WO2000039301A9 - Methods for using mycobacterium tuberculosis molecules as immunological adjuvants - Google Patents
Methods for using mycobacterium tuberculosis molecules as immunological adjuvantsInfo
- Publication number
- WO2000039301A9 WO2000039301A9 PCT/US1999/030975 US9930975W WO0039301A9 WO 2000039301 A9 WO2000039301 A9 WO 2000039301A9 US 9930975 W US9930975 W US 9930975W WO 0039301 A9 WO0039301 A9 WO 0039301A9
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- Prior art keywords
- antigen
- proteins
- protein
- cells
- coding sequence
- Prior art date
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
- A61K2039/53—DNA (RNA) vaccination
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
- A61K2039/55511—Organic adjuvants
- A61K2039/55544—Bacterial toxins
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
- A61K2039/55511—Organic adjuvants
- A61K2039/55561—CpG containing adjuvants; Oligonucleotide containing adjuvants
Definitions
- the present invention relates to immunological adjuvants.
- it relates to
- Mycobacterium tuberculosis proteins which stimulate interleukin- 12 (IL-12) and interferon- gamma (IFN- ⁇ ) production. These proteins and fragments thereof may be used in combination with an antigen for inducing and/or enhancing an immune response to the antigen. Alternatively, polynucleotides encoding such proteins may be ligated with an antigen coding sequence to produce a fusion protein as an immunogen.
- IL-12 interleukin- 12
- IFN- ⁇ interferon- gamma
- An antigen introduced into the immune system first encounters an antigen presenting cell.
- An antigen presenting cell processes the antigen and presents antigenic fragments to helper T cells (TH), which, in turn, stimulate two types of immune responses; i.e., cell-mediated and humoral immune responses.
- TH respond to antigen stimulation by producing lymphokines which "help" or activate other effector cell types in the immune system.
- TH activate B cells to secrete antibodies which function as the major effector molecule in the humoral immune responses.
- Antibodies neutralize foreign antigens or cooperate with other effector cells in mediating antibody-dependent cellular cytotoxicity.
- TH are distinguished from cytotoxic T lymphocytes and B cells by their cell surface expression of a glycoprotein marker termed CD4.
- CD4 glycoprotein marker
- TH can be divided into two subsets on the basis of their secretory products.
- Type 1 helper T cells IL-2
- IFN- ⁇ interleukin-2
- type 2 helper T cells TH2
- TH I Based on the profile of lymphokine production, TH I appear to be involved in promoting the activation and proliferation of other T cell subsets such as cytotoxic T cells, whereas TH2 specifically regulate B cell proliferation and differentiation, antibody synthesis, and antibody class switching.
- Cytotoxic T cells express the CD8 surface marker. Unlike most TH, these cells display cytolytic activity upon direct contact with target cells, although they are also capable of producing certain lymphokines. In vivo, these cells are particularly important in situations where an antibody response alone is inadequate. There is a preponderance of experimental evidence that cytotoxic T cells rather than B cells and their antibody products o play a principal role in the defense against viral infections, intracellular parasitic infections and cancer.
- An immunological adjuvant is an agent that enhances the immune response to an antigen. While an immunogen is capable of eliciting an immune response, the magnitude of the response is often weak, thus an adjuvant is added as an additive or vehicle to enhance the response.
- An adjuvant may function by different mechanisms, including (1) trapping the antigen in the body to cause a slow release, (2) attracting immune cells to migrate to the 0 injection site, (3) stimulating immune cells to proliferate and to become activated, and (4) improving antigen dispersion in the recipient's body.
- adjuvants A number of agents with diverse chemical properties have been used as adjuvants, including oil emulsions, mineral salts, polynucleotides and natural substances.
- One of the most commonly used adjuvants in laboratory animals is Freund's complete adjuvant, which consists of a water-in-oil emulsion and killed M. tuberculosis.
- Other microorganisms have also been added as immunostimulants to the adjuvant formulation, and they include bacille
- Calmette-Guerin an attenuated Mycobacterium used as a tuberculosis vaccine in certain countries
- Corynebacterium parvum an attenuated Mycobacterium used as a tuberculosis vaccine in certain countries
- Bordatella pertussis specific bacterial components have also been included in adjuvant formulations.
- two bacterial cell wall constituents, lipopolysaccharide and muramyldipeptide have been used as adjuvants to stimulate B cells and macrophages, respectfully.
- alum precipitate is the most widely used adjuvant, which is a suspension of aluminum hydroxide onto which an antigen can be adsorbed.
- the present invention relates to methods of using M. tuberculosis proteins and fragments thereof for inducing and/or enhancing an immune response to an antigen.
- it relates to methods of using M. tuberculosis secretory proteins to stimulate cytokine production, thereby enhancing antigen-specific and antigen non-specific immune responses.
- the invention is based, in part, on Applicants' discovery that five secretory proteins of M. tuberculosis, designated DPV, DPAS, 85A, M ⁇ Z2B and Mtb23, stimulated IL-12 production by macrophages.
- DPV also stimulated the production of IFN- ⁇ .
- a large number of other M. tuberculosis-de ⁇ ved proteins did not exhibit such cytokine-inducing activities.
- IL-12 induces CD4 T cells to develop into THI cells which are particularly important in regulating cytotoxic T cell responses
- these five proteins may be used in combination with an antigen to enhance cell-mediated immune responses to the antigen.
- these proteins may stimulate diverse immune cell types, including macrophages and natural killer cells.
- It is an object of the invention to prepare a vaccine or therapeutic composition comprising an antigen plus one or more of the molecules disclosed herein for enhancing an immune response to the antigen.
- a fusion protein between an antigen and one or more of the molecules disclosed herein as an immunogen may be produced by constructing a fusion polynucleotide which is expressed in a host cell followed by protein purification.
- the polynucleotide may be directly administered in a subject as a DNA vaccine to induce and/or enhance an immune response.
- Such cell culture may be used in subsequent adoptive immunotherapy.
- Figure 2 Nucleotide sequence (SEQ ID NO:3) and deduced amino acid sequence (SEQ ID NO:4) of M. tuberculosis antigen DPAS.
- Figure 3 Recombinant DPV and DPAS proteins stimulated IL-12 production.
- M. tuberculosis-de ⁇ ved recombinant proteins and a fusion protein of three M. tuberculosis antigens (Erdl4-DPV-MTI) were incubated with mouse macrophage cell line RAW264.7.
- the proteins were added at 5 and 20 ⁇ g. IL-12 (p40) concentrations were measured by an ELISA.
- Figure 4A & B o Nucleotide sequence (SEQ ID NO:5, Figure 4A) and deduced amino acid sequence (SEQ ID NO: 6, Figure 4B) of M. tuberculosis antigen 85A.
- FIG. 1 Nucleotide sequence (SEQ ID NO:9) and deduced amino acid sequence (SEQ ID NO: 10) of M. tuberculosis antigen Mtb23.
- Mtb23 is shown as MtbLP23.
- the proteins were added at lO ⁇ g/ml.
- Polymixin B was added to inhibit contaminating endotoxin. While polymixin B abrogated the effects of LPS, it did not affect the other molecules.
- the coding sequences for five Mycobacterium molecules were isolated, and their nucleotide and deduced amino acid sequences characterized.
- any nucleotide sequence which encodes the amino acid sequence of the proteins of the invention can be used to generate recombinant molecules which direct the expression of their coding sequences.
- the invention provides purified polynucleotides containing at least 15 nucleotides (i.e., a hybridizable portion) of a coding sequence; in other embodiments, the polynucleotides contain at least 25 (continuous) nucleotides, 50 nucleotides, 100 nucleotides, 150 nucleotides, or 200 nucleotides of a coding sequence, or a full-length coding sequence.
- Nucleic acids can be single or double stranded. Such nucleic acids also encode variant forms of the molecule disclosed herein which retain their IL-12 stimulatory activities.
- the invention also relates to polynucleotides complementary to the foregoing sequences and polynucleotides hybridizable to such complementary sequences.
- polynucleotides are provided which comprise a sequence complementary to at least 10, 25, 50, 100, or 200 nucleotides or the entire coding region of a sequence.
- a polynucleotide which is hybridizable to a DPV (SEQ ID NO: 1), DPAS (SEQ ID NO: 3), 85A (SEQ ID NO:5), Mtb22B (SEQ ID NO:7) or Mtb23 (SEQ ID NO:9) coding sequence or its complementary sequence under conditions of low stringency is provided.
- Hybridizations are carried out in the same solution with the following modifications: 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 ⁇ g/mt salmon sperm DNA, 10% (wt/vol) dextran sulfate, and 5-20 X 10 6 cpm 32 P-labeled probe is used. Filters are incubated in hybridization mixture for 18-20 h at 40°C, and then washed for 1.5 h at 55 °C in a solution containing 2X SSC, 25 mM Tris-HCl (pH 7.4), 5 mM EDTA, and 0.1% SDS. The wash solution is replaced with fresh solution and incubated an additional 1.5 h at 60° C.
- Filters are blotted dry and exposed for autoradiography. If necessary, filters are washed for a third time at 65-68 °C and re- exposed to film.
- Other conditions of low stringency which may be used are well known in the art (e.g., as employed for cross-species hybridizations).
- a polynucleotide which is hybridizable to a coding sequence or its complementary sequence under conditions of high stringency is provided.
- procedures using such conditions of high stringency are as follows: Prehybridization of filters containing DNA is carried out for 8 h to overnight at 65 °C in buffer composed of 6X SSC, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA, and 500 ⁇ g/m ⁇ denatured salmon sperm DNA.
- Filters are hybridized for 48 h at 65° C in prehybridization mixture containing 100 ⁇ g/mf denatured salmon sperm DNA and 5-20 X 10 6 cpm of 32 P-labeled probe. Washing of filters is done at 37°C for 1 h in a solution containing 2X SSC, 0.01% PVP, 0.01% Ficoll, and 0.01% BSA. This is followed by a wash in 0.1X SSC at 50°C for 45 min before auto radiography. Other conditions of high stringency which may be used are well known in the art.
- a polynucleotide which is hybridizable to a coding sequence or its complementary sequence under conditions of moderate stringency is provided.
- procedures using such conditions of moderate stringency are as follows: Filters containing DNA are pretreated for 6 h at 55 °C in a solution containing 6X SSC, 5X Denhart's solution, 0.5% SDS and 100 ⁇ g/mtf denatured salmon sperm DNA. Hybridizations are carried out in the same solution and 5-20 X 10 6 0 cpm 32 P-labeled probe is used.
- Filters are incubated in hybridization mixture for 18-20 h at 55°C, and then washed twice for 30 minutes at 60°C in a solution containing IX SSC and 0.1% SDS. Filters are blotted dry and exposed for autoradiography. Other conditions of moderate stringency which may be used are well-known in the art. Washing of filters is 5 done at 37°C for 1 h in a solution containing 2X SSC, 0.1% SDS.
- Polynucleotides hybridizable to a complementary sequence of a coding sequence may be naturally-occurring or products of in vitro mutagenesis.
- these polynucleotides encode protein products that stimulate IL-12 and/or IFN- ⁇ production.
- a polynucleotide which encodes a polypeptide of c the invention, a mutant polypeptide, peptide fragments thereof, fusion proteins containing one or more of them and a heterologous polypeptide, or functional equivalents thereof may be used to generate recombinant nucleic acid molecules that direct the expression of the protein, peptide fragments, fusion proteins or a functional equivalent thereof, in appropriate host cells.
- DNA sequences which encode substantially the same or a functionally equivalent amino acid sequence may be used in the practice of the invention for the expression of the proteins.
- Altered DNA sequences which may be used in accordance with the invention include deletions, additions or substitutions of different nucleotide residues resulting in a sequence that encodes the same or a functionally equivalent gene product. Additional residues may be added to the amino or carboxyl terminus or both without affecting the activities of a gene product.
- the gene product itself may contain deletions, additions or substitutions of amino acid residues within a sequence, which result in a silent change thus producing a functionally equivalent protein.
- Such conservative amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues involved.
- negatively charged amino acids include aspartic acid and glutamic acid
- positively charged amino acids include lysine, histidine and arginine
- amino acids with uncharged polar head groups having similar hydrophilicity values include
- amino acids with nonpolar head groups include alanine, valine, isoleucine, leucine, phenylalanine, proline, methionine, tryptophan.
- DNA sequences of the invention may be engineered in order to alter a coding
- mutations may be introduced using techniques which are well known in the art, e.g., site-directed mutagenesis, to insert new restriction sites, to alter glycosylation patterns, phosphorylation, etc.
- the coding sequence of the protein could be synthesized in whole or in part, using chemical methods well known in the art. See, e.g., Caruthers et al, 1980, Nuc. Acids Res. Symp. Ser. 7:215-233; Crea and Horn, 180, Nuc. Acids Res. 9(10):233 ⁇ ; Matteucci and Caruthers, 1980, Tetrahedron Letter 27:719; and Chow and Kempe, 1981, Nuc. Acids Res. 9(72 :2807-2817 '.
- the protein itself could be produced using chemical methods to synthesize an amino acid sequence in whole or in part.
- peptides can be synthesized by solid phase techniques, cleaved from the resin, and purified by preparative high performance liquid chromatography. (See
- composition of the synthetic polypeptides may be confirmed by amino acid analysis or sequencing (e.g., the Edman degradation procedure; see Creighton,
- the derivative or analog is functionally active, i.e., capable of exhibiting one or more functional activities associated with a full-length, wild-type protein.
- such derivatives 5 or analogs stimulate IL-12 and/or IFN- ⁇ production.
- a polypeptide containing at least 10 (continuous) amino acids is provided.
- the polypeptide may contain at least 20 or 50 amino acids of the proteins disclosed herein. In specific embodiments, such polypeptides do not contain more than 100, 150 or 200 amino acids.
- Derivatives or analogs include but are not limited to molecules comprising regions that are substantially homologous to the proteins of the invention or fragments thereof (e.g., in various embodiments, at least 60% or 70% or 80% or 90% or 95% identity over an amino acid sequence of identical size or when compared to an aligned sequence in which the alignment is done by a computer homology program known in the art) or product encoded by a polynucleotide is capable of hybridizing to a naturally-occurring coding sequence, under
- the derivatives and analogs of the invention can be produced by various methods known in the art.
- the manipulations which result in their production can occur at the nucleic acid or protein level.
- a cloned coding sequence can be modified by
- the coding sequence can be mutated in vitro or in vivo, to create and/or destroy translation, initiation, and/or termination sequences, or to create variations in coding regions and/or form new restriction endonuclease sites or destroy preexisting ones, to facilitate further in vitro modification.
- Any technique for mutagenesis known in the art can be used, including but not limited to, chemical mutagenesis, in vitro site-directed 5 mutagenesis (Hutchinson, C, et al., 1978, J. Biol. Chem 253:6551), use of TAB® linkers
- Manipulations may also be made at the protein level. Included within the scope of the invention are protein fragments or other derivatives or analogs which are differentially modified during or after translation, e.g., by glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to a cytokine or an antigen. Any of numerous chemical modifications may be carried out by known techniques, including but not limited to specific chemical cleavage by 5 cyanogen bromide, trypsin, chymotrypsin, papain, V8 protease, NaBH 4 ; acetylation, formylation, oxidation, reduction; metabolic synthesis in the presence of tunicamycin; etc.
- analogs and derivatives can be chemically synthesized.
- a peptide corresponding to a portion of a protein disclosed herein which comprises the desired domain or which mediates the desired cytokine stimulatory activities can be synthesized by use of a peptide synthesizer.
- nonclassical amino acids or chemical amino acid analogs can be introduced as a substitution or addition into the sequence.
- Non- classical amino acids include but are not limited to the D-isomers of the common amino acids, ⁇ -amino isobutyric acid, 4-aminobutyric acid, Abu, 2-amino butyric acid, ⁇ -Abu, e-Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric acid, 3 -amino propionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, cysteic acid, t- butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, ⁇ -alanine, fluoro-amino acids, designer amino acids such as ⁇ -methyl amino acids, C ⁇ -methyl amino acids, N ⁇ - methyl amino acids, and amino acid analogs in general.
- the amino acid can be D (dextrorotary) or L (levorotary).
- the derivative is a chimeric, or fusion protein containing one or more proteins of the invention or a fragment thereof (preferably consisting of at least a domain or motif of the protein, or at least 10 amino acids of the protein) joined at its amino- or carboxy-terminus via a peptide bond to an amino acid sequence of a different protein.
- such a chimeric protein is produced by recombinant expression of a nucleic acid encoding the protein (comprising a coding sequence joined in- frame to a coding sequence for a different protein).
- Such a chimeric product can be made by ligating the appropriate nucleic acid sequences encoding the desired amino acid sequences to each other by methods known in the art, in the proper coding frame, and expressing the chimeric product by methods commonly known in the art.
- a chimeric product may be made by protein synthetic techniques, e.g., by use of a peptide synthesizer. Chimeric genes comprising portions of a coding sequence fused to any heterologous protein-encoding sequences may be constructed.
- the derivative is a molecule comprising a region of homology with a protein disclosed herein.
- a first protein region can be considered "homologous" to a second protein region when the amino acid sequence of the first region is at least 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, or 95% identical, when compared to any sequence in the second region of an equal number of amino acids as the number contained in the first region or when compared to an aligned sequence of the second region that has been aligned by a computer homology program known in the art.
- the nucleotide sequence coding for it, or a functional equivalent is inserted into an appropriate expression vector, i.e., a vector which contains the necessary elements for the transcription and translation of the inserted coding sequence.
- an appropriate expression vector i.e., a vector which contains the necessary elements for the transcription and translation of the inserted coding sequence.
- the gene products as well as host cells or cell lines transfected or transformed with recombinant expression vectors can be used for a variety of purposes. Methods which are well known to those skilled in the art can be used to construct expression vectors containing a coding sequence for the proteins disclosed herein and appropriate transcriptional/translational control signals. These methods include in vitro recombinant DNA techniques, synthetic techniques and in vivo recombination/genetic recombination.
- microorganisms such as bacteria transformed with recombinant bacteriophage DNA, plasmid DNA, or cosmid DNA expression vectors containing a coding sequence; yeast transformed with recombinant yeast expression vectors containing a coding sequence; insect cell systems infected with 5 recombinant virus expression vectors (e.g.. baculovirus) containing a coding sequence; plant cell systems infected with recombinant virus expression vectors (e.g.. cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e ⁇ g., Ti plasmid) containing a coding sequence; or animal cell systems. ° The expression elements of these systems vary in their strength and specificities.
- any of a number of suitable transcription and translation elements may be used in the expression vector.
- inducible promoters 5 such as pL of bacteriophage ⁇ , plac, ptrp, ptac (ptrp-lac hybrid promoter; cytomegalovirus promoter) and the like may be used;
- promoters such as the baculovirus polyhedron promoter may be used;
- promoters derived from the genome of plant cells e.g., heat shock promoters; the promoter o for the small subunit of RUBISCO; the promoter for the chlorophyll ⁇ / ⁇ binding protein
- plant viruses e.g..
- the 35S RNA promoter of CaMV; the coat protein promoter of TMV may be used; when cloning in mammalian cell systems, promoters derived from the genome of mammalian cells (e.g.. metallothionein promoter) or from mammalian viruses (e.g.. the adenovirus late promoter; the vaccinia virus 7.5K promoter) may be used; when generating cell lines that contain multiple copies of a coding sequence, SV40-, BPV- and EBV-based vectors may be used with an appropriate selectable marker.
- promoters derived from the genome of mammalian cells e.g.. metallothionein promoter
- mammalian viruses e.g. the adenovirus late promoter
- the vaccinia virus 7.5K promoter e.g. the adenovirus late promoter
- vaccinia virus 7.5K promoter e.g. the vaccinia virus 7.5K promoter
- vectors may be advantageously selected depending upon the use intended for the expressed products. For example, when large quantities of the proteins are to be produced for the generation of antibodies or as therapeutic compositions, vectors which direct the expression of high levels of fusion protein products that are readily purified may be desirable.
- vectors include, but are not limited to, the E. coli expression vector pUR278 (Ruther et al., 1983, ⁇ MBO J. 2:1791), in which a coding sequence may be ligated into the vector in frame with the lacZ coding region so that a hybrid protein is produced; pIN vectors (Inouye & Inouye, 1985, Nucleic acids Res.
- pG ⁇ X vectors may also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST).
- GST glutathione S-transferase
- fusion proteins are soluble and can be purified easily from lysed cells by adsorption to glutathione-agarose beads followed by 0 elution in the presence of free glutathione.
- the pG ⁇ X vectors are designed to include thrombin or factor Xa protease cleavage sites so that the cloned polypeptide of interest can be released from the GST moiety.
- p ⁇ T-17b plasmid is used in BL21 pLysE E. coli to express proteins with six histidine residues at the amino terminus which can be purified by Ni-NTA agarose affinity columns. 5
- a recombinant protein Once a recombinant protein is expressed, it can be identified by assays based on the physical or functional properties of the product, including radioactive labelling of the ° product followed by analysis by gel electrophoresis, immunoassay, ELISA, bioassays, etc.
- the encoded protein may be isolated and purified by standard methods including chromatography (e.g., ion exchange, affinity, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique 5 for the purification of proteins.
- the functional properties may be evaluated using any suitable assay such as stimulation of IL-12 and IFN- ⁇ production.
- the proteins disclosed herein are at least 80% purified from other proteins. It is more preferred that they are at least 90% purified. For in vivo Q administration, it is preferred that the proteins are greater than 95% purified.
- native proteins can be purified from natural sources, by standard methods such as those described above (e.g., immunoaffinity purification).
- the proteins, whether produced by recombinant DNA techniques or by chemical synthetic methods or by purification of native proteins include but are not limited to those containing, as a primary amino acid sequence, all or part of the amino acid sequences substantially as depicted in Figures 1, 2, 4, 5 and 6 (SEQ ID NOS: 2, 4, 6, 8 and 10), as well as fragments and other derivatives, and analogs thereof, including proteins homologous thereto. 5.5. USES OF THE CODING SEQUENCES
- the coding sequence for the proteins disclosed herein may be used to encode a protein product for use as an immunological adjuvant to induce and/or enhance immune responses to an antigen.
- such coding sequence may be ligated with a coding sequence of an antigen to construct a fusion polynucleotide.
- a fusion polynucleotide may also be used to express a recombinant protein for use as an immunogen.
- the polynucleotide may be used in vivo as a DNA vaccine (United States Patent Nos. 5,589,466; 5,679,647; 5,703,055).
- the polynucleotide expresses n its encoded protein in a recipient to directly induce an immune response
- a therapeutic composition comprises a coding sequence that is part of an expression vector that also encodes an immunogenic protein or a fragment thereof.
- a polynucleotide contains a promoter operably linked to the coding region, said promoter being inducible or constitutive, and, optionally, tissue-specific.
- a polynucleotide contains a coding sequence flanked by regions that promote homologous recombination at a desired site in the genome, thus providing for intrachromosomal expression of the coding sequence (Koller and Smithies, 1989, Proc. Natl. Acad. Sci.
- nucleic acid into a subject may be either direct, in which case the subject is directly exposed to the nucleic acid or nucleic acid-carrying vector, or indirect, in which case, cells are first transformed with the nucleic acid in vitro, then transplanted into the subject. These two approaches are known, respectively, as in vivo or ex vivo gene 5 transfer.
- the nucleic acid is directly administered in vivo, where it is expressed to produce the encoded product.
- This can be accomplished by any of numerous methods known in the art, e.g., by constructing it as part of an appropriate nucleic acid Q expression vector and administering it so that it becomes intracellular, e.g., by infection using a defective or attenuated retroviral or other viral vector (see U.S. Patent No.
- microparticle bombardment e.g., a gene gun; Biolistic, Dupont
- coating lipids or cell-surface receptors or transfecting agents, encapsulation in liposomes, microparticles, or microcapsules, or by administering it in linkage to a peptide which is known to enter the nucleus, by administering it in linkage to a ligand subject to receptor-mediated endocytosis (see e.g., Wu and Wu, 1987, J. Biol. Chem. 262:4429-4432) which can be used to target cell types specifically expressing the receptors, etc.
- a nucleic acid-ligand complex can be formed in which the ligand comprises a fusogenic viral peptide to disrupt endosomes, allowing the nucleic acid to avoid lysosomal degradation.
- the nucleic acid can be targeted in vivo for cell specific uptake and expression, by targeting a specific receptor (see, e.g., PCT Publications WO 92/06180 dated April 16, 1992; WO 92/22635 dated December 23, 1992; WO92/20316 dated November 26, 1992; WO93/14188 dated July 22, 1993; WO 93/20221 dated October 14, 1993).
- the nucleic acid can be introduced intracellularly and incorporated within host cell DNA for expression, by homologous recombination (Koller and Smithies, 1989, Proc. Natl. Acad. Sci. USA 86:8932-8935; Zijlstra et al., 1989, Nature 342:435-438).
- a viral vector such as a retroviral vector can be used (see Miller et al., 1993, Meth. Enzymol. 217:581-599).
- Retroviral vectors have been modified to delete retroviral sequences that are not necessary for packaging of the viral genome and integration into host cell DNA.
- An adjuvant protein coding sequence and an antigen coding sequence are cloned into the vector, which facilitates delivery of the genes into a recipient. More detail about retroviral vectors can be found in Boesen et al., 1994, Biotherapy 6:291- 302, which describes the use of a retroviral vector to deliver the mdrl gene to hematopoietic stem cells in order to make the stem cells more resistant to chemotherapy.
- Adenoviruses are other viral vectors that can be used in gene therapy. Adenoviruses are especially attractive vehicles for delivering genes to respiratory epithelia. Adenoviruses naturally infect respiratory epithelia where they cause a mild disease. Other targets for adenovirus-based delivery systems are liver, the central nervous system, endothelial cells, and muscle. Adenoviruses have the advantage of being capable of infecting non-dividing cells. Adeno-associated virus (AAV) has also been proposed for use in in vivo gene transfer (Walsh et al., 1993, Proc. Soc. Exp. Biol. Med. 204:289-300.
- AAV Adeno-associated virus
- Another approach involves transferring a construct to cells in tissue culture by such methods as electroporation, lipofection, calcium phosphate mediated transfection, or viral infection.
- the method of transfer includes the transfer of a selectable marker to the cells. The cells are then placed under selection to isolate those cells that have taken up and are expressing the transferred gene. Those cells are then delivered to a subject.
- the nucleic acid is introduced into a cell prior to administration in vivo of the resulting recombinant cell.
- introduction can be carried out by any method known in the art, including but not limited to transfection, electroporation, microinjection, infection with a viral or bacteriophage vector containing the nucleic acid sequences, cell fusion, chromosome-mediated gene transfer, microcell-mediated gene transfer, spheroplast fusion, etc.
- Numerous techniques are known in the art for the introduction of foreign genes into cells (see e.g., Loeffler and Behr, 1993, Meth. Enzymol. 217:599-618; Cohen et al, 1993, Meth. Enzymol. 217:618-644; Cline, 1985, Pharmac. Ther. 29:69-92) and may be used in accordance with the present invention.
- Purified or partially purified proteins disclosed herein or fragments thereof may be mixed with an antigen for the preparation of a vaccine or therapeutic composition.
- such proteins may be further suspended in an oil emulsion to cause a slower release of the proteins in vivo upon injection.
- the optimal ratios of each component in the formulation may be determined by techniques well known to those skilled in the art.
- an antigen is any molecule that induces an immune response against it.
- it includes, but is not limited to, a molecule derived from an infectious agent such as a bacterium, a virus, a fungus, a protozoan and a parasite.
- a tumor antigen which is preferentially expressed in certain tumor types is also within the scope of the invention.
- a tumor antigen may be of cellular origin or encoded by a virus.
- Such a formulation may be administered to a subject per se or in the form of a pharmaceutical or therapeutic composition.
- Pharmaceutical compositions comprising the proteins may be manufactured by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes. 5
- Pharmaceutical compositions may be formulated in conventional manner using one or more physiologically acceptable carriers, diluents, excipients or auxiliaries which facilitate processing of the protein or biologically active peptides into preparations which can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.
- the proteins may be formulated as solutions, gels, ointments, creams, suspensions, etc. as are well-known in the art.
- Systemic formulations include those designed for administration by injection, e.g. subcutaneous, intravenous, intramuscular, intrathecal or intraperitoneal injection, as well as those designed for transdermal, transmucosal, oral or pulmonary administration. 5
- the proteins may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiological saline buffer.
- the solution may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
- the proteins may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
- penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.
- compositions can be readily formulated by combining the proteins with pharmaceutically acceptable carriers well known in the art.
- pharmaceutically acceptable carriers well known in the art.
- Such carriers enable the proteins to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a subject to be treated.
- suitable excipients include fillers such as sugars, such as lactose, sucrose, mannitol and sorbitol; cellulose preparations 0 such as maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP); granulating agents; and binding agents.
- disintegrating agents may be added, such as the cross-linked polyvinylpyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate. 5
- solid dosage forms may be sugar-coated or enteric-coated using standard techniques.
- suitable carriers, excipients or diluents include water, glycols, oils, alcohols, etc. ° Additionally, flavoring agents, preservatives, coloring agents and the like may be added.
- the proteins may take the form of tablets, lozenges, etc. formulated in conventional manner.
- the proteins for use according to the present 5 invention are conveniently delivered in the form of an aerosol spray from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
- a suitable propellant e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
- the dosage unit may be determined by providing a valve to Q deliver a metered amount.
- Capsules and cartridges of e.g. gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.
- the proteins may also be formulated in rectal or vaginal compositions such as suppositories or retention enemas, e.g, containing conventional suppository bases such as cocoa butter or other glycerides.
- the proteins may also be formulated as a depot preparation. Such long acting formulations may be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection.
- the proteins may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.
- Liposomes and emulsions are well known examples of delivery vehicles that may be used to deliver an
- the proteins may be delivered using a sustained-release system, such as semipermeable matrices of solid polymers containing the therapeutic agent.
- sustained-release materials have been established and are well known by those skilled in the art.
- Sustained-release capsules may, depending on their chemical nature, release the proteins for a few weeks up to over 100 days.
- additional strategies for protein stabilization may be employed.
- An effective dose can be estimated initially from in vitro assays.
- a ° dose can be formulated in animal models to achieve an induction or enhancement of an immune response using techniques that are well known in the art.
- Dosage amount and interval may be adjusted individually.
- a pharmaceutical or vaccine 5 composition contains an adjuvant polypeptide in the range of 1 ⁇ g to 5mg in a volume of 0.1 to 5 ml.
- the amount of each protein administered will, of course, be dependent on the subject being treated, on the subject's weight, the manner of administration and the judgment of the physician. o
- the invention having been described, the following examples are offered by way of illustration and not limitation.
- E. coli strain BL21 (DE3) pLysE (Novagen) was used for high level expression.
- the recombinant (His-Tag) antigens were purified from the soluble supernatant or the insoluble inclusion body of 500 mil of IPTG induced batch cultures by affinity chromatography using the one step QIAexpress Ni-NTA agarose matrix (QIAGEN,
- the inclusion bodies were washed three times in 1% CHAPS in 10 mM Tris-HCl
- Bound protein was eluted with 30 mi of 150 mM immidazole in wash buffer and 5 mi fractions collected. Fractions containing each recombinant antigen were pooled, dialyzed against 10 mM Tris-HCl (pH 8.0) bound one more time to the Ni-NTA matrix, eluted and o dialyzed in 10 mM Tris-HCl (pH 7.8). The yield of recombinant protein varied from 25-150 mg per liter of induced bacterial culture with greater than 98% purity. Recombinant proteins were assayed for endotoxin contamination using the Limulus assay (BioWhittaker) and were shown to contain ⁇ 10 E.U./mg. 5
- C3H SCID mice were maintained at the animal facilities.
- RAW264 cell line was maintained in RPMI containing 10% FBS, 20 ⁇ M 2-mercaptoethanol and 50 ⁇ g/md gentinimicin.
- One mi of cells (at a density of 5 x 10 5 cells per mi) was plated per well using a 24 well plate format and allowed to adhere overnight.
- the media and nonadherent cells were removed by aspiration and the cells replenished with 1.0 ml of media containing 1.2% DMSO and allowed to differentiate for 8 hr.
- the cells were subsequently activated with rIFN- ⁇ (20 ng/mi; R&D Lot#RY 147101) and incubated for about 14 hrs (overnight).
- Recombinant proteins were added at the desired concentration (usually 20 ⁇ g/mi) in the presence or absence of 10 ⁇ g/mi polymixin B.
- Lipopolysaccharide (LPS) was used as a positive control at a concentration of 1 ⁇ g/mi.
- ELISA plates (Corning) were coated with 50 ⁇ l/well of 5 ⁇ g/mi anti-mouse IL-12 0 monoclonal antibody C17.15.10.12, in 0.1 M bicarbonate coating buffer, pH9.6 and incubated for 4 hr at room temperature. Plate contents were shaken out and blocked with PBS-0.05% Tween, 1.0% BSA (200 ⁇ l/well) overnight at 4°C and washed for 6 times in PBS-0.1% Tween. Standards (recombinant mouse IL-12 p40) and supernatant samples from the stimulated RAW 264 cells were diluted in PBS-0.05% Tween, followed by the addition 5 of 0.1% BSA and incubated overnight at 4°C.
- IFN- ⁇ ASSAY o Spleens from C3H/HeJ SCID mice were removed asceptically and single cell suspension prepared in complete RPMI following lysis of red blood cells. 100 ⁇ l of cells (2xl0 5 cells) were plated per well in a 96-well flat bottom microtiter plate. Cultures were stimulated with recombinant proteins for 24h and the supernatant assayed for IFN- ⁇ .
- the levels of supernatant IFN- ⁇ were analyzed by sandwich ELISA, using antibody pairs and procedures available from PharMingen. Standard curves were generated using recombinant mouse cytokines.
- ELISA plates (Corning) were coated with 50 ⁇ l/well (1 ⁇ g/mi, in 0.1 M bicarbonate coating buffer, pH9.6) of a cytokine capture rat anti-mouse IFN- ⁇ monoclonal antibody (PharMingen; Cat. # 18181D), and incubated for 4 hr at room temperature.
- M. tuberculosis antigens were expressed as recombinant proteins in E. coli. After purification, these proteins were tested for their ability to stimulate the production of interleukin- 12 (IL-12) by a mouse macrophage cell line, RAW264. The concentrations of IL-12 in RAW264 culture supernatants were ° measured by an ELISA assay using an antibody to detect the p40 component of the IL-12 heterodimer.
- IL-12 interleukin- 12
- DPV SEQ ID NO:2
- DPAS SEQ ID NO:4
- Figure 5 The stimulatory activities of these proteins were not affected by the inclusion of polymixin B in the cultures, which abrogated the IL-12 stimulatory activities of lipopolysaccharide, a polyclonal B cell mitogen.
- Both DPV and DPAS are secretory proteins of M. tuberculosis. 0 A polynucleotide was constructed to ligate the coding sequences of three M. tuberculosis-de ⁇ ved antigens into a fusion sequence.
- the fusion product (ErD14-DPV- MTI) encoded by such polynucleotide also exhibited IL-12 stimulatory activities.
- DPV was the only IL-12-stimulator when used as an individual antigen. Therefore, the inclusion of DPV in a larger fusion protein did not affect its IL-12 stimulatory activities.
- M. tuberculosis antigens identified three more proteins as capable of stimulating IL-12 production. These antigens are referred to as 85A (SEQ ID NO:6)( Figure 4A and B), Mtb22B (SEQ ID NO:8)( Figure 5) and Mtb23 (SEQ ID NO:10)( Figure 6).
- Recombinant 85A, Mtb22B and Mtb23 stimulated the production of IL- 12 by RAW264 cells in the presence of polymixin B ( Figure 7).
- DPV was further tested for its ability to stimulate IFN- ⁇ production by splenocytes of C3H SCID mice. DPV induced 12.519 ng/mi of IFN- ⁇ as measured by ELISA, whereas total M.
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Abstract
Description
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Priority Applications (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
AU25945/00A AU2594500A (en) | 1998-12-24 | 1999-12-21 | Methods for using mycobacterium tuberculosis molecules as immunological adjuvants |
EP99968552A EP1141312A2 (en) | 1998-12-24 | 1999-12-23 | Methods for using mycobacterium tuberculosis molecules as immunological adjuvants |
CA002356669A CA2356669A1 (en) | 1998-12-24 | 1999-12-23 | Methods for using mycobacterium tuberculosis molecules as immunological adjuvants |
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US22041698A | 1998-12-24 | 1998-12-24 | |
US09/220,416 | 1998-12-24 |
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WO2000039301A2 WO2000039301A2 (en) | 2000-07-06 |
WO2000039301A3 WO2000039301A3 (en) | 2001-05-03 |
WO2000039301A9 true WO2000039301A9 (en) | 2001-10-18 |
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PCT/US1999/030975 WO2000039301A2 (en) | 1998-12-24 | 1999-12-23 | Methods for using mycobacterium tuberculosis molecules as immunological adjuvants |
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EP (1) | EP1141312A2 (en) |
AU (1) | AU2594500A (en) |
CA (1) | CA2356669A1 (en) |
WO (1) | WO2000039301A2 (en) |
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GB0003082D0 (en) * | 2000-02-10 | 2000-03-29 | Glaxo Group Ltd | Vaccine |
AU2001271963A1 (en) * | 2000-07-10 | 2002-01-21 | Colorado State University Research Foundation | Mid-life vaccine and methods for boosting anti-mycobacterial immunity |
NZ519667A (en) | 2002-06-19 | 2005-02-25 | Univ Massey | A putative 23kDa lipoprotein from Mycobacterium paratuberculosis and its use in Johne's disease |
DE60325586D1 (en) * | 2003-08-22 | 2009-02-12 | Axenoll Ag | Mycobacterial protein antigens for cancer therapy and vaccination |
JP4838259B2 (en) | 2004-11-16 | 2011-12-14 | クルセル ホランド ベー ヴェー | Multivalent vaccine containing recombinant viral vectors |
US9297803B2 (en) | 2006-11-01 | 2016-03-29 | Immport Therapeutics, Inc. | Compositions and methods for immunodominant antigens |
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HU225979B1 (en) * | 1995-09-01 | 2008-02-28 | Corixa Corp | Compounds and methods for immunotherapy and diagnosis of tuberculosis |
US6290969B1 (en) * | 1995-09-01 | 2001-09-18 | Corixa Corporation | Compounds and methods for immunotherapy and diagnosis of tuberculosis |
ATE370236T1 (en) * | 1997-04-02 | 2007-09-15 | Statens Seruminstitut | NUCLEIC ACID AND POLYPEPTIDE FRAGMENTS OF M. TUBERCULOSIS |
-
1999
- 1999-12-21 AU AU25945/00A patent/AU2594500A/en not_active Abandoned
- 1999-12-23 WO PCT/US1999/030975 patent/WO2000039301A2/en not_active Application Discontinuation
- 1999-12-23 EP EP99968552A patent/EP1141312A2/en not_active Withdrawn
- 1999-12-23 CA CA002356669A patent/CA2356669A1/en not_active Abandoned
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CA2356669A1 (en) | 2000-07-06 |
AU2594500A (en) | 2000-07-31 |
EP1141312A2 (en) | 2001-10-10 |
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