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Angiopoietin-2 specific binding agents

An angiopoietin-promoting, species-specific technology, applied in cardiovascular system diseases, carrier-bound antigen/hapten components, drug combinations, etc., can solve the problems of not being able to clearly understand the targets of anti-cancer therapy

Inactive Publication Date: 2009-07-29
AMGEN INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Given this information, and because both Ang-1, Ang-2 bind to Tie-2, it is not clear from these studies whether Ang-1, Ang-2, or Tie-2 might be attractive anticancer treatments target

Method used

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  • Angiopoietin-2 specific binding agents

Examples

Experimental program
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Effect test

Embodiment 1

[0313] Express Ang-2 in pathological and normal tissues

[0314] In situ hybridization was used to detect the expression of Ang-2 in normal and pathological tissues. The human Ang-2 sequence fragment (Genbank accession number: AF004327, nucleotides 1274-1726) and mouse Ang-2 fragment (Genbank accession number: AF004326, nucleosides) were amplified from human or mouse fetal lung cDNA by reverse transcription PCR. Acid 1135-1588), and cloned into pGEM-T plasmid vector, confirmed by sequencing. use 33 P-UTP and RNA polymerase are transcribed using linearized plasmid as template 33 P-labeled antisense RNA probe. Tissues blocked by formaldehyde and embedded in paraffin were sectioned (5 μm) and collected on charged glass slides. Before in situ hybridization, the tissue was permeabilized with 0.2M HCl, then digested with proteinase K, and acetylated with triethanolamine and acetic anhydride. The radiolabeled probe was hybridized with the section overnight at 55°C, then digested with R...

example example 2

[0318] Preparation of recombinant mAng-2 protein and rabbit polyclonal anti-Ang-2 antiserum

[0319] Using PCR primers used to amplify the full-length human Ang-2, a 15-day-old mouse embryo cDNA library (Marathon-Ready-cDNA, Cat. #7459-1, Clonetech, Inc.) was passed through PCR (Clontech Advantage PCR Kit, Cat. #K1905-01) to obtain the full-length mouse Ang-2 cDNA with His tag. The PCR product was ligated to the CMV promoter expression vector, and the resulting plasmid was transfected into HT1080 human fibrosarcoma cells (obtained from ATCC) with FuGENE6 transfection reagent (Roche, Cat. #1814443). Stable clones were isolated by G418 selection. The clones expressing mAng-2-his were screened using anti-His tag ELASA and Western blot.

[0320] The recombinant mAng-2 polypeptide was purified from the conditioned medium (C.M.) of these cells. A two-step chromatography was used to purify the conditioned medium containing mAng-2-His. Briefly, Tris buffer (pH 9.5) was added to the cond...

Embodiment 3

[0325] Evaluation of Ang-2 antibody molecular experiments

[0326] Molecular experiments (affinity ELISA, neutralization ELISA, and BIAcore) were developed to directly evaluate antibodies that bind to Ang-2 and related family members, and to evaluate the effects of antibodies on mAng-2:Tie2 interaction. These cell-based in vitro experiments are described below.

[0327] A. Affinity ELISA

[0328] For the initial screening of candidate anti-Ang-2 antibodies, purified human Ang-2 (Rand D Systems, Inc; catalog number 623-AN; where Ang-2 is provided as a mixture of two truncated fragments) or mouse Ang- 2 Polypeptide (prepared according to the method described above). In order to perform confirmatory binding experiments, human 293T cells were transfected with full-length human Ang-2 DNA and cultured in serum-free DMEM (containing about 50 μg / ml of bovine serum albumin (BSA)). Get people Ang-2.

[0329] Using a microtiter plate, add about 100 microliters of Ang-2 to each well and in...

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Abstract

Disclosed are specific binding agents, such as fully human antibodies, that bind to angiopoietin-2. Also disclosed are heavy chain fragments, light chain fragments, and CDRs of the antibodies, as well as methods of making and using the antibodies.

Description

[0001] This article is a partial continuation application of US patent application 10 / 269,805 (application on October 10, 2002) and a partial continuation application of PCT application PCT / US02 / 32613 (application on October 11, 2002). The PCT application requires Priority is granted to US provisional patent application 60 / 328,604 (application on October 11, 2001), all patents are incorporated herein by reference. This document also claims priority to U.S. Provisional Patent Application 60 / 620,161 (filed on October 19, 2004), which is incorporated herein by reference. Invention field [0002] The present invention relates to a specific binding agent that can recognize and bind to Angiopoietin-2 (Ang-2). More specifically, the present invention relates to the production, diagnostic applications and therapeutic applications of polyclonal antibodies, monoclonal antibodies and fragments thereof that can specifically bind to Ang-2. Background of the invention [0003] Angiogenesis, the...

Claims

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Application Information

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IPC IPC(8): C07K16/22C12N15/13C07K16/46C12P21/08C12N15/20A61K39/385A61K39/395A61P35/00A61P9/00G01N33/68G01N33/577
Inventor J·D·奥利纳K·格雷汉
Owner AMGEN INC
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