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The human erythropoietin gene has been isolated from a genomric phage library by using mixed 20-mer and 17-mer oligonucleotide probes. Construction of Oligonucleotide Probes. Purified human urinary Epo'isolated from the urine of patients with aplastic anemia (16) was subjected to tryptic digestion. The resulting fragments were isolated and sequenced by using an Applied Biosystems gas-phase microsequencer (unpublished data). A hexapeptide and a heptapeptide containing the least codon degeneracy were selected for oligodeoxyribonucleotide probe synthesis. The phosphoramidite method was l4sed for oligonucleotide synthesis (19,20). Each' probe mixture contained a pool of 128-oligonucleotide sequences. The probe mixtures were Probe mixture EpV = Val-Asn-Phe-Tyr-Ala-Trp-Lys 3' CAA TTG AAG ATG CGA ACC TT 5'Probe mixture EpQ= Gln-Pro-Trp-Glu-Pro-Leu 3' GTT GGA ACC CTT GGA GA 5'The probe mixtures were labeled at the 5' end with [-32P]ATP, 7500-8000 Ci/mmol (ICN) (1 Ci = 37 GBq), by using T4 polynucleotide kinase (21). Hybridization Procedures. Phage plaques were amplified according to the procedures of Woo (22) except that GeneScreenPlus filters and NZYAM plates [NaCl, 5 g; MgCl2-6H2O, 2 g; NZ-Amine A, 10 g; yeast extract, 5 g; Casamino acids, 2 g; maltose, 2 g; and agar, 15 g (per liter)] were utilized. Phage particles were disrupted and the DNAs were fixed on filters (50,000 plaques per 8.4 x 8.4 cm filter). The air-dried filters were baked at 80'C for 1 hr and then subjected to proteinase K digestion [50 ,ug ofproteinase 'K per ml of buffer solution containing 0.1 M Tris HCl (pH 8.0), 0.15 M NaCl, 10 mM EDTA, and 0.2% NaDodSO4] for 30 min at 550C. Prehybridization with a 1 M NaCl/1% NaDodSO4 solution was carried out at 550C for 4 hr or longer.The hybridization buffer contained 0.025 pmol/ml of each of the 128 probe sequences in 0.9 M NaCl/5 mM EDTA/50 mM sodium phosphate, pH 6.5/0.5% NaDodSO4/100 jg of yeast tRNA per ml. Hybridization was carried out at 480C'for 20 hr by using the EpV probe mixture. This is 2TC below the lowest calculated dissociation temperature (td) (23) for members of the mixture. At the completion of hybridization, the filters were washed three times with 0.9 M NaCl/90 mM sodium citrate, pH 7.0/0. 1% NaDodSO4 at room temperature Abbreviations: Epo, erythropoietin; CHO, Chinese hamster ovary; DHFR, dihydrofolate reductase; kb, kilobase(s); bp, base pair(s); nt, nucleotide(s); SV40, simian virus 40; td, dissociation temperature. 7580The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Angiopoietin-2 (Ang2) exhibits broad expression in the remodeling vasculature of human tumors but very limited expression in normal tissues, making it an attractive candidate target for antiangiogenic cancer therapy. To investigate the functional consequences of blocking Ang2 activity, we generated antibodies and peptide-Fc fusion proteins that potently and selectively neutralize the interaction between Ang2 and its receptor, Tie2. Systemic treatment of tumor-bearing mice with these Ang2-blocking agents resulted in tumor stasis, followed by elimination of all measurable tumor in a subset of animals. These effects were accompanied by reduced endothelial cell proliferation, consistent with an antiangiogenic therapeutic mechanism. Anti-Ang2 therapy also prevented VEGF-stimulated neovascularization in a rat corneal model of angiogenesis. These results imply that specific Ang2 inhibition may represent an effective antiangiogenic strategy for treating patients with solid tumors.

Delivery of protein therapeutics often requires frequent injections because of low activity or rapid clearance, thereby placing a burden on patients and caregivers. Using glycoengineering, we have increased and prolonged the activity of proteins, thus allowing reduced frequency of administration. Glycosylation analogs with new N-linked glycosylation consensus sequences introduced into the protein were screened for the presence of additional N-linked carbohydrates and retention of in vitro activity. Suitable consensus sequences were combined in one molecule, resulting in glycosylation analogs of rHuEPO, leptin, and Mpl ligand. All three molecules had substantially increased in vivo activity and prolonged duration of action. Because these proteins were of three different classes (rHuEPO is an N-linked glycoprotein, Mpl ligand an O-linked glycoprotein, and leptin contains no carbohydrate), glycoengineering may be generally applicable as a strategy for increasing the in vivo activity and duration of action of proteins. This strategy has been validated clinically for glycoengineered rHuEPO (darbopoetin alfa).

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The erythropoietin (EPO) molecule contains four carbohydrate chains. Three contain N-linkages to asparagines at positions 24, 38, and 83, and one contains an O-linkage to a serine at position 126. We constructed human EPO variants that eliminated the three N-glycosylation sites by replacing the asparagines with glutamines singly or in combination. The O-linked carbohydrate chain was removed by replacing the serine with glutamine, valine, histidine, or alanine. A variant with a double mutation (Gln38,83) and another with a triple mutation (Gln24,38,83) were secreted poorly from COS1 and CHO cells even though RNA encoding these variants was present. All other variants with mutations in N-linked glycosylation sites were secreted normally. Removal of any of the N-glycosylation sites reduced the in vivo but not the in vitro biological activity of the EPO molecule. All the mutations at Ser126, the O-glycosylation site, were secreted normally. In vitro activity was also unaffected except for Ala126 which had a 50-fold decrease. The Val126 variant was tested in vivo, and its specific activity was only slightly less than that of the native EPO, which indicates that the O-linked carbohydrate is not essential for activity.

Difficulties associated with in vitro manipulation and culture of the early chicken embryo have restricted generation of transgenic chickens to approaches that use replication-competent retroviruses. The need to produce transgenic chickens in the absence of replicating virus prompted development of a new method of gene transfer into the chicken. Microinjection of the replication-defective reticuloendotheliosis virus (REV) vector ME111 beneath unincubated chicken embryo blastoderms results in infection of germline stem cells. This vector contains genetic information exogenous to the chicken genome, including both the herpes simplex virus type 1 thymidine kinase gene and the Tn5 neomycin phosphotransferase gene. About 8 percent of male birds hatched from injected embryos contained vector DNA in their semen. All four positive males tested passed vector sequences onto their progeny. Analysis of G1 offspring showed that gonads of G0 male birds were mosaic with respect to insertion of vector provirus. Thus, primordial germ cells present in the unincubated chicken embryo blastoderm are susceptible to infection by defective REV vectors.

IL-18-binding protein (IL-18BP) is a natural IL-18 inhibitor. Human IL-18BP isoform a was produced as fusion construct with human IgG1 Fc and assessed for binding and neutralizing IL-18. IL-18BP-Fc binds human, mouse, and rat IL-18 with high affinity (KD 0.3–5 nM) in a BIAcore-based assay. In vitro, IL-18BP-Fc blocks IL-18 (100 ng/ml)-induced IFN-γ production by KG1 cells (EC50 = 0.3 μg/ml). In mice challenged with an LD90 of LPS (15 mg/kg), IL-18BP-Fc (5 mg/kg) administered 10 min before LPS blocks IFN-γ production and protects against lethality. IL-18BP-Fc administered 10 min before LPS blocks IFN-γ production induced by LPS (5 mg/kg) with ED50 of 0.005 mg/kg. Furthermore, IL-18BP-Fc (5 mg/kg) abrogates LPS (5 mg/kg)-induced IFN-γ production even when administered 6 days before LPS but shows no effect when administered 9 or 12 days before LPS. Given 10 min before LPS challenge to mice primed 12 days in advance with heat-killed Propionibacterium acnes, IL-18BP-Fc prevents LPS-induced liver damage and IFN-γ and Fas ligand expression. Given at the moment of priming with P. acnes, IL-18BP-Fc decreases P. acnes-induced granuloma formation, macrophage-inflammatory protein-1α and macrophage-inflammatory protein-2 production and prevents sensitization to LPS. IL-18BP-Fc also prevents Con A-induced liver damage and IFN-γ and Fas ligand expression as well as liver damage induced by Pseudomonas aeruginosa exotoxin A or by anti-Fas agonistic Ab. In conclusion, IL-18BP can be engineered and produced in recombinant form to generate an IL-18 inhibitor, IL-18BP-Fc, endowed with remarkable in vitro and in vivo properties of binding and neutralizing IL-18.