Side-by-side analysis of five clinically tested anti-EpCAM monoclonal antibodies

Background

Epithelial cell adhesion molecule (EpCAM) is frequently and highly expressed on human carcinomas. The emerging role of EpCAM as a signalling receptor and activator of the wnt pathway, and its expression on tumor-initiating cells, further add to its attractiveness as target for immunotherapy of cancer. Thus far, five conventional monoclonal IgG antibodies have been tested in cancer patients. These are murine IgG2a edrecolomab and its murine/human chimeric IgG1 antibody version, and humanized, human-engineered and fully human IgG1 antibodies 3622W94, ING-1, and adecatumumab (MT201), respectively. Here we compared all anti-EpCAM antibodies in an attempt to explain differences in clinical activity and safety.

Methods

We recombinantly produced all antibodies but murine edrecolomab and investigated them for binding affinity, EpCAM epitope recognition, ADCC and CDC, and inhibition of breast cancer cell proliferation.

Results

ING-1 and 3622W94 bound to EpCAM with much higher affinity than adecatumumab and edrecolomab. Edrecolomab, ING-1, and 3622W94 all recognized epitopes in the exon 2-encoded N-terminal domain of EpCAM, while adecatumumab recognized a more membrane proximal epitope encoded by exon 5. All antibodies induced lysis of EpCAM-expressing cancer cell lines by both ADCC and CDC with potencies that correlated with their binding affinities. The chimeric version of edrecolomab with a human Fcγ1 domain was much more potent in ADCC than the murine IgG2a version. Only adecatumumab showed a significant inhibition of MCF-7 breast cancer cell proliferation in the absence of complement and immune cells.

Conclusion

A moderate binding affinity and recognition of a distinct domain of EpCAM may best explain why adecatumumab showed a larger therapeutic window in cancer patients than the two high-affinity IgG1 antibodies ING-1 and 3622W94, both of which caused acute pancreatitis.     

Über die Entwicklung und den feineren Bau des Tapetum lucidum der Feliden

Ohne Zusammenfassung     

The caveolin-binding motif of the pathogen-related yeast protein Pry1, a member of the CAP protein superfamily,is required for in vivo export of cholesteryl acetate

Proteins belonging to the CAP superfamily are present in all kingdoms of life and have been implicated in different physiological processes. Their molecular mode of action, however, is poorly understood. Saccharomyces cerevisiae expresses three members of this superfamily, pathogen-related yeast (Pry)1, -2, and -3. We have recently shown that Pry function is required for the secretion of cholesteryl acetate and that Pry proteins bind cholesterol and cholesteryl acetate, suggesting that CAP superfamily members may generally act to bind sterols or related small hydrophobic compounds. Here, we analyzed the mode of sterol binding by Pry1. Computational modeling indicates that ligand binding could occur through displacement of a relatively poorly conserved flexible loop, which in some CAP family members displays homology to the caveolin-binding motif. Point mutations within this motif abrogated export of cholesteryl acetate but did not affect binding of cholesterol. Mutations of residues located outside the caveolin-binding motif, or mutations in highly conserved putative catalytic residues had no effect on export of cholesteryl acetate or on lipid binding. These results indicate that the caveolin-binding motif of Pry1, and possibly of other CAP family members, is crucial for selective lipid binding and that lipid binding may occur through displacement of the loop containing this motif.     

Iridovirus and microsporidian linked to honey bee colony decline

Background

In 2010 Colony Collapse Disorder (CCD), again devastated honey bee colonies in the USA, indicating that the problem is neither diminishing nor has it been resolved. Many CCD investigations, using sensitive genome-based methods, have found small RNA bee viruses and the microsporidia, Nosema apis and N. ceranae in healthy and collapsing colonies alike with no single pathogen firmly linked to honey bee losses.

Methodology/Principal Findings

We used Mass spectrometry-based proteomics (MSP) to identify and quantify thousands of proteins from healthy and collapsing bee colonies. MSP revealed two unreported RNA viruses in North American honey bees, Varroa destructor-1 virus and Kakugo virus, and identified an invertebrate iridescent virus (IIV) (Iridoviridae) associated with CCD colonies. Prevalence of IIV significantly discriminated among strong, failing, and collapsed colonies. In addition, bees in failing colonies contained not only IIV, but also Nosema. Co-occurrence of these microbes consistently marked CCD in (1) bees from commercial apiaries sampled across the U.S. in 2006–2007, (2) bees sequentially sampled as the disorder progressed in an observation hive colony in 2008, and (3) bees from a recurrence of CCD in Florida in 2009. The pathogen pairing was not observed in samples from colonies with no history of CCD, namely bees from Australia and a large, non-migratory beekeeping business in Montana. Laboratory cage trials with a strain of IIV type 6 and Nosema ceranae confirmed that co-infection with these two pathogens was more lethal to bees than either pathogen alone.

Conclusions/Significance

These findings implicate co-infection by IIV and Nosema with honey bee colony decline, giving credence to older research pointing to IIV, interacting with Nosema and mites, as probable cause of bee losses in the USA, Europe, and Asia. We next need to characterize the IIV and Nosema that we detected and develop management practices to reduce honey bee losses.     

Untersuchungen über die inkretorischen Organe der Fische

Zusammenfassung An Querschnitten durch verschiedene Stadien der Aalmetamorphose wird gezeigt, daß die Schilddrüse in der Metamorphose des Flußaals dieselben Veränderungen wie in der Metamorphose der Frösche aufweist. Daraus wird der Schluß gezogen, daß die Schilddrüse, ähnlich wie bei den Kaulquappen, auch bei den Aalen eine die Metamorphose auslösende und beschleunigende Wirkung hat.     

The perinuclear space of pancreatic acinar cells and the synthetic pathway of zymogen in Scorpaena scrofa L.: ultrastructural aspects

Electron microscopic examination of exocrine pancreatic tissues from the fish Scorpaena scrofa L., probably captured while replenishing the acinar cells, shows two main functional cell morphologies of the same cell type. One cell functional aspect contains numerous well-contrasted small vesicles, the zymogenic vesicles. The other functional morphology is mainly represented by a few cells containing large apical zymogen vesicles with many empty RER cisterns. In our observations, the zymogenic vesicles are always studded with ribosomes. The main cytological finding is to report that zymogenic vesicles can be extruded from the perinuclear space and it confirms the suspected, synthetic activity of this cell compartment. The pool of zymogenic vesicles, maintaining their coat of ribosomes, then fuses and transfers their content into the cis Golgi complex network. Finally, the zymogen vesicles are produced following the classical secretory pathway from the trans Golgi saccular network into the supranuclear, apical region of the acinar cells where the largest vesicles concentrate their content until secretion.     

PR-Set7-dependent lysine methylation ensures genome replication and stability through S phase

PR-Set7/SET8 is a histone H4–lysine 20 methyltransferase required for normal cell proliferation. However, the exact functions of this enzyme remain to be determined. In this study, we show that human PR-Set7 functions during S phase to regulate cellular proliferation. PR-Set7 associates with replication foci and maintains the bulk of H4-K20 mono- and trimethylation. Consistent with a function in chromosome dynamics during S phase, inhibition of PR-Set7 methyltransferase activity by small hairpin RNA causes a replicative stress characterized by alterations in replication fork velocity and origin firing. This stress is accompanied by massive induction of DNA strand breaks followed by a robust DNA damage response. The DNA damage response includes the activation of ataxia telangiectasia mutated and ataxia telangiectasia related kinase–mediated pathways, which, in turn, leads to p53-mediated growth arrest to avoid aberrant chromosome behavior after improper DNA replication. Collectively, these data indicate that PR-Set7–dependent lysine methylation during S phase is an essential posttranslational mechanism that ensures genome replication and stability.     

Down-Regulation of CTLA-4 by HIV-1 Nef Protein

HIV-1 Nef protein down-regulates several cell surface receptors through its interference with the cell sorting and trafficking machinery. Here we demonstrate for the first time the ability of Nef to down-regulate cell surface expression of the negative immune modulator CTLA-4. Down-regulation of CTLA-4 required the Nef motifs DD175, EE155 and LL165, all known to be involved in vesicle trafficking. Disruption of the lysosomal functions by pH-neutralizing agents prevented CTLA-4 down-regulation by Nef, demonstrating the implication of the endosomal/lysosomal compartments in this process. Confocal microscopy experiments visualized the co-localization between Nef and CTLA-4 in the early and recycling endosomes but not at the cell surface. Overall, our results provide a novel mechanism by which HIV-1 Nef interferes with the surface expression of the negative regulator of T cell activation CTLA-4. Down-regulation of CTLA-4 may contribute to the mechanisms by which HIV-1 sustains T cell activation, a critical step in viral replication and dissemination.