The proto-oncogene designated erbB2 or HER2 encodes a 185-kilodalton transmembrane tyrosine kinase (p185erbB2), whose overexpression has been correlated with a poor prognosis in several human malignancies. A 45-kilodalton protein heregulin-alpha (HRG-alpha) that specifically induced phosphorylation of p185erbB2 was purified from the conditioned medium of a human breast tumor cell line. Several complementary DNA clones encoding related HRGs were identified, all of which are similar to proteins in the epidermal growth factor family. Scatchard analysis of the binding of recombinant HRG to a breast tumor cell line expressing p185erbB2 showed a single high affinity binding site [dissociation constant (Kd) = 105 +/- 15 picomolar]. Heregulin transcripts were identified in several normal tissues and cancer cell lines. The HRGs may represent the natural ligands for p185erbB2.

We report identification of interleukin (IL)-17E, a novel member of the IL-17 family of cytokines. IL-17E is a ligand for the recently identified protein termed EVI27/IL-17BR, which we term IL-17 receptor homolog 1 (IL-17Rh1) in light of the multiple reported ligand-receptor relationships. Murine EVI27 was identified through its location at a common site of retroviral integration in BXH2 murine myeloid leukemias. IL-17Rh1 shows highest level expression in kidney with moderate expression in multiple other organs, whereas IL-17E mRNA was detected at very low levels in several peripheral tissues. IL-17E induces activation of NF-B and stimulates production of the proinflammatory chemokine IL-8.

Synthetic genes for human insulin A and B chains were cloned separately in plasmid pBR322. The cloned synthetic genes were then fused to an Escherichia coli #-galactosidase gene to provide efficient transcription and translation and a stable precursor protein. The insulin peptides were cleaved from Pgalactosidase, detected by radioimmunoassay and purified. Complete purification of the A chain and partiaf purification of the B chain were achieved. These products were mixed, reduced, and reoxidized. The presence of insulin was detected by radioimmunoassay.Recently improved methods of DNA chemical synthesis, combined with recombinant DNA technology, permit the design and relatively rapid synthesis of modest-sized genes that can be incorporated into prokaryotic cells for gene expression. The feasibility of this general approach was first demonstrated by the synthesis, and expression in Escherichia col, of a gene for the mammalian peptide somatostatin (1).Following the precursor protein approach used for somatostatin (1), the experimental design for this work was such that the insulin peptide chains would be made in vio as short tails joined by a methionine to the end of ,3-galactosidase. After synthesis, the insulin chains, which contain no methionine, can be cleaved off efficiently by treatment with cyanogen bromide. We deliberately chose to construct two separate bacterial strains, one for each of the two peptide chains of insulin: the 21-amino-acid A chain and the 30-amino-acid B chain. In native insulin, the two chains are held together by two disulfide bonds, and methods have been available for years for joining the chains correctly, in vitro, by air oxidation (2). The efficiency of correct joining has been variable and often low. However, by using S-sulfonated derivatives and an excess of A chain, 50-80% correct joining has been obtained (3).The synthetic plan and chemical synthesis of the DNA fragments coding for the A and B chains of human insulin were described in a previous paper (4) and were summarized in Fig. 1 Enzymes and DNA Preparations. T4 DNA ligase and T4 polynucleotide kinase were purified as described (6). Restriction endonuclease EcoRI was purified by the procedure of Greene et al. (7); HindIII was purified by a method developed by D. Goeddel (unpublished). Restriction endonuclease BamHI was purchased from Bethesda Research (Rockville, MD); E. coli alkaline phosphatase was purchased from Worthington.Plasmids, including pBR322 (8), were isolated by a published procedure (9) with some modifications. The chemical synthesis of the deoxyoligonucleotides (figure 1 of ref. 4) has been described (4). Xplac5 DNA was isolated as described (10).The following reaction buffers were used: kinase buffer, 60 mM Tris-HCl, pH 8/15mM 2-mercaptoethanol/10 mM MgCl2; ligase buffer, 20mM Tris-HCl, pH 7.5/10mM dithiothreitol/10 mM MgCl2; BamHI buffer, 20 mM Tris-HCl, pH 7.5/7 mM MgCl2/2 mM 2-mercaptoethanol; EcoRI-HindIll buffer, BamHI buffer containing 50 mM NaCl; and phosphatase buffer, 50 mM Tris-HCl, pH...

A large-scale effort, termed the Secreted Protein Discovery Initiative (SPDI), was undertaken to identify novel secreted and transmembrane proteins. In the first of several approaches, a biological signal sequence trap in yeast cells was utilized to identify cDNA clones encoding putative secreted proteins. A second strategy utilized various algorithms that recognize features such as the hydrophobic properties of signal sequences to identify putative proteins encoded by expressed sequence tags (ESTs) from human cDNA libraries. A third approach surveyed ESTs for protein sequence similarity to a set of known receptors and their ligands with the BLAST algorithm. Finally, both signal-sequence prediction algorithms and BLAST were used to identify single exons of potential genes from within human genomic sequence. The isolation of full-length cDNA clones for each of these candidate genes resulted in the identification of >1000 novel proteins. A total of 256 of these cDNAs are still novel, including variants and novel genes, per the most recent GenBank release version. The success of this large-scale effort was assessed by a bioinformatics analysis of the proteins through predictions of protein domains, subcellular localizations, and possible functional roles. The SPDI collection should facilitate efforts to better understand intercellular communication, may lead to new understandings of human diseases, and provides potential opportunities for the development of therapeutics.

Binding of hepatocyte growth factor (HGF) to the receptor tyrosine kinase MET is implicated in the malignant process of multiple cancers, making disruption of this interaction a promising therapeutic strategy. However, targeting MET with bivalent antibodies can mimic HGF agonism via receptor dimerization. To address this limitation, we have developed onartuzumab, an Escherichia coliderived, humanized, and affinity-matured monovalent monoclonal antibody against MET, generated using the knob-into-hole technology that enables the antibody to engage the receptor in a one-to-one fashion. Onartuzumab potently inhibits HGF binding and receptor phosphorylation and signaling and has antibody-like pharmacokinetics and antitumor activity. Biochemical data and a crystal structure of a ternary complex of onartuzumab antigen-binding fragment bound to a MET extracellular domain fragment, consisting of the MET Sema domain fused to the adjacent Plexins, Semaphorins, Integrins domain (MET Sema-PSI), and the HGF β-chain demonstrate that onartuzumab acts specifically by blocking HGF α-chain (but not β-chain) binding to MET. These data suggest a likely binding site of the HGF α-chain on MET, which when dimerized leads to MET signaling. Onartuzumab, therefore, represents the founding member of a class of therapeutic monovalent antibodies that overcomes limitations of antibody bivalency for targets impacted by antibody crosslinking.scatter factor | HGFR | MetMAb | OA5D5