acetone 96/4 to 94/6) to afford SI5 (261 mg, 55%) as a colorless oil.

1H NMR (CDCl3, 400 MHz): δ 4.56 (dm, 2JH-F=48.0 Hz, 2H), 3.77 (m, 1H), 3.74-3.68 (m, 3H), 3.64 (m, 4H), 2.50 (t, 3J=6.4 Hz, 2H), 1.45 (s, 9H).

13C NMR (CDCl3, 100 MHz): δ 170.9, 83.1 (d, 1JC-F=168 Hz), 80.5, 70.7, 70.3 (d, 2JC-F=19.8 Hz), 70.3, 66.9, 36.2, 28.0 (3C).

HRMS: calcd for C11H21FO4Na (M+Na+): 259.1316. Found: 259.1315.

3-(2-(2-Fluoroethoxy)ethoxy)propanoic acid (SI6).

To a solution of SI5 (73 mg, 0.31 mmol, 1 eq) in DCM (0.75 mL) was added TFA (176 µL, 1.54 mmol, 5 eq). The mixture was stirred 16 h at room temperature, then concentrated under reduced pressure and dried to afford SI6 (57 mg, quantitative) as a yellow oil which was used without any further purification.

1H NMR (CDCl3, 400 MHz): δ 9.29 (br s, 1H), 4.56 (dm, 2JH-F=47.7 Hz, 2H), 3.80-3.74 (m, 3H), 3.72-3.63 (m, 5H), 2.64 (t, 3J=6.3 Hz, 2H).

13C NMR (CDCl3, 100 MHz): δ 176.9, 83.1 (d, 1JC-F=169 Hz), 70.6, 70.43, 70.38 (d, 2JC-F=19.4 Hz), 66.3, 34.8.

HRMS: calcd for C7H14FO4 (M+H+): 181.0871. Found: 181.0870.

DBCO-F (2)

To a solution of SI6 (22.5 mg, 125 µmol, 1.25 eq) in DCM (2 mL) were added N,N′-disuccinimidyl carbonate (30.7 mg, 120 µmol, 1.2 eq) and DMAP (2.4 mg, 20 µmol, 0.2 eq) at RT. After stirring for 16 h, DBCO-NH2 (27.6 mg, 100 µmol, 1 eq) was added and the mixture was further stirred for 7 h at RT. The mixture was diluted in DCM and the resulting solution was successively washed with a 1 M HCl

S7

solution and with a saturated solution of NaHCO3, dried over Na2SO4 and concentrated under reduced pressure. The crude residue was purified by column chromatography (heptane/acetone 60/40 to 50/50) to afford 2 (37 mg, 84%) as a yellow oil.

1H NMR (CDCl3, 400 MHz): δ 7.67 (d, 3J=7.6 Hz, 1H), 7.43-7.27 (m, 7H), 6.53 (br s, 1H), 5.13 (d, 2J=13.6 Hz, 1H), 4.53 (dm, 2JH-F=47.6 Hz, 2H), 3.75 (m, 1H), 3.71-3.60 (m, 5H), 3.60-3.47 (m, 3H), 3.38-3.21 (m, 2H), 2.48 (ddd, 2J=16.4 Hz, 3J=7.2 Hz, 3J=4.4 Hz, 1H), 2.32 (m, 2H), 1.96 (ddd, 2J=16.4 Hz, 3J=7.2 Hz, 3J=4.4 Hz, 1H).

13C NMR (CDCl3, 100 MHz): δ 172.0, 171.0, 151.0, 148.0, 132.0, 129.0, 128.5, 128.3, 128.2, 127.8, 127.2, 125.5, 123.0, 122.5, 114.7, 107.7, 83.1 (d, 1JC-F=167.6 Hz), 70.5, 70.3 (d, 2JC-F=20.6 Hz), 70.2, 67.1, 55.4, 36.8, 35.1, 34.7.

HRMS: calcd for C25H28FN2O4S (M+H+): 439.2028. Found: 439.2026.

3-(2-(2-(Tosyloxy)ethoxy)ethoxy)propanoic acid (SI7)

To a solution of SI4 (1.16 g, 3.00 mmol, 1 eq) in DCM (10 mL) was added TFA (1.15 mL, 15.0 mmol, 5 eq). The mixture was stirred 6 h at room temperature, then concentrated under reduced pressure and dried to afford SI7 (1.00 g, quantitative) as a colorless oil which was used without any further purification.

1H NMR (CDCl3, 400 MHz): δ 7.79 (d, 3J=8.3 Hz, 2H), 7.38 (br s, 1H), 7.34 (d, 3J=8.3 Hz, 2H), 4.15 (m, 2H), 3.73 (t, 3J=6.2 Hz, 2H), 3.68 (m, 2H), 3.57 (s, 4H), 2.61 (t, 3J=6.2 Hz, 2H), 2.44 (s, 3H).

13C NMR (CDCl3, 100 MHz): δ 176.4, 144.8, 132.8, 129.8 (2C), 127.9 (2C), 70.4, 70.3, 69.2, 68.6, 66.2, 34.7, 21.6.

HRMS: calcd for C14H21O7S (M+H+): 333.1003. Found: 333.1003.

DBCO-OTs (1).

To a solution of SI7 (41.5 mg, 125 µmol, 1.25 eq) in DCM (1.5 mL) were added N,N′-disuccinimidyl carbonate (30.7 mg, 120 µmol, 1.2 eq) and DIPEA (22 µL, 125 µmol, 1.25 eq) at RT. After stirring for 2 h, DBCO-NH2 (27.6 mg, 100 µmol, 1 eq) was added and the mixture was further stirred for 4 h at RT. The mixture was diluted in DCM and the resulting solution was successively washed with a 1 M HCl solution and with a saturated solution of NaHCO3, dried over Na2SO4 and concentrated under reduced

S8

pressure. The crude residue was purified by column chromatography (heptane/acetone 60/40 to 40/60) to afford 1 (53 mg, 90%) as a colorless oil.

1H NMR (CDCl3, 400 MHz): δ 7.78 (d, 3J=8.3 Hz, 2H), 7.65 (d, 3J=7.4 Hz, 1H), 7.42-7.23 (m, 9H), 6.53 (br t, 3J=5.3 Hz, 1H), 5.11 (d, 2J=13.9 Hz, 1H), 4.13 (t, 3J=4.7 Hz, 2H), 3.70-3.63 (m, 3H), 3.62-3.36 (m, 6H), 3.36-3.14 (m, 2H), 2.51-2.41 (m, 4H), 2.29 (m, 2H), 1.94 (ddd, 2J=16.7 Hz, 3J=7.4 Hz, 3J=4.1 Hz, 1H).

13C NMR (CDCl3, 100 MHz): δ 172.0, 170.9, 151.0, 148.0, 144.8, 132.9, 132.0, 129.8 (2C), 129.0, 128.5, 128.3, 128.2, 127.9 (2C), 127.8, 127.2, 125.6, 123.0, 122.4, 114.7, 107.7, 70.4, 70.1, 69.2, 68.6, 67.1, 55.4, 36.8, 35.0, 34.7, 21.6.

HRMS: calcd for C32H35N2O7S (M+H+): 591.2159. Found: 591.2154.

2. Radiosynthesis of [18F]-2

The aqueous solution containing [18F]fluoride anions was automatically transferred to the TRACERLab FX-FN or FX N Pro after the end of irradiation. The irradiated water was then sucked through an anion exchange cartridge (Sep-Pak® Accell Plus QMA Plus Light cartridge, Waters) to fix [18F]fluoride anions and remove the enriched water which was separately collected. The [18F]fluoride anions were eluted from the resin and transferred to the reactor with a K2CO3/K222 solution (water and acetonitrile 30/70, 1 mL) containing 2 mg of K2CO3 and 12 to 15 mg of Kryptofix 222). Finally, the K[18F]F-K222 complex was prepared by evaporation of the solution in two heating steps : (i) first at 60 °C for 7 min under reduced pressure along with a stream of helium and then (ii) at 120 °C for 5 min under vacuum. A solution of precursor 1 (5 mg) in acetonitrile (1 mL) was added to the dried K[18F]F-K222 complex and the resulting mixture was heated to 90 °C for 15 minutes. After cooling to 35 °C, the crude was diluted with HPLC-eluent (1 mL) and the mixture was transferred through an alumina cartridge (Sep-Pak® Alumina N Plus Long, Waters) and collected before HPLC injection. A second portion of HPLC-eluent (2 mL) was added to the reactor and transferred via the alumina cartridge to the crude mixture. The resulting solution was submitted to semi-preparative HPLC purification (Zorbax SB C18 column, Agilent) using an acetonitrile/water 65:35 mixture (0.1% TFA, 5 mL/min) as eluent. Purification was monitored by both UV and radioactivity detection and the desired compound [18F]-2 was collected at a retention time of 28-30 min. For the final formulation, water (20 mL) was added to the collected fraction and this mixture was transferred through a C18 cartridge (Sep-Pak® C18 Plus Short cartridge, Waters) which was then washed with water (10 mL). Desired [18F]-2 was recovered after elution of the cartridge with ethanol (1.5 mL).

In a typical radiosynthesis, an activity of 4.13 GBq of [18F]-2 was obtained from an initial activity of 40.4 GBq of [18F]fluoride after a synthesis time of 80 min (17% RCY). Chemical and radiochemical purities were assessed on an aliquot by analytical HPLC using non-radioactive reference 2 as standard. Molar activity of [18F]-2 was determined from HPLC quantification (UV, triplicate) correlated with the corresponding activity.

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Figure SI. 1. (A) TRACERLab FX-FN synoptic, (B) semi-preparative HPLC chromatogram, (C) analytical HPLC chromatograms.

UV (280 nm)

RAD

UV (290 nm)

RAD

UV (290 nm)

A

B C

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IV. Kinetic studies

Reactions of acid-4-chloro-sydnone with DBCO were carried out in PBS with 4 % DMSO and in plasma with 4 % DMSO at 5 μM concentration of sydnone and 7.5 μM concentration of DBCO using the following procedure. To 3.94 mL of PBS 10 mM (or plasma) was added 20 µL of sydnone (1 mM in DMSO) and 20 µL of the solution of DBCO (1.5 mM in DMSO) in a quartz cuve and the fluorescence of the solution was measured every 10 seconds. λex = 315 nm, λem = 385 nm. This operation was repeated 3 times.

Correspondences between fluorescence and concentrations were established by plotting the calibration curve obtained by measuring the fluorescence at 385 nm (λex = 315 nm) of the click product at different concentration in both media (PBS and plasma, Table SI. 1).

Table SI. 1. Calibration curve of the click product in PBS (A) and in plasma (B).

Second order reaction rate was determined by plotting –ln([A]/[B])/([A]0 – [B]0) versus time and analyzing by linear regression (Equation SI. 1). Second order rate constant corresponds to the determined slope.

− ln[ ]

[ ]

[𝐴] − [𝐵]= 𝑘𝑡 + 𝑐𝑜𝑛𝑠𝑡

Equation SI. 1. [A]—concentration of iminosydnones (M); [B]—concentration of BCN (M); t—reaction time (s); k—reaction rate (M-1·s-1) Fluorescent measurement over time and linear regression curves for the click reaction between the 4-chloro-sydnone and DBCO in both media are illustrated in Table SI. 2.

y = 427736xR² = 0,9953

0,0E+00

5,0E+05

1,0E+06

1,5E+06

2,0E+06

2,5E+06

0,00 1,00 2,00 3,00 4,00 5,00 6,00

Fluo

resc

ence

at 3

85 n

m

Concentration (µM)

(A) Click product in PBS

y = 93447xR² = 0,9926

0,0E+005,0E+041,0E+051,5E+052,0E+052,5E+053,0E+053,5E+054,0E+054,5E+055,0E+05

0,00 1,00 2,00 3,00 4,00 5,00 6,00

Fluo

resc

ence

at 3

85 n

m

Concentration (µM)

(B) Click product in plasma

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Table SI. 2. Kinetic determination of the click reaction between chloro-sydnone and DBCO in PBS and in plasma. λex = 315 nm, λem = 385 nm.

Reaction in PBS k = 292 ± 4 M-1.s-1

Reaction in plasma k = 343 ± 41 M-1.s-1

0,0E+00

2,0E+05

4,0E+05

6,0E+05

8,0E+05

1,0E+06

1,2E+06

1,4E+06

1,6E+06

0 100 200 300 400 500 600 700

Fluo

resc

ence

inte

nsity

Time (s)

0,0E+00

1,0E+05

2,0E+05

3,0E+05

4,0E+05

5,0E+05

6,0E+05

0 100 200 300 400 500 600 700

Fluo

resc

ence

(S1)

Time (s)

y = 293,92x + 155996R² = 0,9892

y = 294,06x + 158561R² = 0,9989

y = 287,7x + 157987R² = 0,9794

160000

165000

170000

175000

180000

185000

190000

195000

0 20 40 60 80 100 120 140

-ln([

SM]/

([SM

]+D

))/D

Time (s)

y = 389,72x + 157095R² = 0,9671

y = 326,31x + 161949R² = 0,9614

y = 311,91x + 161459R² = 0,9791

165000

170000

175000

180000

185000

190000

195000

200000

205000

210000

0 20 40 60 80 100 120 140

-ln([

SM]/

([SM

]+D

))/D

Time (s)

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V. Preparation and in vitro labeling of CTX-Syd conjugate

Scheme SI. 1. Preparation of CTX-Syd.

CTX-Syd was prepared according to a reported procedure.Erreur ! Signet non défini. Briefly, NHS-PEG-Sydnone SI8 was firstly prepared by incubation of HOOC-PEG-sydnone, NHS (1 eq) and DCC (1 eq) in DMF at 25 °C for 1 hour. This solution of SI8 in DMF was then added to a solution of Cetuximab in labeling buffer (2 mg/mL in 0.1 M NaHCO3 pH 8.3). Two SI8/Cetuximab ratios were examined; 50/1 and 100/1 (DMF volume did not exceed 5 % of the total reactional volume). The mixtures were then incubated at 25°C for 4 hours and the resulting CTX-Syd was purified via a minitrap cartridge (500 µL loading, 1 mL elution). The fraction purities were then assessed by size-exclusion chromatography (Superdex® 200 Increase 10/300 GL, Figure SI. 2).

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Figure SI. 2. Size-exclusion analysis of crude mixture (A) and Minitrap-purified fraction (B).

The mean numbers of sydnones moieties grafted per antibody were determined by mass spectrometry analysis (MALDI) (Figure SI. 3). An average number of 4 (50 equivalents) and 8 (100 equivalents) sydnones were incorporated. In vitro and in vivo experiments were carried out with the antibody containing eight sydnone moieties (CTX-Syd).

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Figure SI. 3. MALDI analysis of non-conjugated Cetuximab (A), CTX-Syd conjugate from reaction with 50 eq. NHS-PEG-sydnone SI8 (B) and CTX-Syd conjugate from reaction with 100 eq. NHS-PEG-Sydnone SI8 (C).

In vitro labeling of CTX-Syd with [18F]-2 by SPSAC. A solution of [18F]-2 in ethanol (10, 5 or 1 % v/v) was

added to CTX-Syd (1.48 mg/mL in PBS) and the resulting solutions were incubated at 37 °C for 1 hour.

After 1 hour, reaction mixtures were purified through Minitrap G-25 cartridges (500 µL loading, 1 mL

elution with PBS) to yield [18F]-CTX-Pyr. A control experiment with non-conjugated Cetuximab was

performed to assess specificity of SPSAC reaction (entry 1).

entry conditions

Ab

solution

(µL)

[18F]-2

solution

(EtOH, µL)

Conc.

Ab

(mg/mL)

Ratio

Ab/[18F]-

2

Labelling

yield (%)

Molar

activity

(GBq/µmol)

[18F]-2

incorporated

per Ab

1 control 95 5 1.20 1/0.43 0 - 0

2 10 % EtOH 90 10 1.33 1/0.73 48 27.7 0.35

3 5 % EtOH 95 5 1.41 1/0.37 45 13.0 0.16

4 1 % EtOH 99 1 1.47 1/0.08 45 2.8 0.04

Table SI. 3. In vitro SPSAC experiments. All experiments were carried out at 37 °C in PBS for 1 hour.

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Immunoreactive fraction of [18F]-CTX-Pyr. Determination of the immunoreactive fraction of [18F]-CTX-Pyr was performed in vitro on A431 cells following Lindmo excess antigen method.4 Briefly, 80 ng of the labeled antibody were added to 0.5–5x107 A431 cells in 0.5 ml of medium. The cells were incubated for 60 min at room temperature with continuous mixing to keep the cells in suspension. Cells were harvested by centrifugation, washed once to remove unbound antibody, and the cell pellets were measured in a gamma counter (Cobra II, Canberra-Packard). Percentage binding of [18F]-CTX-Pyr to A431 cells was calculated as the ratio between cpm of the cell pellets and the mean cpm total radioactive antibody. Percentage binding was graphed against inverse cell concentration and immunoreactivities calculated as the y intercept of the inverse plot of both values. An immunoreactive fraction >90% was found for the modified antibody [18F]-CTX-Pyr (Figure SI. 4).

Figure SI. 4. Immunoreactive fraction of [18F]-CTX-Pyr determined with Lindmo methods.

4 Lindmo, T.; Boven, E.; Cuttitta, F.; Fedorko, J.; Bunn, P. A. J. Immunol. Methods 1984, 72, 77–89.

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VI. Cetuximab radiolabeling with 89Zr

Functionalization and radiolabeling of Cetuximab with 89Zr was performed according to a protocol previously described.5 Briefly, to a solution of Cetuximab in saline (NaCl 0.9%, 0.5 mL, 2 mg/mL) was added 0.1 M sodium bicarbonate buffer to reach pH 9.0. A solution of p-isothiocyanatobenzyldesferrioxamine in DMSO (30 mM, 4 equiv) was added to the antibody solution dropwise while mixing vigorously. The reaction was allowed to incubate for 30 min at 37 °C, whereupon the reaction mixture was purified with a PD-10 column using a solution of gentisic acid (5 mg/ml in 0.25 M sodium acetate (pH 5.4–5.6)) as mobile phase. The resulting Cetuximab-DFO conjugate solution was aliquoted and stored at −20 °C un l me of use.

On the day of the imaging process, Cetuximab-DFO conjugate was radiolabeled with 89Zr at 37 °C for 60 min according to the described procedure.5 The final radiolabeled solution was purified by PD-10 column using a solution of gentisic acid (5 mg/ml in 0.25 M sodium acetate (pH 5.4–5.6)) as mobile phase. The radiochemical yield and purity were determined by radio-TLC analysis of instant thin-layer chromatography (ITLC) using 20 mm citric acid (pH 4.9–5.1) as mobile phase. In a typical radiosynthesis, 3.89 MBq of labeled Cetuximab [89Zr]-CTX-DFO (11 µg) was obtained with a radiochemical yield of 82%.

5 Vosjan, M. J. W. D.; Perk, L. R.; Visser, G. W. M.; Budde, M.; Jurek, P.; Kiefer, G. E.; van Dongen, G. A. M. S. Nat. Protocols 2010, 5, 739–743.

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VII. In vivo imaging

Pharmacokinetics (PK) and biodistribution studies in tumor bearing mice. All radiotracers were administered via tail vein injection in <150 µL saline solution. Mice were anesthetized and imaged with a Siemens Inveons small animal PET/CT or PET at dedicated time points post injection. Biodistribution studies were performed via PET imaging analysis with regions of interest delineating the different organs. For the distribution of Cetuximab radiolabeled with zirconium-89, mice bearing tumor were scanned for 30 min at 24 h, 48 h, 72 h and 7 days after injections (0.24MBq/g) and the tumor uptake was determined for each points (Figure SI. 5). The timeline for the pretargeting experiment is represented in Figure SI. 6. For the biodistributions of [18F]-2 alone and in the pretargeting imaging of CTX-Syd (with and without blocking with an excess of Cetuximab), mice were submitted to a dynamic scan during 60 min after injection of [18F]-2 (0.6 MBq/g) and a scan of 30 min at 4 h after injection. The dynamic scan allows a 30 min static PET scan, 30 min after injection. Each mouse was imaged until more than 20 million coincident events were collected. The biodistribution of [18F]-2 in several organs was determined by drawing ROIs on images.

Figure SI. 5. Biodistribution data of [89Zr]-CTX-DFO acquired at different after injection in immunodeficient mice bearing A431 tumor xenograft.

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Figure SI. 6. Design of the imaging process: 1) PET-PK of [18F]-2 alone in mice bearing tumor, 2) PET-PK of [18F]-2 in mice bearing tumor preinjected 3 days before with CTX-Syd mAb. In the last group (2), two cohorts of animals were studied: (i) one preinjected with only CTX-Syd and (ii) a cohort preinjected with CTX-Syd in mixture with a large excess of Cetuximab.

Figure SI. 7. Representative time activity curves of [18F]-2 alone (A.) and with preinjection of CTX-Syd in the pretargeting experiment (B.).

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VII. 1H NMR and 13C NMR spectra

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