Current pretargeting systems use noncovalent biologic interactions, which are prone to immunogenicity. We previously developed a novel approach based on the bioorthogonal reaction between a radiolabeled tetrazine and an antibody-conjugated trans-cyclooctene (TCO). However, the tumor-to-blood ratio was low due to reaction with freely circulating antibody-TCO. Methods: Here we developed 2 tetrazine-functionalized clearing agents that enable rapid reaction with and removal of a TCO-tagged antibody (CC49) from blood. Next, we incorporated this approach into an optimized pretargeting protocol in LS174T-bearing mice. Then we compared the pretargeted 177 Lu-labeled tetrazine with 177 Lu-labeled CC49. The biodistribution data were used for mouse and human dosimetry calculations. Results: The use of a clearing agent led to a doubling of the tetrazine tumor uptake and a 125-fold improvement of the tumor-to-blood ratio at 3 h after tetrazine injection. Mouse dosimetry suggested that this should allow for an 8-fold higher tumor dose than is possible with nonpretargeted radioimmunotherapy. Also, humans treated with CC49-TCO-pretargeted 177 Lu-tetrazine would receive a dose to nontarget tissues 1 to 2 orders of magnitude lower than with directly labeled CC49. Conclusion: The in vivo performance of chemical pretargeting falls within the range of results obtained for the clinically validated pretargeting approaches in mice, with the advantage of potentially allowing for fractionated radiotherapy as a result of a lower likelihood of immunogenicity. These findings demonstrate that biologic pretargeting concepts can be translated to rapid bioorthogonal chemical approaches with retained potential.

Radioimmunotherapy (RIT) of solid tumors is hampered by low tumor-to-nontumor (T/NT) ratios of the radiolabeled monoclonal antibodies resulting in low tumor doses in patients. Pretargeting technologies can improve the effectiveness of RIT in cancer therapy by increasing this ratio. We showed that a pretargeting strategy employing in vivo chemistry in combination with clearing agents, proceeds efficiently in tumor-bearing mice resulting in high T/NT ratios. A dosimetry study indicated that the chemical pretargeting technology, which centered on the bioorthogonal Diels-Alder click reaction between a radiolabeled tetrazine probe and a trans-cyclooctene-oxymethylbenzamide-tagged CC49 antibody (CC49-TCO(1)), can match the performance of clinically validated high-affinity biological pretargeting approaches in mice ( Rossin J Nucl Med. 2013 , 54 , 1989 - 1995 ). Nevertheless, the increased protein surface hydrophobicity of CC49-TCO(1) led to a relatively rapid blood clearance and concomitant reduced tumor uptake compared to native CC49 antibody. Here, we present the in vivo evaluation of a TCO-oxymethylacetamide-tagged CC49 antibody (CC49-TCO(2)), which is highly reactive toward tetrazines and less hydrophobic than CC49-TCO(1). CC49-TCO(2) was administered to healthy mice to determine its blood clearance and the in vivo stability of the TCO. Next, pretargeting biodistribution and SPECT studies with CC49-TCO(2), tetrazine-functionalized clearing agent, and radiolabeled tetrazine were carried out in nude mice bearing colon carcinoma xenografts (LS174T). CC49-TCO(2) had an increased circulation half-life, a 1.5-fold higher tumor uptake, and a 2.6-fold improved in vivo TCO stability compared to the more hydrophobic TCO-benzamide-CC49. As a consequence, and despite the 2-fold lower reactivity of CC49-TCO(2) toward tetrazines compared with CC49-TCO(1), administration of radiolabeled tetrazine afforded a significantly increased tumor accumulation and improved T/NT ratios in mice pretargeted with CC49-TCO(2). In conclusion, the TCO-acetamide derivative represents a large improvement in in vivo Diels-Alder pretargeting, possibly enabling application in larger animals and eventually humans.

The cytoskeletal proteins, FtsZ and tubulin, play a pivotal role in prokaryotic cell division and eukaryotic chromosome segregation, respectively. Selective inhibitors of the GTP-dependent polymerization of FtsZ could constitute a new class of antibiotics, while several inhibitors of tubulin are widely used in antiproliferative therapy. In this work, we set out to identify selective inhibitors of FtsZ based on the structure of its natural ligand, GTP. We found that GTP analogs with small hydrophobic substituents at C8 of the nucleobase efficiently inhibit FtsZ polymerization, whereas they have an opposite effect on the polymerization of tubulin. The inhibitory activity of the GTP analogs on FtsZ polymerization allowed us to crystallize FtsZ in complex with C8-morpholino-GTP, revealing the binding mode of a GTP derivative containing a nonmodified triphosphate chain.

Essential cell division protein FtsZ forms the bacterial cytokinetic ring and is a target for new antibiotics. FtsZ monomers bind GTP and assemble into filaments. Hydrolysis to GDP at the association interface between monomers leads to filament disassembly. We have developed a homogeneous competition assay, employing the fluorescence anisotropy change of mant-GTP upon binding to nucleotide-free FtsZ, which detects compounds binding to the nucleotide site in FtsZ monomers and measures their affinities within the millimolar to 10 nM range. We have employed this method to determine the apparent contributions of the guanine, ribose, and the α-, β-, and γ-phosphates to the free energy change of nucleotide binding. Similar relative contributions have also been estimated through molecular dynamics and binding free energy calculations, employing the crystal structures of FtsZ-nucleotide complexes. We find an energetically dominant contribution of the β-phosphate, comparable to the whole guanosine moiety. GTP and GDP bind with similar observed affinity to FtsZ monomers. Loss of the regulatory γ-phosphate results in a predicted accommodation of GDP which has not been observed in the crystal structures. The binding affinities of a series of C8-substituted GTP analogues, known to inhibit FtsZ but not eukaryotic tubulin assembly, correlate with their inhibitory capacity on FtsZ polymerization. Our methods permit testing of FtsZ inhibitors targeting its nucleotide site, as well as compounds from virtual screening of large synthetic libraries. Our results give insight into the FtsZ-nucleotide interactions, which could be useful in the rational design of new inhibitors, especially GTP phosphate mimetics.

The prokaryotic tubulin homologue FtsZ plays a key role in bacterial cell division. Selective inhibitors of the GTP-dependent polymerization of FtsZ are expected to result in a new class of antibacterial agents. One of the challenges is to identify compounds which do not affect the function of tubulin and various other GTPases in eukaryotic cells. We have designed a novel inhibitor of FtsZ polymerization based on the structure of the natural substrate GTP. The inhibitory activity of 8-bromoguanosine 5'-triphosphate (BrGTP) was characterized by a coupled assay, which allows simultaneous detection of the extent of polymerization (via light scattering) and GTPase activity (via release of inorganic phosphate). We found that BrGTP acts as a competitive inhibitor of both FtsZ polymerization and GTPase activity with a Ki for GTPase activity of 31.8 +/- 4.1 microM. The observation that BrGTP seems not to inhibit tubulin assembly suggests a structural difference of the GTP-binding pockets of FtsZ and tubulin.

The in vitro performance of some of the new tetrazine probes turned out to be comparable to the established lead probe [Lu]Lu-DOTA-PEG-Tz ([Lu]4). However, tumor pretargeting studies in vivo showed lower tumor uptake and increased uptake in non-target organs.

We have recently introduced a 1,4,7-triazacyclononane (TACN) based chelating system with additional five-membered azaheterocyclic substituents for complexation of radioactive Cu ions. In this work, we investigated the complexation properties of these novel chelators with Ga. In labeling experiments, we could show that the penta- and hexadentate imidazole derivatives NODIA-Me 4 and NOTI-Me 1 can be labeled with Ga in specific activities up to ∼30 MBq nmol, while the corresponding thiazole derivative NOTThia 2 did not label satisfactorily under identical conditions. NMR studies on the Ga complexes of 1 and the model compound NODIA-Me-NH-Me 5 revealed formation of rigid 1:1 chelates with a slow macrocyclic interconversion and inert Ga-N bonds to the methylimidazole residues on the NMR time scale. The TACN-derived bifunctional chelator NODIA-Me was furthermore conjugated to a prostate-specific membrane antigen (PSMA) targeting moiety to give the corresponding bioconjugate NODIA-Me-PSMA 7. Serum stability and ligand challenge experiments of Ga-7 confirmed formation of a stable complex for up to 4 h. The remaining coordination site of five-coordinate Ga complexes was found to be occupied by monodentate ligands including hydroxide and chloride anions depending on the conditions. According to density functional theory calculations, coordination of monodentate ligands as well as of the amide group for the bioconjugated ligand are energetically plausible. Finally, the labeled bioconjugateGa-7 exhibited rapid renal clearance in biodistribution studies performed by small animal PET imaging with no indication of transchelation/demetalation in vivo. Altogether, our results provide strong evidence for a stable Ga complexation of our novel TACN-based chelators bearing imidazole arms. Despite the formation of two complexes incorporating different monodentate ligands in vitro, the imidazole type ligands show promise as chelating agents for the future development of gallium based radiopharmaceuticals.

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