Modular Assembly of Multimodal Imaging Agents through an Inverse Electron Demand Diels-Alder Reaction.

The combination of two imaging probes on the same biomolecule gives access to targeted bimodal imaging agents that can provide more accurate diagnosis, complementary information, or that may be used in different applications, such as nuclear imaging and fluorescence guided surgery. In this study, we demonstrate that dichlorotetrazine, a small, commercially available compound, can be used as a modular platform to easily assemble various imaging probes. Doubly labeled tetrazines can then be conjugated to a protein through a biorthogonal IEDDA reaction. A series of difunctionalized tetrazine compounds containing various chelating agents and fluorescent dyes was synthesized. As a proof of concept, one of these bimodal probes was conjugated to trastuzumab, previously modified with a constrained alkyne group, and the resulting dual-labeled antibody was evaluated in a mouse model, bearing a HER2-positive tumor. A significant uptake into tumor tissues was observed in vivo, by both SPECT-CT and fluorescence imaging, and confirmed ex vivo in biodistribution studies.

Site-specific near-infrared fluorescent labelling of proteins on cysteine residues with meso-chloro-substituted heptamethine cyanine dyes.

Near-infrared (NIR) fluorescence imaging is a promising new medical imaging modality. Associated with a targeting molecule, NIR fluorophores can accumulate selectively in tissues of interest and become valuable tools for the diagnosis and therapy of various pathologies. To facilitate the design of targeted NIR imaging agents, it is important to identify simple and affordable fluorescent probes, allowing rapid labelling of biovectors such as proteins, ideally in a site-specific manner. Here, we demonstrate that heptamethine cyanine based fluorophores, such as IR-783, that contain a chloro-cyclohexyl moiety within their polymethine chain can react selectively, at neutral pH, with cysteine residues in proteins to give stable, site-specifically labelled conjugates, that emit in the NIR spectral window. This reaction is exemplified with the labelling of peptides and two protein models: albumin and a Fab' antibody fragment. The resulting fluorescent proteins are stable and suitable for in vivo NIR imaging applications, as shown on a mice model. This straightforward one-step procedure, that does not require the prior derivatisation of the fluorophore with a bioconjugatable handle, should facilitate the production and use of near-infrared labelled proteins in life sciences.

Site-Specific Dual Labeling of Proteins on Cysteine Residues with Chlorotetrazines.

Dual-labeled biomolecules constitute a new generation of bioconjugates with promising applications in therapy and diagnosis. Unfortunately, the development of these new families of biologics is hampered by the technical difficulties associated with their construction. In particular, the site specificity of the conjugation is critical as the number and position of payloads can have a dramatic impact on the pharmacokinetics of the bioconjugate. Herein, we introduce dichlorotetrazine as a trivalent platform for the selective double modification of proteins on cysteine residues. This strategy is applied to the dual labeling of albumin with a macrocyclic chelator for nuclear imaging and a fluorescent probe for fluorescence imaging.

Site-Specific Dual-Labeling of a VHH with a Chelator and a Photosensitizer for Nuclear Imaging and Targeted Photodynamic Therapy of EGFR-Positive Tumors.

Variable domains of heavy chain only antibodies (VHHs) are valuable agents for application in tumor theranostics upon conjugation to both a diagnostic probe and a therapeutic compound. Here, we optimized site-specific conjugation of the chelator DTPA and the photosensitizer IRDye700DX to anti-epidermal growth factor receptor (EGFR) VHH 7D12, for applications in nuclear imaging and photodynamic therapy. 7D12 was site-specifically equipped with bimodal probe DTPA-tetrazine-IRDye700DX using the dichlorotetrazine conjugation platform. Binding, internalization and light-induced toxicity of DTPA-IRDye700DX-7D12 were determined using EGFR-overexpressing A431 cells. Finally, ex vivo biodistribution of DTPA-IRDye700DX-7D12 in A431 tumor-bearing mice was performed, and tumor homing was visualized with SPECT and fluorescence imaging. DTPA-IRDye700DX-7D12 was retrieved with a protein recovery of 43%, and a degree of labeling of 0.56. Spectral properties of the IRDye700DX were retained upon conjugation. 111In-labeled DTPA-IRDye700DX-7D12 bound specifically to A431 cells, and they were effectively killed upon illumination. DTPA-IRDye700DX-7D12 homed to A431 xenografts in vivo, and this could be visualized with both SPECT and fluorescence imaging. In conclusion, the dichlorotetrazine platform offers a feasible method for site-specific dual-labeling of VHH 7D12, retaining binding affinity and therapeutic efficacy. The flexibility of the described approach makes it easy to vary the nature of the probes for other combinations of diagnostic and therapeutic compounds.

(R)-NODAGA-PSMA: A Versatile Precursor for Radiometal Labeling and Nuclear Imaging of PSMA-Positive Tumors.

PURPOSE: The present study aims at developing and evaluating an urea-based prostate specific membrane antigen (PSMA) inhibitor suitable for labeling with 111In for SPECT and intraoperative applications as well as 68Ga and 64Cu for PET imaging. METHODS: The PSMA-based inhibitor-lysine-urea-glutamate-coupled to the spacer Phe-Phe-D-Lys(suberoyl) and functionalized with the enantiomerically pure prochelator (R)-1-(1-carboxy-3-carbotertbutoxypropyl)-4,7-carbotartbutoxymethyl)-1,4,7-triazacyclononane ((R)-NODAGA(tBu)3), to obtain (R)-NODAGA-Phe-Phe-D-Lys(suberoyl)-Lys-urea-Glu (CC34). CC34 was labeled with 111In, 68Ga and 64Cu. The radioconjugates were further evaluated in vitro and in vivo in LNCaP xenografts by biodistribution and PET studies. Biodistribution studies were also performed with 68Ga-HBED-CC-PSMA (HBED-CC: N,N'-bis[2-hydroxy-5-(carboxyethyl)benzyl]ethylenediamine-N,N'-diacetic acid) and 111In-PSMA-617 for comparison. RESULTS: 68Ga-CC34, 64Cu-CC34, and 111In-CC34 were prepared in radiochemical purity > 95%. 68/natGa-CC34, 64/natCu-CC34, 111/natIn-CC34, 68/natGa-HBED-CC-PSMA, and 111/natIn-PSMA-617 exhibited high affinity for the LNCaP cells, with Kd values of 19.3 ± 2.5 nM, 27.5 ± 2.7 nM, 5.5 ± 0.9 nM, 2.9 ± 0.6 nM and 5.4 ± 0.8 nM, respectively. They revealed comparable internalization profiles with approximately 75% of the total cell associated activity internalized after 3 h of incubation. 68Ga-CC34 showed very high stability after its administration in mice. Tumor uptake of 68Ga-CC34 (14.5 ± 2.9% IA/g) in LNCaP xenografts at 1 h p.i. was comparable to 68Ga-HBED-CC-PSMA (15.8 ± 1.4% IA/g) (P = 0.67). The tumor-to-normal tissue ratios at 1 and 2 h p.i of 68Ga-CC34 were also comparable to 68Ga-HBED-CC-PSMA (P > 0.05). Tumor uptake of 111In-CC34 (28.5 ± 2.6% IA/g) at 1 h p.i. was lower than 111In-PSMA-617 (52.1 ± 6.5% IA/g) (P = 0.02). The acquisition of PET-images with 64Cu-CC34 at later time points showed wash-out from the kidneys, while tumor uptake still remained relatively high. This resulted in an increased tumor-to-kidney ratio over time. CONCLUSIONS: 68Ga-CC34 is comparable to 68Ga-HBED-CC-PSMA in terms of tumor uptake and tumor to normal tissue ratios. 64Cu-CC34 could enable high contrast imaging of PSMA positive tissues characterized by elevated expression of PSMA or when delayed imaging is required. 64Cu-CC34 is currently being prepared for clinical translation.

Titanium imido complexes stabilised by bis(iminophosphoranyl)methanide ligands: the influence of N-substituents on solution dynamics and reactivity.

Terminal titanium imido complexes of the general formula [Ti(N(t)Bu)Cl{CH(Ph2PNR)2}] 4 (R = Ph, (i)Pr, (t)Bu) are reported. These compounds were synthesized from the corresponding Li adducts 3 of BIPMH (bis(iminophosphoranyl)methanide) and Mountford's complex [Ti(N(t)Bu)Cl2(Py)3]. The crystal structures of two of the Ti complexes (R = Ph, (t)Bu) and two of the Li compounds (R = (i)Pr, (t)Bu) are reported. Dynamic solution NMR spectroscopy reveals a dynamic isomerisation process in the case of the Ti complex 4c (R = (t)Bu). DFT studies showed that this dynamic process comes from steric repulsion between the imido ligand and the (t)Bu N-substituents on the BIPMH ligand. Complexes 4 were tested in alkyne hydroamination; 4a (R = Ph) displayed modest catalytic activity in the reaction of aniline with phenylacetylene.