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Angiopoietin-2 specific binding agents

A technology of antibodies and variable regions, applied in the field of specific binding agents, can solve problems such as not being clearly seen

Inactive Publication Date: 2010-03-17
AMGEN INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

From this information, and since both Ang-1 and Ang-2 are known to bind Tie-2, it is not clear from these studies that i Ang-1, Ang-2, or Tie-2 would be attractive for anticancer therapy force target

Method used

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  • Angiopoietin-2 specific binding agents
  • Angiopoietin-2 specific binding agents

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0281] Ang-2 expression in pathological and normal tissues

[0282] In situ hybridization was used to examine the expression of Ang-2 in normal and pathological tissues. Amplification of human (Genbank Accession Number: AF004327, nucleotide 1274-1726) and mouse (Genbank Accession Number: AF004326, nucleotide 1135-1588) Ang-2 sequence from human or mouse fetal lung cDNA by reverse transcriptase-PCR The fragment was cloned into the plasmid pGEM-T and confirmed by sequencing. use 33 Transcription of P-UTP and RNA polymerase from linearized plasmid template 33 P-labeled antisense RNA probe. The paraffin-embedded tissue block immobilized with formaldehyde was cut into 5 microns and collected on a charged glass slide. Before in situ hybridization, the tissue was digested with 0.2M HCL, followed by proteinase K, and acetylated with triethanolamine and acetic anhydride. The sections were hybridized with radiolabeled probes overnight at 55°C, then RNase digested, and washed with high ...

Embodiment 2

[0286] Production of recombinant mAng-2 protein and rabbit polyclonal anti-Ang-2 antiserum

[0287] Using PCR primers of full-length human Ang-2, the mouse embryo DNA library (Marathon-Ready-cDNA, Cat.#; 7459-1, Clonetech, Inc.) was obtained by PCR (Clontech Advantage PCRKit, Cat.#K1905-01). .) Obtain a full-length, histidine-tagged Ang-2 cDNA. The PCR product was ligated into the CMV promoter expression vector using FuGENE6 transfection reagent (Roche, Cat.#; 1814443), and the resulting plasmid was transfected into HT1080 human fibrosarcoma cells (obtained from ATCC). Stable clones were isolated by G418 selection. The clones expressing mAng-2-his were screened by anti-histidine labeling and western blotting.

[0288] The recombinant mAng-2 polypeptide was purified from the conditioned medium (C.M.) of these cells. C.M containing mAng-2-His was purified by a two-step chromatography method. Briefly, the conditioned medium was titrated to a final concentration of approximately 20...

Embodiment 3

[0293] Evaluation of molecular assays for Ang-2 antibodies

[0294] Molecular assays (affinity ELISA, neutralization ELISA, and BIAcore) were performed to evaluate direct antibodies that bind to Ang-2 and related family members, and the effects of antibodies on the Ang-2:Tie-2 interaction. These in vitro and cell-based assays are described in detail below.

[0295] A. Affinity ELISA

[0296] For the initial screening of candidate anti-Ang-2 antibodies, purified human Ang-2 (R&D Systems, Inc; catalog number 623-AN; Ang-2 is provided as a mixture of 2 truncated forms) or mouse Ang-2 polypeptide (as above) The preparation). To confirm the binding test, human Ang-2 was obtained from the conditioned medium of human 293T cells transfected with full-length human Ang-2 DNA and tested in serum-free Dulbecco's containing approximately 50 μg / ml bovine serum albumin (BSA). Cultured in modified Eagle medium (DMEM).

[0297] Using a microtiter plate, add approximately 100 μl / well of Ang-2 to e...

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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 wellas methods of making and using the antibodies.

Description

[0001] This application is a divisional application of Chinese patent application 02824877.5 "A specific binding agent for Angiopoietin-2" on October 11, 2002. [0002] This application claims the benefit of U.S. Provisional Application Registration No. 60 / 328,624 filed on October 11, 2001, which is hereby incorporated by reference in its entirety. Technical field [0003] The present invention relates to a specific binding agent that recognizes and binds to Angiopoietin-2 (Ang-2). More specifically, the present invention relates to the preparation, diagnostic use, and therapeutic use of the specific binding agent and fragments thereof that specifically bind to Ang-2. Background technique [0004] Angiogenesis, the formation of new blood vessels from existing blood vessels, is more demanding for many physiological and pathological processes. Generally, angiogenesis is strictly regulated by pro-angiogenic factors and anti-angiogenic factors, but in the case of diseases such as cance...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/22C12N15/13C12N15/63C12N5/10C07K16/46C12P21/08A61K39/395A61P35/00A61P29/00A61P19/08A61P15/00A61P27/02A61P17/06A61P9/00A61P9/10A61P43/00G01N33/53A61K45/00A61P3/04A61P3/10A61P5/00A61P11/06A61P19/02A61P35/02C07K16/18C12N1/15C12N1/19C12N1/21C12N15/09
CPCC07K2317/21C07K16/22A61K2039/505C07K2317/56A61P11/06A61P15/00A61P17/06A61P19/00A61P19/02A61P19/08A61P27/02A61P29/00A61P3/04A61P35/00A61P35/02A61P43/00A61P5/00A61P9/00A61P9/10A61P3/10C07K16/30C07K14/515
Inventor J·D·奥利纳
Owner AMGEN INC
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