The present invention provides a kind of HBV-antigen bound to heat shock proteins (hsps) which comprises core antigen, surface antigen and polymerase antigen. The present invention also provides a complex of HBV antigen bound to heat shock protein gp96 and hsp78, as well as a method for preparing the complex. The complex includes a complex of gp96 and hsp78 non-covalently bound to antigenic polypeptide, as well as a fusion protein of both which results from covalent binding. Such complex can be used to prepare therapeutic vaccine for treating hepatitis B and primary hepatocellular carcinoma.

RELATED APPLICATIONS

This is the U.S. national phase under 35 U.S.C. 371 of International Application PCT/CN01/00295, filed Feb. 27, 2001, designating the US and published in Chinese, which claims priority to Chinese Patent Application 00121270.2, filed Aug. 11, 2000, and Chinese Patent Application 01104060.2, filed Feb. 20, 2001.

FIELD OF THE ART

The present invention relates to a complex of HBV antigenic polypeptide bound to heat shock protein gp96 and hsp78, as well as the application thereof.

BACKGROUND OF THE ART

Heat shock protein (hsp) gp96 (glycoprotein 96), also called as grp94 (glucose-regulated protein 94), is a member of hsp90 family which located on endoplasmic reticulum (ER) membrane of cells, the protein has about 96 KDa of molecuar weight and plays an important role in cellular protein folding and transportation. Recent studies have showed that gp96 is also expressed on the surface of some tumor cells. Gp96 possesses two conserved domains, the C-terminal is a polypeptide-binding domain which can bind a variety of polypeptides with 5-25 amino acids, and the N-terminal is a ATP-binding domain with ATPase activity. Heat shock protein hsp78, a member of hsp70 family in cytoplasma, has about 78 KDa of molecular weight and plays an important role in cellular protein folding and transportation. Hsp78 can bind to a variety of short peptides as molecular chaperone, it has two functional domains: the N-terminal domain with ATPase activity and the C-terminal domain which can bind to polypeptide substrate.

Recent studies demonstrated that immunization with gp96 and hsp78 purified from tumor tissues or cells infected by virus could elicit a specific immunological rejection response against the tumors or viruses. A further study showed that gp96 and hsp78 can bind to all peptide libraries generated in the cells including antigenic polypeptides, this specific immunological response is based on the polypeptides bound to gp96 and hsp78 not the heat shock protein itself, gp96 and hsp78 from tumor tissues or cells infected by virus normally bind to tumor or virus specific polypeptides, gp96 and hsp78 can present the antigenic ploypeptides bound by them to major histocompatibility complex (MHC) molecules and activate cytotoxic T lymphocyte (CTL) to elicit cell immunological response generated by organism. Since gp96 and hsp78 play an important role in antigen presentation in the cells, gp96-ploypeptide complex and hsp78-polypeptide complex can be used to prevent autogenous tumors and some infectious diseases.

Pramod K. Srivastava from University of New York, USA, applied for 6 patents (U.S. Pat. Nos. 6,017,544, 6,017,540, 6,007,821, 5,837,251 and 5,830,464) in USA based on his studies. These patents mainly relate to the use of a complex of heat shock protein (hsp) non-covalently bound to antigen molecular for treating primary and transferred tumors as well as infectious diseases, and activating immunological response generated by organism, wherein the antigen moleculars include the esoteric polypeptides bound to hsp, and also include exoteric antigens or immunogenic fragments that can form complex with hsp in vitro. Hsp mainly comprises hsp70, hsp90 and gp96 protein.

It has been demonstrated that gp96 and hsp78 can bind antigenic polypeptides such as antigenic peptide of vesicular stomatitis virus, ovalbumin antigenic epitope peptide restricted by mouse H-2K.sup.b and an L.sup.d-restricted CTL epitope peptide of a mouse leukemia. Up to now, however, no antigenic ploypeptides bound to hsps have yet been identified in patients tissues infected by virus.

It is estimated that about 350 million individuals worldwide have been infected by HBV, and in China alone the number is about 120 million. HBV is the leading cause of chronic hepatitis, cirrhosis and liver cancer, so it is a kind of infectious diseases that severely disserve the life health of Chinese. The probability of liver cancer developing from hepatitis B is also very high. The HBcAg-positive hepatocytes account for 62.5% in total HCC cells and 29.2% in the totally adjacent liver cells. Cell immunological response elicited by CTL is a major pathway to clear virus and cure hepatitis B. In the patients infected by hepatitis B virus, the HBV antigenic ploypeptide is presented to MHC I molecular after process, and activates specific CTL to elicit cell immunological response. Now, some CTL epitopes on the HBV core protein have been identified, including HLA-A2 restricted HBcAg18-27, HLA-A11 restricted HBcAg88-96 and like. Therefore, it is very important to develop a novel drug for the prevention and treatment of HBV associated hepatitis and primary hepatocellular carcinomas, especially a drug that can elicit actively CTL immunological response generated by organism.

SUMMARY OF THE INVENTION

One object of the invention is to provide a complex of a HBV antigenic molecule bound to gp96 and hsp78. The HBV antigen can be the following amino acid sequences respectively, such as "YVNTNMG" or a variant thereof (derived from core protein of HBV 88-94); "YVNTNMGLK" or a variant thereof (derived from core protein of HBV 88-96); "STLPETTVVRR" or a variant thereof (derived from core protein of HBV 141-151); "FLPSDFFPSV" or a variant thereof (derived from core protein of HBV 18-27); "IPIPSSWAF" or a variant thereof (derived from surface protein of HBV 313-321); "WLSLLVPFV" or a variant thereof (derived from surface protein of HBV 355-363); and "FLLSLGIHL" or a variant thereof (derived from polymerase of HBV 575-583).

The variant sequence is obtained from HBV by one or more replacment, deletion, increment of amino acids or modification of side chains, and has the antigenic sequence of the HBV.

The complex of the present invention consists of the heat shock protein non-covalently and covalently bound to polypeptide.

A further object of the invention is to provide a method for preparing a complex of HBV antigenic polypeptide and heat shock protein gp96 and hsp78 as described above of the present invention.

A still further object of the invention is to provide the application of these complexes of the invention for preparing the drug that can treat hepatitis B and primary hepatocellular carcinomas.

We isolated for the first time a specific 7-mer peptide in the polypeptide bound to heat shock protein gp96 from six human liver cancer tissue infected by HBV and found the 7-mer peptide has the amino acids sequence: "YVNTNMG or YVNVNMG" by sequencing. We searched the GenBank with a BLAST search using the National Center for Biotechnology Information Website and found that the peptide were derived from amino acid residues 88-94 of the core protein of HBV. The complex of gp96-7 mer peptide can be reconstituted in vitro with the synthesized peptide sequence and expressed gp96 protein in vitro. Mice were immunized with 7-mer peptide or gp96-7 mer peptide complex. The results showed that both them can elicit the specific CTL response in mice. Immunization with the gp96-7 mer peptide resulted in >200-fold immunogenicity in comparison to 7-mer peptide only controls. The experiment result showed that the gp96-7 mer peptide complex can be developed as a novel drug for treatment of hepatitis B and primary hepatocellular carcinoma.

The artificially synthesized 7-mer peptide "YVNTNMG, or YVNVNMG" and 7-mer peptide with N-terminal labeled by fluorescein were used to constitute the reaction system of gp96 protein bound to 7-mer peptide labled by fluorescein. The formula is presented as follows:

.times..times..times. ##EQU00001## wherein G is gp96 protein, P is 7-mer peptide, GP is the complex of gp96 protein-7 mer peptide, and k.sub.1 and k.sub.2 represent the forward and the reverse rate constants, respectively. The equilibrium constant (k) of the reaction is given by the following relationship k=k.sub.1/k.sub.2=[GP]/[G][P] wherein [GP], [G] and [P] is the concentration of the reaction product GP and substrate G, P, respectively. In order to find the optimum reaction condition, we assessed several impact factors of the reaction system such as optimum temperature, salt concentration, pH value, additive, catalyst, the optimum reaction concentration rate of gp96 protein and 7-mer peptide and like, and determined the reaction constant.

Synthesis of the gp96 protein-7 mer complex in vitro is performed under the optimum reaction system condition.

The present invention also provides a method for preparing the complex HBV antigen 7-mer peptide and heat shock protein gp96 comprising the gp96 protein and the HBV antigen are incubated for 10 minutes to 30 minutes at 37-39.degree. C. in a low-salt buffer with a maximum concentration of 100 mmol/L comprising 5-10% (v/v) glycerol, wherein the concentration of the gp96 protein is 0.1-0.15 .mu.mol/L and the concentration of HBV antigen is 2.5-3.5 .mu.mol/L.

In the method of the present invention, the temperature is preferably 37.degree. C., and the reaction time is preferably 15 minutes.

In the method of the present invention, the concentration of the gp96 is preferably 0.12 .mu.mol/L, and the concentration of the HBV antigen is preferably 3.0 .mu.mol/L.

The present invention also provides a fusion protein formed by covalent bind of HBV antigen 7-mer peptide and heat shock protein gp96. The sequence of nucleic acid corresponding to the peptide was synthesized artificially, and the sequence was ligated with the 5'-terminal of the gp96 gene by conventional methods of molecular biology and then expressed in the E. coli. For example, after the restriction site of Bgl II is introduced into the 7-mer peptide, the nucleic acid sequence is ligated with 5'-terminal of gp96 gene by T.sub.4 ligase, the restriction sites of two restriction endonucleases BamHI and SacI are introduced into the 5'-terminal and 3'-terminal of the ligation product respectively, then ligated into the expression vector pET30a and expressed in the E. coli. The expression product is the fusion protein of gp96 and 7-mer peptide.

Immunization with gp96 protein-7 mer complex of the invention including the complex formed by the above gp96 non-covalently bound to the 7-mer peptide in vitro and the fusion protein formed by the above gp96 covalently bound to the 7-mer peptide can be performed using any known immunological manner, for example, subcutaneous injection, intradermal injection, intraperitoneal injection and like. The dosage of immunization with gp96 protein-7 mer peptide complex can be, for example, 0.01 nmol, 0.05 nmol, 0.10 nmol and 0.50 nmol. When the immunization is performed only with 7-mer peptide, the dosage of immunization can be 0.2 nmol, 2 nmol, 20 nmol.

Any known adjuvant, such as Freund's adjuvant, chromium alum and like can be used or not when immunization is performed with gp96 protein-7 mer peptide complex.

A 9-mer peptide, "YVNTNMGLK", was also synthesized by our laboratory. A gp96 protein-9 mer peptide complex was synthesized according to the above optimum reaction system in vitro. Immunization can be performed using any known immunological manner, for example, subcutaneous injection, intraderma injection, intraperitonea injection and like. The dosage of immunization with gp96 protein-9 mer peptide complex can be, for example, 0.01 nmol, 0.05 nmol, 0.10 nmol and 0.50 nmol. When the immunization is performed only with 9-mer peptide, the dosage of immunization can be 0.2 nmol, 2 nmol, 20 nmol. Any known adjuvant, such as Freund's adjuvant, chromium alum and like can be used or not when immunization is performed. Both of the gp96-9 peptide complex and the 9-mer peptide can elicit the specific cytotoxic T lymphocytes (CTL) response in mice after immunization. The immunogenicity of the gp96-9 mer peptide is >300-fold in comparison to 9-mer peptide only controls. The experiment results showed that the gp96-9 mer peptide complex can be developed as a novel drug for treatment of hepatitis B and primary hepatocellular carcinoma.

In addition, we also synthesized 11-mer peptide "STLPETTVVRR"; 10-mer peptide "FLPSDFFPSV"; 9-mer peptide "IPIPSSWAF"; 9-mer peptide "WLSLLVPFV", and 9-mer peptide, "FLLSLGIHL". A gp96 protein-polypeptide complex is synthesized according to the above optimum reaction system in vitro. Immunization can be performed using any known immunological manner respectively, for example, subcutaneous injection, intraderma injection, intraperitonea injection and like. The dosage of immunization with gp96 protein-polypeptide complex can be, for example, 0.01 nmol, 0.05 nmol, 0.10 nmol and 0.50 nmol. When the immunization is performed only with polypeptide, the dosage of immunization can be 0.2 nmol, 2 nmol, 20 nmol. Any known adjuvant, such as Freund's adjuvant, chromium alum and like can be used or not when immunization is performed. Both of the gp96-polypeptide complex and the polypeptide can elicit the specific cytotoxic T lymphocyte (CTL) response in mice after immunization. The immunogenicity of the gp96-polypeptide complex is >150-fold in comparison to peptide only controls. The experiment results showed that the gp96-polypeptide complex can be developed as a novel drug for treatment of hepatitis B and primary hepatocellular carcinoma.

According to the optimum reaction system, we synthesized hsp78 protein-polypeptide complexes using the above synthesized multi-peptides including "YVNTNMG"; "YVNTNMGLK"; "STLPETTVVRR"; "FLPSDFFPSV"; "IPIPSSWAF"; "WLSLLVPFV"; "FLLSLGIHL". Immunization can be performed using any known immunological manner respectively, for example, subcutaneous injection, intraderma injection, intraperitonea injection and like. The dosage of immunization with hsp78 protein-polypeptide complex can be, for example, 0.01 nmol, 0.05 nmol, 0.10 nmol and 0.50 nmol. When the immunization is performed only with 7-mer peptide, the dosage of immunization can be 0.2 nmol, 2 nmol, 20 nmol. Any known adjuvant, such as Freund's adjuvant, chromium alum and like can be used or not when immunization is performed. Both of the hsp78-polypeptide complex and the polypeptides can elicit the specific cytotoxic T lymphocyte (CTL) response in mice after immunization. The immunogenicity of the hsp78-polypeptide is >150-fold in comparison to polypeptides only controls. The experiment result showed that the hsp78-polypeptide complex can be developed as a novel drug for treatment of hepatitis B and primary hepatocellular carcinoma.
 

Claim 1 of 9 Claims

1. A composition, comprising a complex of HBV antigen and a human heat shock protein gp96, wherein the HBV antigen consists of the amino acid sequence of: YVNTNMG (SEQ ID NO: 1).

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