Title:  Liposomes comprising peptide antigens derived from X protein of hepatitis B virus

United States Patent:  6,380,359

Assignee:  Mogam Biotechnology Research Institute (Kyonggi-Do, KR)

Filed:  March 30, 1998

PCT NO:   PCT/KR98/00010

102(e) Date:  March 30, 1998

PCT PUB. Date:  July 22, 1999

Foreign Application Priority Data:  Jan 19, 1998[KR] (PCT/KR98/00010)

The present invention relates to liposomes comprising novel peptide antigens which play a role in regulating human immunity against hepatitis B virus, more specifically, to peptide groups corresponding to epitopes of antigens derived from X protein of HBV which induce cytotoxic T lymphocytes against the virus or immunological tolerance to the virus, and pH-sensitive liposomes comprising said peptide groups to induce cellular immunity so that CTLs specific to the virus can be produced. Since peptide antigens derived from X protein such as X3, X4, X5, X6 and X7 activate CTL which can recognize HBV antigens present in human body, and can also be recognized by the CTL, the liposomes can be used for the development of proposed therapeutic agents for the prevention and treatment of HBV-associated diseases.

DETAILED DESCRIPTION OF THE INVENTION

Based on a previous finding that peptides binding to MHC Class I molecules have specific amino acid sequences, peptides having potentials of binding to MHC Class I molecules were sequenced from the full amino acid sequences of HBV X protein. That is, since peptides which can bind to HLA-A2, a human MHC Class I molecule, have leucine, valine or isoleucine at C-terminus and second position from N-terminus(see: Matsumura, M. et al., Science, 257:929-934(1992)), amino acid sequences satisfying the said condition were screened from the full amino acid sequences of HBV X protein, and peptides having potentials of binding to HLA-A2 molecule, i.e., peptide antigens which can induce CTL response by binding to the human MHC Class I molecule, were selected, among peptides produced by degradation of HBV X antigenic protein, based on three-dimensional structure of the molecule(see: Table 1 below). Then, peptides were synthesized chemically according to the amino acid sequences thus determined. Amino acid sequences of peptides derived from HBV X protein, i.e., X3, X4, X5, X6 and X7, are disclosed in Table 1 below.

                             TABLE 1
    Location and amino acid sequences of peptides recognized by CTL among
     peptides produced by degradation of HBV x protein
                    Position of amino Amino acid    Number of amino
    Peptide         acid sequence   sequence*          acid
    x3 (SEQ ID NO: 052-060         HLSLRGLFV            9
    1)
    x4 (SEQ ID NO: 092-100         VLHKRTLGL            9
    2)
    x5 (SEQ ID NO: 102-110         AMSTTDLEA            9
    3)
    x6 (SEQ ID NO: 115-123         CLFKDWEEL            9
    4)
    x7 (SEQ ID NO: 126-134         EIRLKVFVL            9
    5)
    *: Amino acid sequences are abbreviated according to the IUPAC-IBU
     nomenclature system.

The present invention covers not only peptides consisting of 9 amino acids described above but also peptides added with 1 to 5 amino acids to N- or C-terminus of the said peptides and their functional equivalents. In the present invention, the term `functional equivalents` is employed to mean all peptides subsitituted by 1 or 2 amino acids among the amino acid sequences of the said peptides. For example, substitution comprises the combinations such as Gly, Ala; Val, Ile, Leu; Asp, Glu; Asn, Gln; Ser, Thr; Lys, Arg; and, Phe, Tyr.

In order to prepare pH-sensitive liposomes comprising the peptide antigens derived from HBV X protein, phosphatidylethanolamine-.beta.-oleoyl-.gamma.-palmitoyl(POPE) and cholesterol hemisuccinate(CHOH) are mixed in a molar ratio of 6:4 to 8:2, most preferably 7:3, or POPE, phosphatidylethanolamine(PE) and CHOH are mixed in a molar ratio of 3:3:4 to 4:4:2, most preferably 3.5:3.5:3, finally to form thin membrane of phospholipid. And then, the liposomes thus prepared are added to a buffer solution containing the said peptide antigens derived from HBV X protein. In this connection, liposomes comprising peptide antigens derived from HBV X protein are prepared by mixing 1 or more kinds of peptide antigen and phospholipid in a molar ratio of 1:5 to 1:25, most preferably 1:20. Also, the pH-sensitive liposomes prepared according to the present invention may further comprise up to 1 mole % monophosphoryl lipid A as a phospholipid.

The liposomes of the present invention encapsulate peptide antigens inducing human CTL response, and the pH-sensitive liposomes which are used as carriers of the peptides, can also induce CTL response specific to the virus from which the encapsulated peptides are derived. Naturally, it is clearly demonstrated that the pH-sensitive liposomes comprising the said peptides can induce human cellular immunity.

In describing amino acid sequences of the peptides of the invention, one-letter symbols abbreviated by the IUPAC-IUB standards are employed as followings:

Claim 1 of 3 Claims

What is claimed is:

1. A synthetic peptide derived from X protein of hepatitis B virus which is recognized by cytotoxic T lymphocytes to be cytotoxic against hepatitis B virus, whose amino acid sequence consists of (SEQ ID NO:1):

His-Leu-Ser-Leu-Arg-Gly-Leu-Phe-Val.

____________________________________________
If you want to learn more about this patent, please go directly to the U.S. Patent and Trademark Office Web site to access the full patent.