Harriet Struthers

Site‐Specific and Stoichiometric Modification of Antibodies by Bacterial Transglutaminase

The therapeutic efficacy of antibodies can be substantially enhanced by conjugation of cytotoxic compounds such as chemotherapeutics and particle-emitting radionuclides. Intuitively one would assume that the therapeutic index would improve as the number of cytotoxic entities conjugated to the antibody increases. However, recent studies on auristatin–antibody conjugates in mice have demonstrated that a drug/antibody molar ratio of 4:1 results in optimal efficacy and in vivo tolerability. Unfortunately, conventional chemical strategies for protein modification are difficult to control and give rise to heterogeneous populations of immunoconjugates with variable stoichiometries, each of which has its own in vivo characteristics. The introduction of artificial, bio-orthogonal groups for site-specific and stoichiometric protein modification offers a potential solution to this problem. Such strategies are en vogue but are often laborious and still risk product heterogeneity. Transglutaminases (TGs, E.C. 2.3.2.13) catalyze acyltransfer reactions between the g-carboxamide group of glutamine (a side chain, which is otherwise chemically inert under physiological conditions) and the primary e-amino group of lysine, to form catabolically stable isopeptide bonds (Figure 1a). Most TGs are promiscuous with respect to the lysine substrate and accept even simple 5-aminopentyl groups as lysine surrogates. The criteria for a glutamine residue to be recognized by the enzyme, however, are muchmore stringent: it should be both located in a flexible region of the protein and flanked by specific amino acids. Given this inherent selectivity, we hypothesized that TG would be an alternative for the site-specific and stoichiometric functionalization of antibodies. For this study we used bacterial transglutaminase (BTG) because it is robust, inexpensive, and easy to handle. Our group is interested in radioimmunoconjugates for diagnostic and therapeutic applications, where low off-target accumulation of radioactivity is crucial. Earlier studies performed with radiolabeled monoclonal antibodies (mAbs) demonstrated that high numbers of metal chelators adversely affect the biological behavior of radioimmunoconjugates. Therefore, we tested the features of BTG for the preparation of immunoconjugates that are functionalized with different metal chelators and radiolabeled with different diagnostic and therapeutic radionuclides. Deferoxamine (DF, 1), an antidote for metal poisoning, has recently been identified as a suitable chelator for radionuclides such as Ga and Zr. During the course of our studies we recognized that without further derivatization deferoxamine is already a potent BTG substrate. Furthermore, the metal chelating system 4-(1,4,8,11tetraazacyclotetradec-1-yl)methyl benzoic acid (CPTA, 2) was derivatized with a 1,5-diaminopentane (cadaverine) spacer (Figure 1b; see the Supporting Information for details on the synthesis). To probe the scope of the new strategy we investigated other (model) substrates, which are of potential Figure 1. a) TG-mediated modification of Gln (Q) with a substrate containing lysine or a lysine surrogate. b) Substrates used in this study.

Click-to-Chelate : Design and Incorporation of Triazole-Containing Metal-Chelating Systems into Biomolecules of Diagnostic and Therapeutic Interest

The site-specific conjugation of metal chelating systems to biologically relevant molecules is an important contemporary topic in bioinorganic and bioorganometallic chemistry. In this work, we have used the CuI-catalyzed cycloaddition of azides and terminal alkynes to synthesise novel ligand systems, in which the 1,2,3-triazole is an integral part of the metal chelating system. A diverse set of bidentate alkyne building blocks with different aliphatic and aromatic backbones and various donor groups were prepared. The bidentate alkynes were reacted with benzyl azide in the presence of a catalytic amount of CuI to form tridentate model ligands. The chelators were reacted with [ReBr3(CO)3]2- to form well-defined and stable complexes with different overall charges, structures and hydrophilicities. In all cases tridentate coordination of the ligands, including through N3 of the 1,2,3-triazole ring, was observed. The ligand systems could also be quantitatively radiolabelled with the precursor [99 mTc (H2O)3(CO)3]+ at low ligand concentrations. Similarly the alkynes were reacted with an azido thymidine derivative to form a series of compounds, which could be radiolabelled in situ to form single products. Subsequent incubation of the neutral and cationic organometallic 99 mTc thymidine derivatives with human cytosolic thymidine kinase, a key enzyme in tumour proliferation, revealed that only the neutral compounds maintained substrate activity towards the enzyme. Bioconjugation, radiolabelling and enzymatic reactions were successfully performed in a matter of hours. Thus, click chemistry provides an elegant method for rapidly functionalising a biologically relevant molecule with a variety of efficient metal chelators suitable for (radio)labelling with the M(CO)3 core (M=99 mTc, Re), to offer new potential for technetium-99 m in clinical and preclinical tracer development.

DOTA Conjugate with an Albumin-Binding Entity Enables the First Folic Acid–Targeted 177Lu-Radionuclide Tumor Therapy in Mice

The folate receptor (FR) has proven a valuable target for nuclear imaging using folic acid radioconjugates. However, using folate-based radiopharmaceuticals for therapy has long been regarded as an unattainable goal because of their considerable renal accumulation. Herein, we present a novel strategy in which a DOTA–folate conjugate with an albumin-binding entity (cm09) was designed with the aim of prolonging circulation in the blood and therewith potentially improving tumor-to-kidney ratios. Methods: The folate conjugate cm09 was radiolabeled with 177LuCl3, and stability experiments were performed in plasma. Cell uptake studies were performed on FR-positive KB tumor cells, and an ultrafiltration assay was used to determine the plasma protein–binding properties of 177Lu-cm09. In vivo, 177Lu-cm09 was tested in KB tumor–bearing mice using SPECT/CT. The therapeutic anticancer effect of 177Lu-cm09 (20 MBq) applied as a single injection or as fractionated injections was investigated in different groups of mice (n = 5) by monitoring tumor size and the survival time of treated mice, compared with untreated controls. Results: Compound cm09 was radiolabeled at a specific activity of 40 MBq/nmol, a radiochemical yield of more than 98%, and a stability of more than 99% over 5 d in plasma. Ultrafiltration revealed significant binding of 177Lu-cm09 to serum proteins (∼91%) in plasma, compared with folate radioconjugate without an albumin-binding entity. Cell uptake and internalization of 177Lu-cm09 was FR-specific and comparable to other folate radioconjugates. In vivo studies resulted in high tumor uptake (17.56 percentage injected dose per gram [%ID/g] at 4 h after injection), which was almost completely retained for at least 72 h. Renal accumulation was significantly reduced (28 %ID/g at 4 h after injection), compared with folate conjugates that lack an albumin-binding entity (∼70 %ID/g at 4 h after injection). These circumstances enabled SPECT imaging of excellent quality. Radionuclide therapy (1 × 20 MBq) revealed complete remission of tumors in 4 of 5 cases and a significantly prolonged survival time, compared with untreated controls. Conclusion: The modification of a folate radioconjugate with an albumin-binding entity resulted in a significant increase of the tumor-to-kidney ratio of radioactivity, enabling for the first time, to our knowledge, the preclinical application of folic acid–targeted radionuclide therapy in mice.

Synthesis, in vitro, and in silico evaluation of organometallic technetium and rhenium thymidine complexes with retained substrate activity toward human thymidine kinase type 1.

Human cytosolic thymidine kinase (hTK1) has proven to be a suitable target for noninvasive imaging of cancer cell proliferation using radiolabeled substrates such as [ (18)F]fluorothymidine ([ (18)F]FLT). However, a thymidine tracer useful for single photon emission tomography (SPECT) based on the inexpensive radionuclide technetium-99m would be of significant interest. In this work, a series of thymidine derivatives labeled with the organometallic [M(CO) 3] (+) core (M = (99m)Tc, Re) were synthesized. Neutral, cationic, and anionic complexes were readily formed in aqueous media, and all were substrates of recombinant hTK1 when incubated with ATP. The neutral complexes were phosphorylated to a greater extent than the charged complexes. The extent of phosphorylation was further improved by increasing the spacer length separating thymidine and the organometallic core. A molecular dynamics simulation study performed with a modified hTK1 structure supported the experimental findings. In vitro cell internalization experiments performed in a human neuroblastoma cell line (SKNMC) showed low uptake of the charged complexes but significant uptake for the neutral, lipophilic complexes with a log P value >1.

Molecular Assembly of Multifunctional 99mTc Radiopharmaceuticals Using Clickable Amino Acid Derivatives

Synthetic strategies that enable the efficient and selective combination of different biologically active entities hold great promise for the development of multifunctional hybrid conjugates useful for biochemical and medical applications. Starting from side‐chain‐functionalized N(α)‐propargyl lysine derivatives, conjugates containing a 99mTc‐based imaging probe for SPECT and two different moieties (e.g., tumor‐targeting vectors, pharmacological modifiers, affinity tags, or second imaging probes) can be assembled using the CuI‐catalyzed alkyne–azide cycloaddition in efficient one‐pot protocols. This strategy was successfully applied to the preparation of a 99mTc‐labeled conjugate comprising a tumor‐targeting peptide sequence (bombesin(7–14)) and a low‐molecular‐weight albumin binder, a pharmacological modifier that prolongs the blood circulation time of the conjugate. Evaluation of the conjugate in vitro and in vivo provided promising results for its use as an imaging agent for the visualization of tumors positive for the gastrin‐releasing peptide receptor. The methodology presented herein provides an attractive synthetic tool for the preparation of multifunctional 99mTc‐based radiopharmaceuticals with significant potential for a multitude of applications.

Radioiodinated folic acid conjugates: evaluation of a valuable concept to improve tumor-to-background contrast.

Folic acid radioconjugates can be used for targeting folate receptor positive (FR(+)) tumors. However, the high renal uptake of radiofolates is a drawback of this strategy, particularly with respect to a therapeutic application due to the risk of damage to the kidneys by particle radiation. The goal of this study was to develop and evaluate radioiodinated folate conjugates as a novel class of folate-based radiopharmaceuticals potentially suitable for therapeutic application. Two different folic acid conjugates, tyrosine-folate (1) and tyrosine-click-folate (3), were synthesized and radioiodinated using the Iodogen method resulting in [(125)I]-2 and [(125/131)I]-4. Both radiofolates were highly stable in mouse and human plasma. Determination of FR binding affinities using (3)H-folic acid and FR(+) KB tumor cells revealed affinities in the nanomolar range for 2 and 4. The cell uptake of [(125)I]-2 and [(125/131)I]-4 proved to be FR specific as it was blocked by the coincubation of folic acid. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) in vitro assays were employed for the determination of tumor cell viability upon exposure to [(131)I]-4. Compared to untreated control cells, significantly reduced cell viability was observed for FR(+) cancer cells (KB, IGROV-1, SKOV-3), while FR(-) cells (PC-3) were not affected. Biodistribution studies performed in tumor bearing nude mice showed the specific accumulation of both radiofolates in KB tumor xenografts ([(125)I]-2: 3.43 ± 0.28% ID/g; [(125)I]-4: 2.28 ± 0.46% ID/g, 4 h p.i.) and increasing tumor-to-kidney ratios over time. The further improvement of the tumor-to-background contrast was achieved by preinjection of the mice with pemetrexed allowing excellent imaging via single-photon emission computed tomography (SPECT/CT). These findings confirmed the hypothesis that the application of radioiodinated folate conjugates may be a valuable concept to improve tumor-to-background contrast. The inhibitory effect of [(131)I]-4 on FR(+) cancer cells in vitro indicates the potential of this class of radiofolates for therapeutic application.

Synthesis and Evaluation of Bombesin Analogues Conjugated to Two Different Triazolyl-Derived Chelators for 99mTc Labeling

Overexpression of the gastrin‐releasing peptide receptor (GRPR) in a variety of human carcinomas has provided a means of diagnosis and treatment. Previously we reported a metabolically stable (NαHis)Ac‐βAla‐βAla‐[Cha13,Nle14]BBS(7–14) analogue with high affinity for the GRPR. We have also shown that the biodistribution pattern of this fairly lipophilic, radiolabeled peptide can be enhanced by glycation, which is easily carried out by CuI‐catalyzed cycloaddition. Herein, we further elaborate this “click approach” in the synthesis of a new series of triazole‐based chelating systems as alternatives to the (NαHis)Ac chelator for labeling with the 99mTc(CO)3 core. The bombesin analogues, containing these new chelating systems, were evaluated with regard to their synthesis and in vitro and in vivo properties, and were compared with their (NαHis)Ac counterparts. The influence of the chelator on biodistribution properties was less than that of glycation, which clearly improved the tumor‐to‐background ratios.

Organometallic [Re(CO)3]+ and [Re(CO)2(NO)]2+ Labeled Substrates for Human Thymidine Kinase 1

Thymidine was functionalized at position N3 with a tridentate iminodiacetic acid chelating system and a potentially tetradentate mercaptoethyliminodiacetic acid chelating system. Spacers of different lengths (ethyl and butyl) were introduced between the chelators and thymidine. The derivatives were labeled with the [Re(CO)(2)(NO)](2+) and [Re(CO)(3)](+) cores to give isostructural complexes with different overall charges. All complexes were analyzed by NMR, MS, and IR, and in addition, the X-ray structure of a [Re(CO)(2)(NO)](2+) labeled thymidine derivative functionalized at the N3 position was solved. The ligands incorporating the potentially tetradentate mercaptoethyliminodiacetic acid chelating system coordinated tridentately through iminodiacetic acid to both the [Re(CO)(2)(NO)](2+) core and the [Re(CO)(3)](+) core. This was surprising given that the reaction of [NEt(4)][Re(CO)(2)(NO)Br(3)] with the model ligand ethylmercaptoethyliminodiacetic acid led to dissociation of a carbonyl ligand and formation of a monocarbonyl-mononitrosyl complex, as confirmed by X-ray structure analysis. All of the organometallic thymidine derivatives were substrates for human thymidine kinase 1, a key enzyme in (cancer) cell proliferation. Neutral [Re(CO)(2)(NO)](2+) labeled thymidine derivatives revealed substrate activity ranging from 24 to 40%, and the structurally analogous anionic [Re(CO)(3)](+) labeled thymidine derivatives from 20 to 38% compared with the natural substrate thymidine.