Pretargeted radioimmunotherapy and SPECT imaging of peritoneal carcinomatosis using bioorthogonal click chemistry: probe selection and first proof-of-concept

Rationale: Pretargeted radioimmunotherapy (PRIT) based upon bioorthogonal click chemistry has been investigated for the first time in the context of peritoneal carcinomatosis using a CEA-targeting 35A7 mAb bearing trans-cyclooctene (TCO) moieties and several 177Lu-labeled tetrazine (Tz) radioligands. Starting from three Tz probes containing PEG linkers of varying lengths between the DOTA and Tz groups (i.e. PEGn = 3, 7, or 11, respectively, for Tz-1, Tz-2, and Tz-3), we selected [177Lu]Lu-Tz-2 as the most appropriate for pretargeted SPECT imaging and demonstrated its efficacy in tumor growth control. Methods: An orthotopic model of peritoneal carcinomatosis (PC) was obtained following the intraperitoneal (i.p.) injection of A431-CEA-Luc cells in nude mice. Tumor growth was assessed using bioluminescence imaging. Anti-CEA 35A7 mAb was grafted with 2-3 TCO per immunoglobulin. Pretargeted SPECT imaging and biodistribution experiments were performed to quantify the activity concentrations of [177Lu]Lu-Tz-1-3 in tumors and non-target organs to determine the optimal Tz probe for the PRIT of PC. Results: The pharmacokinetic profiles of [177Lu]Lu-Tz-1-3 alone were determined using both SPECT imaging and biodistribution experiments. These data revealed that [177Lu]Lu-Tz-1 was cleared via both the renal and hepatic systems, while [177Lu]Lu-Tz-2 and [177Lu]Lu-Tz-3 were predominantly excreted via the renal system. In addition, these results illuminated that the longer the PEG linker, the more rapidly the Tz radioligand was cleared from the peritoneal cavity. The absorbed radiation dose corresponding to pretargeting with 35A7-TCO followed 24 h later by [177Lu]Lu-Tz-1-4 was higher for tumors following the administration of [177Lu]Lu-Tz-2 (i.e. 0.59 Gy/MBq) compared to either [177Lu]Lu-Tz-1 (i.e. 0.25 Gy/MBq) and [177Lu]Lu-Tz-3 (i.e. 0.18 Gy/MBq). In a longitudinal PRIT study, we showed that the i.p. injection of 40 MBq of [177Lu]Lu-Tz-2 24 hours after the systemic administration of 35A7-TCO significantly slowed tumor growth compared to control mice receiving only saline or 40 MBq of [177Lu]Lu-Tz-2 alone. Ex vivo measurement of the peritoneal carcinomatosis index (PCI) confirmed that PRIT significantly reduced tumor growth (PCI = 15.5 ± 2.3 after PRIT vs 30.0 ± 2.3 and 30.8 ± 1.4 for the NaCl and [177Lu]Lu-Tz-2 alone groups, respectively). Conclusion: Our results clearly demonstrate the impact of the length of PEG linkers upon the biodistribution profiles of 177Lu-labeled Tz radioligands. Furthermore, we demonstrated for the first time the possibility of using bioorthogonal chemistry for both the pretargeted SPECT and PRIT of peritoneal carcinomatosis.


Material for chemical syntheses
Unless otherwise mentioned, all manipulations were performed under argon atmosphere; all reagents were purchased from the following commercial suppliers: Sigma-Aldrich, Acros Organics, Carlo Erba, TCI Europa, Alpha Aesar. DOTA-NHS was purchased from Chematech (Dijon, France). Anhydrous DMF, anhydrous trimethylamine, anhydrous pyridine were purchased from Acros Organics. THF was dried over a Pure Solv™ Micro Solvent Purification System (Sigma-Aldrich) with an alumina column. Dichloromethane was distilled over calcium hydride. Reactions were monitored by thin layer chromatography (TLC) on silica gel (60 F254; Alugram XTra G/UV254; Macherey-Nagel) or alumina gel (Alumina oxide 60A + F254 neutral; Macherey-Nagel) and visualized with UV light (UV lamp Fisher Bioblock Scientific, 365 nm or 254 nm). Purifications by flash column chromatography were performed on silica gel (Chromagel 60 ACC, 40-63 µm, Carlo Erba Reagents). Purifications on RP18 were conducted on a Combiflash EZ prep system (Teledyne Isco) with a RedispSeP C18 column (250 mm x 20 mm, pores 100 Å, particles 5 µm). Sep-Pak C18 light cartridges were purchased from Waters (Milford, MA, USA). Uncorrected melting points (mp) were measured on an IA9100 Digital Melting Point Apparatus. Infrared spectra (IR) were recorded in the range 4000-440 cm -1 on a Nicolet IS10 with attenuated total reflectance (ATR) accessory. Nuclear magnetic resonance (NMR) spectra were acquired on Bruker  or 400 operating at 200 or 400 MHz for 1 H NMR and 50 or 100 MHz for 13 C NMR, respectively. All 1 H and 13 C NMR spectra are reported in δ units, parts per million (ppm). Coupling constants were indicated in Hertz (Hz). The following abbreviations are used for spin multiplicity: s = singlet, d = doublet, t = triplet, quint = quintuplet, m =multiplet, and brs = broad singlet. High resolution mass spectra (HRMS) were recorded on an Alliance 2695 (Waters) liquid chromatography coupled with a Q-ToF micro (Waters/Micromass) spectrometer (UCA-START, Clermont-Ferrand, France).

2-(2-(2-(2-Azidoethoxy)ethoxy)ethoxy)ethan-1-amine (4) 1
To a solution of compound (3) (4.28 g, 17.52 mmol) in a 0.65 M aqueous solution of H3PO4 (23 mL) at RT was added dropwise a solution of PPh3 (15.25 mmol) in diethyl ether (20 mL). The resulting mixture was stirred for 20 h. The reaction mixture was extracted with diethyl ether (3×30 mL). KOH (59.05 mmol) was added to the aqueous layer. Traces of ether were evaporated and the aqueous solution was cooled to 4 °C overnight. The precipitate was filtered off. Then, KOH (165.23 mmol) was added to the filtrate and the solution was extracted with diethyl ether (3×60 mL). The combined organic layer were washed with brine (50 mL), dried over MgSO4, filtered and evaporated under reduced pressure. The crude product was dissolved in CH2Cl2 (80 mL) and the solution was washed with 0.65 M H3PO4 (3×50 mL). The combined aqueous solutions were basified with KOH until pH 14, then were extracted with CH2Cl2 (3×60 mL). The combined organic layers were dried over MgSO4, filtered and evaporated under reduced pressure to afford compound (4) (46%) which was used without further purification in the next step. 1 H NMR (200 MHz, CDCl3)  2.75 (brs, 2H), 3.26-3.31 (m, 2H), 3.39-3.62 (m, 12H). These data are in agreement with those of the literature. 1

Access to TzPEGnDOTA
Compound (19) was synthesized according to published procedure of Rossin et al. 13

Materials for radiolabeling
[ 177 Lu]Lutetium solution was purchased from ITG (Germany) as a [ 177 Lu]LuCl3 solution in 0.04 M aq. HCl. Water was distilled and deionized (18 MΩ/cm) by means of a Milli-Q water filtration system (Millipore). The labeling buffers were treated with Chelex-100 resin (BioRad Laboratories) overnight, then filtered through a "rapid flow" corning with a PES membrane (Thermofisher) and stored at 4 °C. ITLC-SG plates were purchased from Agilent Technologies. ITLC-SG were performed with citrate mobile phase (0.025 M, pH 5) and analyzed with Minigita DUAL radio-TLC scanner. Mobile phase (100 mL) was made up from a combination of citric acid (3.45 g) and sodium citrate (6.94 g) in MilliQ water. Each radiolabeling reaction was monitored by ITLC-SG (free 177 Lu migrated to the solvent front while [ 177 Lu]Lu-Tz derivative remained at the baseline). The purifications by semi-preparative reversed phase-high pressure liquid chromatography (RP-HPLC) were performed on a Perkin Elmer system consisting of a Flexar LC autosampler, a series 200 pump, a Peltier column oven, a vacuum degasser and a PDA and GabiStar Raytest detectors. Semi-preparative RP-HPLC purifications were carried out on a Waters SymmetryPrep TM C18 column (7.8 mm × 300 mm, 7 µm) using the following conditions: flow rate: 2 mL/min, eluent A (Milli-Q water+0.1% TFA), eluent B (CH3CN+0.1% TFA) and detection wavelengths set at 254 and 330 nm. (1) For TzPEG4 derivative, the elution starts with 10% eluent B (0 to 0.5 min), followed by two linear gradients, firstly 10% to 22% eluent B from 0.5 to 20 min, and secondly from 22% to 100% eluent B over 5 min, an isocratic elution with 100% B for 4 min, and finally returned to 10% B over 0.5 min.
(2) For TzPEG8 derivative, the elution begins with 20% eluent B from 0 to 2 min, followed by two successive linear gradients, firstly from 20% to 50% B over 23 min, and secondly from 50% to 100% B over 5 min; 100 % B is kept for 30 to 35 min, and finally returned to 20 % B for 35 to 40 min. Analytical RP-HPLC analyses were performed on an Agilent series 1100 HPLC system equipped with an online degasser, a quaternary pump, an automatic sampler, a DAD detector and coupled to a 500TR series (ULTIMO-FLO TM ). Analytical RP-HPLC analyses were carried out on an Agilent Zorbax extend C18 column (4.6 mm × 150 mm, 5 µm) using the following conditions: analysis time: 20 min, flow rate: 1 mL/min, eluent A (Milli-Q water+0.1% TFA), eluent B (CH3CN+0.1% TFA) and detection wavelengths set at 254 nm and 330 nm. The elution program for TzPEG4 derivative starts with an isocratic elution with 25% eluent B for 3.5 min, followed by a linear gradient from 25% to 50% eluent B over 3.5 min and an isocratic elution with 50% eluent B for 13 min. The optimized elution program for TzPEG8 or TzPEG12 derivatives consists of an isocratic elution with 5% eluent B for 3.5 min, followed by a linear gradient from 5% to 50% eluent B for 3.5 min, and ends with an isocratic elution with 50% B for 13 min. The molar activity (Am) was calculated as following [(%radiochemical purities)/(numbers of moles of cold compound)]*radioactivity, where numbers of moles and radioactivity were expressed in µmol and GBq, respectively. For method A, number of moles of "cold compound" was determined using calibration curve by integrating the corresponding peak detected by UV obtained by analytical chromatography RP-HPLC. The radioactivity corresponds to the radioactivity injected into HPLC. For Method B, number of moles of "cold compound" refers to the initial quantity of precursor and radioactivity corresponds to the final radioactivity after formulation.

Radiolabeling of TzPEGnDOTA derivatives
Method A : To a solution of TzPEGnDOTA derivative (Tz-1-3) (100 µg) in NaOAc buffer (0.025 M, pH 7.0, 100 µL) was added a suitable amount of [ 177 Lu]LuCl3 in 0.04 M HCl (40-500 MBq) and the reaction mixture was heated at 50 °C until completion of the reaction (20 min, ITLC monitoring). The reaction mixture was purified by semi-preparative (radio)-RP-HPLC as mentioned above. The collected radiotracer fraction was evaporated under reduced pressure. The residue was diluted with Milli-Q water (20 mL) and purified by adsorption on a Sep-Pak C18 light cartridge. Elution with 0.6 mL of EtOH followed by concentration under reduced pressure afforded the desired radiotracer which was then formulated in sterile isotonic saline. The final solution contained less than 10 % of EtOH (v/v). The identity and radiochemical purity of radiotracer was confirmed by analytical radio-RP-HPLC. Molar activity (MA) was determined on the basis of a UV/mass calibration curve carried out under analytical radio-RP-HPLC conditions using chromatograms recorded at 330 nm.
Method B : To a solution of TzPEGnDOTA derivative (Tz-1-4) (25µL of 100 µg/100 µL aliquot in DMSO) in NaOAc buffer (0.25 M, pH 5.5, 200 μL) was added a suitable amount of [ 177 Lu]LuCl3 in 0.04 M HCl (40-500 MBq) and the reaction mixture was kept at RT until completion of the reaction (10 min, ITLC). The reaction mixture was then diluted with Milli-Q water (20 mL) and purified by adsorption on a Sep-Pak C18 light cartridge (Waters, Milford, MA, USA). Elution with 0.6 mL of ethanol followed by concentration under reduced pressure provided the desired radiotracer which was then formulated in sterile isotonic saline. The final solution contained less than 10 % of EtOH (v/v). The identity and radiochemical purity of radiotracer were confirmed by analytical radio-RP-HPLC. Molar activity (MA) was determined on the basis of a UV/mass calibration curve carried out under analytical radio-RP-HPLC conditions using chromatograms recorded at 330 nm.