Two is better than one: difunctional high-affinity PSMA probes based on a [CpM(CO)3] (M = Re/99mTc) scaffold.

More than 10% of all men will be given the diagnosis "prostate cancer" during their lifetime. Most of the current radio-diagnostic vehicles involve both expensive and localized production with cyclotrons as well as the use of bulky chelators for the radiometal. We report the use of a new multifunctional cyclopentadiene (Cp) platform to prepare difunctional and monofunctional, PSMA-targeting rhenium and technetium-99m complexes. The Cp-complexes and the free ligands are prepared by straightforward functionalization with either one or two Lys-urea-Glu (LuG) PSMA binding motifs. Cell binding assays revealed that the difunctional rhenium complex displays a dissociation constant (KD = 2.1 nM) that is an order of magnitude lower than the monofunctional compound (KD = 24.2 nM). The 99mTc complexes can be prepared in one step and ≤15 min in high yields. These difunctional Cp-Re(i)/99mTc(i) complexes represent a new class of imaging agents with binding affinities comparable to clinically evaluated compounds. Additionally, this study demonstrates that the Cp-platform can readily be derivatized with amine-containing biomolecules. Extending this work to incorporate both targeting and therapeutic moieties could lead to theranostic systems with Re/99mTc.

Multi-functionalised graphene nanoflakes as tumour-targeting theranostic drug-delivery vehicles.

Graphene nanoflakes (GNFs) consist of a graphene sheet approximately 30 nm in diameter with a pristine aromatic system and an edge terminated with carboxylic acid groups. Their high water solubility and relative ease of functionalisation using carboxylate chemistry means that GNFs are potential scaffolds for the synthesis of theranostic agents. In this work, GNFs were multi-functionalised with derivatives of (i) a peptide-based Glu-NH-C(O)-NH-Lys ligand that binds prostate-specific membrane antigen (PSMA), (ii) a potent anti-mitotic drug (R)-ispinesib, (iii) the chelate desferrioxamine B (DFO), and (iv) an albumin-binding tag reported to extend pharmacokinetic half-life in vivo. Subsequent 68Ga radiochemistry and experiments in vitro and in vivo were used to evaluate the performance of GNFs in theranostic drug design. Efficient 68Ga-radiolabelling was achieved and the particle-loading of (R)-ispinesib and Glu-NH-C(O)-NH-Lys was confirmed using cellular assays. Using dose-response curves and FACS analysis it was shown that GNFs loaded with (R)-ispinesib inhibited the kinesin spindle protein (KSP) and induced G2/M-phase cell cycle arrest. Cellular uptake and blocking experiments demonstrated that GNFs functionalised with the Glu-NH-C(O)-NH-Lys ligand showed specificity toward PSMA expressing cells (LNCaP). The distribution profile and excretion rates of 68Ga-radiolabelled GNFs in athymic nude mice was evaluated using time-activity curves derived from dynamic positron-emission tomography (PET). Image analysis indicated that GNFs have low accumulation and retention in background tissue, with rapid renal clearance. In summary, our study shows that GNFs are suitable candidates for use in theranostic drug design.

Radiolabeled 111In-FGF-2 Is Suitable for In Vitro/Ex Vivo Evaluations and In Vivo Imaging.

Fibroblast growth factor-2 (FGF-2) is a potent modulator of cell growth and regulation, with improper FGF-2 signaling being involved in impaired responses to injury or even cancer. Therefore, the exploitation of FGF-2 as a therapeutic drives the prerequisite for effective insight into drug disposition kinetics. In this article, we present an 111In-radiolabeled FGF-2 derivative for noninvasive imaging in small animals deploying single photon emission tomography (SPECT). 111In-FGF-2 is equally well suitable for in vitro and ex vivo investigations as 125I-FGF-2. Furthermore, 111In-FGF-2 permits the performance of in vivo imaging, for example for the analysis of FGF-2 containing pharmaceutical formulations in developmental or preclinical stages. 111In-FGF-2 had affinity for the low-molecular-weight heparin enoxaparin identical to that of unlabeled FGF-2 (Kd: 0.6 ± 0.07 µM and 0.33 ± 0.03 µM, respectively) as assessed by isothermal titration calorimetry. The binding of 111In-FGF-2 to heparan sulfate proteoglycans (HPSGs) and the biological activity were comparable to those of unlabeled FGF-2, with EC50 values of 12 ± 2 pM and 25 ± 6 pM, respectively. In vivo biodistribution in healthy nude mice indicated a predominant accumulation of 111In-FGF-2 in filtering organs and minor uptake in the retina and the salivary and pituitary glands, which was confirmed by SPECT imaging. Therefore, 111In-FGF-2 is a valid tracer for future noninvasive animal imaging of FGF-2 in pharmaceutical development.

Preclinical imaging of the co-stimulatory molecules CD80 and CD86 with indium-111-labeled belatacept in atherosclerosis.

BACKGROUND: The inflammatory nature of atherosclerosis provides a broad range of potential molecular targets for atherosclerosis imaging. Growing interest is focused on targets related to plaque vulnerability such as the co-stimulatory molecules CD80 and CD86. We investigated in this preclinical proof-of-concept study the applicability of the CD80/CD86-binding fusion protein belatacept as a probe for atherosclerosis imaging. METHODS: Belatacept was labeled with indium-111, and the binding affinity was determined with CD80/CD86-positive Raji cells. In vivo distribution was investigated in Raji xenograft-bearing mice in single-photon emission computed tomography (SPECT)/CT scans, biodistribution, and ex vivo autoradiography studies. Ex vivo SPECT/CT experiments were performed with aortas and carotids of ApoE KO mice. Accumulation in human carotid atherosclerotic plaques was investigated by in vitro autoradiography. RESULTS: (111)In-DOTA-belatacept was obtained in >70 % yield, >99 % radiochemical purity, and ~40 GBq/µmol specific activity. The labeled belatacept bound with high affinity to Raji cells. In vivo, (111)In-DOTA-belatacept accumulated specifically in Raji xenografts, lymph nodes, and salivary glands. Ex vivo SPECT experiments revealed displaceable accumulation in atherosclerotic plaques of ApoE KO mice fed an atherosclerosis-promoting diet. In human plaques, binding correlated with the infiltration by immune cells and the presence of a large lipid and necrotic core. CONCLUSIONS: (111)In-DOTA-belatacept accumulates in CD80/CD86-positive tissues in vivo and in vitro rendering it a research tool for the assessment of inflammatory activity in atherosclerosis and possibly other diseases. The tracer is suitable for preclinical imaging of co-stimulatory molecules of both human and murine origin. Radiolabeled belatacept could serve as a benchmark for future CD80/CD86-specific imaging agents.

ImmunoPET/MR imaging allows specific detection of Aspergillus fumigatus lung infection in vivo.

Invasive pulmonary aspergillosis (IPA) is a life-threatening lung disease caused by the fungus Aspergillus fumigatus, and is a leading cause of invasive fungal infection-related mortality and morbidity in patients with hematological malignancies and bone marrow transplants. We developed and tested a novel probe for noninvasive detection of A. fumigatus lung infection based on antibody-guided positron emission tomography and magnetic resonance (immunoPET/MR) imaging. Administration of a [(64)Cu]DOTA-labeled A. fumigatus-specific monoclonal antibody (mAb), JF5, to neutrophil-depleted A. fumigatus-infected mice allowed specific localization of lung infection when combined with PET. Optical imaging with a fluorochrome-labeled version of the mAb showed colocalization with invasive hyphae. The mAb-based newly developed PET tracer [(64)Cu]DOTA-JF5 distinguished IPA from bacterial lung infections and, in contrast to [(18)F]FDG-PET, discriminated IPA from a general increase in metabolic activity associated with lung inflammation. To our knowledge, this is the first time that antibody-guided in vivo imaging has been used for noninvasive diagnosis of a fungal lung disease (IPA) of humans, an approach with enormous potential for diagnosis of infectious diseases and with potential for clinical translation.

Microbial transglutaminase and c-myc-tag: a strong couple for the functionalization of antibody-like protein scaffolds from discovery platforms.

Antibody-like proteins selected from discovery platforms are preferentially functionalized by site-specific modification as this approach preserves the binding abilities and allows a side-by-side comparison of multiple conjugates. Here we present an enzymatic bioconjugation platform that targets the c-myc-tag peptide sequence (EQKLISEEDL) as a handle for the site-specific modification of antibody-like proteins. Microbial transglutaminase (MTGase) was exploited to form a stable isopeptide bond between the glutamine on the c-myc-tag and various primary-amine-functionalized substrates. We attached eight different functionalities to a c-myc-tagged antibody fragment and used these bioconjugates for downstream applications such as protein multimerization, immobilization on surfaces, fluorescence microscopy, fluorescence-activated cell sorting, and in vivo nuclear imaging. The results demonstrate the versatility of our conjugation strategy for transforming a c-myc-tagged protein into any desired probe.

Delivery of BMP-2 by two clinically available apatite materials: in vitro and in vivo comparison.

Bone morphogenetic proteins (BMPs) are deposited in bone and responsible for osteoinduction. The interplay between delivery system and BMP, resulting in a characteristic release profile, is crucial for clinical success. We here report on two apatite based commercially available granules which could potentially be used in a combination product with recombinant human BMP-2 (rhBMP-2). Regardless of their similar chemistry, their interaction with rhBMP-2 differs. Deproteinized bovine bone matrix (DBBM), a clinically well-established bone substitute, has a high affinity to rhBMP-2 and releases only 50% of the growth factor during the first 2 weeks in vitro. Activity of the physio-adsorbed rhBMP-2 is indicated by an enhanced bone augmentation in vivo. In contrast, all rhBMP-2 delivered in combination with synthetic hydroxyapatite/ß-tricalcium phosphate (HA/TCP) granules is released during the first 24 h. For both HA/TCP and DBBM, the released rhBMP-2 is active in vitro. Our results suggest that the different release behavior from these two apatite granules is due to the 1000-fold higher specific surface area of DBBM compared to HA/TCP.

Mutation of threonine 34 in mouse podoplanin-Fc reduces CLEC-2 binding and toxicity in vivo while retaining antilymphangiogenic activity.

The lymphatic system plays an important role in cancer metastasis and inhibition of lymphangiogenesis could be valuable in fighting cancer dissemination. Podoplanin (Pdpn) is a small, transmembrane glycoprotein expressed on the surface of lymphatic endothelial cells (LEC). During mouse development, binding of Pdpn to the C-type lectin-like receptor 2 (CLEC-2) on platelets is critical for the separation of the lymphatic and blood vascular systems. Competitive inhibition of Pdpn functions with a soluble form of the protein, Pdpn-Fc, leads to reduced lymphangiogenesis in vitro and in vivo. However, the transgenic overexpression of human Pdpn-Fc in mouse skin causes disseminated intravascular coagulation due to platelet activation via CLEC-2. In the present study, we produced and characterized a mutant form of mouse Pdpn-Fc, in which threonine 34, which is considered essential for CLEC-2 binding, was mutated to alanine (PdpnT34A-Fc). Indeed, PdpnT34A-Fc displayed a 30-fold reduced binding affinity for CLEC-2 compared with Pdpn-Fc. This also translated into fewer side effects due to platelet activation in vivo. Mice showed less prolonged bleeding time and fewer embolized vessels in the liver, when PdpnT34A-Fc was injected intravenously. However, PdpnT34A-Fc was still as active as wild-type Pdpn-Fc in inhibiting lymphangiogenesis in vitro and also inhibited lymphangiogenesis in vivo. These data suggest that the function of Pdpn in lymphangiogenesis does not depend on threonine 34 in the CLEC-2 binding domain and that PdpnT34A-Fc might be an improved inhibitor of lymphangiogenesis with fewer toxic side effects.

Anti-L1CAM radioimmunotherapy is more effective with the radiolanthanide terbium-161 compared to lutetium-177 in an ovarian cancer model.

PURPOSE: The L1 cell adhesion molecule (L1CAM) is considered a valuable target for therapeutic intervention in different types of cancer. Recent studies have shown that anti-L1CAM radioimmunotherapy (RIT) with (67)Cu- and (177)Lu-labelled internalising monoclonal antibody (mAb) chCE7 was effective in the treatment of human ovarian cancer xenografts. In this study, we directly compared the therapeutic efficacy of anti-L1CAM RIT against human ovarian cancer under equitoxic conditions with the radiolanthanide (177)Lu and the potential alternative (161)Tb in an ovarian cancer therapy model. METHODS: Tb was produced by neutron bombardment of enriched (160)Gd targets. (161)Tb and (177)Lu were used for radiolabelling of DOTA-conjugated antibodies. The in vivo behaviour of the radioimmunoconjugates (RICs) was assessed in IGROV1 tumour-bearing nude mice using biodistribution experiments and SPECT/CT imaging. After ascertaining the maximal tolerated doses (MTD) the therapeutic impact of 50 % MTD of (177)Lu- and (161)Tb-DOTA-chCE7 was evaluated in groups of ten mice by monitoring the tumour size of subcutaneous IGROV1 tumours. RESULTS: The average number of DOTA ligands per antibody was 2.5 and maximum specific activities of 600 MBq/mg were achieved under identical radiolabelling conditions. RICs were stable in human plasma for at least 48 h. (177)Lu- and (161)Tb-DOTA-chCE7 showed high tumour uptake (37.8-39.0 %IA/g, 144 h p.i.) with low levels in off-target organs. SPECT/CT images confirmed the biodistribution data. (161)Tb-labelled chCE7 revealed a higher radiotoxicity in nude mice (MTD: 10 MBq) than the (177)Lu-labelled counterpart (MTD: 12 MBq). In a comparative therapy study with equitoxic doses, tumour growth inhibition was better by 82.6 % for the (161)Tb-DOTA-chCE7 than the (177)Lu-DOTA-chCE7 RIT. CONCLUSIONS: Our study is the first to show that anti-L1CAM (161)Tb RIT is more effective compared to (177)Lu RIT in ovarian cancer xenografts. These results suggest that (161)Tb is a promising candidate for future clinical applications in combination with internalising antibodies.

Transglutaminase-based chemo-enzymatic conjugation approach yields homogeneous antibody-drug conjugates.

Most chemical techniques used to produce antibody-drug conjugates (ADCs) result in a heterogeneous mixture of species with variable drug-to-antibody ratios (DAR) which will potentially display different pharmacokinetics, stability, and safety profiles. Here we investigated two strategies to obtain homogeneous ADCs based on site-specific modification of deglycosylated antibodies by microbial transglutaminase (MTGase), which forms isopeptidic bonds between Gln and Lys residues. We have previously shown that MTGase solely recognizes Gln295 within the heavy chain of IgGs as a substrate and can therefore be exploited to generate ADCs with an exact DAR of 2. The first strategy included the direct, one-step attachment of the antimitotic toxin monomethyl auristatin E (MMAE) to the antibody via different spacer entities with a primary amine functionality that is recognized as a substrate by MTGase. The second strategy was a chemo-enzymatic, two-step approach whereby a reactive spacer entity comprising a bio-orthogonal thiol or azide function was attached to the antibody by MTGase and subsequently reacted with a suitable MMAE-derivative. To this aim, we investigated two different chemical approaches, namely, thiol-maleimide and strain-promoted azide-alkyne cycloaddition (SPAAC). Direct enzymatic attachment of MMAE-spacer derivatives at an 80 molar excess of drug yielded heterogeneous ADCs with a DAR of between 1.0 to 1.6. In contrast to this, the chemo-enzymatic approach only required a 2.5 molar excess of toxin to yield homogeneous ADCs with a DAR of 2.0 in the case of SPAAC and 1.8 for the thiol-maleimide approach. As a proof-of-concept, trastuzumab (Herceptin) was armed with the MMAE via the chemo-enzymatic approach using SPAAC and tested in vitro. Trastuzumab-MMAE efficiently killed BT-474 and SK-BR-3 cells with an IC50 of 89.0 pM and 21.7 pM, respectively. Thus, the chemo-enzymatic approach using MTGase is an elegant strategy to form ADCs with a defined DAR of 2. Furthermore, the approach is directly applicable to a broad variety of antibodies as it does not require prior genetic modifications of the antibody sequence.

Tumor imaging in patients with advanced tumors using a new (99m) Tc-radiolabeled vitamin B12 derivative.

UNLABELLED: Targeting cancer cells with vitamin B12 (cobalamin) is hampered by unwanted physiologic tissue uptake mediated by transcobalamin. Adhering to good manufacturing practice, we have developed a new (99m)Tc-cobalamin derivative ((99m)Tc(CO)3-[(4-amido-butyl)-pyridin-2-yl-methyl-amino-acetato] cobalamin, (99m)Tc-PAMA-cobalamin). The derivative shows no binding to transcobalamin but is recognized by haptocorrin, a protein present in the circulation and notably expressed in many tumor cells. In this prospective study, we investigated cancer-specific uptake of (99m)Tc-PAMA-cobalamin in 10 patients with various metastatic tumors. METHODS: Ten patients with biopsy-proven metastatic cancer were included. Dynamic imaging was started immediately after injection of 300-500 MBq of (99m)Tc-PAMA-cobalamin, and whole-body scintigrams were obtained at 10, 30, 60, 120, and 240 min and after 24 h. The relative tumor activity using SPECT/CT over the tumor region after 4 h was measured in comparison to disease-free lung parenchyma. Patients 3-10 received between 20 and 1,000 µg of cobalamin intravenously before injection of (99m)Tc-PAMA-cobalamin. The study population comprised 4 patients with adenocarcinomas of the lung, 3 with squamous cell carcinomas of the hypopharyngeal region, 1 with prostate adenocarcinoma, 1 with breast, and 1 with colon adenocarcinoma. RESULTS: The median age of the study group was 61 ± 11 y. Six of 10 patients showed positive tumor uptake on (99m)Tc-PAMA-cobalamin whole-body scintigraphy. The scan was positive in 1 patient with colon adenocarcinoma, in 3 of 4 lung adenocarcinomas, in 1 of 3 hypopharyngeal squamous cell carcinomas, and in 1 breast adenocarcinoma. Renal uptake was between 1% and 3% for the left kidney. Predosing with cobalamin increased the tumor uptake and improved blood-pool clearance. The best image quality was achieved with a predose of 20-100 ug of cold cobalamin. The mean patient dose was 2.7 ± 0.9 mSv/patient. CONCLUSION: To our knowledge, we report for the first time on (99m)Tc-PAMA-cobalamin imaging in patients with metastatic cancer disease and show that tumor targeting is feasible.

Paclitaxel improved anti-L1CAM lutetium-177 radioimmunotherapy in an ovarian cancer xenograft model.

BACKGROUND: Today's standard treatment of advanced-stage ovarian cancer, including surgery followed by a paclitaxel-platinum-based chemotherapy, is limited in efficacy. Recently, we could show that radioimmunotherapy (RIT) with (177)Lu-labelled anti-L1 cell adhesion molecule (L1CAM) monoclonal antibody chCE7 is effective in ovarian cancer therapy. We investigated if the efficacy of anti-L1CAM RIT can be further improved by its combination with paclitaxel (PTX). METHODS: In vitro cell viability and cell cycle arrest of human ovarian cancer cells were assessed upon different treatment conditions. For therapy studies, nude mice (n = 8) were injected subcutaneously with IGROV1 human ovarian carcinoma cells and received a single dose of 6 MBq (177)Lu-DOTA-chCE7 alone or in combination with 600 µg PTX (31.6 mg/kg). Tumour growth delay and survival were determined. To investigate whether PTX can influence the tumour uptake of the radioimmunoconjugates (RICs), a biodistribution study (n = 4) and SPECT/CT images were acquired 120 h post injections of 2 MBq (177)Lu-DOTA-chCE7 alone or in combination with 600 µg PTX. RESULTS: Lu-DOTA-chCE7 in combination with PTX revealed a significantly decreased cell viability of ovarian carcinoma cells in vitro and was effective in a synergistic manner (combination index < 1). PTX increased the RIT efficacy by arresting cells in the radiosensitive G2/M phase of the cell cycle 24 h post treatment start. In vivo combination therapy including (177)Lu-DOTA-chCE7 and PTX resulted in a significantly prolonged overall survival (55 days vs. 18 days/PTX and 29 days/RIT), without weight loss and/or signs of toxicity. Biodistribution studies revealed no significant difference in tumour uptakes of (177)Lu-DOTA-chCE7 72 h post injection regardless of an additional PTX administration. CONCLUSIONS: Combination of anti-L1CAM (177)Lu-RIT with PTX is a more effective therapy resulting in a prolonged overall survival of human ovarian carcinoma-bearing nude mice compared with either monotherapy. The combination is promising for future clinical applications.

Future prospects for SPECT imaging using the radiolanthanide terbium-155 - production and preclinical evaluation in tumor-bearing mice.

INTRODUCTION: We assessed the suitability of the radiolanthanide (155)Tb (t1/2=5.32 days, Eγ=87 keV (32%), 105keV (25%)) in combination with variable tumor targeted biomolecules using preclinical SPECT imaging. METHODS: (155)Tb was produced at ISOLDE (CERN, Geneva, Switzerland) by high-energy (~1.4 GeV) proton irradiation of a tantalum target followed by ionization and on-line mass separation. (155)Tb was separated from isobar and pseudo-isobar impurities by cation exchange chromatography. Four tumor targeting molecules - a somatostatin analog (DOTATATE), a minigastrin analog (MD), a folate derivative (cm09) and an anti-L1-CAM antibody (chCE7) - were radiolabeled with (155)Tb. Imaging studies were performed in nude mice bearing AR42J, cholecystokinin-2 receptor expressing A431, KB, IGROV-1 and SKOV-3ip tumor xenografts using a dedicated small-animal SPECT/CT scanner. RESULTS: The total yield of the two-step separation process of (155)Tb was 86%. (155)Tb was obtained in a physiological l-lactate solution suitable for direct labeling processes. The (155)Tb-labeled tumor targeted biomolecules were obtained at a reasonable specific activity and high purity (>95%). (155)Tb gave high quality, high resolution tomographic images. SPECT/CT experiments allowed excellent visualization of AR42J and CCK-2 receptor-expressing A431 tumors xenografts in mice after injection of (155)Tb-DOTATATE and (155)Tb-MD, respectively. The relatively long physical half-life of (155)Tb matched in particular the biological half-lives of (155)Tb-cm09 and (155)Tb-DTPA-chCE7 allowing SPECT imaging of KB tumors, IGROV-1 and SKOV-3ip tumors even several days after administration. CONCLUSIONS: The radiolanthanide (155)Tb may be of particular interest for low-dose SPECT prior to therapy with a therapeutic match such as the ß(-)-emitting radiolanthanides (177)Lu, (161)Tb, (166)Ho, and the pseudo-radiolanthanide (90)Y.

Enzymatic antibody modification by bacterial transglutaminase.

Enzymatic posttranslational modification of proteins permits more precise control over conjugation site than chemical modification of reactive amino acid side chains. Ideally, protein modification by an enzyme yields completely homogeneous conjugates with improved properties for research or therapeutic use. As an example, we here provide a protocol for bacterial transglutaminase (BTGase)-mediated conjugation of cadaverine-derivatized substrates to an IgG1, resulting in stable bond formation between glutamine 295 of the antibody heavy chain and the substrate. This procedure requires enzymatic removal of N-linked glycans from the antibody and yields a defined substrate/antibody ratio of 2:1. Alternatively, a mutant aglycosylated IgG1 variant may be generated by site-directed mutagenesis. The mutation introduces an additional glutamine and yields a substrate/antibody ratio of 4:1 after coupling. Finally, we describe an ESI-TOF mass spectrometry-based method to analyze the uniformity of the resulting conjugates. The presented approach allows the facile generation of homogeneous antibody conjugates and can be applied to any IgG1 and a wide range of cadaverine-derivatized substrates.

Structural basis for universal corrinoid recognition by the cobalamin transport protein haptocorrin.

Cobalamin (Cbl; vitamin B12) is an essential micronutrient synthesized only by bacteria. Mammals have developed a sophisticated uptake system to capture the vitamin from the diet. Cbl transport is mediated by three transport proteins: transcobalamin, intrinsic factor, and haptocorrin (HC). All three proteins have a similar overall structure but a different selectivity for corrinoids. Here, we present the crystal structures of human HC in complex with cyanocobalamin and cobinamide at 2.35 and 3.0 Å resolution, respectively. The structures reveal that many of the interactions with the corrin ring are conserved among the human Cbl transporters. However, the non-conserved residues Asn-120, Arg-357, and Asn-373 form distinct interactions allowing for stabilization of corrinoids other than Cbl. A central binding motif forms interactions with the e- and f-side chains of the corrin ring and is conserved in corrinoid-binding proteins of other species. In addition, the α- and ß-domains of HC form several unique interdomain contacts and have a higher shape complementarity than those of intrinsic factor and transcobalamin. The stabilization of ligands by all of these interactions is reflected in higher melting temperatures of the protein-ligand complexes. Our structural analysis offers fundamental insights into the unique binding behavior of HC and completes the picture of Cbl interaction with its three transport proteins.

Synthesis and evaluation of biphenyl compounds as kinesin spindle protein inhibitors.

Kinesin spindle protein (KSP), an ATP-dependent motor protein, plays an essential role in bipolar spindle formation during the mitotic phase (M phase) of the normal cell cycle. KSP has emerged as a novel target for antimitotic anticancer drug development. In this work, we synthesized a range of new biphenyl compounds and investigated their properties in vitro as potential antimitotic agents targeting KSP expression. Antiproliferation (MTT (=3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide)) assays, combined with fluorescence-assisted cell sorting (FACS) and Western blot studies analyzing cell-cycle arrest confirmed the mechanism and potency of these biphenyl compounds in a range of human cancer cell lines. Structural variants revealed that functionalization of biphenyl compounds with bulky aliphatic or aromatic groups led to a loss of activity. However, replacement of the urea group with a thiourea led to an increase in antiproliferative activity in selected cell lines. Further studies using confocal fluorescence microscopy confirmed that the most potent biphenyl derivative identified thus far, compound 7, exerts its pharmacologic effect specifically in the M phase and induces monoaster formation. These studies confirm that chemical scope remains for improving the potency and treatment efficacy of antimitotic KSP inhibition in this class of biphenyl compounds.

Fluorinated quinazolinones as potential radiotracers for imaging kinesin spindle protein expression.

Anti-mitotic anti-cancer drugs offer a potential platform for developing new radiotracers for imaging proliferation markers associated with the mitosis-phase of the cell-cycle. One interesting target is kinesin spindle protein (KSP)-an ATP-dependent motor protein that plays a vital role in bipolar spindle formation. In this work we synthesised a range of new fluorinated-quinazolinone compounds based on the structure of the clinical candidate KSP inhibitor, ispinesib, and investigated their properties in vitro as potential anti-mitotic agents targeting KSP expression. Anti-proliferation (MTT and BrdU) assays combined with additional studies including fluorescence-assisted cell sorting (FACS) analysis of cell-cycle arrest confirmed the mechanism and potency of these biphenyl compounds in a range of human cancer cell lines. Additional studies using confocal fluorescence microscopy showed that these compounds induce M-phase arrest via monoaster spindle formation. Structural studies revealed that compound 20-(R) is the most potent fluorinated-quinazolinone inhibitor of KSP and represents a suitable lead candidate for further studies on designing (18)F-radiolabelled agents for positron-emission tomography (PET).

Radioimmunotherapy of fibroblast activation protein positive tumors by rapidly internalizing antibodies.

PURPOSE: Fibroblast activation protein (FAP) is a serine protease that has emerged as a promising target for cancer therapy, either by direct abrogation of its proinvasive activity or by specific targeting of FAP-expressing cells with cytotoxic immunoconjugates. We aimed to select novel human-mouse cross-reactive antibodies and to test suitability for tumor therapy as radioimmunoconjugates in a preclinical model. EXPERIMENTAL DESIGN: Human Fab fragments that bind to human and murine FAP were selected from an antibody phage library. Two candidates (ESC11 and ESC14) were engineered into fully human IgG1 antibodies and further characterized. We investigated the intracellular trafficking of ESC11 and ESC14 in live cells by confocal microscopy and analyzed the biodistribution and therapeutic effects of anti-FAP antibodies labeled with the ß-emitting radionuclide (177)Lu in a melanoma xenograft nude mouse model. Results were compared with vF19, a humanized variant of an anti-FAP antibody that has been previously used in clinical trials. RESULTS: The two antibodies bound selectively to both human and mouse FAP, with affinities in the low nanomolar range. Binding to FAP-expressing melanoma cells resulted in rapid internalization of FAP-antibody complexes. (177)Lu-labeled ESC11 specifically accumulated in melanoma xenografts in vivo, with a higher tumor uptake than ESC14 and vF19. Radioimmunotherapy with 8 MBq (177)Lu-labeled anti-FAP antibodies delayed growth of established tumors, whereas (177)Lu-ESC11 extended mouse survival more pronounced than (177)Lu-ESC14 and (177)Lu-vF19. CONCLUSION: Our results show the potential of ESC11 and ESC14 as potent radioimmunoconjugates or antibody-drug conjugates for diagnostic and therapeutic use in patients with FAP-expressing tumors.

Comparison of recombinant human haptocorrin expressed in human embryonic kidney cells and native haptocorrin.

Haptocorrin (HC) is a circulating corrinoid binding protein with unclear function. In contrast to transcobalamin, the other transport protein in blood, HC is heavily glycosylated and binds a variety of cobalamin (Cbl) analogues. HC is present not only in blood but also in various secretions like milk, tears and saliva. No recombinant form of HC has been described so far. We report the expression of recombinant human HC (rhHC) in human embryonic kidney cells. We purified the protein with a yield of 6 mg (90 nmol) per litre of cell culture supernatant. The isolated rhHC behaved as native HC concerning its spectral properties and ability to recognize both Cbl and its baseless analogue cobinamide. Similar to native HC isolated from blood, rhHC bound to the asialoglycoprotein receptor only after removal of terminal sialic acid residues by treatment with neuraminidase. Interestingly, rhHC, that compared to native HC contains four excessive amino acids (…LVPR) at the C-terminus, showed subtle changes in the binding kinetics of Cbl, cobinamide and the fluorescent Cbl conjugate CBC. The recombinant protein has properties very similar to native HC and although showing slightly different ligand binding kinetics, rhHC is valuable for further biochemical and structural studies.

L1-CAM-targeted antibody therapy and (177)Lu-radioimmunotherapy of disseminated ovarian cancer.

The L1-cell adhesion molecule (L1-CAM) is highly expressed in various cancer types including ovarian carcinoma but is absent from most normal tissue. A chimeric monoclonal antibody, chCE7, specifically binds to human L1-CAM and exhibits anti-proliferative effects on L1-CAM-expressing tumor cells. The goal of this study was to evaluate the efficacy of a novel (177)Lu-chCE7 radioimmunotherapeutic agent and to compare it to a treatment protocol with unlabeled, growth-inhibiting chCE7 in a mouse xenograft model of disseminated ovarian cancer. chCE7agl, an aglycosylated IgG1 variant with improved pharmacokinetics, was conjugated with 1,4,7,10-tetraazacyclododecane-N-N'-N'-N‴-tetraacetic acid (DOTA) and labeled with the low-energy ß-emitter (177)Lu. Tumor growth and survival were assessed after a single i.v. dose of 8 MBq (60 µg) radioimmunoconjugate in nude mice bearing either subcutaneous or intraperitoneal SKOV3.ip1 human ovarian cancer tumors. Therapeutic efficacy was compared with three times weekly i.p. administration of 10 mg/kg unconjugated chCE7. In vivo analysis of (177)Lu-chCE7agl biodistribution demonstrated high and specific accumulation of radioactivity at the tumor site with maximal tumor uptake of up to 48.0 ± 8.1% ID/g at 168 h postinjection. A single treatment with (177)Lu-DOTA-chCE7agl caused significant retardation of tumor growth and prolonged median survival from 33 to 71 days, while administration of a nontargeted (177)Lu-immunoconjugate had no beneficial effect. Three times weekly i.p. application of unlabeled chCE7 10 mg/kg similarly increased survival from 44 to 72 days. We conclude that a single dose of (177)Lu-DOTA-chCE7agl is as effective as repeated administration of nonradioactive chCE7 for treatment of small intraperitoneal tumors expressing L1-CAM.