Optimization of IEDDA bioorthogonal system: Efficient process to improve trans-cyclooctene/tetrazine interaction.

The antibody pretargeting approach for radioimmunotherapy (RIT) using inverse electron demand Diels-Alder cycloaddition (IEDDA) constitutes an emerging theranostic approach for solid cancers. However, IEDDA pretargeting has not reached clinical trial. The major limitation of the IEDDA strategy depends largely on trans-cyclooctene (TCO) stability. Indeed, TCO may isomerize into the more stable but unreactive cis-cyclooctene (CCO), leading to a drastic decrease of IEDDA efficiency. We have thus developed both efficient and reproducible synthetic pathways and analytical follow up for (PEGylated) TCO derivatives, providing high TCO isomeric purity for antibody modification. We have set up an original process to limit the isomerization of TCO to CCO before the mAbs' functionalization to allow high TCO/tetrazine cycloaddition.

Antibody PEGylation in bioorthogonal pretargeting with trans-cyclooctene/tetrazine cycloaddition: in vitro and in vivo evaluation in colorectal cancer models.

Bioorthogonal chemistry represents a challenging approach in pretargeted radioimmunotherapy (PRIT). We focus here on mAb modifications by grafting an increase amount of trans-cyclooctene (TCO) derivatives (0 to 30 equivalents with respect to mAb) bearing different polyethylene glycol (PEG) linkers between mAb and TCO (i.e. PEG0 (1), PEG4 (2) and PEG12 (3)) and assessing their functionality. We used colorectal xenograft (HT29/Ts29.2) and peritoneal carcinomatosis (A431-CEA-Luc/35A7) as tumor cells/mAbs models and fluorescent tetrazines (TZ). MALDI-TOF MS shows that grafting with 2,3 increases significantly the number of TCO per mAb compared with no PEG. In vitro immunofluorescence showed that Ts29.2 and 35A7 labeling intensity is correlated with the number of TCO when using 1,3 while signals reach a maximum at 10 equivalents when using 2. Under 10 equivalents conditions, the capacity of resulting mAbs-1-3 for antigen recognition is similar when reported per grafted TCO and comparable to mAbs without TCO. In vivo, on both models, pretargeting with mAbs-2,3 followed by TZ injection induced a fluorescent signal two times lower than with mAbs-1. These findings suggest that while PEG linkers allow a better accessibility for TCO grafting, it might decrease the number of reactive TCO. In conclusion, mAb-1 represents the best candidate for PRIT.

Synthesis, radiolabeling and preliminary in vivo evaluation of multimodal radiotracers for PET imaging and targeted radionuclide therapy of pigmented melanoma.

Melanin pigment represents an attractive target to address specific treatment to melanoma cells, such as cytotoxic radionuclides. However, less than half of the patients have pigmented metastases. Hence, specific marker is required to stratify this patient population before proceeding with melanin-targeted radionuclide therapy. In such a context, we developed fluorinated analogues of a previously studied melanin-targeting ligand, N-(2-diethylaminoethyl)-6-iodoquinoxaline-2-carboxamide (ICF01012). These latter can be labeled either with (18)F or (131)I/(125)I for positron emission tomography imaging (melanin-positive patient selection) and targeted radionuclide therapy purposes. Here we describe the syntheses, radiosyntheses and preclinical evaluations on melanoma-bearing mice model of several iodo- and fluoro(hetero)aromatic derivatives of the ICF01012 scaffold. After preliminary planar gamma scintigraphic and positron emission tomography imaging evaluations, [(125)I]- and [(18)F]-N-[2-(diethylamino)ethyl]-4-fluoro-3-iodobenzamides ([(125)I]4, [(18)F]4) were found to be chemically and biologically stable with quite similar tumor uptakes at 1 h p.i. (9.7 ± 2.6% ID/g and 6.8 ± 1.9% ID/g, respectively).