WO2012140424A2 - Composition - Google Patents

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Publication number
WO2012140424A2
WO2012140424A2 PCT/GB2012/050802 GB2012050802W WO2012140424A2 WO 2012140424 A2 WO2012140424 A2 WO 2012140424A2 GB 2012050802 W GB2012050802 W GB 2012050802W WO 2012140424 A2 WO2012140424 A2 WO 2012140424A2
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WIPO (PCT)
Prior art keywords
antigen
composition
toxin
immune response
protein
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Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
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PCT/GB2012/050802
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WO2012140424A3 (fr
Inventor
Guillaume Stewart-Jones
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Oxford University Innovation Ltd
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Oxford University Innovation Ltd
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Publication of WO2012140424A3 publication Critical patent/WO2012140424A3/fr
Anticipated expiration legal-status Critical
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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
    • 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
    • A61K39/12—Viral antigens
    • 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
    • A61K39/12—Viral antigens
    • A61K39/21—Retroviridae, e.g. equine infectious anemia virus
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • 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
    • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
    • 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
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins
    • A61K2039/6037—Bacterial toxins, e.g. diphteria toxoid [DT], tetanus toxoid [TT]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16111—Human Immunodeficiency Virus, HIV concerning HIV env
    • C12N2740/16122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16111—Human Immunodeficiency Virus, HIV concerning HIV env
    • C12N2740/16134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • the present invention relates to a composition for use in eliciting an immune response.
  • the invention relates to an immunogenic composition that focuses a B-cell immune response to a specific region of an antigen.
  • the invention also relates to the use of immunogenic compositions and to methods of eliciting immunogenic responses.
  • Immunisation of a human or non-human animal with a protein antigen generally results in a very polyclonal antibody response directed to many of the exposed protein surfaces.
  • a polyclonal response is a natural response which ensures that an antigen is recognised by different antibodies which recognise different epitopes on the antigen.
  • a few rare individuals do have a response that produces broadly neutralising sera, and in these cases the antibody response is more focussed in nature with most antibodies being directed to a specific epitope.
  • epitopes which produce broadly neutralising antisera has been mapped to the CD4 binding site on the gp l 20 protein, as recognised by neutralising antibodies b l 2, VRCO l , HJ16 (Zhou T. et al., Science. 2010 Aug 13 ;329(5993):770-3), and to the V-loops, as recognised by neutralising antibodies PG9 and PG16 (Walker L M et al., PLoS Pathog. 2010 Aug 5;6(8)).
  • the problem Whilst focusing of the immune response has been observed in a few individuals, the problem remains as to how to elicit such a focussed antibody response using a vaccine composition.
  • the problem lies in how to present the epitope or epitopes of interest in the correct confirmation to focus the immune response, to avoid a polyclonal response and to generate neutralising antibodies.
  • Efforts have been made to focus the immune response, in particular with respect to HIV- 1 , where attempts have been made to constrain the structure with disulphide bridges so as to expose the CD4 binding site, or to transplant the binding site structure to other protein scaffolds, but all have proven unsuccessful.
  • An aim of the present invention is to provide one or more compositions that can be used to elicit a focussed, and preferably protective, immune response.
  • the invention provides an immunogenic composition
  • a first antigen i) a first antigen; and ii) a second antigen linked to a toxin, wherein the composition is capable of eliciting an immune response to the first antigen when administered to a human or non-human animal.
  • the second antigen is preferably generated by mutating the first antigen such that there is at least one different epitope between the first and second antigens.
  • the second antigen may be essentially equivalent to the first antigen, for example, an equivalent protein derived from a different but related organism or from an organism carrying a particular mutation, provided the first and second antigens differ in at least one epitope.
  • the second antigen could comprise one or more loop deletions from the first antigen.
  • the second antigen could be identical to the first antigen except that a cluster of amino acids in a single epitope, or in multiple epitopes, on the second antigen may have been changed relative to the first antigen.
  • a cluster of amino acids may comprise at least about 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids.
  • first and second antigens are proteins or peptides they have at least about 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or more sequence identity; preferably at least 75% sequence identity; preferably at least 80% sequence identity; preferably at least 85% sequence identity; preferably at least 90% sequence identity; preferably at least 95% sequence identity.
  • the percentage sequence identity between the first and second antigens is at least about 70%, 75%, 80%, 85%, 90%, 95%, 98% or more; preferably at least 80% sequence identity; preferably at least 85% sequence identity; preferably at least 90% sequence identity; preferably at least 95% sequence identity.
  • the percentage sequence identity between the first and second antigens is preferably at least about 60%, 70%, 75%, 80%), 85%) or more; preferably at least 70% sequence identity; preferably at least 75% sequence identity; preferably at least 80%) sequence identity.
  • 75% sequence identity allows there to be 3 or 4 amino acid changes.
  • the percent sequence identity is determined by standard alignment algorithms such as, for example, Basic Local Alignment Tool (BLAST) described in Altshul et al. (1990) J. Mol. Biol., 215 : 403-410, the algorithm of Needleman et al. (1970) J. Mol. Biol., 48 : 444-453, or the algorithm of Meyers et al. (1988) Comput. Appl. Biosci. , 4: 1 1 - 17.
  • BLAST Basic Local Alignment Tool
  • the first and second antigens are both proteins or peptides.
  • the first antigen may be a wild type protein or peptide, such as a surface protein, or part thereof, from a pathogen.
  • the first antigen may be a tumour antigen, or peptide derived thereform, associated with one or more cancers.
  • the first antigen could be a protein or peptide in a transition state and the second antigen may be the same protein or peptide in its native state.
  • the invention may be used to generate peptide/epitope specific B-cells, either in a human or a non-human animal, which may then be used for the production of therapeutic antibodies, in particular therapeutic monoclonal antibodies.
  • the invention may be used to generate tumour specific B-cells for the production of anti-cancer therapeutic monoclonal antibodies.
  • antibodies raised in this way display increased sensitivity to ADCC (antibody dependent cellular cytotoxicity) and/or promote an immune response to, for instance, the specific binding surface of a receptor coupling site, and not to the rest of the molecule. This may enhance the 'hit-rate' of useful hybridomas, and/or allow for the production of focussed 'polyclonal' antibody sera directed to a specific region of a protein or peptide.
  • ADCC antibody dependent cellular cytotoxicity
  • the invention may also be used to generate catalytic antibodies, in which case the first antigen may be the 'transition state' structure of the antigen, and the second antigen may be the native structure of antigen. This may be used to focus the immune response to the chemical moieties of the transition state groups.
  • the first antigen and/or the second antigen comprise more than one epitope.
  • the first antigen and/or second antigen may independently be isolated from a pathogen or tumour cell, produced recombinantly, or synthetically produced, for example produced by in vitro peptide synthesis or in vitro translation.
  • the first and/or second antigen may be a protein derived from a pathogen or disease state, such as cancer, or may be an analogue thereof.
  • An analogue may include a variant in which one or more residues have been added, deleted, inserted or substituted, while having no material effect on the function of the protein.
  • the addition, deletion, insertion or substitution of a residue may be sufficient to alter one or more of the epitopes presented in the folded protein.
  • a residue, or residues may be added or deleted from one or both end of a naturally occurring protein, or they may be inserted within the protein or substituted for one or more residues within the protein.
  • one or more protein residues may be added, deleted, inserted or substituted while still maintaining the function and structure of the protein, similarly these changes may or may not alter the immunogenic properties of the antigen.
  • the person of ordinary skill in the art would be able to determine what substitutions or changes would affect the immunogenic properties of the antigen.
  • a conservative substitution of one or more residues within a protein may result in a protein analogue.
  • a conservative substitution includes a substitution of one amino acid residue for another having one or more similar chemical properties, such as polarity, charge, hydrophobicity, or aromaticity, for example.
  • the first and/or second antigen may further comprise additional sequence, for example for use in purification of the antigen.
  • the antigen is a recombinant protein
  • the protein may include a tag, such as a his-tag sequence, for use in protein purification.
  • the toxin may be any toxin which on internalisation by a B-cell results in cell non- proliferation and/or cell death.
  • suitable toxins include auristatin, calicheamicin or PE38 (Pseudomonas exotoxin 38) domains 2 and 3.
  • the toxin and second antigen may be linked using any suitable means.
  • the toxin may be coupled to streptavidin and the second antigen may be biotinylated, the two may then be linked via the streptavidin biotin interaction.
  • the toxin may be coupled to biotin and the second antigen may be linked to an avidin group, the two may then be linked via the avidin biotin interaction.
  • the antigen may be expressed as a fusion protein with streptavidin, and a biotin- conjugated toxin may be linked via the streptavidin-biotin interaction to the antigen.
  • the toxin may be linked directly to the antigen using a specific chemical reaction, for example coupling to the antigen's lysine residues or glycan groups.
  • the first antigen may be the HIV- 1 gp l 20 protein, or an immunogenic part thereof.
  • the gp l 20 protein, or an immunogenic part thereof may be produced recombinantly.
  • An immunogenic part of the gp l 20 protein may include any part of the gp l 20 protein which is capable of eliciting an immune response when administered to a human or non-human animal.
  • the antigen may alternatively comprise an analogue of an immunogenic part of the gp l 20 protein.
  • the second antigen may be a mutated form of HIV- 1 gp l 20, or a part thereof, or a gp l 20 protein, or a part thereof, from a different viral strain.
  • the mutation may be in the CD4 binding site. Alternatively or additionally the mutation may be in a V-loop and/or in any other neutralisation target.
  • the second antigen comprises the mutation D368R in the CD4 binding site of the HIV- 1 gp l 20 protein.
  • the mutated gp l 20 protein is linked to the toxin PE38 domains 2 and 3.
  • the second antigen is a mutated form of the first antigen it has been mutated such as to change at least one epitope in the second antigen compared to the first antigen.
  • all B-cells recognising the second antigen are deleted.
  • This deletion preferably arises because upon internalisation of the second antigen the attached toxin causes the death, or at least the non-replication, of the B-cell.
  • the remaining B-cells proliferate and produce neutralising antibodies directed to the epitope or epitopes unique to the first antigen.
  • the production of neutralising antibodies can be engineered.
  • the antibodies may be essentially monoclonal and/or polyclonal/oligoclonal.
  • the antibodies are directed to the epitope in the first antigen which has been mutated in the second antigen.
  • the immune response elicited by a composition of the invention is effective against a pathogen from which the first antigen is derived, the immune response may also be effective against a related pathogen.
  • the immune response elicited by a composition of the invention affects the ability of a pathogen from which the antigen is derived to infect an immunised human or non-human animal.
  • the immune response elicited may impede or prevent the replication of a pathogen from which the first antigen is derived or a related pathogen.
  • the immune response elicited may recognise and destroy a pathogen from which the first antigen is derived or a related pathogen.
  • the immune response elicited may impede or prevent a pathogen from which the first antigen is derived, or a related pathogen, from causing disease.
  • the composition produces an antibody response when administered to human or non-human animals.
  • the immune response elicited produces substantially epitope-specific antibodies which are preferably substantially neutralising.
  • the antibodies are directed to an epitope in the first antigen that has been mutated in the second antigen.
  • the second antigen may be mutated at more than one site, this may result in multiple antibody responses to the non-mutated sites in the first (non- mutated) antigen.
  • the immune response elicited in response to the composition of the invention is a neutralising response.
  • the neutralising response produces antibodies that inhibit or neutralise the biological effect of an antigen, and hence of a pathogen if the antigen is derived from a pathogen.
  • the antibodies are active against a pathogen or disease state that expresses the first antigen.
  • the first antigen may be derived from any pathogenic organism, such as a virus, yeast or bacteria.
  • viruses that the composition of the invention could be used as a vaccine for include, HIV, SIV, H5N1 , influenza viruses, Dengue virus, Hepatitis viruses, Herpes viruses, human papilloma virus, pox viruses, polioviruses etc.
  • bacteria that the composition of the invention could be used as a vaccine for include, Pseudomonas aeringosa, Staphyloccous aureus, Neisseria meningitides, invention could be used to target include HLA-A2-NY-ESO- 1 , HLA-MAGE- 1/3 complexes, VEGF and VEGF receptor.
  • the invention can be readily applied to any disease antigen where the epitope or epitopes for producing a neutralising response are known or predicted.
  • the first antigen is preferably folded into its native configuration.
  • a composition of the invention may comprise a further one or more antigens.
  • the further antigens may be derived from the same or a different pathogen, or from the same or different disease state.
  • composition of the invention may further comprise an adjuvant, wherein an adjuvant enhances the protective efficacy of the composition.
  • adjuvants will be well known to those skilled in the art, and may include emulsifiers, muramyl dipeptides, avridine, aqueous adjuvants such as aluminium hydroxide, chitosan-based adjuvants, monophosphoryl lipid A and any of the various saponins, oils and other substances known in the art, such as, Amphigen, LPS, bacterial cell wall extracts, bacterial DNA, CpG sequences, synthetic oligonucleotides, and combinations thereof.
  • Suitable adjuvants can be formed with an oil component, such as a single oil, a mixture of oils, a water-in-oil emulsion, or an oil-in-water emulsion.
  • the oil may be mineral oil, vegetable oil, or animal oil.
  • Freund' s Complete Adjuvant (FCA) and Freund' s Incomplete Adjuvant (FIA) are two common adjuvants used in vaccine compositions, and are also suitable for use in this invention.
  • FCA Complete Adjuvant
  • FIA Freund' s Incomplete Adjuvant
  • the composition may also comprise polymers or other agents to control the consistency of the composition, or to control the release of the protein from the composition.
  • composition may also comprise other agents such as diluents, which may include water, saline, glycerol or other suitable alcohols etc; wetting or emulsifying agents; buffering agents; thickening agents for example cellulose or cellulose derivatives; preservatives; detergents; antimicrobial agents; and the like.
  • diluents which may include water, saline, glycerol or other suitable alcohols etc; wetting or emulsifying agents; buffering agents; thickening agents for example cellulose or cellulose derivatives; preservatives; detergents; antimicrobial agents; and the like.
  • the active ingredients in the composition are greater than 50% pure, usually greater than 80%> pure, often greater than 90%> pure and more preferably greater than 95%), 98%o or 99%> pure. With active ingredients approaching 100% pure, for example about 99.5% pure or about 99.9%> pure, being used most often.
  • the composition comprises an immunologically effective amount of a first and/or a second antigen, preferably of a first antigen.
  • An "immunologically effective amount" of an antigen is an amount that when administered to an individual, either in a single dose or in a series of doses, is effective for treatment or prevention of infection by a pathogen or related pathogen from which the antigen is derived or is effective for the treatment or prevention of a particular disease state. This amount will vary depending upon the health and physical condition of the individual to be treated and on the antigen. Determination of an effective amount of an immunogenic or vaccine composition for administration to an organism is well within the capabilities of those skilled in the art.
  • a composition according to the invention may be for oral, systemic, parenteral, topical, mucosal, intramuscular, intravenous, intraperitoneal, intradermal, subcutaneous, intranasal, intravaginal, intrarectal, transdermal, sublingual, inhalation or aerosol administration.
  • the composition may be arranged to be administered as a single dose or as part of a multiple dose schedule. Multiple doses may be administered as a primary immunisation followed by one or more booster immunisations. Suitable timings between priming and boosting immunisations can be routinely determined.
  • compositions according to the invention may be used in isolation, or it may be combined with one or more other immunogenic or vaccine compositions, and/or with one or more other therapeutic regimens.
  • Compositions of the invention may be able to induce neutralising antibody responses. These responses are conveniently measured in mice and the results are a standard indicator of vaccine efficacy.
  • compositions of the invention may also, or alternatively, be able to elicit an immune response which neutralises bacterial proteins or other molecules, thereby preventing them from having their normal function and preventing or reducing disease progression without necessarily destroying the pathogen.
  • composition may be used to elicit/produce a protective immune response when administered to a subject.
  • composition may be used as a prophylactic or a therapeutic vaccine directed to a pathogen, or a related pathogen, from which the antigen is derived or to a disease state, for example a cancer.
  • the invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising i) a first antigen; and ii) a second antigen linked to a toxin, and a pharmaceutically acceptable carrier or excipient.
  • the pharmaceutical composition comprises an immunogenic composition according to the first aspect of the invention.
  • the pharmaceutical composition is capable of producing a protective immune response.
  • Suitable excipients and carriers will be well known to those skilled in the art. These may include solid or liquid carriers. Suitable liquid carriers include water and saline.
  • the antigens of the composition may be formulated into an emulsion or they may be formulated into biodegradable microspheres or liposomes.
  • the present invention provides the use of an immunogenic composition
  • an immunogenic composition comprising i) a first antigen; and ii) a second antigen linked to a toxin in the preparation of a medicament for eliciting an immune response.
  • the medicament may be used for the prophylactic or therapeutic vaccination of subjects against pathogens, or pathogens related to the pathogen, from which the first antigen is derived.
  • the medicament may alternatively be used for the prophylactic or therapeutic vaccination of subjects against a disease state, such as cancer, from which the first antigen is derived.
  • the medicament may be a prophylactic or a therapeutic vaccine.
  • the present invention provides a method of protecting a human or non-human animal from the effects of infection by a pathogen comprising administering to the human or non-human animal a composition according to any other aspect of the invention.
  • the composition may be a vaccine.
  • the invention provides a method for raising an immune response in a human or non-human animal comprising administering a pharmaceutical composition according to the invention to the human or non-human animal.
  • the immune response is preferably protective.
  • the method may raise a booster respons e in a patient that has already been primed.
  • the immune response may be prophylactic or therapeutic.
  • One way to check the efficacy of a therapeutic treatment comprising administration of a composition according to the invention involves monitoring for pathogen infection after administration of the composition.
  • One way to check the efficacy of a prophylactic treatment comprising administration of a composition according to the invention involves monitoring immune responses to a pathogen from which the antigen is derived after administration of the composition.
  • the invention provides the use of an immunogenic composition comprising i) a first antigen; and ii) a second antigen linked to a toxin, in the preparation of a medicament for use in the immunisation of human or non-human mammals against infection by a pathogen, or related pathogen, from which the first antigen is derived.
  • the invention provides the use of an immunogenic composition comprising i) a first antigen; and ii) a second antigen linked to a toxin, in the preparation of a medicament for use in the immunisation of human or non-human mammals against a disease from which the first antigen is derived.
  • the invention provides a kit for use in inducing an immune response in an organism, comprising an immunogenic or vaccine composition according to the invention and instructions relating to administration.
  • the invention provides a method of eliciting an immune response in a subject comprising administering a first antigen and a second antigen linked to a toxin to the subject.
  • the first and second antigen may be as described herein.
  • the first antigen and second antigen may be administered in the same or separate compositions. If administered in separate compositions the compositions may be administered simultaneously or sequentially or separately.
  • the subject may be first administered the second antigen linked to a toxin to deplete circulating B-cells with the 'incorrect' specificities, and then subsequently administered either the first antigen alone, or a mix of the first antigen and the second antigen linked to a toxin.
  • the invention provides a method of eliciting an immune response in a subject comprising first administering to a subject a second antigen linked to a toxin, and then subsequently administering to the subject a first antigen.
  • administration of the second antigen linked to the toxin clears the subject of substantially all B cells which recognise the second antigen.
  • the first antigen is administered only B cells which recognise epitopes unique to the first antigen, which are not present on the second antigen, are activated.
  • the first antigen is administered at least 2 hours, preferably at least 4 hours, 8 hours, 12 hours, 24 hours or more after administration of the second antigen and toxin.
  • the first and second antigens may be as described above.
  • first and second antigens are administered simultaneously, or substantially simultaneously but in different compositions.
  • the invention provides a protein-toxin conjugate.
  • the toxin may be as discussed previously, and the protein may be any suitable protein, but preferably as described above with reference to the second antigen.
  • the protein-toxin conjugate serves to delete all B cells specific to the conjugated protein.
  • compositions according to the invention may be useful as diagnostic reagents and as a measure of the immune competence of a vaccine.
  • Figure 1A provides the wild type amino acid sequence of gp l 20 (Seq ID No: 1 ).
  • Figure IB provides the amino acid sequence of D368R (Seq ID No: 2) - the underlined amino acid indicates where the mutation has occurred.
  • Figure 1C provides the amino acid sequence of biotinylated D368R gp l 20 (Seq ID No: 3) - the underlined amino acid indicates where the mutation has occurred, the section underlined and in italics is the biotinylation tag (Avitag).
  • Figure 2 provides the amino acid sequence of His-streptavidin-PE38 Domains 2 and 3 (Seq ID No:4). The sequence in italics is the streptavidin, the sequence underlined is a linker, and the sequence in bold is PE-38 domains 3 and 3.
  • Figure 3 illustrates confocal microscopy images of CD4+ cells incubated with the gp l 20 protein.
  • PE-38 domains 2 and 3 ( Figure 2) can be readily expressed in E. coli using well known recombinant technology and purified for use. Examplary protocols for the preparation and purification of PE-38 domains 2 and 3 can be found in Onda et al Journal of Immunology, 2006, 177: 8822-8834 Pastan et al. Methods Mol. Biol., 2004, 248 : 503-518.
  • Figure 1 A provides the wild type amino acid sequence of mature gp l 20.
  • Figure I B provides the amino acid sequence of D368R gp l 20, this is a mutant form of Figure 1 , in which position 368 has been mutated to arginine.
  • Figure 1 C provides the amino acid sequence of biotinylated D368R gp l 20
  • Figure 3 illustrates by confocal microscopy that when CD4+ cells are incubated with the gp l 20 protein, the protein adheres to the cell and then is internalised.
  • the protein in visualised using an anti-His FITC-anti-mouse Fc antibody (the gp l 20 protein is his-tagged)
  • PE38 is complexed to gp l 20 using the biotin streptavidin interaction and incubated in vitro with B-cell lines and clones, CD4 T-cell lines and CD4-transfected Jurkat cells. Cell death is observed when the toxin complex is taken up by the cells.
  • the toxin is PE38.
  • Groups 8 and 9 in the immunisation lacking any WT gpl20, and containing only antigen linked to toxin, do not generate an immune response as all specific B-cells are deleted.
  • Group 1 in the immunisation generates a very polyclonal response.
  • Groups 2-7 in the immunisation generate more CD4 binding site-specific responses, compared to the WT control, and in particular the responses are increasingly focussed, and specific to the WT gpl20 CD4 binding site.
  • mice are immunised with OVA (ovalbumin), followed by 2 protein boosts with the D368R gp l 20-toxin .
  • OVA ovalbumin
  • CAF01 is used as an adjuvant.
  • ELISAs are used to determine the specificities of the immune responses
  • mice are first administered 10 ug D368R gp l 20-toxin. This is intended to clear the mouse of substantially all B cells which recognise D368R gp l 20 The same mice are then immunised 2 to 7 days later with WT gpl 20, only those B cells specific to the WT region of gp l 20 mutated in D368R gp l 20 are stimulated, resulting in a highly specific response.

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  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biochemistry (AREA)
  • Communicable Diseases (AREA)
  • Hematology (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Abstract

L'invention concerne une composition immunogène comprenant i) un premier antigène ; et ii) un second antigène lié à une toxine, la composition étant apte à susciter une réponse immunitaire au premier antigène lorsqu'elle est administrée à un être humain ou à un animal non humain. L'invention concerne également des utilisations de la composition immunogène et une composition pharmaceutique ou un coffret comprenant la composition immunogène.
PCT/GB2012/050802 2011-04-14 2012-04-12 Composition Ceased WO2012140424A2 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GBGB1106340.1A GB201106340D0 (en) 2011-04-14 2011-04-14 Composition
GB1106340.1 2011-04-14

Publications (2)

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WO2012140424A2 true WO2012140424A2 (fr) 2012-10-18
WO2012140424A3 WO2012140424A3 (fr) 2013-01-24

Family

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PCT/GB2012/050802 Ceased WO2012140424A2 (fr) 2011-04-14 2012-04-12 Composition

Country Status (2)

Country Link
GB (1) GB201106340D0 (fr)
WO (1) WO2012140424A2 (fr)

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6139843A (en) * 1991-04-02 2000-10-31 Albert Einstein College Of Medicine Of Yeshiva University Peptide compositions for the treatment of HIV
TR199701547T1 (xx) * 1995-06-07 1998-03-21 Smithkline Beecham Biologicals S.A. Polisakarit antijen protein e�leni�i i�eren a��.
GB0524408D0 (en) * 2005-11-30 2006-01-11 Glaxosmithkline Biolog Sa Vaccines
GB0524409D0 (en) * 2005-11-30 2006-01-11 Glaxosmithkline Biolog Sa Vaccines

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Title
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ZHOU T. ET AL., SCIENCE, vol. 329, no. 5993, 13 August 2010 (2010-08-13), pages 770 - 3

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GB201106340D0 (en) 2011-06-01
WO2012140424A3 (fr) 2013-01-24

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