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Novel anti-human tie-2 antibody

An antibody and antigen technology, applied in the direction of antibodies, antibody medical components, antibody mimics/scaffolds, etc., can solve problems such as increased retinal cell damage

Active Publication Date: 2020-08-28
ASTELLAS PHARMA INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Meanwhile, increased retinal cell damage has been reported in genetically modified mice with Ang-2-specific overexpression in the retina (Acta. Diabetol. 2010, Vol. 47, pp. 59-64)

Method used

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  • Novel anti-human tie-2 antibody
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  • Novel anti-human tie-2 antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0246] (Example 1: Preparation of anti-human Tie2 antibody-producing hybridomas)

[0247] Antibodies were prepared using "VelocImmune" (VelocImmune Antibody Technology: Regeneron Corporation. (US Patent No. 6596541)) - Human Monoclonal Antibody Development Technology - Mouse. Recombinant human Tie2-Fc chimeric protein (R&D, 313-TI-100) was injected into VelocImmune mice for immunization with adjuvant for eliciting an immune response. According to a usual method, the lymph nodes of the immunized mice were extracted, and lymphocytes were collected and fused with mouse-derived myeloma cells SP2 / 0 (ATCC: CRL-1581) to prepare hybridomas. The hybridomas were monocloned, and each clone was cultured in CD Hybridoma Medium (Invitrogen) as a serum-free medium. Antibodies were purified from the resulting culture supernatant using a protein G column (GE Healthcare). The antibody obtained using the VelocImmune technology is an antibody having the variable region of a human antibody and t...

Embodiment 2

[0248] (Example 2: Cell ELISA assay)

[0249] To measure the antigen-binding activity of the antibodies, the CHO cells expressing human Tie2, CHO cells expressing monkey Tie2, CHO cells expressing rat Tie2, and CHO cells expressing mouse Tie2 were each evaluated by a cellular ELISA assay Binding of antibodies to human Tie2, monkey Tie2, rat Tie2 and mouse Tie2.

Embodiment 3

[0250] (Example 3: Evaluation of Competitive Activity Using Modified Ang-1)

[0251] To assess the Ang-2-competitive activity of the antibody, modified Ang-1 (Proc. Natl. Acad. Sci., 2004, Vol. 101, pp. 5547-5552, also known as COMP-Ang1) was evaluated with Inhibition of Tie2 binding. COMP-Ang1 is a modified Ang-1 in which a site not involved in binding to Tie2 is modified, and the binding ability to Tie2 from COMP-Ang1 is maintained (Proc.Natl.Acad.Sci.2004, Vol.101 , pp.5547-5552), and that Ang-1 and Ang-2 bind to the same site of Tie2 with the same level of affinity (Science, 1997, Vol.277, pp.55-60), it antagonizes The competitive role of Ang-2 can be assessed by evaluating the competitive role against COMP-Ang1.

[0252] The expression vector of COMP-Ang1 was introduced into HEK293 cells. COMP-Ang1 was purified from the culture supernatant of the HEK293 cells and biotinylated. The biotin-labeled COMP-Ang1 was mixed with the purified antibody obtained in Example 1, and...

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Abstract

The present invention aims to provide an anti-human Tie2 antibody, which can prevent or treat diabetic macular edema, diabetic retinopathy and severe limb ischemia by binding to human Tie2 and activating the human Tie2. The present inventors conducted research on an anti-human Tie2 antibody, and thus provided an anti-human Tie2 antibody comprising 4 heavy chain variable regions and 4 light chain variable regions. The heavy chain variable region consists of the amino acid sequence of amino acids 1 to 122 of SEQ ID NO:2, and the light chain variable region consists of the amino acid sequence of amino acids 1 to 113 of SEQ ID NO:4. One of the heavy chain variable regions and one of the light chain variable regions constitutes one antigen-binding site, and the antibody comprises four antigen-binding sites.

Description

technical field [0001] The present invention relates to novel anti-human Tie-2 antibodies. Background technique [0002] Tyrosine kinase with Ig and EGF homology domain 2 (Tie2) is a receptor-type tyrosine kinase. Tie2 is known to be mainly expressed in vascular endothelial cells. As ligands, angiopoietin-1 (Ang-1) and angiopoietin-2 (Ang-2), which are secreted glycoproteins of a multimeric type, are known. [0003] Ang-1 acts as an agonist of Tie2. It has been found that when Tie2 binds to Ang-1, it is autophosphorylated by forming a multimer and sends a signal into the cell, thereby promoting the anti-apoptotic effect of vascular endothelial cells, the blood vessels through the osmotic inhibition of blood vessels Stabilization, maturation and remodeling (Cell, 1996, Vol.87, pp.1171-1180; GenesDev., 1994, Vol.8, pp.1897-1909; Science, 1999, Vol.286, pp.2511- 2514; and Nat. Struct. Biol., 2003, Vol. 10, pp. 38-44). In addition, Ang-1 is also known to exert vasodilating ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/28A61K39/395A61P7/10A61P9/10A61P43/00C07K16/46C12N1/15C12N1/19C12N1/21C12N5/10C12N15/09C12P21/08
CPCC12N2510/02C07K16/2863C07K2317/24C07K2317/33C07K2317/34C07K2317/52C07K2317/55C07K2317/64C07K2317/92C07K2319/00C07K2317/567C07K2317/75A61K2039/505C07K16/40C07K2317/35A61P27/02A61P43/00A61P7/10A61P9/10A61K39/395A61K39/3955C07K16/28C07K16/2896C07K2317/40C07K2317/51C07K2317/515C07K2317/565C07K2317/76C12N5/06C12N15/09C12N15/63
Inventor 蒲原正纯八木繁典石井芳则奈良裕美
Owner ASTELLAS PHARMA INC
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