A monoclonal antibody prevents aggregation of the NBD1 domain of the cystic fibrosis transmembrane conductance regulator

The homozygous deletion of the phenylalanine at position 508 (ΔPhe508) in the first nucleotide-binding domain (NBD1) of the cystic fibrosis transmembrane conductance regulator (CFTR) is the most common CF-causing genetic defect. It has been proposed that the propensity of NBD1 to aggregate may lead to a lower display of the CFTR chloride channel to the cell membrane and to the disease, thus opening an avenue for the pharmacological development of CFTR folding correctors. Here, we show that a human monoclonal antibody fragment specific to the folded conformation of NBD1 inhibits the aggregation of NBD1 in vitro. However, in contrast to the previously published observations, we proved experimentally that NBD1 of wild-type and ΔPhe508 version of CFTR display comparable propensities to aggregate in vitro and that the corresponding full-length CFTR protein reaches the cell membrane with comparable efficiency in mammalian cell expression systems. On the basis of our results, the ‘folding defect' hypothesis seems unlikely to represent the causal mechanism for the pathogenesis of CF. A solid understanding of how the ΔPhe508 deletion leads to the disease represents an absolute requirement for the development of effective drugs against C

Identification of Extracellular Segments by Mass Spectrometry Improves Topology Prediction of Transmembrane Proteins

Transmembrane proteins play crucial role in signaling, ion transport, nutrient uptake, as well as in maintaining the dynamic equilibrium between the internal and external environment of cells. Despite their important biological functions and abundance, less than 2% of all determined structures are transmembrane proteins. Given the persisting technical difficulties associated with high resolution structure determination of transmembrane proteins, additional methods, including computational and experimental techniques remain vital in promoting our understanding of their topologies, 3D structures, functions and interactions. Here we report a method for the high-throughput determination of extracellular segments of transmembrane proteins based on the identification of surface labeled and biotin captured peptide fragments by LC/MS/MS. We show that reliable identification of extracellular protein segments increases the accuracy and reliability of existing topology prediction algorithms. Using the experimental topology data as constraints, our improved prediction tool provides accurate and reliable topology models for hundreds of human transmembrane proteins

Signaling pathways in breast cancer metastasis - novel insights from functional genomics

The advent of genomic profiling technology has brought about revolutionary changes in our understanding of breast cancer metastasis. Gene expression analyses of primary tumors have been used to predict metastatic propensity with high accuracy. Animal models of metastasis additionally offer a platform to experimentally dissect components of the metastasis genetic program. Recent integrated studies have synergized clinical bioinformatic analyses with advanced experimental methodology and begun to uncover the identities and dynamics of signaling programs driving breast cancer metastasis. Such functional genomics studies hold great promise for understanding the genetic basis of metastasis and improving therapeutics for advanced diseases

A monoclonal antibody prevents aggregation of the NBD1 domain of the cystic fibrosis transmembrane conductance regulator

ISSN:1741-0126ISSN:1741-0134ISSN:0269-2139ISSN:1460-213

A comprehensive surface proteome analysis of myeloid leukemia cell lines for therapeutic antibody development

A detailed characterization of the cell surface proteome facilitates the identification of target antigens, which can be used for the development of antibody-based therapeutics for the treatment of hematological malignancies. We have performed cell surface biotinylation of five human myeloid leukemia cell lines and normal human granulocytes, which was used for mass spectrometric analysis and allowed the identification and label-free, relative quantification of 320 membrane proteins. Several proteins exhibited a pronounced difference in expression between leukemia cell lines and granulocytes. We focused our attention on CD166/ALCAM, as this protein was strongly up-regulated on all AML cell lines and AML blasts of some patients. A human monoclonal antibody specific to CD166 (named H8) was generated using phage display technology. H8 specifically recognized AML cells in FACS analysis while demonstrating tumor targeting properties in vivo. After in vitro screening of five potent cytotoxic agents, a duocarmycin derivative was used for the preparation of an antibody-drug conjugate, which was able to kill AML cells in vitro with an IC50 of 8nM. The presented atlas of surface proteins in myeloid leukemia provides an experimental basis for the choice of target antigens, which may be used for the development of anti-AML therapeutic antibodies. BIOLOGICAL SIGNIFICANCE: The ability to discriminate between malignant and healthy, essential cells represents an important requirement for the development of armed antibodies for the therapy of hematological malignancies. Our proteomic study is, to our knowledge, the first large scale comparison of the accessible cell surface proteome of leukemia cells and normal blood cells, facilitating the choice of a suitable target for the treatment of acute myeloid leukemia (AML). An antibody drug conjugate was generated recognizing the CD166 antigen which was found to be strongly up-regulated in all AML cell lines and AML blasts of some patients. This antibody drug conjugate SIP(H8)-Duo might be further characterized in therapy experiments and might lead to a new targeted treatment option for AML