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Novel Anti-human ctgf antibody

An antibody and sequence technology, applied in the direction of antibodies, anti-animal/human immunoglobulins, animal/human proteins, etc., can solve the problems of insufficient binding activity and insufficient CTGF neutralization activity, etc., to achieve the goal of giving Improvement of drug method, improvement of treatment effectiveness and patient compliance, effect of strong anti-fibrotic effect

Active Publication Date: 2014-08-27
ASTELLAS PHARMA INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] However, the binding activity of conventional antibodies to CTGF is not sufficient, and the neutralizing activity to CTGF is not strong enough from the viewpoint of therapeutic effectiveness

Method used

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  • Novel Anti-human ctgf antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0092] (Example 1: the acquisition of CTGF protein from various sources)

[0093]The present inventors obtained human CTGF protein as an antigen for making anti-CTGF antibodies. The full-length gene (sequence number 13) of human CTGF is integrated in the expression vector (pcDNA3.1; Invitrogen Company), the vector that will be made uses FreeStyle MAX Reagent (Invitrogen Company) as gene transfer reagent to FreeStyle293 cell (Invitrogen Company) ) for gene transfer. The cells were cultured in a serum-free culture system using FreeStyle293 expression medium (Invitrogen) to obtain a culture supernatant containing human CTGF protein. Proteins were purified from the obtained culture supernatants using HiTrap heparin columns and CM columns (GE Healthcare Japan), and used for the following experiments. For mouse, rat and monkey CTGF proteins, the same method was used to obtain them.

Embodiment 2

[0094] (Example 2: Immunization to VelocImmune mice)

[0095] Anti-human CTGF antibody was obtained by immunizing VelocImmune mice. In order to increase the diversity of the antibodies obtained, the present inventors conducted research on various immunization methods, administration routes, adjuvants, immunization periods, and the like. As an immunogen, purified human CTGF was used and mixed with an adjuvant for immunization. As the route of administration, plantar administration and intraperitoneal administration were investigated. As an adjuvant, TiterMax Gold (CytRx), complete Freund's adjuvant (Sigma), and incomplete Freund's adjuvant (Sigma) were investigated. As further added immune stimulators, CpG oligonucleotides and aluminum phosphate gel (BRENNTAG) were investigated. As the immunization period, 3 weeks to 14 weeks were studied. After several immunizations, blood was collected from the tail vein of the mice, and the titer was monitored to select VelocImmune mice ...

Embodiment 3

[0097] (Example 3: Production of hybridomas producing anti-human CTGF antibodies)

[0098] Final immunization (intravenous or intraperitoneal administration of the antigen) was performed on mice selected after confirming the increase in antibody titer. According to a conventional method, the spleen, lymph nodes, etc. of the immunized mice were removed, lymphocytes were collected, and they were fused with mouse myeloma cells SP2 / 0 to prepare hybridomas. The hybridomas were subjected to limiting dilution, and on the basis of obtaining single clones, antibodies were purified from the supernatant using a protein A or protein G column (GE Healthcare Japan).

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Abstract

The invention provides an anti-human CTGF antibody having a superior binding activity and / or a superior neutralization activity compared with those of conventional anti-human CTGF antibodies; and a means for preventing or treating various diseases in which human CTGF is involved in the formation of clinical conditions thereof, including renal diseases such as chronic kidney disease and diabetic nephropathy, using the above-mentioned anti-human CTGF antibody. An anti-human CTGF antibody containing a heavy-chain variable region comprising the amino acid sequence represented by SEQ ID NO: 10 and a light-chain variable region comprising the amino acid sequence represented by SEQ ID NO: 4.

Description

technical field [0001] The present invention relates to novel anti-human CTGF antibodies. Specifically, the novel anti-human CTGF antibody of the present invention is an anti-human CTGF antibody superior in binding activity and / or neutralizing activity to conventional anti-human CTGF antibodies. Background technique [0002] CTGF (connective tissue growth factor) is a secreted protein with a molecular weight of about 36 to 38 kDa and rich in cysteine ​​residues belonging to the CCN family (Non-Patent Document 1). TGF-β induction of growth factor (Non-Patent Document 2). Therefore, it is speculated that TGF-β induces CTGF, and the induced CTGF promotes the fibrosis of organs and tissues. CTGF is considered to play an important role in fibrosis, cell proliferation, metabolism of extracellular matrix, angiogenesis, arteriosclerosis, etc. Patent Document 3). [0003] Multiple domains are known to exist in CTGF and interact with other factors. Among them, it has been revealed...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/09C07K16/22C12N1/15C12N1/19C12N1/21C12N5/10C12N15/02C12P21/08
CPCC07K2317/24C07K16/22C07K14/475C12N15/09A61K39/395C12N5/10C07K2317/76A61K2039/505C12N15/02C07K2317/41C07K2317/92A61P13/12
Inventor 岩崎正司守屋隆一吉野正康高仓浩二
Owner ASTELLAS PHARMA INC
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