Antibody-Drug Conjugates for Tumor Targeting-Novel Conjugation Chemistries and the Promise of non-IgG Binding Proteins

Antibody-drug conjugates (ADCs) have emerged as a promising class of anticancer agents, combining the specificity of antibodies for tumor targeting and the destructive potential of highly potent drugs as payload. An essential component of these immunoconjugates is a bifunctional linker capable of reacting with the antibody and the payload to assemble a functional entity. Linker design is fundamental, as it must provide high stability in the circulation to prevent premature drug release, but be capable of releasing the active drug inside the target cell upon receptor-mediated endocytosis. Although ADCs have demonstrated an increased therapeutic window, compared to conventional chemotherapy in recent clinical trials, therapeutic success rates are still far from optimal. To explore other regimes of half-life variation and drug conjugation stoichiometries, it is necessary to investigate additional binding proteins which offer access to a wide range of formats, all with molecularly defined drug conjugation. Here, we delineate recent progress with site-specific and biorthogonal conjugation chemistries, and discuss alternative, biophysically more stable protein scaffolds like Designed Ankyrin Repeat Proteins (DARPins), which may provide such additional engineering opportunities for drug conjugates with improved pharmacological performance.

Tetraamine-derived bifunctional chelators for technetium-99m labelling: synthesis, bioconjugation and evaluation as targeted SPECT imaging probes for GRP-receptor-positive tumours

Owing to its optimal nuclear properties, ready availability, low cost and favourable dosimetry, (99m)Tc continues to be the ideal radioisotope for medical-imaging applications. Bifunctional chelators based on a tetraamine framework exhibit facile complexation with Tc(V)O(2) to form monocationic species with high in vivo stability and significant hydrophilicity, which leads to favourable pharmacokinetics. The synthesis of a series of 1,4,8,11-tetraazaundecane derivatives (01-06) containing different functional groups at the 6-position for the conjugation of biomolecules and subsequent labelling with (99m)Tc is described herein. The chelator 01 was used as a starting material for the facile synthesis of chelators functionalised with OH (02), N(3) (04) and O-succinyl ester (05) groups. A straightforward and easy synthesis of carboxyl-functionalised tetraamine-based chelator 06 was achieved by using inexpensive and commercially available starting materials. Conjugation of 06 to a potent bombesin-antagonist peptide and subsequent labelling with (99m)Tc afforded the radiotracer (99m)Tc-N4-BB-ANT, with radiolabelling yields of >97% at a specific activity of 37 GBq micromol(-1). An IC(50) value of (3.7+/-1.3) nM was obtained, which confirmed the high affinity of the conjugate to the gastrin-releasing-peptide receptor (GRPr). Immunofluorescence and calcium mobilisation assays confirmed the strong antagonist properties of the conjugate. In vivo pharmacokinetic studies of (99m)Tc-N4-BB-ANT showed high and specific uptake in PC3 xenografts and in other GRPr-positive organs. The tumour uptake was (22.5+/-2.6)% injected activity per gram (% IA g(-1)) at 1 h post injection (p.i.). and increased to (29.9+/-4.0)% IA g(-1) at 4 h p.i. The SPECT/computed tomography (CT) images showed high tumour uptake, clear background and negligible radioactivity in the abdomen. The promising preclinical results of (99m)Tc-N4-BB-ANT warrant its potential candidature for clinical translation.

Synthesis and biological activity of new functionalized epothilones for prodrug design and tumor targeting

Epothilones are potent antiproliferative agents, which have served as successful lead structures for anticancer drug discovery. However, their therapeutic efficacy would benefit greatly from an increase in their selectivity for tumor cells, which may be achieved through conjugation with a tumor-targeting moiety. Three novel epothilone analogs bearing variously functionalized benzimidazole side chains were synthesized using a strategy based on palladium-mediated coupling and macrolactonization. The synthesis of these compounds is described and their in vitro biological activity is discussed with respect to their interactions with the tubulin/microtubule system and the inhibition of human cancer cell proliferation. The additional functional groups may be used to synthesize conjugates of epothilone derivatives with a variety of tumor-targeting moieties.

A comprehensive understanding of thioTEPA metabolism in the mouse using UPLC-ESI-QTOFMS-based metabolomics

ThioTEPA, an alkylating agent with anti-tumor activity, has been used as an effective anticancer drug since the 1950s. However, a complete understanding of how its alkylating activity relates to clinical efficacy has not been achieved, the total urinary excretion of thioTEPA and its metabolites is not resolved, and the mechanism of formation of the potentially toxic metabolites S-carboxymethylcysteine (SCMC) and thiodiglycolic acid (TDGA) remains unclear. In this study, the metabolism of thioTEPA in a mouse model was comprehensively investigated using ultra-performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight mass spectrometry (UPLC-ESI-QTOFMS) based-metabolomics. The nine metabolites identified in mouse urine suggest that thioTEPA underwent ring-opening, N-dechloroethylation, and conjugation reactions in vivo. SCMC and TDGA, two downstream thioTEPA metabolites, were produced from thioTEPA from two novel metabolites 1,2,3-trichloroTEPA (VII) and dechloroethyltrichloroTEPA (VIII). SCMC and TDGA excretion were increased about 4-fold and 2-fold, respectively, in urine following the thioTEPA treatment. The main mouse metabolites of thioTEPA in vivo were TEPA (II), monochloroTEPA (III) and thioTEPA-mercapturate (IV). In addition, five thioTEPA metabolites were detected in serum and all shared similar disposition. Although thioTEPA has a unique chemical structure which is not maintained in the majority of its metabolites, metabolomic analysis of its biotransformation greatly contributed to the investigation of thioTEPA metabolism in vivo, and provides useful information to understand comprehensively the pharmacological activity and potential toxicity of thioTEPA in the clinic.

Conformationally Controlled Electron Delocalization in n-Type Rods: Synthesis, Structure, and Optical, Electrochemical, and Spectroelectrochemical Properties of Dicyanocyclophanes

A series of dicyanobiphenyl-cyclophanes 1-6 with various pi-backbone conformations and characteristic n-type semiconductor properties is presented. Their synthesis, optical, structural, electrochemical, spectroelectrochemical, and packing properties are investigated. The X-ray crystal structures of all n-type rods allow the systematic correlation of structural features with physical properties. In addition, the results are supported by quantum mechanical calculations based on density functional theory. A two-step reduction process is observed for all n-type rods, in which the first step is reversible. The potential gap between the reduction processes depends linearly on the cos(2) value of the torsion angle phi between the pi-systems. Similarly, optical absorption spectroscopy shows that the vertical excitation energy of the conjugation band correlates with the cos(2) value of the torsion angle phi. These correlations demonstrate that the fixed intramolecular torsion angle phi is the dominant factor determining the extent of electron delocalization in these model compounds, and that the angle phi measured in the solid-state structure is a good proxy for the molecular conformation in solution. Spectroelectrochemical investigations demonstrate that conformational rigidity is maintained even in the radical anion form. In particular, the absorption bands corresponding to the SOMO-LUMO+i transitions are shifted bathochromically, whereas the absorption bands corresponding to the HOMO-SOMO transition are shifted hypsochromically with increasing torsion angle phi.

A tetrathiafulvalene-functionalized schiff base macrocycle: synthesis, electrochemical, and photophysical properties

Highly selective formation of 2+2 macrocycle 1 from 2,5-bis(3-formyl-2-hydroxyphenyl)-1,3,4-oxadiazole and a diamine-functionalized tetrathiafulvalene derivative is reported. Its electronic properties have been studied experimentally by the combination of electrochemistry and UV-vis-NIR spectroscopy. Particularly, its largely extended pi-conjugation renders this novel macrocycle simultaneously a good multielectron donor and a strong chromophore, which is rationalized on the basis of density functional theory. (C) 2011 Elsevier Ltd. All rights reserved.

Facile double-functionalization of designed ankyrin repeat proteins using click and thiol chemistries

Click chemistry is a powerful technology for the functionalization of therapeutic proteins with effector moieties, because of its potential for bio-orthogonal, regio-selective, and high-yielding conjugation under mild conditions. Designed Ankyrin Repeat Proteins (DARPins), a novel class of highly stable binding proteins, are particularly well suited for the introduction of clickable methionine surrogates such as azidohomoalanine (Aha) or homopropargylglycine (Hpg), since the DARPin scaffold can be made methionine-free by an M34L mutation in the N-cap which fully maintains the biophysical properties of the protein. A single N-terminal azidohomoalanine, replacing the initiator Met, is incorporated in high yield, and allows preparation of "clickable" DARPins at about 30 mg per liter E. coli culture, fully retaining stability, specificity, and affinity. For a second modification, we introduced a cysteine at the C-terminus. Such DARPins could be conveniently site-specifically linked to two moieties, polyethylene glycol (PEG) to the N-terminus and the fluorophore Alexa488 to the C-terminus. We present a DARPin selected against the epithelial cell adhesion molecule (EpCAM) with excellent properties for tumor targeting as an example. We used these doubly modified molecules to measure binding kinetics on tumor cells and found that PEGylation has no effect on dissociation rate, but slightly decreases the association rate and the maximal number of cell-bound DARPins, fully consistent with our previous model of PEG action obtained in vitro. Our data demonstrate the benefit of click chemistry for site-specific modification of binding proteins like DARPins to conveniently add several functional moieties simultaneously for various biomedical applications.

Dog bites man or man bites dog? The enigma of the amino acid conjugations

The proposition posed is that the value of amino acid conjugation to the organism is not, as in the traditional view, to use amino acids for the detoxication of aromatic acids. Rather, the converse is more likely, to use aromatic acids that originate from the diet and gut microbiota to assist in the regulation of body stores of amino acids, such as glycine, glutamate, and, in certain invertebrates, arginine, that are key neurotransmitters in the central nervous system (CNS). As such, the amino acid conjugations are not so much detoxication reactions, rather they are homeostatic and neuroregulatory processes. Experimental data have been culled in support of this hypothesis from a broad range of scientific and clinical literature. Such data include the low detoxication value of amino acid conjugations and the Janus nature of certain amino acids that are both neurotransmitters and apparent conjugating agents. Amino acid scavenging mechanisms in blood deplete brain amino acids. Amino acids glutamate and glycine when trafficked from brain are metabolized to conjugates of aromatic acids in hepatic mitochondria and then irreversibly excreted into urine. This process is used clinically to deplete excess nitrogen in cases of urea cycle enzymopathies through excretion of glycine or glutamine as their aromatic acid conjugates. Untoward effects of high-dose phenylacetic acid surround CNS toxicity. There appears to be a relationship between extent of glycine scavenging by benzoic acid and psychomotor function. Glycine and glutamine scavenging by conjugation with aromatic acids may have important psychosomatic consequences that link diet to health, wellbeing, and disease.

Stimulated platelets use serotonin to enhance their retention of procoagulant proteins on the cell surface

Activated platelets bind numerous adhesive and procoagulant proteins by receptor-mediated processes. Although there is little evidence to suggest that these processes are heterogeneous in platelets, we previously found that platelets co-stimulated with collagen and thrombin express functional alpha-granule factor V only on a subpopulation of cells. Here we show that these cells, referred to as 'COAT-platelets', bind additional alpha-granule proteins, including fibrinogen, von Willebrand factor, thrombospondin, fibronectin and alpha2-antiplasmin. These proteins are all transglutaminase substrates, and inhibitors of transglutaminase prevent the production of COAT-platelets. A synthetic transglutaminase substrate (CP15) also binds to COAT-platelets, and analysis by high performance liquid chromatography/mass spectrometry shows that a product is formed with a relative molecular mass (Mr) equal to CP15 plus 176. Serotonin, an abundant component of platelet-dense granules, has an Mr of 176, and fibrinogen isolated from COAT-platelets contains covalently linked serotonin. Synthetic bovine serum albumin-(serotonin)6 binds selectively to COAT-platelets and also inhibits the retention of procoagulant proteins on COAT-platelets. These data indicate that COAT-platelets use serotonin conjugation to augment the retention of procoagulant proteins on their cell surface through an as yet unidentified serotonin receptor.

Liver repopulation with bone marrow derived cells improves the metabolic disorder in the Gunn rat

BACKGROUND: Reversible ischaemia/reperfusion (I/R) liver injury has been used to induce engraftment and hepatic parenchymal differentiation of exogenous beta2-microglubulin(-)/Thy1(+) bone marrow derived cells. AIM: To test the ability of this method of hepatic parenchymal repopulation, theoretically applicable to clinical practice, to correct the metabolic disorder in a rat model of congenital hyperbilirubinaemia. METHODS AND RESULTS: Analysis by confocal laser microscopy of fluorescence labelled cells and by immunohistochemistry for beta2-microglubulin, 72 hours after intraportal delivery, showed engraftment of infused cells in liver parenchyma of rats with I/R, but not in control animals with non-injured liver. Transplantation of bone marrow derived cells obtained from GFP-transgenic rats into Lewis rats resulted in the presence of up to 20% of GFP positive hepatocytes in I/R liver lobes after one month. The repopulation rate was proportional to the number of transplanted cells. Infusion of GFP negative bone marrow derived cells into GFP positive transgenic rats resulted in the appearance of GFP negative hepatocytes, suggesting that the main mechanism underlying parenchymal repopulation was differentiation rather than cell fusion. Transplantation of wild type bone marrow derived cells into hyperbilirubinaemic Gunn rats with deficient bilirubin conjugation after I/R damage resulted in 30% decrease in serum bilirubin, the appearance of bilirubin conjugates in bile, and the expression of normal UDP-glucuronyltransferase enzyme evaluated by polymerase chain reaction. CONCLUSIONS: I/R injury induced hepatic parenchymal engraftment and differentiation into hepatocyte-like cells of bone marrow derived cells. Transplantation of bone marrow derived cells from non-affected animals resulted in the partial correction of hyperbilirubinaemia in the Gunn rat.

Fenofibrate metabolism in the cynomolgus monkey using ultraperformance liquid chromatography-quadrupole time-of-flight mass spectrometry-based metabolomics

Fenofibrate, widely used for the treatment of dyslipidemia, activates the nuclear receptor, peroxisome proliferator-activated receptor alpha. However, liver toxicity, including liver cancer, occurs in rodents treated with fibrate drugs. Marked species differences occur in response to fibrate drugs, especially between rodents and humans, the latter of which are resistant to fibrate-induced cancer. Fenofibrate metabolism, which also shows species differences, has not been fully determined in humans and surrogate primates. In the present study, the metabolism of fenofibrate was investigated in cynomolgus monkeys by ultraperformance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-QTOFMS)-based metabolomics. Urine samples were collected before and after oral doses of fenofibrate. The samples were analyzed in both positive-ion and negative-ion modes by UPLC-QTOFMS, and after data deconvolution, the resulting data matrices were subjected to multivariate data analysis. Pattern recognition was performed on the retention time, mass/charge ratio, and other metabolite-related variables. Synthesized or purchased authentic compounds were used for metabolite identification and structure elucidation by liquid chromatographytandem mass spectrometry. Several metabolites were identified, including fenofibric acid, reduced fenofibric acid, fenofibric acid ester glucuronide, reduced fenofibric acid ester glucuronide, and compound X. Another two metabolites (compound B and compound AR), not previously reported in other species, were characterized in cynomolgus monkeys. More importantly, previously unknown metabolites, fenofibric acid taurine conjugate and reduced fenofibric acid taurine conjugate were identified, revealing a previously unrecognized conjugation pathway for fenofibrate.

Increasing the Antitumor Effect of an EpCAM-Targeting Fusion Toxin by Facile Click PEGylation

Fusion toxins used for cancer-related therapy have demonstrated short circulation half-lives, which impairs tumor localization and, hence, efficacy. Here, we demonstrate that the pharmacokinetics of a fusion toxin composed of a designed ankyrin repeat protein (DARPin) and domain I–truncated Pseudomonas Exotoxin A (PE40/ETA″) can be significantly improved by facile bioorthogonal conjugation with a polyethylene glycol (PEG) polymer at a unique position. Fusion of the anti-EpCAM DARPin Ec1 to ETA″ and expression in methionine-auxotrophic E. coli enabled introduction of the nonnatural amino acid azidohomoalanine (Aha) at position 1 for strain-promoted click PEGylation. PEGylated Ec1-ETA″ was characterized by detailed biochemical analysis, and its potential for tumor targeting was assessed using carcinoma cell lines of various histotypes in vitro, and subcutaneous and orthotopic tumor xenografts in vivo. The mild click reaction resulted in a well-defined mono-PEGylated product, which could be readily purified to homogeneity. Despite an increased hydrodynamic radius resulting from the polymer, the fusion toxin demonstrated high EpCAM-binding activity and retained cytotoxicity in the femtomolar range. Pharmacologic analysis in mice unveiled an almost 6-fold increase in the elimination half-life (14 vs. 82 minutes) and a more than 7-fold increase in the area under the curve (AUC) compared with non-PEGylated Ec1-ETA″, which directly translated in increased and longer-lasting effects on established tumor xenografts. Our data underline the great potential of combining the inherent advantages of the DARPin format with bioorthogonal click chemistry to overcome the limitations of engineering fusion toxins with enhanced efficacy for cancer-related therapy.