Patent 6,921,534

The present invention features compositions that include a stress protein, or a portion thereof, and an HBV antigen. These compositions are discussed at length below. We note here that their components can be obtained from a variety of sources and their length and content can vary. For example, the stress protein can be one that is naturally expressed by any mammal (e.g. a human or non-human primate) or any other class of organisms that expresses stress proteins (e.g., a bacterium or mycobacterium); the stress protein and/or the HBV antigen can be full length, truncated, or extended by the addition of one or more amino acid residues; and, in addition, the content of the stress protein or HBV antigen can vary (for example, a stress protein, or a portion thereof, and an HBV antigen can contain one or more amino acid substitutions). Any variation must still result, however, in a composition that can induce or enhance an immune response against HBV in a mammal. Preferably, the immune response is substantial enough that an HBV-infected patient experiences an improvement (objective or subjective) in a sign or symptom of the infection. Accordingly, an antigen encompasses full-length and naturally occurring antigens as well as fragments and other variants thereof that, when administered to a subject (e.g., by the methods described herein), elicits an immune response to one or more epitopes present within the fragment or variant.

Similarly, in addition to full-length or naturally occurring stress proteins, the compositions of the invention can include fragments of stress proteins that are immunostimulatory (i.e., fragments that facilitate an immune response to an antigen). The stress protein, or the fragment thereof, facilitates an immune response when the immune response is greater, or in any way superior to, the immune response that typically occurs when the HBV antigen is administered alone.

The immune response can be either a humoral or a cell-mediated response. For example, an antigenic fragment can contain one or more HLA class I peptide antigens, as described herein. A cell-mediated immune response involves antigen specific cells of the immune system, such as cytotoxic T lymphocytes (CTLs) as well as, possibly, T helper lymphocytes (Th) and cells of the innate immune system, such as monocytes, macrophages, dendritic cells, natural killer cells and γδ T cells. One of ordinary skill in the art is well able to detect or otherwise evaluate an immune response, which is evident by, for example, the induction of cytotoxic T lymphocytes (see the Examples below), a cellular proliferative response, induction of cytokines, or a combination of these events.

In particular embodiments, the HBV antigen can be the HBV core antigen or a fragment or derivative thereof. Derivatives of the HBV antigen include variants of the HBV antigen, such as those containing one or more amino acid substitutions (e.g., conservative amino acid substitutions). For example, a variant of an HBV antigen can contain 1-2, 2-5, 5-10, 10-25, or more, substituted amino acid residues. Alternatively, substitutions or other mutations, such as deletions or truncations, can constitute 1-2, 2-5, 5-10, or 10-25% of the sequence of a full-length HBV antigen. Like the antigenic portion of the composition, a variant of a stress protein can contain one or more amino acid substitutions (e.g., conservative amino acid substitutions). For example, a variant of a stress protein can contain 1-2, 2-5, 5-10, 10-25, or more, conservative amino acid substitutions. Here again, substitutions or other mutations, such as deletions or truncations, can constitute 1-2, 2-5, 5-10, or 10-25% of the sequence of a full-length stress protein.

Various combinations of stress proteins and HBV antigens are also within the scope of the invention. For example, the compositions of the invention include those in which a full-length HBV antigen is associated with a full-length stress protein; an antigen that consists of a fragment or other variant of an HBV antigen is associated with a full-length stress protein; a full-length HBV antigen is associated with a fragment or other variant of a stress protein; and a fragment or other variant of an HBV antigen is associated with a fragment or other variant of a stress protein. Of course, as described herein, more than one of each of these components (i.e., more than one HBV antigen and more than one stress protein) may be present, and each of the components may be present in the form of a full-length protein or an immunologically active fragment or variant thereof.

Moreover, in any of the arrangements described herein, the HBV antigen and the stress protein can be associated in any manner. For example, the stress protein and the HBV antigen, can be present in the form of a fusion polypeptide (wherein the stress protein and the HBV antigen are covalently linked during translation of a fused open reading frame). Alternatively, a stress protein and an HBV antigen can be linked by chemical conjugation after each has been translated or synthesized individually. The components can also be non-covalently associated (in, for example, a mixture or a more ordered composition). The terms "polypeptide" and "protein" are used interchangeably to describe a chain of amino acid residues, except where it is clear from the context that a distinct meaning is intended.

While stress proteins are discussed further below, we note here that the stress protein can be a heat shock protein (Hsp). Further, the Hsp can be a mycobacterial Hsp, such as Hsp65 (e.g., Hsp65 of Mycobacterium bovis), or any member of an Hsp family of proteins from any species.

The compositions of the invention can be formulated for administration to a subject in a variety of ways and, optionally, contain an adjuvant. Additional optional components of the composition include pharmaceutically acceptable diluents, excipients, and carriers.

The invention also features methods of treating an HBV infection in a subject (e.g., a mammal, such as a human) by administering a composition of the invention to the subject infected with HBV and methods of preventing (or reducing the likelihood of) an HBV infection in a subject (e.g., a mammal, such as a human) by administering a composition of the invention to the subject before they have been infected with HBV.

The components of the composition need not be directly administered to the subject as polypeptides. Instead, a nucleic acid encoding the stress protein, the HBV antigen, or a fusion protein containing one or more of each can be administered, and the protein, antigen, or fusion protein will be expressed in the subject in vivo. The nucleic acid can be a part of a viral vector, for example, a part of a viral vector genome, or encapsulated in, e.g., liposomes. Alternatively, the nucleic acid can be delivered as a naked nucleic acid, such as plasmid DNA driven by regulatory sequences operable in eukaryotic or mammalian cells. Methods of administering nucleic acid molecules are well known in the art.

The invention further includes the use of compositions of the invention (e.g., HBV-containing fusion proteins, the nucleic acid molecules that encode them, and pharmaceutical compositions containing them) in the manufacture of a medicament for the treatment of hepatitis B virus infection in accordance with the methods described herein.

DETAILED DESCRIPTION OF THE INVENTION

The invention relates to HBV antigen-containing compositions that are useful in treating or preventing HBV infection. The content of the compositions can vary, as described herein, but the compositions comprise a stress protein, or a portion (e.g., a fragment) or derivative thereof, and an HBV antigen. Various materials and procedures suitable for use in the methods of the invention are discussed below.

Because nucleic acid sequences encoding stress proteins and HBV proteins are known and available, nucleic acid constructs encoding them (alone or as a fusion construct) can be readily prepared using methods routinely practiced in the art. For examples of nucleic acids encoding a stress protein (an Hsp) optionally coupled to an antigen see WO 89/12455, WO 94/29459, WO 98/23735, WO 99/07860, and references cited therein. Fusion proteins can be produced not only by recombinant techniques but also by post-translational conjugation of a stress protein (e.g., an Hsp) and an HBV antigen. Conjugation techniques are described, for example, in Hermanson (Bioconjugate Techniques, Academic Press, San Diego, Calif., 1996) Lussow et al. (Eur. J. Immun. 21:2297-2302, 1991), and Barrios et al. (Eur. J. Immun. 22:1365-1372, 1992). Such methods of conjugation include the use of coupling agents such as glutaraldehyde, carbodiimides, and bisdiazobenzidine; the use of heterobifunctional crosslinkers such as M-Maleimidobenzoyl-N-hydroxysuccinimide ester; or the use of cysteine residues (those naturally present and/or those recombinantly inserted) in the stress protein and the antigen to facilitate intermolecular disulfide bond formation.

Any HBV antigen is suitable for inclusion in a fusion protein or composition of the invention. A preferred HBV antigen is the HBV core antigen or a fragment or derivative thereof. To facilitate testing, the HBV antigen can optionally be modified to include known mouse MHC-restricted CTL epitopes such as, for example, mouse H-2K^b-restricted CTL epitopes. An example of such a modification is described in the Examples (for example, in the adw strain of HBV, residue 97 is isoleucine-replacing this with phenylalanine generates a mouse H-2K^b-restricted CTL epitope). In addition, the antigen can be modified to include human HLA epitopes from more than one HBV subtype (e.g. adw, ayw, adr or ayr). For example, a single amino acid substitution from a threonine to a valine at position 91 of the HBV core antigen shown in FIG. 2 would duplicate the sequence of a known HLA-A11-restricted CTL epitope found in both the adw and adr HBV subtypes. Other derivatives of the HBV core antigen include truncations. Such truncations would include, but are not limited to, truncations in which all or part of the C-terminal arginine-rich domain is removed (amino acids 150 to 185 of HBc). Suitable truncated HBc fragments include, but are not limited to, fragments consisting of only the first N-terminal 149 amino acids, or the first 151 N-terminal amino acids of HBc. In any event, a suitable fragment of the HBc antigen (or any suitable HBV antigen) would ideally include one or more B or T cell epitopes (or one or more B cell epitopes and one or more T cell epitopes), preferably one or more CTL epitopes. Additionally, the terminal cysteine of the HBV core antigen can be removed or replaced with a different amino acid. Other modifications to the amino acid sequence could be made. Another example is a substitution in an anchor residue of a known HLA-restricted CTL epitope to enhance the binding affinity of the peptide to the MHC Class I molecule. Although these modified HBV core antigens are suitable for inclusion in bile fusion proteins, they can also be used alone (optionally formulated with an adjuvant) to generate an immune response to HBV.

Additional HBV antigens suitable for use in the present invention include the HBV core antigen, HBV e antigen (HBeAg), x protein (HBx), polymerase polypeptide, and the HBV envelope proteins S, M, and L and fragments thereof (Seeger and Mason, Microbiol. Mol. Biol. Rev. 64: 51-68, 2000; Ganem and Schneider, Hepadnavirdae: The viruses and their replication. In: Knipe, D M and Howley, P M, eds. Fields Virology, Philadelphia: Lippincott Williams & Wilkins, 2001:2923-2969).

As described above, the HBV antigen, the stress protein, or both, can contain one or more amino acid substitutions (e.g., conservative amino acid substitutions). These substitutions can be, but are not necessarily, made at one or more predicted non-essential amino acid residues. A "conservative amino acid substitution" is one in which one amino acid residue is replaced with another amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Regardless of whether the substitution is designed to occur at a predicted non-essential site or is introduced randomly along all or part of an HBV antigen or stress protein coding sequence (such as by saturation mutagenesis), the resultant mutants can be screened for antigenic and inmunostimulatory activity, respectively, to identify mutants that retain biological activity. Following mutagenesis, the encoded protein can be expressed recombinantly and the activity of the protein can be determined.

The HBV antigen can be fused to either the N-terminus or C-terminus of the stress protein, with or without a linker or intervening exogenous sequence. In alternative embodiments, two HBV antigens (which can be naturally occurring or variant, as described herein) can be attached to the stress protein (one at the N-terminus and the other at the C-terminus of the stress protein; both at the N-terminus; or both at the C-terminus). Additionally, one or more HBV antigens (again, naturally occurring or fragments or other variants thereof; from either the same or different HBV proteins) can be attached either to the N-terminus or C-terminus, or both, of the stress protein. Additional alternative arrangements can be made, and will be evident to one of ordinary skill in the art, if more than one stress protein is included.

A stress protein and an HBV antigen (or combinations thereof; for example a stress protein and two or more HBV antigens) can be linked by chemical conjugation after each has been translated or synthesized individually. As noted above, the components can also be non-covalently associated (in, for example, a mixture or a more ordered composition). Compositions containing stress proteins or immunostimulatory fragments thereof that are non-covalently associated with an HPV antigen can be produced as described in U.S. Pat. Nos. 6,048,530; 6,017,544; 6,017,540; 6,007,821; 5,985,270; 5,948,646; 5,935,576; 5,837,251; 5,830,464; or 5,750,119. See also, U.S. Pat. Nos. 5,997,873; 5,961,979; 6,030,618; 6,139,841; 6,156,302; 6,168,793; and International Publication No. WO 97/06821.

Moreover, more than one type of viral antigen can be included in the composition. For example, in addition to the HBV antigen, compositions of the invention can include (or encode; any proteins described herein may be administered directly or by way of nucleic acids) an antigen from a different pathogen. Thus, in addition to an HBV antigen, the compositions can include (or encode) a hepatitis C antigen, a herpes simplex virus (HSV) antigen, a human immunodeficiency virus (HIV) antigen, a cytomegalovirus (CMV) antigen, an Epstein-Barr virus (EBV) antigen, a respiratory syncytial virus (RSV) antigen, a human papillomavirus (HPV) antigen, a herpes virus antigen, or a combination thereof. The same alternatives that have been described for the embodiments in which the compositions contain only HBV as the viral antigen (e.g., the method of association with the stress protein, the inclusion of full-length, fragmented, or variant proteins, the variable number of components, and their arrangement) are applicable to the embodiments in which at least one HBV antigen and at least one other viral antigen are present in (or encoded by) the composition.

Surprisingly, it has also been found that removing the C-terminal arginine-rich domain from the core antigen results in a polypeptide capable of eliciting an immune response to the core antigen, particularly a cellular and/or a CTL immune response. The arginine-rich domain of the core antigen is located between amino acids 150 to 183 of the core antigen (Nassal, J. Virol. 66: 4107-4116, 1992). Suitable core antigen fragments include, but are not limited to, those that lack all or part of this region. For example, suitable core antigen fragments may contain of the first 149 or 151 amino acids (or fewer than 149 or 151 amino acids).

The compositions of the invention can optionally include an adjuvant. Examples of adjuvants that may be effective include, but are not limited to: Freund's complete adjuvant (FCA), Freund's incomplete adjuvant (FIA), SAF, muramyl dipeptide (MDP), lipopolysaccharide (LPS), lipid A, monophosphoryl lipid A (MPL), pertusis toxin (PT), stearyl tyrosine, γ inulin, RIBI (which contains three components extracted from bacteria), Quil-A, saponins (QS21), alum (aluminum hydroxide, aluminum phosphate), calcium phosphate, MF-59, immunostimulatory complexes (ISCOMS), CpG oligonucleotides and cytokines (Gupta and Siber, Vaccine 13: 1263-1276, 1995; Singh and O'Hagan, Nature Biotechnology 17: 1075-1081, 1999).

A suitable fragment or derivative of an HBV antigen will ideally contain at least one B or T cell epitope (or both). In a preferred embodiment, the fragment or derivative will contain at least one CTL epitope.

A variety of stress proteins have been isolated, cloned, and characterized from a diverse array of organisms (Mizzen, Biotherapy 10:173-189, 1998). Any immunostimulatory Hsp or immunostimulatory fragment thereof is suitable for use in the fusion polypeptides and compositions. For example, Hsp70, Hsp60, Hsp20-30 (low molecular weight Hsp), and Hsp10 (the GroES homologue) are among the major determinants recognized by host immune responses to infection by Mycobacterium tuberculosis and Mycobacterium leprae. In addition, Hsp65 of Bacille Calmette Guerin (BCG), a strain of Mycobacterium bovis, was found to be an effective immunostimulatory agent, as described in the example below.

Families of stress genes and proteins for use in the present invention are well known in the art and include, for example, Hsp100-200, Hsp100, Hsp90, Lon, Hsp70, Hsp60, TF55, Hsp40, FKBPs, cyclophilins, Hsp20-30, ClpP, GrpE, Hsp10, ubiquitin, calnexin, and protein disulfide isomerases. See, e.g., Macario, Cold Spring Harbor Laboratory Res. 25:59-70, 1995; Parsell et al., Rev. Genet. 27:437-496, 1993; and U.S. Pat. No. 5,232,833.

Examples of Hsp100-200 proteins include Grp170 (for glucose-regulated protein). Grp170 resides in the lumen of the ER and in the pre-Golgi compartment, and may play a role in immunoglobulin folding and assembly.

Examples of Hsp100 proteins include mammalian Hsp110, yeast Hsp104, and E. coli ClpA, ClpB, ClpC, ClpX agnd ClpY.

Examples of Hsp90 proteins include HtpG in E. coli, Hsp83 and Hsc83 in yeast, and Hsp90alpha, Hsp90beta, and Grp94 (small gp96) in humans. Hsp90 binds groups of proteins that are typically cellular regulatory molecules, such as steroid hormone receptors (e.g., glucocorticoid, estrogen, progesterone, and testosterone receptors), transcription factors, and protein kinases that play a role in signal transduction mechanisms. Hsp90 proteins also participate in the formation of large, abundant protein complexes that include other stress proteins.

Lon is a tetrameric ATP-dependent protease that degrades non-native proteins in E. coli.

Examples of Hsp70 proteins include Hsp72 and Hsc73 from mammalian cells, DnaK from bacteria or mycobacteria such as Mycobacterium leprae, Mycobacterium tuberculosis, and Mycobacterium bovis (such as Bacille-Calmette Guerin; referred to herein as Hsp71), DnaK from E. coli, yeast, and other prokaryotes, and BiP and Grp78. Hsp70 is capable of specifically binding ATP as well as unfolded polypeptides and peptides, and participates in protein folding and unfolding as well as in the assembly and disassembly of protein complexes.

Examples of Hsp60 proteins include Hsp65 from mycobacteria. Bacterial Hsp60 is also commonly known as GroEL. Hsp60 forms large homooligomeric complexes, and appears to play a key role in protein folding. Hsp60 homologues are present in eukaryotic mitochondria and chloroplasts.

Examples of TF55 proteins include Tcpl, TRiC, and thermosome. The proteins typically occur in the cytoplasm of eukaryotes and some archaebacteria, and form multi-membered rings, promoting protein folding. They are also weakly homologous to Hsp60.

Examples of Hsp40 proteins include DnaJ from prokaryotes such as E. coli and mycobacteria and HSJ1, HDJ1, and Hsp40. Hsp40 plays a role as a molecular chaperone in protein folding, thermotolerance and DNA replication, among other cellular activities.

FKBP examples include FKBP12, FKBP13, FKBP25, and FKBP59, Fprl and Nepl. The proteins typically have peptidyl-prolyl isomerase activity and interact with immunosuppressants such as FK506 and rapamycin. The proteins are typically found in the cytoplasm and the endoplasmic reticulum.

Cyclophilin examples include cyclophilins A, B, and C. The proteins have peptidyl-prolyl isomerase activity and interact with the immunosuppressant cyclosporin A.

Hsp20-30 is also referred to as small Hsp. Hsp20-30 is typically found in large homooligomeric complexes or possibly heterooligomeric complexes. An organism or cell type can express several different types of small Hsps. Hsp20-30 interacts with cytoskeletal structures and may play a regulatory role in the polymerization/depoly-merization of actin. Hsp20-30 is rapidly phosphorylated upon stress or exposure of resting cells to growth factors. Hsp20-30 homologues include alpha-crystallin.

ClpP is an E. coli protease involved in degradation of abnormal proteins. Homologues of ClpP are found in chloroplasts. ClpP forms a heterooligomeric complex with ClpA.

GrpE is an E. coli protein of about 20 kDa that is involved in the rescue of stress-damaged proteins as well as the degradation of damaged proteins. GrpE plays a role in the regulation of stress gene expression in E. coli.

Hsp10 examples include GroES and Cpn10. Hsp10 is found in E. coli and in the mitochondria and chloroplasts of eukaryotic cells. Hsp10 forms a seven-membered ring that associates with Hsp60 oligomers. Hsp10 is also involved in protein folding.

Ubiquitin has been found to bind proteins in coordination with the proteolytic removal of the proteins by ATP-dependent cytosolic proteases.

In addition to fill-length stress proteins, any immunostimulatory fragments or derivatives would be useful in the present invention. An immunostimulatory fragment or derivative (e.g., an immunostimulatory fragment of an Hsp) is a fragment or derivative that facilitates an immune response to an antigen. The fragment or derivative can facilitate an immune response in a number of ways. For example, the fragment can induce an immune response that would not otherwise occur or enhance an immune response that would. A number of immunostimulatory fragments have been described. Suitable fragments include, but are not limited to fragments comprising: (a) amino acids 161-370 of mycobacterial Hsp70 (particularly M. tuberculosis Hsp70) (Huang et al., J. Exp. Med. 191:403-408; 2000, U.S. patent application Ser. No. 09/761,534 filed Jan. 16, 2001); (b) the ATPase domain or peptide binding domain of mycobacterial Hsp70 (particularly M. tuberculosis Hsp70) (Young, U.S. Ser. No. 09/025,178 filed Nov. 25, 1997); (c) amino acids 280-385 of murine Hsc70 (the constitutive member of the Hsp70 family) (Udono et al., Int. Immunol. 13: 1233-1242, 2001); (d) amino acids 359-610 of M. tuberculosis Hsp70 (Wand et al., Immunity 15: 971-983, 2001); (e) for (a) to (d), corresponding regions in Hsp70 homologs from other species, and (f) amino acids 1 to 200 of mycobacterial Hsp65 (particularly M. bovis Hsp65) (Chu et al., U.S. Ser. No. 09/613,303 filed Jul. 10, 2000).

The stress proteins useful in the present invention can be obtained from any suitable organism, including, but not limited to: Gram-positive bacteria, Gram-negative bacteria, enterobacteria (e.g., E. coli), mycobacteria (particularly M. leprae, M. tuberculosis, M. vaccae, M. smegmatis, and M. bovis), yeast, Drosophila, and vertebrates (e.g., avians such as chickens, or mammals such as rats, mice, or primates, including humans).

To make a therapeutic (e.g., an immunotherapeutic) composition containing a fusion polypeptide, the polypeptide can be recombinantly produced in bacteria, yeast, plants or plant cells, or animals or animal cells. For example, fusion polypeptides according to the invention can be produced by transformation (transfection, transduction, or infection) of a host cell with a fusion polypeptide-encoding DNA fragment in a suitable expression vehicle. Suitable expression vehicles include plasmids, viral particles, and phage. For insect cells, baculovirus expression vectors are suitable. The entire expression vehicle, or a part thereof, can be integrated into the host cell genome. In some circumstances, it is desirable to employ an inducible expression vector, e.g., the LACSWITCH® Inducible Expression System (Stratagene; La Jolla, Calif.).

Those skilled in the field of molecular biology will understand that any of a wide variety of expression systems can be used to provide the recombinant fusion polypeptide. The precise host cell and vector used is not critical to the invention.

Proteins and polypeptides can also be produced by plant cells. For plant cells, viral expression vectors (e.g., cauliflower mosaic virus and tobacco mosaic virus) and plasmid expression vectors (e.g., Ti plasmid) are suitable. Such cells and vectors are available from a wide range of sources (e.g., the American Type Culture Collection, Manassas, Va.; also, see, e.g., Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1994). The methods of transformation or transfection and the choice of expression vehicle will depend on the host system selected. Transformation and transfection methods are described, e.g., in Ausubel et al., supra. Expression vehicles may be chosen from those provided, e.g., in Pouwels et al., Cloning Vectors: A Laboratory Manual, 1985, Supp. 1987. The host cells harboring the expression vehicle can be cultured in conventional nutrient media adapted as needed for activation or repression of a chosen gene, selection of transformants or amplification of a chosen gene.

Where appropriate or beneficial, the nucleic acid encoding a fusion polypeptide can include a signal sequence for excretion of the fusion polypeptide, e.g., to facilitate isolation of the polypeptide from a cell culture. Specific initiation signals may also be required for efficient translation of inserted nucleic acid sequences. These signals include the ATG initiation codon and adjacent sequences. In some cases, exogenous translational control signals, including, perhaps, the ATG initiation codon, must be provided. Furthermore, the initiation codon must be in phase with the reading frame of the desired coding sequence to ensure translation of the entire insert. These exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic. The efficiency of expression can be enhanced by the inclusion of appropriate transcription or translation enhancer elements (e.g., ones disclosed in Bittner et al., Methods in Enzymol. 153:516, 1987). Additionally the gene sequence can be modified for optimal codon usage in the appropriate expression system, or alternatively, the expression host can be modified to express specific tRNA molecules to facilitate expression of the desired gene.

It would be useful if the fusion polypeptides were soluble under normal physiological conditions. Also within the invention are methods of using fusion proteins (or other configurations of proteins, including covalent and non-covalent complexes and mixtures) in which the stress protein (or an immunostimulatory fragment thereof) and the HBV antigen are fused to (or otherwise associated with) an unrelated third protein or polypeptide to create at least a tripartite protein or mixture of proteins. The third protein may facilitate purification, detection, or solubilization of the fusion or other complex, or it may provide some other function. For example, the expression vector pUR278 (Ruther et al., EMBO J. 2:1791, 1983) can be used to create lacZ fusion proteins. The pGEX vectors can be used to express foreign polypeptides as fusion proteins containing glutathione S-transferase (GST). In general, such fusion proteins are soluble and can be easily purified from lysed cells by adsorption to glutathione-agarose beads, followed by elution in the presence of free glutathione. The pGEX vectors are designed to include thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety.

A fusion protein or covalent complex can be purified using an antibody that specifically binds a portion of the fusion or complex. Alternatively, other properties of the protein included can be exploited for purification (e.g. metal binding). For example, a system described in Janknecht et al. (Proc. Natl. Acad. Sci. USA. 88:8972, 1981) allows for the ready purification of non-denatured fusion proteins expressed in human cell lines. In this system, the gene of interest is subcloned into a vaccinia recombination plasmid such that the gene's open reading frame is translationally fused to an amino-terminal tag consisting of six histidine residues. Extracts from cells infected with recombinant vaccinia virus are loaded onto Ni⁺ nitriloacetic acid-agarose columns, and histidine-tagged proteins are selectively eluted with imidazole-containing buffers. The same procedure can be used for a bacterial culture.

Alternatively, the third protein can be an immunoglobulin Fc domain. Such a fusion protein can be readily purified using an affinity column.

Fusion polypeptides, particularly those containing short antigenic fragments, can also be produced by chemical synthesis (e.g., by the methods described in Solid Phase Peptide Synthesis, 2nd ed., 1984 The Pierce Chemical Co., Rockford, Ill.).

Once isolated, the fusion polypeptide can, if desired, be further purified and/or concentrated, so long as further processing does not impair its ability to elicit (e.g., by inducing or enhancing) an immune response sufficient for implementation of the methods of the invention. A variety of methods for purification and concentration are well known in the art (see, e.g., Fisher, Laboratory Techniques In Biochemistry And Molecular Biology, Work and Burdon, eds., Elsevier, 1980), including ultracentrifugation and/or precipitation (e.g., with ammonium sulfate), microfiltration (e.g., via 0.45 μm cellulose acetate filters), ultrafiltration (e.g., with the use of a sizing membrane and recirculation filtration), gel filtration (e.g., columns filled with Sepharose CL-6B, CL-4B, CL-2B, 6B, 4B or 2B, Sephacryl S-400 or S-300, Superose 6 or Ultrogel A2, A4, or A6; all available from Pharmacia Corp.), fast protein liquid chromatography (FPLC), and high performance liquid chromatography (HPLC).

The polypeptides within the compositions of the invention can include antigenic or immunostimulatory determinants, or the whole protein, of more than one stress protein and/or more than one HBV protein. Optionally, the peptides can include other sequences to which an immune response is desired.

The invention includes immunotherapeutic compositions containing at least one fusion polypeptide as described herein, and, optionally, a pharmaceutically acceptable carrier, such as a diluent, e.g., saline, phosphate buffered saline, or a bicarbonate solution (e.g., 0.24 M NaHCO₃). The carriers used in the composition are selected on the basis of the mode and route of administration, and standard pharmaceutical practice. Suitable pharmaceutical carriers and diluents, as well as pharmaceutical necessities for their use, are described in Remington's Pharmaceutical Sciences. An adjuvant, e.g., a cholera toxin, Escherichia coli heat-labile enterotoxin (LT), liposome, or immune-stimulating complex (ISCOM), can also be included in the immunotherapeutic compositions.

The compositions can be formulated as a solution (suitable for intramuscular, intradermal, or intravenous administration), suspension, suppository, tablet, granules, a powder, a capsule, ointment, or cream. In preparing these compositions, one or more pharmaceutical carriers can be included. Examples of pharmaceutically acceptable carriers or other additives include solvents (e.g., water or physiological saline), solubilizing agents (e.g., ethanol, polysorbates, or Cremophor EL®), agents for rendering isotonicity, preservative, antioxidizing agents, excipients (e.g., lactose, starch, crystalline cellulose, mannitol, maltose, trehalose, calcium hydrogen phosphate, light silicic acid anhydride, or calcium carbonate), binders (e.g., starch, polyvinylpyrrolidone, hydroxypropyl cellulose, ethyl cellulose, carboxy methyl cellulose, or gum arabic), lubricant (e.g., magnesium stearate, talc, or hardened oils), or stabilizers (e.g., lactose, mannitol, maltose, polysorbates, macrogels, or polyoxyethylene-hardened castor oils). If necessary, glycerin, dimethylacetamide, sodium lactate, a surfactant, sodium hydroxide, ethylenediamine, ethanolamine, sodium bicarbonate, arginine, meglumine, or trisaminomethane is added. Biodegradable polymers such as poly-D,L-lactide-co-glycolide or polyglycolide can be used as a bulk matrix if slow release of the composition is desired (see e.g., U.S. Pat. Nos. 5,417,986, 4,675,381, and 4,450,150). As noted above, pharmaceutical preparations such as solutions, tablets, granules or capsules can be formed with these components. If the composition is administered orally, flavorings and colors can be added.

The immunotherapeutic compositions can be administered via any appropriate route, e.g., intravenously, intraarterially, topically, by injection (e.g. intraperitoneally, intrapleurally, subcutaneously, intramuscularly), orally, intradermally, sublingually, intraepidermally, intranasally (e.g., by inhalation), intrapulmonarily, or rectally.

The amount of immunotherapeutic composition administered will depend, for example, on the particular stress protein/antigen composition, whether an adjuvant is co-administered with the composition, the type of adjuvant co-administered, the mode and frequency of administration, and the desired effect (e.g., protection or treatment), as can be determined by one skilled in the art. In general, the immunotherapeutic compositions are administered in amounts ranging between 1 μg and 100 mg per adult human dose. Preferably, between 50 to 10,000 μg (e.g., about 100 to 5000 μg, especially about 500, 1000, 1500 or 2000 μg) of the fusion protein is administered. If adjuvants are administered with the immunotherapeutic, amounts ranging between 1 ng and 100 mg per adult human dose can generally be used. Administration is repeated as necessary, as can be determined by one skilled in the art. For example, a priming dose can be followed by one or more booster doses at weekly or monthly intervals. A booster shot can be given at 3 to 12 weeks after the first immunization, and a second booster can be given at 3 to 12 weeks after the first booster, using the same formulation or a different formulation. Serum, PBLs, or PBMCs, can be taken from the individual for testing the immune response elicited by the immunotherapeutic against the HBV antigen included in the fusion protein. Methods of assaying antibodies or cytotoxic T cells or cytokine-secreting cells against a specific antigen are well known in the art. Additional boosters can be given as needed. By varying the amount of fusion polypeptide in the composition, the immunization protocol can be optimized for eliciting a maximal immune response.

Of course, the polypeptides (alone or as part of a fusion protein) can also be delivered by administering a nucleic acid, such as a viral vector (e.g., a retroviral or adenoviral vector).

The immunotherapeutic of the invention can also be administered in combination with one or more compounds or compositions that have activity against HBV (an HBV antiviral). For example, a patient can first be treated with an HBV antiviral to reduce the severity of the HBV infection (as measured by, for example, reduction or loss of circulating HBe antigen (a marker of HBV replication and high-titre viremia), appearance of anti-HBe antibodies, reduction or disappearance of serum HBV DNA or reduction in alanine aminotransferase (ALT) levels). Once a suitable reduction is achieved, the immunotherapeutic of the invention can then be administered to the patient. Alternatively, the HBV antiviral and the immunotherapeutic can be administered at substantially the same time (keeping in mind that the antiviral and the immunotherapeutic may have different routes of administration), or the immunotherapeutic can be administered first, followed by treatment with the antiviral. Antiviral compounds or compositions suitable for use in such combinations with the immunotherapeutic include, but are not limited to interferon-α2b (Intron A, Schering Plough), pegylated interferon-α2b, and nucleoside analogs such as lamivudine [(-;)-β-L-3′-thia-2 ′,3′-dideoxycytidine or 3TC] (Epivir-HBV, Glaxo Wellcome) and ribavirin (Rebetron™, ICN Pharmaceuticals). There are a number of additional experimental compounds which may be suitable, and these include: hemtricitabine (2′,3′-dideoxy-5′-fluoro-3′-thiacytidine, FTC, coviracil, Triangle Pharmaceuticals), clevudine (2′-fluoro-5-methyl-β-L-arabinofuranosyl uracil, L-FMAU, Triangle), adefovir (9-(2-phosphonylmethyl)-adenine, PMEA, Gilead Sciences), entecavir (Bristol-Myers Squibb), (-;)-beta-D-2, 6-diaminopurine dioxolane (DAPD), β-L-2′,3′-dideoxy-5-fluorocytidine (β-L-FddC), β-L-2′,3′-didehydro-dideoxy-5-fluorocytidine (β-L-Fd4C), and famciclovir.

Claim 1 of 70 Claims

1. A fusion protein comprising (i) a stress protein or an immunostimulatory portion thereof and (ii) a variant of the hepatitis B virus (HBV) core antigen of SEQ ID NQ:2, wherein the variant comprises the amino acid sequence of SEQ ID NO:2 in which at least the isoleucine residue at vosition 97 but not more than 25 of the amino acid residues are substituted, and the fusion protein, when administered to an individual, induces or enhances an immune response against the HBV core antigen.

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