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WO2008142483A2 - Compositions vaccinales et procédés d'utilisation de celles-ci - Google Patents

Compositions vaccinales et procédés d'utilisation de celles-ci Download PDF

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Publication number
WO2008142483A2
WO2008142483A2 PCT/IB2007/004614 IB2007004614W WO2008142483A2 WO 2008142483 A2 WO2008142483 A2 WO 2008142483A2 IB 2007004614 W IB2007004614 W IB 2007004614W WO 2008142483 A2 WO2008142483 A2 WO 2008142483A2
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Prior art keywords
antigen
composition
antibody
polypeptide
oligosaccharide
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PCT/IB2007/004614
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WO2008142483A3 (fr
Inventor
Gerard J. A. Rouwendal
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Plant Research International BV
IQ Corp BV
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Plant Research International BV
IQ Corp BV
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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
    • A61K39/385—Haptens or antigens, bound to carriers
    • 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
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • 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
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising 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
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins
    • A61K2039/6056—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
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6087—Polysaccharides; Lipopolysaccharides [LPS]
    • 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
    • A61K2039/62—Medicinal preparations containing antigens or antibodies characterised by the link between antigen and carrier
    • A61K2039/622—Medicinal preparations containing antigens or antibodies characterised by the link between antigen and carrier non-covalent binding
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the invention relates to compositions and methods of protein vaccines and their use in preventing and treating infection and diseases such as cancer. BACKGROUND OF THE INVENTION
  • Vaccinations involve the administration of one or more immunogens, in the form of live, but weakened (attenuated) infectious agents, which normally are either weaker, but closely- related species (as with smallpox and cowpox), strains weakened by some process or recombinant proteins. In such cases, an immunogen is called a vaccine.
  • Some modern vaccines are administered after the patient already has contracted a disease, as in the cases of experimental AIDS, cancer and Alzheimer's disease vaccines.
  • the present invention provides vaccine compositions containing an antigen having a oligosaccharide and one or more antibodies specific for the oligosaccharide.
  • the composition contains two, three, four, five or more antibodies specific for the oligosaccharide.
  • composition contains an antibody specific for the oligosaccharide specific antibody.
  • the antibody and the antigen form a complex.
  • the antibody is a monoclonal antibody, a polyclonal antibody, a fully human antibody or a humanized antibody.
  • the invention also provides vaccine compositions containing an antigen linked oligosaccharide, were the oligosaccharide is capable of binding a naturally occurring antibody in a subject.
  • the antigen and the antibody component of the vaccine composition are administered concurrently.
  • the antigen component is administered prior to or after the antibody component.
  • the oligosaccharide is directly linked to the antigen.
  • the antigen is operably linked to a second polypeptide having an oligosaccharide.
  • the oligosaccharide is covalently linked to the antigen or polypeptide.
  • the oligosaccharide is an O-linked or a N- linked glycan.
  • the N-linked glycan contains a core bound ⁇ l ,3 fucose, a core bound ⁇ 1 ,2 xylose, a Gal(alphal-3)Gal(betal-4)GlcNAcbetal-R epitope or a Gal(betal- 4)[Fuc(alphal-3)]GlcNAc-R epitope.
  • the antigen is a polypeptide, a carbohydrate or a lipid.
  • the antigen polypeptide and or the second polypeptide, or both is produced in a plant cell, plant part or plant, an animal cell, a fungal cell or a yeast cell.
  • Figure 1 is a line graph showing the lactoferrin-specific IgG titers in serum taken from mice immunized with LF plus alum. Each line represents one mouse.
  • Figure 2 is a line graph showing the antigen specific IgG titers in serum taken from mice immunized with immune complexes of anti-HRP and lactoferrin ( ⁇ ) or lactoferrin only (D). Each line represents one mouse.
  • Figure 3 is a line graph showing the IgG titers in serum taken from mice injected with anti-HRP. Serum from anti-HRP treated mice were tested against both antigens. Each line represents one mouse.
  • Figure 4 is a line graph showing the avidin-specif ⁇ c IgG titers in serum taken from mice immunized with avidin plus alum. Each line represents one mouse.
  • Figure 5 is a line graph showing the IgG titers to avidin in serum taken from mice immunized with immune complexes of anti-HRP and avidin ( ⁇ ) or avidin as such (D). Each line represents one mouse.
  • Figure 6 is a line graph showing the IgG titers to avidin in serum taken from mice immunized with immune complexes of anti-Lewis a and avidin ( ⁇ ) or avidin as such (D). Each line represents one mouse.
  • the invention is based in on the observation that vaccine efficacy can be enhanced by immunization with a vaccine containing an antigen and an antibody specific for the antigen. Practically, this has disadvantages as this requires the development of an antibody specific for every different antigen.
  • the present invention overcomes this disadvantage by generically modifying the antigen or using an antigen with a generic immunogenic moiety, thus allowing the use of a universal antibody in formulating the vaccine composition.
  • the invention provides an antigen containing a carbohydrate moiety (e.g., glycan) that is useful in combination with an antibody specific for that carbohydrate as a vaccine or a vaccine component.
  • the vaccines or vaccine components are useful in methods of inducing an immune response to the antigen in a subject, such as a human, or an animal such as a dog, cat, sheep, horse, cow, or pig.( i.e., immunization).
  • biological component any compound created by or associated with a cell, tissue, bacteria, virus, or other biological entity, including peptides, proteins, lipids, carbohydrates, hormones, or combinations thereof.
  • adjuvant compound is meant any compound that increases an immunogenic response or the immunogenicity of an antigen or vaccine.
  • Antigen is meant any compound capable of inducing an immune response.
  • Antigen Antigens are proteins, carbohydrates or lipids. Exemplary antigens include, toxins, bacteria, foreign blood cells, cancer cells and the cells of transplanted organs. Preferably, the antigen is Hepatitis C, HIV, Hepatitis B, Papilloma virus, Malaria, Tuberculosis, Herpes Simplex Virus, Chlamydia, and Influenza, or a biological component thereof, for example, a viral, bacterial or other polypeptide.
  • immunoglobulin is meant a any polypeptide or protein complex that is secreted by B-cells or B-cell fusions and that functions as an antibody in the immune response by binding with a specific antigen.
  • Immunoglobulins as used herein include IgA, IgD, IgE, IgG, and IgM. Regions of immunoglobulins include the Fc region and the Fab region, as well as the heavy chain or light chain immunoglobulins.
  • antigen presentation is meant the expression of an antigen on the surface of a cell in association with one or more major hisocompatability complex class I or class II molecules.
  • Antigen presentation is measured by methods known in the art. For example, antigen presentation is measured using an in vitro cellular assay as described in Gillis, et al., J. Immunol. 120: 2027 1978.
  • glycoprotein is meant a compound containing carbohydrate (or glycan) covalently linked to protein.
  • proteoglycans is meant a subclass of glycoproteins in which the carbohydrate units are polysaccharides that contain amino sugars. Such polysaccharides are also known as “glycosaminoglycans ".
  • glycopeptide is meant a compound including a carbohydrate linked to an oligopeptide composed of L- and/or D-amino acids.
  • glyco-amino-acid is a saccharide attached to a single amino acid by any kind of covalent bond.
  • glycosyl-amino- acid is a compound consisting of saccharide linked through a glycosyl linkage (O-, N- or S-) to an amino acid. (The hyphens are needed to avoid implying that the carbohydrate is necessarily linked to the amino group.)
  • peptidoglycan' is meant a glycosaminoglycan formed by alternating residues of D-glucosamine and either muramic acid ⁇ 2-amino-3-0-[(R)-l-carboxyethyl]-2-deoxy-D- glucose ⁇ or L-talosaminuronic acid (2-amino-2-deoxy-L-taluronic acid), which are usually N-acetylated or N-glycoloylated.
  • the carboxyl group of the muramic acid is commonly substituted by a peptide containing residues of both L- and D-amino acids, whereas that of L- talosaminuronic acid is substituted by a peptide consisting of L-amino acids only.
  • glycolipd is meant a lipid and containing one or more molecules of a covalently attached sugar.
  • Glycolipids include for example, glycosphingolipids, glycosyl phosphatidylinositols, mannosylphosphoryl dolichol, glucosylphophoryl dolichol, or oligosaccharyl phosphoryl dolichols
  • immunogenicity is meant the ability of a substance to stimulate an immune response. Immunogenicity is measured, for example, by determining the presence of antibodies specific for the substance. The presence of antibodies is detected by methods know in the art, for example, an ELISA assay.
  • immune response is meant a cellular activity induced by an antigen, such as production of antibodies or presentation of antigens or antigen fragments.
  • the immune response can be divided into several phases - the "innate" first response, mediated by cells able to destroy and phagocytose (engulf) a large range of foreign organisms; the secondary, “adaptive” response, characterized by the generation of antibodies and T cells that are specific for the antigen; and a third, “suppression” phase, where the production of immune cells reverts to normal (homeostasis), and the information necessary to mount a future immune response to that antigen is retained in bone marrow memory cells.
  • oligosaccharide is meant a saccharide polymer containing of component sugars, also known as simple sugars. Oligosaccharides are either O- or N-linked to compatible amino acid side chains in proteins or to lipid moieties.
  • proteolytic degradation is meant degradation of the polypeptide by hydrolysis of the peptide bonds. No particular length is implied by the term “peptide.” Proteolytic degradation is measured, for example, using gel electrophoresis.
  • the "cell” includes any cell capable of antigen presentation.
  • the cell is a somatic cell, a B-cell, a macrophage or a dendritic cell.
  • the invention provides an antigen containing a carbohydrate moiety.
  • An antigen includes any compound, cell or tissue to which an immune response is desired.
  • An antigen includes any substance that, when introduced into the body, stimulates an immune response, such as the production of an antibody from a B cell, activation and expansion of T cells, and cytokine expression (e.g., interleukins).
  • B cell or “B lymphocyte” is meant an immune cell that is responsible for the production of antibodies.
  • T cell or “T lymphocyte” is meant a member of a class of lymphocytes, further defined as cytotoxic T cells, helper T cells and regulatory T-cells. T cells regulate and coordinate the overall immune response, identifying the epitopes that mark the antigens, and attacking and destroying the diseased cells they recognize as foreign, or offering help for the induction of cells that attack and destroy or produce antibody.
  • the carbohydrate moiety may be in the form of a monosaccharide, disaccharide(s). oligosaccharide(s), polysaccharide(s), or their derivatives (e.g. sulfo- or phospho-substituted).
  • the carbohydrate is linear or branched.
  • the carbohydrate moiety is N-linked or O-linked to a polypeptide. N-linked glycosylation is to the amide nitrogen of asparagine side chains and O-linked glycosylation is to the hydroxy oxygen of serine and threonine side chains.
  • the carbohydrate moiety is endogenous to the subject being vaccinated. Alternatively, the carbohydrate moiety is exogenous to the subject being vaccinated. All that is requiresd is that the an antibody can bind the carbohydrate moiety.
  • the carbohydrate moiety are carbohydrate moieties that are not expressed on polypeptides or lipids of the subject being vaccinated.
  • the carbohydrate moieties are plant-specific carbohydrates. Plant specific carbohydrate moieties include for example N-linked glycan having a core bound ⁇ l ,3 fucose or a core bound ⁇ 1 ,2 xylose.
  • the carbohydrate moiety contains an alpha gal epitope which is abundantly synthesized on glycolipids and glycoproteins of non-primate mammals and New World monkeys by the glycosylation enzyme alpha l,3galactosyltransferase.
  • Alpha gal epitopes include the Gal(alphal -3)Gal(betal-4)GlcNAcbetal-R epitope or the Gal(betal- 4)[Fuc(alphal-3)]GlcNAc-R epitope.
  • the carbohydrate moiety are carbohydrate moieties that are expressed on polypeptides or lipids of the subject being vaccinate. For example many host cells have been genetically engineered to produce human proteins with human-like sugar attachments.
  • the carbohydrate moiety is linked directly to the antigen.
  • the antigen is a polypeptide the amino acids residues of the antigen are modified to carry the carbohydrate moiety.
  • the antigen is linked, e.g., covalently linked to a second reagent, e.g., a polypeptide or lipid containing a carbohydrate moiety.
  • the second reagent is a glycoprotein, a glycopeptides, glycosyl-amino- acid , a glyco-amino-acid, a peptidoglycan, proteoglycans or a glycolipid.
  • An antigen directly carrying carbohydrate moiety or an antigen linked to a second reagent containing an carbohydrate moiety are refered to herein as an "the antigen moiety".
  • the antigen is linked to the second reagent.
  • “Operatively linked” is intended to indicate that the antigen and second reagent are chemically linked (e.g., via a covalent bond such as a peptide bond)
  • the antigen and the second reagent are linked in a manner that allows for glycosylation of the second reagent.
  • the term operatively linked means that a nucleic acid encoding the antigen polypeptide and the non-antigen polypeptide are fused in-frame to each other.
  • the second reagent is fused to the N-terminus or C-terminus of the antigen polypeptide.
  • the second reagent is linked to the antigen via a covalent bond such as a peptide bond.
  • the antigen is fused to the N-terminus or C-terminus of second reagent.
  • the antigen is fused to an internal amino acid of second reagent.
  • internal amino acid is meant an amino acid that is not at the N-terminal or C-terminal of a polypeptide.
  • the antigen is operably linked to the second reagent, most typically via a covalent bond such as a peptide bond.
  • the antigen moiety is linked to one or more additional moieties.
  • the antigen moiety may additionally be linked to a GST fusion protein in which the mucin-Ig fusion protein sequences are fused to the C-terminus of the GST (i.e., glutathione
  • Such fusion proteins can facilitate the purification of the antigen polypeptide.
  • antibody refers to immunoglobulin molecules and immunologically active portions of immunoglobulin (Ig) molecules, i.e., molecules that contain an antigen binding site that specifically binds (immunoreacts with) an antigen.
  • immunoglobulin immunoglobulin
  • Such antibodies include, but are not limited to, polyclonal, monoclonal, chimeric, single chain, F at> , F ab ' and F( at> ') 2 fragments, and an F ab expression library.
  • an antibody molecule relates to any of the classes IgG, IgM, IgA, IgE and IgD, which differ from one another by the nature of the heavy chain present in the molecule. Certain classes have subclasses as well, such as IgGi, IgG 2 , and others. Furthermore, the light chain may be a kappa chain or a lambda chain. Reference herein to antibodies includes a reference to all such classes, subclasses and types of antibody species. Antibodies that immunospecifically bind the antigen are prepared using standard techniques for polyclonal and monoclonal antibody preparation. The full-length antigen can be used or, alternatively, the invention provides antigenic fragments of the antigen for use as immunogens.
  • any antibody can be used regardless of the method used to generate the antibody.
  • Various procedures known within the art may be used for the production of polyclonal or monoclonal antibodies directed against a protein of the invention, or against derivatives, fragments, analogs homologs or orthologs thereof (see, for example, Antibodies: A Laboratory Manual, Harlow E, and Lane D, 1988, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, incorporated herein by reference). Some of these antibodies are discussed below.
  • the vaccination methods of the present invention employ the use of naturally occurring antibodies already present in the subject to be immunized. For example, the subject may have circulating antibodies against a particular carbohydrate moiety.
  • polyclonal Antibodies For the production of polyclonal antibodies against carbohydrate moieties, various suitable host animals (e.g., rabbit, goat, mouse, fish, birds or other mammal) may be immunized by one or more injections with the native protein carrying a carbohydrate moiety, a synthetic variant thereof, or a derivative of the foregoing.
  • suitable host animals e.g., rabbit, goat, mouse, fish, birds or other mammal
  • the carbohydrate may be conjugated to a protein known to be immunogenic in the mammal being immunized.
  • immunogenic proteins to which the carbohydrate moiety is attached include but are not limited to keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, and soybean trypsin inhibitor.
  • the preparation can further include an adjuvant.
  • adjuvants used to increase the immunological response include, but are not limited to, Freund's (complete and incomplete), mineral gels (e.g., aluminum hydroxide), surface active substances (e.g., lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, dinitrophenol, etc.), adjuvants usable in humans such as Bacille Calmette-Guerin and Corynebacterium parvum, or similar immunostimulatory agents.
  • Additional examples of adjuvants which can be employed include MPL-TDM adjuvant (monophosphoryl Lipid A, synthetic trehalose dicorynomycolate) and CpG dinucleotide motifs (Krieg, A.M. Biochim Biophys Acta
  • the carbohydrate antibody may be a pre-formed naturally occurring antibody that is already present in the subject's blood.
  • the polyclonal antibody molecules directed against the carbohydrate moiety can be isolated from the mammal (e.g., from the blood) and further purified by well known techniques, such as affinity chromatography using protein A or protein G, which provide primarily the IgG fraction of immune serum.
  • the specific antigen which is the target of the immunoglobulin sought, or an epitope thereof may be immobilized on a column to purify the immune specific antibody by immunoaffinity chromatography. Purification of immunoglobulins is discussed, for example, by D. Wilkinson (The Engineer, published by The Engineer, Inc., Philadelphia PA, Vol. 14, No. 8 (April 17, 2000), pp. 25-28).
  • MAb monoclonal antibody
  • CDRs complementarity determining regions
  • Monoclonal antibodies can be prepared using hybridoma methods, such as those described by Kohler and Milstein, Nature, 256:495 ( 1975).
  • a hybridoma method a mouse, hamster, or other appropriate host animal, is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the carbohydrate moiety.
  • the lymphocytes can be immunized in vitro.
  • peripheral blood lymphocytes are used if cells of human origin are desired, or spleen cells or lymph node cells are used if non-human mammalian sources are desired.
  • the lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986) pp. 59-103).
  • Immortalized cell lines are usually transformed mammalian cells, particularly myeloma cells of rodent, bovine and human origin. Usually, rat or mouse myeloma cell lines are employed.
  • the hybridoma cells can be cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, immortalized cells.
  • a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, immortalized cells.
  • the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine ("HAT medium"), which substances prevent the growth of HGPRT-deficient cells.
  • HAT medium hypoxanthine, aminopterin, and thymidine
  • Preferred immortalized cell lines are those that fuse efficiently, support stable high level expression of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium. More preferred immortalized cell lines are murine myeloma lines, which can be obtained, for instance, from the SaIk Institute Cell Distribution Center, San Diego, California and the American Type Culture Collection, Manassas, Virginia.
  • Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies (Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc., New York, (1987) pp. 51-63).
  • the culture medium in which the hybridoma cells are cultured can then be assayed for the presence of monoclonal antibodies directed against the antigen.
  • the binding specificity of monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA). Such techniques and assays are known in the art.
  • the binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson and Pollard, Anal. Biochem., 107:220 (1980).
  • antibodies having a high degree of specificity and a high binding affinity for the target antigen are isolated.
  • the clones can be subcloned by limiting dilution procedures and grown by standard methods. Suitable culture media for this memepose include, for example, Dulbecco's Modified Eagle's Medium and RPMI- 1640 medium. Alternatively, the hybridoma cells can be grown in vivo as ascites in a mammal.
  • the monoclonal antibodies secreted by the subclones can be isolated or purified from the culture medium or ascites fluid by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.
  • the monoclonal antibodies can also be made by recombinant DNA methods, such as those described in U.S. Patent No. 4,816,567.
  • DNA encoding the monoclonal antibodies of the invention can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies).
  • the hybridoma cells of the invention serve as a preferred source of such DNA.
  • the DNA can be placed into expression vectors, which are then transfected into host cells such as simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells.
  • host cells such as simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells.
  • the DNA also can be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains in place of the homologous murine sequences (U.S. Patent No. 4,816,567; Morrison, Nature 368, 812-13 (1994)) or by covalently joining to the immunoglobulin coding sequence all or part of the coding sequence for a non- immunoglobulin polypeptide.
  • non-immunoglobulin polypeptide can be substituted for the constant domains of an antibody of the invention, or can be substituted for the variable domains of one antigen-combining site of an antibody of the invention to create a chimeric bivalent antibody.
  • the antibodies directed against the carbohydrate moiety can further comprise humanized antibodies or human antibodies. These antibodies are suitable for administration to humans without engendering an immune response by the human against the administered immunoglobulin.
  • Humanized forms of antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab')? or other antigen- binding subsequences of antibodies) that are principally comprised of the sequence of a human immunoglobulin, and contain minimal sequence derived from a non-human immunoglobulin.
  • Humanization can be performed following the method of Winter and co- workers (Jones et al., Nature, 321 :522-525 (1986); Riechmann et al., Nature, 332:323-327 ( 1988); Verhoeyen et al., Science, 239: 1534- 1536 ( 1988)), by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. (See also U.S. Patent No. 5,225,539.) In some instances, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies can also comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences.
  • the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the framework regions are those of a human immunoglobulin consensus sequence.
  • the humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin (Jones et al., 1986; Riechmann et al., 1988; and Presta, Curr. Op. Struct. Biol., 2:593-596 (1992)).
  • Fc immunoglobulin constant region
  • Fully human antibodies relate to antibody molecules in which essentially the entire sequences of both the light chain and the heavy chain, including the CDRs, arise from human genes. Such antibodies are termed "human antibodies", or “fully human antibodies” herein.
  • Human monoclonal antibodies can be prepared by the trioma technique; the human B-cell hybridoma technique (see Kozbor, et al., 1983 Immunol Today 4: 72) and the EBV hybridoma technique to produce human monoclonal antibodies (see Cole, et al., 1985 In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96).
  • Human monoclonal antibodies may be utilized in the practice of the present invention and may be produced by using human hybridomas (see Cote, et al., 1983. Proc Natl Acad Sci USA 80: 2026-2030) or by transforming human B-cells with Epstein Barr Virus in vitro (see Cole, et al., 1985 In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96).
  • human antibodies can also be produced using additional techniques, including phage display libraries (Hoogenboom and Winter, J. MoI. Biol., 227:381 (1991); Marks et al., J. MoI. Biol., 222:581 (1991)).
  • human antibodies can be made by introducing human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire. This approach is described, for example, in U.S. Patent Nos. 5,545,807; 5,545,806;
  • Human antibodies may additionally be produced using transgenic nonhuman animals which are modified so as to produce fully human antibodies rather than the animal's endogenous antibodies in response to challenge by an antigen.
  • This animal produces B cells which secrete fully human immunoglobulins.
  • the antibodies can be obtained directly from the animal after immunization with an immunogen of interest, as, for example, a preparation of a polyclonal antibody, or alternatively from immortalized B cells derived from the animal, such as hyb ⁇ domas producing monoclonal antibodies. Additionally, the genes encoding the immunoglobulins with human variable regions can be recovered and expressed to obtain the antibodies directly, or can be further modified to obtain analogs of antibodies such as, for example, single chain Fv molecules An example of a method of producing a nonhuman host, exemplified as a mouse, lacking expression of an endogenous immunoglobulin heavy chain is disclosed in U.S. Patent No. 5,939,598.
  • a method for producing an antibody of interest such as a human antibody, is disclosed in U.S. Patent No. 5,916,771. It includes introducing an expression vector that contains a nucleotide sequence encoding a heavy chain into one mammalian host cell in culture, introducing an expression vector containing a nucleotide sequence encoding a light chain into another mammalian host cell, and fusing the two cells to form a hybrid cell.
  • the hybrid cell expresses an antibody containing the heavy chain and the light chain.
  • vectors preferably expression vectors, containing a nucleic acid encoding the antigen moieties, or derivatives, fragments, analogs or homologs thereof.
  • the vector contains a nucleic acid encoding an antigen moiety or derivatives, fragments analogs or homologs thereof.
  • vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • plasmid which refers to a circular double stranded DNA loop into which additional DNA segments are ligated.
  • viral vector Another type of vector, wherein additional DNA segments are ligated into the viral genome.
  • vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors).
  • Other vectors e.g., non-episomal mammalian vectors
  • certain vectors are capable of directing the expression of genes to which they are operatively-linked. Such vectors are referred to herein as "expression vectors”.
  • expression vectors of utility in recombinant DNA techniques are often in the form of plasmids.
  • plasmid and "vector” is used interchangeably as the plasmid is the most commonly used form of vector.
  • vector e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses
  • viral vectors e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses
  • the recombinant expression vectors of the invention comprise a nucleic acid of the invention in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vectors include one or more regulatory sequences, selected on the basis of the host cells to be used for expression, that is operatively-linked to the nucleic acid sequence to be expressed.
  • "operably-linked" is intended to mean that the nucleotide sequence of interest is linked to the regulatory sequence(s) in a manner that allows for expression of the nucleotide sequence (e.g., in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell).
  • regulatory sequence is intended to includes promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are described, for example, in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN
  • Regulatory sequences include those that direct constitutive expression of a nucleotide sequence in many types of host cell and those that direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, etc.
  • the expression vectors of the invention are introduced into host cells to thereby produce proteins or peptides.
  • the recombinant expression vectors of the invention are designed for expression of antigen moieties in prokaryotic or eukaryotic cells.
  • vaccines are expressed in bacterial cells such as Escherichia coli, insect cells (using baculovirus expression vectors) yeast cells, or mammalian cells (e.g., CHO cells) .
  • bacterial cells such as Escherichia coli, insect cells (using baculovirus expression vectors) yeast cells, or mammalian cells (e.g., CHO cells) .
  • Suitable host cells are discussed further in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990).
  • the recombinant expression vector is transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase.
  • Fusion vectors add a number of amino acids to a protein encoded therein, usually to the amino terminus of the recombinant protein.
  • Such fusion vectors typically serve three purposes: (/) to increase expression of recombinant protein; (//) to increase the solubility of the recombinant protein; and (Ui) to aid in the purification of the recombinant protein by acting as a ligand in affinity purification.
  • a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant protein to enable separation of the recombinant protein from the fusion moiety subsequent to purification of the fusion protein.
  • enzymes, and their cognate recognition sequences include Factor Xa, thrombin and enterokinase.
  • Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith and Johnson, 1988.
  • GST glutathione S-transferase
  • E. coli expression vectors examples include pTrc (Amrann et al, (1988) Gene 69:301-315) and pET 1 Id (Studier et al, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990) 60-89).
  • One strategy to maximize recombinant protein expression in E. coli is to express the protein in a host bacteria with an impaired capacity to proteolytically cleave the recombinant protein. See, e.g., Gottesman, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990) 1 19-128.
  • Another strategy is to alter the nucleic acid sequence of the nucleic acid to be inserted into an expression vector so that the individual codons for each amino acid are those preferentially utilized in E. coli ⁇ see, e.g., Wada, et al, 1992. Nucl. Acids Res. 20: 21 1 1-21 18). Such alteration of nucleic acid sequences of the invention is carried out by standard DNA synthesis techniques.
  • the vaccine or adjuvant polypeptide expression vector is a yeast expression vector.
  • yeast Saccharomyces ce ⁇ visae examples include pYepSec l (Baldari, et al., 1987. EMBO J. 6: 229-234), pMFa (Kurjan and Herskowitz, 1982. Cell 30: 933-943), pJRY88 (Schultz et al., 1987. Gene 54: 1 13-123), pYES2 (Invitrogen Corporation, San Diego, Calif.), and picZ (InVitrogen Corp, San Diego, Calif.).
  • yeast expression systems include Pichia pastoris.
  • vaccine are expressed in insect cells using baculovirus expression vectors.
  • Baculovirus vectors available for expression of proteins in cultured insect cells ⁇ e.g., SF9 cells) include the pAc series (Smith, et al., 1983. MoI Cell. Biol. 3: 2156-2165) and the pVL series (Lucklow and Summers, 1989. Virology 170: 31-39).
  • vaccine polypeptides are expressed in plant cells.
  • the polynucleotide construct is preferable constructed such that it comprises at least and in operable linkage a first promoter that is functional in plants, a nucleotide sequence encoding a protein antigen, and a terminator.
  • the polynucleotide may comprise a gene sequence encoding a selectable or screenable trait operably linked to regulatory sequences for expression.
  • a viral promoter such as the promoter from cassava vein mosaic virus (CVMV) or the promoter from cauliflower mosaic virus (CMV).
  • CVMV cassava vein mosaic virus
  • CMV cauliflower mosaic virus
  • any promoter that provides for expression such as the 35S or the enhanced 35S promoter may be used.
  • the recombinant gene constructs may be inserted into a vector, which may be commercially available, suitable for transforming into plants and suitable for expression of the gene product in the transformed cells.
  • a vector which may be commercially available, suitable for transforming into plants and suitable for expression of the gene product in the transformed cells.
  • binary vectors such as pMOG22, known from Goddijn, O.J.M. et al., 1993, Plant J, 4:863-873 which are useful for plant transformation using Agrobacterium.
  • any transformation method may be used to introduce chimeric DNA according to the invention into a suitable ancestor cell.
  • Methods may suitably be selected from the calcium/polyethylene glycol method for protoplasts, electroporation of protoplasts, microinjection into plant material, (DNA or RNA-coated) particle bombardment of various plant material, infection with (non-integrative) viruses, in planta Agrobacterium twnefaciens mediated gene transfer by infiltration of adult plants or transformation of mature pollen or microspores (EP 0 301 316) and the like.
  • a preferred method according to the invention comprises Agrobacterium-mediated DNA transfer.
  • the so- called binary vector technology as disclosed in EP 0 120 516 and U.S. Patent 4,940,838.
  • a method for production of a transgenic plant or plant part according to the invention may comprise the step of selecting transformed plants or plant parts. Generally after transformation, plant cells or cell groupings are selected for the transfer with the polynucleotide construct comprising the DNA-sequence with the genes encoding the various enzymes, followed by steps know to the skilled person by which the transformed material is regenerated into a whole plant and evaluating the transformed plant for the production of the (glyco)protein antigen. Selectable markers, which may be included as a part of the introduced recombinant
  • DNA are used to select transformed cells (those containing recombinant DNA) over untrans formed cells.
  • suitable markers include genes that provide antibiotic or herbicide resistance. Cells containing the recombinant DNA are capable of surviving in the presence of antibiotic or herbicide concentrations that kill untransformed cells.
  • selectable marker genes include the bar gene which provides resistance to the herbicide Basta; the nptll gene which confers kanamycin resistance; the hpt gene which confers hygromycin resistance; and the cah gene which gives resistance to cyanamid.
  • An entire plant can be generated from a single transformed plant cell through cell culturing techniques known to those skilled in the art.
  • a process for obtaining a transgenic plant according to the invention may in an alternative embodiment comprise introducing a vector according to the invention into an ancestor plant, and then producing said transgenic plant from said ancestor plant.
  • Yet another alternative embodiment for obtaining a transgenic plant according to the invention may comprise introducing a polynucleotide construct according to the invention into a suitable vector for transforming a plant part to produce a transformed plant part, and then regenerating said transgenic plant from said transformed plant part.
  • putatively transformed plants may be evaluated, for instance using Southern analysis, for the presence of the recombinant DNA according to the invention, copy number and/or genomic organization.
  • expression levels of the newly introduced DNA may be undertaken, using Northern and/or Western analysis, techniques well known to persons having ordinary skill in the art.
  • the nucleic acid encoding the antigen proteins may be 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, or in the case of an RNA viral vector, the necessary elements for replication and translation, as well as selectable markers.
  • an appropriate expression vector i.e., a vector which contains the necessary elements for the transcription and translation of the inserted coding sequence, or in the case of an RNA viral vector, the necessary elements for replication and translation, as well as selectable markers.
  • These include but are not limited to a promoter region, a signal sequence, 5' untranslated sequences, initiation codon depending upon whether or not the structural gene comes equipped with one, and transcription and translation termination sequences. Methods for obtaining such vectors are known in the art (see WO 01/29242 for review). Promoter sequences suitable for expression in plants are described in the art, e.g. WO
  • 91/198696 include non-constitutive promoters or constitutive promoters , such as, the nopaline synthetase and octopine synthetase promoters, cauliflower mosaic virus (CaMV) 19S and 35S promoters and the figwort mosaic virus (FMV) 35 promoter (see U.S. Pat. Nos. 5, 352,605 and 6,051,753, both of which are hereby incorporated by reference). Promoters used may also be tissue specific promoters targeted for example to the endosperm, aleurone layer, embryo, pericarp, stem, leaves, tubers, roots, etc.
  • promoters used may also be tissue specific promoters targeted for example to the endosperm, aleurone layer, embryo, pericarp, stem, leaves, tubers, roots, etc.
  • a signal sequence allows processing and translocation of a protein where appropriate.
  • the signal can be derived from plants or could be non-plant signal sequences.
  • the signal peptides direct the nascent polypeptide to the endoplasmic reticulum, where the polypeptide subsequently undergoes post-translational modification.
  • Signal peptides can routinely be identified by those of skill in the art. They typically have a tripartite structure, with positively charged amino acids at the N-terminal end, followed by a hydrophobic region and then the cleavage site within a region of reduced hydrophobicity.
  • the transcription termination is routinely at the opposite end from the transcription initiation regulatory region. It may be associated with the transcriptional initiation region or from a different gene and may be selected to enhance expression.
  • An example is the NOS terminator from Agrobacterium Ti plasmid and the rice alpha-amylase terminator.
  • Polyadenylation tails may also be added. Examples include but are not limited to Agrobacterium octopine synthetase signal, (Gielen et al., 1984, EMBO J. 3:835-846) or nopaline synthase of the same species (Depicker et al., 1982, MoI. Appl. Genet. 1 :561-573).
  • Enhancers may be included to increase and/or maximize transcription of the heterologous protein. These include, but are not limited to peptide export signal sequence, codon usage, introns, polyadenylation, and transcription termination sites ( see WO 01/29242).
  • Markers include preferably prokaryote selectable markers. Such markers include resistance toward antibiotics such as ampicillin, tetracycline, kanamycin, and spectinomycin. Specific examples include but are not limited to streptomycin phosphotransferase (spt) gene coding for streptomycin resistance, neomycin phosphotransferase (nptll) gene encoding kanamycin or geneticin resistance, hygromycin phosphotransferase (hp ⁇ gene encoding resistance to hygromycin.
  • the vectors constructed may be introduced into the plant host system using procedures known in the art (reviewed in WO 01/29242 and WO 01/31045).
  • the vectors may be modified to intermediate plant transformation plasmids that contain a region of homology to an Agrobacterium tumefaciens vector, a T-DNA border region from A. tumefaciens.
  • the vectors used in the methods of the present invention may be Agrobacterium vectors. Methods for introducing the vectors include but are not limited to microinjection, velocity ballistic penetration by small particles with the nucleic acid either within the matrix of small beads or particles, or on the surface and electroporation.
  • the vector may be introduced into a plant cell, tissue or organ. In a specific embodiment, once the presence of a heterologous gene is ascertained, a plant may be regenerated using procedures known in the art.
  • the presence of desired proteins may be screened using methods known in the art, preferably using screening asays where the biologically active site is detected in such a way as to produce a detectable signal.
  • This signal may be produced directly or indirectly.
  • assays include ELISA or a radioimmunoassay.
  • a nucleic acid of the invention is expressed in mammalian cells using a mammalian expression vector.
  • mammalian expression vectors include pCDM8 (Seed, 1987. Nature 329: 840) and pMT2PC (Kaufman, et al, 1987. EMBOJ. 6: 187-195).
  • the expression vector's control functions are often provided by viral regulatory elements.
  • commonly used promoters are derived from polyoma, adenovirus 2, cytomegalovirus, and simian virus 40.
  • the invention pertains to host cells into which a recombinant expression vector of the invention has been introduced.
  • host cell and “recombinant host cell” are used interchangeably herein. It is understood that such terms refer not only to the particular subject cell but also to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.
  • a host cell is any prokaryotic or eukaryotic cell.
  • the adjuvant polypeptides and or vaccines are expressed in bacterial cells such as E. coli, insect cells, yeast or mammalian cells (such as human, Chinese hamster ovary cells (CHO) or COS cells).
  • bacterial cells such as E. coli, insect cells, yeast or mammalian cells (such as human, Chinese hamster ovary cells (CHO) or COS cells).
  • Other suitable host cells are known to those skilled in the art.
  • Vector DNA is introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques.
  • transformation and transfection are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, agrobacterium mediate tranformationor electroporation. Suitable methods for transforming or transfecting host cells are found in Sambrook, et al. (MOLECULAR CLONING: A LABORATORY MANUAL. 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y., 1989), and other laboratory manuals.
  • A. tumefaciens and A. rhizogenes are plant pathogenic soil bacteria which genetically transform plant cells.
  • the Ti and Ri plasmids of A. tumefaciens and A. rhizogenes respectfully, carry genes responsible for genetic transformation of plants. See, for example, Kado, Crit. Rev. Plant Sci., 10: 1-32 ( 1991 ). Descriptions of the Agrobacterium vector systems and methods for Agrobacterium- mediated gene transfer are provided in Gruber et al., supra; and Moloney, et al, Plant Cell Reports, 8: 238-242 ( 1989).
  • a gene that encodes a selectable marker (e.g., resistance to antibiotics) is generally introduced into the host cells along with the gene of interest.
  • selectable markers include those that confer resistance to drugs, such as G418, hygromycin and methotrexate.
  • Nucleic acid encoding a selectable marker is introduced into a host cell on the same vector as that encoding the vaccines containing mucin fusion polypeptides, or are introduced on a separate vector. Cells stably transfected with the introduced nucleic acid are identified by drug selection (e.g., cells that have incorporated the selectable marker gene will survive, while the other cells die).
  • a host cell of the invention such as a prokaryotic or eukaryotic host cell in culture, is used to produce (i.e., express) adjuvant polypeptides and or vaccines.
  • the invention further provides methods for producing adjuvant polypeptides and or vaccines using the host cells of the invention.
  • the method includes culturing the host cell of invention (into which a recombinant expression vector encoding adjuvant polypeptides and or vaccines has been introduced) in a suitable medium such that adjuvant polypeptides and or vaccines s is produced.
  • the method further includes isolating adjuvant polypeptides and or vaccines from the medium or the host cell.
  • the antigen moieties are isolated and purified in accordance with conventional conditions, such as extraction, precipitation, chromatography, affinity chromatography, electrophoresis or the like.
  • antigen moieties according to the invention are chemically synthesized using methods known in the art. Chemical synthesis of polypeptides is described in, e.g., A variety of protein synthesis methods are common in the art, including synthesis using a peptide synthesizer. See, e.g., Peptide Chemistry, A Practical Textbook, Bodasnsky, Ed. Springer- Verlag, 1988; Merrifield, Science 232: 241-247 ( 1986); Barany, et al, Intl. J. Peptide Protein Res. 30: 705-739 ( 1987); Kent, Ann. Rev. Biochem. 57:957-989 ( 1988), and Kaiser, et al, Science 243: 187-198 (1989).
  • the polypeptides are purified so that they are substantially free of chemical precursors or other chemicals using standard peptide purification techniques.
  • the language "substantially free of chemical precursors or other chemicals” includes preparations of peptide in which the peptide is separated from chemical precursors or other chemicals that are involved in the synthesis of the peptide.
  • the language “substantially free of chemical precursors or other chemicals” includes preparations of peptide having less than about 30% (by dry weight) of chemical precursors or non-peptide chemicals, more preferably less than about 20% chemical precursors or non-peptide chemicals, still more preferably less than about 10% chemical precursors or non-peptide chemicals, and most preferably less than about 5% chemical precursors or non-peptide chemicals.
  • Macrocyclization is often accomplished by forming an amide bond between the peptide N- and C-termini, between a side chain and the N- or C-terminus [e.g., with K 3 Fe (CN) 6 at pH 8.5] (Samson et al., Endocrinology, 137: 5182-5185 ( 1996)), or between two amino acid side chains. See, e.g., DeGrado, Adv Protein Chem, 39: 51- 124 (1988). Disulfide bridges are also introduced into linear sequences to reduce their flexibility.
  • compositions can be formulated in pharmaceutical compositions.
  • These compositions may comprise, in addition to one of the above substances, a pharmaceutically acceptable excipient, carrier, buffer, stabilizer or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient.
  • a pharmaceutically acceptable excipient e.g. oral, intravenous, cutaneous or subcutaneous, nasal, intramuscular, intraperitoneal or patch routes.
  • compositions for oral administration may be in tablet, capsule, powder or liquid form.
  • a tablet may include a solid carrier such as gelatin or an adjuvant.
  • Liquid pharmaceutical compositions generally include a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. Physiological saline solution, dextrose or other saccharide solution or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included.
  • the active ingredient will be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability.
  • isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection, Lactated Ringer's Injection.
  • Preservatives, stabilizers, buffers, antioxidants and/or other additives may be included, as required.
  • administration is preferably in a "prophylactically effective amount” or a "therapeutically effective amount” (as the case may be, although prophylaxis may be considered therapy), this being sufficient to show benefit to the individual.
  • a prophylaxis may be considered therapy
  • the actual amount administered, and rate and time-course of administration, will depend on the nature and severity of what is being treated. Prescription of treatment, e.g. decisions on dosage etc, is within the responsibility of general practitioners and other medical doctors, and typically takes account of the disorder to be treated, the condition of the individual patient, the site of delivery, the method of administration and other factors known to practitioners. Examples of the techniques and protocols mentioned above can be found in REMINGTON'S PHARMACEUTICAL SCIENCES, 16th edition, Osol, A. (ed), 1980.
  • targeting therapies may be used to deliver the active agent more specifically to certain types of cell, by the use of targeting systems such as antibody or cell specific ligands. Targeting may be desirable for a variety of reasons; for example if the agent is unacceptably toxic, or if it would otherwise require too high a dosage, or if it would not otherwise be able to enter the target cells.
  • these agents could be produced in the target cells by expression from an encoding gene introduced into the cells, e.g. in a viral vector (a variant of the VDEPT technique - see below).
  • the vector could be targeted to the specific cells to be treated, or it could contain regulatory elements, which are switched on more or less selectively by the target cells.
  • the agent could be administered in a precursor form, for conversion to the active form by an activating agent produced in, or targeted to, the cells to be treated.
  • an activating agent produced in, or targeted to, the cells to be treated.
  • This type of approach is sometimes known as ADEPT or VDEPT; the former involving targeting the activating agent to the cells by conjugation to a cell-specific antibody, while the latter involves producing the activating agent, e.g. a vaccine or fusion protein, in a vector by expression from encoding DNA in a viral vector (see for example, EP-A-415731 and WO 90/07936).
  • the vaccines of the present invention also include one or more adjuvant compounds.
  • Adjuvant compounds are useful in that they enhance long term release of the vaccine by functioning as a depot. Long term exposure to the vaccine should increase the length of time the immune system is presented with the antigen for processing as well as the duration of the antibody response.
  • the adjuvant compound also interacts with immune cells, e.g., by stimulating or modulating immune cells. Further, the adjuvant compound enhances macrophage phagocytosis after binding the vaccine as a particulate (a carrier / vehicle function).
  • Adjuvant compounds useful in the present invention include Complete Freund's Adjuvant (CFA); Incomplete Freund's Adjuvant (IFA); Montanide ISA (incomplete seppic adjuvant); Ribi Adjuvant System (RAS); TiterMax; Syntex Adjuvant Formulation (SAF); Aluminum Salt Adjuvants; Nitrocellulose-adsorbed antigen; Encapsulated or entrapped antigens; Immune-stimulating complexes (ISCOMs); and Gerbu R adjuvant.
  • CFA Complete Freund's Adjuvant
  • IFA Incomplete Freund's Adjuvant
  • Montanide ISA incomplete seppic adjuvant
  • Ribi Adjuvant System Ribi Adjuvant System
  • TiterMax Syntex Adjuvant Formulation
  • SAF Syntex Adjuvant Formulation
  • Aluminum Salt Adjuvants Nitrocellulose-adsorbed antigen; Encapsulated or entrapped antigens
  • Immune-stimulating complexes ISCOMs
  • Gerbu R adjuvant Gerbu
  • the vaccine compositions of the invention do not require additional adjuvant compounds due to the vaccines enhanced function.
  • the vaccines of the present invention have superior immunoprotective and immunotherapeutic properties over other vaccine lacking antibodies.
  • the vaccines have enhanced immunogenicity, safety, tolerability and efficacy.
  • the enhanced immunogenicity of the vaccine of the present invention may be greater than comparative non- carbohydrate antibody-containing vaccines by 1.5-fold, 2-fold, 3-fold, 5-fold, 10-fold, 20- fold, 50-fold, 100-fold or more, as measured by stimulation of an immune response such as antibody production and/or secretion, activation and expansion of T cells, and cytokine expression (e.g., production of interleukins).
  • the invention may reduce the number of vaccinations in a vaccination regimen (i.e., one shot instead of multiple shots).
  • the invention provides a method of immunization, e.g., inducing an immune response, of a subject.
  • a subject is immunized by administration to the subject a first composition containing an antigen moiety described above and a second composition containing a anti-carbohydrate antibody.
  • the first composition and second composition are administered simultaneously.
  • the first composition is administered prior to or after the second composition.
  • the subject is at risk of developing or suffering from an infection, e.g., bacterial, viral or fungal. Infections include, Hepatitis C, HIV, Hepatitis B, Papilloma virus, Malaria, Tuberculosis, Herpes Simplex Virus, Chlamydia, or Influenza, including Influenza A virus subtype H5N1.
  • the subject is at risk of developing or suffering from cancer or any other condition that requires vaccination.
  • the cancer is for example breast, lung, colon, prostate, pancreatic, cervical cancer or melanoma.
  • the methods described herein lead to a reduction in the severity or the alleviation of one or more symptoms of a infection, cancer, Alzheimer's disease, autoimmune disorders such as multiple sclerosis, allergy, dental caries, drug addiction (Hatsukami et al., Clin. Pharmacol. Ther. 78,) weight gain (Zorrilla et al., PNAS 103, 13226-13231, 2006) or any other disease treatable by immunotherapy.
  • a subject requiring immunization is identified by methods know in the art. For example subjects are immunized as outlined in the CDCs General Recommendation on Immunization (51(RR02) ppl-36) Cancer is diagnosed for example by physical exam, biopsy, blood test, or x-ray.
  • the subject is e.g., any mammal, e.g., a human, a primate, mouse, rat, dog, cat, cow, horse, pig, a fish or a bird.
  • the treatment is administered prior to diagnosis of the infection or disorder. Alternatively, treatment is administered after diagnosis. Efficaciousness of treatment is determined in association with any known method for diagnosing or treating the particular disorder or infection. Alleviation of one or more symptoms of the disorder indicates that the compound confers a clinical benefit.
  • the invention provides a method of increasing or stimulating production of antigen- specific B and T cells (e.g., cytotoxic T-cells) and/or secretion of antibodies directed against a target of interest.
  • the cell is an antibody forming cell such as a B-cell.
  • the cell is a cell that augments antibody production by a B cell such as a T-cell (Th and Tc), macrophage, dendritic cell.
  • Antibody secretion is increased by administering a subject a vaccine including an antigen moiety and an anti-carbohydrate antibody or an antigen moiety capable of binding a naturally occurring anti-carbohydrate antibody.
  • Antibody secretion can be increased by stimulating B cells and or T-cells (e.g., helper T-cells). Once activated T cells then stimulate B cells. Increased antibody production and/or secretion is measured by methods known to those of ordinary skill in the art, including ELISA, the precipitin reaction, and agglutination reactions.
  • the invention provides a method of activating or stimulating an immune cell (e.g., a B cell ,a T cell, or an antigen presenting cell).
  • T cell activation is defined by an increase in calcium mediated intracellular cGMP, or an increase in cell surface receptors for IL-2, CD25, CD 30 or CD69.
  • an increase in T cell activation is characterized by an increase of calcium mediated intracellular cGMP and or IL-2 receptors following contacting the T cell with the vaccine, compared to in the absence of the vaccine.
  • Intracellular cGMP is measured, for example, by a competitive immunoassay or scintillation proximity assay using commercially available test kits.
  • Cell surface IL-2 receptors are measured, for example, by determining binding to an IL-2 receptor antibody such as the PC61 antibody. Immune cell activation can also be determined by measuring B cell proliferative activity, polyclonal immunoglobulin (Ig) production, and antigen-specific antibody formation by methods known in the art. Activation of antigen-presenting cells ( macrophages, B-lympocytes, and all cells expressing MHC class II and or class I) is assessed for example by measuring MHC class I or II and or CD80 or CD86 expression
  • EXAMPLE 1 The following experiment shows whether immune complexes (ICs) consisting of plant-produced N-glycosylated lactoferrin and polyclonal rabbit anti-horseradish peroxidase (HRP) antibodies that recognize certain epitopes of these lactoferrin N-glycans will enhance antibody responses to the polypeptide part of the antigen compared to antibody responses using N-glycosylated lactoferrin admininistered as such. It is known that rabbit anti-HRP recognize plant N-glycans containing core-bound 01 ,2-xylose or core-bound ⁇ l ,3-fucose. (Faye et al., Anal Biochem 209, 104-108, 1993).
  • mice Female, specific pathogen-free BALB/c mice (6 weeks old) were obtained from Charles River (Sulzfeld, Germany). They were maintained under barrier conditions in filter-topped macrolon cages with wood chips bedding, at a mean temperature of 23 ⁇ 2°C, 50-55% relative humidity, and 12 h light/dark cycle. Drinking water and standard laboratory food pellets were provided ad libitum. Mice were allowed to settle for a week before start of experiment. The experiments were conducted according to the guidelines of the Animal Experiments Committee of the Veterinary Faculty, Utrecht University (Utrecht, The Netherlands).
  • Antigen for immunization Recombinant, rice-produced human lactoferrin (LF): Sigma-Aldrich under no. L4040.
  • Antigens for ELISA Human lactoferrin: Sigma-Aldrich under no. L0520.
  • Antibodies Rabbit anti-horseradish peroxidase (aHRP): Sigma-Aldrich under no. P7899.
  • Adjuvant Alum: Imject from Pierce (77161), Rockford, IL, USA. Stock solutions were as follows (all in sterile tissue culture grade bi-distilled water):
  • Anti-HRP 2 ml vial, estimated protein content 12 mg/ml, ouchterlony: 1 : 16 vs HRP (1 mg/ml).
  • ELISA-protocol 1 ELISA was used to determine the serum titers of antigen-specific IgG. Costar 3590 hibond microtiter plates were coated overnight at 4 °C with lactoferrin (20 ⁇ g/ml, lOO ⁇ l/well, Sigma-Aldrich L0520) in PBS.
  • IgG levels in serum taken at day 15 were determined. Alum-precipitated antigens induce high antibody titers.
  • Anti-HRP alone did not elicit a specific immune response to avidin of lactoferrin
  • Antibodies used to form complexes with lactoferrin and/or avidin did not elicit antibody responses to the antigens (Figure 3).
  • mice Female, specific pathogen-free BALB/c mice (6 weeks old) were obtained from Charles River (Sulzfeld, Germany). They were maintained under barrier conditions in filter-topped macrolon cages with wood chips bedding, at a mean temperature of 23 ⁇ 2°C, 50-55% relative humidity, and 12 h light/dark cycle. Drinking water and standard laboratory food pellets were provided ad libitum. Mice were allowed to settle for a week before start of experiment. The experiments were conducted according to the guidelines of the Animal Experiments Committee of the Veterinary Faculty, Utrecht University (Utrecht, The Netherlands).
  • Antigen for immunization Recombinant, egg white avidin (Av) produced in maize: Sigma-Aldrich under no. A8706.
  • Antigens for ELISA Egg white avidin: Sigma-Aldrich under no. A9275.
  • Antibodies Rabbit anti-horseradish peroxidase (aHRP): Sigma-Aldrich under no.P7899 Adjuvant:-A ⁇ um: Imject from Pierce (77161 ), Rockford, IL, USA. Stock solutions were as follows (all in sterile tissue culture grade bi-destilled water):
  • Anti-HRP 2 ml vial, estimated protein content 12 mg/ml, ouchterlony: l : 16 vs HRP (l mg/ml).
  • ELISA was used to determine the serum titres of antigen-specific IgG. Coastar 3590 hibond microtiter plates were coated overnight at 4 °C with avidin (20 ⁇ g/ml, 100 ⁇ l/well, Sigma-Aldrich A9275) in PBS. 2. Wells were blocked for 1 h at RT with 150 ⁇ l 0.5% BSA in Tris buffer (5OmM
  • Tris 137 mM NaCl, 2 mM EDTA, 0.5% Tween, pH 7.2
  • IgG levels in serum taken at day 15 were determined.
  • Alum-precipitated antigens induce very high antibody titers.
  • immune complexes of maize-produced N-glycosylated avidin with anti-N- glycan antibodies in the form of rabbit anti-HRP are highly immunogenic, when compared to non-antibody-conjugated antigens. Immune responses might even be higher if optimal ratios of antigen and antibodies would be used.
  • mice Female, specific pathogen-free BALB/c mice (6 weeks old) were obtained from Charles River (Sulzfeld, Germany). They were maintained under barrier conditions in filter-topped macrolon cages with wood chips bedding, at a mean temperature of 23 ⁇ 2°C, 50-55% relative humidity, and 12 h light/dark cycle. Drinking water and standard laboratory food pellets were provided ad libitum. Mice were allowed to settle for a week before start of experiment. The experiments were conducted according to the guidelines of the Animal Experiments Committee of the Veterinary Faculty, Utrecht University (Utrecht, The Netherlands). Antigens and antibodies and preparation of immune complexes
  • Antigen for immunization Recombinant, maize-produced egg white avidin (Av): Sigma-Aldrich under no. A8706.
  • Antibodies Monoclonal anti-Lewis a , clone Tl 74 (aLew): Abeam ab3356, lot no. 015K4817.
  • Adjuvant - A ⁇ um: Imject from Pierce (77161 ), Rockford, IL, USA. Stock solutions were as follows (all in sterile tissue culture grade bi-destilled water):
  • Anti-Lewis a 0.1 mg/ml, contain NaN3 and 1% BSA, recommended dilution 1/40. After mixing as indicated in Table 3, mixtures were incubated for 1 h at 37°C. Then, saline was added to get a final volume of 1 ml. Per animal 100 ⁇ l was injected at tail base. Alum-precipitated avidin was used to obtain antigen-specific positive serum. Alum was added drop wise to antigen solutions, in ratio 1 : 1. After addition of alum the mixture was incubated for 30 minutes while gently shaken.
  • ELISA was used to determine the serum titres of antigen-specific IgG. Coastar 3590 hibond microtiter plates were coated overnight at 4 0 C with avidin (20 ⁇ g/ml, lOO ⁇ l/well, Sigma- Aldrich A9275) in PBS.
  • IgG levels in serum taken at day 15 were determined. Alum-precipitated antigens induce very high antibody titers.
  • immune complexes of maize-produced N-glycosylated avidin with mouse monoclonal anti-Lewis a are highly immunogenic, when compared to non-antibody- conjugated antigens. Immune responses might even be higher if optimal ratios of antigen and antibodies would be used.

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Abstract

La présente invention concerne des compositions vaccinales et des procédés d'immunisation.
PCT/IB2007/004614 2006-12-14 2007-12-14 Compositions vaccinales et procédés d'utilisation de celles-ci Ceased WO2008142483A2 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011047794A3 (fr) * 2009-10-21 2011-10-13 Eth Zurich Utilité médicale des protéines liant les glycanes et des glycanes

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0153871A3 (fr) * 1984-03-01 1987-07-01 Centocor, Inc. Augmentation de l'immunogénéité d'un antigène par anticorps
GB9606425D0 (en) * 1996-03-27 1996-06-05 Binding Site Ltd Improvements in and relating to the production of antibodies and test kits incorporating antibodies
US20070202117A1 (en) * 2005-12-22 2007-08-30 Herman Groen Compositions and Methods Of Modulating the Immune Response

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011047794A3 (fr) * 2009-10-21 2011-10-13 Eth Zurich Utilité médicale des protéines liant les glycanes et des glycanes
JP2013508319A (ja) * 2009-10-21 2013-03-07 イーティーエイチ・チューリッヒ グリカン結合タンパク質およびグリカンの医学的有用性
EP2494980A3 (fr) * 2009-10-21 2013-03-13 ETH Zurich Utilitaire médical de glycanes
JP2015096501A (ja) * 2009-10-21 2015-05-21 イーティーエイチ・チューリッヒ グリカン結合タンパク質およびグリカンの医学的有用性

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