Chemistry: analytical and immunological testing – Biospecific ligand binding assay
Reexamination Certificate
1999-03-12
2002-02-05
Eyler, Yvonne (Department: 1646)
Chemistry: analytical and immunological testing
Biospecific ligand binding assay
C435S005000, C435S007100, C435S069100, C435S252300, C530S350000, C514S002600, C514S04400A
Reexamination Certificate
active
06344362
ABSTRACT:
BACKGROUND OF THE INVENTION
This invention relates to the use of a recombinant protein as receptor for a hepatitis virus, a nucleic acid coding for it, a vector comprising said nucleic acid, a host cell transformed or transfected with said nucleic acid, a new recombinant protein possessing the biological property to act as receptor for a hepatitis virus, and a transgenic animal expressing said recombinant protein.
In particular, the present invention relates to the use of a recombinant protein as receptor for a hepatitis B virus, a nucleic acid coding for it, a vector comprising said nucleic acid, an host cell transformed or transfected with said nucleic acid, a new recombinant protein possessing the biological property to act as receptor for a hepatitis B virus, and a transgenic animal expressing said recombinant protein.
It is known that the hepatitis B disease is caused by the HBV virus (Hepatitis B Virus), belonging to the family named hepadnaviruses, that are viruses possessing a DNA molecule as genome capable to infect differentiated hepatocytes. Such viruses is able to infect humans only and higher primates in the evolutive scale (Ganem D. et al., “Ann. Rev. Biochem.”, 56, 651-693, 1987).
The mechanism by which this non-cytolytic virus infects cells and induces liver damage, has not been completely understood up to now.
It is also known that the HBV has a genome endowed with unusual features of a small DNA molecule that is only partly double-strand and shows a single-strand region. The nucleocapsid, that encompasses the DNA, is essentially constituted by the core protein, also named HBcAg, which is able to interact with the viral DNA, and by a protein kinase able to phosphorylate the HBcAg protein. The virus has an envelope outside the core essentially composed of lipids and of three proteins, named Small (S), Medium (M) and Large (L), that constitute the surface antigen (HBsAg) of said virus (Tiollais P. et al., “Nature”, 317, 489-495, 1985).
The viral envelope plays an essential role in the interaction process of the virus with the receptor protein occurring on the hepatocyte plasma membranes, since the virus/receptor interaction represents the first step of infectious process. In particular, it has been shown that the N terminal region, also named preS1, of the L viral protein is responsible for the virus/receptor interaction and indispensable for the entry of said virus into the hepatocyte (Neurath A. R. et al., “Cell”, 46, 429-436, 1986).
At least 350 million persons are chronic carriers of hepatitis B. The chronic disease occurs, in part, in patients with acute hepatitis B infections and, almost always, in cases of asymptomatic infection. This latter situation occurs in particular in the children infected from the mothers at the birth, where the disease symptoms became evident only when the chronic state is already reached. In about 25-35% of chronic carriers, the disease develops irreversible hepatic complications, due to chronic active hepatitis, cirrhosis or primary hepatocarcinoma, that lead to the death of more than 1 million persons per year.
Up to now, a specific pharmacological therapy to treat hepatitis B patients did not exist (Gitlin N, “Clin. Chem.”, 43, 1500-1506, 1997).
In the case of chronic carriers, for which it is important to intervene to avoid permanent hepatic damage, the only treatment, used up to now, is the administration of &agr;-interferon. &agr;-interferon shows a wide range of properties such as antiviral, antiproliferative, immunomodulatory, cytostatic and/or cytotoxic.
However, &agr;-interferon therapy gives appreciable results only in the 30-40% of treated patients with chronic hepatitis. Furthermore, it is not possible to administer said molecule to all chronic carriers such as, for example, to patients showing high hepatic decompensation or portal hypertension or who are affected by autoimmune diseases.
The high species-specificity of HBV has not allowed developing an experimental model to study the infective process. In fact, the experimental system more utilized to study the HBV/hepatocyte interaction is represented by the use of human hepatocarcinoma cell line and named HepG2 (Knowles B. B. et al., “Nature”, 209, 497-499, 1980).
Cells of HepG2 line have the advantage of not displaying the genome of integrated HBV, the ability to bind an HBV virus and to allow the entry of viral DNA into cytoplasm, thus showing the presence, on the plasmatic membrane, of a receptor molecule able to bind and internalize said virus. (Neurath A. R. et al., “Cell”, 46, 429-436, 1986; Dash S. et al., “J. Med. Virol.”, 37, 116-121, 1992).
However, the use of this cell line as the experimental model has the disadvantage that the virus is not able to replicate itself and thus generate new viral particles.
Given the high need to have a molecule with the biological properties to act as receptor for a hepatitis B virus, up to now, different molecules able to interact with the outer envelope of HVB have been tested, such as, for example, the 6-Interleukin (Neurath A. R. et al, “J. Exp. Med.” 172, 461-469, 1992), the ASGPR receptor (Asialoglycoprotein) (Trechel U. et al., “J. Gen. Virol.”, 75, 3021-3029, 1994), the receptor for the IgA (Fc&agr;R) (Pontisso P. et al.,“J. Gen. Virol.” 73, 2041-2045, 1992), the GAPD (Glyceraldehyde-3-P-Dehydrogenase) (Petit M. A. et al., “Virology”, 187, 211-222, 1992), the pHSA (polymerized Human Sera Albumin) (Pontisso P. et al., “J. Virol.”, 63, 1981-1988, 1989), the HSSP (Human Soluble Serum Protein) (Bubkowska A. et al., “J. Virol.”, 67, 4316-4322, 1993), the endonexin II (Hertogos K. et al., “Virology”, 197, 549-557, 1993), the ApoH (Apolipoprotein H) (Mehdi H. et al., “J. Virol.”, 68, 2415-2424, 1994).
However none of the above mentioned proteins has shown definitive and specific receptorial activity for the viral particles. Indeed a biological assay able to evidence the receptorial function of a protein is not available. Such biological assay should comprise the transfection of a cDNA coding for a protein, in a suitable cell line, and the subsequent verification of the susceptibility to the infection for cells expressing the recombinant receptor (Mendelsohn L. C. et al., “Cell”, 56, 855-865, 1989; Bergelson J. M. et al., “Science”, 275, 1320-1323, 1997).
Now, it has been unexpectably found that the human protein SCCA1 (Squamous Cell Carcinoma Antigen) of
FIG. 1
(SEQ ID NOS: 1 and 3) has the ability to act as HBV receptor.
In addition, the inventors have also found an new allelic variant of SCCA1 (
FIG. 2
; SEQ ID NOS: 2 and 4).
SUMMARY OF THE INVENTION
Therefore, it is a first object of this invention to provide the use of a recombinant protein having, fully or in part, a primary structure as shown in
FIG. 1
(SEQ ID NOS: 1 and 3) or
2
(SEQ ID NOS: 2 and 4), or of any allelic variant thereof, as a receptor for a hepatitis virus.
Preferably, said virus is a hepatitis B virus.
Recently, the methodologies of molecular screening based on combinatorial library have been used to identify new molecules with different biological activity, as new drugs. Typical example of such methodology is reported in Kenan D. L. et al (“Tibs”, 2, 57-64, 1994). For the system HBV virus/receptor, such methodologies allow to the person skilled in the field to select, from a very high number of molecules, those able to neutralize the virus/receptor interaction and to evaluate subsequently their applicability in the pharmacological field.
It is therefore another object of the present invention to provide the use of a recombinant protein having, fully or in part, a primary structure as shown in
FIG. 1
(SEQ ID NOS: 1 and 3) or
2
(SEQ ID NOS: 2 and 4), or of any allelic variant thereof, or of any its derivative having the biological property to act as a receptor for a hepatitis virus, in molecular screening assays.
The inventors' identification of a HBV receptor opens, the way to develop molecules with pharmacological activity able to neutralize the virus/receptor interaction occurring on the plasma membranes of hepatocytes and thus to
De Falco Sandro
Fassina Giorgio
Ruvo Menotti
Verdoliva Antonio
Brannock Michael
Eyler Yvonne
Ministero Dell Universita'E Della Ricerca Scientifica E Tec
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