Gene encoding IGG FC region-binding protein

Details Gene encoding IGG FC region-binding protein Gene encoding IGG FC region-binding protein

1996-11-15

2001-08-07

Navarro, Albert (Department: 1645)

Organic compounds -- part of the class 532-570 series

Organic compounds

Carbohydrates or derivatives

C435S006120, C435S069100, C435S252300, C435S320100, C435S325000

Reexamination Certificate

active

06271362

ABSTRACT:

SPECIFICATION
1. Technical Field
This invention relates to a gene encoding an IgG Fc portion-binding protein [hereinafter referred to as Fc&ggr;BP (Fc&ggr; Binding Protein) or IgG Fc BP), which is a protein specifically binding to the Fc portion of immunoglobulin G (IgG), a recombinant vector containing this gene, host cells transformed by this recombinant vector, a process for producing a recombinant protein which is obtained by incubating these host cells and a protein having a recombinant IgG Fc portion-binding activity which is obtained by the above-mentioned process.
2. Background Art
As one of immunocompetent cells, a macrophage incorporates a foreign invader or a complex thereof with immunoglobulin G (IgG) into the cell via phagocytosis and then digests the same. It is also capable of exerting an antigen presentation effect so as to induce the production of an antibody by lymphocytes. The main entry of this phagocytosis is an Fc receptor [Fc&ggr;R (Fc&ggr; receptor)] of IgG located on the surface of the macrophage cell. The inherent function of this Fc&ggr; receptor, which is a receptor participating in the binding of the Fc portion of IgG, is the elimination of a pathogen or an antigen-IgG immune complex coated with IgG (opposition).
It has been known that Fc&ggr; receptors may be roughly classified into three types including RI, RII and RIII [J. V. Ravetch and J.-P. Kinet, Annu. Rev. Immunol. (1991), 9:457-492]. The cDNA of each of these receptors has been successfully cloned. From 1990 to 1991, furthermore, several groups of workers found out proteins which were associated with these Fc&ggr; receptors and required for the initiation of the incorporation of the binding matters by Fc&ggr; receptors, thus providing a clue to clarify the switch mechanism [L. L. Lanier, G. Yu and J. H. Phillips, Nature (1989), 342:803-805; T. Kurosaki and J. V. Ravetch, Nature (1989), 342:805-807; D. G. Orioff, C. Ra, S. J. Frank, R. D. Klausner and J.-P. Kinet, Nature (1989), 347:189-191; P. Anderson, M. Caligiuri, C. O'Brien, T. Manley, J. Ritz and S. F. Schlossman. Prc. Natl. Acad. Sci. USA (1990), 87:2274-2278; T. Kurosaki, I. Gander and J. V. Ravetch, Proc. Natl. Acad. Sci. USA (1991), 88:3837-3841; L. Azzoni, M. Kamoun. T. W. Salcedo, P. Kanakaraj and B. Perussia, J. Exp. Med. (1992), 176:1745-1750; A. T. Ting, L. M. Karnitz, R. A. Schoon, R. T. Abraham and P. J. Leibson, J. Exp. Med. (1992), 176:1751-1755].
On the other hand, Kobayashi et al., reported a protein Fc&ggr;BP, which could bind specifically to the Fc portion of IgG and was different from the conventionally known Fc&ggr; receptors, occurring in human small intestinal and colonic epithelial cells, in particular, goblet cells. The binding of this protein specific to the IgG Fc portion was confirmed by using horse radish peroxidase-labeled matters. Namely, Fc&ggr;BP bound not to IgGFab, IgA or IgM but exclusively to the Fc portion of IgG. Also, Fc&ggr;BP underwent no cross reaction with antibodies of Fc&ggr; receptors I, II and III [K. Kobayashi, M. J. Blaser and W. R. Brown, J. immunol. (1989), 143:2567-2574].
Further, they partially purified Fc&ggr;BP from human colonic epithelial cells and constructed mouse monoclonal antibodies with the use of this protein as an antigen. Thus it was confirmed that Fc&ggr;BP bound not only to these antibodies but also to mouse IgG [K. Kobayashi, Y. Hamada, M. J. Blaser and W. R. Brown, J. Immunol. (1991), 146:68-74].
The partially purified Fc&ggr;BP was SDS (sodium dodecyl sulfate)-electrophoresed and then subjected to Western blotting with the use of the monoclonal antibodies. As a result, it was found out that Fc&ggr;BP formed an associate of 200 kDa or above containing a protein of 78 kDa [K. Kobayashi, Y. Hamada, M. J. Blaser and W. R. Brown, J. Immunol. (1991), 146:68-74].
The above-mentioned Fc&ggr;BP is identical with the Fc&ggr; receptors in the point of being capable of binding to the Fc portion of IgG. However, the stability and structure of Fc&ggr;BP as a protein and its role in vivo still remained unknown. It is, therefore, highly interesting to analyze Fc&ggr;BP so as to clarify its structure and function.
As will be described hereinafter, it is assumed that Fc&ggr;BP is secreted onto mucosae together with mucus and traps pathogens or viruses invading the body into the mucus to thereby facilitate the excretion of these invaders, thus participating in the mechanism of protection against infection. An autoantibody produced in excess in a mucosa suffering from inflammation activates the complement system or causes cytotoxicity by macrophages, etc., thus worsening the inflammation. It is assumed that Fc&ggr;BP blocks the Fc portion of such an autoantibody to thereby inhibit the progression of the inflammation. Because of having these functions, Fc&ggr;BP might be applicable to drugs, for example, agents for protecting infection, antiinflammatory (antiphlogistic) agents or diagnostic drugs for autoimmune diseases such as ulcerative colitis and Crohn's disease, etc.
To employ Fc&ggr;BP for these medicinal purposes, it is required to obtain Fc&ggr;BP in a large amount and in a uniform state. However, Fc&ggr;BP in a uniform state can be hardly obtained in a large amount by the method wherein Fc&ggr;BP is isolated from an animal tissue per se or a culture supernatant of Fc&ggr;BP-producing cells. It is, therefore, required to produce Fc&ggr;BP in a large amount by using gene recombination techniques.
The present inventors successfully identified the base sequence of a gene encoding Fc&ggr;BP by cloning the cDNA of Fc&ggr;BP with the use of monoclonal antibodies against Fc&ggr;BP.
This cDNA was inserted into an appropriate vector and host cells were transformed by the expression vector thus obtained. Then the obtained transformant was incubated and the target protein thus produced was separated and purified. As a result, it was found out that the protein thus produced had a characteristic of binding specifically to human IgG. Thus, the present inventors have clarified that Fc&ggr;BP can be produced in a large amount and in a uniform state.
DISCLOSURE OF THE INVENTION
Accordingly, the present invention provides a gene encoding Fc&ggr;BP.
The present invention further provides a recombinant vector containing a gene encoding Fc&ggr;BP.
The present invention furthermore provides procaryotic or eucaryotic host cells transformed by a recombinant vector containing a gene encoding Fc&ggr;BP.
The present invention furthermore provides a process for producing Fc&ggr;BP which comprises incubating host cells transformed by a recombinant vector containing a gene encoding Fc&ggr;BP and separating and purifying the protein thus produced.
The present invention furthermore provides a protein showing an IgG Fc region-binding activity which is produced by the above-mentioned process.
The present invention furthermore provides a method for using a gene encoding Fc&ggr;BP or a part of the same as a probe in Northern blotting or in situ hybridization for the identification of the tissue synthesizing Fc&ggr;BP mRNA.


REFERENCES:
patent: 5302527 (1994-04-01), Birkett et al
patent: WO8905351A2 (1989-06-01), None
Lazar et al (Molecular & cellular Bio. vol. 8 Mar. 1988 pp 1247-1252).*
Burgess et al (J. CF cell Bio. vol. III Nov. 1990 pp 2129-2138).*
Salgaller et al (Cancer Immunol. Immunother. vol. 39. 1994 pp 105-116).*
Reeck et al (Cell vol. 50, Aug. 28, 1997 p. 667).*
Kobayashi et al, The Journal of Immunology, vol. 143, No. 8, pp. 2567-2574 (Oct. 15, 1989).
Kobayashi et al, The Journal of Immunology, vol. 146, No. 1, pp. 68-74 (Jan. 1, 1991).
Hamada et al, Immunology, 74:298-303 (1991).
Kobayashi et al, Digestive Diseases and Sciences, vol. 39, No. 3, pp. 526-533 (1994).
van de Winkel et al.,Immunology Today, vol. 14, No. 5, pp.215-221 (May 1993).

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