Anti-human cd69 antibody, and use thereof for medical purposes

a technology of human cd69 and antibody, applied in the field of antihuman cd69 antibody, can solve the problems of substantially impossible inability to evaluate the pharmacological effect of existing antibodies in vivo, and the method of efficiently evaluating in vitro the pharmacological effect of an antibody to human cd69 is not availabl

Inactive Publication Date: 2015-04-30
GENEFRONTIER CORP +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0015]According to the present invention, an anti-human CD69 antibody applicable to the prophylaxis or treatment of allergic diseases and inflammatory diseases is provided. According to the present invention, moreover, an animal model permitting in vivo evaluation of a pharmacological effect of an anti-human CD69 antibody can be provided.

Problems solved by technology

However, since the ligand for CD69 is unknown, a method for efficiently evaluating in vitro a pharmacological effect of an antibody to human CD69 is not available.
Moreover, since existing antibodies to human CD69 do not cross-react with non-human CD69, evaluation of a pharmacological effect of the existing antibodies in vivo is substantially impossible, and even whether or not these antibodies afford a useful pharmacological effect is not clear.

Method used

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  • Anti-human cd69 antibody, and use thereof for medical purposes
  • Anti-human cd69 antibody, and use thereof for medical purposes
  • Anti-human cd69 antibody, and use thereof for medical purposes

Examples

Experimental program
Comparison scheme
Effect test

example 1

Production of Antigen and Antibody

(1) Production of Human CD69 Recombinant Protein

[0112]Since human CD69 forms a homodimer via cysteine 68, cDNA (NM—001781) encoding the extracellular region (amino acid sequences; 62-199) containing cysteine 68 was inserted into a vector for Escherichia coli periplasm expression. Competent cells of Escherichia coli TG1F(−) strain prepared in advance (Z-competent E. Coli Transformation Buffer Set: manufactured by ZYMO RESEARCH) were transformed with this expression vector, and cultured on a LB agar plate containing chloramphenicol (final concentration 34 μg / mL) at 37° C. overnight. This Escherichia coli cells were inoculated in a 2×YT medium, and cultured at 37° C. for 3-5 hr (OD600=0.5-0.8). IPTG (final concentration 0.1 mM) was added, and the mixture was cultured at 25° C. overnight. The cultured Escherichia coli cells were collected by centrifugation, and lysed with lysis buffer (200 mM borate, 160 mM NaCl, 2 mM EDTA, 1 mg / ml lysozyme, pH 8.0), an...

example 2

Effect of Anti-Human CD69 Antibody on Intraalveolar Mononuclear Cell Infiltration

[0119]Mice obtained by crossing BALB / c mouse or CD69 deficient (CD69KO) mouse back-crossed not less than 10 times onto BALB / c (Murata, K. et al. 2003. Int. Immunol. 15: 987-992) with D011.10 transgenic mouse were used. The spleen CD4T cells of these mice were purified by AutoMACS sorter (Miltenyi Biotec) to a purity of >98%. The isolated CD4T cells were cultured with stimulation with immobilized anti-TCR and anti-CD28 monoclonal antibodies under Th2 conditions (IL-2 10 u / ml, IL-4 100 U / ml). Two days after the start of the culture, and human CD69 gene was introduced by a retrovirus vector containing human CD69 (hCD69) gene. The method of introducing human CD69 gene followed the method described previously (Kimura, M. et al. 2001. Immunity 15: 275-287). Five days after the start of the culture, the cultured cells were recovered, and the expression of human CD69 was confirmed by flow cytometry (FIG. 2). 50...

example 3

Selection of High Affinity Anti-Human CD69 Antibody

[0124]Selection of an antibody having higher affinity for human CD69 was tried by introducing a mutation into the light chain CDR3 of the antibodies selected in Example 1. To be specific, the methods described in Prassler J, Steidl S, Urlinger S. Immunotherapy. 2009 July; 1(4):571-83. and Hillig R C, Urlinger S, Fanghanel J, Brocks B, Haenel C, Stark Y, Sulzle D, Svergun D I, Baesler S, Malawski G, Moosmayer D, Menrad A, Schirner M, Licha K. J Mol Biol. 2008 Mar. 14; 377(1):206-19 were employed. The antibody selection round was repeated twice, the base sequences of the light chain CDR3 of the obtained antibody clones were examined and antibody clones having a novel sequence were identified. They were expressed in Escherichia coli, and ELISA was performed for the antigen by using the lysate and the amount of the antibody in the lysate was simultaneously measured by sandwich ELISA. The relative specific binding activity of each clone ...

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Abstract

The present invention provides an antibody that specifically binds to human CD69, has an activity to suppress allergic inflammation, and has cross-reactivity with mouse CD69. In addition, the present invention provides an antibody having high binding affinity for human CD69 and an activity to suppress allergic inflammations. The antibody of the present invention can be a human antibody.

Description

TECHNICAL FIELD[0001]The present invention relates to an anti-human CD69 antibody, and pharmaceutical use thereof.BACKGROUND ART[0002]CD69 is a type II transmembrane protein belonging to the C-type lectin family. Since the expression of CD69 increases within a few hours after stimulation of T cells and B cells, it is widely used as an early activation marker molecule to be an index of lymphocyte activation (non-patent document 1). In addition, the expression is also observed in T cells under selection during differentiation in thymus (non-patent documents 2 and 3). While CD69 is assumed to have a function as a coreceptor to potentiate signal transduction from an antigen receptor, the detail is unknown. Its ligand has not been identified to date. It is constitutively expressed in platelet, and the expression is also observed in activated neutrophils, eosinophils and the like. Therefore, it is assumed to play a role in the expression of function in platelets and topical inflammation r...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C07K16/28
CPCC07K16/2851C07K2317/92C07K2317/33C07K2317/565A61K2039/505A61P29/00A61P37/08C07K2317/21C07K2317/34C07K2317/55
Inventor KOJOH, KANEHISAMIYAKOSHI, AKIRAKATOH, SHIZUETSUIHIJI, KUMIKOHAYAMI, YUKINAKAMURA, MIKIKONAKAYAMA, TOSHINORIIWAMURA, CHIAKI
Owner GENEFRONTIER CORP
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