Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Blood proteins or globulins – e.g. – proteoglycans – platelet...
Reexamination Certificate
1999-03-04
2001-06-12
Saunders, David (Department: 1644)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Blood proteins or globulins, e.g., proteoglycans, platelet...
C530S387100, C530S388200, C435S070210, C435S452000, C435S362000, C435S332000, C435S334000, C435S345000
Reexamination Certificate
active
06245897
ABSTRACT:
BACKGROUND OF THE INVENTION
Field of the Invention
The present invention relates to a monoclonal antibody which recognizes the lipopolysaccharide binding site of macrophage cell surface receptor CD14 and has binding activity to monocyte or macrophage cells, and to a process for producing the same.
It also relates to a method for suppressing the production of an inflammatory mediator in a mammal cell which comprises competitively inhibiting binding of the cell surface receptor CD14 with lipopolysaccharide.
When CD14, which is a cell surface receptor having high affinity for lipopolysaccharide (referred to as “LPS” hereinafter), recognizes a complex of LPS and LPS binding protein (referred to as “LBP” hereinafter) in serum (the complex of LPS and LBP is referred to as “LPS-LBP complex” hereinafter), it causes monocytes or macrophages to excrete various factors such as inflammatory cytokines including TNF and IL-6, and NO to the outside of the cells. It has been reported that these factors play extremely important roles in crisis and worsening of sepsis.
The soluble CD14 antigen may also relate closely to the pathology of sepsis, and various pathological analyses of sepsis utilizing antibodies to CD14 antigen have been attempted. For example, International Patent Application Un-examined Publication in Japanese (KOHYO TOKKYO KOHO) No. Hei 8-510909/1996 discloses an antibody produced by a hybridoma which was obtained by imunizing a mouse or a rabbit with a recombinant CD14 as an antigen. Further, International Patent Application Un-examined Publication in Japanese (KOHYO TOKKYO KOHO) No. Hei 5-501399/1993 discloses use of an anti-CD14 monoclonal antibody for the treatment of sepsis, which competitively inhibits the binding of LPS-LBP complex to CD14.
However, these known antibodies were produced by using a recombinant CD14 as an antigen, and therefore they have problems concerning their specificity. For example, they may not show affinity for actual antigens that are present on cellular surfaces in various forms, antibodies having desired recognition characteristics may not be obtained because the recombinant CD14 used as the antigen may denature during its purification, or other problems may arise. Therefore, they have been insufficient for the application to pathology analysis and treatment of sepsis.
Accordingly, the object of the present invention is to provide a monoclonal antibody recognizing the LPS binding site of the macrophage cell surface receptor, CD14, which is useful for pathology analysis and treatment of sepsis and the like.
SUMMARY OF THE INVENTION
The present inventors prepared hybridomas using cells of animals immunized with monocyte or macrophage cells as antigen, screened only hybridoma clones producing antibodies having binding activity to monocyte or macrophage cells by means of a fluorescence-activated cell sorter (FACS), precisely examined molecular characteristics of antigens reacting with those antibodies, and cloned only antibodies binding with the cell surface CD14 antigen. As a result, they found a novel monoclonal antibody which recognizes the LPS binding site of CD14, and suppresses the production of an inflammation mediator such as TNF, IL-6 or NO by competitively inhibiting the binding of CD14 to LPS. Thus, the present invention has been accomplished.
That is, the present invention provides a monoclonal antibody which recognizes the lipopolysaccharide binding site of macrophage cell surface receptor CD14 and has binding activity to monocyte or macrophage cells.
The monoclonal antibody of the present invention is produced by a hybridoma obtained by fusion of an antibody producing cell of a mammal immunized with monocyte or macrophage cells and a myeloma cell. The hybridoma is the 4C1 hybridoma cell line deposited at the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology, 1-3, Higashi 1 chome Tsukuba-Shi Ibarkai-ken 305-8566 JAPAN under the Budapest Treaty under the Accession No. FERM BP-6924. The hybridoma obtained by fusion of an antibody producing cell and a myeloma cell is proliferated in a culture medium or in a living body, and the hybridoma having an ability to produce continuously said antibody is separated.
Accordingly, the present invention also provides a process for producing the monoclonal antibody of the present invention, which comprises proliferating a hybridoma obtained by fusion of an antibody producing cell and a myeloma cell in a culture medium or in a living body, wherein the antibody producing cell is collected from an animal immunized with monocyte or macrophage cells, and wherein the hybridoma having an ability to produce continuously said antibody is separated.
In the aforementioned monoclonal antibody and the process for producing the same of the present invention, the monocyte or macrophage cells are preferably RAW 264.7 cells derived from mouse (ATCC No. TIB71).
The present invention further provides a method for suppressing the production of an inflammatory mediator in a mammal cell, which comprises competitively inhibiting binding of the cell surface receptor CD14 with lipopolysaccharide by utilizing the aforementioned monoclonal antibody of the present invention.
In the aforementioned method for suppressing the production of the inflammatory mediator of the present invention, the inflammatory mediator is preferably TNF-&agr;, IL-6 or NO.
REFERENCES:
patent: 4701408 (1987-10-01), Koestler
Haslberger et al Journal of Endotoxin Research 4: 431-441, 1997.*
ATTC Cell Lines and Mybridomas Eigth Edition p. 330 Editor May et al, 1994.
Adachi Yoshiyuki
Ohno Naohito
Yadomae Toshiro
DeCloux Amy
Oblon & Spivak, McClelland, Maier & Neustadt P.C.
Saunders David
Seikagaku Kogyo Kabushiki Kaisha (Seikagaku Corporation)
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