The present invention provides a substance which inhibits the binding between E2/NS1 protein of hepatitis C virus and a cell infectious with hepatitis C virus, a cell expressing CD81, or CD81. The present invention can provide a novel medicament which has an anti-viral effects such as an inhibitory action against HCV infection.

DISCLOSURE OF THE INVENTION

HCV is known to be RNA virus and, thus, is highly likely to undergo mutation. This mutation causes mutation in many sites, both in the structural protein and the non-structural protein of HCV. Because of this mutation, the development of a substance for inhibiting HCV infection, for example, an antibody which recognizes a given protein sequence, has been considered to be difficult. However, the present inventors have focused on the point that the neutralizing antibody reported by Ishii et al. inhibits the binding to only one type of E2/NS1 even though the sequence of the infected virus are different for each recovered patient. More specifically, they have considered that a substance recognizing a specific region or a configuration of the antigen can inhibit the binding between E2/NS1 and a human cell infectious with HCV, a CD81 expressing cell, or CD81 and prevent infection regardless of the diversity of viral sequences.

In the present invention, for the purpose of developing an agent for improving the symptoms of hepatitis C patients, the present inventors have conducted concentrated search on substances which inhibit the binding between E2/NS1 and the cell infectious with HCV, the cell expressing CD81, or CD81, and as a result, succeeded in obtaining a substance capable of inhibiting the binding and infection of HCV to a cell infectious with HCV.

Thus, the present invention provides a substance which inhibits the binding between E2/NS1 protein of hepatitis C virus and a cell infectious with hepatitis C virus, a cell expressing CD81, or CD81.

The preferred embodiment of the present invention provides: a substance which inhibits, upon binding to a region having a positively charged regions of E2/NS1 protein of hepatitis C virus, the binding between E2/NS1 protein of hepatitis C virus and a cell infectious with hepatitis C virus, a cell expressing CD81, or CD81; a substance in which E2/NS1 protein of hepatitis C virus is a protein prepared by adding a substance capable of recognizing E2/NS1 protein to E2/NS1 protein of hepatitis C virus; a substance in which E2/NS1 protein of hepatitis C virus is a protein prepared by adding a substance capable of detecting E2/NS1 protein by a fluorescence method or a luminescence method to E2/NS1 protein of hepatitis C virus; a substance in which E2/NS1 protein of hepatitis C virus is a protein prepared by adding a tag to E2/NS1 protein of hepatitis C virus; a substance in which E2/NS1 protein of hepatitis C virus is a protein prepared by adding a tag to the C-terminus of E2/NS1 protein of hepatitis C virus; a substance in which a cell expressing CD81 is a cell which expresses human CD81; a substance in which CD81 is human CD81 and expressed by solubilized form; a substance which has an association constant to E2/NS1 protein of hepatitis C virus is higher than 10.sup.8, or a dissociation constant to E2/NS1 protein of hepatitis C virus is lower than 10.sup.-8; a substance which has an association constant to E2/NS1 protein of hepatitis C virus is higher than 10.sup.9, or a dissociation constant tQ E2/NS1 protein of hepatitis C virus is lower than 10.sup.-9; a substance which is a protein, sulfated polysaccharide, or a low molecular weight compound; a substance in which a protein is an antibody; a substance in which an antibody is derived from the B cell of a patient who recovered from hepatitis C; a substance in which an antibody is derived from genes in the B cell of a patient who recovered from hepatitis C; and a substance in which a patient is recovered from hepatitis C without any antiviral treatment and the B cell is peripheral blood mononuclear cell.

Another aspect of the present invention provides: an antibody in which CDR-1, CDR-2, and CDR-3 of a variable region of the H chain respectively comprise amino acid sequences shown by SEQ ID NOS: 1, 2, and 3 in the Sequence Listing or the amino acid sequences having a deletion, a substitution, or an addition of one or several amino acids in the amino acid sequences shown by SEQ ID NOS: 1, 2, and 3, and which has an affinity to E2/NS1 protein of hepatitis C virus;

said antibody which comprises an amino acid sequence shown by SEQ ID NO: 16 or 34 in the Sequence Listing or the amino acid sequence having a deletion, a substitution, or an addition of one or several amino acids in the amino acid sequence shown by SEQ ID NO: 16 or 34;

an antibody in which CDR-1, CDR-2, and CDR-3 of a variable region of the L chain respectively comprise amino acid sequences shown by SEQ ID NOS: 4, 5, and 6 in the Sequence Listing or the amino acid sequences having a deletion, a substitution, or an addition of one or several amino acids in the amino acid sequences shown by SEQ ID NOS: 4, 5, and 6, and which has an affinity to E2/NS1 protein of hepatitis C virus;

said antibody which comprises an amino acid sequence shown by SEQ ID NO: 17 in the Sequence Listing or the amino acid sequence having a deletion, a substitution, or an addition of one or several amino acids in the amino acid sequence shown by SEQ ID NO: 17;

an antibody in which CDR-1, CDR-2, and CDR-3 of a variable region of the L chain respectively comprise amino acid sequences shown by SEQ ID NOS: 7, 8, and 9 in the Sequence Listing or the amino acid sequences having a deletion, a substitution, or an addition of one or several amino acids in the amino acid sequences shown by SEQ ID NOS: 7, 8, and 9, and which has an affinity to E2/NS1 protein of hepatitis C virus;

said antibody which comprises an amino acid sequence shown by SEQ ID NO: 18 in the Sequence Listing or the amino acid sequence having a deletion, a substitution, or an addition of one or several amino acids in the amino acid sequence shown by SEQ ID NO: 18;

an antibody in which CDR-1, CDR-2, and CDR-3 of a variable region of the L chain respectively comprise amino acid sequences shown by SEQ ID NOS: 10, 11, and 12 in the Sequence Listing or the amino acid sequences having a deletion, a substitution, or an addition of one or several amino acids in the amino acid sequences shown by SEQ ID NOS: 10, 11, and 12, and which has an affinity to E2/NS1 protein of hepatitis C virus;

said antibody which comprises an amino acid sequence shown by SEQ ID NO: 19 in the Sequence Listing or the amino acid sequence having a deletion, a substitution, or an addition of one or several amino acids in the amino acid sequence shown by SEQ ID NO: 19;

an antibody in which CDR-1, CDR-2, and CDR-3 of a variable region of the L chain respectively comprise amino acid sequences shown by SEQ ID NOS: 13, 14, and 15 in the Sequence Listing or the amino acid sequences having a deletion, a substitution, or an addition of one or several amino acids in the amino acid sequences shown by SEQ ID NOS: 13, 14, and 15, and which has an affinity to E2/NS1 protein of hepatitis C virus; and

said antibody which comprises an amino acid sequence shown by SEQ ID NO: 20 in the Sequence Listing or the amino acid sequence having a deletion, a substitution, or an addition of one or several amino acids in the amino acid sequence shown by SEQ ID NO: 20;

A further aspect of the present invention provides:

an antibody in which CDR-1, CDR-2, and CDR-3 of a variable region of the L chain respectively comprise amino acid sequences shown by SEQ ID NOS: 42, 43, and 44 in the Sequence Listing or the amino acid sequences having a deletion, a substitution, or an addition of one or several amino acids in the amino acid sequences shown by SEQ ID NOS: 42, 43, and 44, and which has an affinity to E2/NS1 protein of hepatitis C virus;

said antibody which comprises an amino acid sequence shown by SEQ ID NO: 54 in the Sequence Listing or the amino acid sequence having a deletion, a substitution, or an addition of one or several amino acids in the amino acid sequence shown by SEQ ID NO: 54;

an antibody in which CDR-1, CDR-2, and CDR-3 of a variable region of the L chain respectively comprise amino acid sequences shown by SEQ ID NOS: 45, 46, and 47 in the Sequence Listing or the amino acid sequences having a deletion, a substitution, or an addition of one or several amino acids in the amino acid sequences shown by SEQ ID NOS: 45, 46, and 47, and which has an affinity to E2/NS1 protein of hepatitis C virus;

said antibody which comprises an amino acid sequence shown by SEQ ID NO: 55 in the Sequence Listing or the amino acid sequence having a deletion, a substitution, or an addition of one or several amino acids in the amino acid sequence shown by SEQ ID NO: 55;

an antibody in which CDR-1, CDR-2, and CDR-3 of a variable region of the L chain respectively comprise amino acid sequences shown by SEQ ID NOS: 48, 49, and 50 in the Sequence Listing or the amino acid sequences having a deletion, a substitution, or an addition of one or several amino acids in the amino acid sequences shown by SEQ ID NOS: 48, 49, and 50, and which has an affinity to E2/NS1 protein of hepatitis C virus;

said antibody which comprises an amino acid sequence shown by SEQ ID NO: 56 in the Sequence Listing or the amino acid sequence having a deletion, a substitution, or an addition of one or several amino acids in the amino acid sequence shown by SEQ ID NO: 56;

an antibody in which CDR-1, CDR-2, and CDR-3 of a variable region of the L chain respectively comprise amino acid sequences shown by SEQ ID NOS: 51, 52, and 53 in the Sequence Listing or the amino acid sequences having a deletion, a substitution, or an addition of one or several amino acids in the amino acid sequences shown by SEQ ID NOS: 51, 52, and 53, and which has an affinity to E2/NS1 protein of hepatitis C virus; and

said antibody which comprises an amino acid sequence shown by SEQ ID NO: 57 in the Sequence Listing or the amino acid sequence having a deletion, a substitution, or an addition of one or several amino acids in the amino acid sequence shown by SEQ ID NO: 57 ;

A further aspect of the present invention provides:

an antibody which comprises, as an amino acid sequence of a heavy chain, an amino acid sequences shown by SEQ ID NO: 40 in the Sequence Listing or the amino acid sequences having a deletion, a substitution, or an addition of one or several amino acids in the amino acid sequences shown by SEQ ID NO: 40, and which has an affinity to E2/NS1 protein of hepatitis C virus; and

an antibody which comprises, as an amino acid sequence of a light chain, an amino acid sequences shown by SEQ ID NO: 41 in the Sequence Listing or the amino acid sequences having a deletion, a substitution, or an addition of one or several amino acids in the amino acid sequences shown by SEQ ID NO: 41, and which has an affinity to E2/NS1 protein of hepatitis C virus;

A further aspect of the present invention provides a single-chain antibody having an affinity to E2/NS1 protein of hepatitis C virus, which comprises an amino acid sequence shown by SEQ ID NO: 26, 27, 28, or 29 in the Sequence Listing or the amino acid sequence having a deletion, a substitution, or an addition of one or several amino acids in the amino acid sequence shown by SEQ ID NO: 26, 27, 28, or 29.

A further aspect of the present invention provides a single-chain antibody having an affinity to E2/NS1 protein of hepatitis C virus, which comprises an amino acid sequence shown by SEQ ID NO: 36, 37, 38, or 39 in the Sequence Listing or the amino acid sequence having a deletion, a substitution, or an addition of one or several amino acids in the amino acid sequence shown by SEQ ID NO: 36, 37, 38, or 39.

A further aspect of the present invention provides a single-chain antibody having an affinity to E2/NS1 protein of hepatitis C virus, which comprises an amino acid sequence shown by SEQ ID NO: 62, 63, 64, or 65 in the Sequence Listing or the amino acid sequence having a deletion, a substitution, or an addition of one or several amino acids in the amino acid sequence shown by SEQ ID NO: 62, 63, 64, or 65.

A further aspect of the present invention provides a nucleic acid which encodes the aforementioned antibody of the present invention. Preferably, there is provided: the nucleic acid which comprises a nucleotide sequence shown by any of SEQ ID NO: 21, 22, 23, 24, 25, 35, 58, 59, 60, or 61 in the Sequence Listing; the nucleic acid which comprises a nucleotide sequence shown by SEQ ID NO: 40 or 41 in the Sequence Listing; the nucleic acid which comprises a nucleotide sequence shown by any of SEQ ID NO: 26, 27, 28, or 29 in the Sequence Listing; the nucleic acid which comprises a nucleotide sequence shown by any of SEQ ID NO: 36, 37, 38, or 39 in the Sequence Listing; and the nucleic acid which comprises a nucleotide sequence shown by any of SEQ ID NO: 62, 63, 64, or 65 in the Sequence Listing.

A still further aspect of the present invention provides a method for producing an antibody using any of the above-described nucleic acids.

A further aspect of the present invention provides a recombinant antibody which can be obtained by the above method and has an affinity to E2/NS1 protein of hepatitis C virus. Preferably, a recombinant antibody in which the Fc region is of a human type, and a recombinant antibody which is a single-strand antibody, are provided.

A further aspect of the present invention provides: a medicament which comprises the aforementioned substance of the present invention as an active ingredient; a medicament which comprises the aforementioned antibody of the present invention as an active ingredient; and a medicament which comprises the aforementioned recombinant antibody of the present invention as an active ingredient. Preferably, there are provided a medicament for treating and/or preventing hepatitis C and a medicament for diagnosing hepatitis C. Further, the present invention provides an anti-HCV agent which comprises the aforementioned substance of the present invention as an active ingredient, an anti-HCV agent which comprises the aforementioned antibody of the present invention as an active ingredient, and an anti-HCV agent which comprises the recombinant antibody of the present invention as an active ingredient.

A further aspect of the present invention provides a method for obtaining a sequence of the variable region of the antibody binding to E2/NS1 in which the B cell of a patient who recovered from hepatitis C is stimulated to express mRNA of the antibody against E2/NS1 protein of hepatitis C virus, and thereby mRNA of the antibody and cDNA of the antibody are obtained from the B cell. Preferably, a method for obtaining a sequence of the variable region of the antibody is provided in which a patient who recovered from hepatitis C is one who recovered without antiviral treatment and the B cell is a peripheral blood mononuclear cell.

A further aspect of the present invention provides an antibody having a sequence of the variable region obtained by the above-described method.

A further aspect of the present invention provides a method for screening a substance which inhibits the binding between E2/NS1 of hepatitis C virus and a cell infectious with hepatitis C, a cell expressing CD81, or CD81, which comprises steps of: contacting E2/NS1 of hepatitis C virus with a cell infectious with hepatitis C, a cell expressing CD81, or CD81 in the presence and absence of a test substance; and comparing the bindings between E2/NS1 of hepatitis C virus and the cell infectious with hepatitis C, the cell expressing CD81, or CD81 in the presence and absence of a test substance. Preferably, there are provided: a screening method in which E2/NS1 protein of hepatitis C virus is a protein prepared by adding a substance capable of recognizing E2/NS1 protein to E2/NS1 protein of hepatitis C virus; a screening method in which E2/NS1 protein of hepatitis C virus is a protein prepared by adding a substance capable of detecting E2/NS1 protein by a fluorescence method or a luminescence method to E2/NS1 protein of hepatitis C virus; a screening method in which E2/NS1 protein of hepatitis C virus is a protein prepared by adding a tag to E2/NS1 protein of hepatitis C virus; a screening method in which E2/NS1 protein of hepatitis C virus is a protein prepared by adding a tag to the C-terminus of E2/NS1 protein of hepatitis C virus; a screening method in which the cell expressing CD81 is a cell which expresses human CD81; a screening method in which CD81 is human CD81 and expressed by solubilized form; and a screening method in which the test substance is a protein, sulfated polysaccharides, or a low molecular weight compound.

A still further aspect of the present invention provides a substance which inhibits the binding between E2/NS1 protein of hepatitis C virus and the cell infectious with hepatitis C, the cell expressing CD81, or CD81, which is obtained by any of the above screening methods.

A further aspect of the present invention provides a substance which inhibits a life cycle of hepatitis C virus, which is obtained by the screening method which comprises steps of assaying the binding between a cell infectious with HCV and a cell expressing HCV protein in the presence and absence of the test substance, and comparing it with the binding in the absence of the test substance; and a substance which inhibits a life cycle of hepatitis C virus, which is obtained by the screening method which comprises steps of, after the cell infectious with HCV has bound with the HCV protein expressing cell in the presence or absence of the test substance, assaying the fusion between the cell infectious with HCV and the HCV protein expressing cell, and comparing it with the fusion in the absence of the test substance. Preferably, the test substance is a protein, sulfated polysaccharides, or a low molecular weight compound.

A further aspect of the present invention provides: an anti-HCV agent which comprises the above substance as an active ingredient, and a method for producing the anti-HCV agent which comprises formulating the anti-HCV agent using the above substance. The method for production preferably comprises a step of confirming the inhibition of proliferation of hepatitis C virus by the above screening method.
 

Claim 1 of 5 Claims

1. An antibody comprising a heavy chain (H chain) having a variable region wherein CDR-1, CDR-2, and CDR-3 respectively comprise amino acid sequences shown by SEQ ID NOS: 1, 2, and 3.

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