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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 The resulting fragments were isolated and sequenced by using an Applied Biosystems gas-phase microsequencer (unpublished data).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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