Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1999-06-15
2001-08-21
Stucker, Jeffrey (Department: 1648)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C435S005000, C435S007100, C435S974000, C514S04400A, C530S350000
Reexamination Certificate
active
06277971
ABSTRACT:
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.
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Lui et al.; Binding of HIV-1 Nef to a novel thioesterase enzyme correlates with Nef-mediated CD4 down-regulation. The Journal of Biological Chemistry vol. 272, (1997) p. 13779-13785.*
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Luo T., et al., “Infectivity enhancement by immunodeficiency virus type 1 Nef is independent of its association with a cellular serine/threonine kinase”Journal of Virology, vol. 71, No. 12, 1997, pp. 9524-9530, XP002115666 US.
Rossi F., et al., “HsN3 proteasomal subunit as a target for human immunodeficiency virus type 1 Nef protein”Virology, vol. 237, No. 1, 1997, pp. 33-45, XP002115667, Orlando, US.
Smith B.L., et al., “The HIV Nef protein associates with protein kinase C theta”Journal of Biological Chemistry, vol. 271, No. 28, 1996, pp. 16753-16757, XP002115668, MD US.
Fujii, Y. et al., “Biological Overview of HIV Accessory Protein Nef,”Saibo Kogaku, vol. 16, No. 1, pp. 94-99 (1997).
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Fujii Yoichi
Otake Kaori
Hollander Law Firm, P.L.C.
Nippon Zoki Pharmaceutical Co. Ltd.
Stucker Jeffrey
Winkler Ulrike
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