Tumor necrosis factor (TNF)-soluble high-affinity receptor complex as a TNF antagonist.

A novel high-affinity inhibitor of tumor necrosis factor (TNF) is described, which is created by the fusion of the extracellular domains of TNF-binding protein 1 (TBP-1) to both the alpha and beta chains of an inactive version of the heterodimeric protein hormone, human chorionic gonadotropin. The resulting molecule, termed TNF-soluble high-affinity receptor complex (SHARC), self-assembles into a heterodimeric protein containing two functional TBP-1 moieties. The TNF-SHARC is a potent inhibitor of TNF-alpha bioactivity in vitro and has a prolonged pharmacokinetic profile compared with monomeric TBP-1 in vivo. Consistent with the long half-life, the duration of action in an lipopolysaccharide-mediated proinflammatory mouse model is prolonged similarly. In a collagen-induced arthritis mouse model, this molecule demonstrates improved efficacy over monomeric TBP-1. Based on these results, we demonstrated that inactivated heterodimeric protein hormones are flexible and efficient scaffolds for the creation of soluble high-affinity receptor complexes.

New biological therapies from the human genome.

Over the past 20 years, the biotechnology industry has been extraordinarily successful in bringing a wide variety of new products to the market, including recombinant versions of natural proteins such as growth hormone, insulin and the gonadotropins. The availability of the human genome sequence has given us the chance to identify the entire catalogue of human secreted proteins, often called the secretome. One of the challenges of biotechnology research has been to identify the biological activities of these proteins and to identify if any of them could have a therapeutic or pharmacologic use. The paradigm has effectively been reversed, in that it used to be easy to know the biological activity, but difficult to clone, whereas now the contrary is true. Five years on, it is clear that finding new biological activities is a very difficult process. Much of the ground gained in this period has either been through the development of antibodies as therapies or by the use of protein engineering to produce better versions of the proteins that are already being produced.

A high-throughput mammalian protein expression, purification, aliquoting and storage pipeline to assemble a library of the human secretome.

In the post-human genome-sequencing era, the availability of recombinant proteins has become crucial for the identification of proteins with therapeutic potential. Based upon bioinformatic coding predictions of the genes for putative secreted proteins, we established a high-throughput protein pipeline (HTPP) for the production of a subset of the human secretome. The HTPP was based on a transient expression system in HEK293-EBNA cells at 100 to 500 mL culture scale, combined with an automated affinity purification procedure targeting >75% purity. This was followed by a semi-automated protein sample logistics to provide biologists with quality-controlled and 96 well formatted protein aliquots amenable to cell-based assays. Over a 4-year period, beginning in 2001, we performed over 7,500 transfections representing over 2,200 registered proteins, including both novel and reference proteins, at an average production of 280 proteins/month with a peak production of 320 proteins/month. All these proteins have been tested in more than 50 different cell-based assays. This article describes the major process steps and highlights the optimization required to maintain novel protein production while supporting both stock replenishment and scale-up productions.

Osteopontin is upregulated during in vivo demyelination and remyelination and enhances myelin formation in vitro.

We have used in vitro oligodendrocyte differentiation and the in vivo remyelination model, the cuprizone model, to identify genes regulating oligodendrocyte function and remyelination. One of the genes we identified, osteopontin (opn), is a secreted glycoprotein with cytokine-like, chemotactic, and anti-apoptotic properties that contains an Arg-Gly-Asp (RGD) cell adhesion motif-mediating interactions with several integrins. Both microglia and astrocytes in demyelinating brain regions of cuprizone-fed mice expressed OPN protein. Recombinant OPN protein produced in a baculovirus expression system induced proliferation of both the rat CG-4 and the mouse Oli-neu oligodendrocyte precursor (OLP)-like cell lines in a dose-dependent manner. In addition, recombinant OPN treatment stimulated both myelin basic protein (MBP) synthesis and myelin sheath formation in mixed cortical cultures from embryonic mouse brain, an in vitro primary culture model of myelination. Interestingly, myelinating mixed cultures prepared from OPN(-/-) mice contained significantly less MBP compared to wild-type cultures after 17 days in culture. We propose that in the central nervous system, OPN may act as a novel regulator of myelination and remyelination.

MIG--differential gene expression in mouse brain endothelial cells.

Different diseases of the CNS are associated with blood-brain barrier (BBB) damage and mononuclear cell infiltration. In order to study genes that may play a role in endothelial cell regulation in inflammatory CNS diseases, we performed differential gene expression (DGE) analysis using a mouse brain endothelial cell line. We found that interferon-gamma (IFNgamma)-induced monokine (MIG), a chemokine that plays a role in T lymphocyte and monocyte chemoattraction, is highly expressed in the presence of inflammatory cytokines. We show that MIG, produced by brain endothelial cells in vitro, is biologically active in attractingT lymphocytes and that it is possible to interfere with this mechanism of action using anti-MIG antibodies. We suggest that blocking MIG may be beneficial in CNS inflammation. We detected constitutive expression of the MIG receptor, CXCR3, on the surface of the endothelial cells and therefore hypothesize that it plays a role in maintaining the cytokine gradient at the region of CNS inflammation.

Unconventional organization of the division and cell wall gene cluster of Streptococcus pneumoniae.

The genes responsible for cell wall biosynthesis and cell division (dcw genes) were identified and sequenced in Streptococcus pneumoniae. The genetic organization of the dcw cluster in Streptococcus pneumoniae differed significantly from the clusters of other bacteria reported to date. In particular, the genes corresponding to the 2 min region of the Escherichia coli chromosome were found distributed in three genetically separate regions of the Streptococcus pneumoniae chromosome. The first region contained the expected ftsA and ftsZ cell division genes at one end and pbp2b, ddl and murF at the other end. The murD, murG and divIB genes, always found located upstream of ftsA, were found in a second region separated from the first. A third region contained the yllC, yllD, pbp2x and mraY genes. The chromosomal region downstream of ftsZ was also sequenced and characterized. In Streptococcus pneumoniae this region contains four ORFs, all of unknown function, and an ORF encoding the Bacillus subtilis DivIVA homologue. The gene order and the organization of this region was found to be conserved in Staphylococcus aureus, Streptococcus pyogenes and Bacillus subtilis, raising the possibility that previously unidentified loci may also be involved in division.