Title:  Nef-attachable protein

United States Patent:  6,803,449

Issued:  October 12, 2004

Inventors:  Fujii; Yoichi (Nagoya, JP); Otake; Kaori (Kani, JP)

Assignee:  Nippon Zoki Pharmaceutical Co., Ltd. (Osaka, JP)

Appl. No.:  899863

Filed:  July 5, 2001

Abstract

A monoclonal antibody specific to Nap was prepared by using a crude membrane fraction from a cell line which has a high affinity to Nef as an antigen. A cDNA clone which encodes Nap was obtained by screening a cDNA library of said cell line utilizing the specific antibody. The nucleotide sequence of this cDNA was elucidated whereupon the full amino acid sequences of human Nap was identified. Since the anti-Nap monoclonal antibody inhibits the binding of Nef to Nap, it can be used as a new therapeutic agent for AIDS. In addition, it can be applied for the diagnosis of AIDS by clarifying the relation between the expression of Nap and the development of AIDS. Nap can be utilized for binding experiments to Nef. It is useful for the development of novel therapeutic agents for AIDS based upon a mechanism of action which inhibits the attachment of both factors and development of new inhibitors against the onset of AIDS by a mechanism of action different from that of reverse transcriptase inhibitors or protease inhibitors.

Description of the Invention

FIELD OF THE INVENTION

The present invention relates to a Nef-attachable protein which is useful for diagnosis, therapy and development of a therapeutic agent for AIDS, to a gene encoding the protein and to a monoclonal antibody against said protein.

BACKGROUND OF THE INVENTION

In general, retroviruses have the proteins Gag, Pol, and Env in common. In addition to these proteins, human immunodeficiency virus (HIV) has viral-specific regulatory proteins and accessory proteins. Nef is one of the accessory proteins which is specific to HIV-1, HIV-2 and SIV (simian immunodeficiency virus) and is synthesized in the early stage of the virus replication. The protein was designated Nef at the beginning since it is deemed that Nef is a negative factor which decreases the replication of the virus. However, afterward, it has been suggested that Nef works as a positive regulator for HIV replication by experiments testing the pathogenesis of Nef-deleted SIV mutant in the rhesus monkey. Nef is recognized anew as an important factor for holding a key to developing the pathogenesis of AIDS. See, Fujii et al, Saibo Kogaku, vol.16, No.1, 94-99 (1997)].

As mentioned in detail in the above article by Fujii, et al., the important functions of Nef in vivo are thought to be: (1) promotion of virus replication, (2) down regulation of CD4 molecularly, and (3) cytotoxicity to T cells. With regard to the virus infection, it has been known that Nef has an affinity to cell membranes, is necessary for virus adsorption and the invasion of the virus into cells, and participates in viral DNA synthesis. Concerning the cytotoxicity to T cells, apoptosis of the cells is induced when Nef is bound to the surface of CD4⁺ T cells of intestinal lymph node and peripheral blood and the cross-linking of the Nef molecule is caused by an anti-Nef antibody. Therefore, it is strongly suggested that binding of Nef to a Nef receptor on the CD4⁺ T cells greatly participates in depletion of CD4⁺ T cells in patients with HIV. Namely, pathogenesis of AIDS relates to the binding of Nef and its receptor. In addition, there is a report which suggests that Nef inhibits the production of cytokine resulting in the suppression of the immune system.

Most of the therapeutic agents for AIDS which have been used and developed so far are reverse transcriptase inhibitors such as azidothymidine, ddI, etc. and protease inhibitors, which are drugs which inhibit the growth of virus directly. In addition to these drugs, there are immunopotentiators which activate the function of the immune system depraved by AIDS and chemotherapeutic agents aimed at the treatment of symptoms such as malignant tumors and opportunistic infections caused by AIDS. Based on the above-mentioned findings on Nef, a drug which inhibits T cell apotosis caused by Nef and thereby suppresses the onset of AIDS has aroused public attention as a novel inhibitor for AIDS with a different action mechanism from those of the above-mentioned reverse transcriptase inhibitors and protease inhibitors.

Various kinds of reverse transcriptase inhibitors and protease inhibitors have been developed as a strategy to suppress AIDS. A cocktail therapy where two or more of such inhibitors are combined has been promoted in order to avoid the problems caused by the development of HIV mutation. However, there is still a strong demand for pharmaceutical agents which have a mechanism of action to suppress AIDS in addition to the direct inhibitors of viral growth. It has been recently clarified that Nef participates in invasion of the virus into cells and induces apoptosis of CD4⁺ T cells which play an important role in the disappearance of CD4⁺ cells in patients with HIV. The action is triggered by the binding of Nef to Nef-attachable protein (hereinafter, referred to as Nap) on cell walls. The actual existence of Nap has been anticipated by experiments showing the binding of Nef to CD4⁺ T cells or the like, however, Nap has not been specified yet.

The present invention provides a Nef-attachable protein (Nap) on the CD4⁺ T cell membrane which can recognize and be bound to Nef to play an important role in the onset of AIDS, a gene encoding Nap, and a monoclonal antibody against said protein.

SUMMARY OF THE INVENTION

The present inventors have prepared a specific monoclonal antibody against Nap by using a crude membrane fraction of a cell line as an antigen having high affinity to Nef. They isolated the Nap gene by utilizing said antibody and the DNA sequences of Nap was clarified. Finally, the amino acid sequences of Nap was determined successfully. Proteins having an amino acid sequence represented by SEQ ID NO:1 and functionally active homologues thereof are provided by the present invention. The proteins and DNA molecules which encode the proteins may be used in diagnosing the development of AIDS. A DNA molecule which encodes a protein having an amino acid sequence represented by SEQ ID NO:1 or which encodes a protein which is a functionally active homologue of the protein having an amino acid sequence represented by SEQ ID NO:1 may have a nucleotide sequence represented by SEQ ID NO:2 or a functionally active homologue of SEQ ID NO:2. An anti-Nef-attachable protein monoclonal antibody in accordance with the present invention has a high affinity to Molt-4 clone no.8 cells (human CD4⁺ T cell line) and U937 cells (human macrophage cell line) and does not bind or attach to Raji cells (human B cell line), BT-2 cells (human gliocyte cell line) and Gin-1 cells (human fibroblast cell line). The monoclonal antibody may be a chimeric antibody having a variable region of a mouse monoclonal antibody and a constant region of a human type antibody. It may also be a human type antibody having a complementary-determining region of the mouse monoclonal antibody. The monoclonal antibodies of the present invention may be employed in pharmaceutical compositions in pharmaceutically effective amounts for the treatment of AIDS.

DETAILED DESCRIPTION OF THE INVENTION

The present inventors have prepared a monoclonal antibody specific to Nap by using a crude membrane fraction of a cell line which has high affinity to Nef. A cDNA clone which encodes Nap was obtained by screening a cDNA library of said cell line utilizing the specific antibody. They have elucidated the nucleotides sequence of this cDNA clone and determined the full amino acid sequence of human Nap, thus accomplishing the present invention.

As for an antigen for the preparation of a monoclonal antibody against Nap, the chance of obtaining an anti-Nap specific monoclonal antibody is increased by using the cell which has a high affinity to Nef as an antigen. Thus, high affinity to Nef means that a large number of Naps are expressed on the cell surface. Such cell line is preferred as an antigen for the preparation of the anti-Nap monoclonal antibody of the present invention because the cells contain a large amount of Nap as antigen. For example, Molt-4 clone no.8 cells (human CD4⁺ T cell line) and U937 cells (human macrophage cell line) have been reported to have high affinity to Nef. Although those cell lines are most preferable, other cell lines expressing Nap are applicable as well.

A membrane fraction of the above cells may be used as an antigen to make an affinity column where Nef is immobilized because it is preferable to use a membrane fraction which contains a larger number of Nap. In the present invention, membrane components of Molt-4 clone no.8 cells were roughly fractionated by using recombinant Nef conjugated to an insoluble carrier and used as an antigen for the preparation of anti-Nap monoclonal antibody.

The preparation of the monoclonal antibody may be carried out by using conventional methods. For example, spleen cells of an animal were obtained after the production of antibody was boosted by the sensitization of the animal with the above crude membrane fraction as antigen. Then, preparation of hybridoma and the cloning of hybridoma were carried out. To obtain a hybridoma which produces the desired monoclonal antibody of the present invention, the first selection was carried out by using an ELISA method utilizing the crude membrane fraction of Molt-4 clone no.8 cells employed as an antigen to narrow down the number of potential hybridoma. Then, flow cytometry analysis of antibodies by using Molt-4 clone no.8 cells as antigen was carried out to select a clone having high affinity to the surface of said cells. Three clones were obtained by the selection process. Finally, flow cytometry analysis using various cell lines was carried out to compare the affinity of antibodies to each cell line and a clone which produces anti-Nap monoclonal antibody of the present invention was determined. In addition, an immunoblotting method or the like using each monoclonal antibody is carried out for the confirmation of anti-Nap monoclonal antibody.

Fundamentally, purification and identification of the Nap (Nef-attachable protein) gene of the present invention can be carried out by a panning method using the anti-Nap monoclonal antibody obtained as mentioned above. Thus, in the present invention, mRNA of the Molt-4 clone no.8 cells which were used as antigen for preparation of said anti-Nap antibody is separated and a cDNA library is prepared. cDNA was integrated into plasmids and transfected to cultured cells such as Cos7 cells for the cloning of cDNA. Method for the preparation of mRNA from cells, the preparation of cDNA, integration of cDNA into plasmid, transfection into cultured cells, etc. are well known methods in the field of genetic engineering and may be carried out by setting appropriate conditions. The plasmids which were purified by repeating the above-mentioned panning method several times were transfected to Cos7 cells and the expression of Nap on the cell surface could be confirmed by flow cytometry using the anti-Nap monoclonal antibody.

Nef is strongly believed to participate in invasion of HIV virus into cells and the disappearance of CD4⁺ T cells in patients with HIV. The action of Nef is triggered by binding of Nef to Nap on cell membranes. Thus, it has been anticipated that development of AIDS can be suppressed by inhibiting the binding of Nap to Nef. The anti-Nap monoclonal antibody of the present invention inhibits the binding of Nef to Nap and, therefore, it is a very useful agent as a new therapeutic drug against AIDS.

In the examples "as described in this patent", a specific description is given for a mouse antibody as an anti-Nap monoclonal antibody. However, if mouse antibody is administered to human beings as a pharmaceutical agent, the half-life of the mouse antibody in vivo is short. Moreover, there is concern about the induction of the immune system to exclude the mouse antibody, because the mouse antibody is recognized as a heteroantigen in human. In this case, human antibody against mouse antibody is produced in the human and it induces not only the neutralization of the mouse antibody but also a critical immune reaction such as an anaphylactic shock.

Accordingly, if anti-Nap monoclonal antibody of the present invention is used as a pharmaceutical agent for humans, a chimeric antibody or human-type antibody which does not negatively affect the usefulness of the mouse antibody and does not induce the above-mentioned immune reaction in humans can be used. For example, there have been many reports for methods to manufacture chimeric antibodies with mouse amino acids sequences for the region which recognizes antigen (variable region) and human amino acid sequences for the constant region except for the variable region. It is possible to manufacture chimeric antibodies which can be administered to humans from the monoclonal antibody of the present invention according to known methods such as disclosed in European Patent Publication No. 120694 B and corresponding U.S. Pat. No. 4,816,397, European Patent Publication No. 125023 B and corresponding U.S. Pat. No. 4,816,567, and International Patent Publication WO 86/01533 A, the disclosures of which are herein incorporated by reference in their entireties.

In addition, known methods for preparing human-type antibody by improving the above chimeric antibody may be employed herein. For example, a human-type antibody of the present invention may be prepared from a chimeric antibody of the present invention, by a method such as disclosed in European Patent Publication No. 239400 A, and corresponding U.S. Pat. No. 5,225,539, the disclosures of which are herein incorporated by reference in their entireties. The specific region of human type antibody which recognizes antigen directly (hypervariable region, complementary-determining region, CDR, etc.) is made of the amino acid sequences derived from mouse antibody, while the other region which is necessary for keeping the structure of the antibody is made of the amino acid sequences derived from humans. Therefore, the ratio of the non-human amino acid sequence contained in the human type antibody is lower than that of the chimeric antibody. As a result, it shows a half-life which is similar to that of human antibody in the human body. In addition, it rarely produces human anti-mouse antibody. The anti-Nap monoclonal antibody of the present invention includes said chimeric antibodies and human type antibodies as well.

The anti-Nap monoclonal antibody of the present invention including the above chimeric antibodies and human type antibodies can be characterized based upon its specificity. For example, it can be defined in terms of its specificity of antigen reactivity that: it is strongly bound to Molt-4 clone no.8 cells (human CD4⁺ T cell line) and U937 cells (human macrophage cell line) while it is not bound to Raji cells (human B cell line), BT-2 cells (human gliocyte cell line) and Gin-1 cells (human fibroblast cell line).

Specifically, Nap of the present invention has an amino acid sequence represented by SEQ ID NO:1 and includes proteins having an amino acid sequence wherein one or more amino acid residue(s) in the amino acid sequence represented by SEQ ID NO:1 is/are deleted, added or substituted. The term "protein having an amino acid sequence where one or more amino acid residue(s) in the amino acid sequence represented by SEQ ID NO:1 is/are deleted, added or substituted" means polypeptides (proteins) which are converted without decreasing the functions of human Nap of the present invention and includes naturally produced mutant polypeptides and artificially modified mutant polypeptides. Thus, affinity to Nef is listed as a specific function of Nap and this function can be confirmed by conducting a binding test to Nef using the converted (mutant) peptide.

Thus, embodiments of the present invention, include proteins which are biologically functional homologues of SEQ ID NO: 1, and DNA molecules having nucleotide sequences which encode a protein of SEQ ID NO: 1 or which encode a biologically functional homologue of SEQ ID NO: 1. The DNA molecules may have a nucleotide sequence represented by SEQ ID NO: 2, or may have a nucleotide sequence which is a biologically functional homologue of SEQ ID NO: 2. Biologically functional homologues, or functionally active homologues may be ascertained by those ordinarily skilled in the art without undue experimentation using tests or assays for affinity, attachment or binding to Nef as a specific function of Nap, as described, for example, herein. The deletion, addition, and substitution of amino acid residues in SEQ ID NO: 1 or of the nucleotides in SEQ ID NO: 2 may be conservative, with no significant loss, or with little or no loss of biological activity, such as conservative substitutions described in U.S. Pat. Nos. 5,470,753, 5,559,209, and 5,545,618. The disclosures of U.S. Pat. Nos. 5,470,753, 5,559,209, and 5,545,618 are herein incorporated by reference in their entireties.

In an embodiment of the present invention, the biologically functional homologues or functionally active homologues of the proteins of SEQ ID NO:1 and the nucleotides of SEQ ID NO:2 are produced by conservative substitution of one or more amino acids or is produced by incorporation of one or more synthetic amino acids, amino acid analogs, peptidomimetic compounds or modified amino acids into the protein. For example, the amino acids can be modified or derivitized by phosphorylated, carboxymethylation, acylation, glycosylation and other methods known to the ordinary skilled artisan.

Claim 1 of 8 Claims

What is claimed is:

1. An isolated or purified protein having an amino acid sequence represented by SEQ ID NO:1, wherein one amino acid residue is deleted, added, or substituted, and the protein binds to Nef.

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