World Antibody Drug Conjugate Summit Europe: February 21–23, 2011; Frankfurt,Germany

The World Antibody Drug Conjugate Summit Europe, organized by Biorbis/Hanson Wade was held in Frankfurt, Germany February 21–23, 2011. Antibody drug conjugates (ADCs), also called immunoconjugates, are becoming an increasingly important class of therapeutics as demonstrated by the attendance of nearly 100 delegates at this highly focused meeting. Updates on three ADCs that are in late-stage clinical development, trastuzumab emtansine (T-DM1), brentuximab vedotin (SGN-35) and inotuzumab ozogamicin (CMC-544), were presented by speakers from ImmunoGen, Genentech, Roche, Seattle Genetics and Pfizer. These ADCs have shown encouraging therapeutic effects against solid tumors (T-DM1) and hematological malignancies (SGN-35, CMC-544). The key feature of the new generation of ADCs is the effective combination of the cytotoxicity of natural or synthetic highly potent antineoplastic agents, tumor selective monoclonal antibodies and blood-stable optimized linkers. Early clinical data for ADCs were showcased by Progenics Pharmaceuticals (PSMA ADC), Celldex (CDX-011) and Biotest (BT-062). Takeda, MedImmune and sanofi-aventis outlined their strategies for process development and analytical characterization. In addition, presentations on duocarmycin based-ADCs, α emitting immunoconjugates and antibody-conjugated nanoparticles were given by representatives from Syntarga, Algeta and the University of Stuttgart, respectively.Key words: antibody drug conjugates, immunoconjugates, trastuzumab emtansine, brentuximab vedotin, inotuzumab ozogamicin, oncology, cancer     

Apoptosis of airway epithelial cells in response to meconium

Meconium aspiration syndrome (MAS) is common among newborn children but its mechanism is unclear. The syndrome is known to produce a strong inflammatory reaction in the lungs resulting in massive cell death. In this work we studied lung cell death by apoptosis after meconium aspiration in forty two-week-old rabbit pups. Analyzing lung samples by ISEL-DNA end labeling demonstrated the specific spread of apoptotic bodies throughout the lungs. These bodies were shrunken and smaller in size compared to normal cells and many of them were lacking cell membranes. About 70% of all apoptotic bodies were found among the airway epithelium cell eight hours after meconium instillation. In comparison, among lung alveolar cells, only about 20% cells were apoptotic in the same animals. In meconium-treated lungs and A549 cells, a significant increase of angiotensinogen mRNA and Caspase-3 expression were observed. The pretreatment of cells with Caspase-3 inhibitor ZVAD-fmk significantly inhibited meconium-induced lung cell death by apoptosis. These findings demonstrate the apoptotic process in meconium-instilled lungs or A549 cells in culture. Our results show lung airway epithelial and A549 cell apoptosis after meconium instillation. We suggest that studies of lung airway epithelial cell death are essential to understanding the pathophysiology of MAS and may present a key point in future therapeutic applications.     

Can urine lamivudine be used to monitor antiretroviral treatment adherence?

Patient adherence to treatment is an important factor in the effectiveness of antiretroviral regimens. Adherence to treatment could be monitored by estimation of antiretroviral drugs in biological fluids. We aimed to obtain information on the quantity and duration of excretion of lamivudine in urine following oral administration of a single dose of 300 mg and to assess its suitability for adherence monitoring purposes. Spot urine samples were collected before dosing and at 4, 8, 12, 24, 28, 32, 48, 72, and 96 hours post dosing from 10 healthy subjects, and lamivudine was estimated by high-pressure liquid chromatography (HPLC). Lamivudine values were expressed as a ratio of urine creatinine. About 91% of the ingested drug was excreted by 24 hours, and the concentration thereafter in urine was very negligible. A lamivudine value of 0.035 mg/mg creatinine or less at 48 hours is suggestive of a missed dose in the last 24 hours. The study findings showed that estimation of urine lamivudine in spot specimens could be useful in monitoring patient adherence to antiretroviral treatment. However, this needs to be confirmed on a larger sample size and among patients on once-daily and twice-daily treatment regimens.     

Overexpression of hyaluronan-binding protein 1 (HABP1/p32/gC1qR) in HepG2 cells leads to increased hyaluronan synthesis and cell proliferation by up-regulation of cyclin D1 in AKT-dependent pathway

Overexpression of the mature form of hyaluronan-binding protein 1 (HABP1/gC1qR/p32), a ubiquitous multifunctional protein involved in cellular signaling, in normal murine fibroblast cells leads to enhanced generation of reactive oxygen species (ROS), mitochondrial dysfunction, and ultimately apoptosis with the release of cytochrome c. In the present study, human liver cancer cell line HepG2, having high intracellular antioxidant levels was chosen for stable overexpression of HABP1. The stable transformant of HepG2, overexpressing HABP1 does not lead to ROS generation, cellular stress, and apoptosis, rather it induced enhanced cell growth and proliferation over longer periods. Phenotypic changes in the stable transformant were associated with the increased "HA pool," formation of the "HA cable" structure, up-regulation of HA synthase-2, and CD44, a receptor for HA. Enhanced cell survival was further supported by activation of MAP kinase and AKT-mediated cell survival pathways, which leads to an increase in CYCLIN D1 promoter activity. Compared with its parent counterpart HepG2, the stable transformant showed enhanced tumorigenicity as evident by its sustained growth in low serum conditions, formation of the HA cable structure, increased anchorage-independent growth, and cell-cell adhesion. This study suggests that overexpression of HABP1 in HepG2 cells leads to enhanced cell survival and tumorigenicity by activating HA-mediated cell survival pathways.     

Identification and characterization of a novel Ribose 5-phosphate isomerase B from Leishmania donovani

Leishmaniasis is a group of tropical diseases caused by protozoan parasites of the genus Leishmania. Due to the emergence of resistance to the available antileishmanial drugs there is an immediate need to identify molecular targets on which to base future treatment strategies. Ribose 5-phosphate isomerase (Rpi; EC 5.3.1.6) is a key enzyme of the pentose phosphate pathway (PPP) which catalyses the reversible aldose-ketose isomerization between Ribose 5-phosphate (R5P) and Ribulose 5-phosphate (Ru5P). It exists in two isoforms A and B. These two are completely unrelated enzymes catalyzing the same reaction. Analysis of the Leishmania infantum genome revealed that though the RpiB gene is present, RpiA homologs are completely absent. An absence of RpiBs in the genomes of higher animals makes this enzyme a possible target for the chemotherapy of Leishmaniasis. In this paper, we report for the first time the presence of B isoform of the Rpi enzyme in Leishmania donovani (LdRpiB) by cloning and molecular characterization of the enzyme. An amplified L. donovani RpiB gene is 519 bp and encodes for a putative 172 amino acid protein with a molecular mass of ～19 kDa. An ～19 kDa protein with poly-His tag at the C-terminal end was obtained by heterologous expression of LdRpiB in Escherichia coli. The recombinant form of RpiB was obtained in soluble and active form. The LdRpiB exists as a dimer of dimers i.e. the tetramer form. The polyclonal antibody against Trypanosoma cruzi RpiB could detect a band of ～19 kDa with the purified recombinant RpiB as well as native RpiB from the L. donovani promastigotes. Recombinant RpiB obeys the classical Michaelis-Menten kinetics utilizing R5P as the substrate with a K(m) value of 2.4±0.6 mM and K(cat) value of 30±5.2 s(-1). Our study confirms the presence of Ribose 5-phosphate isomerase B in L. donovani and provides functional characterization of RpiB for further validating it as a potential drug target.